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New views, inputs, and properties: a new look at the renin-angiotensin system

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Content

NEW VIEWS, INPUTS, AND PROPERTIES:

A NEW LOOK AT THE RENIN-ANGIOTENSIN SYSTEM

by

Jung Julie Kang

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(SYSTEMS BIOLOGY AND DISEASES)

May 2008

Copyright 2008 Jung Julie Kang
ii

Epigraph

Although the world is full of suffering, it is full also of the overcoming of it.
Helen Keller
All great things must first wear terrifying and monstrous masks in order to inscribe
themselves on the hearts of humanity.
Friedrich Nietzsche
The eye is meant to see things. The soul is here for its own joy.
Rumi
There are two mistakes one can make along the road to truth...not going all the way,
and not starting.
Buddha
In all things of nature there is something of the marvelous.
Aristotle
Faith is taking the first step, even when you don't see the whole staircase.
Martin Luther King, Jr.
Education is the most powerful weapon which you can use to change the world.
Nelson Mandela
You must be the change you wish to see in the world.
Mahatma Gandhi
iii

Dedication

I could not have made it through this journey without the support of my
family. Daddy, Mommy, Miyun, and Johnny, you gave me unyielding and
unconditional love when I felt lost or hopeless. Each of you is such an inspirational
person and I am so grateful and proud to be in the same family. I have so much
respect for you and trust you so much that when you seemed to have faith in me, I
found the faith and strength to keep moving forward.

Ildiko, your extraordinary mind won me over from the beginning, but your
heart and soul made you like a sister to me. You taught me so much, we shared so
much, and I still think that it’s mostly your fault. But, thank you.

To my friends Nancy, Tina, Sheila, Akane, Jaime, and Jasmin for sharing the
good times that helped me through the bad ones and for believing in me.
iv

Acknowledgements

I would like to thank all of the members of my dissertation committee (Sarah
Hamm-Alvarez, David Hinton, Alicia McDonough, Janos Peti-Peterdi) for offering
me the guidance to follow this project further than I could have imagined it would go
at its inception. I deeply appreciate their encouragement not only of the work, but
also their support of my education and growth as a scientist.
I would also like to thank my lab for their support. To my mentor, Janos, for
giving me so many amazing learning opportunities and the challenges to become a
better person now than when I started. I would also especially like to thank Ildiko
Toma, Sarah Vargas, and Elliott Meer. We have all helped each other so much and
I’m grateful to have worked together on some truly special projects.
I have received permission from the publishers to reprint in my dissertation:

Kang JJ, Toma I, Sipos A, Meer EJ, Vargas SL, Peti-Peterdi J. 2008. The Collecting
Duct is the Major Source of Prorenin in Diabetes. Hypertension. In Press.

Kang, J.J., Toma, I., Sipos, A., and Peti-Peterdi, J. 2008. From In Vitro to In Vivo:
Imaging from the Single Cell to the Whole Organism. Current Protocols in
Cytometry. In Press.

Kang, J.J., Toma, I., Sipos, A., McCulloch, F., and Peti-Peterdi, J. 2006. Imaging the
renin-angiotensin system: an important target of anti-hypertensive therapy. Adv.
Drug Deliv. Rev. 58:824-833.

Kang, J.J., Toma, I., Sipos, A., McCulloch, F., and Peti-Peterdi, J. 2006. Quantitative
Imaging of basic functions in renal (patho)physiology. Am. J. Physiol. Renal.
Physiol. 291:F495–F502.

Toma I, Kang JJ, Peti-Peterdi J. Imaging renin content and release in the living
kidney. Nephron Physiol. 2006; 103:71-74.

February, 2008

v

TABLE OF CONTENTS

Epigraph ii

Dedication iii

Acknowledgements iv

List of Figures vi

Abstract vii

Preface ix

Introduction 1
Introduction Endnotes 14

Chapter 1: Multiphoton in vivo imaging to study the kidney in hypertension 15
Abstract 15
Introduction 17
Materials and Methods 20
Results 24
Discussion 35
References 41

Chapter 2: Sugar metabolism directly activates the renin-angiotensin system 44
Introduction 45
Results 48
Discussion 61
Methods 67
References 74

Chapter 3: Prorenin: new beginnings in the renin-angiotensin system 79
Abstract 79
Introduction 81
Materials and Methods 84
Results 88
Discussion 100
References 105

Conclusion 109
Conclusion Endnotes 121

Alphabetized Bibliography 123
vi

List of Figures

Introduction, Figure 1: The Renin-Angiotensin System in vitro 4

Introduction, Figure 2: The Renin-Angiotensin System 5

Introduction, Figure 3: Single nephron glomerular filtration rate in vivo 9

Introduction, Figure 4: Renin release signals 10

Conclusion, Figure 5: Schematic of Metabolic Activation of RAS 113
Conclusion, Figure 6: Uric acid causes renin release via the macula densa 114

Conclusion, Figure 7: A new in vivo view of RAS: 115
Macula dena and the (pro)renin receptor
Conclusion, Figure 8: Prorenin in the collecting duct, in vivo 117
vii

Abstract
Hypertension is called a silent killer because there are no early symptoms and
it can lead to cardiovascular disease, the leading cause of morbidity worldwide.
There is no cure, but several classes of medications target the renin-angiotensin
system (RAS), a signaling pathway with paramount short and long-term influence
over blood pressure. Renin is a hormone enzyme which controls RAS and leads to
the generation of Ang II, a peptide that regulates blood pressure and electrolyte
balance. RAS activity is tightly controlled to maintain homeostasis, so hypertension
can be taken as a sign of RAS dysregulation due to the loss of inhibition or excessive
stimulation. High sugar diets especially predispose individuals to a form of
hypertension which is responsive to, or whose onset can be delayed by, RAS
inhibition. Accordingly, we hypothesized that there must be a direct mechanism by
which sugar metabolism causes pathological RAS activation. Since the kidney is a
primary target of sugar and blood pressure disorders, the effects of both on
molecular, cellular, tissue, and organ function in the kidneys were examined. We
applied multi-photon fluorescence microscopy to study otherwise undetectable
elements of RAS activity. Chapter 1 characterizes an in vivo imaging based
approach to evaluate renal physiological functions in healthy and hypertensive
animals. Multiphoton microscopy detected perturbations in renin content and
release, single nephron glomerular filtration rate, and cortical blood flow as early
indicators of hypertension even before the onset of symptoms or sequelae. In
Chapter 2, imaging was used to assess the hypertensive and RAS-activating capacity
viii

of a dietary sugar metabolite, uric acid. Determining the mechanism behind the uric
acid-RAS interaction revealed the capability of an environmental factor to
commandeer a physiological pathway to cause pathology. Chapter 3 examines
prorenin, an overlooked component of RAS whose regulation has inauspicious
consequences for persistent RAS activation in a setting of metabolic disequilibrium.
Therefore, this study showed the direct influence of an environmental factor (diet) on
physiological signaling and its potential implications for disease, prompting
reconsideration of the classical understanding of RAS. The kidney is both a cause
and casualty of hypertension, and a critical site for the activity and control of outside
influences on blood pressure.
ix

Preface
The Scientific Revolution began with a restructuring of the universe,
removing the earth from its center to replace it with the sun. As new ideas surfaced,
innovative perspectives for understanding the world came to light. The scientific
method came to prominence, a philosophy which navigated to truth by identifying a
problem, acquiring information, formulating/investigating a hypothesis, and drawing
conclusions which would drive new hypotheses. If the intention is to find answers
changing the body of knowledge, then its achievement inspires new questions. In
modern society, some of the greatest challenges involve discovering the causes of,
and inventing the cures for, disease. Success manifests in an explanation as much as
a solution, revealing previously unknown truths about the way nature works.
If revolutions come first to bring change, I welcome the present challenge to
make a difference and look forward to the future that will have been inspired. Many
of the largest problems facing the world today (global warming, the loss of natural
resources, HIV/AIDS, cancer, etc) may have their solutions in improving our
knowledge and application of science. This process of pursuing a doctor of
philosophy has ignited many revolutions for me. Through some trials, I learned and
accomplished what I had hoped to gain. Other situations forced me to dig deep and
grow. I came to understand that any problem has both a reward and a lesson, if you
look for them. I’ve learned that if you can walk away with an injury and a little more
insight, then you are still moving forward. I have come to believe that one person
can make a difference, and that means that every single person counts.
1

Introduction
Problems in Hypertension
The term epidemic is derived from the Greek words for “upon” and “people,”
and is used to describe a disease which afflicts a population at a greater rate than
would be expected. Historically, it has been used to characterize contagious diseases
which spread due to insufficiencies in both knowledge of the disease and the societal
resources to control the magnitude of infection. Similarly, present-day epidemics
could also be attributed to an incomplete understanding of disease etiology and thus
concomitant social failures in implementing the appropriate preventive measures.
One of the most formidable health challenges of the twentieth century is the
epidemic of hypertension, which has no known cause in the vast majority of cases
and still has no permanent cure. The World Health Organization estimates that there
are at least 600 million hypertension sufferers worldwide.
1
It is not a communicable
disease, but its increased incidence and prevalence in the modern era suggest a role
for environmental, controllable influences in spreading the condition.
Hypertension is a disease of permanently increased blood pressure at a level
which causes organ pathology and increases the probability of cardiovascular events.
Persistent hypertension is one of the most significant risk factors for strokes, heart
attacks, and chronic renal failure. Although it is a comorbidity of other life-
threatening conditions, even moderate elevations of blood pressure alone may
shorten life expectancy.
2
Hypertension can be classified as either essential (primary)
or secondary. Secondary hypertension indicates that the high blood pressure is a
2

result of another problem, such as kidney disease or certain tumors. Essential
hypertension accounts for 90-95% of cases and has no specific medical explanation.
Although the primary insult in essential hypertension is unknown, many
factors are believed to contribute to its progression, including salt sensitivity,
genetics, age, and renin homeostasis. Salt restriction is a common component of
treatment, as salt promotes water retention and thus increases the pressure on blood
vessel walls. However, the control of salt intake is insufficient to permanently
overcome the predisposition towards disease. Genetic heritability in hypertension is
a common clinical observation, and there is a 30% correlation with family history.
3

Nevertheless, in genome-wide scans attempting to implicate specific candidates, it
has been difficult to identify individual genes that are sufficiently influential and
conserved across populations to explain the hypertension statistics. Most studies
suggest that inheritance is most likely multifactorial and that a combination of
genetic defects culminates in elevated blood pressure.
4
Although disease prevalence
increases in older populations, aging appears to be more of an aggravating factor
than a causative factor. In the United States, 1 in 3 adults is hypertensive, but 1 in 5
of all Americans is afflicted, indicating a significant disease presence even in
younger populations.
1
Renin is an enzyme known to have both short and long-term
influences on blood pressure by producing hormones that increase both intravascular
pressure and blood volume. Therefore, renin is a strong candidate for the
pathogenesis of hypertension.

3

The kidney is the organ responsible for the maintenance of blood pressure
and volume homeostasis by filtering the blood, regulating urine production, and
synthesizing hormones which signal vasoconstriction or salt-volume retention. They
readily respond to structural, chemical, and hormonal cues of imbalance, so healthy
kidneys are able to detect disturbances and restore equilibrium. In other words,
intact renal function should serve as the body’s innate remedy for the correction and
prevention of hypertension.
5
However, once hypertension has been sustained,
progressive renal injury impairs the body’s internal defense against the development
of disease and permits unchecked systemic damage. Therefore, protection of the
kidneys is important to preserve the body’s capacity to maintain healthy blood
pressure levels. However, early injuries and dysregulation of renal function in
hypertension suggest that perhaps the kidneys are not merely a target of the disease
but also a factor initiating or exacerbating it. Furthermore, the kidneys are the major
source of renin, a critical enzyme in one of the most significant acute and chronic
regulators of blood pressure, the renin-angiotensin system.
The Canonic Renin-angiotensin System
The renin-angiotensin system (RAS) is one of the most important regulatory
mechanisms of renal tubular salt and water conservation, as well as systemic blood
pressure equilibrium.
6
Tissue RAS components exist in the eyes, ovaries, and brain,
but the constituent with the definitive role in regulating blood pressure is the RAS in
the kidneys. The major structural component of RAS in the kidney is the
juxtaglomerular apparatus ( JGA) which is located at the entry point of the systemic
4

circulation into the basic filtration unit, the nephron. Renin- producing cells reside in
the JGA together with cells of the macula densa, endothelium, vascular smooth
muscle cells, and extraglomerular mesangial cells. The multiple cell types of the
JGA communicate with each
other to perform a complex
array of functions to regulate
filtration, renal hemodynamics
(tubuloglomerular feedback),
and the activity of the renin-
angiotensin system (renin
release).
7
Renin is a hormone Figure 1. The Juxtaglomerular Apparatus (JGA) in vitro.
enzyme which initiates a cascade generating peptides that directly regulate blood
pressure, cell growth, apoptosis, and electrolyte balance. Because of its role as a key
mediator of renal influences on bodily homeostasis, details of the pathway of renin
release have been extensively studied using electron microscopy,
radioimmunoassays, patch-clamp techniques, and multiphoton imaging.
Although the cause of hypertension is unknown in most cases, a universal
final messenger for increased blood pressure is often Angiotensin II (Ang II), one of
the most potent vasoconstrictive peptides known and the final product of RAS.
Although Ang II is the predominant effector molecule, renin is the critical regulator
of the pathway. As the rate-limiting step of RAS activation, renin release from the
JGA is highly regulated. Reductions in renal perfusion pressure, activation of the
5

sympathetic nervous system, local hormones, or reductions in macula densa salt
transport may all promote renin release into the circulation and interstitium. The
released renin may then cleave its substrate, mainly liver-derived angiotensinogen, to
produce Ang I. The lung-derived angiotensin-converting enzyme (ACE) then
converts Ang I to the pathogenic
product Ang II, which has
omnipresent effector sites that
directly cause systemic
vasoconstriction (an immediate
response) and increased blood
volume by sodium/fluid
Figure 2. The Renin-angiotensin system (RAS). retention (a long-term response).
Therefore, RAS can be considered a mode of communication for the endocrine
kidney or the humoral mediator of renal influences over systemic blood pressure. In
turn, abnormal blood pressure can be seen as a sign of RAS dysregulation. Because
of its potency to increase blood pressure, RAS activity is tightly controlled
internally: the end-product Ang II has negative feedback on renin, to prevent its own
formation. However, in hypertension, RAS is often hyperactive because of
persistent stimulation or its unresponsiveness to inhibition.
Most of the current anti-hypertensive therapies target reducing Ang II levels.

ACE inhibitors prevent Ang II production and Ang II receptor blockers (ARBs)

prevent its effects at its receptor. Because renin is the rate-limiting enzyme in this
6

signaling cascade, control of renin activity might be a more efficient and effective
means of limiting pathogenicity via Ang II. However, while the available
pharmacotherapies work systemically and are very useful to disease management,
the gold standard of care is diuretics, which act in the nephron and promote the
excretion of salt and water to lower blood pressure. Clinical experience thus
underscores the critical role of the kidneys in responding to and managing
hypertension.
The involvement of the kidneys in instigating hypertension is further
suggested by the clinical portrait in metabolic disorders. Type II diabetes is
approaching epidemic proportions, and patients are at tremendous risk for the
development of hypertension, supporting the concept that diet (specifically sugar)
has a role in increasing the blood pressure. Protection of the kidneys with ACE
inhibitors or ARBs is a mainstay in the management of diabetes, as the condition is
characterized by increased RAS activity. Despite the clinical evidence that diabetics
eventually develop RAS-dependent hypertension, the direct mechanism linking
excessive sugar levels with RAS activation remains unknown. The risk for
cardiovascular disease is especially high in the metabolic syndrome, a combination
of disorders comprised of hypertension, hyperglycemia, and high triglycerides.
Interestingly, in the metabolic syndrome, high levels of fructose (and its metabolite,
uric acid) are the strongest prognostic indicators of hypertension severity.
8
Again,
dietary sugar is implicated in hypertension by an unknown mechanism, but the
correlation demonstrates that environmental insults do have an impact on blood
7

pressure. Observations in patients have suggested that excessive sugar may lead to
hypertension, but have been unable to provide any explanation for the findings.
Experimentally, it has been difficult to characterize the mechanism driving this
association since most studies capture only snapshots of the interaction and cannot
follow the processes as they occur. However, with advances in multiphoton
imaging, hypertension can be studied before, during, and after the development of
disease.
The Promise of Multiphoton Imaging
Multiphoton microscopy employs techniques in optics, computer science, and
fluorescence labeling to visualize and quantify dynamic physiological events from
the multicellular down to the intracellular levels. This emerging technology offers
impressive spatial and temporal resolution, allowing experimentalists to correlate
microanatomy with physiological functions and their dysfunction in pathology. It
enables the analysis of 4-dimensional (3-D volume in time) structure in organs of
living animals or isolated tissues, measurements of physiological processes, and the
tracking of fluorescence-tagged molecular agents. The technology has led to the
development of novel experimental approaches that permit the isolation, observation,
and analysis of critical structure-function relationships. It has greatly enhanced our
understanding of biology, driving improvements in our management of diseases.
Multiphoton excitation is founded on the concept by physicist Maria
Göppert-Mayer that two low energy photons can synergistically and simultaneously
combine at a fluorophore to emit a photon of equivalent excitation energy as that
8

from the absorption of a single photon of double the energy. The technology as
currently implemented was pioneered by Winfried Denk, who combined the idea of
two-photon absorption with the use of an infrared Ti-sapphire laser beam with a
narrow pulse width (i.e. ~ 100 femtoseconds) and rapid repetition rate (~ 80 MHz) to
enhance the likelihood of an interaction. The longer excitation wavelengths allow
for deeper penetration with less scattering and a higher signal-to-background ratio,
and do not destroy tissue around the plane of interest, which translates into longer
periods of continuous scanning and the recording of videos. Traditional microscopy
captures images at single time points without following the progression of the
processes being studied. In contrast, the sensitivity and specificity of multiphoton
microscopy allow the analysis of dynamic multicellular, integrated processes in vivo
from the beginning through to the end. The exponential rise in publications
employing multiphoton imaging reflects the boundless relevance of the technology
to basic science, medicine, and beyond.
Multiphoton imaging provides heretofore unavailable visual, morphological
and functional information in living specimens, with the benefit of novel quantitative
and integrative methods of data analysis. In studying the living kidney, it offers real-
time imaging of physiological parameters

including single nephron glomerular
filtration rate, glomerular permeability, blood flow, tubular flow, tubular
reabsorption, urinary concentration or dilution, and renin release. All of these factors
may be measured and compared between normotensive and hypertensive animals.
Additionally, high glucose is known to stimulate renin release, but concomitant
9

changes in blood
pressure, cellular
signaling, blood
flow, vascular
expansion, or
filtration function
may also be
measured to assess
the relevance of
acute increases in
glucose to the Figure 3. Single nephron glomerular filtration rate (SNGFR) in vivo.
function of the organ and whole organism. Furthermore, a key advantage of in vivo
imaging is the potential for manipulating the field of interest with experimental
interventions. With the administration of anti-hypertensive medications, their
immediate site-specific effects may be examined and their significance to organ
function assessed. Real-time videos can capture expected findings while also
uncovering unanticipated consequences. For example, in assessing proteinuria with
the injection of a freely filtered fluorescent dye, leakage was visualized from blocked
and purportedly non-functional sclerotic glomeruli rather than the actively
hyperfiltering glomeruli, contrary to the existing paradigm.
9
This experimental
technique has enhanced the understanding of fundamental regulatory mechanisms

10

while also triggering the discovery of new processes. With the careful selection of
probes, nearly any tissue microenvironment can be examined.
In particular, multiphoton imaging is an ideal approach with which to study
key questions in the operation of RAS, unveiling the contributions of specific
mechanisms to complex processes. The application of fluorescence imaging to
cellular studies permits important discoveries about constituents, functions,
responses to the environment, intracellular signaling pathways, and indirectly,
intercellular relationships. As the critical gatekeeper of RAS function, renin is a
particularly important variable to study in the dysregulation of RAS. Acidotropic
fluorophores, including quinacrine and LysoTracker dyes, have been successfully
used to label renin granular contents both in vitro and in vivo. Renin exocytosis has
been visualized in real-time on the individual granule level in response to a number
of physiological stimuli including beta-adrenergic activation, low perfusion pressure
(the baroreflex), and the
macula densa
mechanism. With the
multiphoton approach,
renin release in response
to stimulation of
endothelial, vascular, or
macul a densa cells may
Figure 4. Renin release signals at the JGA be visualized. Furthermore,
11

the specific molecular signals responsible can be determined. Signal transduction
refers to the process by which a cell receives input and relays it to a second
messenger to elicit a response. Hypertension can arise from defects in signal
transduction pathways, whose second messengers (such as calcium) are readily
investigated with multiphoton fluorescence microscopy. With the high
spatiotemporal resolution available, molecular and cellular components of RAS
signaling can be studied and their significance assessed by their influence on the
development of organ dysfunction.
Preserving the structural architecture and native environment of tissues is
fundamental to understanding the physiological relevance of a molecular process.
For example, detecting the production of the potent vasodilator nitric oxide is
immaterial without confirming changes in vascular diameter and blood flow. In
addition to its superlative descriptive power due to highly sensitive imaging of organ
function with extraordinary specificity and resolution, it provides information within
the most realistic and relevant framework. Thus, multiphoton microscopy is
particularly useful for in vivo applications where the ability to visualize events in
three dimensions with feedback mechanisms and humoral signals intact is essential,
as in the kidney. New visual and quantitative data may challenge existing paradigms
in pathophysiology and have the potential to eventually provide novel non-invasive
diagnostic and therapeutic tools for future applications. If sugars cause acute
disruptions in normal physiology, understanding the mechanisms would allow rapid
detection of and response to the injury. Whether isolating and reducing the field of
12

interest to subcellular mechanisms or investigating intricately coordinated processes
and their molecular footprints, multiphoton imaging offers an unparalleled power to
observe isolated parts of phenomena and contextualize their significance in the
setting of the living system.
Multiphoton excitation fluorescence microscopy is an excellent imaging
technique for studying complex physiological mechanisms. Imaging renin content,
release, and activity in the intact kidney provides qualitative as well as quantitative
information from beginning to end, allowing the evaluation of individual steps and
their consequences. It permits the description, discovery, and analysis of otherwise
undetectable phenomena and provides information which will help integrate cellular
processes with their larger effects of organ function. Chapter 1 describes the
characterization of a multiphoton fluorescence microscopy and in vivo imaging
based approach to observe and quantify physiological functions of the living kidney
in healthy and hypertensive animals. Perturbations in renin content and release,
single nephron glomerular filtration rate, and cortical blood flow were established as
early indicators of the onset of hypertension. Accordingly, these parameters were
evaluated to examine the potentially hypertensive effects of a dietary sugar
metabolite, uric acid. In Chapter 2, the technology was harnessed to resolve the
mechanism by which uric acid activates RAS. Ultimately, the consequences of
enduring RAS activation in the diabetic kidney were evaluated in Chapter 3,
revealing new properties and problems of RAS. In explaining how diet has direct
input into a pathway that signals for hypertension, we can begin to understand how
13

to solve the problem. Exploring the story behind this pathological association can
eventually give rise to the innovation of better treatments and preventive strategies
for the epidemic cases of hypertension that are “upon people” and can ultimately be
resolved by people.

14

Introduction Endnotes

1. http://content.nhiondemand.com/psv/HC2.asp?objID=100228&cType=hc

2. Franco OH, Peeters A, Bonneux L, and de Laet C. Blood pressure in
adulthood and life expectancy with cardiovascular disease in men and
women: life course analysis. Hypertension. 2005; 46:280-286.

3. Robinson RF, Batisky DL, Hayes JR, Nahata MC, and Mahan JD.
Significance of Heritability in Primary and Secondary Pediatric
Hypertension. Am J Hypertens. 2005; 18:917–921.

4. Hilbert P, Lindpaintner K, Beckmann,JS, Serikawa T, Soubrier F, Dubay C,
Cartwright P, De Gouyon B, Julier C, Takahasi S, Vincent M, Ganten D,
Georges M, and Lathrop GM. Chromosomal mapping of two genetic loci
associated with blood-pressure regulation in hereditary hypertensive rats.
Nature. 1991; 353:521–529.

5. Guyton AC. Blood pressure control–special role of the kidneys and body
fluids. Science. 1991; 252:1813–1816.

6. MacGregor GA, Markandu ND, Roulston JE, Jones JC, and Morton JJ.
Maintenance of blood pressure by the renin−angiotensin system in normal
man. Nature. 1981; 291:329-331.

7. Guyton AC and Hall JE. The juxtaglomerular complex: its possible control of
multiple nephron functions. Nature. 1979; 277:601–602.

8. Nakagawa T, Tuttle KT, Short RA, and Johnson RJ. Hypothesis: Fructose-
induced Hyperuricemia as a Causal Mechanism for the Epidemic of the
Metabolic Syndrome. Nat Clin Pract Neprol. 2005; 1:80–86.

9. Kang JJ, Toma I, Sipos A, McCulloch F, and Peti-Peterdi J. Quantitative
imaging of basic functions in renal (patho)physiology. Am J Physiol Renal
Physiol. 2006; 291:F495-F502.

15

Chapter 1: Multiphoton in vivo imaging to study the kidney in hypertension

Kang JJ, Toma I, Sipos A, McCulloch F, and Peti-Peterdi J. Quantitative imaging of
basic functions in renal (patho)physiology. Am J Physiol Renal Physiol. 2006;
291:F495-F502.

Abstract
Multi-photon fluorescence microscopy offers the advantages of deep optical
sectioning of living tissue with minimal phototoxicity and high optical resolution.
More importantly, dynamic processes and multiple functions of an intact organ can
be visualized in real-time using non-invasive methods, and quantified. These studies
aimed to extend existing methods of multiphoton fluorescence imaging to directly
observe and quantify basic physiological parameters of the kidney including
glomerular filtration rate (GFR) and permeability, blood flow, urinary
concentration/dilution, renin content and release, as well as more integrated and
complex functions like the tubuloglomerular feedback (TGF)-mediated oscillations
in glomerular filtration and tubular flow. Streptozotocin-induced diabetes
significantly increased single nephron GFR (SNGFR) from 32.4 ± 0.4 to 59.5 ± 2.5
nl/min, and glomerular permeability to a 70 kD fluorophore approximately 8-fold.
The loop diuretic furosemide 2-fold diluted and increased about 10-fold the volume
of distal tubular fluid, while also causing the release of 20% of juxtaglomerular renin
content. Significantly higher speeds of individual red blood cells were measured in
intraglomerular capillaries (16.7 ± 0.4 mm/s) compared to peritubular vessels (4.7 ±
0.2 mm/s). Regular periods of glomerular contraction-relaxation were observed
16

resulting in oscillations of filtration and tubular flow rate. Oscillations in proximal
and distal tubular flow showed similar cycle times (about 45 s) to glomerular
filtration, with a delay of approximately 5-10 and 25-30 s, respectively. These
innovative technologies provide the most complex, immediate and dynamic portrayal
of renal function, clearly depicting and analyzing the components and mechanisms
involved in normal physiology and pathophysiology.
17

Introduction
Recent advances have added microscopic investigative techniques to the
arsenal of tools applicable to gaining a more comprehensive understanding and
quantifiable benchtop assessment of renal function. The development of
micropuncture by Wearn and Richards in 1924 was one of the most significant
advances in renal physiology, leading to the discovery of the phenomena of
filtration, absorption, and secretion in the nephron (24, 29). The localization of these
processes and the determination of the handling of specific substances were deduced
by comparing fluid compositions in the bladder, kidney, and blood (29). Eventually,
manipulation of renal physiological functions was made possible with the
development of the isolated perfused tubule by Burg and colleagues in 1966 (3).
Micropuncture and microperfusion techniques can be used to study the end results of
renal physiological function, but do not permit direct visualization of ongoing
mechanisms or other details of the pathways. Renewed interest in employing
conventional methods to study nephron function in genetically altered animal models
has prompted innovations like the use of FITC-labeled inulin, rather than radioactive
probes, to evaluate single-nephron glomerular filtration rate (SNGFR) and tubular
reabsorption (16). The application of fluorescence microscopy to determine FITC-
inulin signal in micropuncture studies has the advantages of simplicity, precision,
accuracy, and cost effectiveness without consumption of the fluid sample (16). These
long-established, demanding, and invasive methods provide critical snapshots of
renal physiology and have directed countless current methods to discover precise
18

details about the players, interactions, and control mechanisms involved.
Insight into the interplay between different parts of the nephron provides a
necessary order of complexity to evaluate and have a comprehensible understanding
of renal function. Many critical physiological processes in the kidney like the
regulation of glomerular filtration, hemodynamics, concentration, and dilution
involve complex interactions between multiple cell types and customarily
inaccessible structures. For example, rat experiments in the 1980s revealed that
variables of nephron flow exhibited tubuloglomerular feedback (TGF)-mediated
regular oscillations (11, 15). Spontaneously hypertensive rats (SHR) have been
characterized to display irregular TGF-mediated oscillations (11, 31), and recent
studies have attempted to distinguish possible factors that may contribute to the
spectral complexity of the observed oscillations (14). The existence of TGF-
mediated oscillations in nephron flow serves as one example of a complex functional
parameter that could be better examined and also quantified by microscopy.
Multi-photon excitation fluorescence microscopy offers a state-of-the-art
imaging technique superior for deep optical sectioning of living tissue samples. The
higher resolution and minimal phototoxicity of this method permit longer time
periods of continuous tissue scanning with uses in real-time imaging of intact organs.
Using this technique, dynamic processes such as glomerular filtration (9, 32),
proximal tubule endocytosis (23), apoptosis (9), microvascular function (9, 18),
protein expression (27), renal cysts (26), and major functions of the juxtaglomerular
apparatus, including TGF (20, 22) and renin release (21, 22), have been visualized
19

and studied both in vivo and in vitro down to the subcellular level. The capacity to
simultaneously visualize both proximal and distal segments of the nephron permits
observation of the dynamic processes within the living kidney and a quantitative
assessment of the various operations. In fact, a ratiometric intravital two-photon
microscopy technique based on the generalized polarity concept has been recently
applied to quantify glomerular filtration and tubular reabsorption (32). The rapidly
developing field of fluorescence optics and ultra-sensitive detection will fuel further
developments. For example, the construction of novel photonic crystal fibers, and
hence the advent of multi-photon fluorescence endoscopy (1), demonstrates the
potential of this technology for developing non-invasive therapeutic and real-time
diagnostic tools for both clinical and biomedical research applications (33).
Consequently, one of the next steps for multi-photon microscopy is to provide real-
time, quantitative imaging and rapid evaluation of basic organ functions.
The aim of the present study was to extend existing methods of multiphoton
fluorescence imaging to directly observe and quantify basic functional parameters of
the kidney. These include the non-invasive measurement of glomerular filtration
rate, blood flow, urinary concentration/dilution through the course of the nephron,
and renin content and release as well as more integrated and complex functions like
TGF-mediated oscillations in filtration and tubular flow. These innovative
technologies provide the most complex, immediate and dynamic portrayal of renal
function, clearly depicting and analyzing the components and mechanisms involved
in normal physiology and pathophysiology.
20

Materials and Methods
Multiphoton excitation laser scanning fluorescence microscopy
The multiphoton microscope used in these studies consists of a Leica TCS
SP2 AOBS MP confocal microscope system (Leica Microsystems, Heidelberg,
Germany). A Leica DM IRE2 inverted microscope was powered by a wideband,
fully automated, infrared (710-920nm) combined photo-diode pump laser and mode-
locked titanium:sapphire laser (Mai-Tai, Spectra-Physics, Mountain View, CA).
Images were collected in a time (xyt, 20 Hz), or line (xt, 1000 Hz) series, depending
on the purpose of study, with the Leica Confocal Software (LCS 2.61.1537) and
analyzed with the LCS 3D, Process, and Quantify packages. The principles and
advantages of conventional confocal and two-photon microscopy and their
application in imaging renal tissues have been recently reviewed (18, 22).
Three easy-to-use, water-soluble fluorophores were used to label specific
structures in the living kidney. A 70 kD dextran-rhodamine B conjugate was used
(100 µl of a 10 mg/ml stock in iv. bolus, from Invitrogen) to label the circulating
plasma or intra-vascular space. Tubular segments and more specifically the content
of individual renin granules were visualized using quinacrine (100 µl of a 25 mg/ml
stock in iv. bolus, from Sigma) in a manner similar to in vitro applications previously
described (21, 22). In some experiments, the extracellular fluid marker Lucifer
yellow (LY) was used (10 µl of a 10 mg/ml stock in iv. bolus, from Invitrogen).
Because the ionic charge of fluorophores affects glomerular filtration characteristics,
the FITC-conjugate of the gold-standard GFR marker inulin (5 kD), and the likewise
21

neutral rhodamine B–dextran (70 kD) were used. Although the fluid-marker LY is an
anionic compound, it is freely filtered due to its small size (0.45 kD). All three
fluorescent probes were excited using the same, single excitation wavelength of 860
nm (Mai-Tai), and the emitted, non-descanned fluorescent light was detected by two
external photomultipliers (green and red channels) with the help of a FITC/TRITC
filter block (Leica).
Animals
Munich-Wistar male rats (200 g, Harlan, Madison, WI) and C57BL6 mice
(20g, inbred) were anesthetized with thiobutabarbital (Inactin, 130 mg/kg body wt)
alone (for rats) or in combination with 50 mg/kg body wt ketamine (for mice). After

assuring adequate anesthesia, the trachea was cannulated to facilitate breathing. The
left femoral vein and artery were cannulated for dye infusion and blood pressure
measurements, respectively. Subsequently, a 10-15 mm dorsal incision

was made
under sterile conditions and the kidney was exteriorized. The animal was placed on
the stage of an inverted microscope with the exposed kidney placed in a coverslip-
bottomed heated chamber bathed in normal saline and the kidney was visualized
from below as described by Dunn et al (9) using a HCX PL APO 63X/1.4NA oil CS
objective (Leica). High-quality images from the renal cortex were acquired up to 150
micron deep below the surface. During all procedures and imaging, core body
temperature was maintained

with a homeothermic

table. All animal protocols have
been approved by the Institutional Animal Care and Use Committee at the University
of Southern California.
22

In some rats, diabetes was induced by using a single dose of streptozotocin
(STZ) injection (50 mg/kg ip.). Blood glucose levels were measured following STZ
administration by using test strips on blood samples from a tail clip (Freestyle blood
glucose monitoring system, Abbott Laboratories) to verify induction of diabetes.
Two groups of animals with blood glucose levels of 400 mg/ml or higher were used:
one between days 4-6 after STZ injection for SNGFR calculations, and the other
within 4 weeks for glomerular permeability studies.
During in vivo imaging, systemic blood pressure of animals was monitored
through a cannula inserted into the left femoral artery and using an analog single-
channel transducer signal conditioner model BP-1, transducer model BLPR (World
Precision Instruments, Sarasota, FL). Calibration was performed using a Pressure
manometer model PM-015 and data were collected using data acquisition system
QUAD-161.
Chemicals, if not indicated, were purchased from Sigma Chemical Co., St. Louis,

MO, USA.

Glomerular filtration rate
Overall GFR was measured using the fluorescence-based FITC-inulin
method as described before (16). Briefly, anesthetized rats were surgically
instrumented for clearance measurements which included tracheostomy, cannulation
of left femoral artery and vein as described above, as well as the two ureters using a
24G iv. catheter (Terumo). Immediately after surgery, animals were given a bolus (2
23
µl/g body wt) of PBS containing 0.05% FITC-inulin, and 3.5% BSA. This
was followed by a maintenance infusion of the same solution at 50 µl /min, and a 30-
min equilibration period. Renal function was then determined over three consecutive
15-min clearance periods. At the midpoint of each urine collection, an arterial blood
sample (100 µl) was obtained for determination of plasma FITC-inulin. Plasma and
urine samples were diluted 1:100 in HEPES buffer, and FITC fluorescence intensity
was measured at 540 nm in response to excitation at 485 nm in a cuvette-based
fluorometer (Quantamaster-8, PTI, NJ). FITC-inulin concentrations were determined
using FITC standards (16).
Data Analysis
Data are expressed as mean ± SE. Statistical significance was tested using
ANOVA. Significance was accepted at P < 0.05.
24

Results
Measurement of SNGFR. An example of the experimental technique is shown in Fig.
1. A superficial glomerulus was selected which had at least a 100 µm-long initial
segment of the proximal tubule positioned in the same optical section. A single
intravenous bolus of the fluid marker LY was injected into the femoral vein which
appeared in the glomerulus within 5 s and filtered into the Bowman’s space and early
proximal tubule. A supplementary video file showing glomerular filtration of LY is
available at http://ajprenal.physiology.org/cgi/content/full/). Using high temporal
resolution, the fluorescence intensity changes of LY were measured as shown in Fig.
1A-C, within two regions of interest (ROI): one at the opening of proximal tubule
(ROI1), the other approximately 100 µm downstream (ROI2). Internal diameter,
length of the tubule, and transit time of filtrate (time shift shown in Fig. 1C) between
the two areas were measured using the Quantification package of the Leica Confocal
Software. The midpoint of the dye bolus, approximated by the maximal fluorescence
intensity, travels at the same speed as the mean fluid velocity. Thus, the transit time
(shift between ROI1 and ROI2 intensity plots) was calculated at the peak (Fig. 1C).
By calculating tubular fluid volume (length × (diameter/2)
2
× π) the absolute value of
SNGFR was calculated (volume/time). Fig. 1D demonstrates that SNGFR
measurements provided equivalent values if the initial <500 µm segment of proximal
tubule was used. There was no statistically significant correlation between SNGFR
values and distance of ROIs (r=0.07). Also, SNGFR values obtained in the same
nephrons using LY (32.4 ± 0.4 nl/min, n=50) or the gold-standard FITC-inulin (34.2
25
± 0.8 nl/min, n=12) were not statistically different.

Figure 1. Representative images of the technique to measure SNGFR. The
intravascular space is labeled with 70 kD dextran-rhodamine B (red), renal tubules
with quinacrine (green), and the glomerular filtrate (tubular fluid) with Lucifer
yellow (LY, yellow). Glomerulus (G), and proximal tubule (PT) are labeled.
Fluorescence intensity of iv. injected LY was measured within two areas of interest
(ROI) in the early PT fluid: one always at the beginning of the PT (ROI1), the other
about 100 µm downstream (ROI2). Images on panels A-B demonstrate the
downstream movement of filtered LY in the tubular fluid. C: Plot of LY fluorescence
26
intensity changes within the two ROIs. The shift (Δt) indicates the duration of
fluid movement from ROI1 to 2. Scale is 50 µm. D: The effects of positioning ROI2
at different distances from ROI1 on SNGFR calculations (n=18).

To demonstrate the utility of this technique, SNGFR was measured in control
and STZ-diabetic rats. SNGFR in control rats averaged 32.4 ± 0.4 nl/min (n = 50
from 7 different animals) while significantly elevated levels (59.5 ± 2.5 nl/min, n =
15 from 7 different animals) were measured in select, significantly enlarged
glomeruli from diabetic rats. Overall GFR measured with the FITC-inulin clearance
method appeared to be higher in diabetes, but there was no statistically significant
difference between the control (1.13 ± 0.13 ml/min, n=6), and diabetic groups (1.34
± 0.13 ml/min, n=5). Systemic blood pressure was normal in all animals studied; the
mean arterial blood pressure was 102.7 ± 8.6 mmHg.

Estimating renal blood flow. Red blood cell (RBC) velocity as an index of renal
blood flow was measured in peritubular and intraglomerular capillaries by expanding
recently established techniques (13, 19, 33). Peritubular capillary blood flow is
shown in real-time in a supplementary video file available at
http://ajprenal.physiology.org/cgi/content/full/). Using sufficiently high temporal
resolution (1 msec), it was possible to image and characterize the motion of RBCs in
the renal cortex. As shown in Fig. 2A, RBCs exclude the fluorescent dye used to
label the circulating plasma and consequently they appear as dark, non-fluorescent
objects on the images. Acquiring repetitive scans along the central axis of a capillary
27

(called a line-scan), RBC motion leaves dark bands in the data set (Figs. 2A-B).
Figure 2. Illustration of red blood cell (RBC) flow and velocity measurement in
renal capillaries. A: The circulating plasma was labeled with 70 kD dextran-
rhodamine B (red), and proximal (PT) and distal (DT) renal tubules with quinacrine
(green). Note that some rhodamine was filtered through the glomerulus and became
concentrated in the DT as indicated by the red color of tubular fluid. A horizontal
line, about 20 µm long, was positioned through the central axis of a capillary (white
line in the left upper corner), and a line (xt) scan performed at 1msec intervals along
this line continuously for 2s. RBCs appear as unstained dark objects (A) and leave
dark bands on the line scan (B-D) as they move through the capillary (line). Xt line
scan of a peritubular (B), and intraglomerular (C) capillary. RBC velocity as the
slope of the bands (Δx/Δt) was calculated as shown. Note the regular blood flow
(constant slope) in B-C, and higher RBC speed (smaller slope) in C vs. B. D: Line
scan of irregular blood flow in a peritubular capillary, indicated by the variable
slope.
28

Importantly, the slope of the bands is inversely proportional to the velocity, which
was measured as shown in Fig. 2B using the LCS software. In most
peritubular capillaries a regular, steady flow of RBCs was measured, as indicated by
the constant slope of the bands (Figs. 2B-C). Significantly higher speeds of RBCs
were measured in intraglomerular capillaries (16.7 ± 0.4 mm/s) compared to
peritubular vessels (4.7 ± 0.2 mm/s, n = 10 each from 8 different animals) with
similar diameters. In addition, we observed instances of irregular flow in some
peritubular capillaries in which RBC velocity showed fluctuations (Fig. 2D).
Systemic blood pressure was within the normal range throughout these experiments.

Imaging urinary concentration and dilution. We applied multiphoton microscopy to
provide a continuous, real-time measurement of the urinary concentrating and
diluting function of individual renal tubules based on recent work (26). The
technique is demonstrated in Fig. 3. A single iv. bolus of a relatively high molecular
weight fluorescent plasma marker (rhodamine-conjugated 70 kD dextran) remained
in the intravascular space for several hours of imaging due to its large size. Steady-
state levels of the marker in the plasma were evident by the constant level of
fluorescence in the renal vasculature throughout these experiments (Figs. 1-6,
supplementary videos). A microscopic fraction of the marker was filtered in the
glomeruli, and thus only trace amounts of rhodamine were detected in the proximal
tubular fluid (see dark proximal tubule fluid in Fig. 3A). However, due to the
concentrating mechanism, an approximately 7-fold higher level of rhodamine
fluorescence was present in the collecting duct system (Fig. 3A and D).
29

Subsequently, the loop diuretic furosemide was used to demonstrate the capabilities
of this quantitative imaging technique. Effects of a single iv. bolus of 2 mg/kg
furosemide on the morphology and function of the renal cortex are shown in Figs.
3B-D. Within 2 minutes of administration, furosemide caused the enlargement of
distal nephron segments (Fig. 3B). At the peak of furosemide’s effect, the volume of
tubular fluid in the cortical collecting tubule increased approximately 10-fold
compared to control. Obstruction of peritubular capillaries by the enlarged distal
nephron segments was evident (Fig. 3C). Compared to the magnitude of distal
nephron enlargement, no significant morphological changes were observed in
proximal tubule segments (Fig. 3). Simultaneously with tubular enlargement,
furosemide reduced urinary concentration ~50% as measured by the distal/proximal
ratio of fluorescence intensity of rhodamine in the tubular fluid (Fig. 3D).
Figure 3. Visualization
of the concentrating and
diluting function of the
kidney. A: The
circulating plasma was
labeled with 70 kD
dextran-rhodamine B
(red), and proximal
(PT) and distal (*) renal
tubules with quinacrine (green). Note that some rhodamine-dextran was filtered
30

through the glomerulus (G) and was concentrated in the distal vs. proximal tubule as
indicated by the intense red color of tubular fluid. B-C: Effects of the loop diuretic
furosemide. Note the reduced red color and increased volume of distal tubular fluid.
D: Reduction in ratio of distal/proximal tubular fluid rhodamine B fluorescence in
response to furosemide, injected at time zero iv. Same magnification, scale=20 µm.

Physiological oscillations in SNGFR, tubular flow. In the intact kidney, the closed
loop system allows for the observation of myogenic and tubuloglomerular feedback
(TGF)-induced oscillations in glomerular filtration rate and consequently, changes in
tubular flow. The renal cortex was visualized continuously for 5 minutes as
illustrated in Fig. 4. Video of the same preparation is available at
http://ajprenal.physiology.org/cgi/content/full/.
Figure 4. Fluorescence labeling of the
intact living rat kidney. A: Rhodamine
B-conjugated 70 kD dextran (red) was
given iv. to label the cortical
vasculature (plasma). Quinacrine
(green) stained renal tubules
(proximal tubule, PT, cortical
collecting duct, CCD). A small
fraction of plasma dextran is filtered
in the glomerulus (G), and appears in
the concentrated tubular fluid of the
31

CCD (red) more or less intense depending on the high or low phase of flow
oscillations (see supplementary video). B: Spontaneous oscillations in glomerular
filtration, proximal and distal tubular flow detected by changes in rhodamine B
fluorescence in the tubular fluid (or Bowman’s space). Magnitude of changes are not
drawn to scale. Simultaneous measurements are shown (the vertical dotted line
represents time zero), so the delay in PT and CCD oscillations compared to those in
the parent G is easy to read. Scale is 20 µm.

Regular periods of glomerular contraction-relaxation were observed,
resulting in oscillations of filtration and tubular flow rate (Fig. 4B). This was
measured by the changes in fluorescence intensity of the filtered plasma marker 70
kD dextran-rhodamine B in the tubular (Bowman’s space) fluid as well as by
changes in tubular diameter (not shown, see supplementary video). Oscillations
clearly showed two components: a faster (cycle time about 10 s) and a slower (cycle
time about 45 s) mechanism. Tubular flow oscillations were delayed compared to
oscillations in glomerular filtration: an approximately 5-10 s delay was detected in
the proximal tubule, while the delay was measured to be 25-30 s in distal nephron
segments (Fig. 4B). Glomerular and tubular flow oscillations were absent after
furosemide treatment (not shown).

32

Renin content and release. Renin content of juxtaglomerular granular cells was
visualized in vivo in the rat (Fig. 5A) and mouse (Fig. 5B) using quinacrine, a
selective marker of acidic and secretory granules. Quinacrine staining of individual

renin granules in the terminal afferent arteriole was highly specific. However, weak
background staining was observed in all tubule cell types, most likely due to the
accumulation of quinacrine in lysosomes. In addition to the quantification of renin
content (based on the length of the renin-positive segment of afferent arteriole), we
measured the dynamics of renin release. Furosemide was used to trigger
juxtaglomerular renin release. Fig. 5C shows a representative image of four separate
experiments. Minutes after iv. bolus administration of 2 mg/kg furosemide, the renin
content of the afferent arteriole was reduced by approximately 20% as measured by
the reduction in quinacrine fluorescence (Fig. 5C).
Figure 5. In vivo imaging of the juxtaglomerular renin content in the rat (A) and
mouse (B). Circulating plasma is labeled with rhodamine B-conjugated 70 kD
dextran (red), content of individual renin granules with quinacrine (green), nuclei are
stained blue with Hoechst 33342. Note the significant renin content around the
33

juxtaglomerular portion of the afferent (AA), but not the efferent arteriole (EA). G:
glomerulus. Scale is 20 µm. C: Reduction in quinacrine fluorescence intensity (index
of renin release) in response to furosemide, injected iv. at time zero.

Glomerular permeability. In vivo real-time imaging allowed us to observe functional
differences between hyperfiltering and sclerotic glomeruli in the early phase of STZ-
diabetes (within 4 weeks after STZ injection). Sclerotic glomeruli were evident by
their reduced size and the small number of perfusing glomerular capillary loops (Fig.
6). More importantly, increased glomerular permeability was readily apparent in the
sclerotic (approximately 20% of all glomeruli), while rhodamine leakage was
significantly less noticeable in the hyperfiltering enlarged glomeruli as illustrated in
Fig. 6. The behavior of a 70 kD dextran-rhodamine conjugate in the vascular space
was compared. This large molecule (comparable to albumin) was more freely filtered
into the Bowman’s space in the sclerotic than in the hyperfiltering glomeruli.
Figure 6. In vivo imaging of
glomerular permeability in the
diabetic kidney. Sclerotic glomeruli
(arrows) and a hyperfiltering
glomerulus (G) shown. Note the
intense ultrafiltration of the high
molecular weight (70 kD) dextran-
rhodamine B (red) from the plasma
into the Bowman’s space in the sclerotic, but not in the hyperfiltering glomerulus.
34

The ratio of Bowman’s space/intravascular rhodamine fluorescence was 0.1 ±
0.01 in hyperfiltering glomeruli, while it was 0.8 ± 0.1 in sclerotic glomeruli (n=8
each, P<0.05). This finding strongly suggests that the significant proteinuria present
in diabetic nephropathy is predominantly attributable to the sclerotic rather than the
hyperfiltering glomeruli. In mst sclerotic glomeruli, the free downstream passage of
the “red” proximal tubular fluid was persistent and characteristic. Furthermore, LY
given in iv. bolus (as shown in Fig. 1) still cleared from the Bowman’s space and
proximal tubule, evidence that some blood supply and glomerular filtration still
remained.

35

Discussion
Multiphoton microscopy is a state-of-the-art imaging technique which can
provide high resolution images from deep optical sections of the living tissue,
including the kidney (18). However, multiphoton microscopy has applications far
beyond the generation of superior images: dynamic processes, like the multiple
functions of an intact organ can be visualized by real-time imaging in a non-invasive
way and quantified. This study provides further examples of how basic parameters of
renal (patho)physiology can be continuously monitored in the intact rat kidney.
In the pioneering work of Molitoris et al (9, 18, 32) the process of glomerular
filtration has been recently observed in real-time using intravital two-photon
microscopy and a number of fluorescent probes (9, 32). The presently described
methods to quantify the rate of glomerular filtration is based on previous fluorescent
techniques that measured SNGFR using conventional micropuncture (16) and tubular
flow rate with a nonobstructing optical method (7). In our experience, LY (a widely
used extracellular fluid marker) (8) was a better tool to measure SNGFR with this
optical technique than FITC-inulin due to its low molecular weight, excellent water
solubility, and high fluorescent quantum yield. It should be mentioned however, that
LY is not a good GFR marker in the classic physiological sense (i.e. for clearance
studies) because its clearance is most likely greater than that of inulin. Compared to
the demanding conventional micropuncture methods (2, 16, 28), a single iv. bolus of
LY was filtered into the glomeruli, and provided a convenient measure of SNGFR
within 5 s. Another advantage of this non-invasive technique is that tubular flow and
36

macula densa functions remain intact and undisrupted. Tubular dimensions and the
transit time of LY in the early proximal tubule were easily measured using the LCS
software (Fig. 1C) from which SNGFR was calculated. Combined with monitoring
tubular flow rate (Fig. 4B) this technique permits real-time measurement of SNGFR
in the intact kidney. Taking advantage of the high temporal resolution, it was
possible to accurately measure LY transit time within the initial ~500 µm of
proximal tubule. To validate this technique, glomerular filtration was observed and
quantified under normal physiological conditions and in diabetic hyperglycemia.
Consistent with the well-established glomerular hyperfiltration in diabetes (4, 28),
we measured significantly elevated SNGFR levels in select, significantly enlarged
glomeruli in STZ-treated animals. The values of SNGFR are within the same range
as measured by micropuncture techniques in both control and diabetes (2, 28).
In vivo imaging methods permit the observation and measurement of regional
differences in blood flow to the nephron. Cortical blood flow was evaluated by
measuring red blood cell velocity in peritubular and intraglomerular capillaries. Even
with the resistance of the afferent arteriole, glomerular capillary pressure is high and
the postglomerular resistance produces a significant pressure drop from the
glomerular to peritubular capillaries. Thus, as expected, red blood cell velocity was
slower in peritubular capillaries than in glomerular capillaries. The occasionally
observed irregular blood flow (Fig. 2D) is probably due to intermittent circulation in
branches of the peritubular capillary plexus and the dynamic control of vascular
resistance upstream. Consistent with the higher blood flow to the kidneys compared
37

to other organs, the measured RBC speed is higher than in other vascular beds (6,
13).
As a non-invasive alternative to existing methods, in vivo imaging allows
direct and continuous visualization of all cortical segments of the nephron, often in
the same visual field. Such a technique thus permits observation of the ways in
which upstream changes exert effects downstream. For example, fluorescence
intensity of the 75 kD dextran-rhodamine was more pronounced in distal segments of
the nephron, illustrating the concentrating mechanisms present there regionally (Fig.
3). Furosemide, one of the most potent loop diuretics via its ability to block the
Na:K:2Cl cotransporter, elicited a massive fluid loss upon its acute administration
(Fig. 3B-C). Upon treatment with furosemide, the increased distal fluid load inflated
collecting ducts and diluted the distal tubular fluid (Fig. 3D). The enlarged distal
tubular segments appeared to compress peritubular capillaries consistent with
preliminary data on the effect of furosemide to reduce renal blood flow (19). Further
studies with furosemide would hold the potential to visualize morphological and
functional changes occurring in the juxtaglomerular apparatus and glomerulus as
well, directly responding to recent inquiries into the functional significance of
furosemide’s actions on the NKCC1 isoform at these sites (12). Consistent with
recent in vivo and in vitro data on the effect of furosemide on renin release (5), the
present studies detected a 20% release of renin from juxtaglomerular afferent
arterioles in response to acute furosemide treatment (Fig. 5C).

38

The TGF system is a key regulator of filtration rate and of water and
electrolyte delivery to the distal nephron (25). Earlier experiments demonstrated that
renal blood flow and consequently, tubular fluid flow exhibit regular oscillations due
to the myogenic mechanism (6-10 s periods) and TGF (20-50 s periods) (10, 11, 17).
Mathematical models as well indicated that these mechanisms are coupled (17). The
present experimental technique allows non-invasive, real-time, and in vivo
observation and measurement of oscillations in glomerular filtration and tubular flow
simultaneously in both proximal and distal nephron segments (Fig. 4B). Future
studies can directly visualize alterations in this system, for example the irregular
oscillations in hypertensive rats (11, 14, 31) as well as nephron-nephron interactions
(10).
The renin-angiotensin system (RAS) is one of

the most important regulatory
mechanisms of renal tubular salt and water

conservation, and consequent blood
pressure equilibrium. The major structural

component of RAS in the kidney is the
juxtaglomerular apparatus (JGA) located at the vascular pole of the glomerulus.
Renin producing cells of the JGA reside in the wall of the terminal afferent arteriole
and can be visualized (Figs. 5A-B) using multiphoton imaging and quinacrine, a
fluorescent probe selective for individual renin granules (21, 22). This model is a
direct continuation of our recent in vitro work (21), and it is now possible to study
the dynamics of renin content and release in vivo, with high spatial and temporal
resolution down to the individual granule level.

39

The current understanding of diabetes recognizes the disease as a pathology
that begins with hyperfiltration that eventually progresses to loss of filtration
function. The proteinuria characteristically associated with the disease is considered
telltale evidence of concomitant pathology, but the mechanism of its inception was
unknown (4). Some theories implicated the hyperfunctioning glomerulus as the
culprit: the increased blood flow and vascular damage allowed proteins to leak
through (4). Our images and recordings revealed that sclerotizing glomeruli actually
leak into the Bowman’s space and distal collecting segments, whereas the high
molecular weight fluorescence-tagged dextran in the circulating plasma often remain
neatly confined within the hyperfiltering glomerular vasculature (Fig. 6). Diabetes
has already been implicated in epithelial foot process damage (30), a pathological
milestone that would permit proteinuria from the non-functional, and hence non-
discriminate, sclerotic glomeruli may be the source of the proteinuria.
In summary, quantitative imaging of the intact kidney with multiphoton
microscopy provides an excellent non-invasive tool to visualize and study renal
function. With the application of only three fluorescent probes, several basic
parameters of renal physiology can be measured in real-time including glomerular
filtration and permeability, tubular fluid and blood flow, urinary
concentration/dilution, renin content and release. Further studies using this technique
will allow investigations of highly complex and integrative questions in the
processes of renal (patho)physiology. Quantitative imaging of kidney function with

40

multi-photon microscopy may eventually provide a novel non-invasive diagnostic
tool for future clinical applications.

Acknowledgements

This work was supported by grants from NIH DK064324, and AHA EIA 0640056N.
The authors are thankful to Donald J. Marsh for his expert advice and help with the
quantitative analysis of SNGFR.

41

Chapter 1 References
1. Bird D and Gu M. Two-photon fluorescence endoscopy with a mirco-optic
scanning head. Opt Lett. 28:1552–1554, 2003.

2. Brown R, Ollerstam A, and Persson AE. Neuronal nitric oxide synthase
inhibition sensitizes the tubuloglomerular feedback mechanism after volume
expansion. Kidney Int. 65:1349-1356, 2004.

3. Burg M, Grantham J, Abramow M, and Orloff J. Preparation and study of
fragments of single rabbit nephrons. Am J Physiol. 210:1293–1298, 1966.

4. Castellino P, Shohat J, and DeFronzo RA. Hyperfiltration and diabetic
nephropathy: is it the beginning? Or is it the end? Semin Nephrol. 10:228-
241, 1990.

5. Castrop H, Lorenz JN, Hansen PB, Friis U, Mizel D, Oppermann M, Jensen
BL, Briggs J, Skott O, and Schnermann J. Contribution of the basolateral
isoform of the Na-K-2Cl- cotransporter (NKCC1/BSC2) to renin secretion.
Am J Physiol Renal Physiol. 289:F1185-F1192, 2005.

6. Chaigneau E, Oheim M, Audinat E, and Charpak S. Two-photon imaging of
capillary blood flow in olfactory bulb glomeruli. Proc Natl Acad Sci USA.
100: 13081-13086, 2003.

7. Chou CL, and Marsh DJ. Measurement of flow rate in rat proximal tubules
with a nonobstructing optical method. Am J Physiol. 253: F366-F371, 1987.

8. Chu J, Chu S, and Montrose MH. Apical Na+/H+ exchange near the base of
mouse colonic crypts. Am J Physiol Cell Physiol. 283:C358-C372, 2002.

9. Dunn KW, Sandoval RM, Kelly KJ, Dagher PC, Tanner GA, Atkinson SJ,
Bacallao RL, and Molitoris BA. Functional studies of the kidney of living
animals using multicolor two-photon microscopy. Am J Physiol Cell Physiol.
283:C905-C916, 2002.

10. Holstein-Rathlou N-H. Synchronization of proximal intratubular pressure
oscillations: evidence for interaction between nephrons. Pflugers Arch.
408:438-443, 1987.

11. Holstein-Rathlou N-H and Leyssac PP. TGF-mediated oscillations in
proximal intratubular pressure: Differences between spontaneously
hypertensive rats and Wistar-Kyoto rats. Acta Physiol Scand. 126:333-339,
1986.
42

12. Kaplan MR, Plotkin MD, Brown D, Hebert SC, and Delpire E. Expression of
the mouse Na-K-2Cl cotransporter, mBSC2, in the terminal inner medullary
collecting duct, the glomerular and extraglomerular mesangium, and the
glomerular afferent arteriole. J Clin Invest. 98:723-730, 1996.

13. Kleinfeld D, Mitra PP, Helmchen F, and Denk W. Fluctuations and stimulus-
induced changes in blood flow observed in individual capillaries in layers 2
through 4 of rat neocortex. Proc Natl Acad Sci USA. 95:15741-15746, 1998.

14. Layton AT, Moore LC, and Layton HE. Multistability in tubuloglomerular
feedback and spectral complexity in spontaneously hypertensive rats. Am J
Physiol Renal Physiol. 291:F79-F97, 2006.

15. Leyssac PP and Baumbach L. An oscillating intratubular pressure response to
alterations in Henle loop flow in the rat kidney. Acta Physiol Scand.117:415-
419, 1983.

16. Lorenz JN and Gruenstein E. A simple nonradioactive method for evaluating
single-nephron filtration rate using FITC-inulin. Am J Physiol. 276:F172-
F177, 1999.

17. Marsh DJ, Sosnovtseva OV, Chon KH, and Holstein-Rathlou NH. Nonlinear
interactions in renal blood flow regulation. Am J Physiol Regul Integr Comp
Physiol. 288:R1143-R1159, 2005.

18. Molitoris BA, and Sandoval RM. Intravital multiphoton microscopy of
dynamic renal processes. Am J Physiol Renal Physiol. 288:F1084-F1089,
2005.

19. Oppermann M, Castrop H, Briggs J, and Schnermann J. Influence of
furosemide on renal hemodynamics in mice. J Am Soc Nephrol. 16: 393A,
2005.

20. Peti-Peterdi J, Morishima S, Bell PD, and Okada Y. Two-photon excitation
fluorescence imaging of the living juxtaglomerular apparatus. Am J Physiol
Renal Physiol. 283:F197-F201, 2002.

21. Peti-Peterdi J, Fintha A, Fuson AL, Tousson A, and Chow RH. Real-time
imaging of renin release in vitro. Am J Physiol Renal Physiol. 287:F329-
F335, 2004.

22. Peti-Peterdi J. Multiphoton imaging of renal tissues in vitro. Am J Physiol
Renal Physiol. 288:F1079-F1083, 2005.

43

23. Sandoval RM, Kennedy MD, Low PS, and Molitoris BA. Uptake and
trafficking of fluorescent conjugates of folic acid in intact kidney determined
using intravital two-photon microscopy. Am J Physiol Cell Physiol.
287:C517-C726, 2004.

24. Sands JM. Micropuncture: unlocking the secrets of renal function. Am J
Physiol Renal Physiol. 287:F866-F867, 2004.

25. Schnermann J, and Briggs J. Function of the juxtaglomerular apparatus: local
control of glomerular hemodynamics. In: The Kidney, edited by Seldin DW,
and Giebisch G. New York: Raven, 1985, p. 669-697.

26. Tanner GA, Sandoval RM, and Dunn KW. Two-photon in vivo microscopy
of sulfonefluorescein secretion in normal and cystic rat kidneys. Am J Physiol
Renal Physiol. 286:F152-F160, 2004.

27. Tanner GA, Sandoval RM, Molitoris BA, Bamburg JR, and Ashworth SL.
Micropuncture gene delivery and intravital two-photon visualization of
protein expression in rat kidney. Am J Physiol Renal Physiol. 289:F638-
F643, 2005.

28. Thomson SC, Deng A, Komine N, Hammes JS, Blantz RC, and Gabbai FB.
Early diabetes as a model for testing the regulation of juxtaglomerular NOS I.
Am J Physiol Renal Physiol. 287:F732-F738, 2004.

29. Wearn JT and Richards AN. Observations on the composition of glomerular
urine, with particular reference to the problem of reabsorption in the renal
tubules. Am J Physiol. 71:209–227, 1924.

30. Wolf G, Chen S, and Ziyadeh FN. From the periphery of the glomerular
capillary wall toward the center of disease: podocyte injury comes of age in
diabetic nephropathy. Diabetes. 54:1626-1634, 2005.

31. Yip KP, Holstein-Rathlou N-H, and Marsh DJ. Chaos in blood flow control
in genetic and renovascular hypertensive rats. Am J Physiol Renal Physiol.
261:F400-F408, 1991.

32. Yu W, Sandoval RM, and Molitoris BA. Quantitative intravital microscopy
using a Generalized Polarity concept for kidney studies. Am J Physiol Cell
Physiol. 289:C1197-C1208, 2005.

33. Zipfel WR, Williams RM, and Webb WW. Nonlinear magic: multiphoton
microscopy in the biosciences. Nat Biotechnol. 11:1369-1377, 2003.

44

Chapter 2: Sugar metabolism directly activates the renin-angiotensin system
Uric Acid Directly Activates the Renin-Angiotensin System by a Macula Densa-
Dependent Pathway
Manuscript to be submitted to Nature Medicine in April 2008.

Abstract

The renin-angiotensin system has important immediate and long-term
influences on blood pressure, and is therefore a critical target in hypertension
treatment. It is activated by uric acid (a product of fructose metabolism) through an
unknown mechanism, and hyperuricemia is often found in hypertension. In this
study, we discovered that uric acid acutely activates the intra-renal renin-angiotensin
system, and determined the cellular and molecular pathways involved. Real-time
imaging of living tissues showed that renin, the rate-limiting enzyme of the pathway,
was released upon exposure to uric acid in the in vitro microperfused afferent
arteriole-glomerulus experimental model. An acid of similar pKa, L-Histidine, did
not cause renin release. Juxtaglomerular and glomerular endothelial cells, both of
which may stimulate renin release, were not activated by uric acid. However
damage to the macula densa or inhibition of its uptake of uric acid with an organic
anion transporter blocker prevented renin release. Uric acid activated macula densa
MAP kinases to synthesize prostaglandins, potent stimuli of renin release. Direct
activation of the renin-angiotensin system by uric acid at the macula densa provides
a unique mechanism which explains how diet may play a role in instigating
hypertension.
45

Introduction

Nearly 25% of the adults in industrialized nations has the metabolic
syndrome, a combination of disorders including high blood sugar levels, high
triglycerides, low HDL cholesterol, and hypertension, which greatly increases the
risk for cardiovascular disease (1,2). Fructose is an often overlooked component of
the sucrose-rich Western diet, but excessive fructose consumption is actually
associated with the most severe cases of hypertension in the metabolic syndrome
(3,4,5,6). Fructose is readily absorbed and metabolized by the liver, requiring
tremendous amounts of energy to metabolize and leading to the breakdown of
massive quantities of purines like ATP (7). Uric acid is the final product of purine
nucleotide degradation in humans, and high serum uric acid (hyperuricemia) is
associated with many cardiovascular diseases. In particular, the link beween
hyperuricemia and hypertension has inspired much discussion about the etiology of
increased blood pressure (6,8). In the population of adults with untreated
hypertension, 25-40% have hyperuricemia, defined as levels above 6.5 mg/dL, and
over 60% have uric acid levels over 5.5 mg/dL (10). The statistics show strong
correlations between uric acid and hypertension, but do not explain whether it is
merely an associated factor or causative agent contributing to the disease.
In addition to hypertension, hyperuricemia has also been linked with renal
damage including the development of renal arteriolosclerosis, glomerulosclerosis,
and tubulointerstitial injury. The significance of uric acid as an indicator of
pathology is demonstrated by its association with a greater risk for developing
46

chronic renal disease than proteinuria. This may in part explained by the association
of uric acid with an active renin-angiotensin system (RAS), reduced nitric oxide,
endothelial damage, and vascular smooth muscle cell proliferation (11,12,13,14).
The plethora of associated pathologies raises important questions about the
mechanisms behind uric acid’s hypertensive effects. Although crystal deposition
causes substantial renal parenchymal damage, even mild elevations in serum uric
acid levels are harmful. In a model for mild hyperuricemia, rats developed
hypertension, preglomerular vascular disease, renal injury involving RAS activation,
reduced endothelial nitric oxide, and inhibition of the macula densa nitric oxide
synthase (4,13). Interestingly, either reducing uric acid or using pharmacological
RAS inhibitors provides nearly equivalent management of hypertension (15,16).
Therefore, the pathogenicity of uric acid and RAS overlap, and the two components
must interact since management of either entity corrects blood pressure. However,
the mechanism linking uric acid with RAS remains unknown.
The RAS is a critical regulatory cascade in the minute-to-minute and long-
term control of blood pressure. The release of the enzyme renin from the
juxtaglomerular apparatus (JGA), the rate-limiting step of RAS activation, is highly
regulated by chemical, hormonal, and structural signals. Tubular sodium levels,
sympathetic innervation, and renal perfusion pressure all influence renin release,
acting through mediators like prostaglandins and nitric oxide. RAS activation
culminates in the generation of Angiotensin II (Ang II), one of the most potent
vasocontrictive peptides known. Ang II has receptors throughout the body and has
47

pervasive systemic influences. However, control of RAS activation and the
generation of Ang II must begin with renin release in the kidneys.
Hyperuricemic hypertension has been studied extensively in animals
(2,16,17). Hyperuricemia causes afferent arteriolar thickening, mild interstitial
fibrosis, renal cortical vasoconstriction, and glomerular hypertension (17,18). The
RAS-independent effects on renal injury have been attributed to the ability of uric
acid to initiate inflammatory responses or incite damage to the vasculature. The
mechanism by which uric acid activates RAS is unknown (19,20). Since RAS
activation begins in the kidneys and there is abundant evidence for renal damage in
hyperuricemia, we hypothesized that the association must also begin there. Using
our in vivo imaging approach, we first investigated the acute effects of uric acid on
the living animal. Next, we visualized and measured its direct effects on renal
function. The in vitro microperfused afferent arteriole-attached glomerulus
preparation was used to study the influence of uric acid on the rate-limiting step of
RAS, the release of renin. In this study we provide evidence and explanation of the
cellular and molecular pathway responsible for activation of the renin-angiotensin
system by uric acid.

48

Results

Uric acid has acute effects on systemic and renal parameters.
Animal studies have shown that chronic hyperuricemia is associated with
hypertension and renal interstitial damage. We investigated if 200 μL bolus
injections of 0.1 mg/kg bwt uric acid, sufficient to cause transient hyperuricemia, had
acute effects on the whole animal and kidneys using MWF rats. Uric acid caused a
significant increase in mean arterial pressure from 110±9 to 132±6 mm Hg, which
persisted even after 5 minutes fluid injection (Figure 1a). In contrast, the effects of
equivalent volumes of control Krebs Ringer’s solution had no effect on blood
pressure after 5 minutes (Figure 1a). To determine if the rise in blood pressure could
be attributed to RAS activation, the same experiments were conducted in animals
treated with the renin inhibitor, Aliskiren. Neither Krebs Ringer’s nor uric acid
injections had an effect on blood pressure in Aliskiren-treated animals (Figure 1a).
In vivo imaging was then used to observe and quantify the effects of uric acid on the
kidneys. As an index of blood flow to the renal cortex, xt line scans were recorded
along the length of blood vessels. The red blood cell velocities of glomerular and
peritubular capillaries rose after uric acid (Figure 1b), indicating increased cortical
blood flow. Furthermore, there were parallel increases in single nephron glomerular
filtration rate in response to uric acid (Figure 1c). Equivalent volume injections of
control Krebs Ringer’s solution caused no significant changes in any of the previous
parameters, confirming that changes in renal hemodynamic function were not the
effects of volume administration.
49

Figure 1. The acute effects of uric acid on systemic and local renal functions. (A)
Bolus injections of 200 μL of 0.1 mg/kg bwt uric acid (UA) caused a significant
increase in mean arterial pressure 5 minutes after administration: 110±9 vs. 132±6
mm Hg. Equivalent volume injections of control Krebs Ringer’s (KR) solution had
no effect (105±5 vs. 110±3 mm Hg). *P<0.005, n=5 each group. The rise in blood
pressure was Ang II-dependent, as uric acid injections had no effect on Aliskiren-
treated animals: 98±6 vs. 107±12 mm Hg after uric acid, and 102±5 vs. 108±5 mm
Hg after Krebs Ringer. n=3 each group. (B) Quantification of real-time recordings
of xt line scans along the length of a capillary. UA increased the red blood cell
velocities of both glomerular (Δ = 5.0 ± 0.4 μm/ms) and peritubular
50

(Δ=7.8±0.4 μm/ms) capillaries. Equivalent volume control KR injections had no
effect: glomerular (Δ=0.4 ± 0.5 μm/ms), peritubular (Δ=0.9±0.6 μm/ms). *P<0.001,
n=15 each group. (C) Single nephron glomerular filtration rate (SNGFR)
calculations before and after UA injections. UA caused acute 1.5-fold increases in
SNGFR (Δ=18±2 nL/min). Equivalent volume control KR injections had no effect
(Δ=3±2 nL/min). *P<0.001, n=15 each group.

Uric acid directly activates the renin-angiotensin system.
Since the previous experiments detected acute influences of uric acid on the
whole animal and especially the kidneys, we next examined if uric acid had any
direct effects on RAS. The rate-limiting step, the release of renin from the JGA may
be studied in the microperfused afferent arteriole-attached glomerulus preparation
loaded with the fluorescent probe quinacrine to stain renin granules (Figure 2a).
After perfusion with 100 μM uric acid, degranulation of renin was detected within
twenty minutes (Figure 2b). Uric acid caused a 36±6% decrease in quinacrine
fluorescence, compared to an 11±5% reduction with perfusion of control Krebs
Ringer’s solution (Figure 2c). To validate the specificity and examine the effects of
acidity, preparations were perfused with 100 μM L-Histidine, an acid of similar pKa
to uric acid. L-Histidine caused no significant difference in renin release compared
to control [13 ± 2% vs. 11 ± 3%] (Figure 2c), confirming that uric acid specifically
triggers renin release from the JGA to activate RAS.

51

Figure 2. The effects of uric
acid on renin granular
content at the
juxtaglomerular apparatus.
(A) An in vitro
microperfused afferent arteriole(AA)-glomerulus(G) preparation with
juxtaglomerular(JG) renin granular cells of the juxtaglomerular apparatus (JGA).
MD = macula densa. Green= Quinacrine-labeled renin granules. Blue=Hoechst-
stained nuclei. Red= R18 stained endothelial membrane. (B) Perfusion of the AA
with 100 μM UA caused a reduction in quinacrine fluorescence, corresponding to
degranulation of juxtaglomerular renin. (C) Twenty minutes of AA perfusion with
100 μM UA caused a 36 ± 6% reduction in quinacrine intensity, compared to an 11±
5% change with perfusion of control KR solution. Perfusion with 100 μM L-
Histidine, an acid of similar pKa to UA, caused no significant difference (13 ± 2%)
compared to KR control. *P<0.005, n=4 each group.
52

Uric acid does not activate juxtaglomerular renin granular or endothelial cells to
induce renin release.
Renin release occurs when juxtglomerular (JG) granular cells are stimulated,
either directly or by local autocoids from the endothelium or macula densa. We
aimed to determine which of the three cell types were acutely responsive to uric acid.
Cuvette-based spectrofluorometry permits the detection of cellular responses to
stimulation. First, we tested if uric acid could trigger renin release directly from JG
cells. Using a fluorogenic assay, we measured the enzymatic activity of renin from
JG cells exposed to either control Krebs Ringer’s solution or uric acid (Figure 3a).
Uric acid caused no change in renin release or enzymatic activity, signifying that JG
cells are not activated (Figure 3b).
With a direct pathway eliminated, we next evaluated a paracrine signaling
pathway. Nitric oxide (NO) and prostaglandins, especially PGE
2
, are known to be
potent enhancers of renin release. NO and PGE
2
have short half-lives and thus must
be produced locally either by endothelial or macula densa cells, both of which house
the synthetic machinery to produce them. Activation of the endothelium involves a
rise in intracellular calcium concentration, followed by the production of NO or
PGE
2
. To determine if uric acid has an acute effect on the endothelium, changes in
calcium were measured. Spectrofluorometry showed that both 100 μM UA and
control Krebs Ringer’s solution had no effect on endothelial calcium signalling,
assessed by fluorescence intensity of the ratiometric calcium dye, Fura-2 (Figure 3c,
black). Furthermore, neither Krebs Ringer nor uric acid stimulated endothelial NO
53

production, assessed by DAF-FM fluorescence intensity (Figure 3c, gray).
Figure 3. Lack of acute effects of uric acid on juxtaglomerular and endothelial cells.
(A) Spectrofluorometry recording of juxtaglomerular (JG) cell renin enzymatic
activity (EDANS fluorescence). The endogenous renin activity of JG cells in control
Krebs Ringer’s solution from 60-90 seconds and the application of 100 μM UA from
100-130 seconds. (B) Quantitative analysis of previous renin enzymatic activity
measurements. 100 μM UA caused no significant change in the release or activity
from JG cells (before UA: 100±10 U/s, after UA: 101±10 U/s). P=0.96, n= 4
experiments. (C) Spectrofluorometry measurement of the effects of UA on
54

endothelial cell calcium concentration, assessed by fluorescence intensity of the
ratiometric calcium dye, Fura-2. UA did not activate calcium signaling in
endothelial cells (black, n=4 experiments). Spectrofluorometry recording of
endothelial cell nitric oxide (NO) production, assessed by DAF-FM fluorescence
intensity. UA had no effect on endothelial NO synthesis (gray, n=4 experiments).

The macula densa is an integral component of uric acid-mediated renin release.
Since neither JG cells nor endothelium were acutely activated by uric acid,
we next studied cells of the macula densa. In some preparations the macula densa
was dissected off of the JGA-glomerulus complex. In these preparations, uric acid
did not stimulate renin release above control levels (Figure 4). In comparison,
preparations with a completely intact macula densa released significant amounts of
renin (42 ± 2% decrease in quinacrine fluorescence) (Figure 4). Therefore, the
macula densa is a necessary component of uric acid-induced renin release. The
mechanism and directionality of the process were analyzed by pharmacological
inhibition with probenecid, a competitive inhibitor of organic anion transporters
(OATs). An OAT has recently been localized to the basolateral macula densa.
Using 5 mM probenecid added to the bath, we could determine the importance of
uric acid uptake into the macula densa from the basolateral surface. In the presence
of probenecid, uric acid did not stimulate renin release (Figure 4). Therefore, uptake
of uric acid from the basolateral surface of the macula densa is necessary for renin
release.
55

Figure 4. The role
of the macula densa
in uric acid-induced
renin release.
Quantification of
real-time imaging
of renin release from in vitro afferent arteriole-glomerulus preparations.
Preparations were microperfused with 100 μM UA for twenty minutes. UA caused
significant renin release (42±2%) with an intact MD, but there was no effect in
preparations with no MD (17±1%) when compared to control KR perfusions
(12±5%). Preparations were treated with 5 mM Probenecid (an organic anion
transporter blocker) to block MD uptake of UA. UA had no effect on renin release
(16±2%) in probenecid-treated preparations.n=4 experiments each group, *P= 0.001.

Uric acid activates macula densa MAP kinases.
Uric acid has been shown to stimulate NADPH oxidase-dependent reactive
oxygen species, resulting in MAPK activation (21). The mitogen-activated protein
kinase (MAPK) signaling pathway, including p38 and ERK1/2 is a powerful
activator of the inducible cyclooxygenase COX-2, an enzyme responsible for
prostaglandin synthesis. To determine if uric acid activates macula densa ERK1/2, a
mouse macula densa-derived (MMDD1) cell line was studied. MMDD1 cells were
serum-starved to quench endogenous MAPK activity (Figure 5a) and then incubated
56

with 500µM UA for 0, 3, 5, 10, 15, and 30 minutes. Cells were also responsive to
100 and 1000 µM UA, but optimal results were observed with 500 µM.

Figure 5. The effects of uric acid on the MAP kinases, p42/44(ERK1/2), in the
macula densa. MMDD1 cells were serum-starved to qunech endogenous MAPK
activity and then incubated with 100µM UA for 0, 3, 5, 10, and 15 minutes.
Immunocytochemistry of UA-treated MMDD1 cells. Red= phospho p42/44, Blue=
57

DAPI-stained nuclei. (A) At time 0, serum-starved MMDD1 had no p42/44
activation. (B) There is no p42/44 activation after 3 minutes of UA. (C) p42/44
activation begins after 5 minutes of UA. (D) Peak activation of p42/44 occurs after
10 minutes of UA treatment. (E) Activation of p42/44 begins to decay after 15
minutes of UA. (F) Serum was added to cells to confirm intact MAPK pathway
components and provide a reference of low basal activation levels. (G) MMDD1
cells were treated according to the above time course, protein was extracted, and
traditional Western blot shows peak phospho p42/44 activation after 10 minutes of
treatment with UA. The bands at the expected molecular weight of 42 and 44 kDa
were detected by a polyclonal antibody. Pre-incubation with the MAPK inhibitor
(MAPK-I) PD 98059 abolished the effect of UA on MAPK activation. (H) In vitro
preparations were microperfused with Krebs Ringer (KR), 100 μM UA, or UA in the
presence of 10 μM MAPK-I PD 98059. There was a significant difference in renin
release in preparations perfused with control KR (12±5%) vs. UA (42±2%).
Treatment with the MAPK-I PD 98059 reduced the effect of UA to control levels
(17±3%). n= 4 experiments each group, *P= 0.001.

Immunocytochemistry showed the time course of uric acid activation of
p42/44 (ERK1/2) in MMDD1 cells. There was no activation at 3 minutes (Figure
5b), stimulation began at 5 minutes (Figure 5c), peaked at 10 minutes (Figure 5d),
and the signal decayed thereafter (5e). Serum-fed MMDD1 cells were used as a

58

control to compare uric acid-induced activation with baseline MAPK levels (Figure
5f). Traditional Western blot showed that peak p42/44 activation occurred after 10
minutes of treatment with uric acid and the MAPK inhibitor (MAPK-I) PD 98059
abolished the effect of uric acid to control levels (Figure 5g). Furthermore, treatment
of in vitro preparations with the MAPK-I abolished the effect of uric acid on renin
release to control levels (Figure 5h).

Uric acid entry into the macula densa stimulates prostaglandin synthesis.
Finally, we aimed to confirm that MAPK activation in the macula densa
caused the production of prostaglandins, which could ultimately stimulate renin
release. No commercially available dye directly measures prostaglandins, so we
used HEK biosensor cells which have been transfected with the prostaglandin PGE
2

receptor, EP1. When PGE
2
binds to the EP1 receptor, the HEK cell intracellular
calcium concentration increases. Thus, measuring changes in HEK cell calcium with
the dye Fura-2 reports the presence of prostaglandins. Cuvette-based
spectrofluorometry showed that both 500 μM and 100 μM uric acid caused MMDD1
PGE
2
production, but control Krebs Ringer’s solution had no effect (Figure 6a). The
change in Fura-2 ratio could also be converted into changes in the absolute
intracellular calcium concentration. For example, 100 μM uric acid released
sufficient prostaglandins to cause a 70 nM increase in HEK cell calcium (Figure 6b).
To confirm that HEK cell Fura-2 fluorescence was in fact measuring de novo
prostaglandin production, cells were incubated with indomethacin, a COX inhibitor.
59

In the presence of indomethacin, uric acid had no effect on MMDD1 cells, and HEK
biosensor cells were not activated (Figure 6c). L-Histidine, a control for acidity, did
not activate prostaglandin synthesis (Figure 6c). Blocking uptake with probenecid
prevented prostaglandin release, and probenecid alone had no effect (Figure 6c).
Therefore, uric acid must enter the macula densa to promote prostaglandin synthesis.
The time-course of this pathway shows prostaglandin production peaking at 10
minutes, with renin release following shortly thereafter and beginning to plateau at
20 minutes (Figure 6d).

Figure 6. The effects of uric acid on macula densa prostaglandin synthesis. (A)
Cuvette-based spectrofluorometry measurements of the effects of UA on PGE
2

release from MMDD1 cells, assessed by Fura 2 calcium ratio in HEK biosensor
60

cells. HEK biosensor cells were transfected with the prostaglandin PGE
2
receptor,
EP1. PGE
2
binding to the EP1 receptor produced a rise in HEK intracellular
calcium, measured by the ratio of Fura-2 fluorescence. 500 μM UA and 100 μM UA
both caused MMDD1 PGE
2
production, but Krebs Ringer had no effect. n= 4
experiments per group. (B) Comparison of PGE
2
synthesis after uric acid and after
Krebs Ringer. Quantification of changes in HEK cell [Ca
2+
]
i
: 70±13 nM after uric
acid vs. 14±2 nM after Krebs Ringer. (C) Treatment of MMDD1 cells with 5 mM
probenecid abolished the effect of UA on PGE
2
production (black dots, top). COX-2
is responsible for PGE
2
synthesis. MMDD1 cells were treated with the non-selective
COX inhibitor, indomethacin. Perfusion of indomethacin-treated cells with UA
prevented PGE
2
production (second line, black). L-Histidine, an acid of similar pKa
as UA, had no effect on PGE
2
synthesis (dark gray). Treatment with 5 mM
probenecid abolished the ability of both 100 μM UA (light gray) to promote
MMDD1 PGE
2
synthesis. (n=7 for all experimental groups). (D) Time course of
PGE
2
production (black) and renin release (gray). Macula densa PGE
2
production
begins to peak at 10 minutes, with renin release beginning and peaking shortly
thereafter. MMDD1= mouse macula densa-derived cell line. COX=
cyclooxygenase. PGE
2
= prostaglandin E
2
.

61

Discussion
Hypertension is one of the most influential predictors of cardiovascular
disease severity, the primary cause of morbidity in the United States and the world.
In the past century, the prevalence of essential hypertension has increased from 5-
10% of Americans to over 30% today (22). The sweeping rise and epidemic
prevalence of hypertension reflect shortcomings in our ability to prevent disease, as
well as inadequacies in our treatments. Hypertension is a challenge driven by
contributions from genetics, the environment, and the yet unresolved complex
interactions between the two. As diet and exercise are critical components to the
management of hypertension, the clinical portrait suggests that diet/environment has
a profound influence on pathology.
Hyperuricemia can be used as a prognostic indicator for disease severity and
renal injury in hypertension. African-American race, gout, chronic lead intoxication,
obesity/the metabolic syndrome, and diuretic use are indicators for renal progression
in essential hypertension: uric acid has been linked with all of them (23). The tight
association between uric acid and risk factors for disease severity raises much
interest in understanding its contribution to the etiology of hypertension (23,24).
Because RAS inhibition negates the hypertensive effects of hyperuricemia, we aimed
to investigate the relationship between uric acid and RAS.
We hypothesized that uric acid had acute effects on the living animal and,
specifically, in the kidneys. Although increases in blood pressure persisted even 10
minutes after uric acid injection, these effects were not permanent, as blood pressure
62

eventually returned to normal with the clearance of uric acid. Telemetry
measurements have shown that 6-8 weeks of persistent hyperuricemia is necessary to
cause permanent hypertension in animals. The increase we observed was significant
but modest, and did not qualify as an acute induction of hypertension. Quantitative
multiphoton imaging of Munich-Wistar rats assessed uric acid-induced changes in
kidney function. Cortical blood flow increased, as both glomerular and peritubular
capillary red blood cell velocities rose (Figure 1a, 1b). In addition to hemodynamic
changes, renal function changed, as we detected a 150% increase in single-nephron
glomerular filtration rate (Figure 1c). This shows that hyperuricemia may mimic
renal changes in diabetic hypertension: early hyperfunctionality precedes late failure.
Since uric acid had acute effects on renal hemodynamics, we investigated its
influence on one of the kidney’s most important functions: regulation of RAS.
Abundant studies have already demonstrated chronic RAS activation with
hyperuricemia (15,16), without an explanation of the mechanism. Given the in vivo
evidence of the acute effects of uric acid, we hypothesized that uric acid and RAS
intersect in the kidney. RAS activity is controlled by the crucial regulatory step of
renin release from the JGA. Fluorescence confocal microscopy showed that uric
acid caused a 36±6% decrease of JGA renin content (Figure 2). An acid of similar
pKa, L-Histidine, had no effect on renin release, suggesting specificity for uric acid.
Renin release from juxtaglomerular cells is a highly regulated process known
to be stimulated by β-adrenergic innervation, the baroreflex, or autocoids like
prostaglandins and nitric oxide which may be derived from local endothelium or the
63

macula densa. Uric acid did not promote renin release from juxtaglomerular renin
granular cells, eliminating a direct unicellular mechanism (Figure 3b). Therefore, a
paracrine signaling mechanism involving hormones from local cells was examined.
It has been difficult to define the role of uric acid because it may function
beneficially as an antioxidant or detrimentally as a toxin under different
circumstances (25). However, our in vitro approach detects the direct, acute
influences of uric acid on cells. Although endothelial dysfunction has been
described in models of chronic hyperuricemia (11), uric acid did not activate
endothelial calcium signaling or promote the production of renin release signals (i.e.
nitric oxide) from the endothelium (Figure 3c). Therefore, although chronic
hyperuricemia involves endothelial dysfunction, there is no evidence for endothelial
involvement in RAS activation by uric acid.
The macula densa is often called the “brain” of the kidney, because it plays a
critical role in moderating two important renal mechanisms of blood pressure
regulation: tubuloglomerular feedback (TGF) and renin release. The existing
paradigm identifies the macula densa as a tubular salt sensor, whose primary role is
the control of salt concentration by directing changes in filtration rate via TGF or salt
reabsorption via Ang II. However, this study directs the perspective to other sides of
the macula densa: it receives basolateral input from a sugar metabolite found in the
circulation, and its ouput is the activation of a potent hypertensive pathway (RAS).
It presents the macula densa as an important metabolic sensor and effector.

64

In our studies, the macula densa was acutely responsive to uric acid,
producing and releasing prostaglandins hormones known to stimulate renin release
(Figures 4, 6). Several studies have localized an organic anion transporter, OAT3, to
the basolateral aspect of the macula densa (26,27). OATs are known to facilitate uric
acid uptake into cells (28). Using probenecid to inhibit OATs (13), we prevented
uric acid entry into the macula densa and prostaglandin synthesis was inhibited.
Since the overwhelming majority of uric acid is reabsorbed in the proximal tubule
(29), it follows that the apical macula densa (with access to the tubular lumen) would
not be exposed to excessively high uric acid levels. In contrast, the basolateral pole
of the macula densa resides adjacent to the interstitial compartment, namely plasma
which has escaped through afferent arteriole fenestrations. Previous work has
described the presence of rapid rates and significant volumes of fluid flow within the
JGA environment (30). Our study provides an example of the dynamic processes
occurring in this region, and its potential significance to pathology. With plasma
from the afferent arteriole easily entering this interstitial component, there is the
opportunity for persistent macula densa activation by uric acid. The macula densa
may then trigger renin release, some of which enters the systemic circulation and
some of which may re-enter the JGA interstitium. The recent localization of the
(pro)renin receptor to the basolateral macula densa establishes a precarious setting
for RAS overstimulation (31). This suggests the possibility of a short positive-
feedback loop where uric acid activates macula densa-mediated renin release, and
the released renin can then activate its receptor on the macula densa. In essence,
65

monitoring uric acid levels may help control RAS activation, but management of the
many consequences becomes a tremendous challenge once renin is released.
MAP kinases are important mediators of cellular signals for proliferation or
gene regulation, and have been studied extensively in the kidneys. MAPK activation
has been linked with COX-2 upregulation, which influences prostaglandin levels
(32). However, reports show the association of uric acid and MAPK to be disruptive
to normal cellular physiological functions, leading to pathology. For example, uric
acid activates MAPKs, including ERKs, in vascular smooth muscle cells by altering
intracellular redox (34,34). A recent study has shown that MAPK activation
enhances expression of xanthine oxidoreductase, which catalyzes the formation of
uric acid (35). Our study has shown that uric acid activates MAPKs in the macula
densa to promote renin release, and it seems that macula densa MAPK activation
may in turn exacerbate hyperuricemia.
Other studies have reported that metabolic intermediates cause RAS
activation (36, 37). This study provides further support for the hypothesis of a
dietary influence on instigating hypertension. Reducing uric acid levels not only
improves RAS-dependent symptoms, but an Ang II receptor blocker with mildly
uricosuric effects provided even better disease management, suggesting that uric acid
also has significant RAS-independent effects (15). In fact, chronic studies have
shown that it induces inflammation, vascular injury, and interstitial damage
(34,38,39,40). However, acute RAS activation permits the generation of

66

hypertensive autocoids in the short-term which may disrupt normal physiology even
before chronic effects of uric acid cause disease.
Uric acid is more than a symbol associated with underlying pathology, it is an
active agent in propagating the disease. It promotes the production of hypertensive
hormones from the kidneys in the short-term, and eventually causes renal damage.
In humans, only about 10% of the filtered load of uric acid is excreted (41). In other
words, since 90% of uric acid is reabsorbed, it remains in the circulation and may
persistently cause interferences. Uric acid is an unacknowledged and untreated risk
factor in a significant proportion of hypertensive patients. In fact, studies have
shown that hyperuricemia may predict the onset of hypertension in children and
adults (43,43). Our studies provide a mechanistic explanation supporting the
contribution of uric acid to initiating hypertension. Accordingly, the correction of
hyperuricemia may be a valuable component of not only the management, but
possibly even the prevention, of hypertension.

67

Methods
Multi-photon fluorescence microscopy
All preparations were visualized using a two-photon laser scanning fluorescence
microscope (TCS SP2 AOBS MP confocal microscope system, Leica-Microsystems,
Heidelberg, Germany). A Leica DM IRE2 inverted microscope was powered by a
wideband, fully automated, infrared (710-920nm) combined photo-diode pump laser
and mode-locked titanium:sapphire laser (Mai-Tai, Spectra-Physics) for multiphoton
excitation, and/or by red (HeNe 633nm/10mW), orange (HeNe 594nm/2mW), green
(HeNe 543nm/1.2mW) and blue (Ar 458nm/5mW; 476nm/5mW; 488nm/20mW;
514nm/20mW) lasers for conventional confocal microscopy. Images were collected
in time-series (xyt) and analyzed with Leica LCS imaging software.
Animals
All experiments were performed under protocols approved by the Institutional
Animal Care and Use Committee at USC. Male Munich Wistar-Fromter rats (6-8
weeks), purchased from Harlan (Houston, TX), were used for imaging because of
their superficial glomeruli. C57BL6 background mice were bred at USC. Animals
were housed in a temperature-controlled room under a 12h light-dark cycle with
water and standard chow available ad libitum.
In vivo experiments
Male Munich Wistar-Fromter rats were anesthetized with Inactin (120 mg/kg) and
instrumented for multi-photon in vivo imaging of the intact kidney as described
previously (44). Briefly, the femoral artery was cannulated to monitor systemic
68

blood pressure using an analog single-channel transducer signal conditioner model
BP-1 (World Precision Instruments). The left femoral vein was cannulated for dye
infusion of quinacrine (Sigma), rhodamine-conjugated 70,000 MW dextran
(Invitrogen), and Lucifer yellow (Invitrogen). Kidney was exteriorized via a small
dorsal incision. Animal was placed on the stage of the Leica IRE2 inverted
microscope with exposed kidney placed in a coverslip-bottomed heated chamber
bathed in normal saline and visualized from below using a HCX PL APO
63X/1.4NA oil CS objective (Leica). All three fluorescent probes were excited using
the same, single excitation wavelength of 860 nm (Mai-Tai), and the emitted, non-
descanned fluorescent light was detected by two external photomultipliers (green and
red channels) with the help of a FITC/TRITC filter block (Leica). Uric acid (0.1
mg/kg in Kreb’s Ringer, pH 7.4) was given as bolus injection through femoral vein.
In vitro microperfusion
A superficial afferent arteriole with attached glomerulus was microdissected from
freshly harvested mouse kidney slices. The afferent arteriole was cannulated and
perfused using a method similar to those described previously (46,47,47). Briefly,
dissection media was prepared from DMEM mixture F-12 (Sigma) with 3% fetal
bovine serum (Hyclone). Vessel perfusion and bath fluid was a modified Krebs-
Ringer-HCO
3
buffer containing (in mM): 115 NaCl, 25 NaHCO
3
, 0.96 NaH
2
PO
4
,
0.24 Na
2
HPO
4
, 5 KCl, 1.2 MgSO
4
, 2 CaCl
2
, 5.5 D-glucose and 100μM L-Arginine.
Solutions we adjusted to pH 7.4 and aerated with 95% O
2
-5% CO
2
for 45 minutes.
Experimental solution was 100 μM uric acid (20 mg uric acid in 1 mL of 1N NaOH,
69

diluted 1:1000 in Krebs Ringer, pH 7.2) or 100 μM L-Histidine (in Krebs Ringer’s
solution). Each preparation was transferred to a thermoregulated Lucite chamber
mounted on the Leica inverted microscope. Preparations were kept in dissection
media at 4°C until cannulation, and then gradually raised to 37°C for the experiment.
The bath was continuously aerated with 95% O
2
-5% CO
2
. Quinacrine (Sigma), an
intravital fluorescent dye selective for dense-core secretory granules, was used to
label renin granules (48). Membrane stain octadecyl rhodamine B chloride R18 and
nuclear stain Hoechst 33342 were used to visualize the glomerulus and attached
afferent arteriole. For ratiometric calcium imaging, the Fluo-4/Fura Red pair was
used as described before (47,49). DAF-FM was used to monitor changes in nitric
oxide production as described (50). All fluorophores were from Invitrogen.
Isolation of juxtaglomerular (JG) cells
Sacrifice male mice (C57B1/6 background, 6-8 weeks) by Inactin injection (120
mg/kg). Flush aorta with Krebs Ringer’s solution to exsanguinate viscera. Remove
kidneys, decapsulate, and mince in a plastic Petri dish. JG cells were extracted based
on a modification of a previously validated method (51). Transfer tissue paste to a
flask with 30 mL sterile isolation buffer (pH 7.4, 37°C, in mM): 130 NaCl, 5 KCl, 2
CaCl
2
, 10 D-glucose, 20 sucrose, and 10 HEPES, supplemented with 0.1% w/v
collagenase (0.5 U/mL) and 0.25%w/v trypsin (1300 BAEE U/mg). Digest by
shaking horizontally at 250 rpm for 70 min at 37°C. Vacuum-filter the cell
suspension in sterile hood through 20 μm nylon mesh. Flush filter with 2 mL
isolation buffer, filter cells, and flush flask with 10 mL buffer to recover all cells.
70

Transfer cell suspension to sterile 50 mL tubes. Centrifuge filtered cells with 20 mL
isolation buffer at 1000g for 7 minutes, repeat. Resuspend pellet in JG cell medium
(RPMI cell-culture medium, 1 L distilled water, 2.2 g NaHCO
3
, 2% FCS, insulin
0.66 U/mL, 1% Pen/Strep; equilibrate with 95% O
2
and 5% CO
2
, pH 7.2). Seed
cells onto glass coverslips to use for spectrofluorometry studies within one week.
Spectrofluorometry to measure renin enzymatic activity
Renin activity was evaluated by cuvette-based spectrofluorometry (Quantamaster-8,
Photon Technology, Inc., Princeton, NJ) using a FRET-based renin substrate
(Anaspec, San Jose, CA) assay as described previously (46). Substrate is present in
excess, so the initial reaction rate (within 50 seconds of adding sample) estimates
renin activity, using Felix software (PTI). To control for specificity, the renin active
site inhibitor Aliskiren (Novartis, East Hanover, NJ; 50 μmol/L) was incubated with
sample and no renin enzymatic activity was detected.
Cell culture
TsA58 immorto mice glomerular endothelial cells (GENC, generous gift from Dr.
Akis, Istanbul, Turkey) (52) were grown to confluence in 10% DMEM, 25mM
HEPES, 9mM NaHCO3, 7.5% new born calf serum, and 1% penicillin/streptomycin
in a humidified (95% air-5% CO2) incubator at 37°C. Mouse macula densa derived
(MMDD1) cells were a generous gift from Dr. Schnermann (53). MMDD1 cells
were cultured in DMEM nutrient mixture/Ham's F-12 (Invitrogen) supplemented
with 10% FBS, penicillin (100 units/ml), and streptomycin (100 µg/ml) and
incubated at 37°C in a humidified 5% CO
2
atmosphere. Confluent cells were made
71

quiescent with overnight serum starvation prior to studies.
Spectrofluorometry
To detect endothelial nitric oxide synthesis, dissolve 50 μg DAF-FM (Molecular
Probes, Eugene, OR) in 10 μL DMSO. Dilute into 10 mL Krebs Ringer-HCO
3
in the
dark. Load GENC with dye for 10 minutes and wash for 15 minutes. Transfer dye-
loaded coverslip to cuvette with 3 mL of Krebs Ringer-HCO
3
at 37ºC. DAF-FM
fluorescence is measured in a cuvette-based fluorometer with excitation wavelength
of 495 nm and emission of 515 nm. Perfuse cells in cuvette with control Krebs
Ringer for 100 seconds to ensure appropriate baseline counts. The reading plateaus
with cell-bath equilibrium. Switch perfusate to the experimental uric acid solution
and continue recording the change in fluorescence until a plateau is reached again.
To measure GENC calcium, dissolve Fura 2 (Invitrogen) in 3 μL DMSO, dilute into
10 mL Krebs Ringer-HCO
3
. Load cells for 30 minutes, and wash for 20 minutes.
Intracellular [Ca
2+
] of glomerular endothelial cells (GENC) were measured with dual
excitation wavelength fluorometry (Quantamaster-8, Photon Technology, Inc.,
Princeton, NJ, USA) in a cuvette-based system. Fura-2 fluorescence was measured at
510 nm emission in response to excitation wavelengths of 340 and 380 nm. Emitted
photons were detected by a photomultiplier. Autofluorescence-corrected ratios
(340/380) were calculated at a rate of 5points/sec using PTI software.
Immunohistochemistry
MMDD1 cells on glass coverslips were serum-starved overnight and treated with
100 μM uric acid (Sigma) at pH 7.2 or 10 μM MAPK inhibitor PD 98059 (Cell
72

Signal) for varying times. Cells were fixed in 4% formalin for 10 minutes and then
permeabilized with 0.1% Triton-X/PBS for 5 minutes. Sections were blocked with
1:20 goat serum and incubated at 4°C overnight with phosphoERK (p44/42) (1:100,
Cell Signalling) monoclonal antibody (54). Sections were incubated for 1 hour with
AF594 conjugated secondary antibody (1:500, Invitrogen). Sections were mounted
with Vectashield containing DAPI for nuclear staining (Vector Laboratories).
Western blot analysis
Cells were treated as with immunohistochemistry, but protein was extracted.
Western blots against MAPK were performed with 40 μg of protein from MMDD1
lysates. Protein was separated on a 4-20% SDS-PAGE and transferred onto PVDF
membrane. Blots were blocked ≥1 hour with 5% nonfat dry milk in Tris-buffered
saline and tween-20 (TBS-T) at room temp. Blots were incubated ≥1 hour at room
temp with affinity-purified antibodies against phospho p44/42 ERK (1:1000 Cell
Signalling) and ß-actin (1:1000, Abcam). After wash, blots were incubated in 5%
nonfat dry milk in TBS-T with secondary antibody and visualized with Odyssey
Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).
PGE
2
biosensor technique
MMDD1 cells were grown to confluence on 12 x 40 mm coverslips which fit into a
standard quartz cuvette diagonally. Coverslip was bathed in Krebs Ringer with Fura
2-loaded HEK/EP1 biosensor cells. After signal stabilization, uric acid was added,
and prostaglandins detected.
Generation of HEK293/EP1 cells as PGE
2
biosensors and their use has been
73

described before (55). [Ca
2+
]
i
of HEK cells was measured with dual-excitation
wavelength fluorescence microscopy (Photon Technologies, Princeton, NJ) using the
fluorescent probe, Fura 2 (Invitrogen), as described before (49,55). Briefly, Fura 2
fluorescence was measured at 510 nm in response to excitations of 340 and 380 nm,
alternated at 25 Hz by a computer-controlled chopper assembly. Autofluorescence-
corrected ratios (340 nm/380 nm) were calculated at a rate of 5 points/s using. HEK
cells were loaded with acetoxymethyl ester Fura 2 (Fura 2-AM) dissolved in 3 µL
DMSO and diluted in 10 mL Krebs Ringer. Cells were loaded for 30 minutes and
washed for 20 minutes. Increases in [Ca
2+
]
i
normally result in an increase in the 340
nm signal with a parallel decrease in the 380 nm signal. The 340/380 Fura 2 ratios
(R) were converted into [Ca
2+
]
i
values using the equation of Grynkiewicz, et.al. (56):
[Ca
2+
]
i
= K
d
x [(R-R
min
)/(R
max
-R)] x (S
f380
/S
b380
)
where R
max
and R
min
are R values under saturating and Ca
2+
-free conditions,
respectively, and S
f380
and S
b380
are the fluorescence signals (S) emitted by the Ca
2+
-
free and Ca
2+
-bound forms of Fura 2 at 380 nm. In situ calibration was
accomplished after permeabilizing HEK cells with 5 µM ionomycin and measuring
fluorescence at both wavelengths under Ca
2+
-free (2 mM EGTA) or saturating Ca
2+

(1.5 mM CaCl
2
) conditions. Rmax, Rmin, and S
f380
/S
b380
values were 4.0, 0.7, and
4.25, respectively. K
d
, the dissociation constant of Fura 2 for Ca
2+
, was 224 nM.
Statistical Analysis
Data are expressed as mean ± SE. Statistical significance was tested using ANOVA.
Significance was accepted at P < 0.05.
74

Chapter 2 References
1. Ford, E.S., Giles, W.H., and Dietz, W.H. 2002. Prevalence of the metabolic
syndrome among US adults: findings from the third National Health and
Nutrition Examination Survey. JAMA. 287:356-359.

2. Moller, D. 2001. New drug targets for type 2 diabetes and the metabolic
syndrome. Nature. 414:821-827.

3. Basciano, H., Federico, L., and Adeli, K. 2005. Fructose, insulin resistance, and
metabolic dyslipidemia. Nutrition & Metabolism. 2:5.

4. Nakagawa, T., Tuttle, K.T., Short, R.A., and Johnson, R.J. 2005. Hypothesis:
Fructose-induced Hyperuricemia as a Causal Mechanism for the Epidemic of the
Metabolic Syndrome. Nat Clin Pract Neprol. 1:80–86.

5. Nakagawa, T., Hu, H., Zharikov, S., Tuttle, K.R., Short, R.A., Glushakova, O.,
Ouyang, X., Feig, D.I., Block, E.R., Herrera-Acosta, J., Patel, J.M., and Johnson,
R.J. 2006. A causal role for uric acid in fructose-induced metabolic syndrome.
Am J Physiol Renal Physiol. 290:F625–631.

6. Feig, D.I., Mazzali, M., Kang, D.H., Nakagawa, T., Price, K., Kannelis, J., and
Johnson, R.J. 2006. Serum uric acid: a risk factor and a target for treatment? J
Am Soc Nephrol. 17:S69-73.

7. Latta, M., Kunstle, G., Leist, M., and Wendel, A. 2000. Metabolic depletion of
ATP by fructose inversely controls CD95- and tumor necrosis factor receptor 1-
mediated hepatic apoptosis. J Exp Med. 191:1975-1985.

8. Mahomed, F.A. 1879. On chronic Bright’s disease, and its essential symptoms.
Lancet. I: 399–401.

9. Haig A. 1889. On uric acid and arterial tension. BMJ. 1:288–291.

10. Johnson, R.J., Feig, D.I., Herrera-Acosta, J., and Kang, D.-H. 2005. Resurrection
of Uric Acid as a Causal Risk Factor in Essential Hypertension. Hypertension.
45:18-20.

11. Khosla, U.M., Zharikov, S., Finch, J.L., Nakagawa, T., Roncal, C., Mu, W.,
Krotova, K., Block, E.R., Prabhakar, S., and Johnson, R.J. 2005. Hyperuricemia
induces endothelial dysfunction. Kidney Int. 67:1739-1742.

75

12. Sánchez-Lozada, L.G., Nakagawa, T., Kang, D.H., Feig, D.I., Franco, M.,
Johnson, R.J., and Herrera-Acosta, J. 2006. Hormonal and cytokine effects of
uric acid. Curr Opin Nephrol Hypertens. 15:30-33.

13. Kang, D-H, Han, L., Ouyang, X., Kahn, A.M., Kanellis, J., Li, P., Feng, L.,
Nakagawa, T., Watanabe, S., Hosoyamada, M., Endou, H., Lipkowitz, M.,
Abramson, R., Mu, W. and Johnson, R.J. 2005. Uric Acid Causes Vascular
Smooth Muscle Cell Proliferation by Entering Cells via a Functional Urate
Transporter. Am J Nephrol. 25:425-433.

14. Feig, D.I., Kang, D.H., Nakagawa, T., Mazzali, M., and Johnson, R.J. 2006. Uric
Acid and Hypertension. Curr Hypertens Rep. 8:111-115.

15. Mazzali, M., Kannellis, J., Han, L., Feng, L., Xia, Y.-Y., Chen, Q., Kang, D.-H.,
Gordon, K.L., Watanabe, S., Nakagawa, T., Lan, H.Y., and Johnson, R.J. 2002.
Hyperuricemia induces a primary renal arteriolopathy in rats by a blood pressure-
independent mechanism. Am J Physiol Renal Physiol. 282:F991–F997.

16. Mazzali, M., Hughes, J., Kim ,Y.-G., Jefferson, J.A., Kang, D.-H., Gordon, K.L.,
Lan, H.Y., Kivlighn, S., and Johnson, R.J. 2001. Elevated uric acid increases
blood pressure in the rat by a novel crystal-independent mechanism.
Hypertension. 38:1101-1106.

17. Sánchez-Lozada, L.G., Tapia, E., Jimenez, A., Bautista, P., Cristobal, M.,
Nepomuceno, T., Soto, V., Avila-Casado, C., Nakagawa, T., Johnson, R.J.,
Herrera-Acosta, J., and Franco, M. 2007. Fructose-induced metabolic syndrome
is associated with glomerular hypertension and renal microvascular damage in
rats. Am J Physiol Renal Physiol. 292:F423-F429.

18. Sánchez-Lozada, L.G., Tapia, E., Rodriguez-Iturbe, B., Johnson, R.J., Herrera-
Acosta, J. 2005. Hemodynamics of hyperuricemia. Sem in Nephrol. 25:19-24.

19. Perlstein, T.S., Gumieniak, O., Hopkins, P.N., Murphey, L.J., Brown, N.J.,
Williams, G.H., Hollenberg, N.K., and Fisher, N.D. 2004. Uric acid and the state
of the intrarenal renin-angiotensin system in humans. Kidney Int. 66:1465-1470.

20. Zhou, X., Matavelli, L., and Frohlich, E.D. 2006. Uric acid: its relationship to
renal hemodynamics and the renal renin-angiotensin system. Curr Hypertens
Rep. 8:120-124.

21. Sautin, Y.Y., Nakagawa, T., Zharikov, S., Johnson, R.J. 2007. Adverse effects
of the classic antioxidant uric acid in adipocytes: NADPH oxidase-mediated
oxidative/nitrosative stress. Am J Physiol Cell Physiol. 293:C584-C596.

76

22. Feig, D.I., Rodriguez-Iturbe, B., Nakagawa, T., and Johnson, R.J. 2006. Nephron
Number, Uric Acid, and Renal Microvascular Disease in the Pathogenesis of
Essential Hypertension. Hypertension. 48:1-2.

23. Johnson, R.J., Segal, M.S., Srinivas, T., Ejaz, A., Mu, W., Roncal, C., Sanchez-
Lozada, L.G., Gersch, M., Rodriguez-Iturbe, B., Kang, D.H., and Acosta, J.H.
2005. Essential hypertension, progressive renal disease, and uric acid: a
pathogenetic link? J Am Soc Nephrol. 16:1909-1919.

24. Johnson, R.J., Rodriguez-Iturbe, B., Kang, D, Feig, D.I., Herrera-Acosta, J. 2005.
A unifying pathway for essential hypertension. Am J Hypertens. 18:431-440.

25. Hediger, M.A. 2002. Gateway to a long life? Nature. 417:393-395.

26. Bahn, A., Ljubojevic, M., Lorenz, H., Schultz, C., Estifanos, G., Ugele, B.,
Sabolic, I., Burckhardt, G., and Hagos, Y. 2005. Murine renal organic anion
transporters mOAT1 and mOAT3 facilitate the transport of neuroactive
tryptophan metabolites. Am J Physiol Cell Physiol. 289:C1075-C1084.

27. Nilwarangkoon, S., Anzai, N., Shiraya, K., Yu, E., Islam, R., Cha, S.H.,
Onozato, M.L., Miura, D., Jutabha, P., Tojo, A., Kanai, Y., Endou, H. 2007. Role
of Mouse Organic Anion Transporter 3 (mOat3) as a Basolateral Prostaglandin
E2 Transport Pathway. Journal of Pharmacological Sciences. 103:48-55.

28. Price, K.L., Sautin, Y.Y., Long, D.A., Zhang, L., Miyazaki, H., Mu, W., Endou,
H., and Johnson, R.J. 2006. Human Vascular Smooth Muscle Cells Express a
Urate Transporter. J Am Soc Nephrol. 17:1791-1795.

29. Enomoto, A., Kimura, H., Chairoungdua, A., Shigeta, Y., Jutabha, P., Cha, S.H.,
Hosoyamada, M., Takeda, M., Sekine, T., Igarashi, T., Matsuo, H., Kikuchi, Y.,
Oda, T., Ichida, K., Hosoya, T., Shimokata, K., Niwa, T., Kanai, Y., and Endou,
H. 2002. Molecular identification of a renal urate anion exchanger that regulates
blood urate levels. Nature. 417:447-452.

30. Rosivall, L., Mirzahosseini, S., Toma, I., Sipos, A., and Peti-Peterdi, J. 2006.
Fluid flow in the juxtaglomerular interstitium visualized in vivo. Am J Physiol
Renal Physiol. 291:F1241-1247.

31. Nguyen, G. 2006. Increased cyclooxygenase-2, hyperfiltration,
glomerulosclerosis, and diabetic nephropathy: put the blame on the (pro)renin
receptor? Kidney Int. 70:618-620.

32. Harris, R.C., and Breyer, M.D. 2001. Physiological regulation of
cyclooxygenase-2 in the kidney. Am J Physiol Renal Physiol. 281:F1-F11.
77

33. Johnson, R.J., Kang, D., Feig, D., Kivlighn, S., Kanellis, J., Watanabe, S., Rodri-
guez-Iturbe, B., Mazzali, M. 2003. Is There a Pathogenetic Role for Uric Acid in
Hypertension and Cardiovascular and Renal Disease? Hypertension. 41:1183-90.

34. Kanellis, J., Watanabe, S., Li, J., Kang, D., Li, P., Nakagawa, T., Wamsley, A.,
Sheikh-Hamad, D., Lan, H., Feng, L., Johnson, R. 2003.Uric acid stimulates mo-
nocyte chemoattractant protein-1 production in vascular smooth muscle cells via
mitogen-activated protein kinase cyclooxygenase-2. Hypertension. 41:1287-93.

35. Seymour, K., Roberts, L., Fini, M., Oustitch, T., Wright, R. 2006. Stress
activation of mammary epithelial cell xanthine oxidoreductase is mediated by
p38 MAPK CCAAT/enhancer-binding protein-beta. J Biol Chem. 281:8545-58.

36. He, W., Miao, F., Lin, D., Schwandner, R., Wang, Z., Gao, J., Chen, J., Tian, H.,
Ling, L. 2004. Citric acid cycle intermediates as ligands for orphan G-protein-
coupled receptors. Nature. 429:188-193.

37. Hebert, S.C. Orphan detectors of metabolism. 2004. Nature. 429:143-145.

38. Coutinho, T.A., Turner, S.T., Peyser, P.A., Bielak, L.F., Sheedy, P.F., Kullo, I.J.
2007. Associations of serum uric acid with markers of inflammation, metabolic
syndrome, and subclinical coronary atherosclerosis. Am J Hypertens. 20:83-89.

39. Kang, D., Nakagawa, T. 2005. Uric acid and chronic renal disease: possible imp-
lication of hyperuricemia on progression renal disease. Semin Nephrol. 25:43-49.

40. Zoccali, C., Maio, R., Mallamaci, F., Sesti, G., Perticone, F. 2006. Uric acid and
endothelial dysfunction in essential hypertension. J Am Soc Nephrol. 17:1466-71.

41. Cameron, J.S., Moro, F., and Simmonds, H.A. Uric acid and the kidney. Oxford
Textbook of Clinical Nephrology, 2nd ed. Eds. A. Davison, J. Cameron, J. Grün-
feld, D. Kerr, E. Ritz, C.G. Winearls. London: Oxford Medical, 1997.1157–1175.

42. Feig, D.I., Nakagawa, T., Karumanchi, S.A., Oliver, W.J., Kang, D.H., Finch, J.,
and Johnson, R.J. 2004. Hypothesis: Uric acid, nephron number, and the
pathogenesis of essential hypertension. Kidney Int. 66:281-287.

43. Viazzi, F., Leoncini, G., Ratto, E., Falqui, V., Parodi, A., Conti, N., Derchi, L.E.,
Deferrari, G., Pontremoli, R. 2007. Mild hyperuricemia and subclinical renal
damage in untreated primary hypertension. Am J Hypertens. 20:1276-1282.

44. Kang, J.J., Toma, I., Sipos, A., McCulloch, F., Peti-Peterdi, J. 2006. Quantitative
Imaging of basic functions in renal (patho)physiology. Am J Physiol Renal
Physiol. 291:F495–F502.
78

45. Toma, I., Kang, J.J., and Peti-Peterdi, J. 2006. Multiphoton Imaging renin
content and release in the living kidney. Nephron Physiol. 103:71–74.

46. Kang, J.J., Toma, I., Sipos, A., McCulloch, F., Peti-Peterdi, J. 2006. Imaging the
renin-angiotensin system: an important target of anti-hypertensive therapy.
Adv Drug Deliv Rev. 58:824-833.

47. Peti-Peterdi J. 2006. Calcium wave of tubuloglomerular feedback. Am J Physiol
Renal Physiol. 291:F473-480.

48. Peti-Peterdi, J., Fintha, A., Fuson, A.L., Tousson, A., Chow, R.H.2004.Real-time
imaging of renin release in vitro. Am J Physiol Renal Physiol. 287:F329-F335.

49. Peti-Peterdi, J., Bell, P.D. 1999. Cytosolic [Ca2+] signaling pathway in macula
densa cells. Am J Physiol. 277:F472-F476.

50. Kovács G, Komlósi P, Fuson A, Peti-Peterdi J, Rosivall L, Bell PD. 2003.
Neuronal nitric oxide synthase: its role and regulation in macula densa cells. J
Am Soc Nephrol. 14:2475-2483.

51. Della Bruna, R., Pinet, F., Corvol, P., and Kurtz, A. 1991. Regulation of renin
secretion and renin synthesis by second messengers in isolated mouse
juxtaglomerular cells. Cell Physiol Biochem. 1:98–110.

52. Akis, N., and Madaio, M.P. 2004. Isolation, culture, and characterization of
endothelial cells from mouse glomeruli. Kidney Int. 65:2223-2227.

53. Yang, T., Park, J.M., Arend, L., Huang, Y., Topaloglu, R., Pasumarthy, A.,
Praetorius, H., Spring, K., Briggs, J.P., and Schnermann, J. 2000. Low chloride
stimulation of prostaglandin E2 release and cyclooxygenase-2 expression in a
mouse macula densa cell line. J Biol Chem. 275:37922-37929.

54. Zimmermann, S., and Moelling, K. 1999. Phosphorylation and regulation of Raf
by Akt (protein kinase B). Science. 286:1741-1744.

55. Peti-Peterdi, J., Komlosi, P., Fuson, A., Guan,Y., Schneider, A., Qi, Z., Redha,
R., Rosivall, L., Breyer, M.D., Bell, P.D. 2003. Luminal NaCl delivery regulates
basolateral PGE2 release from macula densa cells. J Clin Invest. 112:76-82.

56. Grynkiewicz, G., Ponie, G., Tsien, R.Y. 1985. A new generation of Ca
2+
indicat-
ors with greatly improved fluorescence properties. J Biol Chem. 260:3440-
3450.
79

Chapter 3: Prorenin: New beginnings in the renin-angiotensin system

Kang JJ, Toma I, Sipos A, Meer EJ, Vargas SL, and Peti-Peterdi J. The collecting
duct is the major source of prorenin in diabetes. Hypertension. 2008; in press.

Abstract
In addition to the juxtaglomerular apparatus, renin is also synthesized in renal
tubular epithelium including the collecting duct. Ang II differentially regulates the
synthesis of juxtaglomerular (inhibition) and collecting duct (stimulation) renin.
Since diabetes mellitus, a disease with high intra-renal renin-angiotensin system and
Ang II activity, is characterized by high prorenin levels, we hypothesized that the
collecting duct is the major source of prorenin in diabetes. Renin granular content
was visualized using in vivo multi-photon microscopy of the kidney in diabetic
Munich-Wistar rats. Diabetes caused a 3.5-fold increase in collecting duct renin, in
contrast to less pronounced juxtaglomerular changes. Ang II AT
1
receptor blockade
with Olmesartan reduced collecting duct renin to control levels, but significantly
increased juxtaglomerular renin. Using a fluorogenic renin assay, the prorenin
component of collecting duct renin content was measured by assessing the difference
in enzymatic activity of medullary homogenates before and after trypsin activation of
prorenin. Trypsinization caused no change in control renin activity, but a 5-fold
increase in diabetes. Studies on a collecting duct cell line (M1) showed a 22-fold
increase in renin activity after trypsinization, and a further 35-fold increase with Ang
II treatment. Therefore, prorenin significantly contributes to baseline collecting duct
renin. Diabetes, possibly via Ang II, greatly stimulates collecting duct prorenin and
causes hyperplasia of renin-producing connecting segments. These novel findings
80

suggest that in a rat model of diabetes, prorenin content and release from the
collecting duct may be more important than the juxtaglomerular apparatus, in
contrast to the existing paradigm.
81

Introduction
The renin-angiotensin system (RAS) is one of the most significant
physiological mechanisms of blood pressure regulation, and its dysfunction causes
many pathologies. Renin release is the rate-limiting step of RAS activation and
according to the existing paradigm, renin and its biosynthetic precursor prorenin (1)
are mainly produced in the kidney by granular cells of the juxtaglomerular apparatus
(JGA) in the terminal afferent arteriole (2-5). Recent work, however, has established
that renin transcripts

and protein are also present in renal tubular segments, including
the collecting duct (6-8). The two predominantly studied renal sources of (pro)renin
(a term denoting both renin and prorenin) are granular cells of the JGA and principal
cells of the collecting duct (CD). Since principal cells of the CD synthesize renin de
novo and are far more abundant than the limited population of juxtaglomerular cells,
the CD provides a potentially significant supply of (pro)renin (6-10). Angiotensin II
(Ang II), the potent effector of RAS, oppositely regulates renin synthesis at these
intrarenal locations: Ang II feedback inhibits JGA renin (2-5), but stimulates CD
renin through an AT
1
receptor-dependent mechanism (7,8). Therefore, CD renin
appears to be more relevant to high Ang II states.
The body of evidence has revealed expanded capabilities of RAS, revealing
its complexity compared to the traditional model, in particular in diabetes mellitus.
Although direct data from experimental animals have failed to show significantly
elevated intrarenal Ang II levels in diabetes (11), experimental and clinical studies
showing the efficacy of RAS inhibitors as treatment (12, 13) reveal the activation
82

and importance of some RAS components. While studies have not shown the
expected correlation between renin and RAS activity (14), diabetes has been firmly
associated with vastly increased prorenin levels (15). Prorenin may, in turn, be
proteolytically (enzymatically) or non-proteolytically (by a receptor) activated into
active renin (16). Thus, prorenin may serve as an overlooked and largely undetected
source of Ang II.
Our laboratory recently developed an experimental approach to quantitatively
visualize kidney functions in vivo in the intact kidney and in vitro in the JGA,
including real-time imaging of renin granular content, release and tissue activity (17-
19). This approach applies multi-photon confocal fluorescence microscopy, an ideal
technique for deep optical sectioning of living tissues (18). Quinacrine has been
used extensively to stain the contents of many acidic organelles including renin
granules both in vitro and in vivo (17-22). Although limitations of quinacrine
include its non-specificity for renin, it has been a useful label for renin granular
content because of the large size of granules (in the 2-3 micron range), as opposed to
other acidic vesicular organelles including lysosomes that are much smaller (in the
nanometer range). Also, renin granules are more acidic and therefore trap more
quinacrine (17). The ability of quinacrine and LysoTracker dyes to selectively and
intensely label large renin granules as opposed to small, diffusely distributed
lysosomes has been published (17). Furthermore, colocalization of quinacrine
fluorescence with JGA renin by immunohistochemistry validates the use of this dye
to label JGA renin granules (17).
83

In the present studies, we visualized quinacrine-stained granules in the JGA
and CD as an indication of both renin and prorenin contents in control and diabetic
conditions. Since Ang II differentially regulates JGA and CD (pro)renin, we
hypothesized that the CD could be the major source of prorenin in diabetes. Several
investigations have shown increased synthesis of other RAS components, like
angiotensinogen, in Ang II-rich environments (23, 24). Therefore, we studied if Ang
II was also responsible for augmenting CD renin. Ang II upregulation of CD
prorenin could offer an explanation for the RAS paradox in diabetes: high prorenin
levels and RAS activity, despite JGA renin suppression.
84

Methods
In vivo multi-photon fluorescence imaging of the kidney
A Leica TCS SP2 confocal microscope system (Leica Microsystems, Heidelberg,
Germany) was used to image renal tissues in vivo as described (18-21). Briefly,
Munich-Wistar-Fromter rats (200 g, Harlan, Madison, WI) were used because of
their superficial glomeruli. Diabetes was induced by a single streptozotocin injection
(50 mg/kg ip). Other groups received vehicle injections. Experimental groups
received Olmesartan (Sankyo, Tokyo, Japan) at 5 mg/day in chow for 4 weeks. For
imaging, the animals were anesthetized by thiobutabarbital (Inactin, 130 mg/kg ip).
The left femoral vein and artery were cannulated for dye infusion and blood pressure
measurements. The left kidney was exteriorized through a small dorsal incision and
the animal was placed on an inverted microscope as described (19). Core body
temperature was maintained throughout procedures using a homeothermic

table. All
animal protocols were approved by the Institutional

Animal Care and Use Committee
at the University of Southern

California. Chemicals, if not indicated, were purchased
from Sigma (St.

Louis, MO).
Fluorescent probes
A 70 kDa dextran-rhodamine B conjugate (Invitrogen, Eugene, OR), quinacrine, and
a fluorescence resonance energy transfer (FRET)-based fluorogenic renin substrate
(AnaSpec, San Jose, CA) were used to label the circulating plasma, renin content,
and activity as described previously (17-22).

85

Semi-quantitative analysis of renin content in vivo
The quinacrine-positive length of the afferent arteriole was used as an index of JGA
renin content. An index of CD quinacrine labeling was analyzed as the ratio of
quinacrine fluorescence intensity in principal cells to intercalated cells. Principal
cells were identified by characteristic bulging of their apical poles into the
concentrated, highly fluorescent CD lumen.
Sample Collection
Kidneys were harvested after 4 weeks of diabetes. One kidney was fixed in formalin
for immunohistochemistry as described (17). Polyclonal rabbit antibody against rat
(pro)renin was provided by Dr. Inagami, and used on paraffin-embedded rat sections
or M1 cells. Polyclonal rabbit antibody against proliferating cell nuclear antigen
(PCNA) was from Santa Cruz Biotechnologiy, Inc. (Santa Cruz, CA). Samples were
incubated overnight at 4°C with 1:250 dilution of renin or 1:100 PCNA antibodies,
1:500 Alexa 594-conjugated secondary antibody (Invitrogen, Carlsbad, CA), and
Vectashield mounting medium containing DAPI for nuclear labeling (Vector
Laboratories, Burlingame, CA).
The other kidney was dissected into cortex and medulla. CD renin was analyzed by
homogenizing medullary samples and extracting protein in Protease Inhibitor (BD
Biosciences, San Jose, CA) at 1:50 in Homogenization Buffer (20 mmol/L Tris HCl,
1mmol/L EGTA , pH 7.0), and stored at -80°C until use.
Western blots against

rat renin (1:5000 Fitzgerald, Concord, MA) and ß-actin
(1:1000, Abcam, Cambridge, MA) were performed with 40 μg of protein as
86

described previously (25). Blots were visualized with Odyssey Infrared Imaging
System (LI-COR Biosciences, Lincoln, NE).
Trypsinization Protocol
Prorenin was evaluated as the difference in renin activity before (active renin) and
after trypsinization (total renin). Medullary samples were harvested as described
above. M1 (mixed phenotype collecting duct cell line; ATCC, Manassas, VA) cells
were lysed with 1:50 BaculoGold Protease Inhibitor (BD Biosciences, San Jose, CA)
in Cell Lytic and supernatant protein collected. Trypsinization activates prorenin to
renin (26). Samples were trypsinized (50 g/L) for 60 minutes on ice and reaction
stopped with Soybean Trypsin Inhibitor (100 g/L) for 10 minutes on ice.
Spectrofluorometry to Measure Renin Activity
Renin activity was evaluated by cuvette-based fluorometry (Quantamaster-8, Photon
Technology, Inc., Princeton, NJ) using a FRET-based renin substrate assay as
described previously (20). Substrate is present in excess, so the initial reaction rate
(within 50 seconds of adding 40 μg sample) estimates renin activity, using Felix
software (PTI). Trypsin in the presence of trypsin inhibitor does not interact with
renin substrate. To control for specificity, the renin active site inhibitor Aliskiren
(Novartis, East Hanover, NJ; 50 μmol/L) was incubated with sample for 5 minutes at
25°C.
Cell culture
M1 cells were from ATCC (Manassas, VA). Using previously validated primers
(27), RT-PCR showed de novo renin synthesis, confirmed by sequencing. Ang II
87

concentration of 100 nmol/L was drawn from studies showing intratubular levels
exceeding plasma levels 1000-fold in disease (28). M1 cells were treated with Ang
II twice daily for 5 days, lysed, and protein extracted for renin activity assays.

Statistics
Data are expressed as means ± SE. Statistical significance

was tested using
ANOVA. Significance was accepted at P <

0.05.
88

Results
JGA and CD granular content visualized in vivo
Multiphoton imaging visualized quinacrine-stained granules in the intact
kidney with subcellular resolution. Figure 1A shows renin granules (green) lining
the terminal afferent arteriole at the JGA. CD staining was low in control animals
(Fig. 1B), but we unexpectedly observed significant quinacrine fluorescence in the
CD in diabetes. Closer inspection showed quinacrine labeled granules at both apical
(Fig. 1C) and basolateral poles of principal cells (Figure 1D), having access to the
tubular lumen or systemic circulation via the directly adjacent peritubular capillaries.
This observation
suggested
significant CD
renin synthesis
and inspired the
following
investigations
using
quantitative and
more specific
techniques.

Figure 1. Visualization of quinacrine-labeled granules (green) in the JGA and
89

CD in vivo. Control (A,B) and diabetic kidney (C,D). A dextran-rhodamine B
conjugate (70kDa) labeled the plasma or the concentrated CD lumen (on panel C)
red. A: Quinacrine identified JGA renin granules in the terminal afferent arteriole
(AA). The efferent arteriole (EA) is seen leaving the glomerulus (G), opposite the
AA. Note the absence (B) or abundance (C) of quinacrine-staining in the bulging
apical aspects of CD principal cells in control or diabetes, respectively. D: Arrows
also show quinacrine-stained granules at basolateral poles of the cells, close to
peritubular capillaries (PTC). Bars = 15 µm (B), 20 µm (A, C, D).

Effects of Ang II AT
1
receptor blockade
Diabetes is characterized by an activated RAS and is an ideal model to study
the effects of RAS components at the JGA and CD. Quinacrine fluorescence in the
JGA and CD were visualized and quantified as an index of both renin and prorenin
content in control and diabetic conditions. Figure 2A shows JGA renin in early
diabetes. Interestingly, quinacrine labeling was vastly increased in the CD in
diabetes (Figure 2B), with overflow into the tubular lumen as well as the basolateral
surface towards the interstitium and peritubular capillaries. To distinguish the
effects of Ang II at each region, Ang II binding was prevented by the AT
1
receptor
blocker (ARB), Olmesartan. ARB treatment unmasked the differential regulation of
granular content by Ang II at these two sites. The ARB blocked Ang II-mediated
negative feedback at the JGA, permitting un-inhibited, high renin production (Figure

90

2C). ARB treatment inhibited Ang II-mediated stimulation at the CD, and almost
completely prevented quinacrine labeling (Figure 2D).

Figure 2. Effects of AT
1
receptor blockade. Untreated (A,B) and treated (C, D)
diabetic conditions. A: Quinacrine staining (green) showed slightly elevated
juxtaglomerular apparatus (JGA) renin in early diabetes. B: Quinacrine labeling was
vastly increased in the collecting duct (CD). C: Olmesartan treatment increased
quinacrine staining (a reflection of increased renin granular content) in the JGA. D:
In contrast, olmesartan reduced quinacrine labeling in the CD, reflecting a decrease
91

in granule number. AA: afferent arteriole, EA: efferent arteriole, G: glomerulus. Bar
= 20 µm.

Figure 3 provides a semi-quantitative summary of quinacrine staining at the
JGA and CD under control, diabetic, ARB-treated control and ARB-treated diabetic
conditions. JGA renin (Figure 3A) increased 1.5-fold in early diabetes. ARB
treatment increased JGA renin 3-fold in both control and diabetic animals
(control:47±2 μm VS. diabetic:74±4 μm VS. ARB:127.9±7.6 μm VS.
diabetic+ARB:132±13 μm; n=9, P<0.01). Even more noticeable, quinacrine staining
in the CD (Figure 3B) increased over 3-fold in diabetes and was silenced to or below
control levels with ARB treatment (control:1.3±0.1 VS. diabetic:3.7±0.1 VS.
ARB:1.2±0.1 VS. diabetic+ARB:0.7±0.1; n=9, P<0.001). Thus, the stimulatory
actions of Ang II on CD granules were even more pronounced than its inhibitory
actions at the JGA.
A fluorogenic renin substrate was used to assess renin activity of untreated
and ARB-treated diabetic rat plasma samples before and after trypsin activation of
prorenin (Figure 3C-D). The 5-fold increase in renin activity after trypsinization
confirmed that diabetes was a high prorenin-low renin condition (Fig. 3D). Since
ARB treatment silenced quinacrine staining in the CD, we expected ARBs to reduce
plasma prorenin if the CD is indeed the major source of prorenin in diabetes. Plasma
prorenin in untreated diabetics (806±240 U/s) was 4-fold higher than in ARB-treated

92

diabetic rats (230±59 U/s) and the ratio of plasma prorenin to renin also significantly
decreased with ARB treatment (Fig. 3D).

Figure 3. Summary of the effects of olmesartan on JGA and CD quinacrine
labeling in vivo (A-B) and plasma prorenin (C-D). A: ARB treatment increased
the quinacrine-positive length of the afferent arteriole (AA) in both control and
diabetic animals (n=9 each, *P<0.001). B: Quinacrine-staining in the CD increased
more than 3-fold in diabetes and decreased to below control levels with ARB
treatment in control and diabetic animals (n=9 each, *P<0.001). Values are mean±SE
of 9 measurements/experimental group, 4 animals per group. C: In vitro renin assays
of plasma samples from the same rats. Serum prorenin levels were high in diabetes,
but reduced by ARB treatment. D: Fold-increase in renin activity after trypsinization
using the samples shown in panel C. (n=6 each, *P<0.05)

93

Immunolocalization of increased CD (pro)renin in diabetes
As suggested by in vivo quinacrine labeling, renin expression in the CD was
confirmed by labeling rat kidney sections with a (pro)renin antibody (Figure 4A).
Figure 4B shows that CD (pro)renin expression was substantially elevated in
diabetes, and both granular structures (similar to quinacrine labeling in vivo) and
diffuse intracellular labeling (likely prorenin) were observed. Note the presence of

renin-negative intercalated cells adjacent to renin-rich principal cells which are
packed with granular contents at apical and basolateral poles. Because the renin
antibodies detect both prorenin and renin, immunohistochemistry alone cannot
distinguish which form contributes more substantially to the increased total renin
content.
Figure 4. Immunohistochemical verification of renin granules in the CD. Both
prorenin and renin were detected by a rat (pro)renin-specific antibody (red, Alexa
594). Nuclei were stained with DAPI (blue). A: (Pro)renin is expressed in principal
cells of the CD under control conditions. B: (Pro)renin expression is substantially
94

elevated in diabetes and labeling is present both in granular structures and in a
diffuse, intracellular pattern. CD: collecting duct. Bar =20 µm.

Increased CD prorenin in diabetes – Effects of trypsinization
The pre-trypsinization activity measures endogenous active renin, and the
post-trypsinization activity reflects the activity of both native renin and activated
prorenin. Because JGA renin contaminates the cortex, medullary homogenates
(JGA-free) were used to study CD renin (7,8). Western blot validated trypsinization
as an effective method for prorenin activation. Using antibodies against (pro)renin,
we observed an upper band of approximately 50 kDa pre-trypsinization and a 47 kDa
lower band post-trypsinization, consistent with the literature (29). Figure 5A shows
that trypsinization reduced the intensity of the higher prorenin band and increased
the intensity of the lower renin band. Total prorenin content in diabetic animals
(upper 50 kDa band, lanes 9-12) was significantly increased compared to control
(lanes 1-4). Trypsinization produced even more renin in diabetes (lower 47 kDa
band, lanes 13-16) compared to control (lanes 5-8). Densitometry analysis showed
that diabetes increased prorenin content 3.4-fold compared to control (Figure 5A,
P<0.001). Equal protein loading was confirmed by blotting for β-actin (not shown).
Trypsinization increased the intensity of the 47 kDa renin band (lanes 13-16) and
reduced the intensity of the 50 kDa prorenin band (lanes 9-12) to 72 ± 9% of control
levels, indicating near complete activation of prorenin into renin by trypsin
(P<0.003). The renin assay was specific for renin and not other proteases as pre-
95

incubation of samples (either trypsinized or not) with 50 μM Aliskiren silenced the
signal (Figure 5B). Figure 5C shows that trypsinization increased renin activity
5.1±0.5-fold in diabetic kidney medullary homogenates (n=4 animals, P<0.001),
compared to a statistically insignificant 1.6±0.1 fold change in control animals (n=4
animals). This confirms the significant increase in CD prorenin in diabetes.

Figure 5. Western blot (A) and renin enzyme assays (B-C) provide evidence for
increased CD prorenin in diabetes. A: Renal medullary tissue prorenin was
proteolytically activated by trypsinization, reducing intensity of the upper 50 kDa
prorenin (PR) band (lanes 1-4, 9-12 before trypsinization) and increasing prominence
of the lower 47 kDa renin (R) band (lanes 5-8, 13-16 after trypsinization of the same
samples). Densitometry calculations, normalized to controls, also showed that
prorenin content in diabetic animals (lanes 9-12) is 3.4-fold greater than in controls
(lanes 1-4) (P<0.001). B: Enzyme activity assays were used to further confirm and
96

quantify prorenin content of the same samples. Pre-incubation of medullary
homogenates (either trypsinized or not) with Aliskiren (renin inhibitor) reduced renin
activity, confirming assay specificity for enzymatically active renin. C: There was no
statistically significant increase in renin activity after trypsinization in control kidney
homogenates. There was a 5-fold increase in diabetic animals (n=4 animals,
*P<0.001).
Effects of Ang II on prorenin synthesis by a CD cell line
In order to further study the renal source of prorenin, the M1 CD cell line was
used. Figure 6A shows that control and Ang II-treated M1 cells produce renin de
novo, confirmed by RT-PCR. Ang II feedings were conducted across multiple
concentrations and time courses (data not shown), with optimal effects at 100 nM
and 5 days of treatment. Olmesartan, was used as a control for the integrity of the
Ang II, and it blocked the effects of Ang II and reduced renin to control levels (data
not shown). Immunocytochemistry confirmed that renin is increased in Ang II-
treated cells (Figure 6C) compared to control (Figure 6B). Interestingly, there is a
reticular pattern of staining in Ang II-treated cells (Figure 6C), revealing prorenin
accumulation in the protein synthetic machinery of the endoplasmic reticulum and
the Golgi apparatus. The number and size of renin granules, as well as cytoplasmic
staining, increased in Ang II-treated cells, indicating increased synthesis of both
renin and prorenin. Figure 6D shows that control and Ang II-treated M1 cells
contain endogenous renin activity (n=4 each). M1 renin activity increased 23±2 fold
with trypsinization, showing significant baseline prorenin content in the cell line
97

(Figure 6E). Ang II promoted the accumulation of prorenin, causing a 35±5 fold
increase in renin activity post-trypsinization (Figure 6E, n=4, P<0.01), signifying the
direct upregulation of CD (pro)renin content by Ang II.

Figure 6. Identification of the CD as an Ang II-responsive source of prorenin.
A: RT-PCR confirms de novo renin protein synthesis in M1 cells (lane 2) and even
more in ANG II-fed M1 cells (lane 3). β-actin shows cell viability. B:
Immunocytochemistry confirms renin(red) production in principal cells. Cells
lacking renin immunolabeling are likely intercalated cells. C: Ang II treatment
increased (pro)renin synthesis, producing more renin within large granules as well as
a reticular pattern of staining indicating the accumulation of prorenin in the protein
98

production machinery. Nuclei (blue) were stained with DAPI. D, E: Renin activity
post-trypsinization increased 22-fold in M1 cell lysates. Ang II treatment increased
M1 cell renin activity 35-fold post-trypsinization (n=7 each, * P<0.05).

Connecting segment proliferation in diabetes
The connecting segment (CNT) is a transitional region of variable length
connecting the distal tubule and CD, and it contains the same mixed phenotype of
cells than the CD. CNT is the major site of (pro)renin synthesis (6, 9). CNT-CD
proliferation has been reported in diseases like lithium-induced nephrogenic diabetes
insipidus (30). We discovered CNT proliferation in the cortex of animals with
diabetic mellitus. Figure 7B shows an abundance of renin-positive principal cells
next to renin-negative intercalated cells. CNT has overtaken the field and changed
the population of the cortex, which is normally predominantly comprised of
glomeruli and proximal tubules (Figure 7A). Immunohistochemistry for
proliferating cell nuclear antigen (PCNA) has been used to confirm active mitosis of
cells in the renal cortex (30). PCNA is absent from the quiescent control kidney
(Figure 7C) but present in diabetes (Figure 7D), averaging over 10 PCNA-positive
nuclei per 100 μm X100 μm field (n=5 fields each from 3 animals, for both groups).
Thus, PCNA staining showed CNT hyperplasia in the cortex.

99

Figure 7. CNT proliferation in diabetes. A: The normal architecture of the kidney
cortex is comprised mainly of glomeruli (G), proximal tubule (PT), and various other
nephron segments including the renin-containing connecting segment (CNT).
Red=renin, blue=nuclei. B: There is CNT hyperplasia in diabetes. Renin-rich (red)
principal cells and renin-negative intercalated cells (white arrows) overtake the
region. C: Representative image of control rat section negative for proliferating cell
nuclear antigen (PCNA, red), a marker of cell turnover and hyperplasia. D: Diabetic
rat section shows positive labeling for PCNA (red). Bar =20 µm.
100

Discussion
Conventional knowledge assigns juxtaglomerular renin the role of gatekeeper
for RAS and its end-product, Ang II, with a critical negative feedback function as an
internal control of its activity. However, recent findings have prompted a re-
examination of our standard understanding of the RAS. This study provides
quantitative, functional, and in vivo visual analysis of (pro)renin in the rat kidney.
Quinacrine staining of the JGA and CD was visualized in vivo using multi-photon
microscopy in control and diabetic conditions (Figs. 1-2) and the data suggested
differential regulation of JGA and CD (pro)renin by Ang II. In addition, the present
studies provided further information about the distal RAS (6-9), namely, that
immunoreactive CD renin was actually predominantly the precursor prorenin. In
diabetes, CD (pro)renin content was greatly upregulated, and trypsinization
differentiated prorenin from renin. Treatment of the CD cell line M1 with Ang II
further suggested that the CD may be the source, and Ang II may be the stimulus, to
explain the high prorenin levels and persistent RAS activity in diabetes despite JGA
renin suppression (12-14).
De novo renin synthesis in the CD is established (6,9), and studies have
reported renin upregulation in CD principal cells of Ang II-infused animals by
quantitative real time RT-PCR (7,8). Renin enzymatic activity assays on medullary
homogenates isolated CD renin from contamination by JGA renin. In control
animals, trypsinization of samples caused no significant change in renin activity,
suggesting that prorenin is not normally stored in the CD. Another interpretation
101

would be that the lack of adequate stimulus under control conditions prevents the
accumulation of prorenin, which is constitutively secreted (5). Unlike renin, whose
granular release is highly regulated, prorenin does not respond to acute stimuli,
although chronic stimuli of renin will increase prorenin levels (31). The present
studies found vast CD prorenin upregulation in diabetes, as there was a 5-fold
increase in renin activity post-trypsinization (Fig. 5C). This suggests that intra-renal
Ang II may promote the buildup and release of CD prorenin.
Studies on M1 cells isolated CD from the effects of systemic factors.
(Pro)renin immunolabeling was visualized in the cytoplasm, granules, and
endoplasmic reticulum (ER) (Fig. 6). A recent review (5) shows preprorenin
synthesis in the ER, with post-translational processing into prorenin. Prorenin is
then transported to the Golgi apparatus and sorted to small clear vesicles for
constitutive secretion, or targeted to large dense protease-containing vesicles for
processing into mature renin that is secreted in a regulated fashion. Excessive
stimulation of prorenin synthesis backs up processing, evidenced by visual cues like
increased ER. ER and Golgi proliferation are classic signs of increased protein
synthesis (32). For example, in Type 2 diabetes, persistent stimulation of insulin
secretion increases beta cell ER density and volume (33). We propose an analogous
phenomenon may exist in the kidney: Ang II stimulates CD prorenin synthesis in
diabetes. Both visual (Figure 6C) and functional (Figure 6D,E) experiments
demonstrated intracellular prorenin accumulation.

102

The onset of hyperglycemia in diabetes elicits a pressor response involving a
rise in mean arterial pressure and RAS activation (34). Since the JGA is the primary
source of active renin, JGA renin is the gatekeeper responsible for early Ang II
production (5). However, Ang II then has negative feedback on the JGA. While
Ang II suppresses JGA renin, it activates CD (pro)renin production (7). CD prorenin
may then be released to cause pathological actions at the (pro)renin receptor, or may
be cleaved to serve as a source of renin in the face of JG renin suppression. Our work
supports other studies assigning the kidneys as the primary source of prorenin in the
rat species (35), but work in humans suggests that there are also significant, non-
renal sources of prorenin (36).
(Pro)renin in the CD may enter the tubular lumen (Fig. 1B) to access local
RAS in the distal nephron (9), culminating in intratubular Ang II generation. The
significance of a paracrine RAS in the distal nephron has been confirmed by studies
showing ENaC stimulation

by both Ang I and Ang II in the CD lumen (37,38).
Renin granules also appear to be localized to the basolateral aspect of principal cells,
with access to interstitial RAS components, the systemic circulation via peritubular
capillaries, and the recently localized (pro)renin receptor (39). (Pro)renin receptor
binding increases the catalytic efficiency of Ang I generation four-fold and activates
MAP kinases, illustrating both RAS-dependent and independent effects for
(pro)renin (40).
Although prorenin is a proenzyme, recent evidence questions its ostensible
inactivity. Prorenin may be cleaved into active renin and regulate RAS, or it can
103

exert RAS-independent effects through the (pro)renin receptor (16,40). Thus, distal
prorenin may be an important constituent in the management of Ang II-dependent
and diabetes-associated hypertension. In this regard, the present studies identified a
thus far overlooked potential benefit of ARBs: suppression of CD (pro)renin
synthesis (Fig. 2B,D). Also, we discovered proliferation of the CNT in diabetes
mellitus (Fig. 7). While Ang II stimulates prorenin on the individual cell level,
proliferation of the CNT has larger scale effects by increasing the entire population
of the prorenin-producing principal cells. This further supports our hypothesis that
the major source of plasma prorenin in diabetes is the CD.

Perspectives
The increased incidence and prevalence of hypertension reflect shortcomings
in our management of this epidemic. The majority of cases have unknown etiology,
but the eventual onset of hypertension in diabetics suggests a relationship between
the two pathologies. Despite their comorbidity and hypotheses about their
interaction, the mechanism by which diabetes causes hypertension remains
unresolved. Diabetes is associated with RAS activation despite paradoxically low
plasma renin levels (12-14).
The present studies show that Ang II can renew CD prorenin, providing
substrate for amplification of a hypertensive signal, and serving as a potential target
for disease management. The (pro)renin receptor has been identified and localized to
vascular smooth muscle cells in the heart and kidney, glomerular mesangial cells,
104

and collecting tubular cells of the kidney (39). The proximity of prorenin to the
(pro)renin receptor establishes a volatile setting for persistent RAS activation and
Ang II production. Elevated plasma prorenin correlated with and predicted diabetic
microvascular complications and microalbuminuria (41). Tissue RAS activation and
prorenin levels also contribute to the development and progression of end-organ
damage like cardiac hypertrophy and fibrosis, vascular damage, diabetic
glomerulosclerosis, and nephropathy (42). We postulate that CD (pro)renin, which
is stimulated by Ang II, may be a critical site to target in the treatment of diabetic
hypertension and nephropathy.

SOURCE(S) OF FUNDING
These studies were supported by NIH grants DK64324 and DK74754, an American
Heart Association Established Investigator Award, and by Sankyo Co. (Tokyo,
Japan).

CONFLICT(S) OF INTEREST/DISCLOSURE(S)
None
105

Chapter 3 References

1. Hobart PM, Fogliano M, O'Connor BA, Schaefer IM, Chirgwin JM. Human
renin gene: structure and sequence analysis. Proc Natl Acad Sci U S A. 1984;
81:5026-5030.

2. Schnermann J, Briggs J. Function of the juxtaglomerular apparatus: local control
of glomerular hemodynamics. In: The Kidney, ed Seldin DW, and Giebisch G.
Raven. New York, USA. 1985:669–697.

3. Schnermann J. Juxtaglomerular cell complex in the regulation of renal salt
excretion. Am J Physiol. 1998; 274:R263-R279.

4. Skøtt, O. Renin. Am J Physiol Regul Integr Comp Physiol. 2002; 282:R937-
R939.

5. Schweda F, Friis U, Wagner C, Skott O, Kurtz A. Renin release. Physiology.
2007; 22:310-319.

6. Rohrwasser A, Ishigami T, Gociman B, Lantelme P, Morgan T, Cheng T, Hillas
E, Zhang S, Ward K, Bloch-Faure M, Meneton P, Lalouel JM. Renin and
kallikrein in connecting tubule of mouse. Kidney Int. 2003; 64:2155-2162.

7. Prieto-Carrasquero MC, Harrison-Bernard LM, Kobori H, Ozawa Y, Hering-
Smith KS, Hamm LL, Navar LG. Enhancement of collecting duct renin in
angiotensin II-dependent hypertensive rats. Hypertension. 2004; 44:223-229.

8. Prieto-Carrasquero MC, Kobori H, Ozawa Y, Gutiérrez A, Seth D, Navar LG.
AT1 receptor-mediated enhancement of collecting duct renin in angiotensin II-
dependent hypertensive rats. Am J Physiol Renal Physiol. 2005; 289:F632-F637.

9. Rohrwasser A, Morgan T, Dillon HF, Zhao L, Callaway CW, Hillas E, Zhang S,
Cheng T, Inagami T, Ward K, Terreros DA, and Lalouel JM. Elements of a
paracrine tubular renin angiotensin system along the entire nephron.
Hypertension. 1999; 34:1265–1274.

10. Castrop H, Oppermann M, Weiss Y, Huang Y, Mizel D, Lu H, Germain S,
Schweda F, Theilig F, Bachmann S, Briggs J, Kurtz A, Schnermann J. Reporter
gene recombination in juxtaglomerular granular and collecting duct cells by
human renin promoter-Cre recombinase transgene. Physiol Genomics. 2006;
25:277-285.

106

11. Zimpelmann J, Kumar D, Levine DZ, Wehbi G, Imig JD, Navar LG, Burns KD.
Early diabetes mellitus stimulates proximal tubule renin mRNA expression in the
rat. Kidney Int. 2000; 58:2320-30.

12. Ritz E, Dikow R. Hypertension and antihypertensive treatment of diabetic
nephropathy. Nat Clin Pract Nephrol. 2006; 2:562-567.

13. Gurley SB, Coffman TM. The renin-angiotensin system and diabetic
nephropathy. Semin Nephrol. 2007; 27:144-152.

14. Price DA, Porter LE, Gordon M, Fisher NDL, De'Oliveira JMF, Laffel LMB,
Passan DR, Williams GH, Hollenberg NK. The Paradox of the Low-Renin State
in Diabetic Nephropathy. J Am Soc Nephrol. 1999; 10:2382-2391.

15. Luetscher JA, Kraemer FB, Wilson DM, Schwartz HC, Bryer-Ash M. Increased
plasma inactive renin in diabetes mellitus. A marker of microvascular
complications. N Engl J Med. 1985; 312:1412-1417.

16. Danser AH, Deinum J. Renin, prorenin and the putative (pro)renin receptor.
Hypertension. 2005; 46:1069-1076.

17. Peti-Peterdi J, Fintha A, Fuson AL, Tousson A, Chow RH. Real-time imaging of
renin release in vitro. Am J Physiol Renal Physiol. 2004; 287:F329-F335.

18. Peti-Peterdi J. Multiphoton imaging of renal tissues in vitro. Am J Physiol Renal
Physiol. 2005; 288:F1079-F1083.

19. Kang JJ, Toma I, Sipos A, McCulloch F, Peti-Peterdi J. Quantitative imaging of
basic functions in renal (patho)physiology. Am J Physiol Renal Physiol. 2006;
291:F495-F502.

20. Kang JJ, Toma I, Sipos A, McCulloch F, Peti-Peterdi J. Imaging the renin-
angiotensin system: an important target of anti-hypertensive therapy. Adv Drug
Deliv Rev. 2006; 58:824-833.

21. Toma I, Kang JJ, Peti-Peterdi J. Imaging renin content and release in the living
kidney. Nephron Physiol. 2006; 103:71-74.

22. Alund M. Juxtaglomerular cell activity during hemorrhage and ischemia as
revealed by quinacrine histofluorescence. Acta Physiol Scand. 1980; 110:113-
121.

107

23. Kobori H, Harrison-Bernard LM, Navar LG. Enhancement of angiotensinogen
expression in angiotensin II-dependent hypertension. Hypertension. 2001;
37:1329–1335.

24. Kobori H, Harrison-Bernard LM, Navar LG. Expression of angiotensinogen
mRNA and protein in angiotensin II-dependent hypertension. J Am Soc Nephrol.
2001; 12:431–439.

25. McCulloch F, Chambrey R, Eladari D, Peti-Peterdi J. Localization of connexin
30 in the luminal membrane of cells in the distal nephron. Am J Physiol Renal
Physiol. 2005; 289:F1304-F1312.

26. Suzuki F, Nakagawa T, Kakidachi H, Murakami K, Inagami T, Nakamura Y.
The dominant role of the prosegment of prorenin in determining the rate of
activation by acid or trypsin: studies with molecular chimeras. Biochem Biophys
Res Commun. 2000; 267:577-580.

27. Kim HS, Lee G, John SW, Maeda N, Smithies O. Molecular phenotyping for
analyzing subtle genetic effects in mice: application to an angiotensinogen gene
titration. Proc Natl Acad Sci U S A. 2002; 99:4602-4607.

28. Navar LG, Harrison-Bernard LM, Wang CT, Cervenka L, Mitchell KD.
Concentrations and actions of intraluminal angiotensin II. J Am Soc Nephrol.
1999;10 Suppl 11:S189-S195.

29. Jutras I, Seidah NG, Reudelhuber TL, Brechler V. Two activation states of the
prohormone convertase PC1 in the secretory pathway. J Biol Chem. 1997;
272:15184-15188.

30. Mønster Christensen B, Kim Y-H, Kwon T-H, Nielsen S. Lithium treatment
induces a marked proliferation of primarily principal cells in rat kidney inner
medullary collecting duct. Am J Physiol Renal Physiol. 2006; 291:F39-F48.

31. Toffelmire EB, Slater K, Corvol P, Ménard J, Schambelan M. Response of
plasma prorenin and active renin to chronic and acute alterations of renin
secretion in normal humans. Studies using a direct immunoradiometric assay. J
Clin Invest. 1989; 83:679–687.

32. Sikora-Vanmeter KC, Wierwille RC, Willetts EJ, and Vanmeter WG.
Morphological Evidence for Increased Protein Synthesis in CNS Neurons after
Soman Exposure. Toxicological Sciences. 1987; 8:23-38.

33. Marchetti P, Bugliani M, Lupi1 R, Marselli L, Masini M, Boggi U,Filipponi
F, Weir GC, Eizirik DL, and Cnop M. The endoplasmic reticulum in
108

pancreatic beta cells of type 2 diabetes patients. Diabetologia. 2007;
50:2486-2494.

34. Brands MW, Fitzgerald SM. Arterial pressure control at the onset of type I
diabetes: the role of nitric oxide and the renin-angiotensin system. Am J
Hypertens. 2001; 14:126S-131S.

35. Kim S, Hosoi M, Nakajima K, Yamamoto K. Immunological evidence that
kidney is primary source of circulating inactive prorenin in rats. Am J Physiol.
1991; 260:E526-E536.

36. Hosoi M, Kim S, Tabata T, Nishitani H, Nishizawa Y, Morii H, Murakami K,
Yamamoto K. Evidence for the presence of differently glycosylated forms of
prorenin in the plasma of anephric man. J Clin Endocrinol Metab. 1992; 74:680-
684.

37. Komlosi P, Fuson AL, Fintha A, Peti-Peterdi J, Rosivall L, Warnock DG, Bell
PD. Angiotensin I conversion to angiotensin II stimulates cortical collecting duct
sodium transport. Hypertension. 2003; 42:195-199.

38. Peti-Peterdi J, Warnock DG, Bell PD. Angiotensin II directly stimulates ENaC
activity in the cortical collecting duct via AT(1) receptors. J Am Soc Nephrol.
2002; 13:1131-1135.

39. Nguyen G. Increased cyclooxygenase-2, hyperfiltration, glomerulosclerosis, and
diabetic nephropathy: put the blame on the (pro)renin receptor? Kidney Int. 2006;
70:618-620.

40. Nguyen G, Delarue F, Burcklé C, Bouzhir L, Giller T, and Sraer J-D. Pivotal role
of the renin/prorenin receptor in angiotensin II production and cellular responses
to renin. J Clin Invest. 2002; 109:1417–1427.

41. Deinum J, Rønn B, Mathiesen E, Derkx FH, Hop WC, Schalekamp MA. Increase
in serum prorenin precedes onset of microalbuminuria in patients with insulin-
dependent diabetes mellitus. Diabetologia. 1999; 42:1006-1010.

42. Saris JJ, 't Hoen PA, Garrelds IM, Dekkers DH, den Dunnen JT, Lamers JM, Jan
Danser AH. Prorenin induces intracellular signaling in cardiomyocytes
independently of angiotensin II. Hypertension. 2006; 48:564-571.
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Conclusion
Hypertension is one of the most influential predictors of cardiovascular
disease severity, the primary cause of morbidity in the United States and the world.
In the past century, the prevalence of essential hypertension has increased from 5-
10% of Americans to over 30% today.
1
Despite the development of technological
advances in diagnosing and treating hypertension, its fierce prevalence reveals
insufficiencies in our understanding of the disease and concomitant inadequacies in
our therapeutic approaches. The remedies are symptomatic, aiming to prevent
disease progression without addressing the primary insult initiating the pathology.
Given the propensity for hypertension in patients with metabolic disturbances, the
clinical dogma is to prevent disease onset, or at least mitigate its progression, with
RAS inhibition.
Compared with the decisively pronounced explanations of the perils of salt to
blood pressure, our understanding of the contribution of sugar seems tenuous at best.
Excessive sugar metabolism begets excessive RAS activation, suggesting that sugar
may be the core abnormality generating hypertension in this faction of the
population.
2,3
According to the medical canon, hypertension causes progressive
renal injury, but renal injury also is known to predispose to hypertension.
4
It has
thus been unclear which problem precedes the other because the two are reciprocally
galvanized by the activation of processes like inflammation, disruptions in normal
cellular signaling, the accumulation of reactive oxygen species, and excessive stress
leading to failure. Regardless of whether it is a cause or a casualty of hypertension,
110

the kidney is entrenched in RAS activity and is thus a probable site for the
pathological entry of sugar. We therefore aimed to examine the interactions of sugar
with the intra-renal RAS. Explication of the mechanism binding the two would
enhance general knowledge about environmental inputs into RAS and provide
further support for its early inhibition with the onset of metabolic dysfunction, before
disease symptoms arise. Moreover, it could raise exigency for the limitation of
dietary sugar for all hypertensive patients, even those who do not yet have metabolic
imbalances.
Discoveries Through Multiphoton Imaging/Future Directions
In Chapter 1, the diabetic kidney was explored using multiphoton
fluorescence laser scanning microscopy. Novel, minimally invasive techniques were
developed to visualize, quantify, and continuously monitor renal functions including
glomerular filtration rate, blood flow, renin content and release, urinary
concentration/dilution through the course of the nephron, and tubuloglomerular
feedback-mediated oscillations in filtration. With this experimental approach,
functions may be reduced to smaller components or extrapolated to assess their
contributions larger physiological or pathological processes. In vivo imaging
provides the most true-to-life depiction of reactions undisturbed within their native
environments. Compared to demanding and invasive conventional micropuncture
methods, fluorescence measurements of single nephron glomerular filtration rates
kept tubular flow and macula densa functions undisrupted. Consistent with the well-
established finding of hyperfiltration in diabetes, we measured significantly elevated
111

single nephron glomerular filtration rates in diabetic animals compared to control.
Furthermore, we detected regional differences in blood flow to the nephron. Cortical
blood flow was evaluated by measuring red blood cell velocities in peritubular and
intraglomerular capillaries, both of which were increased significantly in diabetes.
Since all cortical segments of the nephron can often be visualized
simultaneously in the same visual field, in vivo imaging permits observations of
feedback loops in which upstream changes exert effects downstream or vice versa.
For example, tubuloglomerular feedback plays a major role in regulating tubular
fluid flow at an oscillation rate of 20-50 seconds. However, this highly conserved
control is disrupted in disease states, with hypertensive rats exhibiting irregular
oscillations. In addition to detecting these otherwise imperceptible phenomena, in
vivo imaging unearthed unforeseen revelations about the mechanisms driving
pathology. Diabetes is a disease in which early hyperfiltration precedes late failure,
and the characteristic proteinuria is taken as an indication of the beginning signs of
renal damage. Some theories implicated the hyperfunctioning glomerulus as the
culprit: the increased blood flow and pressures facilitate the leakage of proteins.
5

However, our recordings revealed that proteinuria originated from already damaged
sclerotizing glomeruli, whereas the fluorescence-tagged protein typically remained
tightly confined within the vasculature of the hyperfiltering glomeruli.
6
Therefore,
the individual parts of physiological and pathological processes could be observed,
dissected, and coordinated with their functional significance to the operation of the
whole system.
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In addition to its tremendous use for discovery, description, and evaluation,
the implementation of this innovative technology has provided data which have
prompted a reassessment of some principles of renal pathophysiology. For example,
the analysis of uric acid-mediated renin release emphasized the importance of the
basolateral macula densa as a sensor for RAS activation, an often overlooked surface
and functional capacity of the structure. The visualization of massive quantities of
quinacrine-rich granules in the cortical collecting duct of diabetic animals prompted
studies leading to the discovery of de novo prorenin synthesis in the collecting duct.
Moreover, in opposition to its classic inhibitory effect on renin synthesis at the JGA,
Ang II had a stimulatory effect on renin synthesis in the distal nephron.
Whether the objective of the investigation is to study physiological systems
in health or in disease, the relevance of local constituents cannot be convincingly
assessed outside of their systemic contexts. Especially in appraising the pathogenic
capacity of a factor, it would be impractical to estimate its impact isolated from the
potential regulatory or compensatory feedback responses that may exist in a living
organism. For example, uric acid engendered renin release from the in vitro
microperfused JGA-afferent arteriole-macula densa preparations, but in vivo imaging
confirmed that uric acid still had the same capacity in the circulation of a living
animal. Thus, this study showed that uric acid had a direct input into the RAS and
that its effects could be acutely relevant to blood pressure homeostasis. The finding
encourages reconsideration of the traditional perception of RAS activation only by
neural, hormonal, or structural signals: uric acid provides clear evidence of
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environmental (or dietary) crosstalk with mechanisms that are already in place to
induce renin release. The next step for imaging would be visualization of the actual
sites of enzymatic activity of the released renin. The next direction for investigation
would be to study the effects of other metabolic or environmental inputs into RAS.

Figure 5. Schematic of Metabolic Activation of RAS.

The NEW Renin-Angiotensin System
The multiphoton experimental approach uncovered, revised, and added
information by studying the onset and outcomes of RAS activation. Hyperuricemia
is a consequence of the metabolism of excess dietary fructose in patients with the
metabolic syndrome. The efficacy of hyperuricemia as a prognostic indicator for
disease severity in hypertension has raised much interest about its contribution to the

114

etiology of the disease. Because RAS inhibition negates most of the hypertensive
influence of hyperuricemia, we hypothesized that uric acid had direct effects on the
kidneys and specifically, the intra-renal RAS. In addition to acute influences on
blood pressure and renal blood flow, uric acid specifically triggered renin release
from the JGA. The process was dependent on intact basolateral uptake of uric acid
at the macula densa, which activated MAPKs and promoted the production of
prostaglandins, one of the most potent stimuli of renin release. This finding provided
validation for the relevance of the JGA interstitium to physiological processes. The
existing paradigm focuses on the contribution of intravascular or intratubular
compartments to signaling. The discovery of massive fluid flow through
fenestrations in the afferent
arteriole characterized the
JGA as a dynamic
environment.
7
The
characterization of renin
release being triggered from
signaling in this region
provides evidence of the
relevance of the JGA to
Figure 6. Uric acid causes renin release via the macula densa.
8
disease (Chapter 2).
The effects of uric acid also highlight the importance of the macula densa as
a mediator of humoral or systemic inputs into renin release and the RAS. Typically,
115

the macula densa is considered in the context of its role as a salt sensor and its
subsequent influence on filtration and renin release. However, the macula densa is
an especially significant site for its roles as a mediator with the capacity to respond
to various forms of stimuli and as an effector of RAS activation. With the recent
localization of the (pro)renin receptor at its basolateral surface, the macula densa has
developed into a setting for hyperactive stimulation of RAS.
9
There is the potential
for a positive-feedback loop where the macula densa stimulates the release of renin,
which can then bind to its receptor on the macula densa to exert RAS-dependent and
independent effects. The precarious presence of all the machinery for RAS
activation and propagation in
such close proximity at the
macula densa has the potential
to cause pathological
interferences. However, while
positive-feedback loops
cannot be responsible for the
longterm determination of the
equilibrium setpoint of a
physiological process (11), Figure 7. A new in vivo view of RAS: Macula densa and the
disruption of normal enzymatic (pro)renin receptor.
10

signaling pathways with persistent activation may interfere with the system’s ability
to return to normal homeostatic conditions. As a demonstration of this point, Ang II-
116

dependent hypertensive patients on RAS inhibitors return to a state of RAS
dysregulation and elevated blood pressure with the withdrawal of their medications:
once there has been a significant imbalance, RAS cannot return systemic function to
pre-disease status. Furthermore, once RAS has been activated, it is known to be
more sensitive to further activation, and thus may produce heightened responses to
other smaller stimuli. Therefore, while an activating factor (like uric acid) may
disturb the equilibrium but cannot alone permanently control homeostatic blood
pressure levels, normal RAS mechanisms may be sufficiently disrupted enough to
prevent a return to normal baseline levels. Accordingly, the correction of
hyperuricemia with pharmacological interference and dietary caution may be
valuable components of the management and prevention of hypertension.
The RAS is equipped to acutely and chronically address reductions in blood
pressure by producing the vasocontstrictor Ang II, but its activity is intentionally
self-contained by the negative feedback of Ang II on its own production. In other
words, RAS is not an innately pathogenic pathway: it increases blood pressure when
there has been a detectable drop, but its purpose is not to cause a pathological and
persistent elevation of baseline blood pressure. With the release of renin, Ang II
generation rapidly ensues, so the most effective protection against amplification of a
hypertensive signal is to thwart the initial activation. Under normal conditions, a
stimulus for renin release produces Ang II, which inhibits further renin synthesis or
release. Only with fervent induction is it possible for renin release and Ang II
production to continue. Hence, hypertension could be taken as a sign of RAS
117

dysregulation due to an unrelenting activation signal or due to unknown elements of
RAS regulation.
The interface of prorenin with the RAS exemplifies both excessive
stimulation and new properties of its regulation. In vivo imaging revealed intense
quinacrine staining in the collecting duct of diabetic animals (Chapter 3).
Immunological and functional experiments confirmed that the collecting duct was a
robust source of prorenin
in diabetes, a condition
associated with increased
RAS activity. In vitro
studies established that
Ang II stimulated prorenin
synthesis from collecting
duct cells, the opposite of
its inhibitory effects on
Figure 8. Prorenin in the collecting duct, in vivo.
12
JGA renin. Although a
precursor, prorenin is not inactive and exerts pathological effects at the (pro)renin
receptor or may be processed to restore the supply of mature, active renin. We also
visualized proliferation of the entire connecting segment in diabetes mellitus, which
provides a tremendous reserve of renin. Proximity to the (pro)renin receptor
establishes a volatile setting for persistent RAS activation and Ang II production.
Tissue RAS activation and prorenin levels contribute to the development and
118

progression of end-organ damage like cardiac hypertrophy and fibrosis, vascular
damage, diabetic glomerulosclerosis, and nephropathy.
12
Therefore, collecting duct
prorenin, which is stimulated by Ang II, may be a critical new RAS input as well as a
RAS-independent contributor to hypertension, and thus a target in the treatment of
hypertension.
The discovery of the (pro)renin receptor provides even more demand for
determining the etiology of hypertension. Although imperfect, the Ang II-dependent
effects of RAS are at least to some extent managed by medications. However,
limiting the production of Ang II may be inadvertently unfettering renin to cause
other problems via its receptor. With a persistent stimulus for RAS activation, renin
will be released and free to bind its receptor. The (pro)renin receptor has a myriad of
Ang II-independent pathogenic functions including MAPK activation, inflammation,
hyperplasia, and apoptosis.
13,14
Furthermore, when bound to its receptor, renin has
four times its normal Ang II-producing efficacy.
15
Although Ang II inhibition has
been a tremendously useful patch for the management of hypertension, interference
with physiological checks and balances may only ameliorate certain problems while
unintentionally exacerbating new ones. Without targeting the initial seeds which
dispose the body to hypertension, medicine will be mitigating but not curing the
disease. Improvements in treating hypertension may come with shifting the tenet
away from its focus on the terminal RAS effector Ang II towards renin, which is
both an origin of the signal for hypertension as well as an effector of the message.

119

About the Cause and Treatment of Hypertension
As an illustration of the persistent challenge the disease poses, hypertensive
patients must remain on therapy for the rest of their lives and often acquire rebound
hypertension if non-compliant with their medications. According to physiology
doctrine, Ang II is the culprit for RAS-mediated pathology through well-described
effects at the AT
1
receptor: vasoconstriction, tubular salt/water conservation, cardiac
hypertrophy, and interstitial fibrosis. The literature is beginning to explore the
consequences of other receptors and the functions of Ang II metabolites, but these
studies have not yet reached the clinical stages.
16
Unfortunately, the available
pharmacological Ang II inhibition has not cured hypertension. First of all, dosages
increase because the persistent stimuli for renin release and RAS activation remain
unchecked and can override inhibitions on Ang II. Additionally, the body
establishes escape mechanisms, such as producing Ang II via non-ACE dependent
pathways. Most RAS inhibitors ultimately attempt to contain the fire, without
extinguishing the pilot flame. Given the adaptability of the body to overcome
medical interventions to silence Ang II, renin inhibitors were created to block RAS
at the most crucial rate-limiting step. Although this novel class of medications is in
use, long-term data is unavailable if it truly provides a better blockade of RAS. It is
currently being prescribed as a supplemental therapy for patients who are
unresponsive to ACE inhibitors or ARBs. Nevertheless, even renin inhibitors would
prevent overgrowth of the hypertensive signal without confronting the root of the

120

signal. If we accept the clinical evidence, then a constant stimulus for hypertension
will cause the generation of Ang II, even in the face of effective RAS inhibition.
Hypertension is driven by contributions from genetics, the environment, and
the yet unresolved complex interactions between the two. As diet and exercise are
critical components to the management of hypertension, the clinical portrait suggests
that diet/environment has a profound influence on pathology. This study shows one
example of a direct input of the environment into physiological signaling pathways
that are already in place, and there are likely other yet undiscovered environmental
inputs. Furthermore, multiphoton imaging detected disruptions in function even
before the onset of symptomatic disease. Although the data describes a direct link
and synergism between nature and nurture, there is still considerable room for
person-to-person variability. For example, high sugar diets are known to cause Type
II diabetes, but different individuals have varying thresholds of tolerance to sugar
before they acquire disease. Similarly, it seems that the predisposition to
hypertension, even in the setting of a metabolic imbalance, would be different for
different individuals. Ultimately, nature and nurture are not opposite forces at work,
and actually seem to interface in common pathways, such as the RAS, to cause
pathology. However, this may also mean that they may have roles in also
counteracting each other to promote health. Hypertension provides an archetypal
arena to see how the interplay of these checks and balances may trigger disease, and
also poses the ultimate test of how our knowledge and understanding of these forces
will ultimately help us overcome this challenge.
121

Conclusion Endnotes
1. Feig DI, Rodriguez-Iturbe B, Nakagawa T, Johnson RJ. Nephron Number,
Uric Acid, and Renal Microvascular Disease in the Pathogenesis of Essential
Hypertension. Hypertension. 2006; 48:1-2.

2. He, W., Miao, F.J.-P., Lin, D. C.-H., Schwandner, R.T., Wang, Z., Gao, J.,
Chen, J.-L., Tian, H., and Ling, L. Citric acid cycle intermediates as ligands
for orphan G-protein-coupled receptors. Nature. 2004; 429:188-193.

3. Hebert, S.C. Physiology: Orphan detectors of metabolism. Nature. 2004;
429:143-145.

4. Navar LG, Zou L, Von Thun A, Tarng Wang C, Imig JD, Mitchell KD.
Unraveling the Mystery of Goldblatt Hypertension. News Physiol. Sci. 1998;
13:170-176.

5. Castellino P, Shohat J, DeFronzo RA. Hyperfiltration and diabetic
nephropathy: is it the beginning? Or is it the end? Semin. Nephrol. 1990;
10:228-41.

6. Kang JJ, Toma I, Sipos A, McCulloch F, Peti-Peterdi J. Quantitative imaging
of basic functions in renal (patho)physiology. Am J Physiol Renal Physiol.
2006; 291:F495-F502.

7. Rosivall L, Mirzahosseini S, Toma I, Sipos A, Peti-Peterdi J. Fluid flow in
the juxtaglomerular interstitium visualized in vivo. Am. J. Physiol. Renal
Physiol. 2006; 291:F1241-1247.

8. Kang, J.J., Toma, I., Sipos, A., and Peti-Peterdi, J. 2008. From In Vitro to In
Vivo: Imaging from the Single Cell to the Whole Organism. Current
Protocols in Cytometry. In Press.

9. Nguyen G. Increased cyclooxygenase-2, hyperfiltration, glomerulosclerosis,
and diabetic nephropathy: put the blame on the (pro)renin receptor? Kidney
Int. 2006; 70:618-620.

10. Toma I, Kang JJ, Peti-Peterdi J. Imaging renin content and release in the
living kidney. Nephron Physiol. 2006; 103:71-74.

11. Guyton AC. Blood Pressure Control-Special Role of the Kidneys and Body
Fluids. Science. 1991; 252:1813-1816.

122

12. Kang JJ, Toma I, Sipos A, Meer EJ, Vargas SL, Peti-Peterdi J. 2008. The
Collecting Duct is the Major Source of Prorenin in Diabetes. Hypertension.
In Press.

13. Saris JJ, 't Hoen PA, Garrelds IM, Dekkers DH, den Dunnen JT, Lamers JM,
Jan Danser AH. Prorenin induces intracellular signaling in cardiomyocytes
independently of angiotensin II. Hypertension. 2006; 48:564-571.

14. Nguyen G. The (pro)renin receptor: a new kid in town. Semin. Nephrol.
2007; 27:519-23.

15. Nguyen G, Delarue F, Burcklé C, Bouzhir L, Giller T, Sraer J-D. Pivotal role
of the renin/prorenin receptor in angiotensin II production and cellular
responses to renin. J. Clin Invest. 2002; 109:1417–1427.

16. Nicholls J, Peiris M. Good ACE, bad ACE do battle in lung injury, SARS.
Nat Med. 2005; 11:821-822.

123

Alphabetized Bibliography

Akis N, and Madaio MP. Isolation, culture, and characterization of endothelial cells
from mouse glomeruli. Kidney Int. 2004; 65:2223-2227.

Alund M. Juxtaglomerular cell activity during hemorrhage and ischemia as revealed
by quinacrine histofluorescence. Acta Physiol Scand. 1980; 110:113-121.

Bahn A, Ljubojevic M, Lorenz H, Schultz C, Estifanos G, Ugele B, Sabolic I,
Burckhardt G, and Hagos Y. Murine renal organic anion transporters mOAT1 and
mOAT3 facilitate the transport of neuroactive tryptophan metabolites. Am. J.
Physiol. Cell Physiol. 2005; 289:C1075-1084.

Basciano, H., Federico, L., and Adeli, K. Fructose, insulin resistance, and metabolic
dyslipidemia. Nutrition & Metabolism. 2005; 2:5.

Bird D, Gu M. Two-photon fluorescence endoscopy with a mirco-optic scanning
head. Opt Lett. 2003; 28:1552–1554.

Brands MW, Fitzgerald SM. Arterial pressure control at the onset of type I diabetes:
the role of nitric oxide and the renin-angiotensin system. Am J Hypertens. 2001;
14:126S-131S.

Brown R, Ollerstam A, Persson AE. Neuronal nitric oxide synthase inhibition
sensitizes the tubuloglomerular feedback mechanism after volume expansion. Kidney
Int. 2004; 65: 1349-1356.

Burg M, Grantham J, Abramow M, Orloff J. Preparation and study of fragments of
single rabbit nephrons. Am J Physiol. 1966; 210:1293–1298.

Cameron JS, Moro F, and Simmonds HA. “Uric acid and the kidney.” Oxford
Textbook of Clinical Nephrology, 2nd ed. Eds. A.M. Davison, J.S. Cameron, J.P.
Grünfeld, D.N.S. Kerr, E. Ritz, and C.G. Winearls. London: Oxford Medical, 1997.
1157–1175.

Castellino P, Shohat J, and DeFronzo RA. Hyperfiltration and diabetic nephropathy:
is it the beginning? Or is it the end? Semin Nephrol. 1990; 10: 228-241.

Castrop H, Lorenz JN, Hansen PB, Friis U, Mizel D, Oppermann M, Jensen BL,
Briggs J, Skott O, Schnermann J. Contribution of the basolateral isoform of the Na-
K-2Cl- cotransporter (NKCC1/BSC2) to renin secretion. Am J Physiol Renal
Physiol. 2005; 289: F1185-1192.

124

Castrop H, Oppermann M, Weiss Y, Huang Y, Mizel D, Lu H, Germain S, Schweda
F, Theilig F, Bachmann S, Briggs J, Kurtz A, Schnermann J. Reporter gene
recombination in juxtaglomerular granular and collecting duct cells by human renin
promoter-Cre recombinase transgene. Physiol Genomics. 2006; 25:277-285.

Chaigneau E, Oheim M, Audinat E, and Charpak S. Two-photon imaging of
capillary blood flow in olfactory bulb glomeruli. Proc Natl Acad Sci U S A. 2003;
100: 13081-13086.

Chou CL and Marsh DJ. Measurement of flow rate in rat proximal tubules with a
nonobstructing optical method. Am J Physiol. 1987; 253: F366-F371.

Chu J, Chu S, and Montrose MH. Apical Na+/H+ exchange near the base of mouse
colonic crypts. Am J Physiol Cell Physiol. 2002; 283: C358-C372.

Coutinho TA, Turner ST, Peyser PA, Bielak LF, Sheedy PF, 2nd, and Kullo IJ.
Associations of serum uric acid with markers of inflammation, metabolic syndrome,
and subclinical coronary atherosclerosis. Am. J. Hypertens. 2007; 20:83-89.

Danser AH and Deinum J. Renin, prorenin and the putative (pro)renin receptor.
Hypertension. 2005; 46:1069-1076.

Deinum J, Rønn B, Mathiesen E, Derkx FH, Hop WC, and Schalekamp MA.
Increase in serum prorenin precedes onset of microalbuminuria in patients with
insulin-dependent diabetes mellitus. Diabetologia. 1999; 42:1006-1010.

Della Bruna, R., Pinet, F., Corvol, P., and Kurtz, A. Regulation of renin secretion
and renin synthesis by second messengers in isolated mouse juxtaglomerular cells.
Cell Physiol. Biochem. 1991; 1:98–110.

Dunn KW, Sandoval RM, Kelly KJ, Dagher PC, Tanner GA, Atkinson SJ, Bacallao
RL, and Molitoris BA. Functional studies of the kidney of living animals using
multicolor two-photon microscopy. Am J Physiol Cell Physiol. 2002; 283: C905-
C916.

Enomoto A, Kimura H, Chairoungdua A, Shigeta Y, Jutabha P, Cha SH,
Hosoyamada M, Takeda M, Sekine T, Igarashi T, Matsuo H, Kikuchi Y, Oda T,
Ichida K, Hosoya T, Shimokata K, Niwa T, Kanai Y, and Endou H. Molecular
identification of a renal urate anion exchanger that regulates blood urate levels.
Nature. 2002; 417:447-452.

Feig DI, Rodriguez-Iturbe B, Nakagawa T, Johnson RJ. Nephron Number, Uric
Acid, and Renal Microvascular Disease in the Pathogenesis of Essential
Hypertension. Hypertension. 2006; 48:1-2.
125

Feig DI, Kang DH, Nakagawa T, Mazzali M, and Johnson R.J. Uric Acid and
Hypertension. Curr. Hypertens. Rep. 2006; 8:111-115.

Feig DI, Mazzali M, Kang DH, Nakagawa T, Price K, Kannelis J, and Johnson RJ.
Serum uric acid: a risk factor and a target for treatment? J. Am. Soc. Nephrol. 2006;
17:S69-73.

Feig DI, Nakagawa T, Karumanchi SA, Oliver WJ, Kang DH, Finch J, and Johnson
RJ. Hypothesis: Uric acid, nephron number, and the pathogenesis of essential
hypertension. Kidney Int. 2004; 66:281-287.

Ford ES, Giles WH, and Dietz WH. Prevalence of the metabolic syndrome among
US adults: findings from the third National Health and Nutrition Examination
Survey. JAMA. 2002; 287:356-359.

Franco OH, Peeters A, Bonneux L, de Laet C. Blood pressure in adulthood and life
expectancy with cardiovascular disease in men and women: life course analysis.
Hypertension. 2005; 46:280-286.

Grynkiewicz G, Ponie M, and Tsien R.Y. A new generation of Ca2+ indicators with
greatly improved fluorescence properties. J. Biol. Chem. 1985; 260:3440-3450.

Gurley SB and Coffman TM. The renin-angiotensin system and diabetic
nephropathy. Semin. Nephrol. 2007; 27:144-152.

Guyton AC and Hall JE. The juxtaglomerular complex: its possible control of
multiple nephron functions. Nature. 1979; 277:601–602.

Guyton AC. Blood pressure control–special role of the kidneys and body fluids.
Science. 1991; 252:1813–1816.

Haig A. On uric acid and arterial tension. BMJ. 1889; 1:288–291.

Harris RC and Breyer MD. Physiological regulation of cyclooxygenase-2 in the
kidney. Am. J. Physiol. Renal Physiol. 2001; 281:F1-F11.

He W, Miao FJ-P, Lin DC-H, Schwandner RT, Wang Z, Gao J, Chen J-L, Tian H,
and Ling L. Citric acid cycle intermediates as ligands for orphan G-protein-coupled
receptors. Nature. 2004; 429:188-193.

Hebert SC. Physiology: Orphan detectors of metabolism. Nature. 2004; 429:143-
145.

126

Hediger MA. Gateway to a long life? Nature. 2002; 417:393-395.

Hilbert P, Lindpaintner K, Beckmann JS, Serikawa T, Soubrier F, Dubay C,
Cartwright P, De Gouyon B, Julier C, Takahasi S, Vincent M, Ganten D, Georges M,
Lathrop GM. Chromosomal mapping of two genetic loci associated with blood-
pressure regulation in hereditary hypertensive rats. Nature. 1991; 353:521–529.

Hobart PM, Fogliano M, O'Connor BA, Schaefer IM, Chirgwin JM. Human renin
gene: structure and sequence analysis. Proc Natl Acad Sci U S A. 1984; 81:5026-
5030.

Holstein-Rathlou N-H and Leyssac PP. TGF-mediated oscillations in proximal
intratubular pressure: Differences between spontaneously hypertensive rats and
Wistar-Kyoto rats. Acta Physiol Scand. 1986; 126: 333-339.

Holstein-Rathlou N-H. Synchronization of proximal intratubular pressure
oscillations: evidence for interaction between nephrons. Pflugers Arch. 1987; 408:
438-443.

Hosoi M, Kim S, Tabata T, Nishitani H, Nishizawa Y, Morii H, Murakami K,
Yamamoto K. Evidence for the presence of differently glycosylated forms of
prorenin in the plasma of anephric man. J. Clin. Endocrinol. Metab. 1992; 74:680-
684.

http://content.nhiondemand.com/psv/HC2.asp?objID=100228&cType=hc

Johnson RJ, Feig DI, Herrera-Acosta J, and Kang D-H. Resurrection of Uric Acid as
a Causal Risk Factor in Essential Hypertension. Hypertension. 2005; 45:18-20.

Johnson RJ, Kang D-H, Feig D, Kivlighn S, Kanellis J, Watanabe S, Tuttle KR,
Rodriguez-Iturbe B, Herrera-Acosta J, and Mazzali M. Is There a Pathogenetic Role
for Uric Acid in Hypertension and Cardiovascular and Renal Disease? Hypertension.
2003; 41:1183-1190.

Johnson RJ, Rodriguez-Iturbe B, Kang D-H, Feig DI, and Herrera-Acosta J. A
unifying pathway for essential hypertension. Am. J. Hypertens. 2005; 18:431-440.

Johnson RJ, Segal MS, Srinivas T, Ejaz A, Mu W, Roncal C, Sanchez-Lozada LG,
Gersch M, Rodriguez-Iturbe B, Kang D-H, and Acosta JH. Essential hypertension,
progressive renal disease, and uric acid: a pathogenetic link? J. Am. Soc. Nephrol.
2005; 16:1909-1919.

127

Jutras I, Seidah NG, Reudelhuber TL, Brechler V. Two activation states of the
prohormone convertase PC1 in the secretory pathway. J Biol Chem. 1997;
272:15184-15188.

Kanellis J, Watanabe S, Li JH, Kang DH, Li P, Nakagawa T, Wamsley A, Sheikh-
Hamad D, Lan HY, Feng L, and Johnson RJ. Uric acid stimulates monocyte
chemoattractant protein-1 production in vascular smooth muscle cells via mitogen-
activated protein kinase and cyclooxygenase-2. Hypertension. 2003; 41:1287-1293.

Kang D-H and Nakagawa T. Uric acid and chronic renal disease: possible
implication of hyperuricemia on progression of renal disease. Semin. Nephrol. 2005;
25:43-49.

Kang D-H, Han L, Ouyang X, Kahn AM, Kanellis J, Li P, Feng L, Nakagawa T,
Watanabe S, Hosoyamada M, Endou H, Lipkowitz M, Abramson R, Mu W, and
Johnson RJ. Uric Acid Causes Vascular Smooth Muscle Cell Proliferation by
Entering Cells via a Functional Urate Transporter. Am. J. Nephrol. 2005; 25:425-
433.

Kang JJ, Toma I, Sipos A, Meer EJ, Vargas SL, Peti-Peterdi J. The Collecting Duct
is the Major Source of Prorenin in Diabetes. Hypertension. 2008; in press.

Kang, JJ, Toma I, Sipos A, and Peti-Peterdi J. From In Vitro to In Vivo: Imaging
from the Single Cell to the Whole Organism. Current Protocols in Cytometry. 2008;
in press.

Kang JJ, Toma I, Sipos A, McCulloch F, and Peti-Peterdi J. Imaging the renin-
angiotensin system: an important target of anti-hypertensive therapy. Adv. Drug
Deliv. Rev. 2006; 58:824-833.

Kang JJ, Toma I, Sipos A, McCulloch F, and Peti-Peterdi J. Quantitative Imaging of
basic functions in renal (patho)physiology. Am. J. Physiol. Renal. Physiol. 2006;
291:F495–F502.

Kaplan MR, Plotkin MD, Brown D, Hebert SC, and Delpire E. Expression of the
mouse Na-K-2Cl cotransporter, mBSC2, in the terminal inner medullary collecting
duct, the glomerular and extraglomerular mesangium, and the glomerular afferent
arteriole. J Clin Invest. 1996; 98:723-730.

Khosla UM, Zharikov S, Finch JL, Nakagawa T, Roncal C, Mu W, Krotova K,
Block ER, Prabhakar S, and Johnson RJ. Hyperuricemia induces endothelial
dysfunction. Kidney Int. 2005; 67:1739-1742.

128

Kim HS, Lee G, John SW, Maeda N, and Smithies O. Molecular phenotyping for
analyzing subtle genetic effects in mice: application to an angiotensinogen gene
titration. Proc Natl Acad Sci U S A. 2002; 99:4602-4607.

Kim S, Hosoi M, Nakajima K, and Yamamoto K. Immunological evidence that
kidney is primary source of circulating inactive prorenin in rats. Am. J. Physiol.
1991; 260:E526-E536.

Kleinfeld D, Mitra PP, Helmchen F, and Denk W. Fluctuations and stimulus-induced
changes in blood flow observed in individual capillaries in layers 2 through 4 of rat
neocortex. Proc Natl Acad Sci U S A. 1998; 95:15741-15746.

Kobori H, Harrison-Bernard LM, and Navar LG. Enhancement of angiotensinogen
expression in angiotensin II-dependent hypertension. Hypertension. 2001; 37:1329–
1335.

Kobori H, Harrison-Bernard LM, and Navar LG. Expression of angiotensinogen
mRNA and protein in angiotensin II-dependent hypertension. J Am Soc Nephrol.
2001; 12:431–439.

Komlosi P, Fuson AL, Fintha A, Peti-Peterdi J, Rosivall L, Warnock DG, and Bell
PD. Angiotensin I conversion to angiotensin II stimulates cortical collecting duct
sodium transport. Hypertension. 2003; 42:195-199.

Kovács G, Komlósi P, Fuson A, Peti-Peterdi J, Rosivall L, Bell PD. Neuronal nitric
oxide synthase: its role and regulation in macula densa cells. J. Am. Soc. Nephrol.
2003; 14:2475-2483.

Latta M, Kunstle G, Leist M, and Wendel A. Metabolic depletion of ATP by fructose
inversely controls CD95- and tumor necrosis factor receptor 1-mediated hepatic
apoptosis. J. Exp. Med. 2000; 191:1975-1985.

Layton AT, Moore LC, and Layton HE. Multistability in tubuloglomerular feedback
and spectral complexity in spontaneously hypertensive rats. Am J Physiol Renal
Physiol. 2006; 291:F79-97.

Leyssac PP and Baumbach L. An oscillating intratubular pressure response to
alterations in Henle loop flow in the rat kidney. Acta Physiol Scand. 1983; 117: 415-
419.

Lorenz JN and Gruenstein E. A simple nonradioactive method for evaluating single-
nephron filtration rate using FITC-inulin. Am J Physiol. 1999; 276: F172-77.

129

Luetscher JA, Kraemer FB, Wilson DM, Schwartz HC, and Bryer-Ash M. Increased
plasma inactive renin in diabetes mellitus. A marker of microvascular complications.
N Engl J Med. 1985; 312:1412-1417.

MacGregor GA, Markandu ND, Roulston JE, Jones JC, and Morton JJ. Maintenance
of blood pressure by the renin−angiotensin system in normal man. Nature. 1981;
291:329-331.

Mahomed FA. On chronic Bright’s disease, and its essential symptoms. Lancet.
1879; I: 399–401.

Marchetti P, Bugliani M, Lupi1 R, Marselli L, Masini M, Boggi U,Filipponi F, Weir
GC, Eizirik DL, and Cnop M. The endoplasmic reticulum in pancreatic beta cells of
type 2 diabetes patients. Diabetologia. 2007; 50:2486-2494.

Marsh DJ, Sosnovtseva OV, Chon KH, and Holstein-Rathlou NH. Nonlinear
interactions in renal blood flow regulation. Am J Physiol Regul Integr Comp Physiol.
2005; 288: R1143-1159.

Mazzali M, Hughes J, Kim Y-G, Jefferson JA, Kang D-H, Gordon KL, Lan HY,
Kivlighn S, and Johnson RJ. Elevated uric acid increases blood pressure in the rat by
a novel crystal-independent mechanism. Hypertension. 2001; 38:1101-1106.

Mazzali M, Kannellis J, Han L, Feng L, Xia Y-Y, Chen Q, Kang D-H, Gordon KL,
Watanabe S, Nakagawa T, Lan HY, and Johnson RJ. Hyperuricemia induces a
primary renal arteriolopathy in rats by a blood pressure-independent mechanism. Am.
J. Physiol. Renal. Physiol. 2002; 282:F991–997.

McCulloch F, Chambrey R, Eladari D, Peti-Peterdi J. Localization of connexin 30 in
the luminal membrane of cells in the distal nephron. Am J Physiol Renal Physiol.
2005; 289:F1304-F1312.

Molitoris BA and Sandoval RM. Intravital multiphoton microscopy of dynamic renal
processes. Am J Physiol Renal Physiol. 2005; 288: F1084-1089.

Moller D. New drug targets for type 2 diabetes and the metabolic syndrome. Nature.
2001; 414:821-827.

Mønster Christensen B, Kim Y-H, Kwon T-H, and Nielsen S. Lithium treatment
induces a marked proliferation of primarily principal cells in rat kidney inner
medullary collecting duct. Am J Physiol Renal Physiol. 2006; 291:F39-F48.

130

Nakagawa T, Hu H, Zharikov S, Tuttle KR, Short RA, Glushakova O, Ouyang X,
Feig DI, Block ER, Herrera-Acosta J, Patel JM, and Johnson RJ. A causal role for
uric acid in fructose-induced metabolic syndrome. Am. J. Physiol. Renal. Physiol.
2006; 290:F625–631.

Nakagawa T, Tuttle KT, Short RA, and Johnson RJ. Hypothesis: Fructose-induced
Hyperuricemia as a Causal Mechanism for the Epidemic of the Metabolic Syndrome.
Nat. Clin. Pract. Neprol. 2005; 1:80–86.

Navar LG, Harrison-Bernard LM, Wang CT, Cervenka L, and Mitchell KD.
Concentrations and actions of intraluminal angiotensin II. J Am Soc Nephrol. 1999;
10 Suppl 11:S189-195.

Navar LG, Zou L, Von Thun A, Tarng Wang C, Imig JD, and Mitchell KD.
Unraveling the Mystery of Goldblatt Hypertension. News Physiol. Sci. 1998; 13:170-
176.

Nguyen G, Delarue F, Burcklé C, Bouzhir L, Giller T, and Sraer J-D. Pivotal role of
the renin/prorenin receptor in angiotensin II production and cellular responses to
renin. J. Clin Invest. 2002; 109:1417–1427.

Nguyen G. Increased cyclooxygenase-2, hyperfiltration, glomerulosclerosis, and
diabetic nephropathy: put the blame on the (pro)renin receptor? Kidney Int. 2006;
70:618-620.

Nguyen G. The (pro)renin receptor: a new kid in town. Semin. Nephrol. 2007;
27:519-523.

Nicholls J and Peiris M. Good ACE, bad ACE do battle in lung injury, SARS. Nat
Med. 2005; 11:821-822.

Nilwarangkoon S, Anzai N, Shiraya K, Yu E, Islam R, Cha SH, Onozato ML, Miura
D, Jutabha P, Tojo A, Kanai Y, and Endou H. Role of Mouse Organic Anion
Transporter 3 (mOat3) as a Basolateral Prostaglandin E2 Transport Pathway. Journal
of Pharmacological Sciences. 2007; 103:48-55.

Oppermann M, Castrop H, Briggs J, and Schnermann J. Influence of furosemide on
renal hemodynamics in mice. J Am Soc Nephrol. 2005; 16: 393A.

Perlstein TS, Gumieniak O, Hopkins PN, Murphey LJ, Brown NJ, Williams GH,
Hollenberg NK, and Fisher ND. Uric acid and the state of the intrarenal renin-
angiotensin system in humans. Kidney Int. 2004; 66:1465-1470.

131

Peti-Peterdi J. Calcium wave of tubuloglomerular feedback. Am. J. Physiol. Renal
Physiol. 2006; 291:F473-480.

Peti-Peterdi J. Multiphoton imaging of renal tissues in vitro. Am J Physiol Renal
Physiol. 2005; 288:F1079-F1083.

Peti-Peterdi J and Bell PD. Cytosolic [Ca2+] signaling pathway in macula densa
cells. Am. J. Physiol. 1999; 277:F472-F476.

Peti-Peterdi J, Fintha A, Fuson AL, Tousson A, and Chow RH. Real-time imaging of
renin release in vitro. Am J Physiol Renal Physiol. 2004; 287:F329-F335.

Peti-Peterdi J, Komlosi P, Fuson AL, Guan Y, Schneider A, Qi Z, Redha R, Rosivall
L, Breyer MD, and Bell PD. Luminal NaCl delivery regulates basolateral PGE2
release from macula densa cells. J. Clin. Invest. 2003; 112:76-82.

Peti-Peterdi J, Morishima S, Bell PD, and Okada Y. Two-photon excitation
fluorescence imaging of the living juxtaglomerular apparatus. Am J Physiol Renal
Physiol. 2002; 283: F197-201.

Peti-Peterdi J, Warnock DG, and Bell PD. Angiotensin II directly stimulates ENaC
activity in the cortical collecting duct via AT(1) receptors. J Am Soc Nephrol. 2002;
13:1131-1135.

Price DA, Porter LE, Gordon M, Fisher NDL, De'Oliveira JMF, Laffel LMB, Passan
DR, Williams GH, and Hollenberg NK. The Paradox of the Low-Renin State in
Diabetic Nephropathy. J Am Soc Nephrol. 1999; 10:2382-2391.

Price KL, Sautin YY, Long DA, Zhang L, Miyazaki H, Mu W, Endou H, and
Johnson RJ. Human Vascular Smooth Muscle Cells Express a Urate Transporter. J.
Am. Soc. Nephrol. 2006; 17:1791-1795.

Prieto-Carrasquero MC, Harrison-Bernard LM, Kobori H, Ozawa Y, Hering-Smith
KS, Hamm LL, and Navar LG. Enhancement of collecting duct renin in
angiotensin II-dependent hypertensive rats. Hypertension. 2004; 44:223-229.

Prieto-Carrasquero MC, Kobori H, Ozawa Y, Gutiérrez A, Seth D, and Navar LG.
AT1 receptor-mediated enhancement of collecting duct renin in angiotensin II-
dependent hypertensive rats. Am J Physiol Renal Physiol. 2005; 289:F632-F637.

Ritz E and Dikow R. Hypertension and antihypertensive treatment of diabetic
nephropathy. Nat Clin Pract Nephrol. 2006; 2:562-567.

132

Robinson RF, Batisky DL, Hayes JR, Nahata MC, and Mahan JD. Significance of
Heritability in Primary and Secondary Pediatric Hypertension. Am. J. Hypertens.
2005; 18:917–921.

Rohrwasser A, Ishigami T, Gociman B, Lantelme P, Morgan T, Cheng T, Hillas E,
Zhang S, Ward K, Bloch-Faure M, Meneton P, and Lalouel JM. Renin and kallikrein
in connecting tubule of mouse. Kidney Int. 2003; 64:2155-2162.

Rohrwasser A, Morgan T, Dillon HF, Zhao L, Callaway CW, Hillas E, Zhang S,
Cheng T, Inagami T, Ward K, Terreros DA, and Lalouel JM. Elements of a paracrine
tubular renin angiotensin system along the entire nephron. Hypertension. 1999;
34:1265–1274.

Rosivall L, Mirzahosseini S, Toma I, Sipos A, and Peti-Peterdi J. Fluid flow in the
juxtaglomerular interstitium visualized in vivo. Am. J. Physiol. Renal Physiol. 2006;
291:F1241-1247.

Sánchez-Lozada LG, Nakagawa T, Kang D-H, Feig DI, Franco M, Johnson RJ, and
Herrera-Acosta J. Hormonal and cytokine effects of uric acid. Curr Opin Nephrol
Hypertens. 2006; 15:30-33.

Sánchez-Lozada LG, Tapia E, Jimenez A, Bautista P, Cristobal M, Nepomuceno T,
Soto V, Avila-Casado C, Nakagawa T, Johnson RJ, Herrera-Acosta J, and Franco M.
Fructose-induced metabolic syndrome is associated with glomerular hypertension
and renal microvascular damage in rats. Am. J. Physiol. Renal. Physiol. 2007;
292:F423-429.

Sánchez-Lozada LG, Tapia E, Rodriguez-Iturbe B, Johnson RJ, and Herrera-Acosta
J. Hemodynamics of hyperuricemia. Sem. in Nephrol. 2005; 25: 19-24.

Sandoval RM, Kennedy MD, Low PS, and Molitoris BA. Uptake and trafficking of
fluorescent conjugates of folic acid in intact kidney determined using intravital two-
photon microscopy. Am J Physiol Cell Physiol. 2004; 287: C517-526.

Sands JM. Micropuncture: unlocking the secrets of renal function. Am J Physiol
Renal Physiol. 2004; 287: F866-867.

Saris JJ, 't Hoen PA, Garrelds IM, Dekkers DH, den Dunnen JT, Lamers JM, and
Jan Danser AH. Prorenin induces intracellular signaling in cardiomyocytes
independently of angiotensin II. Hypertension. 2006; 48:564-571.

Sautin YY, Nakagawa T, Zharikov S, and Johnson RJ. Adverse effects of the classic
antioxidant uric acid in adipocytes: NADPH oxidase-mediated oxidative/nitrosative
stress. Am. J. Physiol. Cell Physiol. 2007; 293:C584-596.
133

Schnermann J and Briggs J. “Function of the juxtaglomerular apparatus: local
control of glomerular hemodynamics.” The Kidney. Eds. DW Seldin and G
Giebisch. New York: Raven, 1985. 669-697.

Schnermann J. Juxtaglomerular cell complex in the regulation of renal salt excretion.
Am J Physiol. 1998; 274:R263-R279.

Schweda F, Friis U, Wagner C, Skott O, and Kurtz A. Renin release. Physiology.
2007; 22:310-319.

Seymour KJ, Roberts LE, Fini MA, Parmley LA, Oustitch TL, and Wright R.M.
Stress activation of mammary epithelial cell xanthine oxidoreductase is mediated by
p38 MAPK and CCAAT/enhancer-binding protein-beta. J. Biol. Chem. 2006;
281:8545-8558.

Sikora-Vanmeter KC, Wierwille RC, Willetts EJ, and Vanmeter WG. Morphological
Evidence for Increased Protein Synthesis in CNS Neurons after Soman Exposure.
Fundam Appl Toxicol. 1987; 8:23-38.

Skøtt, O. Renin. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2002; 282:R937-
R939.

Sipos A, Toma I, Kang JJ, Rosivall L, and Peti-Peterdi J. Advances in renal
(patho)physiology using multiphoton microscopy. Kidney Int. 2007; 72:1188-1191.

Suzuki F, Nakagawa T, Kakidachi H, Murakami K, Inagami T, and Nakamura Y.
The dominant role of the prosegment of prorenin in determining the rate of activation
by acid or trypsin: studies with molecular chimeras. Biochem Biophys Res Commun.
2000; 267:577-580.

Tanner GA, Sandoval RM, and Dunn KW. Two-photon in vivo microscopy of
sulfonefluorescein secretion in normal and cystic rat kidneys. Am J Physiol Renal
Physiol. 2004; 286: F152-160.

Tanner GA, Sandoval RM, Molitoris BA, Bamburg JR, and Ashworth SL.
Micropuncture gene delivery and intravital two-photon visualization of protein
expression in rat kidney. Am J Physiol Renal Physiol. 2005; 289: F638-643.

Thomson SC, Deng A, Komine N, Hammes JS, Blantz RC, and Gabbai FB. Early
diabetes as a model for testing the regulation of juxtaglomerular NOS I. Am J
Physiol Renal Physiol. 2004; 287: F732-738.

134

Toffelmire EB, Slater K, Corvol P, Ménard J, and Schambelan M. Response of
plasma prorenin and active renin to chronic and acute alterations of renin secretion in
normal humans. Studies using a direct immunoradiometric assay. J Clin Invest.
1989; 83:679–687.

Toma I, Kang JJ, and Peti-Peterdi J. Imaging renin content and release in the living
kidney. Nephron Physiol. 2006; 103:71-74.

Viazzi F, Leoncini G, Ratto E, Falqui V, Parodi A, Conti N, Derchi LE,
Tomolillo C, Deferrari G, and Pontremoli R. Mild hyperuricemia and subclinical
renal damage in untreated primary hypertension. Am. J. Hypertens. 2007; 20:1276-
1282.

Wearn JT and Richards AN. Observations on the composition of glomerular urine,
with particular reference to the problem of reabsorption in the renal tubules. Am J
Physiol. 1924; 71: 209–227.

Wolf G, Chen S, and Ziyadeh FN. From the periphery of the glomerular capillary
wall toward the center of disease: podocyte injury comes of age in diabetic
nephropathy. Diabetes. 2005; 54: 1626-1634.

Yang T, Park JM, Arend L, Huang Y, Topaloglu R, Pasumarthy A, Praetorius H,
Spring K, Briggs JP, and Schnermann J. Low chloride stimulation of prostaglandin
E2 release and cyclooxygenase-2 expression in a mouse macula densa cell line. J.
Biol. Chem. 2000; 275:37922-37929.

Yip KP, Holstein-Rathlou N-H, and Marsh DJ. Chaos in blood flow control in
genetic and renovascular hypertensive rats. Am J Physiol Renal Physiol. 1991; 261:
F400-408.

Yu W, Sandoval RM, and Molitoris BA. Quantitative intravital microscopy using a
Generalized Polarity concept for kidney studies. Am J Physiol Cell Physiol. 2005;
289: C1197-1208.

Zhou X, Matavelli L, and Frohlich ED. Uric acid: its relationship to renal
hemodynamics and the renal renin-angiotensin system. Curr. Hypertens. Rep. 2006;
8:120-124.

Zimmermann S and Moelling K. Phosphorylation and regulation of Raf by Akt
(protein kinase B). Science. 1999; 286:1741-1744.

Zimpelmann J, Kumar D, Levine DZ, Wehbi G, Imig JD, Navar LG, and Burns KD.
Early diabetes mellitus stimulates proximal tubule renin mRNA expression in the rat.
Kidney Int. 2000; 58:2320-2330.
135

Zipfel WR, Williams RM, and Webb WW. Nonlinear magic: multiphoton
microscopy in the biosciences. Nat. Biotechnol. 11: 1369-77, 2003.

Zoccali C, Maio R, Mallamaci F, Sesti G, and Perticone F. Uric acid and endothelial
dysfunction in essential hypertension. J. Am. Soc. Nephrol. 2006; 17:1466-1471.

Abstract (if available)

Abstract Hypertension is called a silent killer because there are no early symptoms and it can lead to cardiovascular disease, the leading cause of morbidity worldwide. There is no cure, but several classes of medications target the renin-angiotensin system (RAS), a signaling pathway with paramount short and long-term influence over blood pressure. Renin is a hormone enzyme which controls RAS and leads to the generation of Ang II, a peptide that regulates blood pressure and electrolyte balance. RAS activity is tightly controlled to maintain homeostasis, so hypertension can be taken as a sign of RAS dysregulation due to the loss of inhibition or excessive stimulation. High sugar diets especially predispose individuals to a form of hypertension which is responsive to, or whose onset can be delayed by, RAS inhibition. Accordingly, we hypothesized that there must be a direct mechanism by which sugar metabolism causes pathological RAS activation. Since the kidney is a primary target of sugar and blood pressure disorders, the effects of both on molecular, cellular, tissue, and organ function in the kidneys were examined. We applied multi-photon fluorescence microscopy to study otherwise undetectable elements of RAS activity.

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Asset Metadata

Creator Kang, Julie Jung (author)

Core Title New views, inputs, and properties: a new look at the renin-angiotensin system

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Systems Biology

Publication Date 05/06/2008

Defense Date 02/19/2008

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Diabetes Mellitus,hypertension,in vivo imaging,juxtaglomerular apparatus,multiphoton fluorescence microscopy,OAI-PMH Harvest,prorenin,renal physiology,renin-angiotensin system,uric acid

Language English

Advisor Peti-Peterdi, Janos (committee chair), Hamm-Alvarez, Sarah F. (committee member), Hinton, David (committee member), McDonough, Alicia A. (committee member)

Creator Email jungkang@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1229

Unique identifier UC183742

Identifier etd-Kang-20080506 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-68245 (legacy record id),usctheses-m1229 (legacy record id)

Legacy Identifier etd-Kang-20080506.pdf

Dmrecord 68245

Document Type Dissertation

Rights Kang, Julie Jung

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu