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Connexins and pannexins in the kidney: a study of their expression, regulation, and function
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Connexins and pannexins in the kidney: a study of their expression, regulation, and function
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Content
CONNEXINS AND PANNEXINS IN THE KIDNEY:
A STUDY OF THEIR EXPRESSION, REGULATION, AND FUNCTION
by
Fiona P Hanner
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(SYSTEMS BIOLOGY AND DISEASE)
May 2010
Copyright 2010 Fiona P Hanner
ii
EPIGRAPH
The scientist is not a person who gives the right answers; he’s one who asks the right
questions.
Claude Lévi-Strauss
We sometimes tend to think we know all we need to know to answer these kinds of
questions but sometimes our humble hearts can help us more than our proud minds. We
never really know enough until we recognize that God alone knows it all.
- 1 Corinthians 13:4-13 (The Message)
iii
DEDICATION
This dissertation is dedicated to my family and friends, who are a constant source
of love, encouragement, and laughs. To my husband Frank, thank you for patiently
listening to me and doing your best to understand what it is I actually do. I am truly
blessed everyday to be your wife. To my parents Ross and Pamela, thank you for loving
me, believing in me, and supporting my education for far too many years. If I could pick
anyone to have as parents, I would pick only you. And to my friends, especially to
Bonnie, Bethany, and Peg, thank you for encouraging me unceasingly and in my quest
for answers, keeping me focused on what truth is all about.
iv
ACKNOWLEDGEMENTS
I would like to thank the members of my dissertation committee for their
encouragement, input, and guidance. Each have given generously of their time and shared
their expertise. I am so grateful for this support and how it has shaped my PhD education.
Judy Garner
Agnieszka Kobielak
Janos Peti-Peterdi
Alapakkam Sampath
Alan Yu
Thank you to my mentor Janos for many years of teaching, listening, and
encouraging me to go beyond what I thought I was capable of accomplishing. I would
also like to thank all the members, both past and present, of the Peti-Peterdi lab. It has
been a great privilege to work alongside, to learn from, and to get to know such an
impressive and committed group of scientists. I have also had the honor of being a part of
several international collaborations and I am grateful to our collaborators both here and in
Europe for their contributions to this work.
Finally, I am deeply indebted to those who have supported this research beyond
the lab. Thank you to the American Heart Association for their financial support and to
William Wood and Lee Stein-Wood for generously granting me the David Stein research
award. Finally, I would like to acknowledge the hard work and dedication of those behind
v
the scenes at USC, especially those who provide administration support and those who
care for our animals. Without their work, this research would not have come to fruition.
These studies were supported by by an AHA Western Affiliate Predoctoral
Research Fellowship to Fiona Hanner, and by grants DK64324 and DK74754 from NIH,
an ASN Carl. W. Gottschalk Research Scholar Award, and by an Established Investigator
Award 0640056N from the American Heart Association to Janos Peti-Peterdi. Work in
the Bonn laboratory was supported by grants of the German Research Association (SFB
645, B2 and Wi270/29-1) to Klaus Willecke. Work in the Eladari lab was supported by
the Institut de la Santé et de la Recherche Médicale.
The following articles are used with permission from the American Physiological
Society:
Hanner F, Sorensen CM, Holstein-Rathlou NH, Peti-Peterdi J. Connexins and the kidney.
Am J Physiol Regul Integr Comp Physiol. In press.
Hanner F, von Maltzahn J, Maxeiner S, Toma I, Sipos A, Kruger O, Willecke K, Peti-
Peterdi J. Connexin45 is expressed in the juxtaglomerular apparatus and is involved in
the regulation of renin secretion and blood pressure. Am J Physiol Regul Integr Comp
Physiol 295: R371-R380, 2008.
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 289: F1304-
F1312, 2005.
I am requesting permission from the publishers of Cell Communication and
Adhesion to reprint from the following article:
Hanner F, Schnichels M, Zheng-Fischhofer Q, Yang LE, Toma I, Willecke K,
McDonough AA, Peti-Peterdi J. Connexin 30.3 is expressed in the kidney but not
regulated by dietary salt or high blood pressure. Cell Commun Adhes 15: 219-230, 2008.
vi
TABLE OF CONTENTS
Epigraph ii
Dedication iii
Acknowledgements iv
List of Tables viii
List of Figures ix
Abstract xi
Introduction 1
Introduction references 14
Chapter 1: The renal expression and regulation of Cx30
Chapter 1 Abstract 19
Introduction 21
Materials and Methods 23
Results 29
Discussion 37
Chapter 1 References 43
Chapter 2: The renal expression and regulation of Cx30.3
Chapter 2 Abstract 47
Introduction 49
Materials and Methods 51
Results 57
Discussion 64
Chapter 2 References 69
Chapter 3: The expression of Cx45 in the juxtaglomerular apparatus and its role in
calcium signaling, renin regulation, and blood pressure homeostasis
Chapter 3 Abstract 72
Introduction 74
Materials and Methods 76
Results 84
Discussion 93
Perspectives and Significance 99
Chapter 3 References 100
vii
Chapter 4: The expression and function of Pannexin1 in the renal tubular epithelium
Chapter 4 Abstract 104
Introduction 106
Materials and Methods 108
Results 116
Discussion 125
Chapter 4 References 132
Conclusion 137
Conclusion References 149
Alphabetized Bibliography 152
viii
LIST OF TABLES
Table 1: Summary of renal Cxs 4
Table 1.1: Summary of localization and expression of Cx30 in 37
rat, rabbit and mouse kidney tissue.
ix
LIST OF FIGURES
Figure 1: Connexins hexamers form gap junctions and hemichannels. 1
Figure 2: Expression of Cx isoforms along the nephron. 137
Figure 3: The model of Cx30-dependent collecting duct transport regulation 139
by flow-induced purinergic signaling.
Figure 1.1: Detection of Cx30 mRNA in the mouse and protein in the rat kidney. 29
Figure 1.2: Immunoperoxidase staining of Cx30 in rat kidney sections. 31
Figure 1.3: Immunofluorescence labeling of Cx30 in the rabbit kidney. 32
Figure 1.4: Pre-incubation of the Cx30 antibody with its blocking peptide resulted 33
in no Cx30 labeling.
Figure 1.5: Immunofluorescence labeling of Cx30 in the mouse kidney. 34
Figure 1.6: Regulation of Cx30 expression in the rat inner medullary collecting duct. 34
Figure 1.7: Immunoblotting analysis of Cx30 in rat tissue under various dietary 35
salt conditions.
Figure 1.8: Regulation of Cx30 expression in cultured cell lines by high salt in the 36
medium.
Figure 2.1: Detection of connexin 30.3 (Cx30.3) mRNA and protein in the mouse 57
kidney.
Figure 2.2: Renal Cx30.3 expression analyzed by staining for nuclear lacZ reporter 58
gene expression in heterozygous Cx30.3+/lacZ adult mouse kidneys.
Figure 2.3: Detection of Cx30.3 in the mouse kidney by immunofluorescence. 59
Figure 2.4: Immunofluorescence labeling of Cx30.3 in rat and rabbit kidney sections. 60
Figure 2.5: Schematic representation of Cx30.3-expressing nephron segments in 61
both the mouse, rat, and rabbit kidney.
Figure 2.6: Immunoblotting analysis of Cx30.3 expression in the rat kidney under 62
various dietary salt conditions.
x
Figure 2.7: Immunoblotting analysis of Cx30.3 expression in the kidneys of 63
normotensive and hypertensive rats.
Figure 3.1: Localization of Cx45 transcripts in Cx45
+/-
mouse kidney sections by 84
X-Gal staining.
Figure 3.2: Detection and localization of EGFP in Cx45fl/fl:Nestin-Cre mouse 85
kidney sections.
Figure 3.3: Renal renin expression, plasma renin activity, and systemic blood 87
pressure in C57BL/6 and Cx45fl/fl:Nestin-Cre mice.
Figure 3.4: Characterization of vascular smooth muscle cell primary cultures from 88
C57BL/6 and Cx45fl/fl:Nestin-Cre mice.
Figure 3.5: Calcium wave propagation in vascular smooth muscle cells from 92
C57BL/6 and Cx45fl/fl:Nestin-Cre mice.
Figure 4.1: Expression of Panx1 in the renal tubules. 116
Figure 4.2: Co-labeling of Panx1 with epithelial cell markers in the mouse kidney. 118
Figure 4.3: Detection of Panx1 in renal epithelial cell cultures. 120
Figure 4.4: Measurement of ATP release and dye uptake in Panx1 inhibitor-treated 121
MDCK and M1 cells.
Figure 4.5: Measurement of ATP release and dye uptake in Panx1 124
siRNA-transfected MDCK and M1 cells.
xi
ABSTRACT
Connexins are the constituent proteins of a gap junction, a non-selective channel
that links together two cells, allowing the passage of molecules directly between them. In
addition, connexins are thought to form discrete “hemichannels” in uncoupled cells. This
conformation permits molecular exchange between the intra- and extracellular
environments. Pannexins are a structurally similar, but genetically unrelated class of
proteins that appears to share the hemichannel-like structure and function of connexins,
but does not engage in gap junction formation. Both connexins and pannexins are
ubiquitously expressed and regulate intercellular communication, both through direct
cell-cell coupling and via the release of secondary messengers such as ATP to the
extracellular fluid. Gap junction coupling is well-established in the kidney and
intercellular communication pathways that are critical to controlling major renal
regulatory mechanisms are known to involve connexins and pannexins in other cell types.
However, a systematic study of the expression and function of these proteins in the
kidney has been absent. Therefore we sought to examine connexin and pannexin
localization within the kidney and, based on these findings, investigate how they facilitate
the signaling mechanisms and physiology at work in these regions. Based on initial
reports of mRNA expression, we focused on four select isoforms. Chapters 1 and 2 delve
into the expression and regulation of two connexins, Cx30 and Cx30.3, along the distal
nephron. Finding expression of both isoforms in the apical membrane of the renal
epithelial cells suggested a function for Cx30 and Cx30.3 as an ATP release mechanism
which may regulate salt and water transport in the distal nephron through purinergic
xii
signaling. Chapter 3 details the expression of Cx45 in the juxtaglomerular apparatus.
After observing Cx45 localization in this region, we tested the hypothesis that Cx45 plays
a role in regulating renin release and blood pressure by facilitating calcium wave
propagation in the afferent arteriole vascular smooth muscle cells. Finally, Chapter 4
addresses the renal expression of the pannexin isoform Panx1 and presents data from
renal epithelial cell cultures that demonstrate a role for Panx1 in the regulation of ATP
release in the kidney. These studies have provided new insights into how connexins and
pannexins are expressed in the kidney and suggest a significant role for these proteins in
facilitating renal signaling mechanisms. Due to the importance of intercellular
communication in the kidney, connexins and pannexins may ultimately have a profound
downstream effect on key renal physiological phenomena.
1
INTRODUCTION
Connexins
Connexins (Cxs) are the family of transmembrane proteins that form gap
junctions, which are critical to intercellular communication in nearly all cell types. In
1977, the structure of gap junctions was elucidated from x-ray crystallographic data. In
this model a hexamer of Cx proteins (known as a connexon) forms a non-selective pore
in the cell membrane.
1-2
A gap junction forms when two connexons dock to each other in
adjacent cell membranes. These channels allow the passage of inorganic ions, including
Ca
2+
and Na
+
and secondary messengers such as cAMP and IP
3
(Figure 1).
3-5
After
discovering the structure of gap junctions, a model for how these channels function
needed to be developed.
The next major
breakthrough in the gap junction
body of knowledge was the
confirmation that these channels
could form open or closed
configurations, therefore
controlling the passage of small molecules and secondary messengers between coupled
cells. Along with this discovery, it was observed that the same small molecules could
alter gap junction conductance.
6
More recent data has pointed to the existence of
functional, uncoupled connexon channels. These so-called hemichannels are thought to
Figure 1: Connexins hexamers form gap junctions and
hemichannels. Gap junctions (left); hemichannels (right).
2
behave much like gap junctions, allowing molecular exchange between the intra- and
extracellular environment (Figure 1).
7
Both gap junctions and hemichannels are regulated at several levels. In addition to
the effects of small molecules on channel conductance, gating is also altered by metabolic
inhibition, transjunctional voltage, and mechanical stress.
8-11
The Cx proteins themselves
are preprogrammed to undergo rapid turnover, with typical half-lives of a few hours.
12
This constant renewal is thought to be an important aspect of gap junction regulation.
Once synthesized and inserted into the cell membrane, hemichannels of different Cx
isoforms can dock to each other forming a heterotypic gap junction. Additionally, the
connexon hexamer can be composed of more than one Cx isoform, referred to as a
heteromeric channel.
13
The many possible combinations that can result lead to an
incredible diversity in channel function as each Cx isoform has different gating
properties.
14
This ability to regulate Cx expression and function appears to be important
to many cellular and physiological processes.
15
For example, in the cardiovascular system, several Cx isoforms play important
roles in morphogenesis and electrical coupling.
16-17
Pathological conditions, such as
diabetes and hypertension are associated with changes in Cx regulation,
18
while genetic
mutation of Cx genes can have deleterious effects on cardiac function.
16
Clearly the
regulation of Cxs and cardiac physiology are reciprocal. Cx are of similar significance to
normal development and physiology in a diverse range of organs, including the nervous
system, reproductive organs, and the skin.
19-21
3
Renal expression of connexins
While the presence of gap junctions within the kidney has long been known,
22
it is
only recently that the specific details of renal Cx expression have become apparent.
Using mRNA-based methods, immunohistochemistry, and transgenic reporter mice,
significant advances have been made in identifying and localizing renal Cxs (Table 1).
This knowledge is advantageous for several reasons. First, since each Cx isoform exhibits
unique gating and permeability profiles, determining which Cx is expressed in a region of
the kidney is critical to understanding the physiological significance of renal gap
junctions. Additionally, identifying the subcellular localization of a Cx points to how it
functions. Localization along apical or basal, but not lateral, membranes suggests that a
Cx isoform is functioning as a hemichannel, while Cx expression between cells suggests
gap junction coupling. Finally, several Cx mutations manifest as systemic and severe
pathology, which may be related to renal development and physiology.
23
Knowing how
and where a Cx isoform is expressed within the kidney may explain these phenotypes.
One of the major limitations in identifying Cx localization within the kidney is the
availability of specific antibodies. The use of Cx knockout mice provides a suitable
negative control for antibodies, but not all isoforms produce viable knockouts. To address
this issue, researchers have frequently attempted to confirm immunological localization
with RNA expression-based techniques, such as RT-PCR of microdissected tissue.
However, different approaches have frequently led to different results, making it difficult
to obtain a consensus. One alternative approach that has arisen is the use of transgenic
mice that express reporter genes under the control of Cx promoters, which allows the
4
Table 1: Summary of renal Cxs ATL: Ascending thin limb of the loop of Henle; A: afferent arteriole;
CD: Collecting duct; CNT: connecting tubule; DCT: Distal convoluted tubule; EA: efferent arteriole;
IA: interlobular artery. JGA: juxtaglomerular apparatus; TAL: thick ascending limb of the loop of
Henle; VSMC: vascular smooth muscle cell. * indicates conflicting localization data;
5
direct visualization of gene expression on a cellular level. Using a combination of these
techniques increases confidence in establishing intrarenal Cx expression, but Cx
expression in the kidneys should still be thought of as an evolving picture.
Four Cx isoforms have classically been considered vascular: Cx37, 40, 43, and 45.
They follow a pattern of expression within the vasculature with Cxs37 and 40 typically
expressed in endothelial cells and Cxs43 and 45 found in vascular smooth muscle
(VSMC). A more complicated distribution is found in the kidney however. Cx40 is the
predominant endothelial cell isoform, with strong expression in the preglomerular
vasculature and the renin-producing JG cells. Both Cx37 and Cx43 have been detected in
endothelial cells, but the specific region of expression as well as the level of expression
varies between reports. The picture is even less clear in terms of VSMCs. There are
conflicting reports of Cx37 and 43 expression in VSMC and Cx45 had received little
attention as a potential isoform, despite its predominance in other vascular systems.
23
The glomerulus, which filters the renal blood supply, generating the tubular fluid, is
comprised of several cell types including endothelial cells, mesangial cells, and
podocytes. Each has a unique function in the filtration process and there is abundant
evidence of gap junctions in each cell types. The cellular distribution of Cx isoforms is
again a complex situation. In the endothelium, studies are split between whether or not it
expresses Cx40. Mesangial cells express Cx40, while Cx37 expression is limited to the
vascular pole only. Cx37 expression has been found podocytes, where Cx45 expression
has also been reported.
23
6
Several Cx isoforms have been localized in the tubules and collecting duct. MRNA
expression studies have found many Cx isoforms including Cxs 36, 37, 43, 45, and 50 in
these regions.
24-25
Cx immunohistochemistry studies have shown tubular expression of
Cx26, 30, 30.3, and 37.
26-28
In many cases however, data from immunohistochemistry
experiments have either failed to confirm earlier results or presented conflicting data. For
example in-situ RT-PCR detected Cx43 expression at high levels in the collecting duct
with lower levels of expression found in the tubules.
25
Labeling kidney sections with a
Cx43 antibody failed to show any expression of the protein in the nephron.
29
But in a
renal proximal tubule primary cell culture, Cx43 protein expression was not only
identified, but functional Cx43 hemichannels were shown to exist too.
30
Similar conflict
exists over the expression of Cx45.
24,29
As such the localization of certain Cxs in the
tubules remains equivocal.
While the expression profiles of Cxs in the kidney continue to emerge, they have also
presented opportunities to address the next question – what is the functional significance
of Cxs in renal physiology? Whether as gap junctions or hemichannels, the fundamental
purpose of Cx proteins is to facilitate cell to cell communication. Therefore, it is
imperative to understand how and why intracellular signaling occurs in the kidney and
how this may relate to Cx expression. In other cell types, Cxs are known to play a role in
two major signaling mechanisms that also occur in the kidney: purinergic signaling and
calcium propagation.
7
Renal signaling mechanisms: purinergic signaling
The major function of the renal tubules and collecting duct is to process the
glomerular filtrate by reabsorbing and secreting solutes and water. Through the dynamic
regulation of proteins along the nephron, reabsorption and secretion can be modulated to
maintain fluid homeostasis in the body. Renal function itself is also regulated in the
tubules through the process of tubuloglomerular feedback (TGF), whereby conditions in
the cortical thick ascending limb (cTAL) affect the glomerular filtration rate. In many
cases, changes in ion concentration or flow are “sensed” by the epithelial cells along the
nephron, triggering intracellular signal transduction which alters protein function
downstream.
31-33
Purinergic signaling is one such mechanism at work in the kidney.
Purinergic signaling occurs when extracellular purines bind to and activate
transmembrane purinergic receptors in an autocrine/paracrine manner. Purinergic
receptors are classified by both their ligand and their activity. P1 receptors are activated
by the binding of adenosine, while P2 receptors bind nucleotides with varying specificity.
P2 receptors are sub-classified as either P2X receptors, which are ligand-gated ion
channels, or P2Y receptors, which are G protein-coupled receptors. Activation of either
type of channel results in downstream signaling in the cell, either through changes in
intracellular ion concentration or secondary messengers, such as cAMP. Both P2X and
P2Y receptors have been found in nearly all types of tissue. In the nephron, various P2
receptor isoforms have been identified along the tubules, in blood vessels, and the
glomerulus. In epithelial cells, purinergic receptors isoforms tend to be segregated to
either the apical or basolateral membrane.
34
8
Functionally, purinergic signaling in the tubules alters reasborption and secretion by
activation or inhibition of various transport mechanisms. This is best established in the
principal cells of the collecting ducts, where apical P2Y2 receptor activation triggers the
inhibition of sodium transport via the ENaC channel and potassium via the ROMK
channel.
35
Purinergic signaling in the collecting duct is regulated by flow, suggesting that
it functions as a negative feedback loop, mediating salt and water reabsorption in
response to tubular conditions.
36
The other major role of purinergic signaling in the
kidney is as a mediator of the renal autoregulation of glomerular filtration rate through its
two components.
37-38
In the myogenic mechanism, ATP is released from endothelial cells
in response to increased renal perfusion pressure, binds P2X receptors, and triggers
vasoconstriction of the afferent arterioles. In TGF, ATP is released from macula densa
cells in response to tubular conditions and either directly, or indirectly as adenosine,
causes afferent arteriole vasoconstriction. Beyond its physiological role in the kidneys,
aberrant purinergic signaling has also been associated with renal pathologies such as
hypertension and polycystic kidney disease.
39
Despite the well-established role of purinergic signaling in regulating renal epithelial
transport and blood flow, a major question remains: how does the ligand, ATP, move
across the cell membrane? Many transport mechanisms have been hypothesized to play a
role but no consensus has been reached on which mechanism(s) are physiologically
correct.
40
A candidate for a renal ATP transporter must meet several requirements. First,
purinergic signaling must occur in an autocrine/paracrine manner due to the activity of
local nucleotidases. Therefore, the site of ATP release must be close to P2 receptors and
9
consequently, have similar regulation of expression. Also, ATP release can occur in
response to mechanical stress,
36-37
so the ideal ATP release channel would be
mechanosensitive. Cx hemichannel-mediated release of ATP is a mechanism that fits this
paradigm. ATP passage via hemichannels has been documented in various cell cultures,
such as glial cells.
10
The localization of Cx isoforms at sites of renal purinergic signaling
provides an additional basis to investigate the hypothesis that Cxs are an ATP release
mechanism in the kidney.
Renal signaling mechanisms: the calcium wave
Calcium wave propagation is a type of signal transduction found in non-excitable
cells, whereby an increase in intracellular calcium spreads among coupled cells. This
permits the synchronizing of adjacent cells, allowing them to function together.
Propagation of the wave is coordinated by Cxs and occurs by one of two methods:
directly, with calcium moving from cell to cell via gap junctions or indirectly, by the
downstream effects of purinergic signaling via ATP released from Cx hemichannels. As
with purinergic signaling, calcium wave propagation often occurs as a result of
mechanical stimulation.
41
In the kidney, calcium waves mediate renin release, TGF, and vascular
conduction and Cxs play a role in each of these processes. Calcium’s role in the
regulating renin is particularly unique. It inhibits the release of renin from the JG cells,
contrary to its behavior in other cells.
42
The JG cells are coupled via Cx40 gap junctions
to each other and to the adjacent endothelial cells. Recent data has shown that interfering
with Cx40 causes a loss of the negative inhibition of renin. Since renin is a key player in
10
blood pressure homeostasis, Cx40 loss also results in hypertension.
23
This suggests that
the signal mediating renin inhibition, calcium, is dependent on Cx40. In the
juxtaglomerular apparatus (JGA), a calcium wave propagates through the region in
response to TGF activation at the macula densa. The TGF calcium wave is dependent on
both gap junctions and extracellular ATP.
43
Finally, the renal arterioles are known to
engage in the propagation of vasoconstriction by rapid increases in intracellular calcium
that spreads along the vessel. Conduction of this response appears to be Cx37-dependent
in mice and requires gap junction communication in rats.
44-45
Many Cx isoforms are expressed in the vasculature, JGA, and tubules where they
mostly likely function in facilitating cell signal transduction, responding to changes in
flow and pressure and therefore playing a role in the regulation of blood flow and fluid
homeostatsis. Cxs, however, may not be the only player in these signaling cascades. The
recent discovery of pannexins presents an alternative paradigm that may better resolve
the question of how ATP is released to the extracellular region.
Pannexins:
Pannexins are proteins that are structurally similar to Cxs, but unrelated by
sequence homology. As with Cxs, they are 4 pass transmembrane proteins with an
extracellular C-terminus, cysteine-containing extracellular loops, and an intracellular N-
terminus. They also polymerize as hexamers to form a non-selective membrane pore.
Because of this remarkable resemblance in structure, it was initially thought that
pannexins were the evolutionary connection between Cxs, which are exclusively found in
vertebrates, and innexins, their invertebrate counterpart. However, pannexins fail to show
11
alignment with either Cxs or innexins and appear to represent a unique family of
proteins.
46
Three pannexin isoforms have been identified, Panx1, 2, and 3. Panx1 appear
to be the only functional isoform, while Panx2 seems to serve a support role in Panx1
channel formation.
47
Panx3 distribution and function remain to be determined.
In terms of their function, it was predicted that pannexins would behave much like
Cxs and innexins based on their structure. Early studies in Panx1-transfected oocytes
showed gap junction coupling when oocytes were paired and hemichannel activity in
unpaired oocytes, supporting the initial hypothesis.
46
However, as studies expanded
beyond the oocyte model, there was little evidence to support cell coupling by pannexins
in-vivo. Based on these findings, a new paradigm has been proposed: Cxs and pannexins
are not redundant in function, but instead each serves a unique purpose. Cxs form gap
junctions, while pannexins form hemichannel-like pores, named pannexons.
48
But this proposal presents a direct challenge to the concept of the Cx
hemichannel. While there is evidence that Cx hemichannels do exist, questions have been
raised about whether they can open under physiological conditions. They also appear to
be inhibited by increases in intracellular calcium, which contradicts their involvement in
calcium wave propagation by ATP release. Pannexons lack these limitations. They are
open at physiological extracellular calcium concentrations and membrane depolarization.
Additionally, their opening is triggered by increased intracellular calcium and there is
strong evidence that ATP directly passes through pannexons.
40
Finally, they form
complexes with purinergic receptors.
49
Taken together, the properties of Panx1 channels
fit the model of extracellular calcium wave propagation by purinergic signaling.
12
Since both purinergic signaling and calcium wave signaling are important cell-cell
communication mechanisms in renal physiology, there is a need to evaluate the
possibility that Panx1 is expressed in the kidney and facilitates renal ATP release. Panx1
is thought to be ubiquitously expressed and there is evidence of Panx1 mRNA expression
in the kidney.
50
A detailed picture of which renal cells and regions express Panx1 remains
to be established. Beyond a localization study, the physiological significance of Panx1 in
the kidney would need to be determined and reconciled with the function of renal Cxs.
Despite its ubiquitous expression, investigating Panx1 function in terms of whole
tissue physiology is a relatively unexplored field. Due to the recent discovery of Panx1,
most work has focused on establishing how it behaves on a cellular level in transfected
cell lines. The major limitation in going beyond these models has been the lack of
specific inhibition. All gap junction inhibitors block both Cx hemichannels and
pannexons.
51
Therefore, the ability to distinguish Cx gap junctions from Cx hemichannels
from pannexons is hampered. Genetic modification of the Panx1 gene, in either a mouse
or cell model remains to be established. However, the use of siRNA specific to Panx1
and the recent discovery of probenecid,
52
an organic anion transporter inhibitor that also
inhibits Panx1 but not Cxs have lowered the barrier to conducting Panx1 research beyond
the level of the cell.
Cxs and pannexins show global expression throughout the body and facilitate cell
to cell communication through direct and indirect means. Despite the awareness of renal
gap junctions for several decades, details about the expression of renal Cxs have only
recently been delved into. Given that Cxs mediate signaling mechanisms that regulate
13
kidney function, evaluating renal Cxs also provides insights into renal (patho)physiology.
In Chapter 1, the renal expression of Cx30 is detailed by immunofluorescence and
Western blotting techniques. Its localization within the apical membrane of the distal
nephron, where salt and water transport are regulated, prompted an evaluation of how
dietary salt effects Cx30 expression. The renal localization of an unrelated isoform,
Cx30.3, is similarly detailed in Chapter 2. The relationship between Cx30.3 and blood
pressure and salt intake were also addressed. Cx45, the predominant Cx in VSMCs, had
yet to be studied in the kidney, despite the importance of afferent arteriole VSMC
coupling in renal hemodynamics. The expression and function of Cx45 were tested in
Chapter 3 using a novel mouse model, Cx45fl/fl:Nestin-Cre. This mouse model had a
conditional knockout of Cx45 in the kidney and instead expressed the reporter gene
eGFP, which allowed the investigation of Cx45 localization without using antibodies.
Due to the critical role of calcium signaling in regulating vasomotor function and renin
release in the JGA, the systemic effects of deleting Cx45 in the kidney were measured.
Finally, the dependence of the calcium wave on Cx45 was directly tested in an afferent
arteriole VSMC primary culture. The recent discovery of Panx1 channels which not only
function in physiological conditions, but are also permeable to ATP, challenges the
hypothesis that Cx hemichannels are a major ATP release mechanism. Therefore chapter
4 presents the first study on renal Panx1 expression and tests the hypothesis that ATP is
released from renal epithelial cells via Panx1. Panx1 protein localization was performed
by immunofluorescence. Using two renal epithelial cells lines that endogenously express
Panx1, both channel permeability and ATP release via Panx1 were investigated.
14
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18
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7
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19
CHAPTER 1: THE RENAL EXPRESSION AND REGULATION OF CX30
Published in the American Journal of Physiology: Renal Physiology December 2005
Localization of connexin 30 in the luminal membrane of cells in the distal nephron
Fiona McCulloch*, Régine Chambrey, Dominique Eladari, János Peti-Peterdi
*published under maiden name
CHAPTER 1 ABSTRACT
Several isoforms of the gap junction protein connexin (Cx) have been identified in
a variety of tissues that communicate intercellular signals between adjacent cells. In the
kidney, Cx37, Cx40, and Cx43 are localized in the vasculature, glomerulus, and tubular
segments in a punctuate pattern, typical of classical gap junction channels. We performed
immunohistochemistry in the mouse, rat, and rabbit kidney to study the localization of
Cx30 protein, a new member of the connexin family. The vasculature, glomerulus, and
proximal nephron segments were devoid of staining in all three species. Unexpectedly,
Cx30 was found throughout the luminal membrane of select cells in the distal nephron.
Expression of Cx30 was highest in the rat which also showed some diffuse cytosolic
labeling, continuous from the medullary thick ascending limb to the collecting duct
system, and with the highest level in the distal convoluted tubule. Labeling in the mouse
20
and rabbit was much less, limited to intercalated cells in the connecting segment and
cortical collecting duct, where the apical signal was particularly strong. High salt
containing diet and culture medium up-regulated Cx30 expression in the rat inner
medulla and in M1 cells, respectively. The distinct, continuous labeling of the luminal
plasma membrane and up-regulation by high salt suggests that Cx30 may function as a
hemichannel involved in the regulation of salt reabsorption in the distal nephron.
21
INTRODUCTION
Connexin (Cx) proteins are the building blocks of gap junction channels that
allow electric coupling and the cell-to-cell diffusion of ions and signaling molecules
between the cytoplasm of two adjacent cells. Gap junctions are common features in a
variety of tissues and cell types, and are essential components of intercellular
communication in many physiological processes. Apart from the classic gap junction
between two cells, Cx proteins can form large, non-selective ion channels on a single
cell’s surface. The existence of single connexons (a hexamer of Cx proteins) in the non-
junctional plasma membrane raised the intriguing possibility that Cx proteins may
function as transmembrane ion channels in addition to serving as precursors for the
formation of gap junction channels (8, 11). It is now widely acknowledged that these so-
called hemichannels indeed exist and are functional. They are permeable to a variety of
small metabolites (including nucleotides, ATP, and NAD
+
) and function in paracrine
signaling in many cell types (8).
In humans, there are at least 20 Cx genes. Some of the ubiquitous Cx isoforms,
(Cx37, Cx40, Cx43 and Cx45) have been identified in the kidney and localized to mainly
vascular and glomerular components (2, 3). For example, intercellular communication
between the cells of the juxtaglomerular apparatus (JGA) involves an ATP release-
mediated calcium wave that regulates contractile function as well as renin secretion (14,
33, 34). Although most cells of the JGA are interconnected via gap junctions composed
of Cx40, 43, and 45, spreading of this calcium wave does not appear to require classical
gap junction channels or physical contact between cells (33). This paracrine, ATP-
22
dependent and purinergic receptor-mediated intercellular calcium wave (34) may involve
hemichannel function, since Cx hemichannels are top candidates for the plasma
membrane ATP channel. However, the exact molecular identity of this channel is still to
be determined (14). Also, the most ubiquitous Cx isoform, Cx43, has been localized to
tubule segments, including the rat collecting duct system (3). However, the classical gap
junction channels, in electronmicroscopy terms, are reportedly absent (3). Activation of
Cx43 hemichannels may cause cell damage of renal proximal tubule (PT) cells in culture
(31).
Cx30 is a newly identified member of the connexin gene family. Cx30 was
isolated by screening a mouse genomic library with a rat Cx26 probe (6). The Cx30
protein is highly homologous to Cx26 and it has an additional 37 amino acids at its C-
terminus. Cx30 is considered the adult form of Cx26, since their expression patterns are
clearly distinct. Specifically, Cx30 is highly expressed in adult skin and brain. However,
it cannot be detected in the embryonic and fetal brain (6, 22). On the other hand, Cx26 is
highly expressed in prenatal brain and decreases after birth. Importantly, the renal
expression of Cx30 has been recently suggested (13).
This paper provides evidence that the Cx30 protein, perhaps in the form of
luminal hemichannels, is expressed in renal tubular epithelial cells and suggests that it
may have a potential role in the regulation of salt reabsorption in the distal nephron.
23
MATERIALS AND METHOD
Animals
Sprague-Dawley rats (200g, Harlan, Madison, WI) were fed standard chow (0.3%
NaCl), high salt (TD 92012: 8% NaCl, Harlan Teklad, Madison, WI) or low salt (TD
90228: 0.01% NaCl, Harlan) rodent diet for one week. Rats fed the high salt diet also
received 0.45% NaCl (w/v) containing drinking water. Swiss-albino mice (20g, in house
bred) and New- Zealand White rabbits (500g, Irish Farm, Norco, CA) were maintained
on standard diet with normal water. All animal protocols have been approved by the
Institutional Animal Care and Use Committee at the University of Southern California
and INSERM.
Antibodies
Mouse monoclonal anti-Cx26 and rabbit polyclonal anti-Cx30 antibodies were
purchased from Zymed Laboratories Inc. (San Francisco, CA). The Cx30 blocking
peptide was kindly provided by Zymed. These antibodies have been previously
characterized (20,21,22,24,32) and the Cx30 antibody used in the present studies has
been demonstrated to recognize Cx30 in the brain and cochlea using both immunoblot
and immunohistochemistry of mouse and rat tissue (22, 24, 32). The mouse monoclonal
anti-pendrin antibody was purchased from MBL International, Woburn, MA. Rabbit
polyclonal anti-aquaporin-2 antibody (23) was a generous gift from Dr. Mark Knepper.
Rabbit polyclonal antibody to the thiazide-sensitive NaCl cotransporter (NCC) was a gift
from Dr. D.H. Ellison (Oregon Health and Science University, Portland, Oregon) and has
also been previously extensively characterized (9, 27, 28). The mouse monoclonal
24
antibody to rat anion exchanger AE1 (1) was kindly provided by Dr. Daniel Biemesderfer
(Yale University, New Haven, CT). The NCC, pendrin, AE1 and AQP-2 antibodies are
from the original batch of immunization, that were initially used and characterized in
previous publications (4,17,23).
Cell Cultures
Renal medullary interstitial cells (RMIC) were a kind gift from Dr. Christine
Maric (Georgetown University, Washington, DC) and have been previously characterized
(19). The M1 cell line has a mixed phenotype, representative of both intercalated and
principle cells of the collecting duct. These cells were originally purchased from ATCC
and have been described by Fejes-Toth et al. (10).
Immunoblotting of rat tissue
Rats were anesthetized with 100mg/mL Inactin and kidneys were perfused
retrograde
with ice-cold PBS to remove blood, and then removed. Slices of cortex and
inner medulla were manually dissected and tissue was homogenized with a rotor-stator
homogenizer in a buffer containing 20mM Tris-HCl, 1mM EGTA, pH 7.0, and a protease
inhibitor cocktail (BD Bioscience, San Jose, CA). Samples were centrifuged at low speed
to pellet cellular debris and supernatant was collected and assayed. Forty micrograms of
protein were run
per lane, separated on a 4-20% SDS-polyacrylamide gel, and then
transferred to a polyvinyledene difluoride membrane (Biorad, Hercules CA). After
blocking the membrane in 5% non-fat dry milk, immunoblotting was performed with a
rabbit polyclonal
antibody to Cx30 at a dilution of 1:250 (Zymed). Reactivity was
detected
by a horseradish peroxidase-labeled goat anti-rabbit (1:1000 dilution, BD
25
Biosciences, San Diego, CA) or donkey anti-goat (1:1000 dilution, Santa Cruz
Biotechnology) secondary antibody. An enhanced
chemiluminescence kit (Amersham
Biosciences, Little Chalfont, England) was used to visualize the secondary
antibody. The
blot was stripped and reprobed with a goat polyclonal antibody to Actin at a dilution of
1:1000
(Santa Cruz Biotechnology) to test for protein loading and quality of transfer.
Densitometric analysis of blots was performed using ImageJ (NIH) software. The
data was then normalized against the control sample and an average for each group (n =
4) was calculated. Statistical significance
was tested using ANOVA and data are shown
as mean+SE.
Immunoblotting of cultured cells
RMIC and M1 cells were grown to confluence in plates as previously described
(10, 19). Plates were then treated for 16 hours with either 110mM NaCl-supplemented
media (high salt) or 220mM-mannitol supplemented media (to control for osmolality).
Cells were lysed using CellLytic-M lysis buffer (Sigma) according to manufacturer’s
instructions and assayed for protein concentration by a modified Bradford method (Quick
Start Bradford protein assay, Biorad). Samples were blotted and analyzed for Cx30 and
actin as described earlier.
Immunoperoxidase labeling of kidney tissue
Rat kidneys were fixed in situ by perfusion of 4% paraformaldehyde in
Dulbecco’s Modified Eagle’s/F12 medium (Invitrogen, Carlsbad, CA). Coronal kidney
sections containing all kidney zones were then post-fixed for 4-6 h at 4°C in 4%
paraformaldehyde and then embedded in paraffin. Subsequently, 4-µm sections of the
26
paraffin block were deparaffinized in toluene and rehydrated through graded ethanol.
Rehydratation was completed in Tris-buffered saline pH 7.6 (TBS). Slides were then
placed in plastic tank filled with Target Retrieval Solution (Dako Corp.) and heated 3 x 5
min in a microwave with medium (450W) heat. These steps unmasked antigens and
allowed immunostaining on paraformaldehyde-fixed paraffin sections, as determined in
preliminary experiments (not shown). To reduce nonspecific binding, sections were
rinsed in TBS for 10 min and preincubated for 15 min with 20% normal goat serum
followed by treatment with background reducing buffer (Dako Corp.) for 20 min. Rat
kidney sections were then labeled with the rabbit polyclonal Cx30 antibody as follows:
anti-Cx30 was applied for 1 h at room temperature. After three washes, sections were
incubated with a 1:600 dilution (in background reducing buffer) of goat anti-rabbit IgG
coupled to horseradish peroxidase, (Vector Laboratories, Burlingame, CA) in TBS, 30
min at room temperature,
followed by three TBS washes. Peroxidase activity was
revealed with 3-amino-9-ethylcarbazole (AEC) which gives a red-brown precipitate.
To ascertain the presence of Cx30 in distal convoluted tubules (DCT),
immunostainings of the same cells with antibodies to Cx30 and to the thiazide-sensitive
NaCl cotransporter (NCC) were performed in two consecutive 4-µm thick sections.
Consecutive sections were also stained with an antibody to pendrin to identify connecting
tubules (CNT) and cortical collecting ducts (CCD), and with an antibody to the chloride
bicarbonate exchanger AE1 to identify medullary collecting duct (MCD). Labeling for
these three antibodies were performed using the same method as for Cx30 except that the
antigen retrieval procedure consisted of a 40 min heating at 96-98°C in a water bath of
27
the sections in 1 mM EDTA. Sections were then incubated with a 1:200 dilution of a
rabbit anti-NCC, or 1:200 dilution of rabbit anti-AE1 antibody containing serum in place
of the anti-Cx30 peptide antibody.
After staining, sections were counterstained with hematoxylin. Glass coverslips
were mounted after applying liquid-phase faramount solution (Dako) to the tissue
sections. Sections were examined with a Zeiss microscope.
Immunofluorescence labeling of kidney tissue
A method similar to the one described above was used to prepare animal tissue for
immunofluorescence labeling. Blocking with
goat anti-rabbit Fab IgG for rabbit tissue
(1:100, Jackson ImmunoResearch Laboratories, West Grove, PA) for 40 min was used to
reduce nonspecific
binding with a rabbit polyclonal antibody. Sections were then
incubated with Cx30 antibody at a 1:50 dilution overnight and washed in PBS. Sections
were then incubated with HRP-conjugated goat anti-rabbit IgG and enhanced with Alexa
Fluor 594 labeled tyramide signal amplification according to the manufacturer’s
instructions (Molecular Probes, Eugene, OR). Some rabbit tissue sections were double-
labeled with an anti-pendrin monoclonal antibody overnight at a 1:50 dilution and the
secondary and TSA steps were repeated as above, except using a HRP-conjugated goat
anti-mouse IgG and Alexa Fluor 488 TSA (Molecular Probes). Following a wash step,
sections were mounted with Vectashield mounting media containing the nuclear stain
DAPI (Vector Laboratories) and examined with a Leica TCS SP2 confocal microscope.
All sections were labeled in parallel.
28
Reverse Transcription-PCR
Total RNA was purified from whole mouse kidney samples using a Total RNA
Mini Kit in accordance with manufacturer’s instructions (Biorad, Hercules, CA). RNA
was then quantified using spectrophotometry and reverse-transcribed to single-stranded
cDNA using avian reverse-transcriptase and random hexamers according to
manufacturer’s instructions (Thermoscript RT-PCR system, Invitrogen). 2µ L of cDNA
was amplified using a master mix containing Taq polymerase (Invitrogen) and the
following primers: Connexin 30 sense, 5’-GGCTTGGTTTTCAGAGATAG-3’;
Connexin 30 antisense, 5’-GAGTTGTGTTACCTGCTGC-3’; β-Actin sense, 5’-
GGTGTGATGGTGGGAATGGGTC-3’, β-Actin antisense 5’-
ATGGCGTGAGGGAGAGCATAGC-3’; each at a final concentration of 200µ M.
Connexin 30 oligonucleotides were based on previously published primer sequences (30).
Previous work using the same Cx30 primer sequences demonstrated the presence of Cx30
mRNA in the mouse brain (6). β-Actin oligonucleotide sequence was determined using
published sequence and confirmed with sequencing. The PCR reaction was carried out
for 30 cycles of the following: 94
o
C for 30 seconds, 55.4
o
C for 30 seconds, and 72
o
C for
30 seconds. The PCR product was analyzed on a 2% agarose gel stained with ethidium
bromide to identify fragments of approximately 369 bp for Connexin 30 (30) and 400 bp
for β-Actin.
29
RESULTS
Detection of Cx30 mRNA and protein in the kidney
RNA was isolated from whole mouse kidney and the presence of Cx30 mRNA
was detected using RT-PCR (Fig. 1.1A). A negative control (lack of template in the PCR
reaction) and a positive control (β-actin) were also employed. Using ethidium bromide,
we observed a band, approximately 369bp in size, for Cx30 sample, as expected.
Negative control samples produced no visible bands and β-actin samples produced bands
of the expected size (400bp). Western blot (Fig. 1.1B) using a Cx30 antibody resulted in
a single band around
the expected
molecular weight of
30 kD, within the 25-
62 kD range of
known connexin
isoforms.
Immunolocalization of Cx30 in the rat kidney
Localization of Cx30 in rat kidney was investigated by indirect
immunoperoxidase on paraformaldehyde-fixed, paraffin-embedded kidney sections.
Staining for Cx30 was restricted to some tubular structures and was observed in all major
ctrl ctrl Cx30 Cx30 β-actin β-actin
10-
25-
37-
75-
200-
Cx30
A B
Figure 1.1: Detection of Cx30 mRNA (A) in the mouse and protein (B) in the
rat kidney. RNA and protein were isolated from whole kidney samples. Using
RT-PCR and oligonucleotides designed to amplify Cx30 and β-actin, bands at
369bp and 400bp were observed respectively as shown (A). Absence of
template served as a negative control (ctrl). Western blotting (B) using a Cx30
antibody resulted in a single, intense band around the expected molecular
weight of 30 kD.
30
anatomical parts of the kidney: within the cortex, outer medulla, and inner medulla (Fig.
1.2A-C). In the cortical labyrinth (Fig. 1.2A) and the medullary ray (Fig. 1.2B-C), anti-
Cx30 antibody strongly labeled the apical pole of tubules with additional, less intense
staining of the cytosol (shown in inserts). No basolateral staining was observed in any
nephron segments. These Cx30-positive tubule segments were devoid of brush-border
membrane and with morphological features of cortical thick ascending limb (cTAL),
DCT, CNT, CCD, and MCD. Weak apical Cx30 staining was also observed in macula
densa (MD) cells (Fig. 1.2A). Proximal tubules, the thin limbs of the loop of Henle,
glomeruli and the vasculature were devoid of staining.
Cx30-positive tubular segments were next identified in consecutive sections (Fig.
1.2D-I) using well-known tubular markers. Staining consecutive sections with an anti-
NCC antibody that identified the apical membrane of DCT (Fig. 1.2D) demonstrated that
not only the DCT, but its downstream segment CNT both expressed Cx30 (Fig. 1.2E).
Similarly, AE1 staining labeled the basolateral membrane of type A intercalated cells in
the outer MCD (OMCD) (Fig. 1.2F). Staining of a consecutive section with the Cx30
antibody confirmed that the OMCD as well as the medullary TAL (MTAL) both
expressed Cx30 (Fig. 1.2G). In the last pair of consecutive sections, pendrin, a marker of
type B and non-A, non-B intercalated cells identified the CNT and CCD (Fig. 1.2H).
These nephron segments also expressed Cx30 (Fig. 1.2I).
The same pattern of labeling was seen in rat sections using immunofluorescence
techniques (not shown).
31
Immunolocalization of Cx30 in the rabbit kidney
Localization of Cx30 in the rabbit kidney was studied by immunofluorescence.
Staining for Cx30 was restricted to certain tubular and interstitial structures in the cortex
and outer medulla (Figs. 1.3B-F). No staining was observed in the inner medulla (not
shown), in vascular structures and in the glomerulus. The most intense Cx30 labeling was
F
G
A B C
D
*
E
*
H
*
I
*
F
G
A A B B C C
D
*
D
*
E
*
E
*
H
*
H
*
I
*
I
*
Figure 1.2: Immunoperoxidase staining of Cx30 in rat kidney sections. Views of the cortex (A), outer
medulla (B) and inner medulla (C) with magnified areas of the DCT (A), and the MCD (B and C) on
inserts. Mostly apical labeling is present. A: Position of the cTAL and MD is indicated by an arrow. Co-
localization of Cx30 (E, G, I) with NCC in the DCT (D), with AE1 in the OMCD (F), and with pendrin in
the CCD (H) on consecutive sections. =CD, #=MTAL. D: Note the transition of DCT to CNT, an abrupt
disappearance of apical NCC labeling (arrow). E: Cx30 labeling was found in all NCC positive tubules
(i.e. DCT) but also in the CNT. F: Basolateral AE1 staining appeared in a subpopulation of cells within
the OMCD indicating type A intercalated cells. No labeling was observed in the MTAL. G: Cx30 was
found in the MTAL and OMCD. H: pendrin labeling identified type B, and non-A, non-B intercalated cells
of the CNT and CCD. I: Cx30 was found in these pendrin-positive segments. Magnification is 20X for B
and C, 40X for A, and D-I.
32
#
G
*
G
PT
A B C
D E F
#
G
*
G
PT
A B C
D E F
observed at the apical plasma membrane of select cells in the CNT and CCD (Fig. 1.3B).
These cells were identified as intercalated cells, since in some of these cells, Cx30 was
co-localized with pendrin (Fig. 1.3A,C), an apical membrane anion exchanger of type B
and non-A, non-B intercalated cells. Intercalated cells showed continuous luminal plasma
membrane staining for both pendrin (Fig. 1.3A) and Cx30 (Fig. 1.3B).
Figure 1.3: Immunofluorescence labeling of Cx30 in the rabbit kidney. Pendrin (A, green) and Cx30 (B,
red) staining in the apical membrane of select cells in the CNT and CCD. C: Merged image of A and B
demonstrates co-localization of Cx30 and pendrin (yellow) in type B and non-A, non-B intercalated cells
of the CNT and CCD (*). Apical membrane of the cTAL (arrow) next to the glomerulus (G) is also
positive for Cx30. D: MD cells in the rabbit kidney (arrow) are devoid of Cx30 staining. Cells of the
cTAL across the MD however, are labeled at the apical membrane. E: Cx30 labeling in renal medullary
interstitial cells (arrowhead) was mainly cytosolic but also observed at the end of cell processes (arrows),
appearing to make contact with tubular segments (MTAL, #). F: Scattered and punctuate Cx30 labeling
was observed very rarely between various cells of the renal cortex and medulla as exemplified here by a
contact between a proximal tubule (PT) cell and an interstitial cell. Nuclei are stained with DAPI (blue).
DIC background was added and merged with fluorescence in C, D, and F. Scales are 40 µ m for A-C, 20
µ m for D-F.
33
Additional, weak Cx30 labeling was observed at the apical membrane of the
cTAL, however the MD was devoid of staining (Fig. 1.3D). In addition, renal medullary
interstitial cells in the outer medulla showed expression of Cx30 (Fig. 1.3E). While most
of these cells displayed a cytosolic staining pattern, some cells showed Cx30 localization
at the end of the cell processes making contact with tubular cells. Weak cytosolic Cx30
staining was observed in the MTAL (Fig. 1.3E). In addition to the luminal plasma
membrane labeling, while very rare, we observed highly scattered and punctuate labeling
in both the cortex and medulla between
cells of the tubular epithelium, vasculature,
and glomerulus, consistent with the
antibody recognizing gap junction proteins
(Fig. 1.3F). Pre-incubation of the Cx30
antibody with the blocking peptide resulted
in absolutely no labeling (Fig. 1.4).
Using a Cx26 specific antibody, no
labeling was found in the adult rabbit
kidney, consistent with Cx26 being an
embryonic isoform (not shown).
Immunolocalization of Cx30 in the mouse kidney
Localization of Cx30 in the mouse kidney was studied by immunofluorescence.
Staining for Cx30 was present only in the cortex and restricted to the apical membrane of
certain tubular cells of the CCD (Fig. 1.5). Double-labeling with an AQP-2 antibody
B
*
G
Figure 1.4. Pre-incubation of the Cx30 antibody
with its blocking peptide resulted in no Cx30
labeling. Rabbit tissue, arrow points at the MD and
cTAL, *: CCD, G: glomerulus. Nuclei are stained
with DAPI (blue). DIC background was added and
merged with fluorescence. Scale is 40 µ m.
34
identified the apical membrane of principal cells in the CCD (Fig. 1.5). Principal cells
were devoid of Cx30 staining, but all other cells of the CCD showed intense, apical Cx30
labeling (Fig. 1.5). Similar to the rabbit
(Fig. 1.4), pre-incubation of the Cx30
antibody with the blocking peptide
resulted in absolutely no labeling in the
mouse (not shown).
Regulation of Cx30 protein
expression by dietary salt in rat
kidney: an immunofluorescence
approach
To help ascertain a functional
role for Cx30 in the kidney, we tested
whether changes in dietary salt content
A
*
Figure 1.5. Immunofluorescence labeling of Cx30 in the
mouse kidney. Double-labeling of AQP-2 identified the
apical membrane of principal cells (green) in the CCD
(*). All AQP-2 negative cells (intercalated cells) were
stained for Cx30 (red) at the apical membrane. Scale is
20 µ m.
Figure 1.6: Regulation of Cx30 expression in the rat inner medullary collecting duct. Kidney sections
stained with Cx30 and imaged with confocal microscopy from rats on A: control diet, B: high salt diet, C:
low salt diet. The expression of Cx30 was up-regulated in rats fed a high salt diet. High salt rat tissue also
exhibited an increase in the luminal membrane vs. cytosolic signal in the inner medullary collecting duct
when compared with low salt and control experimental groups. All images were captured using the same
instrument settings. Scale is 40 µ m.
35
regulate the expression of Cx30 in the rat kidney. Using confocal microscopy and
immunofluorescence, we observed a sharp upregulation of Cx30 expression in the rat
IMCD from high salt-fed rats (Fig. 1.6B), when compared to control (Fig. 1.6A) and low
salt rats (Fig. 1.6C) under the same instrument imaging settings. Particularly in the high
salt kidney, Cx30 labeling was localized at the apical pole of cells in the IMCD (Fig.
1.6B).
Regulation of Cx30 protein expression by dietary salt in rat kidney: an
immunoblotting approach
Using tissue samples from
four rats per experimental group, we
performed Western blotting using a
Cx30 antibody, as well as an actin
antibody as a loading control
(Figure 1.7A-B). After
densitometric analysis, a significant
up-regulation of Cx30 (approximate
molecular weight of 30kD) was
observed in samples from the inner
medulla of high salt diet rats when
compared with control and low salt
groups (Fig. 1.7C). Inner medulla
low salt samples were not
Figure 1.7: Immunoblotting analysis of Cx30 in rat tissue
under various dietary salt conditions. A: A representative blot
of rat tissue blotted with connexin 30 (approximate molecular
weight of 30kD). B: The same blot was stripped and reprobed
with β-actin to demonstrated even loading. C: Densitometric
analysis of immunoblots indicates that Cx30 was significantly
up-regulated in high salt conditions when compared to control
(p<0.01). No significant difference between control and low
salt groups was observed. Shown is average + SE of 4 rats
per experimental group.
36
significantly different from control. While a difference between groups was occasionally
seen in the cortical samples, it was not found to be statistically significant.
Regulation of Cx30 protein expression by salt in RMIC and M1 cell lines
To further investigate the
role that dietary salt plays in the
expression of Cx30, RMIC and M1
cell lines were treated with a
control, high salt-, or mannitol-
supplemented media for 16 hours
before lysis and immunoblotted for
Cx30 (Fig. 1.8A-B). The mannitol-
supplemented media served as
control for osmolality. RMIC cells
showed no significant increase in
Cx30 levels under high salt
conditions and there was no
significant difference between
control and mannitol treatment
groups (Fig. 1.8C). In M1 cell
lines, we observed increased
expression of Cx30 due to increased salt levels and again, the mannitol group showed no
significant increase when compared to control (Fig. 1.8C).
Figure 1.8: Regulation of Cx30 expression in cultured cell
lines by high salt in the medium. A: Confluent RMIC cells
were treated with standard, high salt, or mannitol-
supplemented media and blotted for Cx30. B: Similar
treatments were used for confluent M1 cells. C: Densitometric
analysis of immunoblots showed no significant difference
between the three groups of RMIC cells. High salt treated M1
cells showed a significant up-regulation (p<0.01) when
compared to control cells and there was no significant
difference between control and mannitol treated groups.
Shown is average + SE of 3 samples per experimental group.
37
DISCUSSION
The present study describes the intra-renal localization of a novel gap junctional
protein isoform, Cx30 in three species, with each species showing varying levels of
expression (Table 1.1). The order of Cx30 expression level was rat>>rabbit>mouse. In
the rat, Cx30 was present continuously along the entire distal nephron from the MTAL to
the IMCD with the highest expression in the DCT. In the rabbit and mouse, Cx30 was
restricted to cortical segments of the distal nephron, and the highest level of expression
was observed in intercalated cells
of the CCD. Particularly in the
rabbit and mouse, Cx30 was
localized in the apical cell
membrane.
The recent cloning of Cx30
provided conflicting results
whether or not it is expressed in
the kidney (6, 13). Studies using
Northern-blot techniques
suggested it is undetectable in the mouse kidney (6) or it is present in human kidney in
very low amounts compared to the brain (13). This information turns out to be misleading
for at least two reasons. From the present work, we now know that Cx30 mRNA is
indeed present in the mouse kidney and also that renal expression of Cx30 protein is
regulated by certain factors. So it is possible that in the previous study, down-regulation
Table 1.1: Summary of localization and expression of Cx30 in
rat, rabbit and mouse kidney tissue. Legend: Intensity levels of
Cx30 are as follows: -, absence of signal; +, low; ++,
medium; +++, high.
38
in the particular kidney tissue sample accounted for the low expression levels. Also, it is
now apparent that other species express higher levels of Cx30 in the kidney than the
mouse, which shows Cx30 labeling exclusively in intercalated cells of the collecting
tubule (Fig. 1.5 and Table 1.1). The extremely tiny fraction of intercalated cells compared
to the whole kidney mass can also explain why Cx30 was difficult to detect.
Since immunostaining of gap junction channels usually gives a punctuate pattern
between cells, localization of Cx30 throughout the apical plasma membrane of distal
tubular nephron segments is an unexpected finding. However, this work used specific,
commercially available antibodies (Zymed) that have been well characterized by many
other investigators (20,21,22,24,32). In our hands, immunoblotting using whole kidney
homogenate and the Cx30 antibody produced an intense band around the expected
molecular weight of 30 kD. This single band was within the 25-62 kD range of known
connexin isoforms, indicating that the Cx30 antibody reacted with a single Cx species in
the kidney. Further supporting specificity is that in addition to the apical label, although
extremely rarely, we observed highly scattered and punctuate labeling between cells of
the tubular epithelium, vasculature, and glomerulus, consistent with the antibody
recognizing gap junction proteins.
Polarity, namely the apical location of the continuous plasma membrane labeling
is even more unexpected. Co-localization of Cx30 with a well-known apical membrane
transport protein pendrin (25), further supports its presence in the luminal plasma
membrane (Fig. 1.3A-C). Also, Cx30 staining in intercalated cells was continuous with
and similar to the apical AQP-2 labeling in neighboring principal cells (Fig. 1.5). We
39
used confocal optical sectioning techniques that exclude the possibility of detecting
signals from lateral membranes or tight junctional localization. At this location however,
Cx30 is in contact with only the tubular fluid and not with another cell as usual for
classical gap junctions. This suggests that Cx30 may function as a hemichannel in the
distal nephron. In addition to serving as precursors in the formation of gap junction
channels, connexins may function as transmembrane ion channels (8). These
hemichannels can open under certain conditions and may release small metabolites such
as ATP and NAD
+
, which are involved in paracrine signaling (8).
Despite the obvious differences in Cx30 localization between the rat, rabbit and
mouse, all three species expressed Cx30 in the luminal membrane of distal tubular
epithelium. Cx30 was consistently present in intercalated cells, suggesting that it may be
involved in acid/base homeostasis and tubular ion transport. Increased expression of
Cx30 in the rat may be consistent with the higher number of purinergic P2X and P2Y
receptors along the entire collecting duct system in this species (29), as discussed below.
It remains to be shown what molecules can pass through these hemichannels and
how their gating is regulated. One of the likely candidates is ATP, since other connexins
are known to be involved in ATP release and in purinergic cell-cell signaling in several
cell types, including the glomerular mesangium (34), juxtaglomerular cells (33), and
astrocytes (5). The presence of a luminal ATP channel in the distal nephron would be
very consistent with the purinergic autocrine and/or paracrine regulation of salt and water
reabsorption or perhaps with the pressure-natriuresis phenomenon. Supporting evidence
is the co-localization of AMP degrading enzymes, the ecto-5’nucleotidase and purinergic
40
receptors, particularly at the luminal membrane of intercalated cells (15, 26, 16). These
cells have been suggested to be capable of producing large changes in cell volume (15)
which may stimulate ATP release (26). We find it very intriguing that it is exactly the
intercalated cell apical membrane where Cx30 is localized in all species studied. The
molecular identity of the ATP permeable channels including the maxi-chloride channel
has been controversial (13), but we believe Cx hemichannels are likely candidates.
In order to ascertain a functional role for the Cx30 protein, we examined the
effects of dietary salt on Cx30 expression. The present finding that high salt diet or high-
salt containing media increased the amount of Cx30 in tubular epithelial cells where it is
expressed supports the possible role of Cx30 in the regulation of distal tubular salt
reabsorption (Figs. 1.7 and 1.8). If Cx30 can indeed release ATP, then it may play a role
in the autocrine and paracrine inhibition of sodium reabsorption and stimulation of
chloride secretion in the distal nephron in response to high salt diet and elevations in
tubular fluid flow rate (18). Up-regulation of the Cx30 protein in response to high salt
appears to be specific for salt absorbing epithelial cells. Rat IMCD cells in the native
tissue, or M1 cells in culture that possess mixed principal and intercalated cell phenotype,
showed increased Cx30 expression in response to high salt diet or high salt containing
medium. In contrast, RMIC that also express Cx30, did not regulate this protein.
Additionally, using Western blotting, cortical samples did not show the same regulation
as inner medulla samples. This may be due to the limited amounts of Cx30 in the cortex
relative to the medulla and the limitation of this technique, rather than a lack of regulation
41
by dietary salt. Taken together, these results further support that Cx30 may be involved in
the regulation of renal salt reabsorption.
A possible association between Cx30 function and chloride-secretion is evidenced
by its co-localization with pendrin, an apical Cl-HCO
3
anion exchanger in type B and
non-A, non-B intercalated cells (Fig. 1.2H-I and Fig. 1.3A-C). At present, there are no
data that Cx hemichannels regulate ion transport in the major reabsorbing epithelia (i.e.
gastrointestinal and renal epithelia). Interestingly, in the cochlea of the inner ear, Cx30
also co-localizes with pendrin in the epithelial marginal cells on the lateral wall (12).
These cells also serve as a source of ATP for the endolymphatic fluid that regulates ion
transport of the cochlea via purinergic receptors (12). Furthermore, deletion of pendrin
(Pendred-syndrome) or knocking out Cx30 is characterized by a similar phenotype,
including non-syndromic hearing loss, or deafness (13, 25). The similarities in both Cx30
and pendrin localization and ion-transport regulatory function may indicate that ion
transport in the renal collecting duct system is similarly regulated than in the inner ear.
This interesting association further necessitates the exploration of Cx30 function and its
possible regulation of ion transport in more detail. Because of the lack of selective
inhibitors, Cx30 hemichannel function could be best studied using the available Cx30
knockout mouse model (7).
In summary, we localized a novel connexin isoform, Cx30 in the luminal cell
membrane of select cells in the distal nephron. The presence of this junctional protein at
the apical membrane in contact with only the tubular fluid suggests that it may function
as a hemichannel, permeable to certain molecules that are yet to be identified. Cx30 may
42
be involved in the regulation of distal tubular salt reabsorption which function needs to be
further investigated. Emerging understanding of the gap-junctional proteins and new
tools for their investigation now offer the opportunity to explore the vital role that Cx
molecules may play in the regulation of renal blood flow, the filtration process, renin
release, and tubular reabsorption.
43
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46
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47
CHAPTER 2: THE RENAL EXPRESSION AND REGULATION OF CX30.3
Published in Cell Communication and Adhesion May 2008
Connexin 30.3 is expressed in the kidney but not regulated by dietary salt or high
blood pressure
Fiona Hanner, Marc Schnichels, Qingyi Zheng-Fischhöfer, Li E. Yang, Ildikó Toma,
Klaus Willecke, Alicia A. McDonough, János Peti-Peterdi
CHAPTER 2 ABSTRACT
Several isoforms of connexin (Cx) proteins have been identified in a variety of
tissues where they play a role in intercellular communication, either as the components of
gap junctions or as large, non-selective pores known as hemichannels. This investigation
seeks to identify the localization and regulation of Cx30.3 in mouse, rat and rabbit kidney
using a Cx30.3
+/lacZ
transgenic approach and immunofluorescence. Cx30.3 was detected
in all three species and predominantly in the renal medulla. Both the nuclear lacZ staining
indicative of Cx30.3 expression and indirect immunohistochemistry provided the same
results. Cx30.3 immunolabeling was mainly punctate in the mouse, typical for gap
junctions. In contrast, it showed continuous apical plasma membrane localization in
certain tubule segments in the rat and rabbit kidney, suggesting that it may also function
as hemichannels. In the cortex, Cx30.3 was localized in the intercalated cells of the
48
cortical collecting duct, since the immunoreactive cells did not label for AQP2, a marker
for principal cells. In the medulla, dense Cx30.3 staining was confined to the ascending
thin limbs of the loop of Henle, since the immunoreactive cells did not label for AQP1, a
marker of the descending thin limbs. Immunoblotting studies indicated that Cx30.3
expression was unchanged in response to either high or low salt intake or in
spontaneously hypertensive rats (SHR). Cx30.3 appears to be constitutively expressed in
certain renal tubular segments and cells and its role in overall kidney function remains to
be investigated.
49
INTRODUCTION
The connexin (Cx) family of proteins is comprised of a group of structurally-
related, transmembrane proteins found in a diverse range of cell types. Individual Cx
proteins form hexameric structures known as connexons. In adjacent cells, these
connexons form gap junctions, which participate in intercellular communication between
the two cells by allowing the passage of ions, metabolites and secondary messengers up
to 1-2 kD in size (Spray, 2006). There is also evidence that Cx proteins may remain in the
cell membrane as single hexamers or hemichannels (Ebihara, 2003). If the hemichannel is
localized to a membrane that does not form a junction with another cell, it allows for the
transport of molecules between the cell and the extracellular environment. The opening
of the channel is regulated by several factors, including pH, [Ca
2+
], and metabolic and
mechanical stresses (Trexler et al, 1999; John et al, 1999; Gomes et al, 2005). A number
of studies have indicated that hemichannels maybe capable of releasing ATP into
extracellular fluid and could therefore participate in purinergic signaling (John et al,
1999; Cotrina et al, 1998).
Several Cx isoforms have been identified in the kidney including Cx30, 37, 40,
43, 45 (Arensbak et al, 2001; Barajas et al, 1994; McCulloch et al, 2005; Butterweck et
al, 1994), and Cx40 was recently found to play an important role in renal hemodynamics
(Wagner et al, 2007). Previous studies have identified Cx30.3 mRNA in the kidney
(Tucker et al, 1994), however Cx30.3 protein localization is yet to be established. Cx30.3
has mostly been associated with the skin disease erythrokeratodermia variabilis
(Common et al, 2005). Recent work by Zheng-Fischhöfer utilized a transgenic mouse
50
model where the Cx30.3 gene was replaced with the lac-z reporter gene to study its
expression in the skin, olfactory organs as well as in the kidney (Zheng-Fischhöfer et al,
2007). This study found evidence that Cx30.3 was expressed at least in the inner medulla
of the kidney, specifically in cells of the thin ascending limb of the loop of Henle.
However, detailed renal and intracellular localization of Cx30.3 has not been
investigated.
Here we report that Cx30.3 is expressed at both the mRNA and protein levels in
the kidney of mice, rats and rabbit, and we characterize in detail its renal localization in
these species using both a Cx30.3
+/lacZ
transgenic approach and Cx30.3
immunohistochemistry.
51
MATERIALS AND METHODS
Animals:
New- Zealand White rabbits (500g, Irish Farm, Norco, CA) were maintained on standard
diet with normal water. Sprague-Dawley rats, spontaneously hypertensive rats (SHR) and
C57Bl/6 mice were age and weight matched for all experiments. Transgenic mice with
the coding region of the Cx30.3 gene replaced by the lac-Z reporter gene with a nuclear
localization signal (NLS-lacZ) under the control of the Cx30.3 promoter were developed
and previously described by Zheng-Fischhöfer et al (2007). The physiological parameters
of the SHR animals, including blood pressure measurements have been previously
published (Yang et al, 2007). All animal protocols have been approved by the
Institutional Animal Care and Use Committee at the University of Southern California.
Salt-Adjusted Diet:
Male Sprague-Dawley rats (200g, Harlan, Madison, WI) were fed standard chow
(0.3% NaCl), high salt (TD 92012: 8% NaCl, Harlan Teklad, Madison, WI) or low salt
(TD 90228: 0.01% NaCl, Harlan) rodent diet for one week. Rats fed the high salt diet
also received 0.45% NaCl (w/v) containing drinking water.
M1 Cells:
The M1 cell line was previously characterized (Fejes-Toth et al, 1992) and have a
phenotype representative of both the intercalated and principal cells of the collecting
duct. Cells were purchased from American Type Culture Collection (Manassas, VA).
52
Antibodies:
Rabbit polyclonal anti-Cx30.3 antibodies were purchased from Zymed
Laboratories Inc. (San Francisco, CA). A mouse monoclonal anti-beta actin antibody was
purchased from Abcam (Cambridge, MA) and a mouse monoclonal anti-villin antibody
was purchased from Immunotech (Chicago, IL). The mouse monoclonal anti-aquaporin 2
(AQP2) antibody was kindly provided by Dr. Mark Knepper, is from the original batch of
immunization and was characterized in a previous publication (Nielsen et al, 1993). A
mouse monoclonal antibody against aquaporin 1 (AQP1) was purchased from Novus
Biologicals (Littleton, CO).
Genotyping of Transgenic Mice:
DNA was extracted from mouse tail tips using the ZR Genomic DNA II kit
according to the manufacturer’s protocol (Zymo Research, Orange, CA). 2µ L of purified
DNA was amplified by PCR using a master mix containing Taq polymerase (Platinum
PCR kit, Invitrogen, Carlsbad, CA) and the following primers: Cx30.3 wild-type sense:
5’-GGCCAAGGTTCAAGACCACCTGTG-3’; LacZ sense 5’-
AACGACGGGATCATCGCGAGCCAT-3’Cx30.3; antisense (shared): 5’-
CCCCTCTTCTTGCTCAGGTTGCTG-3’. Primers sequences were previously published
(Zheng-Fishhöfer et al, 2007). The PCR reaction was carried out for 45 cycles of the
following: 94
o
C for 30 seconds, 60
o
C for 45 seconds, and 72
o
C for 60 seconds. The PCR
product was analyzed on a 2% agarose gel stained with ethidium bromide to identify
fragments of approximately 672 bp for the wild-type allele and 359 bp for the mutant
allele.
53
Immunoblotting of Mouse and Rat Tissue:
Mice and rats were anesthetized with 100mg/mL Inactin and kidneys were
perfused retrograde
with ice-cold PBS to remove blood. Slices of whole kidney (mouse)
or cortex (rat) were manually dissected and tissue was homogenized with a rotor-stator
homogenizer in a buffer containing 20mM Tris-HCl, 1mM EGTA, pH 7.0, and a protease
inhibitor cocktail (BD Bioscience, San Jose, CA). Samples were centrifuged at low speed
to pellet cellular debris and supernatant was collected and assayed. 40 µ g of protein were
separated
per lane, along with a protein standard molecular weight marker (Biorad,
Hercules, CA) on a 4-20% SDS-polyacrylamide gel, and then
transferred to a
polyvinyledene difluoride membrane (Millipore, Billerica, MA). After blocking the
membrane in blocking buffer (Li-Cor, Lincoln, NE), immunoblotting was performed with
the rabbit polyclonal
antibodies to Cx30.3 (Zymed) at a dilution of 1:2500 overnight. A
mouse monoclonal antibody to β-Actin (Abcam) (1:2500 dilution) was used for a loading
control for salt-adjusted samples. A mouse monoclonal anti-villin antibody (Immunotech)
(1:1000 dilution) was used as a loading control for SHR samples as has been previously
published (Yang, 2007). Reactivity of the primary antibodies was detected
by either
IR680-labeled goat anti-rabbit or IR800-labeled goat anti-mouse (1:15,000 dilutions, Li-
Cor) secondary antibodies. Blots were imaged and quantified using Odyssey Infrared
Imaging
System (Li-Cor) and accompanying software. The data was then normalized
against the control sample and an average for each group was calculated. Statistical
significance
was tested using an unpaired t-test and data are shown as mean±SE.
54
Lac-Z Staining of Kidney Tissue
Kidneys from Cx30.3
+/lacZ
mice and wild-type littermates were frozen on dry ice,
embedded in Tissue-Tec (Sakura, Zoeterwoude, The Netherlands), sectioned (10–20 mm)
on a cryostat (MICROM HM500 OM) and transferred onto superfrost plus slides
(Menzel, Braunschweig, Germany). Sections were fixed with 0.2% glutaraldehyde in
PBS, rinsed three times in lacZ washing buffer (0.1M phosphate buffer, pH 7.4, 1.25mM
MgCl2, 5mM EGTA, 0.2% Nonidet P-40, 0.01% sodium deoxycholate) and stained in
lacZ substrate buffer overnight (lacZ washing buffer supplemented with 0.4 mg/ml X-Gal
[5-brom-4-chloro-3-indolyb-D-galactopyranoside], 5mM potassium ferrocyanide, 5mM
potassium ferricyanide) at 37
o
C. Sections were then washed in PBS, stained in 0.1%
eosin for 5 min, rinsed in water, and mounted in Entellan (Merck, Darmstadt, Germany).
Immunofluorescence Labeling of Kidney Tissue
Kidneys were fixed in situ by perfusion of 4% paraformaldehyde. Coronal kidney
sections containing all kidney zones were then post-fixed overnight at 4°C in 4%
paraformaldehyde and then embedded in paraffin. Subsequently, 4-µm sections of the
paraffin block were deparaffinized in toluene and rehydrated through graded ethanol. To
retrieve antigens, slides were heated for 2 x 10 min in a microwave with medium heat in
PBS and allowed to cool for 40 minutes. Sections were then incubated for 30 min with
5% normal goat serum in PBS to block non-specific binding. An additional block with
goat anti-rabbit Fab IgG for rabbit tissue (1:100, Jackson ImmunoResearch Laboratories,
West Grove, PA) for 40 min was used to reduce nonspecific
binding with rabbit
polyclonal antibodies. Rat and rabbit sections were then incubated with Cx30.3
55
antibodies overnight at a 1:50 dilution and washed in PBS. Sections were then incubated
with HRP-conjugated goat anti-rabbit IgG and enhanced with Alexa Fluor 594 labeled
tyramide signal amplification according to the manufacturer’s instructions (Molecular
Probes, Eugene, OR). Some rat tissue sections were double-labeled with an anti-AQP2
monoclonal antibody overnight at a 1:50 dilution and the secondary and TSA steps were
repeated as above, except using a HRP-conjugated goat anti-mouse IgG and Alexa Fluor
488 TSA (Molecular Probes). A similar method was employed for mouse sections,
however primary antibody incubation was limited to one hour and TSA amplification was
not utilized. Additionally, some mouse sections were double-labeled with an anti-AQP1
monoclonal antibody for 1 hour (1:100 dilution). All antibody dilutions and incubation
times were experimentally determined. Following a final wash step, all sections were
mounted with Vectashield mounting media containing the nuclear stain DAPI (Vector
Laboratories) and examined with a Leica TCS SP2 confocal microscope. All sections
were labeled in parallel.
Reverse Transcription-PCR
Total RNA was purified from whole mouse kidney samples or confluent M1 cells
using a Total RNA Mini Kit in accordance with manufacturer’s instructions (Biorad,
Hercules, CA). RNA was then quantified using spectrophotometry and reverse-
transcribed to single-stranded cDNA using avian reverse-transcriptase and random
hexamers according to manufacturer’s instructions (Thermoscript RT-PCR system,
Invitrogen). 2µ L of cDNA was amplified using a master mix containing Taq polymerase
(Invitrogen) and the following primers: Connexin 30.3 sense, 5’-
56
GGCCAAGGTTCAAGACCACCTGTG-3’; Connexin 30.3 antisense, 5’-
CCCCTCTTCTTGCTCAGGTTGCTG-3’; β-Actin sense, 5’-
GGTGTGATGGTGGGAATGGGTC-3’, β-Actin antisense 5’-
ATGGCGTGAGGGAGAGCATAGC-3’; each at a final concentration of 200µ M.
Connexin 30.3 and β-Actin oligonucleotides were based on previously published primer
sequences (Zheng-Fischhöfer et al, 2007; McCulloch et al, 2005). The PCR reaction was
carried out for 45 cycles of the following: 94
o
C for 30 seconds, 60
o
C for 30 seconds, and
72
o
C for 30 seconds. The PCR product was analyzed on a 2% agarose gel stained with
ethidium bromide to identify fragments of approximately 672 bp for Connexin 30.3 and
350 bp for β-Actin.
57
Figure 2.1. Detection of connexin 30.3 (Cx30.3)
mRNA (A) and protein (B) in the mouse kidney. A:
mRNA isolated from mouse whole kidney (lane 1) or
from a renal collecting duct cell line (M1, lanes 3, 5)
was amplified by RT-PCR to detect Cx30.3 and β-
actin (lanes 2, 4, 6). Bands approximately at the
predicted sizes of 672bp and 350bp respectively were
observed in both samples. B: Western blotting with
Cx30.3 antibodies produced a band of approximately
37kD in wild-type mouse kidney homogenate, as
indicated by a protein standard (lane 1) (WT, n=4).
Tissue homogenate from Cx30.3 knockout mice (KO,
n=4) failed to produce a signal. C: Incubation with a
β-actin antibody confirmed equal protein loading
amounts.
RESULTS
Detection of Cx30.3 mRNA and Protein in the Kidney
RNA was isolated from whole mouse kidney or from a mouse renal cell line (M1,
mixed phenotype with both the principal
and intercalated cells of the collecting duct)
and using RT-PCR, the presence of Cx30.3
mRNA was detected (Fig. 2.1A). The
amplification of β-actin served as a positive
control. We observed bands of the
predicted size of 672 bp for Cx30.3 and 350
bp for β-actin in both tissue and cell culture
samples. Immunoblotting kidney
homogenate from wild-type mice with
Cx30.3 antibodies produced a band around
37kD, as was expected. Tissue from the
Cx30.3 knockout mouse was used as a
negative control and when probed with the
same antibodies it failed to produce
detectable bands (Fig. 2.1B). β-actin was
used as a control for loading (Fig. 2.1C).
58
Nuclear lacZ reporter gene
expression in transgenic
Cx30.3
+/lacZ
mouse kidneys
The intra-renal
localization of Cx30.3 was
analyzed first using sections of
heterozygous Cx30.3
+/lacZ
adult
mouse kidneys by staining for
nuclear lacZ reporter gene
expression (Fig. 2.2). Wild-
type kidney samples served as
negative controls (Fig. 2.2B,
F). LacZ staining was specific
and most intense in the inner
medulla (IM) followed by the
weakly labeled outer medulla
(OM) (Fig. 2.2A). LacZ
staining in the renal cortex was
very weak (Fig. 2.2A). Higher
magnification revealed that the
dense nuclear lacZ staining in
the IM region was confined to
Figure 2.2. Renal Cx30.3 expression analyzed by staining for
nuclear lacZ reporter gene expression (blue) in heterozygous
Cx30.3+/lacZ adult mouse kidneys (A, C-E). A-B: Cross section
through the whole kidney. A: The most intense lacZ staining was
found in the inner medulla (IM). Labeling was weak in the outer
medulla (OM) while the renal cortex (C) appeared to be devoid of
lacZ staining. B, F: Wild-type kidney samples served as negative
controls. C-F: High magnification of the IM (C), OM (D, F), and
C (E) kidney regions. C: In the IM, cell nuclei of thin tubular
structures stained positive, while cells of the large collecting ducts
(*) were not labeled. D: In the OM, only a few tubular structures
showed lacZ staining (*), but the dominant structure of the region,
the medullary thick ascending limbs (mTAL) were not labeled. E:
In the cortex, only select cells of branching tubular structures,
reminiscent of the collecting duct (*) were positive for lacZ. Other
tubules, the vasculature and glomeruli (G) were negative. F: High
magnification of the OM region in a wild-type kidney shows no
labeling. Bars: 100 µm.
59
long, thin-walled tubular structures reminiscent of the loop of Henle. Most cells of the
large collecting ducts were not labeled (Fig. 2.2C). Only a few, similar thin tubular
structures showed lacZ staining in the OM region (Fig. 2.2D). In the cortex, only a very
few, select cells of branching tubular structures, suggestive of the collecting duct were
positive for lacZ. Other tubules, the vasculature and glomeruli were negative (Fig. 2.2E).
Immunolocalization of Cx30.3 in the Mouse Kidney
Immunofluorescence studies
were conducted in kidney sections
from both wild-type and Cx30.3
knockout mice to confirm the
localization of Cx30.3 in the kidney
and to further test the specificity of
the Cx30.3 antibodies in
immunohistochemical techniques.
Specific labeling was detected in
wild-type mouse sections (Fig.
2.3A), since Cx30.3 knockout
tissues were negative for antibody
labeling (Fig. 2.3C). In the mouse
tissue, the pattern of Cx30.3
immunofluorescence was
predominately punctate and
Figure 2.3. Detection of Cx30.3 in the mouse kidney by
immunofluorescence (red). A, B, D: Specific staining was
observed in wild-type mouse sections labeled with Cx30.3
antibodies. C: Labeling of Cx30.3 knockout mouse tissue
failed to produce a signal. Mostly punctate and cytosolic
labeling was observed in only a few cells in the renal cortex,
in select cells of the cortical collecting duct (CCD)(A).
Occasionally, some staining resembling apical membrane
localization was also observed (B). Other cortical structures
including the proximal tubule (PT) were devoid of staining.
In the renal medulla, dense labeling of thin-walled tubular
structures was found. The Cx30.3-positive tubules (*) did not
label with AQP1 (green), a marker of the descending thin
limb of the loop of Henle (#). Nuclei are stained with 4’, 6’-
diamidino-2-phenylindole (DAPI; blue). Bars: 10 µm.
60
intracellular (Fig. 2.3A). In some tubules, however, Cx30.3 did appear to localize to the
apical plasma membrane (Fig. 2.3B). Consistent with the lacZ expression data above,
majority of the renal cortex, the vasculature, glomeruli and proximal tubules were devoid
of labeling. Only select cells of the cortical collecting duct were Cx30.3-positive (Fig.
2.3A-B). Significantly more structures were immunoreactive in the renal medulla, all of
which appeared as thin-walled tubules with no red blood cells in their lumen. These
tubules were later identified as the
thin ascending limb of the loop of
Henle, based on their lack of labeling
for the water channel aquaporin 1
(AQP1), a marker of the descending
thin limbs (Fig. 2.3D). No other
major structures were labeled in the
renal medulla.
Immunolocalization of Cx30.3 in
the Rat and Rabbit Kidney
Localization of Cx30.3 in the
rat and rabbit kidney was studied by
immunofluorescence of
paraformaldehyde-fixed, paraffin-
embedded kidney sections. The
localization and pattern of Cx30.3
Figure 2.4. Immunofluorescence labeling of Cx30.3 in rat
(A-B) and rabbit (C-D) kidney sections. In the rat cortex
(A), apical membrane localization was apparent in select
cells of the cortical collecting duct (CCD). No other
cortical structures were labeled including the proximal
tubule (PT). B: In the rat outer medulla, the Cx30.3-
positive cells of the CCD (arrow) did not label with AQP2
(green, arrowheads), a marker of the principal cells of the
CCD. Fluorescent labeling was highest in the inner
medulla, and localized in thin-walled tubular structures in
the loop of Henle (C). Higher magnification with DIC
overlay revealed apical membrane labeling in these
tubular structures (*) (D). Most parts of the large
medullary collecting ducts (#) were devoid of staining (C-
D). Nuclei are stained with DAPI (blue). Bars: 10 µm.
61
immunolabeling was essentially identical in these two species. Similar to the mouse,
staining for Cx30.3 was observed mainly in the inner medulla. However, the pattern of
labeling was different from the one observed in the mouse kidney. In addition to some
punctate staining which is typical for gap junctions, majority of the Cx30.3
immunolabeling was continuous and localized to the apical plasma membrane of certain
tubular segments. Like in the mouse, the renal cortex was mainly negative with the
exception of a few, select cells of the cortical collecting duct (Fig. 2.4A). These select
Cx30.3-positive cells of the collecting duct were found in both the cortex and medulla
and were later identified as intercalated cells, since they did not label for the apical
membrane water channel aquaporin 2 (AQP2), a marker of the principal cells of the CCD
(Fig. 2.4B). Cx30.3 immunolabeling was highest
in the inner medulla, and localized in thin-walled
tubular structures (Fig. 2.4C). Higher
magnification with DIC overlay revealed
continuous apical membrane labeling in these
tubules (Fig. 2.4D).
Fig. 2.5 summarizes the localization of
Cx30.3 expression in different renal tubular
segments/cells.
Figure 2.5. Schematic representation of
Cx30.3-expressing nephron segments (bold
line) in both the mouse, rat, and rabbit
kidney. G: glomerulus, PT: proximal
tubule, DTL: descending thin limb, ATL:
ascending thin limb, TAL: thick ascending
limb of the loop of Henle, DT: distal
tubule, CNT: connecting tubule, CCD:
cortical collecting duct, MCD: medullary
collecting duct. Cx30.3 labeling was found
in the ATL and in select (intercalated) cells
of the CCD and MCD.
62
Cx30.3 Expression in Rats with
Salt-adjusted Diets
The effects of changes in
dietary salt intake on Cx30.3
protein expression were examined
in rat kidneys to help determine a
functional role for Cx30.3.
Animals were fed salt-adjusted
diets and cortical kidney tissue
homogenate samples from each rat
experimental group were
immunoblotted for Cx30.3 (Fig.
2.6A). The amount of protein
loaded was confirmed by
concurrently probing the blots
with β-Actin (Fig. 2.6B). Band
intensity was quantified using
densitometric analysis and
normalized against protein loading amounts, as determined by β-actin levels (Fig. 2.6C).
The percent change from control was as follows: high salt: 1.6%; low salt: 16.8%; (n= 4
rats per group). There was no statistically significant difference in Cx30.3 expression
levels between the control group and either of the salt-adjusted diet groups.
Figure 2.6. Immunoblotting analysis of Cx30.3 expression in
the rat kidney under various dietary salt conditions. A:
Representative Cx30.3 blots of rat cortical tissue from
animals fed a control, high, or low salt diet (n=4 rats per
experimental group). B: Blots were probed with β-actin to
demonstrate even loading. Specific bands for Cx30.3 and β-
actin were detected around 37 and 42 kDa, respectively. C:
Densitometric analysis of Cx30.3 expression. No significant
difference between control and high salt (p=0.92) or control
and low salt groups was observed (p=0.09). Shown is mean ±
SE of 4 rats per experimental group.
63
Cx30.3 Expression in Sprague-
Dawley Rats and SHRs
To determine whether
hypertension had an effect on the
expression of Cx30.3 in the rat kidney,
samples from SHR rats were compared
to Sprague-Dawley control samples
(Fig. 2.7A). Villin was used to
demonstrate even protein loading (Fig.
2.7B) before calculating Cx30.3
expression levels using densitometric
analysis (Fig 2.7C). The percent change
in Cx30.3 expression in SHR compared
to SD rats was 5.3% (n=6 per group).
This difference was not statistically
significantly significant.
.
Figure 2.7. Immunoblotting analysis of Cx30.3
expression in the kidneys of normotensive (Sprague-
Dawley, control) and hypertensive (SHR) rats. A: A
representative blot of control and SHR samples probed
with Cx30.3 B: The same blot probed with villin
antibodies to demonstrate even loading. Specific bands
for Cx30.3 and villin were detected around 37 and 92
kDa, respectively. C: Densitometric analysis of Cx30.3
expression. No statistically significant difference in
Cx30.3 expression was observed between the two
groups (p=0.85). Shown is mean ± SE of 6 rats per
experimental group
64
DISCUSSION
Here we report on the expression and localization of Cx30.3 in the kidneys of
mice, rats, and rabbits. The protein was found in the kidney predominantly in the renal
medulla, with the same level of expression and localization in each species (Fig. 2.5).
Cx30.3 immunolabeling was mainly punctate in the mouse which pattern of labeling was
expected, since connexins predominantly form gap junctions. In contrast, it showed
continuous apical plasma membrane localization in certain tubule segments in the rat and
rabbit kidney. Since this part of the cell membrane interfaces with the lumen of the tubule
and is non-junctional, these findings raise the possibility that that Cx30.3 may also
function as hemichannels.
Both the Cx30.3
+/lacZ
transgenic and immunohistochemical approaches found
essentially the same results, confirming their specificity. Both techniques identified the
ascending thin limb of the loop of Henle and the intercalated cells of the collecting duct
as the only two cell types within the kidney which express Cx30.3 (Figs. 2.2, 2.3, and
2.4). Specificity of the Cx30.3 antibody was also confirmed by the lack of
immunoreactive signals in Cx30.3 knockout kidneys using either western blotting or
immunohistochemistry (Figs. 2.1 and 2.3).
The apical membrane localization was found in the loop of Henle and the
collecting duct, tubular segments that are both active in salt and water reabsorption,
suggesting that Cx30.3 may contribute to this function. Cx30.3 was consistently present
in intercalated cells, suggesting that it may be involved in acid/base homeostasis. To help
ascertain a renal function for Cx30.3, kidney homogenate was first obtained from rats
65
treated with high- or low-salt diets. Neither group showed a significant difference from
control in the level of Cx30.3 expression, which was surprising, since Cx30.3 expressing
nephron segments, the loop of Henle and the collecting duct control salt reabsorption.
This also marked a deviation from the renal expression seen with Cx30 (McCulloch et al,
2005),
which showed similar expression patterns, but did show altered expression due to
altered salt-intake. Since the same changes to dietary salt induced changes in Cx30
expression, it appears that the unaltered expression of Cx30.3 is not due to a lack of
sensitivity in our approach. We also examined kidney samples from SHRs since other
groups have proposed a role for other connexins in hypertension and have noted changes
in expression levels of some Cxs as a result (Rummery et al, 2002; Li et al, 2002,
Wagner et al, 2007). Again, no significant deviation from the levels of Cx30.3 expression
found in the control rat was observed. These results however do not exclude the
possibility of functional changes in Cx30.3. One limitation of this technique is that whole
cell immunoblots cannot detect changes in synthesis, processing and transport of proteins
within organelles. It is possible that changes within these connexin pools could result in
functional changes in Cx30.3 at the plasma membrane.
While it is easy to suggest that Cx30.3 plays a role in renal physiology based on
its localization, determining the mechanisms through which this occurs remains a
challenge. One possible mechanism of action is through purinergic signaling, which is
implicated in several renal functions, including ion transport (Unwin et al, 2003). For
example, luminal levels of ATP are known to be high in the proximal part of the nephron
including the loop of Henle (Vekaria et al, 2006). Given the significant amount of
66
nucleotidase activity found along the apical membrane (Le Hir et al, 1993; Leipziger et
al, 2003), it follows that ATP must be secreted along the luminal membrane to produce
detectable levels in the tubular fluid (Vekaria et al, 2006). In CCDs, ATP has been shown
to inhibit the SK potassium channel of principal cells (Lu et al, 2000). Since purinergic
signaling occurs in an autocrine/paracrine manner (Schwiebert et al, 2001), it follows that
ATP would be released from neighboring intercalated cells. However, the exact
mechanism of ATP release in the tubules has not yet been elucidated, although several
candidates exist (Schwiebert et al, 2001). Among them are connexin hemichannels,
which have been shown to release ATP in other cell types (Gomes et al, 2005; Cotrina et
al, 1998). Given its localization in the ascending thin limb of the loop of Henle and the
collecting ducts, Cx30.3 hemichannels could be involved in ATP release there.
Another criterion in determining whether Cx30.3 hemichannels are involved in
renal physiology is the regulation of their conformation. It is clear that hemichannels
cannot be constitutively open, but instead must be gated. If hemichannels do function in
the kidney, it needs to be determined whether they can be opened under the (patho)-
physiological conditions. Several factors have been proposed to open hemichannels,
including metabolic and mechanical stress (John et al, 1999; Gomes et al, 2005). Both
factors are potentially at work in the kidney and it is interesting to speculate what
influence they may have in the regions where we have found Cx30.3. The intercalated
cells of the CCD have been shown to respond to mechanical stress from increased flow
with changes in the [Ca
2+
]
i
and it is known that purinergic signaling can increase [Ca
2+
]
i
through P2Y receptors (Liu et al, 2003).
Luminal release of ATP through connexin
67
hemichannels in intercalated cells may provide the link between increased flow in the
CCD and increased intracellular Ca
2+
. Another finding that suggests hemichannel versus
gap junction function of Cx30.3 in the collecting duct intercalated cells is that electron
microscopy was not able to detect classical gap junctions in this part of the nephron (Liu
et al, 2003). The presence of a luminal ATP channel in the loop of Henle and the
collecting duct would be very consistent with the purinergic autocrine and/or paracrine
regulation of salt and water reabsorption or perhaps with acid/base homeostasis.
Supporting evidence is the co-localization of ATP degrading enzymes, the ecto-
5’nucleotidase and purinergic receptors, particularly at the luminal membrane of
intercalated cells (Le Hir et al, 1993, Leipziger et al, 2003, Schwiebert et al, 2001).
These cells have been suggested to be capable of producing large changes in cell volume
(Le Hir et al, 1993) which may stimulate ATP release (Schwiebert et al, 2001). We find
it very intriguing that it is exactly one of the only two cell types in the kidney, the
intercalated cell apical membrane where Cx30.3 was localized in all species studied. The
unusual and interesting apical membrane localization of Cx30.3 and the overlap of its
expression with those of ATP degrading enzymes further necessitate the exploration of
Cx30.3 function in renal (patho)physiology in future work. Because of the lack of
selective inhibitors, Cx30.3 hemichannel function could be best studied using the recently
established Cx30.3 knockout mouse model (Zheng-Fischhöfer et al, 2007).
In the medulla, a previous study identified Cx30.3 in the ascending thin limb
(Zheng-Fischhöfer et al, 2007). The rhythmic contractions of the pelvic muscles which
cause periodic squeezing (compressions) of the renal pelvis could serve as mechanical
68
stimulus for the opening of Cx30.3 hemichannels strongly expressed in the ascending
thin limb. Tubuloglomerular feedback-induced tubular flow oscillations are also
transmitted to this nephron segment and beyond (Kang et al, 2006) which could also
provide mechanical stimulus for hemichannel opening.
In summary, this study described the detailed localization of Cx30.3 in mouse, rat,
and rabbit kidneys. Cx30.3 was found in the ascending thin limb of the loop of Henle and
in the intercalated cells of the collecting duct. Labeling was mostly punctate in the mouse
(typical of gap junction localization), however apical cell membrane localization was
evident in the rat and rabbit kidneys which may suggest hemichannel function. Cx30.3
expression was unchanged in response to either high or low salt intake or in
spontaneously hypertensive rats (SHR). Cx30.3 appears to be constitutively expressed in
certain renal tubular segments and cells and its role in overall kidney function remains to
be resolved.
69
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72
CHAPTER 3: THE EXPRESSION OF CX45 IN THE
JUXTAGLOMERULAR APPARATUS AND ITS ROLE IN CALCIUM
SIGNALING, RENIN REGULATION, AND BLOOD PRESSURE
HOMEOSTASIS
Published in the American Journal of Physiology: Regulatory, Integrative, and
Comparative Physiology August 2008
Connexin45 is expressed in the juxtaglomerular apparatus and is involved in the
regulation of renin secretion and blood pressure
Fiona Hanner, Julia von Maltzahn, Stephan Maxeiner, Ildiko Toma, Arnold Sipos,
Olaf Krüger, Klaus Willecke, János Peti-Peterdi
CHAPTER 3 ABSTRACT
Connexin (Cx) proteins are known to play a role in cell to cell communication via
intercellular gap junction channels or transiently open hemichannels. Previous studies
have identified several connexin isoforms in the juxtaglomerular apparatus (JGA), but the
vascular connexin isoform Cx45 has not yet been studied in this region. The present work
aimed to identify in detail the localization of Cx45 in the JGA and to suggest a functional
role for Cx45 in the kidney using conditions where Cx45 expression or function was
altered. Using mice expressing LacZ coding DNA under the control of the Cx45
promoter, we observed β-galactosidase staining in cortical vasculature and glomeruli,
with specific localization to the JGA region. Renal vascular localization of Cx45 was
73
further confirmed using conditional Cx45-deficient (Cx45fl/fl:Nestin-Cre) mice which
express EGFP instead of Cx45 only in cells that during development expressed the
intermediate filament nestin. EGFP fluorescence was found in the afferent and efferent
arteriole smooth muscle cells, renin producing JG cells and in the extra- and
intraglomerular mesangium. Cx45fl/fl:Nestin-Cre mice exhibited increased renin
expression and activity, as well as higher systemic blood pressure. The propagation of
mechanically-induced calcium waves was slower in cultured vascular smooth muscle
cells (VSMCs) from Cx45fl/fl:Nestin-Cre mice as well as in control VSMC treated with a
Cx45 blocker. VSMCs allowed the cell-to-cell passage of the gap junction permeable dye
lucifer yellow and calcium wave propagation was not altered by addition of the ATP
receptor blocker suramin, suggesting that Cx45 regulates calcium wave propagation via
direct gap junction coupling. In conclusion, the localization of Cx45 to the JGA and
functional data from Cx45fl/fl:Nestin-Cre mice suggest that Cx45 is involved in the
propagation of JGA vascular signals and in the regulation of renin release and blood
pressure.
74
INTRODUCTION
The juxtaglomerular apparatus (JGA) is an important anatomical component of
the renin-angiotensin system (RAS) and it plays a major role in regulating body fluid and
electrolyte homeostasis and blood pressure. The JGA consists of a tubular component
(the macula densa, MD), the extraglomerular mesangium, and a vascular component
which includes the terminal part of the afferent arteriole containing the renin producing
juxtaglomerular (JG) cells. Two major regulatory functions are performed by the JGA:
the high distal tubular [NaCl]-induced afferent arteriolar vasoconstriction
(tubuloglomerular feedback, TGF) and the low tubular [NaCl]-induced renin release.
Several connexin (Cx) isoforms have already been studied in the JGA. Cx37,
Cx40, and Cx43 have all been identified in the endothelium of the proximal afferent
arteriole (2, 17). Only Cx43 has been found within the efferent arteriole endothelium
(42). JG cells express Cx37 and Cx40 and these Cxs are also found in the intra- and
extraglomerular mesangial cells (42). Cx43 is also expressed within the glomerulus (35).
These 3 isoforms, along with Cx45, are considered to be the predominant vascular
connexins (39), yet Cx45 has not been studied within the renal vasculature and JGA.
Cx45 is expressed in several organ systems during embryogenesis and is essential
for the proper development of the cardiac and vascular system (19, 20). In the adult
animal, expression is reduced (1, 7) but is known to continue both in conductive
cardiomyocytes (14) and distinct neuronal subpopulations in the adult brain (33).
Although Cx45 has been previously found in the kidney, these studies focused on its
expression in the developing kidney and in kidney derived cell lines (5, 32). In the mature
75
kidney however, exact and detailed cellular localization of Cx45 was hampered by the
lack of specific detection methods. Recently, transgenic mouse techniques have been
developed that provide not only a localization tool but also tissue or cell-specific deletion
of connexins (19, 23). To aid in localization of Cx45 in all organ systems, a transgenic
mouse in which one copy of the Cx45 coding DNA is replaced by the LacZ reporter
coding DNA (Cx45
+/-
) was developed and studied (19). Additionally, since general
deletion of Cx45 proves to be lethal during embryogenesis (19, 20), the Cre/loxP
technique was recently used to generate a mouse with a Cx45 gene deletion that is
restricted to cells expressing the protein nestin during development (23). This method has
previously been used to achieve conditional expression of a variety of genes in the kidney
(4, 15). By creating a mouse with the Cre recombinase gene under the control of kidney-
specific promoters, gene deletions have been produced in renal structures including the
vasculature and glomeruli, both of which express nestin during development(8).
The present work details the localization of Cx45 in the JGA of adult mouse
kidney using genetic techniques. We then went on to identify its relevance in renal
(patho)physiology. Our data show that Cx45 is expressed in the vascular component of
JGA and is involved in the propagation of JGA vascular signals and in the regulation of
renin release and blood pressure.
76
MATERIALS AND METHODS
Animals
C57BL/6 mice were bred in house and were fed standard diets (Harlan Teklad,
Madison WI) and provided drinking water ad libitum or water supplemented with
500mg/L captopril for one week . Cx45
+/+
and Cx45
+/-
mice, where the coding region of
the Cx45 gene was replaced with the β-galactosidase (LacZ) reporter gene, were
previously generated and described (17). Cx45fl/fl:Nestin-Cre mice were kindly provided
by Dr. Marla Feller at the University of California in San Diego and had previously been
described (23). The Cx45fl/fl:Nestin-Cre mice were developed from two mouse strains:
Cx45fl/fl mice which were generated in C57BL/6 mice and backcrossed to C57BL/6
mice for at least 3 generations and Nestin-Cre mice which were generated in B6SJLF2
mice and backcrossed to C57BL/6 mice for at least 6 generations (The Jackson
Laboratory, Bar Harbor, ME), resulting in approximately 87% of the C57BL/6 genetic
background in Cx45fl/fl:Nestin-Cre mice (23). In these mice, the Cx45 coding DNA has
been replaced by the EGFP reporter gene in cells which expressed nestin during
development by way of the Cre/loxP site specific recombination system. Due to this
recombination, EGFP gene transcription comes under the control of the Cx45 promoter.
Therefore EGFP staining will only occur in cells that expressed nestin during
development and that normally express Cx45. All animal protocols have been approved
by the Institutional Animal Care and Use Committee at the University of Southern
California.
77
β β β β-Gal staining
Kidneys from Cx45
+/+
and Cx45
+/-
mice were frozen on dry ice, embedded in
Tissue-Tec (Sakura, Zoeterwoude, The Netherlands), sectioned (10-20 µm) on a cryostat
(MICROM HM 500 OM) and transferred onto superfrost plus slides (Menzel,
Brunswickm Germany). Sections were fixed with 0.2% glutaraldehyde in 0.1 M
phosphate buffered saline (PBS), rinsed three times in LacZ washing buffer (0.1 M
phosphate buffer, pH 7.4, 1.25 mM MgCl
2
, 5 mM EGTA, 0.2% Nonidet P-40 and 0.01%
sodium doxycholate) and stained in LacZ substrate buffer (LacZ washing buffer
supplemented with 0.4 mg/ml X-Gal [5-brom-4-chloro-3-indoly-β-D-glactopyranoside],
5 mM potassium ferrocyanide, and 5 mM potassium ferricyanide) overnight at 37°C.
Sections were then washed in phosphate buffered saline (PBS), stained in 0.1% eosin for
5 minutes, rinsed in water, and mounted in Entellan (Merck, Darmstadt, Germany).
Antibodies
The rabbit polyclonal renin antibodies for immunofluorescence studies were
provided by Dr. Joël Menard (INSERM, Paris, France), while rabbit polyclonal renin
antibodies for immunoblots were a kind gift from Dr. Tadashi Inagami (Vanderbilt
University, Nashville, TN). Both renin antibodies were characterized in previous
publications (6, 25). Rabbit polyclonal anti-Cx45 was kindly provided by Dr. Ulrike
Janssen-Bienhold, University of Oldenburg, Germany, and was previously characterized
(10).
78
Immunofluorescence labeling of Cx45fl/fl:Nestin-Cre mouse kidney tissue
Kidneys were fixed in situ by perfusion with periodate-lysine-paraformaldehyde
(PLP). Coronal kidney sections were incubated overnight in PLP at 4
o
C before overnight
cryoprotection in 2.3 M sucrose. Tissue was embedded in O.C.T. embedding medium
(Sakura, Torrance, CA) over dry ice. Thin sections were cut on a Leica CM cryostat
(Leica Microsystems, Bannockburn, IL). Sections were fixed with 4% paraformaldehyde
for 10 min, permeabilized for 10 min with 0.1% Triton-X in PBS, and subsequently
incubated in a solution of 5% normal goat serum in PBS for 30 min to block non-specific
binding. Additionally, some sections were also probed with antibodies against renin, at a
dilution of 1:100 for 1 hour, followed by incubation with secondary Alex Fluor 594 goat-
anti rabbit antibodies (Invitrogen, Carlsbad, CA) at a 1:500 dilution for 1 hour. Following
a final wash step, all sections were mounted with Vectashield mounting media containing
the nuclear stain DAPI (Vector Laboratories, Burlingame, CA) and examined with a
Leica TCS SP2 confocal microscope.
Measuring plasma renin activity
Renin activity of mouse plasma was measured using a fluorescence resonance
energy transfer (FRET)-based 5-FAM-conjugated renin substrate (Anaspec, San Jose
CA) and a cuvette-based spectrofluorometer (Quantamaster-8, PTI, Birmingham, NJ). In
the FRET-peptide’s native state, the fluorescence of 5-FAM is quenched by QXL-520.
Upon cleavage of the substrate into two fragments by renin, 5-FAM will fluoresce. A
similar method utilizing a FRET-based EDANS-conjugated renin substrate has been
described before (16, 35). Briefly, 0.33 µ M of the renin substrate in Krebs ringer (pH 7.4)
79
was loaded into the cuvette and heated to 37
°
C. After taking a baseline reading, 30 µ L of
mouse plasma were mixed with the renin substrate in the 37
°
C chamber and the emitted
fluorescence signal as an index of ANG I generation was measured at 520 nm in response
to excitation at 490 nm for a period of 800 sec. The initial rate of the increase in 5-FAM
fluorescence was then analyzed as a measure of renin activity using the FeliX32 software
(PTI).
Blood pressure measurement
C57BL/6 mice and Cx45fl/fl:Nestin-Cre mice were anesthetized with a
combination of Inactin
(100 mg/kg body wt) and Ketamine (100 mg/kg body wt)
intraperitoneally. To measure systemic blood pressure, a cannula was inserted into the
left carotid artery and using an analog single-channel transducer signal conditioner and
transducer, data was collected using data acquisition system QUAD-161 (World
Precision Instruments, Sarasota, FL). Statistical significance was tested using an unpaired
t-test and data are shown as mean + SE.
Isolation and culture of vascular smooth muscle cells from mouse kidneys
Kidneys were collected from C57BL/6 and Cx45fl/fl:Nestin-Cre mice
anesthetized with 100 mg/kg body weight Inactin. The terminal afferent arteriole was
manually dissected on ice under a microscope from sagittal slices of kidney in DMEM
culture medium containing 3% fetal bovine serum (FBS) (Invitrogen). The afferent
arteriole was cut into short segments and transferred to tissue culture dishes containing
circular glass coverslips. Explants gave rise to vascular smooth muscle cells (VSMC)
approximately 2-3 days after attachment. Isolated VSMC were then grown to 90%
80
confluence on the glass coverslips in the following media: DMEM with 25 mM D-
glucose with the addition of 3.7 g NaHCO
3
, 20% FBS, and 1% Penicillin-Streptomycin.
Cells were bathed in a modified Krebs-Ringer HCO
3
buffer during dye incubation and
subsequent experiments. This buffer was composed of: 115 mM NaCl, 5 mM KCl, 25
mM NaHCO
3
, 960 µ M NaH
2
PO
4
, 240 µ M Na
2
HPO
4
, 1.2 mM MgSO
4
, 2 mM CaCl
2
, 5.5
mM D-glucose, and 100 µ M L-arginine. All solutions were adjusted to pH7.4 using HCl
and NaOH.
Immunoblotting of C57BL/6 and Cx45fl/fl:Nestin-Cre mouse and rat kidneys
Mice were anesthetized with 100mg/kg Inactin and kidneys were perfused with
ice-cold PBS to remove blood. Tissue was then homogenized with a rotor-stator
homogenizer in a buffer containing 20mM Tris-HCl, 1mM EGTA, pH 7.0, and a protease
inhibitor cocktail (BD Bioscience, San Jose, CA). Samples were centrifuged at low speed
to pellet cellular debris and supernatant was collected and assayed. Forty micrograms of
protein were run
per lane, separated on a 4-20% SDS-polyacrylamide gel (Biorad,
Hercules CA), and then
transferred to a polyvinyledene difluoride membrane (Millipore,
Billerica, MA). After blocking the membrane in blocking buffer (Li-Cor, Lincoln, NE)
blots were probed with rabbit polyclonal
antibodies to Cx45 at a dilution of 1:1000
overnight. Reactivity of the primary antibodies was detected
with IR680-labeled goat
anti-rabbit antibodies (1:15,000 dilutions, Li-Cor). Blots were imaged using the Odyssey
Infrared Imaging
System (Li-Cor) and accompanying software. The blot was reprobed
with a mouse monoclonal antibody to GAPDH (Ambion, Austin, TX) at a dilution of
1:4000 for 1 hour (Santa Cruz Biotechnology, Santa Cruz, CA) to test for protein loading
81
and quality of transfer. Labeling was detected and imaged with an IR800-labeled goat
anti-mouse antibody as described above (Li-Cor system).
Immunoblotting of vascular smooth muscle cells
Afferent arteriole VSMCs isolated from C57BL/6 and Cx45fl/fl:Nestin-Cre mice
were grown to confluence in plates as described above. Cells were removed from the
plates by scraping and lysed using CellLytic-M lysis buffer (Sigma) according to
manufacturer’s instructions. Protein concentration was assayed by a modified Bradford
method (Quick Start Bradford protein assay, Biorad). Seven microgram samples were
blotted and analyzed for Cx45 and GAPDH as described above.
RT-PCR of vascular smooth muscle cells
Total RNA was purified from confluent afferent arteriolar vascular smooth
muscle cells derived from C57BL/6 and Cx45fl/fl:Nestin-Cre mouse kidneys using a
Total RNA Mini kit in accordance with manufacturer’s instructions (Bio-Rad). RNA was
then quantified using spectrophotometry and reverse-transcribed to single-strand cDNA
using avian reverse transcriptase and random hexamers according to manufacturer’s
instructions (Thermoscript RT-PCR systems, Invitrogen). Two microliters of cDNA were
amplified using a master mix containing Taq polymerase (Invitrogen) and the following
primers: Cx37 forward: 5’-GGC TGG ACC ATG GAG CCG GT-3’, Cx37 reverse: 5’-
TTT CGG CCA CCC TGG GGA GC-3’; Cx40 forward: 5’-CCA GCT TTT AAT GCC
GAG AG-3’, Cx40 reverse: 5’-CCA GCT TTT AAT GCC GAG AG-3’; Cx43 forward:
5’-TAC CAC GCC ACC ACT GGC-3’, Cx43 reverse: 5’-AAT CTC CAG GTC ATC
AGG-3’; β-actin sense: 5’-GGTGTGATGGTGGGAATGGGTC-3’, β-actin anti-sense:
82
5’-ATGGCGTGAGGGAGAGCATAGC-3’. All primer sequences were based on
previous publications (36, 24).
Measurement of calcium wave propagation in vascular smooth muscle cells
Coverslips containing the VSMC monolayer were mounted to a chamber of the
Leica TCS SP2 confocal microscope system and imaged in the absence of any dyes to
establish any EGFP fluorescence (excitation at 488 nm, emission at 520 ± 20nm).
VSMCs were then loaded for 20 minutes with the ratiometric calcium dyes Fluo-4 AM
(excitation at 488 nm, emission at 520 ± 20nm) and Fura red AM (excitation at 488 nm,
emission at >600nm) (Invitrogen) at a final concentration of 1 µ M and 5 µ M
respectively. A transmitted light detector and differential interference contrast imaging
(DIC) was used to visualize the position of the pipette prior to and during mechanical
stimulation. All experiments were performed using the same instrument settings and data
acquisition and analysis were done using the Leica LCS imaging software (LCS
2.61.1537). Calcium wave velocity was calculated using the formula: velocity =
distance/time, where distance was defined as the length between the point of mechanical
stimulation and the center of a cell with increased [Ca
2+
]
i
. For each experimental group,
n=6. Statistical significance was calculated by a one-way ANOVA analysis followed by
Dunnett’s post-hoc comparison with data shown as mean + SE.
Mechanical stimulation of vascular smooth muscle cells
A single VSMC of the monolayer was stimulated with a glass micropipette
(Drummond Scientific Company, Broomall, PA) pulled to 2-3 µ m diameter using a
micropipette puller (PP-830, Narishige, Japan). A micromanipulator (ROE-200, Sutter
83
Instruments, Novato, CA) was used to position and lower the micropipette to contact the
monolayer.
Pharmacological treatment of vascular smooth muscle cells
In cell calcium wave experiments, the gap junction uncoupling agent 18α-
glycyrrhetinic acid (18α-GA, 25 µ M) (Sigma-Aldrich, St. Louis, MO) was used as a non-
specific gap junction inhibitor. To specifically block Cx45 in the same experiments, a
Cx45 gap mimetic peptide of sequence QVHPFYVCSRLPCPHK (amino
acids 202-217)
was synthesized (USC/Norris Cancer Center DNA Core Facility, Los Angeles, CA)
based on the work of Li and Simard (21). Cell monolayers were incubated with the gap
mimetic peptide at a concentration of 500 µΜ for 3 hours at 37°C, as previously
described. The non-selective purinergic receptor antagonist suramin was applied to cell
monolayers at a concentration of 50 µ M for 10 minutes at 37°C.
Dye spreading assay
Coverslips containing a confluent VSMC monolayer were mounted to a chamber
of the Leica confocal microscope system and bathed with 1mL modified Krebs-Ringer
HCO
3
buffer. Hoechst 33342 (10µ M, Invitrogen) was added to the bath prior to the
experiment to identify nuclei. A single cell within the VSMC monolayer was then
injected with a micropipette loaded with Lucifer Yellow (700 µ M, Invitrogen) and the
dye was allowed to diffuse to adjacent cells for five minutes. Images were recorded every
15 seconds. Both Hoechst 33342 (emission between 400-450nm) and Lucifer Yellow
(emission > 550nm) were excited using two-photon excitation at 800nm by a MaiTai
laser (Spectra-Physics, Mountain View, CA).
84
RESULTS
LacZ localization in the renal cortex of Cx45
+/-
mice
Kidneys from Cx45
+/-
mice, where one copy of the Cx45 gene is replaced by the
LacZ reporter gene were sectioned
and stained with X-Gal, in order to
determine Cx45 renal expression.
Expression of a transgenic reporter
gene was used for localization
instead of immunohistochemistry,
since specific antibodies were not
available. LacZ staining was found
in the renal cortex, specifically in
blood vessels and glomeruli (Fig
3.1A). While these results confirm
previously published data on the
gross renal localization of Cx45
(19), Cx45 expression within these
structures was not investigated in
earlier studies. Using higher power
magnification, we were able to
further elucidate the localization of Cx45 in the JGA. Both afferent and efferent arterioles
were positive for LacZ, as well as the extra- and intraglomerular mesangium (Fig 3.1B
Figure 3.1: Localization of Cx45 transcripts in Cx45
+/-
mouse kidney sections by X-Gal staining. A: β-gal was
detected in the renal cortex, predominately in vasculature
(arrow) and glomeruli (arrowhead). B and C: Within the
juxtaglomerular apparatus (JGA), β-gal staining was
observed in the afferent (AA) and efferent arterioles (EA) as
well as the extra- and intraglomerular mesangial cells
(arrows). D: No β-gal staining was detected in sections from
Cx45
+/+
mouse kidneys; G: glomerulus. Bar = 50µ m.
85
and 3.1C). No LacZ labeling was detected in sections stained in parallel from Cx45
+/+
mice, which served as a negative control for endogenous galactosidase activity. (Fig
3.1D). These results indicate that Cx45 is expressed throughout the vascular components
of JGA.
EGFP localization in the JGA of Cx45fl/fl:Nestin-Cre mice
To provide a further line of evidence that Cx45 is expressed in the JGA, the
expression of EGFP in kidney sections of Cx45fl/fl:Nestin-Cre mice was analyzed (Fig
3.2). In these mice, the coding region of Cx45 was flanked by loxP sites. Additionally,
these mice expressed cre recombinase in cells expressing the intermediate filament
protein nestin during development. In the nestin-positive cells, cre recombinase excised
the floxed Cx45 coding region, leaving the EGFP reporter gene under the control of the
Cx45 promoter. Therefore EGFP expression indicates Cx45 expression only in cells that
express nestin during development and express Cx45 in the adult.
At the JGA, strong EGFP signals were observed in vascular smooth muscle cells
of both the afferent and efferent arterioles (Fig 3.2A-B), and in extraglomerular and
Figure 3.2: Detection and localization of EGFP in Cx45fl/fl:Nestin-Cre mouse kidney
sections. EGFP staining is controlled by the Cx45 promoter only in cells that expressed
nestin during development. A: EGFP (green) was detected in the afferent and efferent
arterioles (AA, EA) and in the extraglomerular mesangial cells. G: glomerulus; Arrow:
macula densa. B: Sections were labeled for renin (red) in a consecutive serial section. EGFP
was co-localized to the renin positive region of the AA. C: EGFP was also found in the
extraglomerular mesangium (arrow). Bars = 20µ m
86
intraglomerular mesangial cells (Fig 3.2C). To verify JGA localization, a consecutive
section was co-labeled with renin antibodies (Fig 3.2B). The renin labeling was
consistent with the anticipated JGA location in the afferent arteriole. As predicted, the
EGFP signal overlapped with the renin labeling confirming the expression of Cx45 in
renin producing JG cells. EGFP labeling was not detected in any endothelial cells.
Effects of Cx45 on renin expression and blood pressure
Since Cx45 was expressed in the JGA, it may play a role in renin expression and
blood pressure regulation. Samples of whole kidney homogenate from Cx45fl/fl:Nestin-
Cre mice (n=4) and C57BL/6 mice (control) (n=5) were run on SDS-PAGE gels,
transferred and blotted for renin (Fig 3.3A). Densitometry analysis (Fig.3. 3B) revealed a
significant up-regulation (approximately 50%) of renin expression in Cx45fl/fl:Nestin-
Cre mice when compared to C57BL/6 mice (p<0.05).
Using spectrofluorometry, plasma renin activity was analyzed in C57BL/6 and
Cx45fl/fl:Nestin-Cre mice (Fig. 3.3C). Plasma samples were mixed with a fluorogenic
renin substrate and the emitted FRET-signal (representative of the generation of AngI)
was detected. Renin activity (AngI generation) was plotted as a function of time and the
initial activity was quantified. Plasma renin activity was significantly higher by
approximately 70% in Cx45fl/fl:Nestin-Cre mice compared to control mice (control: 121
± 10; Cx45fl/fl:Nestin-Cre: 204 ± 24, n=5, p<0.05). This increase in plasma renin activity
was comparable to the increase observed in captopril-treated mice (194 ± 14, p<0.05 vs.
control, p>0.05 vs. Cx45fl/fl:Nestin-Cre).
87
The blood pressure of control and Cx45fl/fl:Nestin-Cre was measured by pressure
transducer catheterization. C57BL/6 mice had an average mean arterial blood pressure
(MAP) of 90 ± 2 mmHg (n=3), while Cx45fl/fl:Nestin-Cre had an average MAP of 116 ±
5 mmHg (n=3) (Fig. 3D). The 28% increase in MAP in Cx45fl/fl:Nestin-Cre mice was
found to be significant (p<0.05).
Figure 3.3: Renal renin expression, plasma renin activity, and systemic blood pressure in C57BL/6 and
Cx45fl/fl:Nestin-Cre mice. A: C57BL/6 (control, n=5) and Cx45fl/fl:Nestin-Cre (n=4) kidney homogenate
samples were blotted with renin antibodies. B: Densitometric analysis of immunoblot indicated that the
expression of renin in Cx45fl/fl:Nestin-Cre mice was significantly higher than that of C57BL/6 mice
(*p<0.05). C: Plasma renin activity was measured in real-time using a FRET-based fluorogenic renin
substrate. Renin activity was significantly higher in Cx45fl/fl:Nestin-Cre mice compared to C57BL/6 mice
and this increase was comparable to the increase observed in captopril-treated mice (*p<0.05, n=5 per
group). D: Mean arterial blood pressure was significantly increased by approximately 30% in
Cx45fl/fl:Nestin-Cre mice (*p<0.05, n=3 per group). Values are mean+SE.
88
Characterization of vascular
smooth muscle cells from
C57BL/6 and
Cx45fl/fl:Nestin-Cre mice
VSMCs derived from
Cx45fl/fl:Nestin-Cre mice
were positive for EGFP (Fig
4A), indicating that the Cx45
coding region was excised in
these cells. Under the same
microscope and laser settings,
no EGFP signal was observed
in VSMCs cultured from
C57BL/6 (control VSMC)
mice (Fig 3.4B).
In order to quantify the
excision of the Cx45 coding
region from Cx45fl/fl:Nestin-
Cre VSMCs, protein extracts
from C57BL/6 (control) and
Cx45fl/fl:Nestin-Cre
(Cx45fl/fl) VSMCs were run
Figure 3.4: Characterization of vascular smooth muscle cell
(VSMC) primary cultures from C57BL/6 and Cx45fl/fl:Nestin-Cre
mice. A: EGFP expression (green) was detected in VSMC derived
from Cx45fl/fl:Nestin-Cre mice. B: Under the same microscope and
laser settings VSMCs derived from C57BL/6 mice (control VSMC)
were negative for the EGFP signal. Differential interference-
contrast (DIC) background was added and merged with
fluorescence in A and B. 40x magnification. C: Immunoblots with
protein samples from C57BL/6 VSMCs and kidney (control) and
Cx45fl/fl:Nestin-Cre VSMCs and kidney (Cx45fl/fl) probed for
Cx45 and the loading control GAPDH. Cx45 expression was
detected in all four samples, but expression was reduced in
Cx45fl/fl:Nestin-Cre VSMCs and kidney extracts. D: Cxs 37, 40,
and 43 mRNA was detected in C57BL/6 (control) and
Cx45fl/fl:Nestin-Cre (Cx45fl/fl) VSMCs by RT-PCR. mRNA from
all three Cxs at their expected bands sizes (344bp, 323bp, and
394bp respectively) was observed. β-actin (400bp) served as a
positive control.
89
on SDS-PAGE gels, transferred to membranes and probed with Cx45 antibodies (Fig
3.4C). Whole kidney homogenate from C57BL/6 and Cx45fl/fl:Nestin-Cre mice were
also analyzed by immunoblot to provide a means to compare Cx45 expression in a cell
culture with total renal Cx45 expression. A single band approximately 50 kDa in size
appeared on all blots probed with Cx45 antibodies. Cx45 expression was approximately
50% lower in both VSMCs and kidneys from the Cx45fl/fl:Nestin-Cre mouse compared
to control VSMCs and kidneys based on densitometry (not shown), indicating effective
Cx45 excision in a significant cell population of Cx45fl/fl:Nestin-Cre VSMCs and
kidneys. Both blots were also probed with GAPDH as a confirmation of equal protein
loading (Fig. 3.4C).
Since VSMCs are subject to phenotypic changes in culture, we sought to identify
any differences in Cx expression that may have arisen during the culture process. Total
RNA was purified from confluent C57BL/6 (control) and Cx45fl/fl:Nestin-Cre
(Cx45fl/fl) VSMC and amplified by RT-PCR with primers for Cxs 37, 40 and 43. Bands
at the predicted band size (344bp, 323bp and 391bp) were detected for both control and
Cx45fl/fl:Nestin-Cre VSMC samples (Fig 3.4D). Amplification with a β-actin primer
pair, which served as a positive control for the experiment, also produced bands of the
expected size (400bp) for both cell culture types.
Effects of Cx45 on calcium wave propagation in vascular smooth muscle cells
When control and Cx45fl/fl:Nestin-Cre VSMCs were loaded with Fluo-4/Fura red
and one VSMC in the center of the microscope field was mechanically stimulated with a
90
glass micropipette, an increase in [Ca
2+
]
i
in the stimulated cell was observed (Fig 3.5A ).
This increase in [Ca
2+
]
i
propagated to adjacent cells (Fig 3.5A).
The propagation speed (µ m/s) of the calcium wave was analyzed in control,
Cx45fl/fl:Nestin-Cre, and pharmacologically treated control VSMCs (n=6 for all
experimental groups) (Fig 3.5B). In control VSMCs, the propagation speed was 17 ± 5
µ m/s. The speed of propagation in Cx45fl/fl:Nestin-Cre VSMCs was significantly lower
(7 ± 1 µ m/s, p<0.05). The non-specific gap junction inhibitor 18α-GA failed to
significantly lower the speed of calcium wave propagation in control VSMCs (12.4 ± 2.0
µ m/s, p>0.05), however control VSMCs treated with a Cx45-specific gap mimetic
peptide (GAP) did exhibit a significant reduction in calcium wave propagation speed (5.7
± 1.6 µ m/s, p<0.05).
Since these experiments pointed to a role for Cx45 in calcium wave propagation
we sought to establish whether Cx45 regulates calcium wave propagation indirectly by an
extracellular agent such as ATP or directly via intercellular coupling. Generally, calcium
wave speeds above 100 µ m/s are thought to be due to direct coupling between cells (28,
31), while slower calcium wave speeds (as we observed) are associated with the release
of ATP via Cx hemichannels (9, 36). To test whether ATP played a role in the calcium
wave propagation, control VSMCs were treated with the non-selective purinergic
receptor antagonist suramin (Fig 3.5B). This treatment did not significantly reduce
calcium propagation speed when compared to control (10.3 ± 2.4 µ m/s, p>0.05). To
determine whether VSMCs were directly coupled, a dye spreading assay was conducted.
A single VSMC was loaded by microinjection with Lucifer Yellow (Fig 3.5C). Lucifer
91
Yellow, which does not cross cell membranes but does permeate gap junctions, spread to
adjacent VSMCs within 60 seconds of injection, indicating that cultured VSMCs were
coupled by gap junctions.
92
Figure 3.5: Calcium wave propagation in vascular smooth muscle cells (VSMC) from C57BL/6 and
Cx45fl/fl:Nestin-Cre mice. A: Representative pseudocolor Fluo-4/Fura red ratio images from control
VSMC shown at different time points (0-8 s) after the stimulation of a single cell (indicated by X). B: Speed
of calcium wave propagation in control VSMC, Cx45fl/fl:Nestin-Cre VSMC, and control VSMC treated
with 18α- glycyrrhetinic acid (18α-GA), Cx45 gap mimetic peptide (GAP), or suramin. Calcium wave
propagation was significantly slower in Cx45fl/fl:Nestin-Cre and GAP-treated VSMC (n=6, *p<0.05).
Bar= 20µ m. C: The gap junction permeable dye Lucifer Yellow was loaded by microinjection into a single
VSMC (indicated by X) and spread to adjacent cells within 60 seconds. Nuclei were labeled blue. Bar=
10µ m
93
DISCUSSION
Here we report that Cx45 is found in the renal cortex of the adult mouse kidney,
specifically in the vasculature and glomeruli. In the JGA, Cx45 was expressed in the
mesangium and the smooth muscle cells of the afferent and efferent arterioles, and in
renin producing JG cells. Both renal renin expression and plasma renin activity were
markedly increased in Cx45fl/fl:Nestin-Cre mice, which are considered deficient in their
JGA expression of Cx45. VSMCs cultured from afferent arterioles of Cx45fl/fl:Nestin-
Cre mice had reduced calcium wave propagation speed when compared to VSMCs
cultured from C57BL/6 mice. This decrease in speed was replicated in control VSMCs
treated with a Cx45-specific gap mimetic peptide. The non-specific gap junction blocker
18α-GA did not significantly reduce propagation speed.
Due to the lack of specific antibodies against Cx45, two transgenic mice with
reporter gene constructs were studied to determine the intrarenal localization of Cx45.
Using mice expressing the LacZ gene under the control of the Cx45 promoter, Cx45 was
detected in glomeruli and vascular structures of the renal cortex, which affirms previous
findings in these mice (19) (Fig. 3.1A). In addition, we observed β-gal staining under
higher magnification and identified positive structures as the afferent and efferent
arterioles, glomeruli, and mesangial cells (Figs. 3.1B-C). We utilized a second method to
substantiate these findings. Cx45fl/fl:Nestin-Cre mice have a deletion of the Cx45 coding
region in cells expressing nestin during embryogenesis. While nestin is typically thought
of as a neuronal marker, it is also expressed during development by the metanephric
mesenchyme, the progenitor to all renal cell types, except collecting duct epithelial cells
94
(8). Therefore Cx45 is functionally knocked-out in JGA cells and its expression is
replaced by EGFP expression. By detecting EGFP signal in Cx45fl/fl:Nestin-Cre mice we
observed a continuous Cx45 labeling in vascular smooth muscle cells of the afferent and
efferent arteriole and the adjacent extraglomerular mesangial cells (Figs. 3.2A-C). This
localization data suggests the possibility of fast and direct coupling between the afferent
and efferent arterioles and the mesangium. There is indeed evidence for simultaneous
propagation of the TGF calcium wave into these areas (26). The colocalization of EGFP
with renin in the afferent arteriole (Fig. 3.2B) suggests that this Cx isoform may also play
a role in renin synthesis and release.
The importance of Cx45 to the vasculature was already evident from previous
studies. Cx45-deficient mice are characterized by abnormal development of the
vasculature and this genetic modification proves to be lethal (19). Functional Cx45 gap
junctions have also been discovered in the vascular smooth muscle cells of cerebral
arteries, where it has been suggested that they play a role in regulating blood flow in the
nervous system (21, 22). However, the appearance of Cx45 in the renal vasculature does
not allow us to presume a functional role for the protein.
Therefore, to ascertain the physiological relevance of Cx45 in the kidney, in-vivo
and in-vitro experiments were performed with Cx45fl/fl:Nestin-Cre mice. These mice
had increased renin expression and activity and increased mean arterial pressure (Fig.
3.3). These changes in blood pressure and the renin-angiotensin system (RAS), as well as
the localization, mirror those observed in another Cx transgenic mouse, the Cx40
deficient mouse (Cx40
-/-
) (18, 38). Cx40
-/-
mice have renin-dependent hypertension.
95
Wagner et al (38) concluded that this hypertension is due to a failure to properly engage
the AngII and intrarenal blood pressure negative feedback loops on renin. Clearly, both
mouse model studies indicated that Cxs exert an effect on renal blood pressure regulation.
There are also differences between our study and those performed in the Cx40
-/-
model. While Cx45fl/fl:Nestin-Cre mice had an significant elevation in blood pressure,
they were not hypertensive. This limited blood pressure increase may be due to a
restriction of the gene knockout to nestin-expressing cells, as opposed to the systemic
knockout in the Cx40
-/-
mouse model. As with Cx40
-/-
, the elevated blood pressure we
observed does appear to be RAS-dependent in nature, given the increase in renin levels
and activity. However, the precise feedback mechanism through which renin is
dysregulated in Cx45fl/fl:Nestin-Cre mice requires further study. Blood pressure
regulation and renin secretion can also be regulated by the renal sympathetic nerve (12).
Therefore, we cannot exclude the possibility that the observed elevations in blood
pressure and renin were due to the effects of a conditional knockout in other Cx45- and
nestin-expressing cells, including those of the nervous system (33, 39, 43). However, the
effects of Cx45 loss in isolated VSMC primary cultures on calcium waves does suggest
that Cx45 plays a role in JGA function at the local level and independent of sympathetic
innervation. In addition to in-vivo and in-vitro analysis of Cx45fl/fl:Nestin-Cre mice, we
also attempted to block Cx45 function pharmacologically. Surprisingly, the non-specific
gap junction inhibitor 18α-GA did not significantly reduce VSMC calcium wave
propagation in control VSMC. Recently published data in mouse embryonic stem cells
suggests that 18α-GA may not inhibit Cx45 (40). In these cells (which express Cx31, 43,
96
and 45), inhibition of gap junction intracellular communication by 18α-GA required the
expression of Cx43. It has been established that Cx45 and Cx43 can interact together to
form functional gap junctions (11, 40), but only Cx43 mRNA, and not protein, has been
found in the smooth muscle cells of the renal vasculature (2, 42). Therefore, it seems
unlikely that Cx43 and Cx45 form heteromeric channels in VSMCs and the lack of these
channels could explain the inability of 18α-GA to reduce calcium wave propagation we
observed.
A previously developed Cx45 gap mimetic peptide was also used to inhibit of
Cx45 (21). The Cx45 peptide was designed to be homologous to earlier identified Cx43
and Cx40 blocking peptides. By patch-clamping smooth muscle cell pairs, it was
demonstrated that the Cx45 peptide altered conductance in a manner that was consistent
with channel blockade (21). In applying the Cx45 peptide to control VSMC, we were
able to reduce calcium wave propagation to levels observed with Cx45fl/fl:Nestin-Cre
VSMC. However, our study marks the first attempt to use this mimetic peptide in a cell
culture model and the results therefore should be interpreted with caution.
One possible mechanism through which Cx45 could affect renin regulation and
blood pressure is calcium signaling. Basolateral ATP released from the macula densa (3)
initiates a propagating calcium wave in the JGA and beyond (26) during TGF that
controls renal blood flow and glomerular filtration rate (29). The increase in [Ca
2+
]
i
accomplishes two mechanisms: inhibition of renin release from JG cells (30) and
contraction of VSMCs in the afferent arterioles (41). Gap junctions are known to be
instrumental in calcium wave propagation in several cells types (9, 13, 34) and it has
97
been previously demonstrated that the calcium wave of TGF can be abolished by gap
junction blockers (26).
Our observation that calcium wave propagation in VSMCs is dependent on the
expression and function of Cx45 (Fig 3.5B) suggests that Cx45 may play a role in the
propagation of TGF calcium wave (26). Calcium propagation involving Cxs occurs by
two mechanisms: either by intercellular gap junction communication or via the release of
an extracellular mediator such as ATP. Calcium wave propagation speeds above 100
µ m/s have previously been found in intact preglomerular smooth muscle cells (28). In the
present study however, the VSMC calcium propagation speeds measured were at least 5-
fold slower suggesting that the calcium wave in cultured VSMCs does not rely solely on
fast and direct gap junctional coupling, but instead involves an extracellular mediator,
such as ATP. A recently published paper from Toma et al (36) examined the role of Cx40
on calcium wave propagation in a glomerular endothelial cell (GENC) culture. Calcium
wave propagation speeds similar to those we presently observed in VSMCs were
recorded in GENCs and the authors concluded that Cx40’s control of calcium wave
propagation was mediated by ATP (36). It is well established that extracellular ATP can
cause cell-to-cell calcium signaling via Cxs (9).
However, when we tested this slow wave hypothesis in cultured VSMCs, our data
pointed towards gap junction intercellular communication as the mechanism behind the
calcium wave. The purinergic receptor antagonist suramin failed to significantly reduce
calcium wave propagation speeds and a dye spreading assay using lucifer yellow
provided evidence of direct VSMC coupling. Our findings are supported by several other
98
studies that have reported intercellular coupling in both smooth muscle cultures and
preparations (27, 31). The reason for the slow calcium wave propagation in VSMCs in
spite of the presence of direct gap junctional coupling is unknown, but the discrepancy
could be possibly explained by the different techniques (cultured VSMCs versus intact
vessels) used. Future investigation of calcium signaling in intact preglomerular vessels
from control and Cx45fl/fl:Nestin-Cre mice could further support the physiological
significance of Cx45 in the propagation of calcium waves in the JGA, including that of
TGF.
In comparing our findings to the recent work on Cx40 in GENCs (36), Cxs appear
to be a critical factor for calcium signaling in the JGA, but they seem to use at least two
different mechanism to achieve calcium wave propagation: direct gap junctional coupling
(Cx45) and cell-to-cell signaling via extracellular ATP (Cx40). The different
characteristics of the two cell types (endothelial cells vs. smooth muscle cells) or the
functional differences that exist between the Cx40 and 45 isoforms may explain why both
indirect and direct methods of calcium wave propagation occur in the JGA.
99
PERSPECTIVES AND SIGNIFICANCE
In conclusion, in this study we reported the localization of Cx45 to the renal
cortical vasculature, glomeruli, and the JGA region. In the JGA, the afferent and efferent
arterioles, and intra- and extraglomerular mesangial cells were all Cx45-positive. Renin
expression, plasma renin activity, and blood pressure were all increased significantly in
Cx45fl/fl:Nestin-Cre, which have reduced JGA Cx45 expression. The speed of calcium
wave propagation in VSMC cultured from Cx45fl/fl:Nestin-Cre mice was significantly
lower than in control VSMCs. Treatment of control VSMCs with a Cx45-specific gap
mimetic peptide also reduced calcium wave propagation. Blockade of purinergic
receptors failed to reduce calcium wave propagation, while a dye spreading assay
provided evidence of cell-to-cell coupling between VSMCs. The localization of Cx45, its
effects on renin and calcium wave propagation all suggest a role for Cx45 in TGF, renin
regulation and systemic blood pressure maintenance. While, the precise mechanism
through which Cx45 controls these regulatory systems remains to be determined, a model
that utilizes intercellular gap junction communication is likely involved.
100
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104
CHAPTER 4: THE EXPRESSION AND FUNCTION OF PANNEXIN1 IN THE
RENAL TUBULAR EPITHELIUM
To be submitted to the American Journal of Physiology: Renal Physiology
Pannexin1 is expressed in the renal tubular epithelium and facilitates ATP release in
renal epithelial cells
Fiona Hanner, Mien T.X. Nguyen, Alan Yu, János Peti-Peterdi
CHAPTER 4 ABSTRACT
In the renal tubules, ATP released from epithelial cells stimulates purinergic
receptors, regulating salt and water reabsorption. However, the mechanism by which
ATP is released into the tubular lumen remains to be determined. Pannexin1 (Panx1) is a
ubiquitously expressed protein that forms connexin-like hemichannels in the plasma
membrane which have been demonstrated to function as a mechanosensitive ATP
conduit. Here we report on the expression of Panx1 in the mouse kidney. Using
immunofluorescence, we observed Panx1 localization in thin descending limbs, S2 and
S3 segments of the proximal tubules, and cortical collecting duct apical membranes. No
expression was detected in distal convoluted tubules, renal vasculature, or the
juxtaglomerular apparatus. Additionally, we demonstrated for the first time that Panx1
hemichannels expressed in renal epithelial cells facilitate ATP release upon mechanical
105
stimulation. Inhibition of Panx1 pharmacologically or by siRNA decreased ATP release
in MDCK cells, but did not alter dye uptake. ATP release was also reduced in M1 cells
treated with chemical Panx1 inhibitors. Based upon these results, we propose that Panx1
hemichannels may regulate ATP release in the tubular lumen and suggest that they play a
role in mediating renal epithelial transport.
106
INTRODUCTION
Purinergic signaling is a major signaling mechanism in the kidney and regulates
several important renal mechanisms including tubuloglomerular feedback and renin
release. In the renal tubules, purinergic receptor activation regulates epithelial transport
(23). ATP concentrations consistent with P2 receptor activation have been found in the
tubular lumen (51). Additionally, the identity of several P2X and P2Y receptors along the
renal epithelium, as well as their effect on ion transport, has already been determined
(23). However, the mechanism by which ATP, the ligand, is released into the tubular
lumen remains contentious.
In the renal tubules, ATP may be released both by vesicular and channel-based
ATP release mechanisms. Several different transporters have been proposed as ATP
conduits, including the cystic fibrosis transmembrane conductance regulator and the
P2X7 receptor, but each has significant limitations (36). It has also been suggested that
connexin proteins (Cx) can form hemichannels, or connexons, which allow ATP release
to the extracellular fluid (9, 12, 14). Recently, a study by Sipos et al (47) provided
evidence that Cx30 is required for luminal ATP release in the cortical collecting ducts
and for pressure natriuresis. However there is limited evidence that Cxs form functional
hemichannels under physiological conditions (49).
Another candidate mechanism for renal ATP release is the pannexin hemichannel.
Pannexins are a class of proteins that also form hexameric transmembrane channels,
similar to those formed by connexins (6). Despite having similar structures, Cx and
pannexins have no sequence homology. While their ability to form connexin-like gap
107
junctions between two cells is controversial (4, 18, 46), there is overwhelming evidence
that pannexins form functional hemichannels under physiological conditions, allowing
the passage of small molecules between the intra- and extracellular regions (5, 10, 43).
Three pannexin isoforms have been identified: Panx1, Panx2, and Panx3. Of these three,
only Panx1 forms functional hemichannels in-vivo. Panx2 seems to function in a support
role to Panx1 (23). Panx1 hemichannels are mechanosensitive, open at physiological
extracellular [Ca
2+
], and activated by increased intracellular [Ca
2+
]. As such, Panx1
hemichannels have been shown to facilitate ATP release in several cells types, including
epithelial cells in the lungs and taste buds (2, 19, 25, 39). Additionally, studies have
found Panx1 mRNA expression in the kidney (3, 40).
Therefore, we hypothesized that Panx1 is expressed in the epithelial cells of the
renal tubules where it functions to facilitate ATP release from these cells. To test this
hypothesis, we first used immunofluorescence to localize Panx1 protein within the kidney
and identify its specific cellular expression. Then, two renal epithelial cell culture
models, MDCK and M1, which express Panx1 endogenously, were treated with Panx1
inhibitors in order to determine whether Panx1 plays a role in ATP release from these
cells. Finally, Panx1 activity was tested with a dye uptake assay in MDCK and M1 cells.
108
MATERIALS AND METHODS
Animals
C57BL/6 mice (bred in house) were fed standard diets (Harlan, Madison, WI) and
drinking water ad libitum. All animal protocols have been approved by the Institutional
Animal Care and Use Committee at the University of Southern California.
Antibodies
The chicken polyclonal Panx1 antibodies (4515) for immunofluorescence and
immunoblotting studies were provided by Dr. Gerhard Dahl (University of Miami Miller
School of Medicine, Miami FL) and have been characterized in previous publications
(25). Rabbit polyclonal aquaporin-1 (AQP1) and aquaporin-2 (AQP2) antibodies were
kind gifts from Dr. Dennis Brown and Dr. Mark Knepper respectively. The rabbit
polyclonal sodium-chloride cotransporter NCC antibody was a kind gift from Dr David
Ellison. The AQP1, AQP2, and NCC antibodies were characterized in previous
publications (29, 42, 44).
Immunofluoresence labeling of kidney tissue
Kidneys were fixed in situ by perfusion of 4% paraformaldehyde and coronal
kidney sections containing all kidney zones were then post-fixed overnight at 4°C in
fixative. Kidney sections were then embedded in paraffin and 4-µm sections of the
paraffin block were cut, deparaffinized in toluene, and rehydrated through xylene and
graded ethanol. To retrieve antigens, slides were heated for 2 x 10 min in a microwave
with medium heat in PBS and allowed to cool for 40 minutes. Sections were then fixed
with 4% paraformaldehyde for 10 min and permeablized for 10 min with 0.1% Triton-X
109
in PBS. 10% BlockHen (Aves, Tigard OR) in PBS was applied to section for 1 hour to
block non-specific binding. Sections were then probed with Panx1 chicken polyclonal
antibodies, at a dilution of 1:100 overnight followed by incubation with secondary Alexa
Fluor 594 goat-anti chicken antibodies (Invitrogen, Carlsbad, CA) at a 1:500 dilution for
1 hour. Additionally, some sections were also probed overnight with the following rabbit
polyclonal antibodies: AQP1 (1:50), AQP2 (1:500), and NCC (1:500). These sections
were incubated for 1 hour with goat anti-rabbit secondary Alexa Fluor 488 antibodies at a
dilution of 1:500. Following a final wash step, all sections were mounted with
Vectashield mounting media containing the nuclear stain DAPI (Vector Laboratories,
Burlingame, CA) and examined with a Leica TCS SP2 confocal microscope.
Cell cultures
Madin-Darby canine kidney type 1 cells (MDCK) are epithelial cells
representative of the distal nephron and were cultured in the following media: Dulbecco’s
modified Eagle’s medium (D7777, Sigma, St. Louis, MO) supplemented with 3.7 g
NaHCO
3
, 5% fetal bovine serum, and 1% penicillin/streptomycin. The M1 cell line has a
mixed phenotype, representative of both intercalated and principle cells of the collecting
duct. These cells were purchased from ATCC and have been previously described (13).
M1 cells were cultured in the following media: Dulbecco’s modified Eagle’s
medium/nutrient mixture F-12 Ham (D8900, Sigma) supplemented with 1.2 g NaHCO
3
,
5% fetal bovine serum, 0.005 mM dexamethasone, and 1% penicillin/streptomyocin.
110
Immunoblotting of cultured cells
MDCK and M1 cells were grown to confluence on 100 mm plates, collected by
trypsinization, and washed with PBS. Cells were lysed using CellLytic-M lysis buffer
(Sigma) supplemented with a protease inhibitor cocktail (BD Bioscience, San Jose, CA)
according to manufacturer’s instructions. Protein concentration was determined with a
modified Bradford assay (Quick Start Bradford protein assay, Biorad, Hercules, CA).
Twenty micrograms of protein were run
per lane, separated on a 4-20% SDS-
polyacrylamide gel (Biorad), and then
transferred to a polyvinyledene difluoride
membrane (Millipore, Billerica, MA). After blocking the membrane in blocking buffer
(Li-Cor, Lincoln, NE) blots were probed with chicken polyclonal
antibodies to Panx1 at a
dilution of 1:1000 overnight. Reactivity of the primary antibodies was detected
with
IR680-labeled goat anti-chicken antibodies (1:15,000 dilutions, Li-Cor). Blots were
imaged using the Odyssey Infrared Imaging
System (Li-Cor) and accompanying
software. The blot was reprobed with a mouse monoclonal antibody to GAPDH
(Ambion, Austin, TX) at a dilution of 1:4000 for 1 hour to test for protein loading and
quality of transfer. Labeling was detected and imaged with an IR800-labeled goat anti-
mouse antibody.
Densitometric analysis of blots was performed using Odyssey software. The data
was then normalized against the GAPDH positive control. Statistical significance
was
calculated by paired Student’s t-test analysis with data shown as mean ± SE.
111
Immunoblotting of tissue
Samples were taken from previously frozen dog and mouse kidneys and
homogenized with a rotor-stator homogenizer in a buffer containing 20 mM Tris-HCl, 1
mM EGTA, and a protease inhibitor cocktail (BD Bioscience). Protein concentration was
determined with a modified Bradford assay and 40-80 µ g of protein were then run on
SDS-PAGE gels, transferred to PVDF membranes, probed, and imaged as described
above.
Immunocytochemistry
MDCK and M1 cells were cultured to confluence on glass coverslips in their
respective media. Coverslips were washed in PBS to remove media and were then fixed
with 4% paraformaldehyde for 10 min and permeablized for 10 min with 0.1% Triton-X
in PBS. 5% normal goat serum in PBS was applied to section for 30 min to block non-
specific binding. Slides were probed with chicken Panx1 antibodies and goat-anti
chicken secondary AF 594 antibodies, mounted, and imaged as described in
immunofluorescence methods.
Reverse Transcription-PCR
Total RNA was purified from whole mouse kidney, dog kidney, M1, or MDCK
cells using a Total RNA Mini Kit in accordance with manufacturer’s instructions
(Biorad). RNA was then quantified using spectrophotometry and reverse-transcribed to
single-stranded cDNA using avian reverse-transcriptase and random hexamers according
to manufacturer’s instructions (Thermoscript RT-PCR system, Invitrogen). Reactions
were also performed without reverse-transcriptase to control for the presence of genomic
112
DNA. 2µ L of cDNA was amplified using a master mix containing Taq polymerase
(Invitrogen) and the following primers: canine Panx1 F 5’-AAT GAG AAC ATG GGG
CAA AG-3’; R 5’-CTT GAT GCT GCA CAC GAA CT-3’; murine Panx1 F: 5’-GGC
CAC GGA GTA TGT GTT CT-3’; R: TAC AGC AGC CCA GCA GTA TG-3’; murine
Cx30 F: 5’-GAG TTG TGT TAC CTG CTG C-3’; R: 5’-GGC TTG GTT TTC AGA
GAT AG-3’; canine Cx43 F: 5’-ACC CAA CAG CAG CAG ACT TT-3’; R: 5’-TGG
AGT AGG CTT GGA CCT TG-3’; murine Cx43 F: 5’-GGA CAT GCA CTT GAA GCA
GA-3’; R: 5’-CAG CTT GTA CCC AGG AGG AG-3’; canine Cx45 F: 5’-ATG GTG
TTA CAG GCC TTT GC-3’; R: 5’-TGC TAG ATC CAA GCG TTC CT-3’; murine
Cx45 F: 5’-TTG GGA AAG CAA CAA ACA AA-3'; R: 5’-GGA GTT GCA ACC AGG
ATG AT-3’; β-actin F: 5’-CTG AAC CCT AAG GCC AAC CGT G-3’; R: 5’-ATG GGC
CAT CTC CTG CTC CAA G-3’; each at a final concentration of 200µ M. All
oligonucleotide sequences were determined using published sequence and Primer3
software and designed to be intron-spanning when possible. The PCR reaction was
carried out for 30 cycles of the following: 94
o
C for 30 seconds, 60
o
C for 30 seconds, and
72
o
C for 30 seconds. The PCR product was analyzed on a 2% agarose gel stained with
ethidium bromide to identify fragments of 222 bp for canine Panx1, 247 bp for murine
Panx1, 369 bp for murine Cx30, 496 bp for canine Cx43, 200 bp for murine Cx43, 350
bp for canine Cx45, 349 bp for murine Cx45, and 360 bp for β-actin.
Panx1 short interfering RNA
M1 and MDCK cells were grown to confluence, trypsinized, and counted.
Approximately 1 x 10
5
cells were then resuspended in 75 µ L of siPORT siRNA
113
electroporation buffer (Ambion, Austin, TX) and transferred to a sterile 0.4 cm
electroporation cuvette (Biorad). 2 µ g of one of the following siRNAs was added to the
cuvette: Silencer GAPDH siRNA (human, mouse, and rat), Silencer negative control #2
siRNA, or Silencer Select predesigned Panx1 siRNA (id#: s79965). All siRNAs were
designed by and purchased from Ambion. Cells were then transfected by electroporation
using a Gene Pulser XL electroporator (Biorad) and the following protocol: one 300V,
150 µ s, 1 pulse followed by two rounds of 25V, 25 ms, 15 pulses with 100-ms interval
between pulses (11). Following electroporation, cells were incubated for 10 min at 37ºC,
then resuspended in standard media and plated. To test the efficiency of transfection,
GAPDH siRNA-transfected cells were assayed for GAPDH activity and compared to
non-transfected and negative control siRNA-transfected cells using a KDalert GAPDH
assay kit (Ambion) 72 hours post-transfection. After optimizing transfection conditions,
Panx1 siRNA-transfected and negative control siRNA-transfected cells were cultured for
72 hours and then either collected for immunoblotting or cultured for use in the luciferin-
luciferase ATP or dye-uptake assays.
Luciferin-luciferase ATP assay
MDCK and M1 cells were cultured to 70% confluence on 24-well tissue culture
plates. To chemically inhibit Panx1 hemichannels, cells were treated with media
supplemented with either probenecid (Sigma, 1 mM final concentration) or
carbenoxolone (Sigma, 50 µ M final concentration) for 2.5 hours based on previous
studies (24, 48). Panx1 hemichannel expression was also inhibited by siRNA (see above).
114
Prior to ATP stimulation, cells were incubated in serum-free DMEM
supplemented with 20 mM HEPES buffer (DMEH) for 30 minutes at 37º C. To induce
mechanical stimulation of ATP, media was aspirated from the plate and replaced with
500 µ L of fresh media as previously described (30). After a 2 minute incubation period,
100 µ L samples were collected from each well and assayed by luciferin-luciferase ATP
assay kit (Sigma) in accordance with manufacturer’s instructions. Luminescence was
detected using a cuvette-based spectrofluorometer (Quantamaster-8, PTI, Birmingham,
NJ) and data was analyzed using FeliX32 software (PTI). For each experimental group,
n≥6. Statistical significance
was calculated by a one-way ANOVA analysis followed by
Bonferroni’s correction post hoc analysis (Panx1 inhibitors) or paired Student’s t-test
(Panx1 siRNA) with data shown as mean ± SE.
Dye uptake assay
Dye uptake by MDCK and M1 cells was measured using the cell-impermeable
nucleic acid dye YoPro-1 (Invitrogen; ex: 491 nm, em: 509 nm) in a cuvette-based
spectrofluorometer (PTI). MDCK and M1 cells were cultured to confluence on glass
cover slips in their respective standard media. Panx1 hemichannels were either
chemically inhibited with probenecid or carbenoxolone (per ATP assay protocol) or with
Panx1-siRNA. Cells were incubated in a quartz cuvette in 2mL of modified Krebs-Ringer
HCO
3
buffer (115 mM NaCl, 5 mM KCl, 25 mM NaHCO
3
, 960 µM NaH
2
PO
4
,
240 µM
Na
2
HPO
4
, 1.2 mM MgSO
4
, 2 mM CaCl
2
, 5.5 mM D-glucose,
and 100 µM L-arginine,
adjusted to
pH 7.4) along with 10 µ L YoPro-1 (final concentration 5 µ M) and a baseline
measurement was taken for 5 minutes prior to stimulation. Panx1 hemichannels were
115
then stimulated by either mechanically (induced with a magnetic stir bar) or with 1 mM
ATP and the emitted
fluorescence signal was measured for
an additional 5 minutes. The
difference between post- and pre-stimlulation peak fluorescence intensity was analyzed
as a measure of dye uptake
using the FeliX32 software (PTI). For each experimental
group, n = 5. Statistical significance
was calculated by a one-way ANOVA analysis
followed by Dunnett's
post hoc comparison (Panx1 inhibitors) or paired Student’s t-test
(Panx1 siRNA) with data shown as mean ± SE.
116
RESULTS
Expression of Panx1 within the mouse kidney
While previous studies have shown evidence of Panx1 mRNA in the kidney, no
details regarding the specific renal localization of Panx1 were presented. Therefore,
immunofluorescence studies were performed to establish where Panx1 protein is
expressed within the kidney. Paraffin mouse kidney sections were labeled with chicken
polyclonal Panx1 antibodies and imaged using confocal fluorescent microscopy (Figure
4.1). Panx1 labeling was observed in the S2 and S3 segments of the proximal tubules
(Figure 4.1A), thin-walled structures in the Loop of Henle (Figure 4.1B), and cortical
collecting ducts (Figure 4.1C). Apical membrane localization was most apparent in the
collecting ducts, where only select cells were positive for Panx1. No signal was detected
in the vasculature or juxtaglomerular apparatus.
Figure 4.1: Expression of Panx1 in the renal tubules. A: Panx1 was found in the brush borders (inset,
arrows) of the S2 and S3 proximal tubules. B: In the medulla, Panx1 was observed in the thin limbs of the
Loop of Henle. C: Labeling along the apical membrane of the cortical collecting ducts was also found.
Nuclei were stained with DAPI (blue). Bar = 20 µ M.
117
Co-labeling of Panx1 in the mouse kidney with renal epithelial cell markers
Mouse kidney sections were labeled with Panx1 antibodies along with several
antibody markers of different epithelial cells in the renal tubules to confirm the initially
observed Panx1 labeling (Figure 4.2). To ascertain the identity of the Panx1-positive
region in the Loop of Henle, we co-labeled sections with Panx1 (red) and aquaporin-1
(AQP1) (green), a marker of the descending thin limbs. Panx1 labeling was detected in
all AQP1-positive cells of the Loop of Henle (Figure 4.2A). Since some, but not all, cells
of the cortical collecting duct expressed Panx1 (red), co-labeling with the principal cells
marker AQP2 (green) was used to distinguish which collecting duct cell type was
positive for Panx1 (Figure 4.2B). Panx1 labeling was detected in both AQP2-positive and
–negative cells, indicating that Panx1 is expressed in the principal and intercalated cells.
Panx1 and NCC co-labeling was performed to confirm that the cortical labeling observed
in Figure 4.1A was specific for the proximal tubules and not the distal convoluted tubules
(Figure 4.2C). NCC is a known marker of the distal convoluted tubules. Panx1-positive
tubules (red) consistently lacked any NCC expression (green), indicating that Panx1 is
not expressed in the distal convoluted tubules.
Expression of Panx1 in MDCK and M1 cell lines
In order to assess their feasibility as models for testing Panx1 function in-vitro,
two renal epithelial cell lines, MDCK and M1, were tested for the expression of
endogenous Panx1. RNA was isolated from confluent MDCK and M1 cells and amplified
by RT-PCR with primers for Panx1. Panx1 mRNA was detected in both MDCK and M1
118
cells, producing appropriately sized bands (Figure 4.3A). β-actin primers were used as a
Figure 4.2: Co-labeling of Panx1 with epithelial cell markers in the mouse kidney. A: In the loop of Henle,
Panx1 (red) was expressed in AQP1-positive thin limbs (green). B: Panx1 (red) was co-expressed with
AQP2 (green), a marker of the apical membranes of principal cells in collecting ducts (*). Labeling was
also observed in the AQP2-negative intercalated cells (arrowhead). DIC background was added and
merged with fluorescence. Nuclei were stained with DAPI (blue). C: Co-labeling of the distal convoluted
tubule marker NCC (green) (*) and Panx1 (red) (arrowhead) failed to show any overlap in signal. A-C: left
panel: Panx1; center panel: epithelial cell marker; right panel: merge. Bar=20 µ M.
119
positive control and produced bands of expected size. No bands were observed in
the negative control reverse-transcriptase-free reactions.
Previous studies have shown evidence of Cx hemichannels functioning as ATP
conduits in several cell types. Therefore we sought to determine whether Cxs were also
expressed in MDCK and M1 cells. cDNA from both cell types was amplified using
primers for the three other renal tubular Cx isoforms: Cx30, Cx43, and Cx45 (Figure 3B).
The Cx30 isoform has not been identified in the canine genome and therefore MDCK
cDNA was not amplified with Cx30 primers. Cx30 mRNA was not detected in M1 cells
while both cell types expressed Cx43 mRNA. Cx45 mRNA was detected in M1, but not
MDCK cells. RT-PCR was performed using mRNA extracted from mouse and dog
kidney as a positive control for each primer and appropriately sized bands were detected
in each case (Figure 4.3C).
Panx1 protein expression was analyzed in MDCK and M1 cells by
immunoblotting. Cell homogenates from MDCK and M1 cells were collected and
separated by SDS-PAGE, then transferred to PVDF membranes and probed with Panx1
antibodies (Figure 4.3D). Double bands characteristic of Panx1 (25, 33) were detected
between 40 and 55 kD for both cell types in all samples. Protein samples from mouse and
dog kidneys were also probed for Panx1 and double bands around 50 kD were detected in
each sample (Figure 4.3E). Protein expression was confirmed by immunocytochemistry
(Figure 4.3F). Labeling was detected in both cell types, with a strong signal observed
along cell membranes.
120
Figure 4.3: Detection of Panx1 in renal epithelial cell cultures. A: mRNA isolated from MDCK cells
and M1 cells was amplified by RT-PCR to detect Panx1 and β-actin. Bands of appropriate size were
detected in both cell types. No bands were detected in samples amplified without reverse-transcriptase
(RT-). B: primers for renal epithelial connexins were used to amplify MDCK and M1 cell cDNA. In M1
cDNA, bands for Cx43 (mCx43 +) and Cx45 (mCx45 +) were detected, while in MDCK cells, a band
was detected for Cx43 (dCx43 +), but not Cx45 (dCx45 +). No bands were observed in reverse-
transcriptase-free reactions (-). C: Mouse (m) and dog (d) kidney cDNA samples were used as positive
controls for Cx and Panx1 primers. Bands of the expected size were observed for each primer in both
species. D: MDCK and M1 cell protein lysate samples were blotted with Panx1 antibodies. A double
band around 50 kD was detected in both cell types. E: 40-80 µ g of mouse and dog kidney homogenate
samples were probed with Panx1 antibodies as a positive control and similar bands were observed. F:
Fixed MDCK and M1 cells were labeled with Panx1 antibodies (red) with staining observed in all cells
and along cell membranes. Nuclei are stained with DAPI (blue). Bars = 20 µ m.
121
Effect of chemical inhibition of Panx1 hemichannels in MDCK and M1 cell lines
MDCK and M1 cells were cultured to 70% confluence in 24-well plates and ATP
concentration of the cell culture media was assayed by a luciferin-luciferase ATP assay
Figure 4 4: Measurement of ATP release and dye uptake in Panx1 inhibitor-treated MDCK and M1
cells. A: ATP release was detected by luciferin-luciferase assay and flow-stimulated ATP
concentrations were normalized against baseline ATP levels. Stimulated ATP release significantly
lowered by probenecid and carbenoxolone (CBX) treatment in both MDCK (* p< 0.05, n ≥ 6 per
group) and M1 (# p< 0.05, n = 6 per group) cells. B: Uptake of YoPro-1 dye was assayed by
spectrofluorometry in MDCK and M1 cells and the difference between flow-stimulated and baseline
peak fluorescence intensity (∆ peak RFU) was compared among control, probenecid-treated, and
CBX-treated cells (n=5 per group). No significant difference was found between the groups. Values
are means ± SE.
122
(Figure 4.4A). ATP release was stimulated by media change and the measured ATP
concentrations were normalized against baseline ATP concentrations. Mechanical
stimulation induced a 1.89-fold increase in the concentration of released ATP in MDCK
cells ([ATP]
media
stimulated: 189.63 ± 30.32 nM; [ATP]
media
baseline: 100.25 ± 26.96 nM;
n=6, p<0.05). In M1 cells mechanical stimulation led to a 3.13-fold increase in released
ATP concentration ([ATP]
media
stimulated: 71.17 ± 10.05 nM; [ATP]
media
baseline: 22.69
± 6.69 nM; n=6, p<0.05). Treatment with carbenoxolone (CBX) significantly inhibited
the effect of mechanical stimulation on ATP release in both MDCK and M1 cells,
reducing [ATP] to near baseline levels (fold change vs. baseline: MDCK: 1.09 ± 0.07;
M1: 1.17 ± 0.03; n=6, p<0.05 vs. control). Probenecid treatment resulted in a similar
inhibition on stimulated ATP release (fold change vs. baseline: MDCK: 1.17 ± 0.02, n=9;
M1: 1.20 ± 0.03, n=6; p<0.05 vs. control).
Confluent MDCK and M1 cells cultured on glass coverslips were assayed for
YoPro-1 dye uptake using spectrofluorometry (Figure 4.4B). Stimulation by flow led to a
significant increase in fluorescence over baseline levels in MDCK cells, but not in M1
cells (MDCK: baseline RFU: 24316 ± 6107; stimulated RFU: 54049 ± 4882, n=5 p<0.05;
M1: baseline RFU: 21181 ± 3285; stimulated RFU: 23646 ± 3840, n=5 p>0.05). Neither
treatment with probenecid or CBX resulted in a significant reduction in stimulated
fluorescence levels in MDCK cells or M1 cells (MDCK: control ∆ RFU: 29733 ± 7084;
probenecid ∆ RFU: 31727 ± 31380, CBX ∆ RFU: 33643 ± 18695, n=5, p>0.05; M1:
control ∆ RFU: 2465 ± 3776; probenecid ∆ RFU: 2910 ± 1094, CBX ∆ RFU: 11786 ±
6206, n=5, p>0.05).
123
Effect of Panx1 expression knockdown by siRNA in MDCK and M1 cell lines
MDCK and M1 cells were transfected with either Panx1 or a negative control
siRNA by electroporation. Efficacy of Panx1 siRNA knockdown was determined by
immunoblotting. Blots were probed with Panx1 and GAPDH antibodies (Figure 4.5A)
and analyzed by densitometry with Panx1 expression normalized against GAPDH
expression (Figure 4.5B). Panx1 siRNA transfection significantly decreased Panx1
protein expression in MDCK and M1 cells, with 42.47 ± 10.78 % remaining Panx1
expression in MDCK cells and 67.72 ± 6.80 % remaining Panx1 expression in M1 cells
versus negative control siRNA-transfected cells (n=4 per group, p<0.05 vs negative
control siRNA-transfected groups).
After confirming knockdown by Panx1 siRNA, transfected cells were either
assayed for ATP release or dye uptake as described above. MDCK cells transfected with
Panx1 siRNA released ~30% less ATP than MDCK cells transfection with negative
control siRNA (negative control siRNA [ATP]
media
: 74.43 ± 7.42 nM; Panx1 siRNA
[ATP]
media
: 54.07 ± 6.47 nM; n=6, p<0.05) (Figure 4.5C). No significant difference in
ATP release was detected between Panx1 siRNA-treated and negative control siRNA-
treatment M1 cells (negative control siRNA [ATP]
media
: 54.70 ± 5.74 nM; Panx1 siRNA
[ATP]
media
: 64.80 ± 6.03 nM; n=6, p>0.05) (Figure 4.5C). YoPro-1 dye uptake was
stimulated by purinergic receptor activation by 1 mM ATP as previously reported (26).
Dye uptake was not significantly altered by Panx1 siRNA transfection in either MDCK
or M1 cells when compared to the respective negative-control siRNA-transfection cells
(MDCK: negative control siRNA ∆ RFU: 2158 ± 2317; Panx1 siRNA ∆ RFU: 12933 ±
124
5934; M1: negative control siRNA ∆ RFU: 3768 ± 2451; Panx1 siRNA ∆ RFU: 4100 ±
2855; n=5, p>0.05) (Figure 4.5D).
Figure5. 5: Measurement of ATP release and dye uptake in Panx1 siRNA-transfected MDCK and M1 cells.
A: Panx1 siRNA-transfected cells (Panx1) and negative control siRNA-transfected cells (NC) were
immunoblotted and probed with Panx1 antibodies to assay the efficiency of siRNA knockdown. GAPDH
antibody was used as a loading control. B: densitometric analysis of the immunoblot indicated that Panx1
siRNA significantly reduced the expression of Panx1 in both cell types. Data expressed as a percent of
Panx1 expression in equivalent NC cells (n=4, * p<0.05 vs. NC cells). C: ATP release induced by
mechanical stimulation was significantly reduced by Panx1 siRNA in MDCK, but not M1 cells (n=6, *
p<0.05 vs. negative control). D: No significant difference was found between Panx1 and negative control
siRNA-transfected cells in YoPro-1 dye uptake after stimulation by 1 mM ATP (∆ peak RFU) (n=5 per
group). Values are means ± SE.
125
DISCUSSION
Here we present the first known study of Panx1 expression within the kidney.
Panx1 was localized to the renal tubular epithelium. Specifically, Panx1 expression was
detected in the proximal tubules, thin descending limb, and the apical membrane of the
cortical collecting duct. Panx1 was not detected in the juxtaglomerular apparatus and or
in the renal vasculature. Panx1 expression was also lacking in the distal convoluted
tubules. Apical membrane localization was most prominent in the collecting ducts,
suggesting that Panx1 could serve as a hemichannel in this region. In the proximal
tubules, brush border localization was observed in some regions, while a more diffuse
staining was common in the thin limbs, similar to what has been described with Cx30.3
(16).
Additionally, we confirmed the endogenous expression of Panx1 in two renal
epithelial cell lines, MDCK and M1. Panx1 protein expression in MDCK cells had been
previously reported (34), while the discovery of Panx1 in M1 cells, which is considered
to represent the collecting duct, is a novel finding. Both cell types are known to respond
to mechanical stimulation such as increased flow by releasing ATP (50, 54). Therefore
the discovery of mechanosensitive Panx1 channels in these cells strengthens the
argument that Panx1 allows the release of ATP in renal epithelial cells, as has been
shown in other epithelial cell cultures.
RT-PCR analysis of the renal tubule Cx isoforms Cx30, 43, and 45 (7, 15, 28)
confirmed expression of Cx43 in both cell types. Cx43 is thought to form hemichannels
(8) and a recent study by Kang et al provided the first direct evidence that Cx43
126
hemichannels are permeable to ATP (22). This raises the possibility that the ATP release
we observed may be due to Cx43 hemichannels and not Panx1. However, in the present
study, ATP release was inhibited by probenecid, which is not known to inhibit Cx43
hemichannels, and by Panx1 siRNA. Furthermore, new evidence has shown that ATP
release in astrocytes does not occur via Cx43 hemichannels, as had long been reported,
but by Panx1 instead (21). An interaction between Cx43 and Panx1 via the P2X7 receptor
has also been proposed (20), which suggests that if Cx43 hemichannels release ATP, this
may occur in a Panx1-dependent manner. Cx45 was expressed in M1 cells only, but there
is a lack of evidence that it forms ATP-permeable hemichannels. No Cx30 mRNA was
detected in M1 cells. Therefore, we conclude that in MDCK and M1 cells, ATP release is
in part due to Panx1, not Cx, hemichannel activity.
Chemically inhibiting Panx1 activity with either probenecid or CBX significantly
reduced ATP released in both cell types in response to mechanical stimulation,
suggesting that Panx1 hemichannels facilitate ATP release in renal epithelial cells.
Although chemical inhibition of Panx1 caused an almost complete loss of stimulated
ATP release, the level of constitutive ATP release was similar to that seen in control
samples. We speculate that another mechanism, perhaps vesicular, may cause ATP
release under unstimulated conditions, while Panx1 hemichannels only release ATP
under conditions that promote channel opening, including increased flow. Moreover,
reducing Panx1 expression by siRNA treatment significantly reduced stimulated ATP
release in MDCK cells, but not M1 cells. This discrepancy between the two cell types
was likely due to the less-efficient knockdown of Panx1 expression in M1 cells. It is also
127
notable that the effect of Panx1 siRNA on ATP release was less prominent than that of
chemical inhibition methods, again probably because the knockdown of Panx1 protein
expression is only partial.
Reducing either Panx1 activity with probenecid or CBX, or Panx1 expression
with siRNA in MDCK and M1 cells did not cause a significant decrease in YoPro-1 dye
uptake. The current literature presents a mixed picture with regard to the regulation of
dye uptake by pannexin hemichannels, with different cell types exhibiting varying
responses to stimuli (32, 38, 41). Since neither MDCK nor M1 cells have been assayed
by this method before, we used two common methods of inducing Panx1 dye uptake –
mechanical stress and P2 receptor activation (28, 48). Both cells types express P2Y
receptors and are known to respond to flow (35, 37, 50, 54). However, we were unable to
detect an inhibition of dye uptake by Panx1 inhibition with either method. High
variability was present, both between cell types and within experimental groups. MDCK
showed a greater response to flow-induced dye uptake than M1 cells, perhaps due to
different flow-sensing mechanism in the cells (50, 54). Stimulating cells by ATP
produced less dye uptake in both cells types which may be due to their expression of P2Y
and not P2X receptors. Since other cell types where dye uptake assays have successfully
shown an effect of Panx1 inhibition expressed the P2X7 receptor, which complexes with
Panx1 (30, 31), we propose that a different, flow-dependent mechanism may be at work
in renal epithelial cells.
The use of probenecid as a specific pannexin inhibitor was only recently
demonstrated and this is the first application of this drug as a Panx1 inhibitor in renal
128
cells of which we are aware (48). Probenecid is more commonly known for its inhibitory
effect on organic anion transporters (OAT), including those in the kidney. Therefore the
possibility that the effect of probenecid on ATP release and dye uptake observed is due to
OAT inhibition should be considered. On the other hand, previous studies have shown a
lack of effect of probenecid on OAT activity in MDCK cells (55) and M1 cell have not
shown demonstrable levels of the renal isoform OAT1. But since both the proximal and
distal nephron express OAT isoforms that are known to be inhibited by probenecid (27),
it is not likely an appropriate Panx1 inhibitor for studies involving renal tissue.
The finding of functional, ATP-releasing Panx1 channels in the renal tubular
epithelium adds to the growing evidence that Panx1 is an ATP conduit that plays a role in
purinergic signaling throughout the body. This role of Panx1 has been demonstrated in
several cell types and in other epithelia (10, 19, 25). In airway epithelial cells, Panx1
expression was found at the apical membrane, similar to the localization we observed in
the collecting duct, and inhibition of Panx1 reduced ATP release (39). Interestingly,
airway epithelia and renal epithelia share additional key characteristics. Both respond to
mechanical stress by releasing ATP and have ciliated cells which may act as a flow
sensor. P2 receptor activation by the released ATP regulates transport in both regions (17,
23, 52). In the collecting duct specifically, purinergic signaling inhibits salt reabsorption
via ENaC, water reabsorption via AQP2, and potassium secretion by ROMK. There is
also a flow-dependent component to the regulation of this transport, suggesting an ATP
release mechanism that can be mechanically activated (23). Our study provides new
129
evidence that Panx1, as a mechanosensitive ATP channel, could regulate purinergic
signaling in renal tubular epithelial cells.
The suggestion that Panx1 hemichannels could function as an ATP conduit in
cortical collecting ducts presents an interesting question about its role alongside that of
Cx30. We previously showed that Cx30 is expressed exclusively along apical membranes
in the collecting ducts of mice, presumably as Cx hemichannels (28). Additional work by
Sipos et al demonstrated that Cx30 is necessary for ATP release and therefore salt and
water transport regulation by purinergic signaling (47). Presently, however, there is no
direct evidence of ATP passage via Cx30 hemichannels. A model where Cx30 and Panx1
interact in the collecting ducts is one possibility that would explain both the shared
localization of both proteins and reconcile the dependence of collecting duct ATP release
on Cx30 with the results we present here. Interestingly, Panx1 and Cx30 mRNA have
also been found together in taste epithelium and human airway epithelia (19, 53). The
proposed binding between Panx1 and Cx43 via P2X7R also seems to support a possible
interaction between Cx30 and Panx1 (20). Analysis of the effect of Panx1 inhibition on
renal function as well as a more direct study of Cx30 and Panx1 protein-protein
interaction would be necessary however to validate this hypothesis.
Based on these finding, we speculate that Panx1’s role in renal physiology is to
facilitate renal transport in the tubular epithelium and therefore regulate fluid
homeostasis. If, as we predict, Panx1 is regulating ATP release in the collecting duct, an
in-vivo inhibition of Panx1 would show a similar physiological effect as that seen in
Cx30 knockout mice, namely a blunted pressure natriuresis response resulting in a salt-
130
retention phenotype. In the proximal tubule, luminal purinergic receptor activation
inhibits acidification (1) and relatively high levels of ATP have been found in the lumen
(51), suggesting that an ATP release mechanism is located along the brush border
membrane of proximal tubules. The localization of Panx1 in the proximal tubule provides
one possible explanation for these earlier findings.
A wide range of roles have been speculated for Panx1 in pathophysiology, but to
date, there is limited in-vivo evidence to support these ideas (4, 39, 43). In terms of renal
pathology, the discovery that Panx1 channels facilitate ATP release in MDCK cells
supports a role for Panx1 in regulating cysts development in autosomal dominant
polycystic kidney disease (ADPKD), the most common genetic cause of renal failure.
ADPKD is characterized by the presence of multiple cysts in the kidney which become
enlarged over time. In MDCK cells induced to form cysts, this enlargement is due to
P2YR activation by extracellular ATP (55). Additionally, ATP release rates are increased
in epithelial cells from polycystic kidneys (45), however the ATP release mechanism in
ADPKD is unknown. Panx1 could be responsible for this increase in ATP that stimulates
cyst growth and presents a possible therapeutic target. It remains to be determined
whether the cyst-forming models of MDCK also express Panx1 endogenously.
In summary, our results show that Panx1 hemichannels facilitate ATP release in
renal epithelial cells cultures and taken along with the localization results, we conclude
that Panx1 may be a mechanism by which ATP is released into the tubular lumen. As has
been shown with Cx30, this points to a role for Panx1 in the regulation of renal transport
mechanisms in the collecting duct by purinergic signaling. In-vivo studies using either
131
chemical inhibition or genetic modification of Panx1 expression are needed to establish
the role Panx1 plays in renal (patho)-physiology.
132
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CONCLUSION
Cx proteins comprise both gap junctions and hemichannels, both of which are
important mechanisms in intercellular communication in most organ systems. But details
about the nature of Cx expression and activity in the kidney were, until recently, lacking.
In the past five years however, much progress has been made in confirming earlier
reports of Cx localization within the nephron (Figure 2). Consequently, the functional
significance of renal
Cxs has also come to
light. In the JGA,
Cx40 has been
established as
essential for proper
regulation of renin
release and therefore
has been implied in
the maintenance of
systemic blood pressure. Gap junctions are known to facilitate the JGA calcium wave
which regulates TGF and as presented here Cx45 is one isoform now recognized as part
of this process. In the vasculature, various Cx isoforms are expressed and may be
required for the conduction of vasoconstriction and vasodilation responses.
1
Finally, in
the tubules, while multiple isoforms are expressed, so far only the function of Cx30 has
been established.
Figure 2: Expression of Cx isoforms along the nephron.
138
Cxs 30 and 30.3: regulating distal nephron function
Evidence of Cx isoform expression in the renal tubules was limited to studies of
mRNA expression in either cell lines or microdissected tissue. There was an additional
lack of functional data to suggest a role for Cxs along the renal epithelium. The
expression of two unrelated isoforms, Cx30 and Cx30.3, was detailed in the distal
nephron in chapters 1 and 2. Renal sections labeled by immunofluorescence showed that
Cx30.3 is expressed in the ascending thin limb of the loop of Henle while Cx30 has been
detected in the cortical thick ascending limb and both isoforms have been found in the
distal nephron. The expression of Cx30.3 in the loop of Henle was in agreement with an
earlier study of the global distribution of Cx30.3 using a transgenic reporter mouse model
where the LacZ gene is under the control of the Cx30.3 promoter.
2
Interestingly in the
cortical collecting duct both isoforms are found in the apical membrane of intercalated
cells, which play a role in acid-base transport. The adjacent principal cells do not express
either isoform. This expression along an unopposed membrane suggested that in this
context Cx30 and Cx30.3 are acting as hemichannels and not gap junctions. Previous
studies have shown a lack of gap junction coupling in the collecting duct cells, which
again supports the hemichannel theory.
3
By pooling these expression studies together with P2 receptor localization data, a
pattern emerges: Cxs are co-expressed with purinergic receptors. For example, P2Y2
receptors and Cx30 and 30.3 are all found on the apical surface of the collecting duct.
Moreover, oocyte studies have revealed that Cx hemichannels are gated by mechanical
139
stress.
4
This lends credence to the hypothesis that Cx hemichannels could be the long-
sought mechanosensitive ATP conduit in the kidney.
The Cx30 study also
presented information
about the regulation of
renal Cx30 protein
expression by salt levels,
with high-salt diet
correlated with an
increase in expression.
Based on this finding, it was suggested that Cx30 mediates salt and water reabsorption in
the distal nephron via purinergic signaling. New evidence from a study by Sipos et al has
tested this hypothesis and affirms a role for distal nephron Cx30 hemichannels in salt and
water handling.
5
Using an ATP biosensor technique, significant ATP release was
detected from the luminal membrane of isolated CCDs upon mechanical stimulation. A
loss of ATP release in Cx30 knockout mice suggested that ATP release in the CCD was
dependent on Cx30 hemichannels. Cx30 knockout mice also could not correct for acute
blood pressure increases by increasing salt and water excretion, indicating that Cx30
hemichannels play a role in the pressure-natriuresis phenomenon. Accordingly, high salt
diet increased blood pressure in these mice, which could be corrected for by
administration of an epithelial sodium channel inhibitor. These findings have led to the
establishment of a new model combining Cx-mediated ATP release and purinergic
Figure 3: The model of Cx30-dependent collecting duct transport
regulation by flow-induced purinergic signaling.
140
signaling in the distal nephron (Figure 3). In this model, increased pressure and tubular
flow triggers Cx30 hemichannel opening. ATP is then released into the lumen, where it
binds P2Y2 receptors in an autocrine/paracrine manner, inhibiting salt and water
reabsorption. The resulting increase in urinary output corrects increased pressure,
returning the body to homeostasis. The loss of Cx30 removes this inhibitory pathway,
leaving sodium reabsorption unchecked and the pressure-natriuresis phenomenon fails to
occur.
This research has demonstrated a clear, physiological function for Cx30 in renal salt
and water handling, but it also invites new speculation about how these Cx hemichannels
might be regulated by additional factors, both in terms of their expression and gating.
Examining gap junctions in astrocytes has led to the hypothesis that gap junction proteins
and P2 receptors form a functional unit, where their expression is co-regulated.
6
P2Y2
receptors in the collecting duct are known to be regulated by hydration states.
7
Cx30
expression may be changed under similar circumstances. How might Cx30 hemichannel
opening be regulated? It has been proposed that Cx30 hemichannel gating may involve a
flow sensor, suggested to be a cilia or microvilli, on the apical surface of either the
principal or intercalated cells of the collecting duct.
5,8
Our examination of Cx30.3
regulation failed to show an effect of either dietary salt or hypertension on the level of
protein expression and thus the role of Cx30.3 in the collecting duct also remains to be
resolved.
141
Cx45: calcium, renin, and the JGA
While evidence of Cx45 expression in both the developing and mature kidney has
been available for some time, a detailed description of Cx45’s localization within the
kidney was missing.
9
In Chapter 3, by using transgenic mice that express reporter genes
under the control of the Cx45 promoter, we were able to discern the Cx45-positive
regions of the kidney, circumventing the challenge of antibody specificity that plagues
studies of this isoform. Cx45 was expressed in the afferent and efferent arterioles and the
intra- and extraglomerular mesangium. The discovery of Cx45 in the JGA, a major
regulator of kidney function, suggests that Cx45 could be of significance to renal
physiology. Within the afferent arteriole, Cx45-positive vascular smooth muscle cells
were identified, confirming what others have observed in vasculature outside the
kidney.
10-12
Additionally, co-localization of the Cx45 signal with a renin antibody led to the
conclusion that Cx45 is expressed within the renin-producing JG cells. A different study
using an immunohistochemical approach confirmed the smooth muscle expression of
Cx45, but failed to find evidence of Cx45 in JG cells in the adult mouse.
13
The authors
did however observe Cx45-renin co-localization during embryogenesis. Both these
studies suggest that Cx45 is involved in renin cell regulation, challenging paradigms that
have typically regarded Cx40 as the “JG-cell Cx”.
1
Whether that role is developmental in
nature or occurs in the mature JGA, it is important to resolve the discrepancy in
expression pattern before drawing conclusions about Cx45’s function in the kidney.
142
At the molecular level, Cx45 may play a role in calcium wave propagation, a
fundamental JGA signaling pathway. Since Cx mediation of calcium wave propagation is
well-documented,
14-16
it was hypothesized that Cx45 could regulate calcium signaling in
the JGA. The JGA calcium wave is generated by ATP released from the macula densa
that propagates through the JGA in a gap junction-dependent manner.
16
Calcium in the
JGA regulates renal blood flow and glomerular filtratation by inhibiting renin release
from the JG cells and causing contraction of vascular smooth muscle cells in the afferent
arteriole.
17
When calcium signaling was studied in afferent arteriole vascular smooth
muscle cells (AA VMSC) isolated from mice with a conditional knockout of Cx45, a
reduction in propagation speed was observed, indicating that Cx45 mediates the calcium
wave in the afferent arteriole. Pretreatment of the cells with a Cx45 gap mimetic peptide
produced the same results. Generally, Cx-mediate calcium propagation can occur due to
either direct gap junction communication or purinergic signaling via ATP released from
Cx hemichannels. The purinergic antagonist suramin did not inhibit calcium signaling in
the AA VSMCs and a dye-transfer assay indicated direct cell coupling. Therefore it
seems that direct coupling via gap junctions is mostly likely the mechanism at work in
the afferent arteriole. These findings suggest that Cx45 could be mediating vasomotor
function in the afferent arteriole. However, in another study, when isolated renal
arterioles were treated with a Cx45 gap mimetic peptide, it failed to inhibit calcium
conduction along the vessels, implying that either another Cx or another mechanism is
involved.
18
These mixed results regarding Cx45’s role in calcium signaling in the JGA
may be due in part to the different models used. An in-vivo model that looks at
143
vasomotor function in relation to Cx45 expression might help clarify the mechanism that
occurs in the renal vasculature.
How might the actions of Cx45 at the cellular level translate to changes in
physiology? One possible role for Cx45 at the systemic level is in blood pressure
regulation. In accordance with the JGA’s role in modulating blood pressure, Cx45 in the
kidney may alter blood pressure through the regulation of the renin-angiotensin-
aldosterone system (RAAS). In investigating Cx45 conditional knockout mice, increased
renal renin content and plasma renin activity, as well as increased blood pressure was
observed. It has been speculated that the increased blood pressure is due to dysfunctional
feedback on the RAAS, as is the case with Cx40 knockout mice.
19-20
However, a detailed
mechanism explaining how Cx45 gap junction coupling in the JGA alters blood pressure
regulation still remains to be developed.
Because gap junctions are regulated on many levels, uncovering this mechanism is a
complex task that must take into account several factors. Just as Cxs can regulate
physiological processes, different physiological states can alter Cx expression and
function. For example, in cerebral arterial smooth muscle, Cx45 gap junction expression
and conductivity is altered in spontaneously hypertensive rats.
10
It is not yet known if
Cx45 expression or function is also affected by changes in blood pressure regulation.
Another factor to consider in analyzing Cx45’s role in renal physiology is the
intriguing possibility that Cx45 is interacting with other JGA Cxs. As previously
discussed, Cx40 is often considered the major JGA Cx, but this study shows that Cx45 is
expressed in this region too. Loss of either Cx40 or Cx45 shows dysfunctional regulation
144
of renin and increased blood pressure.
19-20
A new paradigm that incorporates these results
may be based on uncovering how these two isoforms interact. Newly released data has
examined this possibility in yet another transgenic mouse model, the Cx40KICx45 mouse
where the Cx40 coding sequence is replaced by the Cx45 coding region so that Cx45
expression replaces Cx40. Cx40KICx45 mice had lower blood pressure than Cx40KO
mice, but still had impaired vasodilator function.
21
Cx40KICx45 mice also regain the
RAAS feedback mechanisms that are missing in Cx40KO mice.
22
This data suggests that
Cx40’s role in renin release can be substituted for by Cx45. Moreover, it establishes that
the function of Cxs in the JGA does not depend on electrical properties, since Cx40 and
45 have different conductivities as well as sensitivities to cAMP, a well-known regulator
of renin release.
23
The ability of Cx45 to replace Cx40 function in calcium wave
propagation and vasomotor function within the JGA still needs to be assessed.
In light of this research, how might Cx40 and Cx45 function together in the JGA?
One potential explanation is that the two isoforms are functionally redundant. But if the
expression and function of Cx45 and Cx40 overlap, why do they not compensate for each
other in their respective knockout models? Also, the study of Cxs in the renal
vasculature/JGA relies heavily on transgenic mouse models, which begs the question
whether genetic knockout of one Cx isoform may change the expression of another.
Could different Cx isoforms be directly interacting? Studies in other cell types have
shown that Cx45 can form heterotypic channels with Cx40 and Cx43 and that each
combination has unique properties.
24-27
While Cx45 and Cx43 are not co-expressed in the
JGA, Cx45 and Cx40 are both found in the mesangium and the JG cells, making the
145
formation of heterotypic channels a distinct possibility. Clearly the discovery of Cx45 in
the JGA has raised many interesting questions that will hopefully be answered by future
studies.
Panx1: an alternative ATP release mechanism in the renal epithelium
Chapter 4 presented the first study of Panx1 expression in the kidney. Using
immunofluorescence, Panx1 was localized along the renal epithelium, from the proximal
tubules to the collecting ducts. Since Panx1 has been shown to act as a mechanosensitive
ATP channel in other cell types, two renal epithelial cell lines, MDCK and M1, were
identified as expressing Panx1 endogenously and used to test its function. ATP release
was induced by flow and was reduced when Panx1 channels were inhibited. The
functional aspect of this study provides the initial evidence that Panx1 channels may
release ATP in the tubular lumen in response to stress and suggests that they mediate
purinergic signaling in the nephron.
Full assessment of the physiological role of Panx1 in the kidney requires an
investigation beyond the cellular level, ideally in a whole animal model where Panx1
expression or activity can be modulated. Currently there is no Panx1 transgenic mouse
model and use of the Panx1 inhibitor probenecid is inappropriate, as it also inhibits the
organic anion transporter OAT1. Another option is to adapt the siRNA approach used in
this study on cells to systemically inhibit Panx1 in either mice or rats. This technique
could also be used in a whole kidney model, which would increase target specificity and
reduce the confounding effects of inhibiting Panx1 in other organs.
146
If indeed Panx1 functions as the ATP channel in the renal epithelium, then there
is a need to explain these findings in light of the Cx30 model of distal nephron purinergic
signaling.
5
ATP release in response to flow in isolated cortical collecting ducts was
reduced but not completely abolished in Cx30 mice, suggesting that Cx30 and Panx1 may
be redundant in function in this region of the kidney. Alternatively, Cx30 and Panx1 may
interact. Cx30 was shown to be required for ATP release and purinergic signaling in the
kidney, but the permeability of Cx30 hemichannels to ATP has not been directly proven.
Moreover, Panx1 channels lack several key limitations to the hypothesis that Cx
hemichannels release ATP under physiological conditions. Flow in the collecting duct,
the ATP release stimulus, causes an increase in intracellular calcium levels, which
inhibits Cx hemichannel opening.
28
Physiological extracellular calcium levels also reduce
Cx hemichannel opening. Finally, Panx1 was expressed in both intercalated cells, where
Cx30 is expressed, and principal cells, where transport is altered by P2 receptor
activation.
29
Therefore, it is also feasible that ATP may enter the collecting duct lumen
via Panx1 channels and that this transport requires the expression of Cx30 in intercalated
cells.
Beyond the collecting duct, Panx1 was expressed in the proximal tubules and thin
descending limb of the loop of Henle. This expression in the early renal tubules coincides
with expression of Cx37 and Cx43, but was not specifically found at the apical
membrane. P2 receptor expression has been found in both segments of the nephron in
apical and basolateral poles and significant levels of ATP have been detected in the
proximal tubules.
30
Given the expression of ectonucleotidases in this region, ATP must
147
be locally released in the proximal tubules and Panx1 may be the ATP conduit. In
concordance with the expression of Cx30 and Panx1 in the collecting duct, there is
evidence of Cx43 hemichannels in proximal tubule cells.
31
Additionally, Cx43 and Panx1
have been shown to interact via the P2X7 receptor, which again suggests that Panx1 and
Cx hemichannels may interact.
6
This study of Panx1 in the renal tubules provides preliminary insights into the role
of pannexins in the kidney and the field of pannexins itself is still in its infancy. As the
field moves beyond experiments at the cellular level and into whole organ and whole
animal studies, the relevance of pannexins to physiological mechanism can be better
ascertained. However, the finding that flow-induced ATP release is dependent on Panx1
in renal epithelial cells suggests that it is an important player in renal purinergic signaling
and therefore future developments should focus on how Panx1 alters epithelial transport
mechanisms.
In summary, the significance of Cxs to renal physiology has recently begun to
emerge. Renal Cxs contribute to intercellular communication as either gap junctions that
directly link cells or as hemichannels that release signaling molecules like ATP which
propagate messages in the extracellular milieu. The examinations of Cx30 and Cx45 in
the kidney provide examples of both types of Cx communication. In the distal nephron
Cx30 expression is regulated by salt and later work has now shown it to be required for
flow-stimulated ATP release. In the JGA, Cx45 behaves as a gap junction, coupling
VSMCs and propagating calcium waves among these cells. Its expression in the renin-
producing JG cells suggests it may play a role in calcium signaling in these cells as well.
148
The downstream effects of signaling via these Cxs on renal mechanisms is profound, with
Cx30 required for pressure-natriuresis and proper regulation of renin and blood pressure
depending on Cx45 expression in the kidneys. The physiological significance of kidney
Cx30.3 has not been established by this work, but its expression in apical membranes of
the distal nephron also points to it functioning as a hemichannel. Finally, pannexins
present a compelling alternative to Cxs as the renal ATP conduit, with this work
demonstrating that renal epithelial cell ATP release is under the control of Panx1.
149
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Abstract (if available)
Abstract
Connexins are the constituent proteins of a gap junction, a non-selective channel that links together two cells, allowing the passage of molecules directly between them. In addition, connexins are thought to form discrete “hemichannels” in uncoupled cells. This conformation permits molecular exchange between the intra- and extracellular environments. Pannexins are a structurally similar, but genetically unrelated class of proteins that appears to share the hemichannel-like structure and function of connexins, but does not engage in gap junction formation. Both connexins and pannexins are ubiquitously expressed and regulate intercellular communication, both through direct cell-cell coupling and via the release of secondary messengers such as ATP to the extracellular fluid. Gap junction coupling is well-established in the kidney and intercellular communication pathways that are critical to controlling major renal regulatory mechanisms are known to involve connexins and pannexins in other cell types. However, a systematic study of the expression and function of these proteins in the kidney has been absent. Therefore we sought to examine connexin and pannexin localization within the kidney and, based on these findings, investigate how they facilitate the signaling mechanisms and physiology at work in these regions. Based on initial reports of mRNA expression, we focused on four select isoforms. Chapters 1 and 2 delve into the expression and regulation of two connexins, Cx30 and Cx30.3, along the distal nephron. Finding expression of both isoforms in the apical membrane of the renal epithelial cells suggested a function for Cx30 and Cx30.3 as an ATP release mechanism which may regulate salt and water transport in the distal nephron through purinergic signaling. Chapter 3 details the expression of Cx45 in the juxtaglomerular apparatus.
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Creator
Hanner, Fiona P
(author)
Core Title
Connexins and pannexins in the kidney: a study of their expression, regulation, and function
School
Keck School of Medicine
Degree
Doctor of Philosophy
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Systems Biology
Publication Date
04/01/2010
Defense Date
03/15/2010
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University of Southern California
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calcium signaling,epithelial transport,gap junction,hemichannel,intercellular communication,OAI-PMH Harvest,purinergic signaling,renal hemodynamics
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Peti-Peterdi, Janos (
committee chair
), Garner, Judy A. (
committee member
), Kobielak, Agnieszka (
committee member
), Sampath, Alapakkam P. (
committee member
), Yu, Alan S. L. (
committee member
)
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fiona.hanner@gmail.com,fmccullo@usc.edu
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Tags
calcium signaling
epithelial transport
gap junction
hemichannel
intercellular communication
purinergic signaling
renal hemodynamics