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Regulation of potassium homeostasis during acute potassium loading
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Regulation of potassium homeostasis during acute potassium loading
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Content
Regulation of potassium homeostasis during acute
potassium loading
By
Srinivas Rengarajan
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
AUGUST 2013
Copyright 2013 Srinivas Rengarajan
ii
TABLE OF CONTENTS
Acknowledgements ……………………………………………………………………………………………………………………………….iii
List of Figures …………………………………………………………………………………………………………………………………………iv
List of Tables ………………………………………………………………………………………………………………………………………….vi
List of Abbreviations ……………………………………………………………………………………………………………………………..vii
Abstract ………………………………………………………………………………………………………………………………………………….ix
Introduction ……………………………………………………………………………………………………………………………………………1
Scope of the dissertation ………………………………………………………………………………………………………………………12
Materials and Methods …………………………………………………………………………………………………………………………13
Results ………………………………………………………………………………………………………………………………………………….19
Discussion …………………………………………………………………………………………………………………………………………….30
References ……………………………………………………………………………………………………………………………………………34
iii
ACKNOWLEDGEMENTS
McDonough lab My parents
Dr. Alicia McDonough Rengarajan Seshadri
Donna Lee Vasantha Rengarajan
Mien Nguyen
Nikhil Kamat
Muhammad Madkour
Youn lab
Dr. Jang Youn
Dr. Young Oh
Committee members
Dr. Zoltan Tokes
Dr. Vijay Kalra
Dr. Janos Peti-Peterdi
Dr. Vito Campese
Dr. Lorraine Turcotte
Cannon lab
Dr. Paula Cannon
Dr. Colin Exline
Dr. Su Yang
Kevin Haworth
Program co-ordinators
Dawn Burke
Raquel Gallardo
Karissa Nguyen
iv
LIST OF FIGURES
Figure 1: Schematic diagram of the potassium homeostasis maintenance system
Figure 2: Feedback and feedforward mechanisms of regulating potassium excretion
Figure 3: Differential regulation of potassium excretion
Figure 4: Major ion transport proteins along the nephron
Figure 5: Regulation and distribution of NCC
Figure 6: Effects of consuming a high K
+
diet on renal transporters
Figure 7: Integrated view of the effects of a high K
+
meal on renal transporters
including potential regulatory signals
Figure 8: Regulation of NKCC and NCC phosphorylation (activity) by the WNK-SPAK
kinase cascade
Figure 9: Timeline for diet feeding experiments comparing the effects of a 3hr meal
of 0% or 2% K+ diet on renal transporter regulation
Figure 10: Schematic diagram showing the experimental setup used to perform the
KCl infusion experiments with real time plasma sampling
Figure 11: Time course of change in urinary Na
+
, K
+
and tissue kallikrein after a meal.
Figure 12: Urine volume, urinary and plasma Na
+
, K
+
concentrations after a meal.
Figure 13: Effect of 2% versus 0% K+ meal on NCC, NKCC abundance and
phosphorylation in renal cortex homogenates.
Figure 14: Effect of 2% K
+
meal versus 0% K
+
meal on subcellular distribution of NCC
and NCCp.
Figure 15: Effect of 2% K+ meal versus 0% K+ meal on SPAK (Ste20/SPS1-related
proline/alanine-rich kinase) abundance and phosphorylation in renal cortex
and medulla homogenates.
Page 2
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Page 11
Page 14
Page 15
Page 19
Page 20
Page 21
Page 22
Page 23
v
Figure 16: Effect of 2% K+ meal versus 0% K+ meal on cortical collecting duct ROMK
and ENaC abundance in renal cortex homogenates.
Figure 17: Timecourse of plasma [K+] during KCl infusion
Figure 18: Effects of raising plasma [K
+
] by tail vein infusion on urinary electrolytes in
conscious rats.
Figure 19: Effects of raising plasma [K+] to 5.5 mEq by tail vein infusion on NCC and
NKCC abundance and phosphorylation in renal cortex homogenates.
Figure 20: Effects of raising plasma [K+] to 5.5mEq by tail vein infusion on SPAK
abundance and phosphorylation in renal cortex homogenates.
Page 24
Page 26
Page 26
Page 28
Page 29
vi
LIST OF TABLES
Table 1: Summary of physiology data after a 3hr meal
Table 2: Effects of 3hr KCl infusion
Page 25
Page 27
vii
LIST OF ABBREVIATIONS
ACE: Angiotensin converting enzyme
ASDN: Aldosterone sensitive distal nephron
ATPase: Adenosine triphosphatase
BK: Big potassium channel
BSA: Bovine serum albumin
CCD: Cortical collecting duct
DCT: Distal convoluted tubule
Disodium EDTA: Disodium ethylenediaminetetraacetic acid
ECF: Extracellular fluid
ENaC α/β/γ: Epithelial sodium channel α/β/γ subunit
Hct: Hematocrit
ICF: Intracellular fluid
ICM: Intracellular membrane
Mu: Mouse
NCC-t, NCC-pS71, NCC-pS89, NCC-pT53: Sodium chloride cotransporter-total/phosphorylated at Serine
71/Serine 89/Threonine 53
Nedd4-2: Neural precursor expressed, developmentally downregulated
NHE3-t, NHE3-pS552: Sodium hydrogen exchanger 3-total/phosphorylated at Serine 552
NKCC2-t, NKCC2-pT96T101: Sodium potassium chloride cotransporter type 2-total/phosphorylated at
both Threonine 96 and 101
OD: Optical density
OSR1-pS325: Oxidative stress response 1 phosphorylated at Serine 325
PM: Plasma membrane
Rb: Rabbit
ROMK: Renal outer medullary potassium ion channel
viii
S/D rats: Sprague/Dawley rats
SDS-PAGE: Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
SE: Standard error of mean
Sh: Sheep
So: Supernatant
SPAK-2, FL-SPAK, KS-SPAK, SPAK-pS373: STE20/SPS1-related proline/alanine-rich kinase type 2 (FL-full
length, KS-kidney specific, pS373-phosphorylated at Serine 373)
TALH: Thick ascending loop of Henle
TBST: Tris buffered saline with tween 20
UKV: Total urinary potassium excreted in a volume V
UNaV: Total urinary sodium excreted in a volume V
UV: Total urinary volume excreted
WNK, KS-WNK1: With no lysine kinase (KS-kidney specific)
ix
ABSTRACT
Potassium is important for the maintenance of acid-base balance, electrolyte homeostasis and optimal
cellular excitability. It is vital that the plasma K
+
level is maintained within a narrow window to prevent
adverse health effects. On a day to day basis, the greatest challenge to the maintenance of K
+
homeostasis is the periodic dietary intake of boluses of K
+
. The kidneys, muscles and gut work in a co-
ordinated manner to regulate the output of K
+
from the body and match it to the intake. The kidney is
the most important of these components in the maintenance of K
+
homeostasis. It reacts rapidly to
incoming K
+
loads and is able facilitate filtration and excretion of K
+
via the urine by regulating the
abundance and activity of various ion transporters in the nephron. Currently, there is a lack of complete
understanding about the source of the signal which responds to disturbances in the K
+
homeostasis and
communicates to the kidneys to regulate its ion transporters accordingly to maintain homeostasis.
Dietary potassium loading results in rapid kaliuresis, natriuresis and diuresis. We found that a 3 hour 2%
K
+
meal caused a modest increase in plasma K
+
from 4.01±0.12mM to 5.18±0.16mM in Sprague Dawley
rats (n=8). This was accompanied by rapid decrease in NCC-P potentially regulated via a decrease in
cortical SPAK-P abundance. However there was no effect on CCD transporters ENaC or ROMK within 3
hours. Similar effects on NCC-P and SPAK-P abundance were observed when the plasma K
+
level was
raised by direct intravenous KCl infusion to the same level as by diet feeding. Thus an acute and minimal
increase in the plasma [K
+
] is a sufficient signal to trigger renal kaliuretic mechanisms via NCC
downregulation.
1
INTRODUCTION
Potassium is an important nutrient for maintaining acid balance, cell function and electrolyte
homeostasis. The extracellular fluid (ECF) concentration of potassium is responsible for maintaining
membrane potential and thus vital for nerve and muscle cell function. In turn this affects cardiovascular
health (Bia and DeFronzo 1981; Kurtzman, Gonzalez et al. 1990). The physiological range of ECF [K
+
]
concentrations is about 3.6-5mEq in mammals. It is important to maintain the ECF [K
+
] in this range as
hypokalemia and hyperkalemia can cause cell membranes to hyperpolarize or depolarize respectively,
leading to improper functioning of excitable cells and cardiovascular distress (Cohen, Madhavan et al.
2001; Macdonald and Struthers 2004; Aburto, Hanson et al. 2013).
K
+
handling disorders are a serious real world problem. The incidence of hyperkalemia ranges from 1.1%
to 10% in all hospitalized patients. Cardiac arrhythmias, nervous symptoms such as tingling of the skin,
numbness of the hands or feet, weakness, or flaccid paralysis, are characteristic of hyperkalemia and
hypokalemia. Several genetic disorders such as Bartter’s syndrome, Gitelman’s syndrome,
Pseudohypoaldosteronism type-1 and Liddle’s syndrome are caused due to mutations in the renal
electrolyte transport proteins and their related regulatory proteins (Rossier, Staub et al. 2013). These
disorders manifest hyperkalemia or hypokalemia which can lead to secondary health problems.
There are several large populations at risk. For example: 15% of type 1 diabetics have hyperkalemia due
to the lack of insulin which causes defects in muscle uptake of potassium. The prevalence of
hyperkalemia ranges from 5% to 10% in patients with end-stage renal disease (Jarman, Kehely et al.
1995). Another population which is at significant risk are people suffering from hypertension and taking
prescription angiotensin-converting enzyme (ACE) inhibitors which are one of the most prescribed drugs
in the world (IMS Institute for Healthcare Informatics report, 2011). Hyperkalemia occurs in 9% to 12%
2
of these patients (Anderson "Hyperkalemia" Brenner & Rector's The Kidney). A thorough understanding
of the body’s K
+
handling mechanism is necessary to alleviate disorders due to improper K
+
homeostasis.
The ECF contains about 2% of body’s stored K
+
content. However, there is a huge reservoir (>90% of
total body K
+
) of intracellular fluid (ICF) [K
+
] where the concentration is 120–140 mM. Most of this
reservoir of potassium is stored in muscle cells (Giebisch and Wang 1996; Thompson, Choi et al. 1999).
K
+
has a very high turnover rate. Potassium consumed through the diet constantly needs to be excreted
and this poses a homeostatic challenge to the body. The body is extremely efficient at clearing dietary
potassium after a meal and also at maintaining ECF [K
+
] during fasting or K
+
poor diet. This keeps the
plasma K
+
relatively constant. The gut, kidneys and muscle cells work in concert to maintain the ECF K
+
homeostasis (Figure 1: adapted from Youn, McDonough 2009).
Figure 1: Schematic diagram of the potassium homeostasis maintenance system
The diagram depicts the roles of the gut, muscle cells and kidney in the maintenance of extracellular
potassium concentration. The figure also shows the distribution of potassium within the
intracellular and extracellular fluid of the body via Na, K ATPase pumps (grey arrows). The red
arrows show the path taken by potassium consumed as part of the diet. (Youn and McDonough
2009)
3
The K
+
homeostatic system includes 2 major methods of controlling the ECF K
+
levels. One is the
regulation of K
+
distribution between the ECF and the ICF via the Na, K
+
ATPase pumps on the muscle cell
surface. These serve to shunt sudden boluses of excess K
+
in the ECF into the large ICF pool via the Na
+
pumps to provide fast protection against spiking of ECF K
+
concentration.
The other major component of the K
+
homeostasis machinery is the regulation of K
+
excretion. The
kidneys are the most vital organs in this system (Rutledge and Rabinowitz 1987). They process about
25% of the cardiac output and are primarily responsible for filtering and excreting or retaining K
+
in the
ECF in order to maintain homeostasis. Both the components of the K
+
homeostatic machinery depend
on the K
+
sensing mechanism in order to function effectively.
There are 2 possible methods of detecting a K
+
load in the ECF (Figure 2: adapted from Youn,
McDonough 2009). One is the traditional feedback mechanism by which any increase or decreases in the
plasma K
+
concentration would trigger the components of the homeostatic machinery and enable
suitable regulation of potassium excretion to adjust the ECF K
+
concentration back to normal levels.
The second mechanism that was proposed by Rabinowitz et al (Rabinowitz 1988; Calo, Borsatti et al.
1995) is the feedforward mechanism wherein an incoming load of K
+
is sensed before it enters the ECF.
In this manner a rapid response can be triggered preparing the regulatory machinery to receive a bolus
of K
+
and be primed to rapidly excrete or remove the K
+
from the ECF as it appears. This mechanism
allows even transient increases in the ECF K
+
to be avoided. There is evidence that K
+
sensing mechanism
exists in the splanchic bed which facilitates K
+
clearance by the kidney via urine (Lee, Oh et al. 2007; Oh,
Oh et al. 2011). This is one possible feedforward regulation mechanism.
4
The kidneys continuously match K
+
excretion to dietary K
+
intake (Rutledge and Rabinowitz 1987; Wang
and Giebisch 2009; Youn and McDonough 2009). They do this by modulating the amount of K
+
excretion.
They control 90% of K
+
excretion (Giebisch and Wang 1996; Wang 2004) from the body. The ICF pool of
K
+
also serves as a buffer for maintaining the ECF K+ concentration by rapidly shifting K
+
between the
ECF and the ICF pools. The total ECF pool of K
+
is only about 70mEq and the consumption of K
+
rich foods
can add more than 70mEq of K
+
into the body system in a very short span of time. This may potentially
double the ECF [K
+
] if there were not rapid adjustments to either transfer the K
+
to the ICF compartment
or excrete it. After a meal, insulin is secreted and it stimulates muscle and liver to take up glucose and
the K
+
that is not excreted in the short term by the kidneys (DeFronzo, Felig et al. 1980). Later the K
+
is
released into the ECF from the large ICF pool and then excreted via the urine.
Figure 2: Feedback and feedforward mechanisms of regulating potassium excretion
Changes in ECF [K
+
] after dietary intake drives regulation of K
+
excretion (blue arrow) in
the traditional feedback control mechanism. Whereas feedforward control is dependent
on sensing of the incoming K
+
load (purple arrow) before it is delivered to the ECF. (Youn
and McDonough 2009)
5
In the opposite scenario, fasting or consuming a low-K
+
diet causes the urinary excretion of K
+
to be
reduced to almost zero as the kidneys reabsorb more K
+
(Giebisch and Wang 1996; Weiner and Wingo
1997). Despite the highly efficient renal conservation of K
+
there is loss in the stools and via sweat which
over time could cause the ECF K
+
pool to deplete due to relatively small size. To prevent this from
happening and to keep the ECF K
+
concentration constant muscle cells donate ICF [K
+
] to the ECF
(Norgaard, Kjeldsen et al. 1981; Thompson, Choi et al. 1999). In such a scenario the sensing of ECF K
+
status and rapid responses are required to maintain a ratio of ECF to ICF [K
+
] and ensure the normal
functioning of nerves and maintain suitable muscle cell excitability. This sensing and control mechanism
may also occur by feedback or feedforward control of ECF K
+
.
Electrolyte balance is regulated by modulation of the abundance and activity of a variety of ion
transporters in the renal nephron. These determine the complement of electrolytes that must be
reabsorbed from the renal filtrate. There is evidence that the transporters in the proximal nephron are
passive and their activity level is constant both in conditions of high and low K
+
diet consumption (Figure
3: Stanton and Koeppen, Renal Physiology, 4
th
edition, 2006)
Figure 3: Differential regulation of
potassium excretion
Under conditions of normal K
+
intake, the
distal tubule (DT) and the cortical
collecting duct (CCD) combine to secrete
upto 80% of K
+
flowing through the
nephron into the urine. During K
+
depletion, they secrete only about 1% of
K
+
flowing through, reabsorbing most of
the K
+
passing through. The proximal
nephron has a similar reabsorption profile
under both conditions. (Stanton and
Koeppen, Renal Physiology, 4
th
edition,
2006)
6
The transporters that have been found to be most important for regulating K
+
excretion are present in
the aldosterone sensitive distal nephron (ASDN) which is comprised of the distal convoluted tubule
(DCT) and the cortical collecting duct (CCD). The Na-Cl cotransporter (NCC) in the DCT reabsorbs Na
+
and
Cl
-
from the renal filtrate. It meters the amount of Na
+
that is sent to the distal collecting tubule. The
major transporters in the collecting tubule are the renal outer medullary K
+
channel (ROMK), BK
channels and the epithelial Na
+
channel (ENaC). Figure 4 below illustrates the major ion transporters
along the nephron (Mullins, Bailey et al. 2006).
Figure 4: Major ion transport proteins along the nephron
(B) The Na, H exchanger 3 (NHE3) in the proximal tubule is responsible for bulk of the Na
+
reabsorption in this region. (C) The Na, K, Cl cotransporter 2 (NKCC2) reabsorbs Na
+
and K
+
in the
thick ascending loop (TAL) of Henle. The absorbed K
+
is recycled out via constitutively active renal
outer medullary K (ROMK) channels. (D) The Na, Cl cotransporter (NCC) in the distal convoluted
tubule (DCT) reabsorbs Na and Cl. (E) The epithelial Na channel (ENaC) reabsorbs Na in the cortical
collecting duct (CCD) and creates the driving force to excrete K via the ROMK channels. (Mullins,
Bailey et al. 2006)
7
The major mechanism of NCC regulation is by differential phosphorylation. NCC is phosphorylated by
SPAK (STE20/SPS1-related proline/alanine-rich kinase). NCC has a long N-terminal intracellular domain
on which multiple sites of phosphorylation have been identified (Dimke 2011). The T58 site in rats is the
master phosphorylation site which regulates the phosphorylation of other sites on NCC and the
activation of the protein (Figure 5A).
Phosphorylated (active) NCC is present on the apical plasma membrane of distal convoluted tubule cells.
They are not present in the sub apical cytoplasmic membranes (Lee, Maunsbach et al. 2013). Whereas,
total NCC is distributed between both the apical plasma membrane and the sub apical intracellular
vesicles (Figure 5B)
A
B
Figure 5: Regulation and distribution
of NCC
Figure (A) shows multiple sites of
phosphorylation in the intracellular N-
terminal domain of NCC in humans.
The T60 site is the main site of
phosphorylation which controls the
phosphorylation of other downstream
sites and regulates NCC activity. Only
NCCp is active. (Dimke 2011)
Figure (B) is an immunoelectron
micrograph showing the distribution of
NCCpT58 (secondary antibody labeled
with 15 nm Au particles-horizontal
arrows) and NCC-total (secondary
antibody labeled with 5nm Au
particles-vertical arrows). NCCpT58 is
found only on the apical plasma
membrane surface while the NCC-total
is found on the plasma membrane and
the subapical cytoplasmic vesicles
(Lee, Maunsbach et al. 2013)
8
It is postulated that when a high K
+
meal is consumed, the NCC transporter activity in the DCT is
downregulated, causing an increased load of Na
+
to be delivered to the collecting duct. This creates an
electrochemical gradient favoring the reabsorption of Na
+
via the ENaC channel generating the driving
force of K
+
secretion in the opposite direction through the ROMK and BK channels (Figure 6) (Wang and
Giebisch 2009; Holtzclaw, Grimm et al. 2011). Recently, Sorensen et al (Sorensen, Grossmann et al.
2013) observed that feeding a high K
+
diet or a K
+
gavage in the short term in mice caused a rapid
dephosphorylation of NCC (only the phosphorylated form of NCC is active) to support this hypothesis. In
the longer term Vallon et al also observed NCC-t/p reduction (Vallon, Schroth et al. 2009). The
mechanism of action is similar to that of a thiazide diuretic since dietary K loading diminishes the
natriuretic response to a thiazide diuretic (Shirley, Skinner et al. 1987). While these groups investigate
the effector components of the renal K
+
regulatory mechanism, they do not consider the signaling
elements or source (gut or plasma) that may be responsible for priming the observed transporter
adaptations.
Figure 6: Effects of consuming a high K
+
diet on renal transporters
After a high K
+
meal, signals from the splanchic bed or due to increase in ECF [K
+
] cause the
downregulation of NCC activity in the DCT. Increased Na
+
delivery to the CCD causes increased
uptake of Na
+
through the ENaC channels and creates a suitable electrochemical gradient to drive
the excretion of K
+
through the ROMK channels in the CCD.
9
In this study we are especially interested in the renal regulation of K
+
excretion after the consumption of
a K
+
rich meal and the signaling events (Figure 7) that immediately precede the renal transporter
adaptations that facilitate rapid K
+
excretion. One possible signal is the gut K
+
sensing factor that has
been shown to influence efficient K
+
excretion after K
+
loading (Rabinowitz, Sarason et al. 1985;
Rabinowitz 1988; Rabinowitz, Green et al. 1988; Lee, Oh et al. 2007; Oh, Oh et al. 2011). The gut sensing
factor would act through the feedforward regulation model for K
+
excretion suggested by Rabinowitz et
al. the second possible signal is increases in ECF [K
+
] which would work through the traditional feedback
regulation mechanism. It has been shown that raising the plasma K
+
by infusion causes a linear increase
in K
+
excretion (Rabinowitz, Sarason et al. 1985). Several studies have attempted to describe the effect
of K
+
loading on the kidney, but there have always been confounding factors (long term diet feeding, ex
vivo work: this does not account for any neuro-humoral response stimulated by plasma K
+
in an intact
animal etc.) that prevented investigators from making conclusions about the acute role of plasma K
+
as a
signal for renal transporter adaptations. This paper aims to address this factor directly and without any
such confounders. A third factor that may be considered is increased plasma aldosterone which
influences the transporters in ASDN and stimulate K
+
excretion by concerted regulation of ENaC, ROMK
and Na
+
,K
+
ATPase (Wang and Giebisch 2009; Holtzclaw, Grimm et al. 2011). However, this process
occurs by regulation of transcription and would occur over a longer time frame. Also there is evidence
that K
+
loading causes increased natriuresis which is contrary to the effects of high plasma aldosterone.
Aldosterone(Sorensen, Grossmann et al. 2013) has been shown to be not an important factor though
since they observed NCC-P reduction before plasma aldosterone levels increased in wild type mice. They
also observed similar NCC-P reduction in aldosterone synthase
-/-
mice after K
+
loading by gavage or
through diet feeding. Finally, kallikrein has been shown to be protective against K
+
loads in an
aldosterone independent manner (El Moghrabi, Houillier et al. 2010). Kallikrein is produced by
connecting tubule cells which connect the DCT and the CCD. The kallikrein is released into the lumen of
10
the nephron and flows out into the urine. Kallikreins have also been shown to have a role in the
activation of ENaCs (Picard, Van Abel et al. 2005; Chambrey and Picard 2011; Palmer, Patel et al. 2012;
Patel, Chao et al. 2012) by proteolysis as they move down the nephron. The activation of ENaC by
kallikrein is a potential signal to initiate renal transporter adaptations to allow increased K
+
excretion in
an acute time period.
Figure 7: Integrated view of the effects of a high K
+
meal on renal transporters including potential
regulatory signals
The figure shows 4 possible sources of regulatory signals transmitting information from the K
+
sensing system to the regulatory machinery that controls the activation of renal ion transporters and
ultimately the excretion of potassium though the urine.
11
We postulate that these are the possible signals that may cause the renal transporter adaptation to an
acute high K
+
load. These signals may act via various intermediate effector molecules like WNK-4 (With
No lysine-isoform 4), WNK-1, Full Length- SPAK (STE20/SPS1-related proline/alanine-rich kinase) and
Kidney specific-SPAK (Kahle, Wilson et al. 2003; O'Reilly, Marshall et al. 2006; Liu, Xie et al. 2011;
McCormick, Mutig et al. 2011; Hoorn and Ellison 2012). SPAK is the kinase that is responsible for
phosphorylating and activating NCC (Figure 8).
Figure 8: Regulation of NKCC and NCC phosphorylation (activity) by the WNK-SPAK kinase
cascade
FL-SPAK is responsible for the phosphorylation and activation of TALH and DCT transporters
NKCC2 and NCC respectively. FL-SPAK activity is in turn regulated by a complicated cascade of
kinases including Wnk-1, Wnk-4 and KS-SPAK. Only the phosphorylated species of NKCC2 and
NCC are active
12
SCOPE OF THE DISSERTATION
K
+
homeostasis is extremely important for cellular function, excitability and cardiovascular health. The
biggest challenges faced on a day to day basis for the maintenance of K
+
homeostasis is the consumption
of K
+
rich meals. The kidney has been shown to be vital in regulating K
+
homeostasis. NCC-P
downregulation in response to an acute load of dietary potassium has been documented (Sorensen,
Grossmann et al. 2013). However, the signaling pathway connecting potassium sensing machinery to the
transporter regulation in the kidney is unclear.
Gut sensing of dietary potassium has been shown to be important for efficient potassium handling (Lee,
Oh et al. 2007; Oh, Oh et al. 2011). However there is no proof that the gut sensing of dietary potassium
is responsible observed renal transporter adaptations. It is also unclear if changes exclusively in the
plasma [K
+
] are sufficient to bring about changes in renal ion transporter activities.
We hypothesize that the kidney matches the K
+
excretion to the dietary input by downregulating NCC
activity in the DCT so that more Na
+
is delivered to the CCD and reabsorbed by ENaC creating the
electrochemical gradient to facilitate K
+
excretion through the ROMK channel. Related to these events,
we aimed to determine
1) The increase in plasma [K
+
] following a single K
+
rich meal in rats and whether there were
accompanying changes in NCC, NKCC or SPAK abundance and phosphorylation that could drive
increased K
+
excretion.
2) Whether tissue kallikrein accumulation and ENaC activation occur after a single K
+
rich meal
3) Whether a rise in plasma [K
+
] equivalent to that observed in response to the single K
+
rich meal
can drive the acute kaliuresis
13
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.
Experiments were performed on male Sprague-Dawley rats (225–280 g wt) purchased from Harlan
Laboratories (San Diego, CA).
Diet feeding experiments: Animals were divided into two groups (n=8 each). The animals initially
received standard rat chow pellets (0.74% NaCl and 2% KCl) up to 3pm on the day before the
experiment was conducted. At this point, the rats were weighed and moved into metabolic cages
(Techniplast) with free access to water but no food. Food was withdrawn over 16 hours till 7am on the
day of the experiment to clear potassium from the rats’ urine and to prime them to consume the diet
used in the experiment. At 6am, the rats were given access to saccharin (Sweet n Low 0.5g/100ml)
sweetened water to stimulate fluid intake but no food. At 7am, the rats were fed with diets prepared
from potassium-deficient powdered rat chow (cat. no.TD 88239; Harlan-Teklad, Madison, WI), which
was supplemented with KCl to the following percentages (in dry weight): 4% KCl and 0.74% NaCl (2% K
+
diet); 0% KCl and 0.74% NaCl (0%K
+
diet). To gel the diets, 25 g of Difco Agar Noble was dissolved by
heating in 835 ml of deionized water and added to the dry diet. These diets were stored at -20°C in meal
size blocks until use. The rats had access to the food and sweetened water for about 3 hours before the
rats were withdrawn from their metabolic cages for sacrifice.
14
Both overnight (16 hour) and 3 hour urine samples were collected in metabolic cages (Techniplast). The
rats were then anesthetized intramuscularly with ketamine-xylazine, blood samples were collected from
tail clipping and cardiac puncture, plasma was prepared, and kidneys were removed and weighed. The
urine present in the rats’ bladder was extracted using a syringe and added to the collected 3hr urine
sample. Urine volume was recorded in graduated cylinders, urine and plasma [Na
+
] and [K
+
] were
measured by flame photometry (Radiometer FLM3).
Potassium infusion experiments: The Sprague-Dawley rats were placed in specially made cages with
their tails tethered for a 4 days acclimatization period. On the day prior to the experiment, they were
shifted to a new cage at 3pm following tail vein cannulation as described before (Lee, Oh et al. 2007; Oh,
Oh et al. 2011) and were allowed to continue consuming vivarium rat chow ad lib with free access to
water. The rats were fasted with free access to water starting 8pm on the day before the experiment. At
7am on the day of the experiment, the rats’ tail artery cannulation was done as described earlier (Lee,
Oh et al. 2007; Oh, Oh et al. 2011) and the rats were allowed to recover for 4 hours before the
Figure 9: Timeline for diet feeding experiments comparing the effects of a 3hr meal of 0% or 2%
K
+
diet on renal transporter regulation
15
experiment in a cage with a wire gauze bottom. The rats were divided into 3 groups (n=5) for the
experiment. KCl infusion and saline control group rats received a saline pre-infusion for 10 minutes (at
4ml/hr) prior to the start of the experiment. The KCl infusion group received 150mM KCl solution at a
rate based on previous studies (Lee, Oh et al. 2007; Oh, Oh et al. 2011). The KCl infused rats’ plasma was
sampled every 15 minutes via the tail artery catheter and real-time flame photometry was done to
determine the plasma [K
+
] and [Na
+
]. The KCl infusion rate was adjusted to keep the plasma [K
+
] at
approximately 5.2mM. The isoosmotic saline infusion rate was concurrently varied to keep the overall
infusion rate constant at 4ml/hr over 3 hours. The saline only infusion group was matched pairwise to
have matching saline infusion profiles to the KCl infusion group. The third group was the no infusion
control. After the 3 hour infusion period the kidneys were collected as described earlier (Nguyen, Lee et
al. 2013). The urine excreted during the 3 hour infusion period was collected from the bottom of the
cage and pooled with the bladder urine collected at sacrifice.
Figure 10: Schematic diagram showing the experimental setup used to perform the KCl infusion
experiments with real time plasma sampling
16
Baseline animals were the no treatment controls. KCl infusion and saline control rats received a saline
pre-infusion for 10 minutes (at 4ml/hr) prior to the start of the experiment. Following this, the KCl
infusion group rats received 150mM KCl solution at a rate based on previous studies (Lee, Oh et al.
2007; Oh, Oh et al. 2011) for the first hour. The rate of infusion of isoosmotic saline along with the KCl
solution was adjusted throughout so that the total infusion rate was a constant at 4ml/hr. the saline
infusion was done so as to dilute the KCl and reduce the discomfort caused to the rats at the point of
infusion. The saline control group received only saline matched pairwise to the rate of infusion of saline
in the KCl infusion rats. At the end of 3 hours of infusion during which the excreted urine was collected
at the bottom of the cage, the animals were sacrificed as before and their kidneys were processed as
before. The rats had free access to water throughout the course of the experiment.
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, 0.5mM disodium EDTA, and 5mM histidine-imidazole buffer, pH 7.5, with the addition of
0.2mM PMSF, 9µg/ml aprotinin, and 5µl/ml phosphatase inhibitor cocktail (Sigma)]. Each sample was
homogenized for 5 min at a low-speed setting with an Ultra-Turrax T25 (IKA-Labortechnik) and then
centrifuged at 2,000 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 as before, and pooled with the first
supernatants. The 2,000g supernatant (So) protein concentrations were determined using the Pierce
BCA kit (Thermo Scientific). The samples were aliquoted and stored at -80°C. So protein concentrations
were ~10 mg/ml for cortex and ~3 mg/ml for medulla. The samples were then assayed by immunoblot.
Differential fractionation of intracellular membranes vs. plasma membranes: In a subset of samples,
intracellular (ICM) and plasma membranes (PM) were enriched as described by Sachs et al (Sachs,
Pisitkun et al. 2008). In brief, the 2,000g supernatant, prepared as above, was spun at 17,000g; the
17
resultant 17,000g pellet, enriched in PM, was re-suspended in isolation buffer (see Homogenate
preparation). The 17,000g supernatant was spun at 150,000g for 80 min, and the pellet, enriched in ICM,
was re-suspended in isolation buffer. Aliquots of the PM and ICM fractions were frozen at -80°C pending
assay.
Quantitative immunoblotting: Cortical and medullary homogenates were denatured in SDS-PAGE
sample buffer for 20 min at 60°C and then resolved on SDS-polyacrylamide gels (Laemmli 1970). For
each sample, one-half the amount of protein was loaded adjacent to the full amount of protein to verify
linearity of the detection system. 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 (Immobilon-FL; Millipore, Temecula, CA), blocked
(bløk-FL; Millipore), and then probed with one of the following antibodies (diluted in TBST: 1% BSA, 15
mM NaN3): Rb anti-NHE3 (1:2000, McDonough); Mu anti-NHE3-pS552 (1:1000, Santa Cruz); Mu
monoclonal anti-NKCC2 (1:1,000; C. Lytle, UCR); Rb polyclonal NKCC2pT96T101 (1:2,000; B. Forbush,
Yale University), Rb anti-NCC (1:5,000; McDonough); Rb anti-NCCpS71 (1:5000; Loffing, Zurich), Rb anti-
NCC-pT53 (1:5000; Loffing, Zurich), Rb anti-NCCpS89 (1:5000; Loffing, Zurich), Rb anti-ENaCα (1:5000;
Loffing, Zurich), Rb anti-ENaCβ, (1:15000; Loffing, Zurich); Rb anti-ROMK (1:2000, Alomone), Rb anti-C
term SPAK (1:3000, Delpire) and Sh SPAK-pS373/OSR1-pS325 (1:1000; DSTT, Dundee, UK)
Primary antibodies were recognized by either Alexa Fluor 680 (Invitrogen)- or IRDye 800 (LI-COR,
Lincoln, NE)-labeled goat anti-rabbit, goat anti-mouse, or donkey anti-sheep secondary antibodies.
Signals were detected with Odyssey Infrared Imaging System (LI-COR) and quantified by accompanying
software
Quantification and statistical analysis: The range for linearity of signal intensity with sample loading
was established for each protein and on each blot by loading one and one-half amounts of each sample
18
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 2% K
+
diet groups and defined as 1.0. The
normalized values for the one and one-half protein loading lanes were averaged. The difference in total
abundance and phosphorylation of transporters and their associated proteins was assessed by unpaired
two-tailed Student’s t-test, assuming unequal variance. Data were expressed as means ± SE. Differences
were regarded significant at P < 0.05.
Kallikrein assay: The assay was done based on previously described protocols (Amundsen, Putter et al.
1979; Bonner and Marin-Grez 1981). The exact conditions were as follows. 5μl of undiluted urine, 10 μl
of 0.8mg/ml aprotinin (only in the paired controls) and 200mM Tris-HCl pH 8.2 to a volume of 120μl
were added to each well of a 96 well plate. The reaction mixture was pre-incubated at 37⁰C for 10
minutes. 80μl of 0.2mM S2266 kallikrein substrate tripeptide (Bachem AG, Bubendorf) was added
rapidly to each well and the plate was incubated at 37⁰C for 30 minutes. The plate was read with the
415nm filter on a 96-well plate reader. The ΔOD between the sample and paired +aprotinin control was
proportional to the kallikrein activity of the sample. The assay was done in duplicate on 2 separate
plates and the mean reading was used for calculations.
19
RESULTS
Effect of a 3 hour 2% K
+
meal on the plasma and urinary electrolyte concentrations
Male Sprague-Dawley rats were placed in metabolic cages and fed either a 2% K
+
or 0% K
+
containing
diet for 3 hours following an overnight fast. A 12 hour timecourse for Na
+
and K
+
excretion and urinary
kallikrein levels after a 2% K
+
meal was recorded in 2 hour windows.
Figure 11: Time course of change in urinary Na
+
, K
+
and tissue kallikrein after a meal.
Rats were fasted overnight and then fed a 2% K containing gelled diet meal for 3 hr. Urine was
collected overnight and over 2 hr intervals for 12 hr. A) Urinary K
+
excretion ([K
+
] x volume) and Na+
excretion ([Na
+
] x volume) measured by flame photometry indicated as means SEM (n=8).
B) Urinary tissue kallikrein (TK) activity x urine volume measured with a chromogenic microplate
assay indicated as means SEM (n=8).
20
We noted that the plasma K levels was 4.01±0.12mM for the 0% K
+
diet fed group as compared to
5.18±0.16mM for the 2% K
+
diet fed group after 3 hours (n=8; p<0.01). However there was no difference
in the plasma Na
+
levels between the two groups (133±3mM for the 0% K
+
diet group versus 135±2mM
for the 2% K
+
diet group; n=8; p=0.71). There was no significant difference in the 16 hour fasting urine
electrolyte levels between the two groups (data not shown). There was a significant difference in the 3
hour feeding urine K
+
levels between the two groups (0% K
+
vs 2%K
+
: urine [K
+
] was 18.25±3.3µmoles/hr
vs 226±16µmoles/hr; n=6; p<0.01).The excreted urine volume was higher in the 2% K
+
group at
3.19±0.34ml/3hr vs 1.55±0.55ml/3hr in the 0% K
+
group. The 3 hour feeding urine Na
+
level tended to
increase in the 2% K
+
group but did not reach significance.
Figure 12: Urine volume, urinary and plasma Na
+
, K
+
concentrations after a meal.
Rats were fasted overnight and then fed either 2% K
+
meal or 0% K
+
meal in metabolic cages before
sacrifice. A) Individual plasma [K
+
] and [Na
+
] values along with mean ±SE, n=8 per group. B)
Individual urinary electrolytes and volume measured in urine collected in metabolic cage + bladder,
individual values along with mean ±SE, n=8 per group.
21
A 2% K
+
meal causes a reduction in total abundance and phosphorylation of NCC
Cortical NCC, NCC-PT53 and NCC-PS71 abundance were decreased by 26±5% (p<0.01), 61±6% (p<0.01)
and 63±4% (p<0.01) respectively in 2% K
+
versus 0% K
+
fed rats as assayed by western blot.
Figure 13: Effect of 2% versus 0% K
+
meal on NCC, NKCC abundance and phosphorylation in renal
cortex homogenates.
A) Immunoblots of homogenate samples. To ensure linearity of the detection system, 1.0 and 0.5
amounts of each sample were loaded for each animal (only one amount shown): 30, 60µg for NCC-
total and NCC-pS71; 40, 80µg for NCC-pT53; 10, 20µg for NKCC-total, NKCCpT96T101. Density
values, normalized to mean density of 0%K
+
group defined as 1.00, are displayed under respective
blots as mean ±S E. *P<0.05 vs. 0%K
+
group.
B) Individual normalized values displayed with means, *P<0.05 vs. 0%K
+
group.
22
2% K
+
meal causes NCC to shift from the plasma membrane to the intracellular membrane
Cortex homogenates of rats fed either a 0 or 2% K
+
diet were fractionated into plasma membrane and
intracellular membrane fractions as described in the materials and methods section. These fractions
were then run on an SDS-PAGE gel and the abundance of NCC in each fraction was calculated using
quantitative western blotting. Feeding a 2% K
+
meal for 3 hours caused no changes in the distribution
pattern of NCC-pS71 between the plasma membrane and the intracellular membrane. However, it
caused 4.6±1.55% of total NCC to shift from the surface plasma membrane to the intracellular
membrane of the cell indicating possible degradation of NCC.
Figure 14: Effect of 2% K
+
meal versus 0% K
+
meal on subcellular distribution of NCC and NCCp.
The fraction of NCC total and NCCpS71 in plasma membranes (PM) versus intracellular
membranes (ICM) (n=8) was assessed after a differential fractionation protocol that enriches for
PM vs. ICM (Sachs, Pisitkun et al. 2008). Immunoblots were run at a constant amount of
protein/lane (6μg) of PM and ICM. The percentage of transporter in PM vs. ICM was calculated
from the signal intensity corrected for the recovery of PM and ICM membrane protein in each
sample (average recovery of 9.3 ± 1.3 mg of PM and 2.8 ± 0.8 mg of ICM from 34.4 1.2 mg
starting homogenate), and expressed as percentage of transporter abundance in total kidney
homogenates. Using this method, > 90% of the NCCpS71 was localized to PM, confirming
immunohistochemistry findings (Lee, Maunsbach et al. 2013) and validating this approach. After
the 2% K
+
meal, a lower percentage of NCC total localized to PM and a higher percentage of NCC
total localized to ICM enriched pools. Mean ±S E. *P<0.05 vs. 0%K
+
group.
23
A 2% K
+
meal causes a reduction in phosphorylation of cortical SPAK
Cortical SPAK-PS373 abundance were decreased by 38±5% in 2% K
+
versus 0% K
+
fed rats as assayed by
western blot. Medullary SPAK was identical in both the groups.
Figure 15: Effect of 2% K
+
meal versus 0% K
+
meal on SPAK (Ste20/SPS1-related proline/alanine-
rich kinase) abundance and phosphorylation in renal cortex and medulla homogenates.
A) Immunoblots of homogenate samples. To ensure linearity of the detection system, 1.0 and 0.5
amounts of each sample were loaded for each animal (only one amount shown): 10, 20 µg for SPAK-
total and 40, 80 g for SPAKpS373 and OSR1pS325 (detected with the same antiserum). Density
values, normalized to mean density of 0%K
+
group defined as 1.00, are displayed under respective
blots as mean ±S E. *P<0.05 vs. 0%K
+
group. B) Individual normalized values of SPAK and
SPAKpS373 displayed with means, *P<0.05 vs. 0%K
+
group.
24
2% K
+
meal does not activate ENaC or ROMK beyond basal levels or increase its abundance
There was no significant difference in ENaCα subunit abundance (cleaved and full length) between 0%
and 2% K
+
diet fed rats. This is in agreement with the previous data showing no significant change in the
abundance of urinary kallikrein (a marker of ENaC activation by proteolytic cleavage) in the first 4 hours
after a 2% K
+
meal was consumed. The abundance of the core and fully glycosylated forms of ROMK
(Frindt and Palmer 2010; Wade, Fang et al. 2011) was identical in both the diet groups.
Figure 16: Effect of 2% K
+
meal versus 0% K
+
meal on cortical collecting duct ROMK and ENaC
abundance in renal cortex homogenates.
To ensure linearity of the detection system, 1.0 and 0.5 amounts of each sample were loaded for
each animal (only one amount shown): 30, 60µg for ROMK and ENaC-β ; 40, 80µg for ENaC-α
were loaded. Density values, normalized to mean density of 0%K
+
group defined as 1.00, are
displayed under respective blots as mean ±SE. Arrows indicate ROMK that is core glycosylated
(Core glycos) and putative fully glycosylated ROMK band (Fully glycos) based on expected
mobility. LS indicates a sample from a rat fed nominally Na
+
free diet for 1 week to increase ENaC
expression and cleavage.
25
TABLE 1: SUMMARY OF PHYSIOLOGY DATA AFTER A 3HR MEAL
Effect of intravenous KCl infusion on plasma and urinary electrolyte concentrations
We were able to increase the plasma [K
+
] to above 5mM within an hour and maintain it between 5 and
5.5mM throughout the course of the 3 hour infusion period via tail vein infusion. The concurrent Na
infusion did not cause any change in the plasma [Na
+
] in this period (Figure 17). The rats in the K
+
infusion group excreted 559±68μmoles of K
+
over the 3 hour infusion period compared to 76±14μmoles
by the saline control rats and 88±18μmoles by the un-infused control rats. The K
+
infusion group rats
also showed a marked natriuresis compared to the saline control excreting 545±49μmoles versus
173±72μmoles for the saline control. The un-infused baseline group rats excreted 133±23μmoles of Na
+
over 3 hours. There was a marked diuresis and corresponding reduction in the urine concentration as
measured by an osmometer (not shown) in the KCl infused rats compared to the other groups.
Values expressed as mean ±SE. * P<0.05 vs. 0% KCl diet fed group
26
Figure 17: Timecourse of plasma [K+] during KCl infusion
Plasma [K
+
] measured at 15 minute intervals in rats infused over 3 hrs with 150mM KCl at a variable
rate to raise plasma [K
+
] to 5.5mEq, mimicking the changes observed after feeding 2% K diet. The
Na
+
infused group was infused with 150mM saline matched to the volume of saline infused into the
K
+
infused group. The volume infused and total mmol electrolytes infused are detailed in Table 2.
The un-infused group received no infusion. All three groups had no food and free access to drinking
water (n=4-5/group).
Figure 18: Effects of raising plasma [K
+
] by tail vein infusion on urinary electrolytes in conscious
rats. Rats were fasted overnight and then infused with either 150 mM KCl or NaCl (NaCl matched to
that infused in the KCl infused group) or uninfused over a period of 3 hrs. The rats had free access
to drinking water but continued fasting. A) Individual urine K
+
excretion
and B) individual [Na
+
]
excretion values along with mean ±SE, n=5 per group.
27
TABLE 2: EFFECTS OF 3hr KCl INFUSION
Moderate plasma [K
+
] increase by KCl infusion without a meal provokes dephosphorylation of NCC
There was no significant difference in NCC-P abundance between the saline control rats and the
uninfused control rats (not shown). NCC-PT53, NCC-PS71 and NCC-PS89 species were significantly
downregulated at various phosphorylation sites in the K
+
infusion rats compared to the saline control
rats by 50±9%, 57±8% and 71±4% respectively. Total and phosphorylated NKCC abundances were
identical in the KCl infused and matched saline infused groups.
Values expressed as mean ±SE, n=5/group. * P<0.05 vs. NaCl infused
28
Figure 19: Effects of raising plasma [K
+
] to
5.5 mEq by tail vein infusion on NCC and
NKCC abundance and phosphorylation in
renal cortex homogenates.
A) Immunoblots of homogenate samples.
To ensure linearity of the detection system,
1.0 and 0.5 amounts of each sample were
loaded for each animal (only one amount
shown): 30, 60µg for NCC-total and
NCCpT53, 15, 30µg for NCCpS71, 40, 80µg
for NCCpS89, 10, 20µg for NKCC-total, and
NKCC-pT96T101 were loaded. Density
values were normalized to mean density of
the NaCl infused group (defined as 1.00)
displayed under respective blots as
means±SE. *P<0.05 vs. NaCl infused group.
B) Individual normalized values displayed
with means, *P<0.05 vs. NaCl infused
group.
29
Moderate plasma [K
+
] increase by KCl infusion without a meal provokes dephosphorylation of SPAK
We observed that the FL-SPAK abundance was not significantly different between the 3 groups (not
shown). However the SPAK-PS373 species was significantly downregulated in the K
+
infusion rats
compared to the saline control rats by 38±5%. There was no significant difference in SPAK-PS373
abundance between the saline control rats and the un-infused control rats (not shown).
Figure 20: Effects of raising plasma [K
+
] to 5.5mEq
by tail vein infusion on SPAK abundance and
phosphorylation in renal cortex homogenates.
A) Immunoblots of homogenate samples. To
ensure linearity of the detection system, 1.0 and
0.5 amounts of each sample were loaded for each
animal (only one amount shown): 10,20 µg for
SPAK-total and 40, 80 g for SPAKpS373 and
OSR1pS325 (detected with the same antiserum).
Density values, normalized to mean density of NaCl
infused group defined as 1.00, are displayed under
respective blots as mean ±S E. *P<0.05 vs. NaCl
infused group; ^ and # indicates sample from SPAK
wildtype and knockout kidney sample s,
respectively ,(Delpire lab). B) Individual normalized
values of SPAK and SPAKpS373 displayed with
means, *P<0.05 vs. NaCl infused group.
30
DISCUSSION
Effects of 2% K
+
diet feeding on NCC regulation
The reduction of NCC-P abundance in rats extends the findings of Sorensen et al made in mice, and does
so with a much less pronounced increase in the plasma K
+
concentration. The experiments were also
performed in a larger rat animal model compared to the mice used by Sorensen et al. The decrease in
the abundance of cortical but not medullary SPAK-P (a kinase that is responsible for NCC
phosphorylation and activation) with no change in the total SPAK abundance points to a possible
mechanism by which the 2% K
+
meal is able to cause a reduction in NCC phosphorylation. The decrease
in the cortical SPAK-P but not the medulla SPAK-P is consistent with the presence on NCC only in the
kidney cortex and not in the medulla. The differential regulation of SPAK-P in the cortex and medulla is
along similar lines as the differential regulation of NKCC2 and SPAK seen by our lab recently in
Sprague/Dawley rats infused with Angiotensin II for 2 weeks (Nguyen, Lee et al. 2013). Alternately, there
may be a phosphatase that decreases the phosphorylation of both NCC and SPAK.
Sorensen’s paper (Sorensen, Grossmann et al. 2013) shows that just increasing the extracellular K
+
concentration from 5 to 10mM in ex vivo studies with freshly isolated renal tubules does not cause any
change in NCC-P status. Also, their addition of a phosphatase inhibitor Calyculin A to the tubule with
5/10mM K
+
solution prevented the dephosphorylation of NCC. This suggests that there is possibly
another phosphatase involved in the dephosphorylation of NCC. Therefore there are potentially two
mechanisms working in concert to keep NCC-P low. One is the reduction in activation of the NCC
phosphorylating kinase SPAK to prevent activating new NCC molecules and a phosphatase enzyme like
PP4 (Glover, Mercier Zuber et al. 2010) which is able to dephosphorylate the existing NCC-P.
There is evidence that NCC can be turned over rapidly in the yeast expression system (Needham,
Mikoluk et al. 2011). The reduction in total NCC abundance could be due to ubiquitination and
31
degradation of the NCC by Nedd4-2 (Arroyo, Lagnaz et al. 2011; Gamba 2012; Ronzaud, Loffing-Cueni et
al. 2013). The shift of NCC from the plasma membrane to the intracellular membrane supports the
possibility of NCC being withdrawn from the cell surface into intracellular vesicles after
dephosphorylation where they can be ubiquitinated as a marker for degradation. Another possibility is
the expulsion of NCC from the plasma membrane of the DCT cells into the lumen of the nephron in
urinary exosomes (Esteva-Font, Wang et al. 2010; van der Lubbe, Jansen et al. 2012).
2% K
+
meal does not activate ENaC or ROMK beyond basal levels or increase its abundance
While the dephosphorylation of NCC supports the initial hypothesis that more Na
+
is allowed to reach
the CCD where it can be taken up by ENaC and drive K
+
secretion via ROMK, there is no evidence for
enhanced ENaC activation or increase in its abundance within 3 hours of feeding as seen in our study.
Other studies (Sorensen, Grossmann et al. 2013) have also not found any evidence for ENaC activation
up to 6 hours after K
+
gavage.
However, K
+
excretion via urine increases almost 10 fold after a 2% K
+
meal within 3 hours. Sorensen et
al have showed that K
+
, Na
+
excretion increases within an hour of oral K
+
gavage in mice. This would
imply that there are other alternate mechanisms at play which effect this excretion of K
+
without the
activation of ENaC. It is also possible that the basal level of activation of the ENaC channels in the CCD is
sufficient to handle the increased load of Na
+
being delivered to it.
We also did not see any increase in the abundance of the core or fully glycosylated form of ROMK
channel in the plasma membrane when we fed either a 0 or 2% K
+
diet. The number of active ROMK
channels in the plasma membrane is likely sufficient to handle the increased K
+
load resulting from the
consumption of a 2% K
+
meal.
32
It is also possible for the K
+
to be excreted via the BK channels (Holtzclaw, Grimm et al. 2011) which are
flow activated. This is possible as we see marked diuresis in rats fed a K
+
rich diet. A final possibility to
consider would be the impaired absorption of K
+
in the proximal portion of the nephron causing it to be
excreted out.
Effect of intravenous KCl infusion on plasma and urinary electrolyte concentrations
The KCl infusion was done so that the plasma [K
+
] closely matched the average plasma K
+
concentration
in rats that consumed a 2% K
+
meal over 3 hours.
Previous studies with 2% K
+
meal feeding indicated that the plasma [K
+
] rises continuously for about 3
hours after consumption of the meal (in Wistar rats, Youn et al, unpublished). This allowed us to make
the assumption that the plasma [K
+
] recorded in rats 3 hours after they ate a 2% K
+
meal was the highest
level it would reach within the 3 hour period
Other groups have previously shown that a meal induced gut factor is an essential signal for effective
urinary K
+
clearance (Calo, Borsatti et al. 1995; Lee, Oh et al. 2007; Oh, Oh et al. 2011). In this study we
show that it is sufficient to have increased plasma [K
+
] independent of a meal to trigger signaling to the
kidney. Similar renal transporter adaptations as a K
+
rich meal are observed when only the plasma [K
+
] is
increased which may indicate that both the gut factor mediated signaling and the increased plasma [K
+
]
mediated signaling may work via similar mechanisms.
It is also possible that both the mechanisms be completely independent with the plasma [K
+
] increase
being the reason for SPAK-P and NCC-P decrease seen by us (Sorensen et al first reported the decrease
in NCC-P), while the gut factor signal could trigger responses in the proximal portion of the nephron or
uptake by muscles. The role of plasma [K
+
] in the regulation of K
+
excretion is not marginal as previously
33
thought. Even a minor increase in the plasma [K
+
] may be sufficient to initiate NCC-P and SPAK-P
downregulation
In summary, we have determined that the maintenance of K
+
homeostasis by the kidney after an acute
K
+
load either as a diet or by direct infusion into the plasma is accomplished by downregulating NCC
phosphorylation, a correlate of NCC activity. There are 2 mechanisms by which downregulation of NCC
activity may occur: dephosphorylation of NCC which reduces NCC activity and by the degradation of the
unphosphorylated NCC in the intracellular membrane which reduces the total pool of available NCC for
activation. The NCC dephosphorylation is reduced by a decrease in the abundance of SPAK-P which is
responsible for phosphorylating NCC. We also hypothesize that an unknown phosphatase might be
involved in dephosphorylating NCC and SPAK rapidly allowing for a system of opposing kinases and
phosphatases to exist. This seems to be a logical way by which the kidney can adapt to rapid changes in
K
+
retention requirements. We have no direct evidence for the degradation of NCC at this time; this can
be investigated by assaying for the increase in ubiquitination of NCC (a marker for degradation).
We have also determined that feedback regulation is also extremely important in the maintenance of K
+
homeostasis and probably works synergistically with gut sensing of dietary potassium to regulate the
renal transporter activity. Gut sensing of a meal causes rapid secretion of insulin which stimulates the
uptake of K
+
from the plasma into skeletal muscles by Na
+
K
+
ATPaseα2 pump activation. To determine if
this is a vital signal in preventing a post-prandial increase in plasma [K
+
], we plan to perform similar
experiments as described above in mice lacking the Na
+
K
+
ATPaseα2 pump in the near future.
34
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Abstract (if available)
Abstract
Potassium is important for the maintenance of acid-base balance, electrolyte homeostasis and optimal cellular excitability. It is vital that the plasma K⁺ level is maintained within a narrow window to prevent adverse health effects. On a day to day basis, the greatest challenge to the maintenance of K⁺ homeostasis is the periodic dietary intake of boluses of K⁺. The kidneys, muscles and gut work in a co-ordinated manner to regulate the output of K⁺ from the body and match it to the intake. The kidney is the most important of these components in the maintenance of K⁺ homeostasis. It reacts rapidly to incoming K⁺ loads and is able facilitate filtration and excretion of K⁺ via the urine by regulating the abundance and activity of various ion transporters in the nephron. Currently, there is a lack of complete understanding about the source of the signal which responds to disturbances in the K⁺ homeostasis and communicates to the kidneys to regulate its ion transporters accordingly to maintain homeostasis. ❧ Dietary potassium loading results in rapid kaliuresis, natriuresis and diuresis. We found that a 3 hour 2% K⁺ meal caused a modest increase in plasma K⁺ from 4.01±0.12mM to 5.18±0.16mM in Sprague Dawley rats (n=8). This was accompanied by rapid decrease in NCC-P potentially regulated via a decrease in cortical SPAK-P abundance. However there was no effect on CCD transporters ENaC or ROMK within 3 hours. Similar effects on NCC-P and SPAK-P abundance were observed when the plasma K⁺ level was raised by direct intravenous KCl infusion to the same level as by diet feeding. Thus an acute and minimal increase in the plasma [K⁺] is a sufficient signal to trigger renal kaliuretic mechanisms via NCC downregulation.
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Rengarajan, Srinivas
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Core Title
Regulation of potassium homeostasis during acute potassium loading
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Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Publication Date
08/06/2013
Defense Date
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Publisher
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homeostasis,ion transport,Kidney,metabolic cage,NCC,OAI-PMH Harvest,phosphorylation,plasma potassium,potassium,SPAK
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potassium
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