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Integrated regulation of transporters along the nephron to maintain electrolyte and fluid homeostatis

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Integrated regulation of transporters along the nephron to maintain electrolyte and fluid homeostatis

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Content INTEGRATED REGULATION OF TRANSPORTERS ALONG THE NEPHRON TO
MAINTAIN ELECTROLYTE AND FLUID HOMEOSTASIS

by

Mien Thi Xuan Nguyen

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

August 2013

Copyright 2013 Mien T.X. Nguyen
ii

Table of Contents
List of abbreviations iii
Abstract iv
Chapter 1: Introduction
A. Role of kidneys in K
+
homeostasis 1
B. Role of the renin-angiotensin system in regulating Na
+
transport
and extracellular fluid volume
B.1. The systemic renin-angiotensin system 3
B.2. The intrarenal renin-angiotensin system 5

Chapter 2: Effects of K
+
-deficient diets with and without NaCl
supplementation on Na
+
, K
+
, and H
2
O transporters’ abundance
along the nephron
Abstract 7
Introduction 8
Materials and Methods 10
Results 13
Discussion 31
Chapter 3: Differential regulation of Na
+
transporters along
nephron during AngII-dependent hypertension: Distal
stimulation counteracted by proximal inhibition
Abstract 40
Introduction 41
Materials and Methods 42
Results 45
Discussion 58
Chapter 4: Renal responses to short-term non-pressor angiotensin II
with and without angiotensin-converting enzyme inhibition in rats
Abstract 64
Introduction 65
Materials and Methods 67
Results 69
Discussion 80
Chapter 5: Overall summary and future directions 85
Bibliography 90
iii

List of abbreviations
AngII: Angiotensin II
ACE: Angiotensin-converting enzyme
AQP2: aquaporin-2
BP: blood pressure
CD: collecting duct
CNT: connecting tubule
DCT: distal convoluted tubule
ECFV: extracellular fluid volume
ENaC: epithelial Na
+
channel
NaPi2: Na
+
/Pi cotransporter type 2
NCC: Na
+
-Cl
-
co-transporter
NHE3: Na
+
/H
+
exchanger isoform 3
NKCC: Na
+
-K
+
-2Cl
-
co-transporter
OSR1: oxidative stress responsive kinase-1
PT: proximal tubule
RAS: renin-angiotensin system
ROMK: renal outer medullary K
+
channel
SPAK: Ste20/SPS-1 related proline-alanine rich kinase
TALH: thick ascending limb of Henle’s loop
U
K
V: urinary K excretion
U
Na
V: urinary Na excretion
U
osm
V: urinary osmolality excretion
UV: urine volume
WNK: with-no-lysine kinase

iv

Abstract
The kidneys play an essential role in regulating blood pressure (BP) and maintaining fluid and
electrolyte homeostasis. The balance is achieved by an integrative network of hormones and
nerves that signal changes in Na
+
and K
+
transporters along the nephron under various stimuli,
such as hypokalemia, elevated circulating plasma AngII concentration, and high BP. The
regulation could be either acute or chronic via trafficking, covalent modification, or changes
in total transporter abundance. Taking an integrative approach to generate transporter profiles
along the nephron, this dissertation aimed to determine 1) the effects of K
+
-deficient diets
with and without NaCl supplementation on Na
+
, K
+
, and H
2
O transporters’ abundance; 2) how
transporters are differentially regulated in AngII-dependent hypertension model: a balance
between blood pressure vs. AngII; and 3) the renal responses at the early stage of AngII
infusion without accompanying hypertension. Results: 1) Despite profound hypokalemia and
renal K
+
conservation with K
+
-deficient diets, most transporters’ abundance was not altered as
previously reported in the literature; there was primarily a decrease in the sodium channel
whose activity drives K
+
secretion, but no change in the K
+
transporter. We predict that
changes in their distribution and activity are likely more important for conserving K
+
than
changes in total pool size, since they need to act quickly when an animal suddenly switches
from a K
+
-deprived mode to a K
+
-replete mode. 2) In AngII-dependent hypertension, we
reported a differential regulation of Na
+
transporters along the nephron: distal stimulation
counteracted by proximal inhibition to obtain a homeostatic balance. We postulate that
starting from the cortical TALH through the medullary CD, AngII has an anti-natriuretic
action on Na
+
transporters to increase Na
+
reabsorption in the distal region of the nephron,
while pressure-natriuresis acts along the proximal nephron, starting from the PT through the
v

medullary TALH, to reduce Na
+
reabsorption. 3) At the early stage of AngII infusion, we
demonstrated that Na
+
transporters are activated in both the proximal tubule and distal
nephron, creating a pre-hypertensive state, which likely increases Na
+
reabsorption in these
regions to contribute to the development of hypertension observed with longer infusion
periods. Overall, these studies illustrate that a determination of changes in ion transporter
along the entire nephron are necessary to ascertain how fluid and electrolyte homeostasis is
maintained in the face of changes in intake or experimental hypertension.

1

Chapter 1. Introduction
A. Role of kidneys in regulating K
+
homeostasis
Plasma K
+
level has to be tightly controlled since it is the major determinant of cell membrane
action potential and excitability. To maintain the plasma K
+
level in the normal range, the
kidneys are very efficient at balancing the amount of K
+
excreted to match with the dietary K
+

intake. Many studies have investigated the renal adaptations to conserve K
+
when the intake is
less than output and to quickly stimulate K
+
secretion when K
+
intake increases (review in
(34)). Along the nephron, the reabsorptive and secretory components of K
+
transport in the
loop of Henle and further distal nephron segments are subject to regulation while transport in
proximal tubule does not control urinary K
+
excretion (Fig. 1.1). During a prolonged fasting
period or a K
+
-deficient diet, there is an increased fractional reabsorption of K
+
in the early
nephron and decreased K
+
delivery to the distal nephron (98), causing urinary K
+
excretion to
fall dramatically, evidence of renal adaptation. On the other hand, when a high K
+
diet is
consumed, the kidneys quickly excrete K
+
to bring plasma K
+
level back to the normal
physiological range. A major mechanism for K
+
secretion is the generation of a lumen-
negative potential by the reabsorption of Na
+
through the apical epithelial Na
+
channel
(ENaC) located within the same cells. I was intrigued by the question of how the body of a
carnivorous mammal in the wild punctuating long fasting periods responded after the animal
successfully hunted down and consumed another K
+
-rich mammal and how K
+
homeostasis is
achieved quickly enough to prevent the animal from disturbances affecting cardiac and
nervous functions.
Hypokalemia is a common electrolyte disorder that can be life threatening when severe
([K
+
] < 2.5 mEq). It has been estimated that > 20% of hospitalized patients develop
2

hypokalemia. Common causes of chronic hypokalemia include inappropriate renal loss
(diuretics) or gastrointestinal loss (diarrhea). Prolonged fasting, famine, and anorexia can also
lead to hypokalemia. Previous studies have reported the effects of chronically removing K
+

from the diet and hypokalemia, including decreased body weight gain and marked kidney
hypertrophy (38), as well as multiple changes in Na
+
and K
+
transporters along the nephron
(13, 15, 22, 45, 56, 59). Interestingly, the results have not been consistent among groups,
which could be attributed to various methods of sample preparation from homogenates to
fractionated membranes or the differences in diet composition between control regular and
K
+
-deficient diet. Chapter 2 of my thesis aimed to explore renal responses to K
+

restriction/hypokalemia by examining the total pool size of transporters (including active and
reserved pools) along the nephron, rather than membrane fractions, to understand how K
+

homeostasis is achieved in these settings.

Figure 1.1. Potassium transport along a simplified nephron. PCT, proximal convoluted
tubule; PST, proximal straight tubule; DTL, descending thin limb of Henle’s loop; ATL,
ascending thin limb of Henle’s loop; MTAL and CTAL, medullary and cortical portions of
the thick ascending limb; DCT, distal convoluted tubule; CNT, connecting tubule; ICD, initial
collecting duct; CCD, cortical collecting duct; OMCD and IMCD, outer and inner medullary
collecting duct. The arrow size represents relative magnitudes of K+ secretion and
reabsorption. (34).
3

B. Role of the renin-angiotensin system (RAS) in regulating Na
+
transport and
extracellular fluid volume (ECFV)
B.1. The systemic renin-angiotensin system
Hypertension, defined as BP above 140 (systolic) / 90 (diastolic) mmHg, affects more than
30% United States adults and costs ~ $156 billion
(http://www.hhs.gov/news/press/2012pres/05/20120502a.html). It increases risks of
cardiovascular diseases, such as stroke, heart failure, and atherosclerosis, as well as kidney
diseases. The RAS has been a key regulator of the ECFV and BP, since medications targeting
different components of the RAS, including angiotentensin-converting enzyme (ACE)
inhibitors and AngII receptor blockers, have been very effective in lowering blood pressure in
hypertensives. The active hormone synthesized from this pathway is angiotensin II (AngII)
(Fig. 1.2), which is known to be a potent vasoconstrictor and stimulate Na
+
transporters along
the nephron, thus, increase overall Na
+
reabsorption. As a result, ECFV is expanded causing
an increase in cardiac output and elevated BP. As a compensatory mechanism, BP elevation
provokes pressure-natriuresis to reduce Na
+
reabsorption and restore ECFV (summarized in
Fig. 1.3).

Figure 1.2. The renin-angiotensin system.
In the past, the McDonough laboratory has been focusing on the effects of acute AngII
infusion on Na
+
transport via proximal tubule NHE3 and NaPi2 and distal convoluted tubule
NCC and the molecular mechanisms responsible for the interdependence of BP, ECFV and
RAS. 20 min of AngII infusion in anesthetized rats without raising BP redistributes PT NHE3
4

from the base to the body of the microvilli, presumably to increase Na
+
transport (81), and
provokes acute trafficking of NCC from subapical vesicles into the plasma membrane (86),
without changing the total abundance of either NHE3 or NCC. On the other hand, blocking
AngII formation with an ACE inhibitor, captopril, retracts NHE3 out of the body of the
microvilli into the base of the microvilli and NaPi2 into endosomes, presumably to depress PT
reabsorption (49), and provokes NCC redistribution from the plasma membrane into subapical
vesicles to decrease DCT Na
+
reabsorption (86).

Figure 1.3. Feedback relationship between BP, AngII, sympathetic nervous system
(SNS), and ECFV (58)

Chronic elevation in circulating AngII level produces sustained hypertension, thus,
chronic AngII infusion makes a great experimental hypertension model. The interesting
question is how the kidneys balance the effects of AngII (anti-natriuresis) versus elevated BP
(natriuresis) to maintain electrolyte and fluid homeostasis. Many groups have investigated
how Na
+
transporters are regulated focusing on specific regions in AngII-dependent
hypertension: increased DCT NCC and CCD ENaC (5, 25, 95) and decreased PT NHE3 and
5

TALH NKCC2 (31), suggesting anti-natriuresis in the distal nephron and pressure-natriuresis
in the proximal nephron. Using an integrative approach to study the cortex separated from the
medulla, chapter 3 sought to address where along the nephron Na
+
transport is stimulated by
AngII and depressed by elevated BP.
B.2. The intrarenal renin-angiotensin system
In recent years, the idea of a complete RAS within the kidneys has emerged (review in (66));
specifically, when plasma AngII is elevated, angiotensinogen is synthesized by the proximal
tubule cells, released into tubular lumen, and spilled over into the distal nephron; renin is
secreted from the principle cells of the collecting duct. Together with the presence of ACE in
the distal nephron, AngII is formed locally and binds to AngII receptor type 1 (AT1R) along
the nephron to increase Na
+
reabsorption by increasing phosphorylation of NKCC2 in the
TALH and of NCC in the DCT (Fig. 1.4). Therefore, chronic AngII infusion further activates
intrarenal RAS, and the increase in local AngII formation may induce hypertension.
With longer AngII infusion period, hypertension develops and sustains, presumably
due to the activation of the intrarenal RAS that increases Na
+
reabsorption in the distal
nephron. Elevated BP would in turns down-regulate transporters in the early nephron via
pressure-natriuresis mechanism. The interesting questions are what happen during the earlier
time course of AngII infusion (3 days) before hypertension and pressure-natriuresis develop,
and will intrarenal RAS play an important role in regulating Na
+
transporters at this early time
point?
6

Figure 1.4. AngII infusion stimulates intrarenal RAS production. Increased AngII in
tubular fluid acts on AT1R to activate regulatory kinases and Na
+
transporters along the
nephron. Even when intrarenal RAS is inhibited AngII stimulates ENaC activation (25).

Chapter 4 of my dissertation investigated the renal responses to a short-term non-
pressor dose of AngII, to ensure that they are purely AngII-mediated, and not affected by
pressure-natriuresis. It also aimed to determine which transporter(s) would be dependent on
the activation of local RAS at this early time course.
Using the integrative approach to study Na
+
transporters along the nephron, the overall
aim of my dissertation is to understand how the kidneys adapt to various stimuli, including
hypokalemia, chronic AngII infusion with and without accompanying hypertension, which are
crucial for the body to maintain overall fluid and electrolyte homeostasis.

7

Chapter 2. Effects of K
+
-deficient diets with and without NaCl supplementation on
Na
+
, K
+
, and H
2
O transporters’ abundance along the nephron (Nguyen,
M.T., et al. Am J Physiol Renal Physiol. 303(1): F92–F104, 2012 July 1.
PMID: 22496411)

Abstract
Dietary potassium (K
+
) restriction and hypokalemia have been reported to change the
abundance of most renal Na
+
and K
+
transporters and AQP2, but results have not been
consistent. The aim of this study was to re-examine Na
+
, K
+
and H
2
O transporters’ pool size
regulation in response to removing K
+
from a diet containing 0.74% NaCl, as well as from a
diet containing 2% NaCl (as found in American diets) to blunt reducing total diet electrolytes.
Sprague-Dawley rats (n = 5-6) were fed for 6 days with one of these diets: 2% KCl, 0.74%
NaCl (2K1Na, control chow) compared to 0.03% KCl, 0.74% NaCl (0K1Na), or 2% KCl,
2%NaCl (2K2Na) compared to 0.03% KCl, 2% NaCl (0K2Na, Na
+
replete). In both 0K1Na
and 0K2Na there were significant decreases in: plasma [K
+
] (< 2.5 mM); urinary K
+
excretion
(< 5% of control); urine osmolality and plasma [aldosterone], as well as an increase in urine
volume and medullary hypertrophy. The 0K2Na group had the lowest [aldosterone] (172.0 ±
17.4 pg/ml) and lower blood pressure (93.2 ± 4.9 versus 112.0 ± 3.1 mmHg in 2K2Na).
Transporter pool sizes regulation was determined by quantitative immunoblotting of renal
cortex and medulla homogenates. The only differences measured in both 0K1Na and 0K2Na
groups were: a 20-30% decrease in cortical β-ENaC, 30-40% increases in KS-SPAK, and a
40% increase in medullary sodium pump abundance. The following proteins were not
significantly changed in both the 0K groups: NHE3, NKCC, NCC, OSR1, ROMK, ARH, c-
Src, AQP2 or renin. Thus, despite profound hypokalemia and renal K
+
conservation, we did
not confirm many of the changes that were previously reported. We predict that changes in
8

transporter distribution and activity are likely more important for conserving K
+
than changes
in total abundance.
KEYWORDS: hypokalemia, aldosterone, ROMK, Na
+
transporters, blood pressure

Introduction
Plasma potassium must be closely controlled because it is a key determinant of membrane
potential and excitability. This is critical for both cardiac action potential and CNS
excitability (69). Among the major electrolytes, K
+
has the highest ratio of daily intake to
extracellular pool size (i.e., turnover), that is, the most significant homeostatic challenge
(108). To meet this challenge, the kidneys are very efficient at clearing K
+
in proportion to
K
+
intake and the kidneys and muscles are efficient at maintaining extracellular fluid (ECF)
[K
+
] between meals. During prolonged fasting or a K
+
deficient diet, urinary K
+
excretion
falls dramatically, evidence of renal adaptation. Eventually, plasma K
+
falls indicating that
muscle K
+
stores cannot fully compensate when K
+
output chronically exceeds K
+
input (92).
In this setting, renal adaptations and compensations are key to reducing K
+
excretion in order
to prevent K
+
losses from the body.
Many studies have investigated the renal adaptations to conserve K
+
when intake is less than
output. Along the nephron, reports include reduced glomerular filtration rate (GFR),
increased fractional reabsorption of K
+
in the early nephron, decreased K
+
delivery to the
distal nephron (98), and polyuria. Abundance of the K
+
secretion pathway in the distal
nephron principal cell, the renal outer medullary K
+
channel (ROMK), has been reported to
both decrease (22, 61) and to not change (97), yet ROMK has been reported to redistribute out
of the apical membranes secondary to an increase in protein tyrosine kinase (PTK) and c-Src
9

mediated phosphorylation of ROMK (99). Abundance of intercalated cell H,K-ATPases has
been reported to increase to facilitate active K
+
reabsorption (13, 45). Many other effects of
chronically removing K
+
from the diet and hypokalemia have also been reported, including
decreased weight gain and marked kidney hypertrophy (38), multiple changes in Na
+

transporters including: a four-fold increase in Na
+
/H
+
exchanger isoform 3 (NHE3) abundance
along with profound decreases in Na
+
-K
+
-2Cl
-
co-transporter (NKCC), Na
+
-Cl
-
cotransporter
(NCC) and epithelial sodium channel (ENaC) (15), as well as no change in NCC (22, 94),
increased Na,K-ATPase abundance in the distal nephron (59), decreased aquaporin 2 isoform
(AQP2) abundance in cortex and medulla (56), and recruitment of renin along the afferent
arterioles (77).
Previous studies of the effects of K
+
deficiency on transporter abundance utilized a
variety of starting samples from homogenates to fractionated membranes. The first aim of
this study was to measure the pool size of Na
+
, K
+
and H
2
O transporters in unfractionated
homogenates prepared from renal cortex and medulla in hypokalemic versus control rats
using quantitative immunoblotting. Previous studies compared control vivarium chow (~ 2%
KCl, 0.74% NaCl), to KCl deficient chow (< 0.03% KCl, 0.74% NaCl), which yields a diet
with low overall electrolyte content. A second aim of this study was to test the effect of
elevating NaCl to partially compensate for the decrease in KCl to rule out this potential
complication. Specifically, we compared the effects of normal dietary KCl versus K
+

deficiency in diets that contained either control dietary NaCl (0.74%) or NaCl increased to
2%, which is the amount of salt found in the typical American diet (93). A third aim was to
carefully control regular versus K
+
deficient diet composition by starting with a single
powdered diet and varying only the KCl and NaCl content. Thus, we aimed to examine the
10

effects of hypokalemia per se on physiologic parameters as well as the total pool size of Na
+
,
K
+
and H
2
O transporters. We discovered that while most physiologic parameters previously
reported were replicated in this study, only ENaC β and medullary sodium pump abundance
were significantly altered during hypokalemia in both the 0K diet fed groups.

Materials and Methods
Animal protocols. All animal procedures were approved by the Institutional Animal Care and
Use Committee of the Keck School of Medicine of USC and were conducted in accordance
with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Experiments were performed on male Sprague-Dawley rats (225−250 g) purchased from
Harlan Laboratories (San Diego, CA).
Animals were divided into 4 groups of n = 5-6 and fed with diets prepared from
potassium-deficient powdered rat chow (TD. 88239, Harlan-Teklad, Madison, WI) which was
supplemented with KCl and/or NaCl to the following percentages (in dry weight): 0.03% KCl
and 0.74% NaCl (0K1Na), 2% KCl and 0.74% NaCl (2K1Na), 0.03% KCl and 2% NaCl
(0K2Na), and 2% KCl and 2% NaCl (2K2Na). To gel the diets, 25 g of Difco Agar Noble was
dissolved by heating in 835 ml of deionized water and added to 500 g of dry diet. Diet was
stored at -20
o
C in meal size blocks until use. Rats were provided with 60−70 g of gelled
diets per rat per day and free access to water for 6 days. To increase ENaC and SPAK
expression, a subset of rats was fed a pelleted sodium-deficient diet for 6 days (TD.90228,
Harlan-Teklad, Madison, WI).
11

Physiologic measurements. At the end of the 6 day dietary treatment period, urine was
collected in metabolic cages (Techniplast) overnight (16−18 hrs) and animals were weighed.
Rats were anesthetized intraperitoneally with Inactin (Sigma; 100 mg/kg), body temperature
maintained thermostatically at 37
0
C, and cannulas inserted in the jugular for fluid infusion
(0.9% NaCl + 4% BSA, 50 μl/min) and into carotid artery for blood pressure measurement.
After blood pressure was stabilized and recorded, blood samples were collected, plasma
prepared, and kidneys removed and weighed.
Urine volume was recorded in graduated cylinders, urine and plasma [Na
+
] and [K
+
]
measured by flame photometry (Radiometer FLM3), and osmolality with an osmometer
(Precision Systems µOsmette). Plasma aldosterone levels were determined by
125
I
radioimmunoassay (Coat-A-Count
®
, TKAL kit, Siemens Healthcare Diagnostics).
Homogenate preparation. Cortex and medulla (outer and inner) from both kidneys of each rat
were dissected, diced and suspended separately: cortex in 5 ml and medulla in 3 ml of
isolation buffer (5% sorbitol, 0.5 mM disodium EDTA and 5 mM histidine- imidazole buffer,
pH = 7.5, with the addition of 0.2 mM phenylmethylsulfonyl fluoride, 9 μg/ml aprotinin and 5
µl/ml phosphatase inhibitor cocktail (Sigma)). Each sample was homogenized for 5 min at a
low speed setting with an Ultra-Turrax T25 (IKA
®
- Labortechnik) then centrifuged at 2,000 x
g for 10 min. Supernatants were retained and the cortex (not medulla) pellets were re-
homogenized in another 5 ml of isolation buffer, re-centrifuged and pooled with the first
supernatants. The 2,000 x g supernatant (S
o
) protein concentrations were determined using
the Pierce BCA kit (Thermo Scientific, IL). The samples were aliquoted and stored at -80
o
C.
S
o
protein concentrations were ~ 10 mg/ml for cortex and ~ 3 mg/ml for medulla. The low
12

speed pellets, assayed by immunoblot, contained only negligible amounts of NHE3 and NCC
(not shown) and were discarded.
Differential fractionation of ICM vs. PM. In a subset of samples, intracellular and plasma
membranes were enriched as described by Sachs et. al. (83). In brief, the 2,000 x g
supernatant prepared above was spun at 17,000 x g; the resultant 17,000 x g pellet, enriched in
plasma membranes (PM), was resuspended in isolation buffer (see Homogenate preparation).
The 17,000 x g supernatant was spun at 150,000 x g for 80 min and the pellet, enriched in
intracellular membranes (ICM), was resuspended in isolation buffer. Aliquots of the PM and
ICM fractions were frozen at -80
o
C pending assay.
Quantitative immunoblotting. Cortical and medullary homogenates were denatured in SDS-
PAGE sample buffer for 20 min at 60
0
C then resolved on SDS-polyacrylamide gels (47). For
each sample, one-half amount of protein was loaded adjacent to the full amount of protein to
verify linearity of the detection system, as evident in the figures. Additionally, loading gels
were run and stained with Coomassie blue, and random bands were quantified to verify that
total protein loading was uniform. Gels were transferred to polyvinylidene difluoride
membranes (Millipore Immobilon-FL), blocked (bløk-FL, Millipore) and then probed with
one of the following antibodies (diluted in: TBST, 1% BSA, 15 mM NaN
3
): polyclonal anti-
NHE3 (1:2,000; Millipore, Temecula, CA); monoclonal anti-NHE3 phosphorylated at Ser552
(NHE3pS552, 1:1,000; (44)); anti-NaPi2 (1:1,000; McDonough lab (104)); monoclonal anti-
NKCC2 (1:1,000; C. Lytle, Univ. of California Riverside (53)); polyclonal anti-NKCC2
phosphorylated at Thr96, Thr101 (NKCC2pT96T101, 1:2,000; B. Forbush, Yale Univ. (19));
anti-NCC (1:1,000; D. Ellison, Oregon Health and Science Univ.(6)); anti-NCC
phosphorylated at Ser71 (NCCpS71, 1:1,500; S. Bachmann lab (63)); anti-α-ENaC, anti-β-
13

ENaC, and anti-γ-ENaC (all 1:1,000; L. Palmer, Weill Cornell Univ. (16)); anti-ROMK
(1:2,000; Alomone Labs, Israel); anti-c-Src (1:500; Santa Cruz Biotechnology); anti-AQP2
(H-40) (1:1,000; Santa Cruz Biotechnology); monoclonal anti-NKAα
1
(464.6, 1:200; M.
Kashgarian, Yale Univ.); anti-NKAβ
1
(1:500; McDonough lab); anti-renin (1:2000; AnaSpec,
San Jose, CA); anti-ARH (1:2000; Abcam, Cambridge, MA); anti-SPAK (1:1000; E. Delpire,
(73)); anti-OSR1 (1:1,000; Division of Signal Transduction Therapy, Dundee, UK). Primary
antibodies were recognized by either Alexa Fluor 680- (Invitrogen) or IRDye 800- (LI-COR)
labeled goat anti-rabbit, goat anti-mouse, or donkey anti-sheep secondary antibodies. Signals
were detected with Odyssey Infrared Imaging System (LI-COR, Lincoln, NE) and quantitated
by accompanying software.
Quantitation and statistical analysis. The range for linearity of signal intensity with sample
loading was established for each protein and on each blot by loading 1 and ½ amounts of each
sample side by side to verify doubling of signal intensity with doubling of sample volume.
Absorbance values of 0K diet groups were normalized to mean intensity of paired 2K diet
groups defined as 1.0 (0K1Na versus 2K1Na and 0K2Na versus 2K2Na). The normalized
values for the 1 and ½ protein loading lanes were averaged. Difference in total abundance
and phosphorylation of transporters and their associated proteins was assessed by unpaired
two-tailed Student’s t-test, not assuming equal variance. Data were expressed as means ±
SEM. Differences were regarded significant at P < 0.05.

Results
Effects of K
+
deficient diets with 0.74% NaCl (0K1Na) and with 2% NaCl (0K2Na) on
physiological parameters. To test whether the overall level of dietary electrolytes influences
14

the responses to K
+
deficiency, Sprague-Dawley rats (n = 5-6/group) were fed one of the
following diets for 6 days. 1) K
+
deficient diet with the sodium level typically found in control
chow: 0.03% KCl, 0.74% NaCl (0K1Na); 2) diet with Na
+
and K
+
levels typically found in
control chow: 2% KCl, 0.74% NaCl (2K1Na); 3) K
+
deficient diet with NaCl level raised to
2%, that found in the typical American diet: 0.03% KCl, 2% NaCl (0K2Na), and 4) diet with
control level of KCl and NaCl elevated to 2%: 2% KCl, 2% NaCl (2K2Na). Six days on the
K
+
deficient diets reduced plasma [K
+
] from control levels of 3.6 - 3.8 mM to 2.5 ± 0.1 mM in
the 0K1Na group and 2.4 ± 0.1 mM in the 0K2Na group (Table 2.1). Plasma [Na
+
] was
unchanged, remaining at 137 - 140 mM. Similar effects of K
+
deficient diet have been
previously reported (15, 38). Recently, Abu Hossain et al. (1) reported that 6 day K
+
deficient
diet did not significantly reduce plasma [K
+
]: [K
+
] was 5.1 mEq versus 4.9 mEq in control
versus K
+
deficient diet fed rats. It is not obvious whether diet, method of anesthesia or
sacrifice, collection or measurement accounts for this difference. Plasma aldosterone, which
is increased by both high K
+
diets and low Na
+
diets, was predictably highest in the group fed
with 2K1Na (831 ± 88 pg/ml), lower in the groups fed 2K2Na and 0K1Na (417 ± 19 and 413
± 21 pg/ml, respectively), and lowest in the group fed 0K2Na (172 ± 17 pg/ml). Interestingly,
the 0K2Na group with the lowest concentration of aldosterone had significantly lower blood
pressure (measured directly at the carotid under Inactin anesthesia): 93.2 ± 4.9 mmHg in
0K2Na versus 112.0 ± 3.1 mmHg in 2K2Na (P < 0.05) while no significant difference in
blood pressure was detected in the groups fed 0K1Na versus 2K1Na. Urine was collected
overnight before sacrifice. Urine volume was doubled in the 0K1Na group versus 2K1Na
group (1.37 ± 0.24 versus 0.69 ± 0.13 ml/hr, P < 0.05), as previously reported (3, 38, 56).
The volumes excreted in the 0K2Na vs. 2K2Na groups followed the same pattern (1.56 ± 0.22
15

versus 1.01 ± 0.11 ml/hr) but the difference did not reach statistical significance. Removing
K
+
from the diet did not alter Na
+
excretion: Na
+
excreted in the 0K2Na and 2K2Na groups
was roughly two-fold higher than Na
+
excreted in the 0K1Na and 2K1Na groups, indicating
that NaCl output corresponded to intake. Similarly, urinary K
+
excretion reflected the amount
of KCl in the diets: The 0K1Na and 0K2Na groups had K
+
excretion rates of 3.0 ± 0.7 and
9.3 ± 0.6 μmol/hr, respectively, while the 2K1Na and 2K2Na groups had excretion rates of
127.7 ± 12.4 and 183.8 ± 18.6 μmol/hr, respectively.
K
+
depletion has been reported to significantly lower urine osmolality (3, 15, 38)
which was confirmed in this study: urine osmolality was lower in 0K1Na versus 2K1Na
(331.8 ± 43.3 versus 1159.9 ± 149.4 mOsm) and in 0K2Na versus 2K2Na (507.0 ± 81.9
versus 1020.8 ± 52.0 mOsm) (Table 2.1). When corrected for volume, the UosmV values
were also significantly lower in both 0K fed groups, and higher in the 2Na versus the 1Na
groups. Overall, the amount of solutes excreted per hour correlated to the total salt intake
(Na
+
+ K
+
) with the highest excretion rate in 2K2Na and the lowest in 0K1Na group.
As previously described by us and others (77, 92), the 0K1Na group had a weight gain
per day of only 1/3 of that measured in the 2K1Na group (1.92 ± 0.48 versus 5.83 ± 0.39
g/day, P < 0.05, Table 2.1). Elevating dietary Na
+
to 2% increased the rate of weight gain in
both 2Na groups and the 0K2Na group had a weight gain that was 2/3 of that in the 2K2Na
group (5.77 ± 0.86 versus 8.63 ± 0.65 g/day, P < 0.05). Kidney weight per 100 g body
weight was also increased in both groups of 0K fed rats as previously reported (59, 77),
indicating that K
+
deficiency induces renal hypertrophy, independent of dietary NaCl content.

16

Table 2.1. Effects of K
+
deficient diets with 0.74% NaCl (0K1Na) (n = 6) and 2% NaCl
(0K2Na) (n = 5), compared to their 2% KCl controls (2K1Na and 2K2Na, respectively)
on physiological parameters.
Parameter 2K1Na (n = 6) 0K1Na (n = 6) 2K2Na (n = 5) 0K2Na (n = 5)
Diet % KCl:% NaCl (dry wt)
2.0 : 0.74 0.05 : 0.74 2.0 : 2.0 0.05 : 2.0
P
K
, mM
3.6 ± 0.2 2.5 ± 0.1 * 3.8 ± 0.1 2.4 ± 0.1 *
P
Na
, mM
133.9 ± 3.1 139.4 ± 3.5 145.5 ± 1.6 137.2 ± 3.9
P
Aldosterone
, pg/ml
831.2 ± 88.1 413.2 ± 20.8 * 417.4 ± 18.9 172.0 ± 17.4 *
MAP, mmHg 117.5 ± 8.6 109.8 ± 8.4 112.0 ± 3.1 93.2 ± 4.9 *
U
V
, ml/hr
0.69 ± 0.13 1.37 ± 0.24 * 1.01 ± 0.11 1.56 ± 0.22
U
Na
V, μmol/hr
127.8 ± 18.7 137.4 ± 35.9 212.0 ± 16.3 218.2 ± 12.7
U
K
V , μmol /hr
127.7 ± 12.4 3.0 ± 0.7 * 183.8 ± 18.6 9.3 ± 0.6 *
U
osm
, mosmol/kgH
2
O
1159.9 ± 149.4 331.8 ± 43.3 * 1020.8 ± 52.0 507.0 ± 81.9 *
U
osm
V , μosmol/hr
705.3 ± 71.0 413.3 ± 34.7 * 1014.0 ± 79.0 722.0 ± 26.1 *
Initial body wt, g 240.7 ± 2.3 239.8 ± 4.9 224.2 ± 3.8 252.6 ± 10.2 *
Body wt at sacrifice, g 275.7 ± 2.1 251.3 ± 4.2 * 276.0 ± 4.7 287.2 ± 5.9
Body wt gain per day, g 5.83 ± 0.39 1.92 ± 0.48 * 8.63 ± 0.65 5.77 ± 0.86 *
Kidney wt/100g body wt 0.40 ± 0.01 0.49 ± 0.03 * 0.42 ± 0.01
0.48 ± 0.001
♮

Values represent means ± SEM. P
Na
, plasma Na; P
K
, plasma K; P
Aldosterone
, plasma
aldosterone; U
V
, urine volume; U
Na
V, urinary Na
+
excretion rate; U
K
V, urinary K
+
excretion
rate; U
osm
, urine osmolality; U
osm
V, urine osmolality excretion rate.
♮
, measurements on 2 rats.
* P < 0.05 vs. respective controls, assessed by unpaired two-tailed Student’s t-test, assuming
unequal variance.

Effects of K
+
depletion on proximal tubule (PT) sodium transporters. A previous study
reported a 7-fold increase in NHE3 in a pooled sample of cortex plus outer medulla (proximal
tubule plus cortical thick ascending limbs) in rats fed K
+
deficient chow for 4 days (15). To
address whether the total abundance and phosphorylation of NHE3 were altered under the
condition of K
+
depletion ± Na
+
supplementation, kidney cortex and medulla S
o
samples were
immunoblotted with antibodies against NHE3 total and NHE3pS552 on the same blot (Fig.
2.1). An increase in NHE3pS552 correlates with a less active NHE3 located at the base of the
17

microvilli (43). 0K1Na changed neither NHE3 nor NHE3pS552 abundance as compared to
2K1Na. However, in the rats fed 0K2Na, NHE3pS552 was increased slightly (1.26 ± 0.05)
versus 2K2Na (1.00 ± 0.09, P < 0.05) and the NHE3pS552/NHE3 total ratio was increased
50% (to 1.57 ± 0.09, P < 0.05) suggesting an increase in NHE3 localized to the base of the PT
microvilli in the 0K2Na group as compared to the 2K2Na group. Abundance of medullary,
i.e. thick ascending limb, NHE3 and NHE3pS552 increased significantly with 0K1Na diet as
compared to the 2K1Na diet (1.79 ± 0.29, 2.09 ± 0.37 and 1.00 ± 0.08, 1.00 ± 0.14,
respectively) without a change in NHE3pS552/NHE3 total ratio, in agreement with the
previous study reporting increased medullary NHE3 (15). However, in rats fed 0K2Na there
was neither increase in medullary NHE3 nor NHE3pS552 as compared to 2K2Na, despite a
similar degree of hypokalemia, indicating that the increases observed in the 0K1Na group
were not driven by hypokalemia per se. Thus, this study does not support the previous report
that K
+
deficiency resulting in hypokalemia provokes increases in NHE3 in cortex or medulla.
Decreased sodium-phosphate cotransporter activity and increased urinary phosphate
excretion have also been reported during dietary K
+
deficiency accompanied by an increase in
brush border membrane Na-phosphate cotransporter type 2 (NaPi2) total abundance (110). In
the current study, neither 0K1Na nor 0K2Na diets altered total NaPi2 abundance (Fig. 2.1A)
as compared to their respective controls; we did not assay trafficking of NaPi2 between
microvilli and endocytic vesicles that could explain phosphaturia.
18

Figure 2.1. Abundance of total and phosphorylated NHE3 and NaPi2 in renal cortex (A) and
medulla (B) of rats fed K
+
deficient diets with either 0.74% NaCl (0K1Na,) (n = 6) or 2%
NaCl (0K2Na) (n = 5), compared to control 2% KCl fed controls (2K1Na and 2K2Na,
respectively). Immunoblots of homogenate samples are shown. To ensure linearity of the
detection system, 1 and ½ amounts of each sample are loaded in adjacent lanes for each
animal: 30, 15 μg for cortex and 34, 17 μg for medulla NHE3 and NHE3pS552; 30, 15 μg for
cortex NaPi2. Density values were normalized to mean density of control K
+
groups (= 1.00).
Values are displayed as dot-plots over a bar at the mean value (white bars for 1Na groups,
shaded bars for 2Na groups). * P < 0.05 versus respective 2K group control.

Effects of K
+
depletion on loop of Henle (LH) and distal convoluted tubule (DCT) sodium
transporters and their regulatory kinases. Overall, we would predict that increasing sodium
reabsorption upstream from the region where K
+
secretion is coupled to Na
+
reabsorption in
19

the distal nephron (i.e., by increasing NKCC, NCC abundance and/or phosphorylation) would
promote K
+
conservation. However, previous reports have concluded that K
+
deficiency
either decreases expression of NKCC and NCC by ~50% (15, 61) or has no effect on NCC
abundance (22, 94). In this study, homogenates were subjected to immunoblotting with both
anti-total NKCC2 and anti-NKCC2pT96T101 antibodies on the same blot. Phosphorylation
of NKCC2 at these sites is associated with increased transport activity (19),(80). In cortex,
neither 0K1Na nor 0K2Na altered the total abundance or phosphorylation of NKCC2 (Fig.
2.2A). No significant decrease in medullary NKCC2 abundance or phosphorylation was
observed with 0K1Na diet (Fig. 2.2B). However, in the animals fed 0K2Na diet, the NKCC2
appeared to decrease more than 50% (which could contribute to the lower blood pressure in
these animals) but this did not reach statistical significance. However, the
NKCC2pT96T101/NKCC2 total ratio (measured on the same blot) increased nearly 3 fold
(2.87 ± 0.58 versus 2K2Na 1.00 ± 0.29, P < 0.05). Whether overall NKCC transport activity
is elevated by this increased phosphorylation was not determined, however, increased Na
+

reabsorption in this region would reduce Na
+
delivery downstream to the region where K
+
is
secreted.
We did not observe a significant change in NCC abundance with either 0K1Na or
0K2Na, compared to their respective control groups in agreement with other recent studies
(22, 94) that failed to reproduce the decreases previously reported with K
+
deficient diets (15).
Phosphorylated NCC is localized to the apical membrane alone (72) and associated with
increased transporter activity (62). In this study we did not detect a significant change in
NCCpS71 in either 0K1Na or 0K2Na diets implying no significant change in NCC transporter
activity during 0K diets. This is in contrast to a recent report of a significant increase in renal
20

cortex NCCpS71 after K
+
deficient diet feeding (94); this difference may be due to the use of
a membrane fraction versus the total homogenate used in this study.
To determine whether kinases responsible for activating NKCC2 and NCC were
increased by K
+
deficient diets, we analyzed total abundance of Ste20/SPS1-related
proline/alanine-rich kinase (SPAK) and oxidative stress response kinase-1 (OSR1) (Fig. 2.2A
and 2.2B) (78, 80). McCormick et al. recently reported that, in addition to full length SPAK
(FL-SPAK), a kidney-specific SPAK isoform (KS-SPAK), which lacks the kinase domain
and inhibits the effects of FL- SPAK to phosphorylate NCC and NKCC2, is highly expressed
along the TAL, and less so in the DCT (57). These authors also found that FL- and KS-SPAK
are differentially regulated in response to changes in ECF volume (57). To verify the
mobility of the SPAK isoforms on SDS-PAGE, Fig. 2.2C demonstrates detection of FL- and
KS- SPAK in rats fed with control and low salt diet for 6 days using mouse testis as a positive
control for FL-SPAK and SPAK knockout kidney homogenates (from E. Delpire) as a
negative control. FL-SPAK abundance increased in both cortex and medulla of rats fed with
a low salt diet (Fig. 2.2C), confirming the role of SPAK in activating NKCC2 and NCC under
low ECF volume condition (11). KS-SPAK decreased in the medullas of rats on the low salt
diet, as reported (57), consistent with the hypothesis that there is less inhibition of FL-SPAK
by KS-SPAK in the TAL and DCT with low salt diet.
In cortex, K
+
depletion increased FL- SPAK abundance in 0K2Na and tended to
increase in 0K1Na (p=0.05). In both 0K1Na and 0K2Na groups, KS-SPAK also significantly
increased. Using the ratio of FL- to KS-SPAK as an indication of activity, the ratio increased
in the 0K2Na group, consistent with higher SPAK activity, despite the lowest aldosterone
levels in this group; the ratio was not significantly increased in 0K1Na diet group. In the
21

medulla, FL-SPAK was not significantly changed but tended to increase in 0K1Na; we were
unable to clearly identify medullary KS-SPAK. Medullary OSR1 abundance was unchanged
by K
+
depletion while cortical OSR1 was increased in the 0K2Na but not significantly in the
0K1Na group, which indicates that the response was not due to hypokalemia per se. Whether
there is physiological significance to the changes in SPAK and OSR1 expression is not
readily apparent as there was no accompanying increase in NCC and/or NKCC
phosphorylation.

22

Figure 2.2. Abundance of total and phosphorylated NKCC2, NCC, and abundance of their
regulatory kinases SPAK, OSR1 in renal cortex (A) and medulla (B) of rats fed K
+
deficient
diets with either 0.74% NaCl (0K1Na) (n = 6) or 2% NaCl (0K2Na) (n = 5), compared to
control 2% KCl fed controls (2K1Na and 2K2Na, respectively). Immunoblots of homogenate
samples are shown. To ensure linearity of the detection system, 1 and ½ amounts of each
sample are loaded in adjacent lanes for each animal: in cortex: 15, 7.5 μg for NKCC2,
NKCC2pT96T101 and SPAK; 30, 15 μg for NCCpS71 and OSR1; 60, 30 μg for NCC; in
medulla: 10, 5 μg for NKCC2 and NKCC2pT96T101 and SPAK; and 6, 3 μg for OSR1. #:
anti-OSR1 antibody recognizes tubulin . Density values were normalized to mean density of
control K
+
groups (= 1.00). Values are displayed as dot-plots over a bar at the mean value
(white bars for 1Na groups, shaded bars for 2Na groups). * P < 0.05 versus respective 2K
group control.
Panel C: Immunoblots demonstrating the detection of SPAK in: cortex (30 μg) and medulla
(15 μg) of rats fed with either control (C) or low salt (LS) diet, in mouse testis homogenate (1
μg), in wild-type (WT) and SPAK knockout (KO) total kidney homogenates (10 μg); FL-
SPAK, full length SPAK; KS-SPAK, kidney-specific SPAK.
23

Effects of K
+
depletion on ENaC subunits’ abundance (α-, β-, and γ-ENaC). ENaC activity is
one of the most important determinants of K
+
secretion because by reabsorbing a Na
+
from the
tubular fluid without removing an anion, it provides a driving force for K
+
secretion into the
tubular fluid. A decrease in ENaC activity, mediated by decreasing abundance or proteolytic
processing; therefore, decreases the driving force for K
+
secretion in the distal nephron and
promotes K
+
conservation. Cortical and medullary homogenates were probed for the 3
subunits of ENaC as previously described (16) (Fig. 2.3A and 2.3B). To illustrate the ability
to use the antibodies to detect processing and proteolysis of all 3 subunits, we analyzed
samples of cortex and medulla from Sprague-Dawley rats fed with a low salt diet to activate
ENaC (16) versus normal diet fed rats (2K1Na). As previously illustrated, activation of
ENaC under salt depletion is associated with an increase in full-length and putative cleaved
forms of α-ENaC, increased total β-ENaC expression and β hyperglycosylation, and increased
γ-ENaC cleavage (Fig. 2.3C) (16).
In the cortex of 0K1Na fed animals both α- and β-ENaC were significantly decreased
versus 2K1Na diet fed animals (0.66 ± 0.07 versus 1.00 ± 0.04; and 0.65 ± 0.04 versus 1.00 ±
0.02, respectively) (Fig. 2.3A). Likewise, in cortex of 0K2Na fed animals, β-ENaC abundance
decreased (0.78 ± 0.07 versus 2K2Na 1.00 ± 0.04) and there was a similar, albeit
insignificant, decrease in mean α-ENaC abundance. These findings confirmed previous
reports (15, 22). The abundance of γ-ENaC in cortex, and the cleaved form of α-ENaC (30-
kD band) in cortex and medulla were too low to be detected in homogenates, as evident in
Fig. 2.3C as well. Unlike cortex, in medulla, neither K
+
deficient diets reduced ENaC protein
levels; in fact, α-ENaC abundance was slightly increased in 0K1Na (Fig. 2.3B) indicating
differential regulation of ENaC in cortex compared to medulla during K
+
deficiency. Overall,
24

the results are consistent with the concept that reduced ENaC activity in cortical collecting
duct during K
+
deficiency will contribute to K
+
conservation

Figure 2.3. Total abundance of ENaC subunits in renal cortex (A) and medulla (B) of rats fed
K
+
deficient diets with either 0.74% NaCl (0K1Na) (n = 6) or 2% NaCl (0K2Na) (n = 5),
compared to control 2% KCl fed controls (2K1Na and 2K2Na, respectively). Immunoblots of
homogenate samples are shown. To ensure linearity of the detection system, 1 and ½
amounts of each sample are loaded in adjacent lanes for each animal: 60, 30 µg for cortex α-
ENaC; 30, 15 μg for cortex β-ENaC, (cortex γ-ENaC signal too low to quantitate); 34, 17 μg
for medulla α-, β-, and γ-ENaC. Density values were normalized to mean density of control
K
+
groups (= 1.00). Values are displayed as dot-plots over a bar at the mean value (white bars
for1Na groups, shaded bars for 2Na groups). * P < 0.05 versus respective 2K group control.
Panel C: Immunoblots demonstrating the detection of ENaC subunits in the cortex and
medulla of rats fed control (C) versus low salt (LS) diets (60 μg of kidney homogenate/lane).

25

Effects of K
+
deficient diets on the abundance of the secretory K
+
channel (ROMK), c-Src and
ARH. K
+
restriction has been reported to decrease distal nephron principal cell apical ROMK
expression by provoking retraction from the apical membrane leading to a reduction in K
+

secretion and excretion (99). Whether K
+
deficiency also provokes a decrease in ROMK
abundance is unclear: decreased abundance has been reported in some studies (22, 61) and not
in others (97). In the current study, ROMK abundance was not significantly depressed by
either 0K diet in either cortex or medulla (Fig. 2.4A and 2.4B). The ROMK regulator c-Src
has been reported to increase during K
+
deficient diets, phosphorylating ROMK and
provoking its internalization (100). In this study we did not observe any change in total
homogenate c-Src expression during 0K diets in cortex or medulla (Fig. 2.4A and 2.4B). The
clathrin adaptor molecule autosomal recessive hypercholesterolemia (ARH) has been reported
to interact with ROMK and facilitate its internalization via clathrin-coated pits. It has also
been reported in mice that in the transition between high K
+
(10% K
+
) diet and K
+
deficient
diet, ARH abundance increases approximately 70% (18). In this study, ARH total abundance
was not changed in either cortex or medulla in the transition between control K
+
and 0K diet
(Fig. 2.4A and 2.4B). This leads us to speculate that the increase in ARH abundance
measured in the transition between high and low K
+
fed mice (18) is due to an increase in
ARH abundance in the transition between high and control K
+
diet, not an increase in the
transition from control to 0K diet fed rats. These results indicate that changes in cell surface
expression and activity of ROMK are central to K
+
conservation and that changes in ROMK,
c-Src and ARH abundance are not required for K
+
conservation.
26

Figure. 2.4. Total abundance of ROMK, c-Src, and ARH in renal cortex (A) and medulla (B)
of rats fed K
+
deficient diets with either 0.74% NaCl (0K1Na) (n = 6) or 2% NaCl (0K2Na) (n
= 5), compared to control 2% KCl fed controls (2K1Na and 2K2Na, respectively).
Immunoblots of homogenate samples are shown. To ensure linearity of the detection system,
1 and ½ amounts of each sample are loaded in adjacent lanes for each animal: in cortex: 30,
15 μg for ROMK and c-Src; 5, 2.5 μg for ARH; in medulla: 34, 17 μg for ROMK; 15, 7.5 μg
for c-Src; 6, 3 μg for ARH; Density values were normalized to mean density of control K
+

groups (= 1.00). Values are displayed as dot-plots over a bar at the mean value (white bars
for1Na groups, shaded bars for 2Na groups). * P < 0.05 versus respective 2K group control.

Effects of K
+
deficient diets on aquaporin-2 (AQP2) water channel abundance. It has been
previously reported that the polyuria observed during K
+
deficiency is associated with a >
50% decrease in abundance of the apical water channel AQP2 detected in total membranes
prepared from either renal cortex or medulla (3, 56). In this study, urine volume doubled (P <
27

0.05) in the animals fed 0K1Na vs. 2K1Na and tended to increase (insignificant 50%
increase) in the 0K2Na vs 2K2Na fed rats. AQP2, detected by immunoblot after SDS-PAGE,
runs as two major bands at 29- and 37-kDa (3, 56). In renal cortex homogenates, there was no
change in either major band in the 0K fed groups versus the control 2K fed groups (Fig. 2.5),
in contrast to previous reports. In renal medulla homogenates, the 37-kDa band decreased
30% in the 0K1Na but not the 0K2Na group and the 29-kDa band increased in the 0K1Na
group (Fig. 2.5). In this study, the length of time on the K
+
deficient diets as well as the
method of sample preparation (homogenates versus membranes) are different from the
previous studies (3, 56). Nonetheless, this study replicates a decrease in the mature
glycosylated form of AQP2 in medulla associated with polyuria in the 0K1Na fed animals, as
previously reported. It appears that the change in AQP2 and perhaps the polyuria, are blunted
in animals fed additional NaCl (0K2Na) despite profound hypokalemia.

28

Figure 2.5. Total abundance of AQP2 in renal cortex (A) and medulla (B) of rats fed K
+

deficient diets with either 0.74% NaCl (0K1Na) (n = 6) or 2% NaCl (0K2Na) (n = 5),
compared to control 2% KCl fed controls (2K1Na and 2K2Na, respectively). Immunoblots of
homogenate samples are shown. To ensure linearity of the detection system, 1 and ½
amounts of each sample are loaded in adjacent lanes for each animal: 75, 37.5 μg for cortex
and 34, 17 μg for medulla. Both 37-kDa and 29-kDa bands were quantified. Density values
were normalized to mean density of control K
+
groups (= 1.00). Values are displayed as dot-
plots over a bar at the mean value (white bars for1Na groups, shaded bars for 2Na groups). *
P < 0.05 versus respective 2K group control.

Effects of K
+
deficient diets on abundance of Na,K-ATPase α
1
and β
1
subunits. A number of
previous studies, including our own, have reported that hypokalemia provokes hypertrophy of
the outer medulla (77) and an increase in Na,K-ATPase activity and α
1
and β
1
subunit
abundance in that region (15, 59). In this study, neither subunit was increased in cortex
during K
+
deficient diets, and medullary α
1
was increased 40% in animals fed with either 0K
diet (Fig 2.6A and 2.6B), confirming previous studies and providing evidence of a response to
K
+
deficiency per se or secondary to K
+
deficiency provoked renal hypertrophy. Medullary
β
1
was also increased in the 0K1Na group, but not in the 0K2Na group, despite hypokalemia
and renal hypertrophy (Fig 2.6A and 2.6B).
Effect of K
+
deficient diets on abundance of renal cortical renin. We hypothesized that the
previous report of increased renin expression along afferent arterioles in 0K fed rats, detected
by immunohistochemistry (77), might be a consequence of a diet low in overall electrolyte
content stimulating the renin angiotensin system. In this study we measured total abundance
of renin in cortical homogenates (Fig. 2.6A). In a preliminary study of mice with a clip
around one renal artery to raise renin abundance, we found that a set of bands migrating
around 37-kDa was markedly increased compared to controls (not shown). We quantitated
29

the bands migrating at the same mobility in the rats fed 0K versus 2K diets (Fig. 2.6A) but did
not observe a difference in renal cortical renin.

Figure 2.6. Total abundance of Na,K-ATPase (NKA) α
1
and β
1
subunits in renal cortex (A)
and medulla (B) and cortex renin (A) of rats fed K
+
deficient diets with either 0.74% NaCl
(0K1Na) (n = 6) or 2% NaCl (0K2Na) (n = 5), compared to control 2% KCl fed controls
(2K1Na and 2K2Na, respectively). Immunoblots of homogenate samples are shown. To
ensure linearity of the detection system, 1 and ½ amounts of each sample are loaded in
adjacent lanes for each animal: 1, 0.5 μg for NKAα
1
; 10, 5 μg for NKAβ
1
; and 60, 30 μg for
renin. For renin, all the bands in the box were quantified together. Density values were
normalized to mean density of control K
+
groups (= 1.00). Values are displayed as dot-plots
over a bar at the mean value (white bars for1Na groups, shaded bars for 2Na groups). * P <
0.05 versus respective 2K group control.

Subcellular distribution of NCC, ROMK and AQP2. The analysis of transporter abundance
along the nephron in response to K
+
deficient diets (Figures 2.1-2.6) arrives at the prediction
30

that changes in transporter distribution and activity are likely more important for conserving
K
+
than changes in total abundance of transporters. To illustrate the importance of subcellular
distribution of renal transporters, three each of the 2K1Na and 0K1Na homogenates were
fractionated into plasma membranes (PM) and intracellular membranes (ICM) using a simple
differential centrifugation protocol developed to study AQP2 trafficking (83). Equal protein
amounts PM and ICM were analyzed by immunoblot (Fig. 2.7). NHE3 was restricted to the
PM as expected (104), NCC was enriched ~ 2 fold in PM vs. ICM, while ROMK was
enriched 2-3 fold in ICM vs. PM and AQP2 was enriched ~ 10 fold in ICM vs. PM. These
distribution patterns are not unexpected in rats that have been provided with water but no food
for a couple of hours before they are anesthetized and infused with saline for an hour: 1)
hours after feeding, even in the normal K fed rats, ROMK would likely retract to ICM to
conserve K
+
, 2) AQP2 would likely retract to the ICM to allow urine volume flow rate to
compensate for volume infusion, and 3) NCC resides predominantly in the PM and its activity
is regulated by phosphorylation (63, 85, 105). These findings indicate that there are very
large intracellular pools of all three transporters, and indicate that assessing the effect of K
+

deplete vs. replete diets on transporter distribution would best be analyzed in post-prandial
rats without prolonged anesthesia or infusions.
31

Figure 2.7. Subcellular distribution of NHE3, NCC, ROMK, and AQP2 determined by
differential centrifugation of 2K1Na and 0K1Na homogenates (n=3 each) at 17,000 x g to
pellet enriched plasma membranes (PM) and centrifugation of the 17,000 x g supernatants at
150,000 x g to pellet enriched intracellular membranes (ICM)(83). Equal protein amounts of
PM and ICM, as indicated, were analyzed by immunoblot . NHE3 is restricted to PM as
previously reported (104). The results illustrate significant differences in the PM/ICM ratio
of NCC, ROMK and AQP2.

Discussion
Hypokalemia is a common electrolyte disturbance that can be life threatening when severe
([K
+
] < 2.5 mEq). Acute hypokalemia can develop as a consequence of redistribution of K
+

from the small extracellular pool to the much larger intracellular K
+
pool in response to
insulin or adrenergic receptor stimulation (12). Chronic hypokalemia develops as a
consequence of K
+
excretion rising above K
+
intake. Common causes include inappropriate
renal loss (diuretics) or gastrointestinal loss (diarrhea). Fasting, famine and anorexia, if
prolonged, can lead to hypokalemia even though the kidneys have the capacity to greatly
reduce K
+
excretion when K
+
intake is reduced, as evident in Table 2.1. It has been estimated
that >20% of hospitalized patients develop hypokalemia. Since severe hypokalemia can
provoke cardiac dysfunction, it is important to understand not only why it develops but also
the homeostatic mechanisms that help to minimize the fall in extracellular K
+
(108).
32

We aimed to specifically determine whether hypokalemia changed the total pool size
of Na
+
, K
+
and H
2
O transporters along the nephron. During a K
+
deficient diet, the kidney
adapts to reduce urinary K
+
to match the fall in intake (Table 2.1). This occurs without a
noticeable change in Na
+
excretion. Nonetheless, Na
+
transport along the nephron can impact
the renal K
+
conservation response because Na
+
reabsorption via ENaC provides a driving
force for K
+
secretion via ROMK in the principal cells of the connecting segment (CNT) and
cortical collecting ducts (CCD) just past the DCT (21, 101). Inhibiting Na
+
reabsorption in
the LH or DCT with diuretics not only increases Na
+
excretion but also increases Na
+
delivery
and reabsorption in the CNT and CCD, thus, increasing K
+
secretion to the point that these
diuretics can provoke hypokalemia. Extending this logic, decreasing Na
+
delivery to the CNT
and CCD, for example by increasing Na
+
reabsorption proximal to this region, would decrease
the driving force for K
+
secretion and promote K
+
conservation without affecting Na
+
balance.
The subunit abundance and activity of ENaCs in the CNT and CCD has a direct impact on
Na
+
reabsorbed, thus, K
+
secreted. For these reasons, we measured the total abundance of
ENaC subunits, ROMK and associated regulators, abundance and phosphorylation status of
NHE3, NKCC and NCC and Na,K-ATPase subunits (Fig. 2.8).
Our study was also designed to test whether removal of K
+
, the primary salt in the
diet, would lead to changes provoked by low dietary electrolyte content per se, separate from
effects provoked by dietary K
+
deficiency. We compared a 0K diet containing 0.74% NaCl
(0K1Na) to a 0K diet containing 2% NaCl (0K2Na), the amount of NaCl found in the average
American diet, which partially compensated for the removal of the KCl found in control diet.
We postulated that any effects due to K
+
deficiency per se would be evident in both of the 0K
fed groups, while effects due to low electrolytes would be evident in the 0K1Na but not
33

0K2Na fed group. By 6 days, plasma [K
+
] fell from control levels of 3.6-3.8 mM to 2.5 mM
in both 0K1Na and 0K2Na groups and the rate of weight gain was less in both 0K groups
versus 2K groups, suggesting that dietary K
+
is rate limiting for growth. Weight gain/day was
higher in the 0K2Na than the 0K1Na group suggesting that higher electrolyte content may
also played a role in weight gain, however, this parameter may be influenced by the initial
body weight, which was about 15% higher in the 0K2Na group. We postulated that effects
that did not appear by six days were unlikely to be primary responses to hypokalemia. Plasma
aldosterone levels were reduced to half both when dietary Na
+
was increased to 2% (2Na
versus 1Na groups) and when K
+
was removed from the diet (0K versus 2K groups) (Table
2.1). Low Na
+
intake (via activation of renin-angiotensin system (RAS)) and high K
+
intake
independently stimulate the transcription and translation of enzymes in the aldosterone
synthesis pathway in the zona glomerulosa of the adrenal glands (32). It was difficult for us to
locate previous studies addressing how low K
+
intake changes aldosterone production. In this
study, removing K
+
from the diet reduced aldosterone levels about 50%, whether NaCl in the
diet was 0.74% or 2% (Table 2.1). While evidence from both clinical and animal studies
indicate that dietary K
+
restriction significantly increases blood pressure (46, 77), in this study
the 0K2Na group had the lowest blood pressure. We postulate that combined effects of
reduced RAS with the 2% NaCl and reduced aldosterone with 0K might reduce Na
+

reabsorption along the nephron and reduce blood pressure set point over this 6-day time
course.
Dietary K
+
restriction has been the most common protocol implemented to study
responses to hypokalemia in experimental animals (3, 15, 38, 45, 56, 59, 61, 77, 94, 97). Our
study did not replicate most of the previously reported effects of 0K diets, including increased
34

NHE3, decreased NKCC and NCC (15), decreased ROMK (61), increased c-Src (100),
increased ARH (18) and decreased AQP2 (56). The changes we observed in both the 0K diet
groups were: 1) decreased cortical β-ENaC and αENaC (decrease in 0K1Na (P< 0.05),
marginal decrease in α-ENaC in 0K2Na (P=0.05)), 2) increased medullary Na,K-ATPase, 3)
increased cortical KS-SPAK and perhaps 4) FL-SPAK. Assuming reduced α and β-ENaC
leads to less ENaC in the apical membranes of the CNT and CCD, this response could
decrease Na
+
reabsorption in this region and the driving force for stimulation of K
+
secretion.
Frindt et al. reported that low K
+
diet decreased α- β- and γ(cleaved)-ENaC subunits in
membranes prepared from the superficial cortex (22). Our results also indicate a decrease in
ENaC subunits in the cortex, especially in the 2K1Na vs. 0K1Na groups (Fig. 2.3A). In
contrast, medullary αENaC increased in the 0K1Na vs. 2K1Na groups suggesting differential
regulation in cortical vs. medullary collecting ducts. Perhaps lower aldosterone levels (Table
2.1) contribute to the reductions in cortical ENaC. Whether there is an analogous decrease in
principal cell Na,K-ATPase activity cannot be determined in cortical homogenates due to
much higher levels of Na,K-ATPase in other cortical tubules such as PT and DCT (59).
The increase in medullary sodium pump has been reported previously by us and others
(15, 59). It is difficult to provide a physiologic rationale for the increase, which is associated
with hypertrophy of the outer medulla (77). Likewise, it is difficult to assign a physiologic
rationale for an increase in FL- and KS- SPAK expression during 0K diets except to speculate
that elevated SPAK activity might increase apical expression of NCC and NKCC
(independent of transporter phosphorylation) and reduce Na
+
delivery to the CNT and CCD
where Na
+
reabsorption drives K
+
secretion, or that the changes in SPAK may indirectly
35

facilitate ROMK retraction from the apical surface. These are important questions for further
analysis.
Preserving the abundance of key renal transporters, perhaps by retracting to
intracellular pools, would help to maintain potassium homeostasis over highly varied levels of
intake. Consider a carnivorous mammal in the wild punctuating long periods of fast with
feasts after the animal successfully hunts down and consumes another K
+
-rich mammal. The
homeostatic challenge immediately switches from conserving K
+
to secreting a large K
+
load.
The switch would be sluggish if it depended on changing transporter biosynthetic rates, and
optimized by simply activating existing transporters by redistribution or covalent
modification. Likewise, changes in K
+
dietary status may alter AQP2 subcellular distribution
(rather than abundance as previously reported (56)) contributing to the lower urinary
osmolality apparent in this and other previous studies.
A number of potential explanations could account for the differences reported in this
study versus previous studies of the effect of 0K diets. The past studies discussed were
conducted in both Sprague Dawley (3, 15, 38, 45, 56, 59, 61, 77, 94, 97, 110) and Wistar (3,
15, 38, 45, 56, 59, 61, 77, 94, 97) rats as well as in mice (60, 94, 97), thus, species and strain
differences should be taken into account. Another potential explanation is that the
composition of the control versus 0K diets may not be closely matched between studies. In
this study “normal rodent chow” is 2% KCl and 0.74% NaCl but these percentages vary
slightly between “control” chows depending on vendor. In past studies either a commercial
K
+
deficient diet was paired to the normal vivarium chow or two similar diets were purchased
from a single vendor. We could identify only one lab, before this current study, to substitute
NaCl for the KCl removed (110). To eliminate unidentified differences between the control
36

and K
+
deficient diets as experimental variables, we purchased a single powered synthetic diet
and manipulated only the potassium and sodium salts. Finally, and likely more significant
than strain differences or diet, sample preparation varies between studies. Transporter activity
can be regulated by changing total abundance, subcellular distribution between plasma
membranes and intracellular membranes, covalent modification (e.g. phosphorylation) or
protein-protein association. Previous investigations of transporter abundance regulation
during K
+
deficiency have utilized a number of preparations, such as: Triton-SDS extracted
membranes (18, 97), brush border membranes (110), total membranes pelleted at 150-200,000
x g (3, 15, 61) or membranes pelleted at 16-18,000 x g (22, 94), which would enrich for
plasma membranes over intracellular membranes (83). We chose to study unfractionated
renal homogenates after a low speed spin (2,000 x g, 10 min) to remove unhomogenized
materials, nuclei and mitochondria. Since we determined that the low speed pellet contained
only insignificant amounts of NHE3 or NCC (not shown), our starting sample contains
virtually all the transporters and eliminates concern about differential recoveries. Third, we
paid careful attention to establishing the linearity of the signal with the abundance. Linearity
was also aided by the use of a stable detection system rather than a chemiluminescence
detection system in which the signal is transient. Stable detection systems also allow probing
a single blot with multiple secondary antibodies conjugated to fluorescent probes emitting at
distinct wavelengths (e.g. Alexa 680 and IRDye 800). Thus, we were able to probe for total
and phosphorylated forms of a transporter on the same blot to assess changes in the
phosphorylation status.
Supplementing NaCl in 0K diet to 2% (0K2Na) increased NHE3 phosphorylation in
the cortex. NHE3 phosphorylation does not change NHE3 transport activity, but it is a marker
37

for redistribution of NHE3 to the base of the microvilli where activity is reduced (43).
Reduced proximal tubule Na
+
reabsorption could contribute to the lower blood pressure
observed in the 0K2Na group (Table 2.1); the increased NHE3 phosphorylation/total ratio
appears to be independent of hypokalemia because it is not seen in 0K1Na group.
We did not assess all known transporters key to K
+
homeostasis for lack of antibody probes
that could reproducibly detect their targets in renal homogenates. Intercalated cells of the
distal nephron express BK channels that facilitate flow induced K
+
secretion, important for
secreting a K
+
load (35); there is no suggestion that they would be regulated in response to K
+

deprivation. Intercalated cells also express H,K-ATPase (HKA) isoforms that are postulated
to participate in K
+
conservation by actively reabsorbing K
+
from tubular fluid (13, 45). Two
isoforms of HKA are expressed in kidney: the gastric isoform (gHKA) expressed from CCD
to IMCD, and the colonic isoform (cHKA) expressed from TAL to IMCD (30). During
dietary K
+
restriction or hypokalemia, it has been reported that cHKA increases in whole
kidney (45) or selectively in medulla (13). We were unable to identify antibodies that could
clearly discriminate between the HKA isoforms in renal homogenates. However, previous
studies suggest that HKA isoforms may not be central to K
+
conservation during K
+
deficiency: 1) cHKA
-/-
mice exhibit the same profound drop in urinary K
+
excretion with K
+

deficient diets (and with the same time course) as cHKA
+/+
mice (60). 2) gHKA protein
appears to be expressed constitutively along the collecting duct (89) and regulation has not
been evaluated as thoroughly as the colonic isoform (30), however, there is no notable renal
phenotype reported for the gHKA
-/-
mice. 3) Recent analysis of cHKA, gHKA double
knockout mice indicate that they have significantly higher plasma [K
+
] compared to wild type
38

mice (28). All three studies are consistent with the lack of a significant role of HKA in renal
K
+
conservation.
In summary, the only consistent change in transporter abundance that could contribute
to a decrease in K
+
secretion was a decrease in ENaC abundance. Replacing K
+
with
additional Na
+
reduced aldosterone levels and blood pressure, but otherwise, did not
significantly affect the responses to hypokalemia. Our findings suggest that previous studies
measuring large differences in transporter abundance (cited above) may have been influenced
by assay of membrane fractions rather than unfractionated homogenates, thus, reflect
distribution in apical membranes rather than total homogenates. There is already clear
evidence for retraction of ROMK from the apical membranes of the CNT and CCD during K
+

deficient diets (51) and the simple subcellular fractionation results in this study (Fig. 2.7)
suggest most of the ROMK is in intracellular pools hours after feeding even in normalkalemic
animals. Reducing Na
+
delivery to the CNT and CCD could further reduce K
+
secretion.
Since we did not measure increases in total abundance or phosphorylation of Na
+
transporters
proximal to this region, we postulate that any decrease in Na
+
delivery to CNT and CCD
would involve subcellular redistribution or covalent modification of transporters. We
previously determined that high Na
+
diets provoke retraction of Na
+
transporters along the
nephron from low density apical membranes into the base of the microvilli or into subapical
vesicles (105). Determining whether redistribution of Na
+
transporters into apical membranes
occurs during 0K diets will require similar subcellular fractionation assays.
39

Figure 2.8. Schematic representation of transporter and regulator distributions along the rat
nephron. PT, proximal tubule; Thin DL, thin descending limb; Thin AL, thin ascending limb;
TALH, thick ascending limb of Henle’s loop; MD, macula densa; DCT, distal convoluted
tubule; CNT, connecting tubule; CD, collecting duct; NHE3, Na
+
/H
+
exchanger isoform 3;
NCC, Na
+
-Cl
-
cotransporter; gHKA, gastric H,K-ATPase; cHKA, colonic H,K-ATPase (30);
NaPi2, Na
+
/phosphate cotransporter type 2; NKCC2, Na
+
-K
+
-2Cl
-
cotransporter type 2; BK,
large conductance, calcium activated K
+
channel (35); SPAK, Ste20/SPS1-related
proline/alanine-rich kinase (76); Renin (65); AQP2, water channel aquaporin-2; ENaC,
epithelial Na
+
channel; ROMK, renal outer medullary K
+
channel (23); ARH, clathrin adaptor
molecule autosomal recessive hypercholesterolemia (18); c-Src, Src family protein tyrosine
kinase (52); NKA, Na,K-ATPase; OSR1, oxidative stress response kinase-1 (76).

40

Chapter 3. Differential Regulation of Na
+
Transporters along Nephron during AngII-
Dependent Hypertension: Distal Stimulation Counteracted by Proximal
Inhibition (Nguyen, M.T., et al. Am J Physiol Renal Physiol. (resubmitted for
2
nd
review)

Abstract
During angiotensin II (AngII)-dependent hypertension, AngII stimulates, while hypertension
inhibits, Na
+
transporter activity to balance Na
+
output to input. This study tests the
hypothesis that Ang II infusion activates Na
+
transporters in the distal nephron while
inhibiting transporters along the proximal nephron. Male Sprague-Dawley rats were infused
with AngII (400 ng/kg/min) or vehicle for 2 weeks. Kidneys were dissected (cortex versus
medulla) or fixed for immunohistochemistry (IHC). AngII increased mean arterial pressure
by 40 mmHg, urine Na
+
by 1.67-fold, and urine volume by 3-fold, evidence for hypertension
and pressure natriuresis. Na
+
transporters’ abundance and activation (assessed by
phosphorylation (-P) or proteolytic cleavage), were measured by immunoblot. During AngII
infusion: Na
+
/H
+
exchanger 3 (NHE3) abundance decreased in both cortex and medulla; Na-
K-2Cl cotransporter 2 (NKCC2) decreased in medullary thick ascending loop of Henle
(TALH) and increased, along with NKCC2-P, in cortical TALH; Na-Cl cotransporter (NCC)
and NCC-P increased in the distal convoluted tubule; epithelial Na
+
channel subunits and their
cleaved forms were increased in both cortex and medulla. Like NKCC2, STE20/SPS1-related
proline alanine-rich kinase (SPAK) and SPAK-P were decreased in medulla and increased in
cortex. By IHC, during Ang II: NHE3 remained localized to PT microvilli at lower
abundance and the differential regulation of NKCC2 and NKCC2-P in cortex versus medulla
was evident. In summary, AngII-infusion increases Na
+
transporter abundance and activation
from cortical TALH to medullary collecting duct while the hypertension drives a natriuresis
41

response evident as decreased Na
+
transporter abundance and activation from proximal tubule
through medullary TALH.
KEYWORDS – pressure natriuresis, NHE3, NCC, NKCC2, SPAK, ENaC

Introduction
Hypertension is the leading cause of stroke and cardiovascular diseases and is the leading risk
factor for global disease burden (50). Evidence suggests that the renin angiotensin system
(RAS) is activated during hypertension since inhibitors of AngII production or action are
effective at lowering blood pressure in most hypertensives. Chronic AngII-infusion, a well-
studied model that mimics the RAS activation of Goldblatt hypertension (25, 31), stimulates
both Na
+
reabsorption and vascular contractility, which elevates both extracellular fluid
volume (ECFV) and blood pressure, triggering a pressure-natriuresis response to normalize
ECFV (31). How the Ang II-stimulated anti-natriuretic responses and the hypertension-
stimulated natriuretic responses are integrated along the nephron during chronic AngII
infusion has not been investigated systematically; however, studies of specific regions provide
evidence for increased abundance and/or activity of distal convoluted tubule (DCT) NCC and
cortical collecting duct (CD) epithelial sodium channel (ENaC) (5, 9, 25, 31, 95, 112), and
decreased abundance of PT NHE3 and TALH NKCC2 (31).
Recent studies have demonstrated activation of sodium transporters by
phosphorylation and by proteolytic cleavage. SPAK and the oxidative-stress responsive
kinase 1 (OSR1) are homologous kinases that can phosphorylate and, thus, activate NKCC2
and NCC (78, 80). AngII infusion increases total abundance and phosphorylation of SPAK,
but not OSR1 (9, 25, 95) and SPAK, in turn, is regulated by With-No-Lysine (WNK) kinases
42

(9). The AngII dependent activation of NCC and SPAK is independent of aldosterone
stimulation (95), but depends on an intact intrarenal RAS (25). ENaCs (from late DCT
through CD) are activated by extracellular proteolytic cleavage of the α and γ subunits by
tubular fluid proteases (41, 70) and AngII infusion increase α and γ proteolysis (25).
In this study we tested the hypothesis that during AngII dependent hypertension there
is nephron-region specific stimulation of sodium transporters by Ang II balanced by nephron
specific inhibition of sodium transporters by hypertension, evidenced by respective increases
and decreases in transporter abundance, phosphorylation and cleavage. The results indicate
that during AngII hypertension there is inhibition of transporters from PT through medullary
TALH (inhibition of cortical and medullary NHE3, medullary NKCC2, SPAK and Na,K-
ATPase) and stimulation of transporters from cortical TALH through medullary CD
(stimulation of cortical NKCC2, NCC, SPAK and both cortical and medullary ENaC). These
findings define the regions that provoke the rise in Na
+
reabsorption, extracellular fluid
volume (ECFV) and blood pressure and also define the regions where these elevations in
ECFV and/or blood pressure can override the effects of AngII to suppress Na
+
reabsorption
and normalize ECFV.

Materials and Methods
Animal protocols. All animal procedures were approved by the Institutional Animal Care and
Use Committee of the Keck School of Medicine of the University of Southern California and
were conducted in accordance with the National Institutes of Health Guide for the Care and
Use of Laboratory Animals. Male Sprague-Dawley rats (225-250 g body weight) obtained
from Harlan Laboratories (San Diego, CA) were anesthetized intramuscularly with 200 μl of
43

ketamine and xylazine (mixed at a 1:1 volume ratio), randomized to two groups (n = 8 each)
and implanted with osmotic minipumps (Alzet, model 2002, Cupertino, CA) subcutaneously
containing either vehicle (5% acetic acid, “Control”) or AngII (400 ng/kg/min; Sigma; “AngII
infused”). Infusion was continued for 14 days during which the rats had free access to normal
vivarium diet and drinking water.
Physiological measurements. Rats were placed in metabolic cages overnight (16 hrs) for urine
collection both before minipump implantation and before sacrifice. Urine volumes were
measured by graduated cylinders, urinary [Na
+
] and [K
+
] were measured by flame photometry
(Radiometer FLM3), and osmolality was measured with an osmometer (Precision Systems,
µOsmette). Urinary angiotensinogen was assessed by immunoblot in a constant fraction
(0.02%) of the overnight urine volume.
After two weeks of AngII or vehicle infusion, rats were anesthetized intraperitoneally
with Inactin (125 mg/kg; Sigma), body temperature was maintained thermostatically at 37 °C,
and cannulas were inserted into the jugular vein for fluid infusion (0.9% NaCl + 4% BSA, 50
µl/min) to maintain euvolemia and into the carotid artery for blood pressure recording. Mean
arterial pressure (MAP) was calculated as the sum of ⅓ systolic blood pressure and ⅔
diastolic blood pressure. After stable blood pressure was recorded, renal arteries were
clamped, kidneys excised and placed in iced saline, and a blood sample was collected from
the carotid cannula, hearts removed, flushed with PBS, blotted and weighed. Plasma samples
were prepared from the blood sample by centrifugation for electrolyte measurements.
Homogenate preparation and quantitative immunoblotting. Kidney cortex and medulla were
immediately dissected manually and separately diced and homogenized as described in detail
previously (67), quick frozen in aliquots in liquid N
2
; protein concentration was determined
44

by BCA assay (Pierce Thermo, Rockford, IL). Cortical and medullary homogenates were
denatured in SDS-PAGE sample buffer for 20 min at 60°C (67). To verify uniform protein
concentration, 10 µg of protein from each sample was resolved by SDS-PAGE, stained with
Coomassie blue, and multiple random bands quantified and determined to be uniform (if not,
protein reassessed and gel re-run). For immunoblot, each sample was run at both one and
one-half amounts to verify linearity of the detection system on each immunoblot (only one
amount is shown in Figures). Antibodies used in this study, dilutions and vendors are
catalogued in Table 3.1. Blots were never stripped and reprobed. Signals were detected with
Odyssey Infrared Imaging System (Li-COR) and quantified by accompanying software.
Arbitrary density units collected were normalized to mean intensity of control group, defined
as 1.0. Since the samples were run twice (at 1 and ½), the normalized values were averaged
and mean values compiled for statistical analysis.
A new anti-NCC antibody was generated by immunizing rabbits with a peptide
sequence from N-terminal amino acids 74-96 (PGEPRKVRPTLADLHSFLKQEG) of NCC
(74) at Antibodies Incorporated (Davis, CA). Serum was collected and tested for specificity
by immunoblot against 40 μg of kidney homogenates from rat cortex (which contains NCC),
medulla (which does not contain NCC but does contain NKCC to asses cross reactivity), and
mouse whole kidney. Blots were probed with antibody diluted to 1:5,000. A frozen slice of
renal cortex was probed with the new anti-NCC antibody as well as an antibody directed to
NCCpT58.
Immunohistochemistry. In a separate set of control versus AngII infused rats (n = 4), kidneys
were perfusion-fixed via the dorsal aorta as previously described (81). The fixed tissues were
cryoprotected by overnight incubation with 30% sucrose in PBS, embedded in Tissue-Tek
45

O.C.T. compound (Sakura Finetek, Torrance, CA), and frozen on dry ice. Cryosections (5
µm) from control versus AngII animals were sliced and transferred to the same Superfrost
Plus-charged glass slide (Fisher) for direct side-by-side processing and viewing. For
immunofluorescent labeling, the sections were rehydrated, washed, and blocked with 1%
BSA/PBS before antibody incubation. All antibodies were diluted in 1% BSA/PBS (Table
3.1). The sections were mounted in Prolong Antifade containing DAPI (Invitrogen) and air
dried overnight. Samples were viewed with a ZEISS LSM 510 confocal system. Fluorescence
excitation and detector settings were the same for imaging sections from Control and AngII
treated kidneys.
Statistical analysis. Differences in physiological parameters and in protein total abundance
and phosphorylation were assessed by unpaired two-tailed Student’s t-test. Difference in
urinary angiotensinogen between control and AngII groups before and after 2 weeks of
infusion was assessed by two-way ANOVA analysis. Data were expressed as means ± SEM.
Differences were regarded significant at P < 0.05.

Results
Effects of AngII-dependent hypertension on physiological parameters. Baseline body weight,
urine volume (V), [Na
+
], [K
+
] and osmolality were similar before treatment in both control
and pre-AngII groups (Table 3.2A). As summarized in Table 3.2B, AngII infusion had the
following effects: increased MAP measured from the carotid artery, increased heart weight,
reduced rate of weight gain as reported previously (87), increased overnight urine volume
more than 3-fold and increased urinary Na
+
, K
+
and osmolar excretion indicative of chronic
pressure-natriuresis and diuresis. Kaliuresis and increased solute excretion could be attributed
46

to the reduced skeletal muscle mass (87) or increased food consumption (not measured).
Interestingly, AngII infusion significantly reduced plasma [Na
+
], likely associated with the
dipsogenic effects of AngII (90) which is also reflected in the 3-fold increase in urine volume.
AngII infusion increased urinary angiotensinogen levels more than 10 fold (Fig. 3.1),
evidence for activation of intrarenal RAS (25).

47

Antibody (Ab)
target
Primary Ab
supplier
Protein /
lane ( µg)
for cortex
Protein /
lane ( µg)
for medulla
Ab
host
Dilution
Inc.
time
Secondary
Ab supplier
Ab host
and
target
Dilution
Inc.
time
IMMUNOBLOTS
AMPK Cell Signaling 80 37 Rb 1:1500 O/N Invitrogen GAR680 1:5000 1hr
AMPKpT172 Cell Signaling 150 37 Rb 1:1500 2hrs Invitrogen GAR680 1:5000 1hr
Angiotensinogen Sernia 20 10 Sheep 1:5000 O/N Invitrogen DAS680 1:5000 1hr
NCC McDonough 60 Rb 1:5000 2hrs Invitrogen GAR680 1:5000 1hr
NCCpS71 Loffing (Zurich) 30 Rb 1:5000 2hrs Invitrogen GAR680 1:5000 1hr
NCCpS89 Loffing (Zurich) 30 Rb 1:5000 2hrs Invitrogen GAR680 1:5000 1hr
NCCpT53 Loffing (Zurich) 60 Rb 1:5000 2hrs Invitrogen GAR680 1:5000 1hr
NHE3 McDonough 30 40 Rb 1:2000 O/N Invitrogen GAR680 1:5000 1hr
NHE3pS552 Santa Cruz 30 40 Mu 1:1000 2hrs LiCor GAM800 1:5000 1hr
NKA α
1

Kashgarian (Yale) 2 2 Mu 1:200 2hrs Invitrogen GAM680 1:5000 1hr
NKA β
1

McDonough 20 2 Rb 1:500 O/N Invitrogen GAR680 1:5000 1hr
NKCC C.Lytle (UCR) 20 8 Mu 1:6000 O/N Invitrogen GAM680 1:5000 1hr
NKCCpT96T101 Forbush (Yale) 20 8 Rb 1:2000 2hrs LiCor GAR800 1:5000 1hr
OSR1 DSTT, Dundee, UK 80 40 Sheep 1:1000 O/N Invitrogen DAS680 1:5000 1hr
SPAK Delpire (Vanderbilt) 20 4 Rb 1:3000 O/N Invitrogen GAR680 1:5000 1hr
SPAKpS373/
OSR1pS325 DSTT, Dundee, UK
80 40
Sheep 1:1000 2hrs Invitrogen DAS680 1:5000 1hr
αENaC Loffing (Zurich) 80 37 Rb 1:5000 O/N Invitrogen GAR680 1:5000 1hr
βENaC Loffing (Zurich) 80 37 Rb 1:15000 2hrs Invitrogen GAR680 1:5000 1hr
γENaC Palmer (Cornell) 80 25 Rb 1:2500 O/N Invitrogen GAR680 1:500 O/N
IMMUNOHISTOCHEMISTRY
NCC McDonough Rb 1:5000 1.5hrs Mol. Probes GAR488 1:500 1hr
NCCpT58 DSTT, Dundee, UK Sheep 1:1600 1.5hrs Mol. Probes DAS568 1:500 1hr
NHE3 McDonough Rb 1:100 1.5hrs Mol. Probes GAR488 1:500 1hr
NKCC2 C.Lytle (UCR) Mu 1:2000 1.5hrs Mol. Probes GAM555 1:500 1hr
NKCCpT96T101 Forbush (Yale) Rb 1:500 1.5hrs Mol. Probes GAR488 1:500 1hr

Table 3.1. Immunoblot and Immunofluorescence antibody details. Inc. time = incubation
time; Mu = mouse; Rb = rabbit; O/N = overnight. GAR= goat anti rabbit, GAM= goat anti
mouse, DAS = donkey anti sheep.

48

Figure 3.1. Chronic AngII (400 ng/kg/min)
infusion increases urinary angiotensinogen. A.
Representative immunoblots of angiotensinogen in
urine of rats infused with either vehicle (Control) or
AngII (n =8 each) assayed in 0.02% of overnight
urine volume before (baseline) and after 2 weeks of
AngII infusion (final). B. Relative abundance
displayed as individual records with means ± SEM.
Density values normalized to mean density of
baseline values from their respective groups. * P <
0.05: final AngII versus final Control; ♯ P < 0.05:
final AngII versus baseline AngII; assessed by two-
way ANOVA analysis.

Effects of AngII-dependent hypertension on NHE3. NHE3 is expressed in the apical
membranes of epithelial cells along the entire proximal tubule (PT) as well as in the thick
ascending loop of Henle (TALH); both tubule segments span cortex and medulla. AngII
infusion decreased NHE3 abundance in both regions (Fig. 3.2A, B): to 0.78 ± 0.06 of control
in cortex and to 0.54 ± 0.04 of control in medulla. NHE3 phosphorylation (NHE3pS552), a
marker for its transit to the base of the microvilli (43), was unchanged in cortex and reduced
in medulla. Kidney sections from cortex of control and AngII-infused rats were placed on the
same slide, co-stained with NHE3 (green), and the microvilli marker villin (red) and imaged
at the same settings (Fig. 3.2C). In both Control and AngII infused groups, NHE3 was
49

resident in the microvilli overlapping with villin, and, as in the immunoblots, NHE3 staining
was lower after AngII-infusion, evidence for a pressure-natriuresis response.

Figure 3.2. AngII infusion decreases NHE3 abundance. A. Immunoblots of total and
phosphorylated NHE3 in renal cortex and medulla of rats infused with either vehicle (Control)
or AngII (n =8 each). Protein/lane in Table 3.1. B. Relative abundance displayed as
individual records with means ± SEM. * P < 0.05. C. Indirect immunofluorescence
microscopy of NHE3 and villin in renal cortex. Sections were processed identically on same
slide, and imaged with the same settings. Antibody labelling specifics provided in Table 3.1.
Bar, 20 μm.

Effects of AngII-dependent hypertension on NKCC2. NKCC2 is expressed in the apical
membranes of TALH epithelial cells from medulla to cortex. The anti-NKCC antibody
reagent used in this study recognizes not only NKCC2 but also the basolateral secretory
NKCC1 isoform that is expressed in a subset of outer medullary principal cells (24). The lack
of detectable basolateral staining by immunohistochemistry with this anti-NKCC in cortex
and medulla (Fig. 3.3) indicates that the abundance of NKCC1 in kidney is quite low
compared to NKCC2, and, with this caveat, we will refer to the signals detected as NKCC2.
NKCC2 phosphorylation at threonine 96 and 101 (NKCC-P) is indicative of transporter
50

activation (19, 80). During AngII infusion, total NKCC2 and NKCC2-P were differentially
regulated in cortex versus medulla (Fig. 3.3A,B): total NKCC2 increased in cortex (to 1.71 ±
0.26 of control) and decreased in medulla (to 0.46 ± 0.06 of control); phosphorylated NKCC2
increased in cortex (to 1.74 ± 0.23 of control) and was unchanged in the medulla. Frozen
sections from both groups were processed and analyzed on the same slide with same settings
and co-labeled with antibodies against NKCC2 (red) and NKCC2-P (green) (Fig. 3.3C). In
cortex, location of NKCC2 was confirmed by apical localization and proximity to glomeruli.
Ang II increased the fluorescent intensities of both NKCC2 and NKCC2-P in cortex, while in
medulla Ang II decreased the intensities of both NKCC2 and NKCC-P compared to Controls
stained on the same slide. These findings suggest decreased NKCC2 transporter activity in the
medullary TALH, consistent with a pressure-natriuresis effect, and increased NKCC2 activity
in the cortical TALH, indicative of AngII stimulation.

51

Figure 3.3. AngII infusion provokes differential regulation of NKCC2 in cortex versus
medulla. A. Immunoblots of total NKCC2 and NKCC2-P in cortex and medulla of rats
infused with either vehicle (Control) or AngII (n =8 each). Protein per lane in Table 3.1. B.
Relative abundance displayed as individual records with means ± SEM. * P < 0.05. C.
Indirect immunofluorescence microscopy of NKCC2 and NKCC2-P. Kidneys from 2 rats
were fixed for each condition. Sections were processed identically on same slide, and imaged
with the same settings. Two sections were fully examined and the representative images were
chosen with glomeruli for identification of the cortex. Antibody labelling specifics provided
in Table 3.1. Bar, 20 μm. G, glomerulus.

52

Effects of AngII-dependent hypertension on sodium pump. Na,K-ATPase (NKA) is expressed
ubiquitously in the basolateral membranes of tubular cells. In cortex, the levels are high in
both PT and DCT while in medulla NKA is primarily abundant in the TALH and low in other
medullary segments (59). During AngII infusion both NKAα
1
and β
1
decreased in medulla, to
0.77 ± 0.02 and 0.63 ± 0.05 of control, respectively (Fig. 3.4A, B), suggesting that apical
NKCC2 and basolateral NKA decrease in parallel in the medullary TALH during AngII
infusion, evidence of a pressure-natriuresis response. Cortical NKA α
1
and β
1
abundance,
which reflects expression in both the proximal and distal tubules, was unchanged during
AngII infusion.

Figure 3.4. AngII infusion decreases sodium
pump subunits’ abundance in the medulla.
Immunoblots of: A. NKAα
1
and B. NKAβ
1
in
renal cortex and medulla of rats infused with
either vehicle (Control) or AngII (n =8). Protein
per lane in Table 3.1.

Relative abundance is
displayed as individual records with means ±
SEM. * P < 0.05.

53

Effects of AngII-dependent hypertension on NCC. It has been established that AngII infusion
increases NCC abundance and phosphorylation (9, 25, 95). NCC-P is indicative of apical
membrane localization (48) and activation (79). This study confirms that AngII infusion
increases NCC total (to 1.93 ± 0.16 of control) using a new polyclonal antibody
(characterized in Figure 3.5) as well as increases NCC phosphorylated at the following three
sites: NCCpT53 by 5.2-fold, NCCpS71 by 2.3-fold and NCCpS89 by 3.3-fold (Fig. 3.5).

Figure 3.5. Chronic AngII (400 ng/kg/min) infusion increases NCC abundance and
phosphorylation. A. Immunoblots of NCC total, NCCpT53, NCCpS71 and NCCpS89 in
renal cortex of rats infused with either vehicle (Control) or AngII (n =8 each). Protein per lane
in Table 3.1. B. Relative abundance is displayed as individual records with means ± SEM.
Density values were normalized to mean density of Control group. * P < 0.05. C.
Characterization of a new anti-NCC antibody produced in rabbits against N-terminal amino
acids 74-96 (PGEPRKVRPTLADLHSFLKQEG). 40 µg of homogenate from untreated rat
cortex (contains NCC and NKCC), medulla (contains NKCC) and mouse whole kidney were
resolved and probed with the new anti-NCC diluted 1:5000. The antibody detects a band
54

around 150 kDa in cortex and very little in medulla, indicating that it detects NCC and not
NKCC2. The band at 100 kDa in the medulla is non-specific since there should be no NCC in
the medulla, and the band at 70 kDa in mouse is unknown. D. Frozen slice of renal cortex
was probed with the new anti-NCC antibody as well as an antibody directed to NCCpT58.
The antibodies both detect apical NCC, with little non-specific labeling. Antibody labelling
specifics provided in Table 3.1.

Effects of AngII-dependent hypertension on kinases. SPAK and the related kinase OSR1 co-
localize with TALH NKCC2 and DCT NCC where they can phosphorylate the transporters
and stimulate their transport activity; likewise, increased phosphorylation of SPAK and OSR1
are indicators of increased kinase activity (78, 80). Abundance and phosphorylation of SPAK
and OSR1 were measured to assess whether they were differentially regulated by AngII
infusion in cortex versus medulla. SPAK is expressed as three isoforms: full-length (FL-
SPAK), SPAK2, and kidney-specific SPAK (KS-SPAK). KS-SPAK is reported to exert a
dominant negative effect on both SPAK and OSR1 (57). We have previously identified FL-
and KS-SPAK isoforms in rat kidney cortical and medullary homogenates, and showed that
the major isoform in the medulla is FL-SPAK (67). In this current study, AngII infusion
increased FL-SPAK in cortex to 2.07 ± 0.40 fold over control and decreased FL-SPAK in
medulla to 0.61 ± 0.03 of control (Fig. 3.6). KS-SPAK was unchanged with AngII infusion
(1.03 ± 0.13) compared to control (1.00 ± 0.12); medullary KS-SPAK as well as SPAK-2 in
cortex and medulla were too low to quantitate (data not shown). SPAK phosphorylated at
Ser373 (SPAKpS373), an indicator of kinase activation, was increased in cortex during AngII
infusion to 1.82 ± 0.17 over control but was not significantly altered by AngII in medulla,
mimicking the pattern of NKCC2-P regulation (Fig. 3.3). Neither OSR1 total abundance nor
OSR1 phosphorylated at Ser325 (detected with the same antibody that detects SPAKpS373)
were altered by AngII infusion (Fig. 3.7). Likewise, AMP-activated protein kinase (AMPK),
55

reported to interact with and phosphorylate NKCC2 (20), was not regulated during AngII
infusion (not shown).

Figure 3.6. AngII infusion differentially regulates SPAK and SPAK-P abundance in
cortex versus medulla. A. Immunoblots of SPAK and SPAK-P in renal cortex and medulla
of rats infused with either vehicle (Control) or AngII (n =8 each). Protein per lane in Table
3.1. B. Relative abundance displayed as individual records with means ± SEM. * P < 0.05.
FL, full-length SPAK; KS, kidney-specific SPAK.

56

Figure 3.7. Chronic AngII (400 ng/kg/min) infusion does not change the abundance or
phosphorylation of regulatory kinase oxidative stress response-1 (OSR1) in cortex or
medulla. A. Immunoblots of total OSR1 and OSR1-P in renal cortex and medulla of rats
infused with either vehicle (Control) or AngII (n =8). OSR1-P is detected by the same
antibody used for SPAK-P. Protein per lane in Table 3.1. B. Relative abundance displayed as
individual records with means ± SEM. * P < 0.05. ♮, tubulin recognized by anti-OSR1
antibody (76).

Effects of AngII-dependent hypertension on ENaC. Epithelial Na
+
channels, ENaC (made up
of α, β and γ subunits), are located in the apical membranes of epithelia from the late DCT
through CD. AngII directly stimulates ENaC activity in the cortical collecting duct (CD)
(55), increases αENaC protein abundance in kidney cortex (5), and increases Na
+
reabsorption
in the distal nephron (112). Proteolytic cleavage of α and γ has been shown to increase
channel activity (37). Figure 3.8 demonstrates that AngII infusion increases the abundance of
the cleaved forms of α and γ in cortex (by 1.78 ± 0.23 and 1.67 ± 0.23 of control,
respectively) and in medulla (by 2.23 ± 0.32 and 1.44 ± 0.13 of control, respectively). In
addition, AngII infusion increased full-length α (by 1.33 ± 0.11 in cortex and 1.83 ± 0.15 in
medulla) and β subunit (by 1.24 ± 0.06 of control in cortex). These findings suggest
57

activation of both cortical and medullary ENaC during AngII infusion, confirming studies
conducted previously in mouse (25).

Figure 3.8. AngII infusion increases proteolytic cleavage of the ENaC subunits in both
cortex and medulla. Immunoblots of: A. αENaC, B. βENaC, and C. γENaC in renal cortex
and medulla of rats infused with either vehicle (Control) or AngII (n =8 each). Protein per
lane in Table 3.1. Relative abundance displayed as individual records with means ± SEM. * P
< 0.05. FL, full-length; Cleaved, proteolytic cleavage product; # non-specific band above FL-
αENaC (91).

58

Discussion

During AngII-dependent hypertension, there is a homeostatic balance between AngII
stimulation of Na
+
transporters (which may be direct or indirect via aldosterone stimulation),
which raises ECF volume and blood pressure, and hypertension driven inhibition of Na
+

transporters, which restores ECF volume. If the pressure natriuresis response was not
appropriately activated, ECF volume would be significantly expanded during AngII infusion.
Table 3.2B provides evidence for both hypertension (elevated mean arterial pressure and
increased heart weight) and for pressure-natriuresis and diuresis (elevated urine volume and
urine Na
+
excretion) in this study. By separating cortex from medulla and analyzing all the
sodium transporters along the nephron, this study was able to determine that AngII infusion
stimulates transporters (by increasing abundance, phosphorylation and/or proteolytic
cleavage) from the cortical TALH through the medullary CD, and that transporters are
inhibited (presumably by the hypertension or ECF volume signals) from the PT through the
medullary TALH (Fig. 3.9).
Previous studies demonstrated stimulation of post-macula densa sodium transporters
during AngII hypertension, namely increased NCC abundance and phosphorylation and
increased ENaC abundance and proteolytic cleavage (5, 9, 25, 95, 112). A component of the
transporter stimulation may be secondary to stimulation of aldosterone secretion during AngII
infusion (68). However, recent studies (reviewed in (96)) demonstrate AngII stimulates NCC
in adrenalectomized rats, mediated, at least in part, by the WNK4-SPAK-dependent cascade
that is independent of aldosterone, while stimulation of ENaC is via a pathway that is additive
to the effects of aldosterone. In this study, we confirmed that AngII infusion: increased
abundance of the regulatory kinase SPAK and of phosphorylated SPAK (an indicator of
59

SPAK activation) in renal cortex, increased abundance of NCC, and NCC phosphorylated at
multiple sites (a surrogate marker of NCC activation in apical membranes (41, 48)) in renal
cortex (Figs. 3.5,3.6), and activated ENaC subunits, evidenced by their proteolytic cleavage
(41, 48) (Fig. 3.8).
The TALH NKCC2 is also regulated by SPAK and, unlike NCC, is found in both
cortex and medulla (Figs. 3.3, 3.9). We recently reported that AngII infusion in mice
decreased total NKCC2 while increasing NKCC2-P, and increased SPAK-P without changing
total SPAK, all measured in whole mouse kidney (25). This pattern of disparate regulation is
clarified by the results of the present study. By analyzing kidney medulla separately from
cortex, differential regulation along the TALH was revealed: cortical NKCC2 and NKCC2-P
were stimulated by AngII infusion while medullary NKCC2 abundance was decreased
(NKCC2-P was not significantly decreased) (Fig. 3.3). Similarly, cortical SPAK and SPAK-P
were stimulated by AngII while medullary SPAK was decreased (SPAK-P was not
significantly decreased) (Fig. 3.6). More than 90% of NKCC2-P is estimated to be localized
to the plasma membrane (25), evidence of NKCC2 activation in the cortex. These findings
indicate that the medullary TALH SPAK and NKCC2 are not stimulated by AngII infusion
when it is accompanied by hypertension, rather, the pool sizes of both are depressed and their
phosphorylation is not increased, evidence for changes that facilitate the pressure-natriuresis
response. Conversely, cortical TALH SPAK and NKCC2 and their phosphorylation are
stimulated by AngII. In contrast to SPAK, our analyses did not provide any evidence for
regulation of OSR1 (Fig. 3.7) or AMPK, kinases reported to stimulate NKCC
phosphorylation (20), in either cortex or medulla.
60

Recent studies in genetically modified mouse models provide some insight into the
role of SPAK in regulating NKCC2: SPAK
T243A/T243A
knockin mice (with inactive SPAK,
intact KS-SPAK) exhibit less NKCC2 phosphorylation than wild-type mice (76), SPAK KO
(also lack KS-SPAK) exhibit increased NKCC2 total abundance (106) and phosphorylation
(29, 57, 106) as well as increased OSR1 phosphorylation (57, 106) consistent with the lack of
the dominant negative influence of KS-SPAK on SPAK and OSR1 activation. Together with
our in vivo observations in rats in this study, we postulate that as long as SPAK is present, it
can play a significant role in regulating NKCC2.
At the basolateral side of the TALH cell the sodium pump drives salt reabsorption; it
accounts for most medullary NKA (59). The decrease in NKA α
1
and NKA β
1
abundance in
medulla (Fig. 3.4), whether primary or secondary to decreased apical NKCC2 activity, is
another likely molecular mechanism contributing to the pressure-natriuretic response in the
face of AngII infusion with hypertension. The design of this study does not allow
determination of NKA regulation in nephron specific regions because of the heterogeneity of
tubules with different levels of NKA. However, previous studies have reported that AngII
stimulation alone rapidly increases proximal tubule apical membrane Na,K-ATPase (107),
and that acute hypertension alone decreases Na,K-ATPase activity (111) indicating that
cortical Na,K-ATPase is likely regulated in a region specific manner during AngII dependent
hypertension.
AngII stimulation (without accompanying hypertension) has been reported to
stimulate pre-macula densa NHE3 and NKCC2 (4, 81, 88). Specifically, blocking AngII
production with an ACE inhibitor redistributes PT NHE3 from body to the base of the
microvilli and decreases PT Na
+
reabsorption (49, 103) and AngII infusion (without
61

hypertension) redistributes NHE3 into the microvilli and activates transport (81). In contrast,
NHE3 redistributes to the base of the microvilli in response to acute hypertension without
AngII stimulation (58). In the current study the PT NHE3 is subjected to the simultaneous
opposing forces of AngII and hypertension. Interestingly, the AngII stimulation appears to
retain NHE3 in the body of the microvilli, yet there is a compensatory decrease in NHE3
abundance (by both immunoblot and immunohistochemistry) (Fig. 3.2). Nonetheless, the
persistent localization of NHE3 in the microvilli during AngII infusion may blunt the
magnitude of the natriuresis for a given increase in blood pressure and contribute to the rise in
blood pressure. Whether the decreased pool size of NHE3 during AngII hypertension is due
to depressed synthesis or elevated degradation remains to be determined. It will also be
interesting to learn whether the NHE3 abundance increases initially in response to AngII
alone prior to the development of hypertension and subsequently decreases after the blood
pressure goes up. In an earlier study with Gurley et al., we reported that elimination of the
AngII-receptor type 1
A
from the mouse PT improved the pressure-natriuresis response during
AngII-dependent hypertension, evident as larger decreases in NHE3 abundance and lower
blood pressure, thus, illustrating the important role of the PT in blood pressure regulation
(31).
Differential regulation of NKCC2 in medullary versus cortical TALH may be a
function of ion transport characteristics of NKCC2 isoforms. Isoform NKCC2F is expressed
mainly in medullary TALH while NKCC2B is expressed mainly in cortical TALH (10, 71).
Both furosemide administration and chronic water loading alter expression of NKCC2 in an
isoform specific manner (7), suggesting that splicing machinery may be influenced by the
filtered load and/or the intracellular [Cl
-
]. During AngII hypertension, the decrease in PT
62

NHE3 would contribute to increased flow into the TALH which could alter the expression of
NKCC2 splice variants along the TALH and activate parallel changes in NKA. However, we
have previously established that increased flow out of the PT (during acute hypertension or in
response to a PT diuretic) actually increases NKA activity in the medulla (54). Since the
upstream kinase SPAK, localized to the TALH and DCT, also responds to AngII infusion
with a differential pattern (increase in cortical SPAK, SPAK-P, and decrease in medullary
SPAK), SPAK is more likely a target for effecting differential regulation of NKCC2.
Potential candidates, regulated by Ang II and/or by tubular flow, that could suppress
SPAK, NKCC2 and NKA in medullary TALH would include changes in WNKs (36), nitric
oxide and reactive oxygen species (8). High blood pressure has been shown to stimulate
release of the cytochrome P-450 metabolite 20-hydroxyeicosatetraenoic acid, 20-HETE (102),
which inhibits Na
+
transport and NKA activity in the PT and TALH (75, 109) and NKCC
transport activity in isolated medullary TALH (17). AngII hypertension stimulates release of
20-HETE in rat kidney (2) suggesting the possibility that 20-HETE could counteract the anti-
natriuretic influence of AngII from the PT through the medullary TALH by suppressing
NHE3, NKA and NKCC2.
What is the significance of these findings? Evidence indicates that AngII can
stimulate transporter abundance and/or activity all along the nephron. However, if AngII
stimulation is accompanied by hypertension, compensatory natriuretic responses override the
anti-natriuresis of AngII. This study determined that AngII hypertension increases
transporters’ abundance and activation from the cortical TALH to the medullary CD
(NKCC2, NCC, ENaC and regulatory kinase SPAK) and that this stimulation is balanced by a
compensatory inhibition of transporters (NHE3 and medullary: NKCC2, NKA, SPAK) from
63

PT through medullary TALH, presumably driven by elevated blood pressure. That is,
hypertension overrides the effects of AngII from PT through medullary TALH. Futures
studies should recognize and consider that the AngII dependent hypertension model generates
opposing signals to maintain ECF volume homeostasis. On a practical note, region-specific
regulation of NKCC and SPAK will not be evident in analyses of whole kidney homogenates:
it is important to dissect cortex from medulla to discriminate regions of anti-natriuretic
response versus natriuretic response. Understanding where AngII stimulates Na
+
transporters
along the nephron is crucial for targeting with anti-hypertensive therapies. Natriuretic
mediators that counteract the effects of AngII warrant further investigation.

Figure 3.9. Na
+
transporter regulation along the nephron during AngII-dependent
hypertension. AngII infusion activates distal nephron Na
+
transporters (highlighted in red)
consistent with increased Na
+
reabsorption, elevated ECFV and higher blood pressure.
Hypertension provokes pressure-natriuresis to normalize ECFV by decreasing: NHE3,
medullary NKCC and regulatory kinase SPAK (highlighted in green). See text for
abbreviations.

64

Chapter 4. Renal responses to short-term non-pressor angiotensin II with and
without angiotensin-converting enzyme inhibition in rats

Abstract
Long-term (14 days) angiotensin II (AngII) infusion augments intrarenal AngII production,
activates renal transporters from the cTALH to the CD and raises blood pressure. Angiotensin
converting enzyme (ACE) inhibition during AngII infusion blunts these responses implicating
local tissue RAS production of AngII. This study aimed to determine 1) the renal responses to
a short-term (3 days) non-pressor dose of AngII and 2) whether the responses were blunted by
ACE inhibition implicating local AngII production. Male Sprague-Dawley rats were infused
via osmotic minipumps with either vehicle (control) or AngII (200 ng/kg/min) with and
without ACE inhibitor enalapril (30 mg/kg/day in drinking water). Overnight urine was
collectd in metabolic cages and kidney cortex and medulla were homogenized separately, and
sodium transporter abundance and phosphorylation, and their regulatory kinases were
determined by immunoblotting. There were no significant differences in body weight gain,
overnight urine volume, or urinary Na
+
and K
+
excretion among the groups. The non pressor
AngII infusion significantly increased cortical transporter abundance and phosphorylation
along the nephron as follows: proximal tubule (PT) Na
+
/H
+
exchanger 3 (NHE3), thick
ascending loop of Henle (TALH) Na-K-2Cl cotransporter 2 (NKCC2) and phosphorylated
NKCC2 (NKCC2-P), distal convoluted tubule (DCT) Na-Cl cotransporter (NCC) and
phosphorylated NCC (NCC-P), phosphorylated STE20/SPS1-related proline alanine-rich
kinase (SPAK-P), and collecting duct (CD) β subunit of the epithelial Na
+
channel (βENaC),
while medullary transporters were not altered. There was no evidence for activation of sodium
transporters by enalapril treatment to inhibit tissue RAS. In conclusion, 3-day AngII infusion
65

increases cortical NHE3, NKCC2, NCC and SPAK-P, consistent with activation of Na
+
at
early stage of the development of hypertension independent of activation of local renin-
angiotensin system
KEYWORDS – NHE3, NCC, NKCC2, SPAK, ENaC, intrarenal RAS, ACE inhibition

Introduction
The renin-angiotensin system (RAS) affects sodium balance, extracellular fluid volume, and
renal and vascular resistance. RAS blockers, including angiotensin-converting enzyme (ACE)
inhibitors and angiotensin II (AngII) receptor blockers, have been widely used to reduce
blood pressure in hypertensive subjects. How AngII regulates Na
+
transport at the molecular
levels has been a topic of research interest by many groups including our own. Acute AngII
infusion (20 min) in anesthetized rats without raising blood pressure provokes proximal
tubule (PT) NHE3 and Na
+
-P
i
cotransporter 2 (NaPi2) redistribution into the microvilli where
they likely contribute to rapid increase in PT salt and water reabsorption (81). In the distal
convoluted tubule (DCT), acute AngII infusion provokes trafficking of NCC from subapical
vesicles to the plasma membrane, which likely increases Na
+
reabsorption (86). In contrast,
acute ACE inhibition by captopril redistributes NHE3 out of the body of microvilli into the
base of the microvilli and NaPi2 into a subapical vesicular compartment (49), and provokes
acute NCC trafficking out of the plasma membrane (86). In these studies, acute AngII
stimulation regulates Na
+
transport via redistribution of transporters, rather than changing
their total pool size or covalent modification by phosphorylation.
Chronic AngII infusion is a well-studied model of hypertension. Two weeks of AngII
infusion provokes anti-natriuresis by increasing cortical NKCC and NCC total abundance and
66

phosphorylation, and increasing ENaC abundance and subunit proteolytic cleavage (Chapter
3, (9, 25, 95), (5)). In contrast, the proximal part of the nephron, including the PT and
medullary TALH, respond to AngII hypertension with a pressure-natriuresis response by
downregulating PT NHE3 and medullary TALH NKCC2 total abundance (Chapter 3, (31)).
In contrast to a pure pressure response, NHE3 is retained within the microvilli, presumably by
AngII action, while simply raising the BP drives NHE3 to the base of the microvilli (Chapter
3). Renal responses during the early phase of chronic AngII infusion before hypertension and
pressure-natriuresis ensues have not been investigated systemically. The first aim of this study
was to test the hypothesis that in the early stages of AngII infusion, AngII activates Na
+

transporters all along the nephron, by increasing total pool size and/or covalent modification,
and contribute to the subsequent development of chronic hypertension observed with longer
infusion.
In addition to systemic RAS, intrarenal RAS contributes a major role to blood pressure
control (66). Chronic AngII infusion activates intrarenal RAS (27), which further stimulates
Na
+
transporters along the distal nephron (25). ACE inhibition blunts the rise in blood
pressure in AngII-dependent hypertension model by blocking the activation of intrarenal RAS
(26). Mice lacking kidney ACE showed blunted responses to AngII infusion in the induction
of Na
+
and water retention as well as the activation of renal Na
+
transporters, suggesting that
even with an elevated systemic AngII, renal ACE is required to activate transporters in the
distal nephron (25). The second aim of this study was to test the hypothesis that blocking local
production of AngII with an ACE inhibitor at the early time course of AngII infusion will
prevent activation of transporters that cause the rise in blood pressure.
67

Our results indicate that 1) short-term low-dose AngII infusion (200 ng/kg/min for 3
days) creates a pre-hypertensive state in which Na
+
transporters are activated in the PT and
distal nephron and 2) blocking tissue RAS activation with ACE inhibitor enalapril does not
prevent transporter activation. These findings contribute to our understanding of how
hypertension is developed during AngII-dependent hypertension at the molecular level.

Materials and Methods
Animal protocols. All animal procedures were approved by the Institutional Animal Care and
Use Committee of the Keck School of Medicine of the University of Southern California and
were conducted in accordance with the National Institutes of Health Guide for the Care and
Use of Laboratory Animals. Male Sprague-Dawley rats (240-260 g body weight) obtained
from Harlan Laboratories (San Diego, CA) were anesthetized intramuscularly with 200 μl of
ketamine and xylazine (mixed at a 1:1 volume ratio), randomized to three groups and
implanted with osmotic minipumps (Alzet, model 2002, Cupertino, CA) subcutaneously at
day 0 containing either vehicle (5% acetic acid, “Control”), AngII (200 ng/kg/min; Sigma)
without (“AngII”) or with ACE inhibitor enalapril (30 mg/kg/day in drinking water pretreated
for 4 days; Sigma) (“AngII + enalapril”). Infusion was continued for 3 days during which the
rats had free access to normal vivarium diet.
Physiological measurements. Rats were placed in metabolic cages overnight (16 hrs) for urine
collection both before minipump implantation (day -1) and before sacrifice (day 2). Urine
volumes were measured by graduated cylinders, urinary [Na
+
] and [K
+
] were measured by
flame photometry (Radiometer FLM3). Urinary angiotensinogen was assessed by immunoblot
in a constant fraction (0.02%) of the overnight urine volume.
68

After 3 days of AngII or vehicle infusion, rats were anesthetized intramuscularly with
ketamine/xylazine, kidneys were excised rapidly and placed in iced saline, removed capsules
and weighed. Blood samples were collected by cardiac puncture, hearts removed, flushed with
PBS, blotted and weighed. Plasma samples were prepared from the blood samples by
centrifugation for electrolyte measurements.
Homogenate preparation and quantitative immunoblotting. Kidney cortex and medulla were
immediately dissected manually, separately diced and homogenized as described in detail
previously (67), aliquoted and quick frozen in liquid N
2
; protein concentration was
determined by BCA assay (Pierce Thermo, Rockford, IL). Cortical and medullary
homogenates were denatured in SDS-PAGE sample buffer for 20 min at 60°C (67). To verify
uniform protein concentration, 10 µg of protein from each sample was resolved by SDS-
PAGE, stained with Coomassie blue, and multiple random bands quantified and determined to
be uniform (if not, protein reassessed and gel re-run). For immunoblot, each sample was run
at both one and one-half amounts to verify linearity of the detection system on each
immunoblot. Antibodies used in this study, dilutions and vendors are catalogued in Table 4.1.
Signals were detected with Odyssey Infrared Imaging System (Li-COR) and quantified by
accompanying software. Arbitrary density units collected were normalized to mean intensity
of control group, defined as 1.0. Since the samples were run twice (at 1 and ½), the
normalized values were averaged and mean values compiled for statistical analysis.

69

Antibody
target
Primary
Ab supplier Ab host Dilution
Incubation
time/temp
NHE3 total McDonough Rb 1:2,000
O/N 4
0
C
NHE3pS552 Santa Cruz Mu 1:1,000 2hrs RT
NKCC total C. Lytle (UCR) Mu 1:6,000
O/N 4
0
C
NKCCpT96/T101 Forbush (Yale) Rb 1:2,000 2hrs RT
NCC total McDonough Rb 1:5,000
O/N 4
0
C
NCCpS71 Loffing (Zurich) Rb 1:5,000 2hrs RT
βENaC Loffing (Zurich) Rb 1:15,000 2hrs RT
SPAK total Delpire (Vanderbilt) Rb 1:3,000
O/N 4
0
C
SPAKpS373 DSTT (Dundee) Sheep 6ug/25ml 2hrs RT
Angiotensinogen Sernia (Queensland) Sheep 1:5,000 2hrs RT
Hsp70 Duncan (USC) Mu 1:5,000
O/N 4
0
C
Table 4.1. Immunoblot antibody details. Rb = rabbit, Mu = mouse, O/N = overnight

Statistical analysis. Differences in physiological parameters and in protein total abundance
and phosphorylation were assessed by one-way ANOVA analysis. Difference in urinary
angiotensinogen before and after 3 days of infusion was assessed by two-way ANOVA
analysis. Bonferroni’s corrections were performed after ANOVA between control vs. AngII
and between AngII vs. AngII + enalapril. Data were expressed as means ± SEM. Differences
were regarded significant at P < 0.05.

Results
Short-term non-pressor AngII with or without enalapril did not alter physiological
parameters. Baseline body weight, urine volume (UV), urinary Na
+
and K
+
excretion (U
Na
V
and U
K
V, respectively) were similar before treatment in all 3 groups. After 3 days of AngII
70

infusion, neither above parameters was significantly different among the groups (Table 4.2),
indicating there was no AngII-mediated reduced weight gain as reported previously (87), as
well as no pressure diuresis and natriuresis. We have not observed a difference in heart weight
or hematocrit (Table 4.2), suggesting there was no cardiac hypertrophy or extracellular fluid
volume expansion, as reported previously (14, 68). AngII infusion with or without enalapril
did not significantly change plasma Na
+
and K
+
levels (Table 4.2), indicating all animals
maintained their electrolyte balance. No significant differences in physiological parameters
among 3 groups demonstrated that this short-term low-dose AngII infusion was a pre-
hypertensive state.
Physiological
parameters
Control
(n = 7)
AngII
(n = 7)
AngII + Enalapril
(n=6)
Hematocrit 0.45 ± 0.01 0.45 ± 0.01 0.43 ± 0.01
Plasma Na
+
, mM
135.6 ± 2.1 138.4 ± 1.0 140.2 ± 1.3
Plasma K
+
, mM
4.5 ± 0.1 4.7 ± 0.1 4.5 ± 0.1
Body wt gain per day, g 4.82 ± 0.54 4.75 ± 0.51 3.33 ± 0.67
Heart wt/100 g body wt 0.29 ± 0.01 0.31 ± 0.005 0.30 ± 0.004
Kidney wt/100 g body wt 0.44 ± 0.01 0.43 ± 0.01 0.42 ± 0.01
Baseline UV , ml 12.3 ± 0.9 11.3 ± 1.0 14.5 ± 0.9
Baseline U
Na
V , mmol 1.9 ± 0.1 1.8 ± 0.1 2.0 ± 0.1
Baseline U
K
V , mmol 3.2 ± 0.1 3.1 ± 0.1 3.3 ± 0.2
Final UV , ml 13.3 ± 1.0 14.1 ± 1.3 15.0 ± 0.9
Final U
Na
V , mmol 2.1 ± 0.1 2.1 ± 0.1 2.0 ± 0.2
Final U
K
V , mmol 3.5 ± 0.1 3.3 ± 0.1 3.4 ± 0.1
Table 4.2. Short-term low dose AngII infusion (200 ng/kg/min, 3 days) and enalapril
treatment do not significantly alter physiological parameters. UV, 16-hr overnight urine
volume; U
Na
V, urinary Na
+
excretion, UKV, urinary K
+
excretion. “Baseline” urine was
collected the night before minipump implantation. “Final” urine was collected the night
before sacrifice.

Effects of short-term low-dose AngII with and without enalapril on cortical NHE3. NHE3 is
expressed in the apical membranes of epithelial cells along the entire proximal tubule (PT) as
71

well as in the thick ascending loop of Henle (TALH). We have previously shown that acute
AngII infusion without raising blood pressure provokes redistribution of NHE3 into proximal
tubule microvilli, without a change in its total abundance (81). In this study, the low-dose
AngII infusion after 3 days significantly increased cortical NHE3 abundance (Fig. 4.1A, B) to
1.40 ± 0.11 of control. This observation is in contrast to what we have reported in the
previous chapter: with a high dose (400 ng/kg/min) and long term (2 weeks) AngII infusion,
NHE3 total was significantly lower compared to control, evidence for pressure-natriuresis.
NHE3 phosphorylation (NHE3pS552), a marker for its transit to the base of the microvilli
(43), was unchanged (Fig. 4.1A, B). Treating AngII-infused rats with enalapril did not alter
the AngII response.

Figure 4.1. Short-term low dose AngII infusion increases cortical PT NHE3 abundance.
A. Immunoblots of total and phosphorylated NHE3 of rats infused with either vehicle
(Control, ○) or AngII (n) (n=7) or AngII + Enalapril (▲) (n=6). 30/15 μg protein were loaded
per lane. NHE3 total and NHE3-P signals were detected on the same blot. B. Relative
abundance displayed as individual records with means ± SEM. * P < 0.05.

72

Effects of short-term low-dose AngII with and without enalapril on medullary TALH NKCC2
and SPAK. NKCC2 is expressed in the apical membranes of TALH epithelial cells from
medulla to cortex. SPAK co-localizes with, phosphorylates and activates NKCC2 (80, 106).
SPAK phosphorylated at serine 373 (SPAKpS373) is an indicator of kinase activation. During
short-term low-dose AngII infusion, medullary NKCC2 total and NKCC2 phosphorylated at
threonine residues 96 and 101 (NKCC2-P) were not different from control (Fig. 4.2A, B), nor
were SPAK total and SPAKpS373 (Fig. 4.2C, D). In comparison, 2 weeks of AngII infusion
with high dose (400 ng/kg/min) significantly reduced medullary NKCC2 and SPAK total
abundance, indicative of a pressure-natriuresis effect. Enalapril did not significantly alter
either NKCC2 or SPAK expression compared to AngII group.

73

Figure 4.2. Short-term low dose AngII infusion does not alter medullary TALH NKCC2
(A, B) or SPAK (C, D) abundance and phosphorylation. Immunoblots of total and
phosphorylated NKCC2 (A) and SPAK (C) of rats infused with either vehicle (Control, ○) or
AngII (n) (n=7) or AngII + Enalapril (▲) (n=6). 21/10.5 μg protein were loaded per lane.
NKCC2 total and NKCC2-P signals were detected on the same blot, SPAK total and
SPAKpS373 were detected on the same blot. Relative abundance displayed as individual
records with means ± SEM (B, D). * P < 0.05.

Effects of short-term low-dose AngII with and without enalapril on cortical TALH NKCC2.
Similar to the effects of long-term AngII infusion, this study also showed that cortical TALH
NKCC2 total abundance and phosphorylation were significantly higher in AngII group (1.55
± 0.19 and 1.72 ± 0.14 vs. controls, respectively) (Fig. 4.3A, B). There were no significant
differences in NKCC2 or NKCC2-P with enalapril treatment, compared to AngII infusion
alone; suggesting the activation of cortical NKCC2 is independent of RAS activation.
74

Figure 4.3. Short-term low dose AngII infusion increases cortical TALH NKCC2
abundance and phosphorylation. A. Immunoblots of total and phosphorylated NKCC2 of
rats infused with either vehicle (Control, ○) or AngII (n) (n=7) or AngII + Enalapril (▲)
(n=6). 20/10 μg protein were loaded per lane. NKCC2 total and NKCC2-P signals were
detected on the same blot. B. Relative abundance displayed as individual records with means
± SEM. * P < 0.05.

Effects of short-term low-dose AngII with and without enalapril on DCT NCC. We have
previously reported that acute AngII infusion (20 min) in anesthetized rats provokes rapid
trafficking of NCC from subapical vesicles to plasma membranes without changing the total
abundance, which likely increases Na
+
transport in the DCT (86). Two weeks of AngII
infusion increases NCC abundance and phosphorylation (25). NCC-P is located exclusively in
the apical membrane (48) and is indicative of activation (79). This study showed that after 3
days of AngII infusion with a low dose, both NCC total and NCC phosphorylated at serine
residue 71 (NCCpS71) were increased (to 1.25 ± 0.04 and 1.23 ± 0.07 of controls,
respectively) (Fig. 4.4A, B). NCC phosphorylated at serine 89 was also significantly
increased in AngII group (1.41 ± 0.07 of control, not shown). Treating AngII-infused rats
75

with enalapril prevented the AngII stimulation of NCC, but did not prevent NCCpS71 (1.01 ±
0.04 vs. 1.25 ± 0.04 of AngII) (Fig. 4.4A, B.

Figure 4.4. Short-term low dose AngII infusion increases cortical DCT NCC abundance
and phosphorylation and the response was blunted by enalapril. A. Immunoblots of total
and phosphorylated NCC of rats infused with either vehicle (Control, ○) or AngII (n) (n=7) or
AngII + Enalapril (▲) (n=6). 60/30 μg and 30/15 μg protein were loaded per lane for NCC
total and NCCpS71, respectively. B. Relative abundance displayed as individual records with
means ± SEM. * P < 0.05.

Effects of short-term low-dose AngII with and without enalapril on cortical TALH and DCT
SPAK. In addition to its co-localization with NKCC2 in the TALH, SPAK also co-localizes
with DCT NCC where it can phosphorylate and increase NCC activity (79, 106). We
previously showed that chronic AngII infusion in rats significantly increased cortical SPAK
total and SPAKpS373. We next examined its abundance with a lower dose of AngII after 3-
day infusion: there was no significant difference in SPAK total among three groups while
SPAKpS373 was significantly increased in AngII-infused rats to 1.31 ± 0.11 of control (Fig.
76

4.5A, B). Enalapril did not have a significant effect on AngII-mediated activation of SPAK,
suggesting that SPAK activation is tissue RAS independent at this time point.

Figure 4.5. Short-term low dose AngII infusion increases cortical TALH and DCT
SPAK phosphorylation. A. Immunoblots of total and phosphorylated SPAK of rats infused
with either vehicle (Control, ○) or AngII (n) (n=7) or AngII + Enalapril (▲) (n=6). 20/10 μg
and 80/40 μg protein were loaded per lane for SPAK total and SPAKpS373, respectively. B.
Relative abundance displayed as individual records with means ± SEM. * P < 0.05.

Effects of short-term low-dose AngII with and without enalapril on cortical CD βENaC.
Epithelial Na
+
channels (ENaC) are located in the apical membranes of epithelia from the late
DCT through CD. AngII directly stimulates ENaC activity in the cortical collecting duct
(CD) (55) and increases Na
+
reabsorption in the distal nephron (112). We have demonstrated
that chronic pressor dose of AngII after 2 weeks increased the total abundance of the β
subunit of ENaC (βENaC). Similarly, figure 4.6 showed that short-term low-dose AngII
infusion increased the abundance of βENaC (by 1.27 ± 0.07 of control). There was no
77

significant change with enalapril treatment. These findings suggest βENaC is activated during
earlier time course of AngII infusion, and the activation is tissue RAS independent.

Figure 4.6. Short-term low dose AngII infusion increases cortical CD βENaC. A.
Immunoblots of βENaC of rats infused with either vehicle (Control, ○) or AngII (n) (n=7) or
AngII + Enalapril (▲) (n=6). 60/30 μg protein were loaded per lane. B. Relative abundance
displayed as individual records with means ± SEM. * P < 0.05.

Effects of short-term low-dose AngII with and without enalapril on cortical angiotensinogen
expression and urinary angiotensinogen excretion. In AngII-hypertension, intrarenal RAS is
activated causing further increase in AngII level in the kidney (64). Previous study on mice
infused with 400 ng/kg/min of AngII for 12 days showed that angiotensinogen mRNA and
protein expression in kidney cortex were significantly increased compared to uninfused mice
(27). Chronic AngII infusion increases urinary angiotensinogen excretion rate, suggesting that
it is a specific index of intrarenal angiotensinogen production (42). We previously showed
that infusing rats with AngII (400 ng/kg/min) for 2 weeks significantly raised the amount of
angiotensinogen detected in the urine by immunoblotting. We demonstrated here that short-
78

term low-dose AngII infusion did not alter renal cortical angiotensinogen protein expression
(Fig. 4.7A, B). We found no significant difference in urinary angiotensinogen excretion
before and after AngII infusion among three groups (Fig. 4.7C, D). Both angiotensinogen
levels in kidney cortex and urine were not altered by enalapril treatment, indicating that there
was no evidence for stimulation of intrarenal RAS at this early phase of AngII infusion.

Figure 4.7. Short-term low dose
AngII infusion does not alter renal
cortical (A, B) nor urinary
angiotensinogen excretion (C, D).
Immunoblots of cortical (A) and
urinary (C) angiotensinogen of rats
infused with either vehicle (Control,
○) or AngII (n) (n=6-7) or AngII +
Enalapril (▲)(n=6). 20/10 μg protein
were loaded per lane (A), 0.02% of
overnight urine volume before
minipump implantation (day -1) and
before sacrifice (day 2) (C). Relative
abundance (B) and signal intensities
(D) displayed as individual records
with means ± SEM. * P < 0.05.

79

Effects of short-term low-dose AngII with and without enalapril on cortical Hsp70. Ishizaka et
al. have shown that continuous infusion of AngII (0.7 mg/kg/day = 4861 ng/kg/min) with
accompanied hypertension increased the expression of heat shock protein 70 (Hsp70) in the
kidneys starting at day 3 and provide evidence that this is a neo-antigen that attracts immune
infiltration (39). In this study, we observed a similar response with a lower dose of AngII (200
ng/kg/min) after 3-day infusion: Hsp70 was significantly increased in the cortex of AngII-
infused group (1.31 ± 0.09 of control) (Fig. 4.8A, B). Enalapril did not significantly alter
AngII-mediated Hsp70 overexpression.

Figure 4.8. Short-term low dose AngII infusion increases cortical Hsp70. A. Immunoblots
of Hsp70 of rats infused with either vehicle (Control, ○) or AngII (n) (n=7) or AngII +
Enalapril (▲) (n=6). 20/10 μg protein were loaded per lane. B. Relative abundance displayed
as individual records with means ± SEM. * P < 0.05.

Discussion
Our previous work focused on the effects of chronic AngII infusion with accompanying
hypertension on renal transporters and demonstrated a homeostatic balance between AngII
stimulation of Na
+
transporters in the distal nephron, which raises ECF volume and blood
80

pressure, and hypertension driven inhibition of Na
+
transporters in the proximal nephron,
which restores ECF volume (Chapter 3). The progression of hypertension in AngII infusion
model has not been extensively investigated at the molecular level. This study examined renal
responses early in the time course of AngII infusion (3-day) and the role of intrarenal AngII
production before hypertension and pressure-natriuresis ensue. No significant differences in
physiological parameters were observed among three groups (Table 4.2), suggesting that at
this level of AngII (200 ng/kg/min), the animals maintained their fluid and electrolyte balance
and showed no evidence of hypertension or pressure-natriuresis and diuresis. By separating
cortex from medulla and analyzing all the sodium transporters along the nephron, this study
was able to determine that in the early stages of AngII infusion, before hypertension and
pressure-natriuresis establish, AngII activates Na
+
transporters in the proximal tubule (NHE3)
and distal nephron (cortical NKCC2, NCC and ENaC) (by increasing abundance and
phosphorylation), and blocking tissue RAS activation with enalapril prevented the increase in
NCC abundance caused by AngII infusion.
Acute AngII infusion for 20 min (without accompanying hypertension) has been reported
to stimulate NHE3 redistribution into the microvilli, presumably to increase Na
+
transport in
the PT (81), and blocking AngII production acutely with an ACE inhibitor redistributes NHE3
from the body to the base of the microvilli to decrease Na
+
reabsorption (49, 103). In addition,
we showed that NHE3 redistributes to the base of the microvilli in response to acute
hypertension without AngII stimulation (58). In these acute studies, the total abundance of
NHE3 was not altered with either stimulus. During chronic AngII infusion with hypertension,
AngII retains NHE3 in the body of the microvilli, while there is a significant decrease in
NHE3 abundance (Chapter 3), indicative of the compensatory pressure-natriuresis. In the
81

current study, the PT NHE3 abundance increases after 3 days of low-dose AngII infusion with
the assumption that this time point preceeds the hypertension (Fig. 4.1). We postulate that
increased NHE3 abundance stimulates Na
+
reabsorption at the early stage of AngII infusion,
contributing to the development of hypertension. Blocking local AngII production chronically
with enalapril did not reduce NHE3 total to the control level (Fig. 4.1), suggesting AngII
infusion alone stimulates Na
+
transport in the PT independent of local tissue RAS activation.
We predict that this short term sub-pressor AngII infusion retains NHE3 localization within
the microvilli in addition to increasing its total pool size, and that animals with enalapril will
also could drive NHE3 from the body to the base of the microvilli.
The TALH NKCC2 is found in both cortex and medulla and is regulated by SPAK via
phosphorylation (80). We recently reported that AngII infusion in mice decreased total
NKCC2 while increasing NKCC2-P, and increased SPAK-P without changing total SPAK
(25). In a more recent study in rats (Chapter 3), we were able to separate kidney cortex from
medulla and demonstrated that cortical NKCC2 and NKCC2-P were stimulated by AngII
infusion while medullary NKCC2 abundance was decreased (NKCC2-P was not significantly
decreased). Similarly, cortical SPAK and SPAK-P were stimulated by AngII while medullary
SPAK was decreased (SPAK-P was not significantly decreased) (Chapter 3). In this present
study, without accompanying hypertension, medullary TALH SPAK and NKCC2 were not
altered by short-term AngII infusion, or with enalapril treatment (Fig. 4.2), emphasizing that
medullary SPAK and NKCC2 are responsive to elevated blood pressure, and not to AngII
stimulation, independently from intrarenal RAS activation. Conversely, cortical TALH SPAK
and NKCC2 and their phosphorylation are stimulated by both chronic AngII infusion
(Chapter 3) as well as short-term low-dose AngII infusion (Fig. 4.3). The activation of
82

NKCC2 in the cortical TALH could be attributed by the increased phosphorylation of SPAK
(an indicator of SPAK activation) (Fig. 4.5) in the cortex. Taken together, we postulate that at
the early stage of AngII infusion, NKCC2 and SPAK are activated to increase Na
+

reabsorption in the cortical TALH, and the activation is sustained throughout the course of
AngII infusion to contribute to the development of hypertension seen at the end of the long-
term infusion (Chapter 3). It appears that blocking local tissue RAS generation with enalapril
did not affect the phosphorylation of both NKCC2 and SPAK at early time points, indicating
that their activation is independent from intrarenal RAS activation.
Previous studies demonstrated stimulation of post-macula densa sodium transporters
during AngII hypertension, namely increased NCC abundance and phosphorylation and
increased ENaC abundance (25, 95). This study explored the effects of AngII stimulation at
the earlier time course. We showed that AngII infusion increased abundance of NCC, and
NCC phosphorylation (a surrogate marker of NCC activation in apical membranes (48)), as
well as SPAK phosphorylation (the upstream kinase known to directly phosphorylate and
activate NCC (79)) in renal cortex (Figs. 4.4, 4.5), and increased the abundance of βENaC
subunit (Fig. 4.6). These findings demonstrate that Na
+
transporters along the distal nephron
are activated as early as 3 days after AngII infusion; thus, likely contributing to the AngII-
hypertension observed with longer infusion (Chapter 3). Interestingly, blocking tissue RAS
activation with enalapril significantly prevented the increase in NCC abundance caused by
AngII infusion (Fig. 4.4), suggesting that intrarenal RAS activation (or lowering whole body
AngII or aldosterone production) is required for the increased NCC activity at the early stage
of AngII infusion. The molecular mechanism(s) responsible for this ACE inhibition sensitive
accumulation of NCC remain to be determined.
83

In AngII-dependent hypertension, increased intrarenal angiotensinogen expression and
urinary angiotensinogen excretion are indicators of activation of intrarenal RAS;
subsequently, more AngII is produced and spilled over in the tubular fluid (42, 66). Increased
local AngII level in the kidney is essential to stimulate Na
+
transport in the distal nephron and
induce hypertension (25). We examined the activation of intrarenal RAS at the early phase of
AngII infusion and demonstrated that both cortical angiotensinogen and urinary
angiotensinogen excretion were not significantly increased after 3-day AngII infusion (Fig.
4.7) indicating that intrarenal RAS might not be activated at this early time point.. AngII has
been known to stimulate cellular oxidative stress (33, 40), which further increases the
expression of endogenous antigens such as Hsp70 to recruit adaptive immune response via
perivascular T-cell infiltration (reviewed in (82)). In accord with the previous study by
Ishizaka et al., we found that AngII infusion stimulates an increase in Hsp70 protein
expression in the cortex (Fig. 4.8)(39), which was not prevented by enalapril. We postulate
that at the early stage and low dose of AngII infusion, Na
+
transporters in the PT and in the
distal nephron are activated directly by AngII and that the accompanying increase in AngII
provoked ROS generation that stimulates the oxidative stress-Hsp70 pathway. When AngII is
sustained in the system for a longer period (2 weeks), positive feedback loop generating more
AngII in the tubular fluid via intrarenal RAS system will maintain the activation of
transporters in the distal nephron (Chapter 3, (25)) to further increase Na
+
reabsorption in this
region contributing to hypertension.
In conclusion, this study demonstrated that AngII can stimulate transporter abundance
and/or activity all along the nephron as early as after 3 days of AngII infusion without
accompanying hypertension, specifically PT NHE3, cortical TALH NKCC2, DCT NCC, and
84

cortical CD ENaC, are activated and may increase Na
+
reabsorption and contribute to the
AngII hypertension observed with longer infusion (Chapter 3). Together with the previous
study, we have a better understanding of how Na
+
transporters are regulated in AngII-
dependent hypertension model: in the pre-hypertensive state, there are increases in Na
+

transporters’ abundance along the nephron (without changing medullary TALH NKCC2
abundance); and an increase in the neo antigen Hsp 70 that can recruit T-cells to the kidney
and stimulate intrarenal RAS. Once hypertension ensues, pressure-natriuresis is provoked and
overrides the anti-natriuresis of AngII via suppression of PT and medullary TALH Na
+

transporters (Chapter 3) while maintaining the activation of transporters in the distal nephron
(Fig. 4.9). Understanding the time course of AngII action and where it stimulates Na
+

transporters along the nephron is crucial for targeting with anti-hypertensive therapies.

Figure 4.9. Proposed model of AngII-dependent hypertension. Red, increased
transporters’ abundance and/or activity; Green, decreased transporters’ abundance and/or
activity.

85

Chapter 5. Overall summary and future directions
The kidneys play an important role in maintaining fluid and electrolyte homeostasis by
regulating Na
+
and K
+
transport along the nephron. When there is a disturbance in Na
+
and
water reabsorption or K
+
balance, the kidneys develop compensatory mechanisms to achieve
new homeostatic states. The studies in this dissertation applied an integrative approach to
investigate transporters along the nephron to give a big picture of how fluid and electrolyte
balance is obtained.
Chapter 2 has shown that it is crucial to preserve the abundance of key renal
transporters under K
+
restriction, perhaps by retracting to intracellular pools. The only
differences measured in both K
+
-deficient groups (with and without NaCl supplementation)
were: a 20-30% decrease in cortical β-ENaC, 30-40% increases in KS-SPAK, and a 40%
increase in medullary sodium pump abundance. These are perhaps key adaptations that the
kidneys develop to conserve K
+
under hypokalemic condition. We have not confirmed
changes in other transporters reported by various groups (NHE3, NKCC, NCC, AQP2, and
ROMK). The differences may derive from different methods of sample preparation or from
the inconsistency of the diets. We emphasized in this study the importance of conserving the
total pool size of transporters that could be activated rapidly. Consider a carnivorous mammal
in the wild punctuating long periods of fast with feasts after the animal successfully hunts
down and consumes another K
+
-rich mammal (Fig. 5.1). The homeostatic challenge
immediately switches from conserving K
+
to secreting a large K
+
load. The switch would be
sluggish if it depended on changing transporter biosynthetic rates, and optimized by simply
activating existing transporters by redistribution or covalent modification.
86

Figure 5.1. Sudden switch from a K
+
-deprived to a K
+
-replete mode requires rapid activation
of transporters to prevent disturbances to cardiac or nervous systems

The next questions to be addressed are: 1) what is the molecular mechanism of
transporters’ activation responsible for rapid actions? (trafficking and/or covalent
modification by phosphorylation or proteolytic cleavage); 2) how do the kidneys sense the
drastic increase in plasma K
+
and is there a gut factor involved?
AngII is known to acutely stimulate Na
+
reabsorption in the PT, DCT and CCD in an
acute setting, and we previously showed that inhibition of the endogenous production of
AngII increases flow out of the PT and triggers the redistribution of NHE3 and NaPi2 to the
base of the microvilli and endosomes, respectively, and retracts NCC from plasma membrane
to the intracellular vesicles. In chapter 3, we explored the molecular mechanism of Na
+

transport in chronic AngII infusion hypertension model. When AngII stimulation is
accompanied by hypertension, compensatory natriuretic responses override the anti-
natriuresis of AngII. Previous studies reported the effects of AngII in this model using whole
kidney homogenates. By separating cortex from the medulla, this study determined that there
were increases transporters’ abundance and activation from the cortical TALH to the
medullary CD (NKCC2, NCC, ENaC and regulatory kinase SPAK) and that this stimulation
87

was balanced by a compensatory inhibition of transporters (NHE3 and medullary: NKCC2,
NKA, SPAK) from PT through medullary TALH, presumably via pressure-natriuresis
response. That is, hypertension overrides the effects of AngII from PT through medullary
TALH.
The observation that NKCC2 are differentially regulated in the cortex versus the
medulla leads to an interesting question: what is the molecular mechanism(s) responsible for
this differential regulation? We discovered that SPAK, a kinase that binds to and
phosphorylate NKCC2 directly (80), is also regulated in the same manner as NKCC2
(decreased abundance in the medulla and increased abundance and phosphorylation in the
cortex), indicating that SPAK regulation could be the key determinant responsible for NKCC2
activity in both cortical and medullary TALH. The upstream cascade should be explored
further. In recent years, the role of the WNK kinase network in regulating transporters,
especially those along the distal nephron, has been uncovered. This network is of pathological
relevance since Pseudohypoaldosteronism type II (PHAII) is an autosomal dominant disorder
characterized by hyperkalemia and hypertension, caused by mutations in two members of the
WNK kinase family, WNK1 and WNK4. WNK1 mutations are believed to increase WNK1
expression while the effect of WNK4 mutations remains controversial. It has been reported
that AngII action on the NCC occurs via a WNK4-SPAK-dependent signaling pathway (9,
84), and in vitro study has revealed that the activation of WNK1-SPAK cascade promotes the
phosphorylation of NKCC2 (80). It would be interesting to confirm whether WNK kinases are
also differentially regulated in the cortex versus medulla in AngII-hypertension model, which
could provide potential therapeutics for hypertension.
88

An important question to address is how the pressure natriuresis response counteracts
the anti-natriuretic influence of AngII infusion. This will require defining the signaling
pathway connecting the rise in blood pressure to the inhibition of sodium transport, discussed
in Chapter 3.
In chapter 4 of this dissertation, we studied the effects of short-term chronic AngII
infusion without accompanying hypertension to tease out the role of AngII per se. We
determined that AngII can stimulate transporter abundance and/or activity all along the
nephron, specifically PT NHE3, cortical TALH NKCC2, DCT NCC, and cortical CD ENaC,
as early as after 3 days of AngII infusion, to increase Na
+
reabsorption in these regions,
contributing to hypertension observed with longer infusion. In this study, the medullary
TALH NKCC2 and SPAK abundance were not altered with AngII infusion, in comparison to
the significant decreases seen with AngII-hypertension (Chapter 3). It is possible that the
medullary TALH is only responsive to elevated blood pressure, rather than AngII, and that
NKCC2 in the medullary TALH does not play a vital role in the development of hypertension.
Acute AngII infusion without increasing BP promotes NHE3 trafficking from the base
of the microvilli into the body of the microvilli (81). In chapter 3, we reported that chronic
AngII infusion with accompanying hypertension retains NHE3 within the microvilli while
pressure-natriuresis compensates by decreasing the total abundance, emphasizing the
important role of AngII in maintaining NHE3 localization in the microvilli. With short-term
low-dose AngII infusion, we reported a significant increase in NHE3 total abundance. The
next questions to be addressed are: 1) what is the localization of NHE3 in this model, and 2)
how does the transition from short to long term AngII treatment decrease the NHE3
abundance. We hypothesize that at the early phase of AngII infusion without the presence of
89

hypertension, NHE3 is also localized in the PT microvilli, together with increased total
abundance promoting an increase in Na
+
transport in the PT. The renal responses to short-
term low-dose AngII are similar to those in long-term AngII infusion starting from the
cortical TALH through the CD, suggesting that the distal nephron is affected by anti-
natriuretic action of AngII to contribute to the development and maintenance of hypertension
in AngII-dependent hypertension model.
These studies again emphasize the roles of kidneys to maintain electrolyte and fluid
homeostasis under various stimuli. A lion eating a gazelle after a long fasting period does not
suffer from a heart attack, and a hypertensive individual does not walk around with a body
full of excess fluid. Taking an integrative approach, rather than focusing on specific regions
of the nephron, provided us with a complete picture of stimulatory versus compensatory
mechanisms and how they interphase with each other throughout the nephron. Considering
counter-regulatory regulatory pathways concurrently (high versus low K
+
diets, hypertension
versus pressure natriuresis is crucial to improving our knowledge of the molecular
mechanisms of body homeostasis is maintained.

90

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Abstract (if available)

Abstract The kidneys play an essential role in regulating blood pressure (BP) and maintaining fluid and electrolyte homeostasis. The balance is achieved by an integrative network of hormones and nerves that signal changes in Na⁺ and K⁺ transporters along the nephron under various stimuli, such as hypokalemia, elevated circulating plasma AngII concentration, and high BP. The regulation could be either acute or chronic via trafficking, covalent modification, or changes in total transporter abundance. Taking an integrative approach to generate transporter profiles along the nephron, this dissertation aimed to determine 1) the effects of K⁺-deficient diets with and without NaCl supplementation on Na⁺, K⁺, and H₂O transporters’ abundance

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Asset Metadata

Creator Nguyen, Mien Thi Xuan (author)

Core Title Integrated regulation of transporters along the nephron to maintain electrolyte and fluid homeostatis

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Integrative Biology of Disease

Publication Date 07/21/2013

Defense Date 05/14/2013

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

Tag angiotensin II,hypertension,hypokalemia,OAI-PMH Harvest,transporters

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor McDonough, Alicia A. (committee chair), Chow, Robert H. (committee member), Okamoto, Curtis Toshio (committee member), Peti-Peterdi, Janos (committee member)

Creator Email duodenum07@gmail.com,miennguy@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-294063

Unique identifier UC11293690

Identifier etd-NguyenMien-1805.pdf (filename),usctheses-c3-294063 (legacy record id)

Legacy Identifier etd-NguyenMien-1805.pdf

Dmrecord 294063

Document Type Dissertation

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Rights Nguyen, Mien Thi Xuan

Type texts

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

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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