A Dissertation Entitled Na/K-Atpase Mediates Renal Sodium Handling
A Dissertation
Entitled
Na/K-ATPase Mediates Renal Sodium Handling
By Yanling Yan
Submitted to the Graduate Faculty as partial fulfillment of the requirements for the
Doctor of Philosophy Degree in Biomedical Sciences
Dr. Joseph Shapiro, Committee Chair
Dr. Jiang Liu, Committee Member
Dr. Nader Abraham, Committee Member
Dr. Deepak Malhotra, Committee Member
Dr. Bina Joe, Committee Member
Dr. Zijian Xie, Committee Member
Dr. Patricia Komuniecki, Dean
College of Graduate Studies
The University of Toledo August 2012
Copyright 2012, Yanling Yan This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author.
An Abstract of
Na/K-ATPase Mediates Renal Sodium Handling
by
Yanling Yan
Submitted to the Graduate Faculty as partial fulfillment of the requirement for the Doctor of Philosophy Degree in Biomedical Sciences
The University of Toledo August 2012
Hypertension, affecting over one billion people worldwide, is one of the leading risk factors for heart attack and stroke. Kidney cross-transplantation studies between hypertensive and normotensive people and strains of rats provide the most compelling evidence for the fundamental role of the kidney in the pathogenesis of hypertension.
Recently, there is growing evidence supporting Arthur Guyton’s hypothesis that a
common characteristic feature of hypertension is impaired renal sodium excretion.
However, the exact molecular mechanism responsible for the impaired renal sodium
excretion is not clearly defined. The overall aim of this dissertation is to improve our
understanding of Na/K-ATPase and sodium proton exchanger 3 (NHE3) trafficking
regulation and determine molecular mechanisms of renal proximal tubular sodium
iii
handling, which might contribute to the impaired sodium excretion associated with hypertension and results may help to develop effective therapy for hypertension.
Renal proximal tubules (RPTs) responsible for 65-70% of filtered sodium and water reabsorption have profound effects on renal and body fluid balance associated with hypertension. Studies from our lab were the first to demonstrate that in renal proximal tubular cells, binding of cardiotonic steroids (CTS) such as ouabain to Na/K-ATPase stimulates Na/K-ATPase signaling cascade and induces the redistribution of basolateral
Na/K-ATPase and apical NHE3, leading to a net increase in urinary sodium excretion.
The first manuscript entitled “Ouabain-stimulated trafficking regulation of the
Na/K-ATPase and NHE3 in renal proximal tubule cells” improves our understanding of Na/K-ATPase and NHE3 trafficking regulation. Three renal proximal tubular cell lines (human HK-2, porcine LLC-PK1, and AAC-19 originated from LLC-PK1 cells in which the pig α1 was replaced by ouabain- resistant rat α1) were employed to compare ouabain-induced regulation of the α1 subunit and NHE3 as well as transcellular 22Na+ transport. Data indicate that our previous observations that ouabain-induced redistribution of Na/K-ATPase α1 subunit and NHE3 is not species-specific. In LLC-PK1 cells, ouabain also inhibited the endocytic recycling of internalized NHE3, but has no significant effect on recycling of endocytosed α1 subunit.
The second manuscript entitled “Impairment of Na/K-ATPase signaling in renal proximal tubule contributes to Dahl salt-sensitive hypertension” defines a
iv
novel mechanism of salt-sensitive hypertension. Studies from Sprague Dawley and
Dahl salt-resistant and Dahl salt-sensitive rats demonstrate that impaired Na/K-
ATPase signaling and consequent regulation of Na/K-ATPase and NHE3 in renal proximal tubule may contribute to salt induced hypertension in the Dahl S rat.
Given there is no difference in the Na/K-ATPase α1 gene (Atp1a1) coding and α1 sensitivity to ouabain between Dahl salt-resistant and Dahl salt-sensitive rats, other factors must be present to affect the activation of Na/K-ATPase/c-Src signaling.
Reactive oxygen species (ROS) play a critical role in the pathogenesis of hypertension. Manuscript 3 entitled “Redox modulation of the Na/K-ATPase signaling and renal proximal tubular sodium handling” present that ouabain- stimulated Na/K-ATPase signaling and subsequent redistribution are redox- sensitive, and ouabain induces direct carbonylation of proline/threonine residues in the actuator (A) domain of the Na/K-ATPase α1 subunit. These data indicate that a proper redox modulation of renal proximal tubular Na/K-ATPase signaling is critical in renal sodium handling and might play an important role in counteracting volume expansion mediated high blood pressure.
All in all, this dissertation not only improves our understanding of Na/K-
ATPase signaling regulation, but also defines a novel mechanism involved in salt- sensitive hypertension, and also demonstrates ouabain-activated Na/K-ATPase signaling is redox sensitive, which might play a crucial role in the regulation of renal sodium handling and blood pressure.
v
Dedication
I dedicate this dissertation to my lovely family, particularly to my understanding and patient husband, Dengyue, who has put up with my so many years of research overseas and has supported my every step of the way, and to our precious daughter Shuyan, who is the joy of our lives, and to my loving parents who have never failed to give me moral support and encouragement.
vi
Acknowledgments
I wish to extend my sincere appreciation to all the persons who made my
dissertation possible. I would never have been able to finish my dissertation without the
guidance of my committee members, help from friends and support from my family.
I would like to express my sincere gratitude to Dr. Joseph Shapiro, my major advisor for giving me a chance to join his excellent lab and providing me a fascinating research topic. I cannot thank him enough for his patience, his solicitude, his kindness, his wisdom and support. His inspiring words and excellent guidance will sway my life deeply and make me achieve what I have dreamt of.
I am very grateful to Dr. Jiang Liu, for his thoughtfulness and generosity with time and energy, for his sharing meticulous research and insights with me that support and expand my thesis work, for risking his health to help me with those critical data.
I am also indebted to my committee members, Dr. Nader Abraham, Dr. Bina Joe,
Dr. Deepak Malhotra, and Dr. Zijian Xie, for serving on my dissertation committee, for their expert suggestions, contributions and good-natured support throughout the production of my research and dissertation.
vii
I would also like to thank my fellow lab members, past and present, for their assistance and support over the years: Dr. Steven Haller, Dr. David J. Kennedy, Dr. Jiang
Tian, Dr. Christopher Drummond, Sandeep Vetteth, Shalini Gupta, Mohammad Taleb,
Vinai Katragadda, Anna Shapiro, Chiamaka Mbaso, Georgy Budnyy, Imad Hariri, Sayed
Moustafa Helmy, Eric Kim. I really enjoy being with all of you in the lab. Special thanks
go to Dr. Sankaridrug Periyasamy, our previous lab manager, and his wife for their support and assistance when I first came to the lab.
Many thanks must also go to Ms. Carol Woods, and Ms. Jenelle Thurn for their research assistance; to Dr. Robert Blumenthal for his timely help with my graduate admission process, to Dr. Randall Ruch for his support and assistance as an assistant dean
for Admissions, Biomedical Graduate Program, to Dr. Andrew Beavis for his course and
research help as a director of CVMD track; to Dr. Lijun Liu, Dr. Jean H. Overmeryer and
Dr. John David Dignam for their kind, unselfish help to make my experiments go well, to
Dr. Nancy Collins, my mentor in Graduate Student Professional Mentoring Program, for
her valuable advice in many different ways, to Madge Levinson, my English tutor, for her
encouragement and guidance with my English language study.
I would like to gratefully acknowledge the support, encouragement and friendship
of my good friends, classmates and co-workers over the years: Lauren Stanoszek, Erin
Crawford, Xiaolu Zhang, Jiyoun Yeo, Jieying Wang, Shuai Dong, Ran Lu, Shuo Geng,
Marjie Gable, Jian Wu, Yan Bai, Daxiang Li, Yiliang Chen, Zhichuan Li, Qiying Ye,
Zhen Jiang, Fangfang Lai, Katye Smedlund, Jean-Yves Tano, Joshua Waldman, Qiang
Mei, Jun Lu, Jie Zhou. viii
Additionally, I wish to thank the members of Dr. Nader Abraham, Dr. Bina Joe, Dr.
Lijun Liu and Dr. Zijian Xie’s lab for sharing their valuable thoughts with me, helping me get through difficult experiment.
I would like to thank my parents, my family and all my friends for their steadfast support and encouragement throughout the years. Lastly, but most importantly, I would like to thank my loving husband, Dengyue Sun, and daughter, Shuyan Sun for never failing to lift my spirits. Your love and support have been with me through all the good
times and bad, and helping to keep me sane.
ix
Contents
Abstract iii
Dedication vi
Acknowledgments vii
Contents x
List of Abbreviations xiii
Chapter 1 – Literature Review 1
Chapter 2 – “Ouabain-Stimulated Trafficking Regulation of the Na/K-ATPase and
NHE3 in Renal Proximal Tubule Cells” (Manuscript 1accepted for publication) 55
2.1 Abstract 56
2.2 Introduction 57
2.3 Experimential Methods 58
2.4 Results 64
2.5 Discussion 68
2.6 Manuscript References 73
2.7 Figure Legends 79 x
2.8 Tables and Figures 82
Chapter 3– “Impairment of Na/K-ATPase Signaling in Renal Proximal Tubule
Contributes to Dahl Salt-Sensitive Hypertension” (Manuscript 2) 90
3.1 Abstract 91
3.2 Introduction 92
3.3 Materials and Methods 92
3.4 Results 97
3.5 Discussion 100
3.6 Manuscript References 105
3.7 Figure Legends 109
3.8 Tables and Figures 112
Chapter 4 – “Redox Modulation of the Na/K-ATPase Signaling and Renal Proximal
Tubular Sodium Handling ” (Manuscript 3 to be submitted) 122
4.1 Abstract 123
4.2 Introduction 124
4.3 Materials and Methods 125
4.4 Results 131
4.5 Discussion 139
4.6 Manuscript References 144
4.7 Figure Legends 152
4.8 Figures 156 xi
Chapter 5 – Summary and Conclusions 174
xii
List of Abbreviations
BP------Blood pressure
CAT------Catalase
C2-9------Caveolin-1 knock out cells
CO------Carbon oxide
CoPP------Cobalt protoporphyrin
CTS------Cardiotonic steroids
Dahl R rat------Dahl salt-resistant rat
Dahl S rat------Dahl salt-sensitive rat
EGFR------Epithelial growth factor receptor
GPx------Glutathione peroxidase
G.O. ------Glucose oxidase
Grb2 ------Growth factor receptor-bound protein 2
HO-1------Heme oxygenase 1
HS------High salt
LLC-PK1------Porcine renal proximal tubule cell lines
LS------Low salt
MAPK------Mitogen-activated protein kinase
MBG------Marinobufagenin
MEK------MAPK-ERK activating kinase
NAC------N-acetyl cystein xiii
NHE3------Sodium proton exchange 3
NKA------Na/K-ATPase
NO------Nitric oxide
PKC------Protein kinase C
PLC------Phospholipase C
PI3K------Phosphoinositide 3’ kinase
PY-17 cells------NKA knock down cells
RPT------Renal proximal tubules
ROS------Reactive oxygen species
Shc------Src homology collagenlike protein
SNS------Sympathetic nervous system
SOD------Superoxide dismutase
SOS------Son of sevenless
SYF------Mouse fibroblast cells with deficient Src kinase
SYF+c-Src------Mouse fibroblast cells overexpressing Src kinase
xiv
Chapter 1-Literature Review
1. Na/K-ATPase (EC 3.6.3.9, also known as Na pump)
Na/K-ATPase, originally reported by Skou (Skou 1957), is ubiquitous and crucial important membrane protein that maintains the high internal K+ and low internal Na+ concentrations via coupling the transmembrane transport of three Na+ outward and two
K+ inward utilizing energy derived from ATP hydrolysis (Post, Hegyvary et al. 1972;
Lingrel and Kuntzweiler 1994; Blanco and Mercer 1998). In addition to its classical
pumping function, The Na/K-ATPase acts as a signal transducer that relays extracellular
messages into the cell through regulation of protein-protein interactions (Xie 2003;
Schoner and Scheiner-Bobis 2005), which can subsequently up or down-regulate various cellular processes such as gene activation, motility, cell-cell contact, cell proliferation and apoptosis, as well as transcellular transport processes in intestine, glands, and kidney
(Schoner and Scheiner-Bobis 2007; Bagrov and Shapiro 2008; Newman, Yang et al.
2008).
1.1 Na/K-ATPase structure
P-type ATPases including Na+, K+-ATPase, Ca2+-ATPas (SERCA) and gastric
H+, K+-ATPase are responsible for the active transport of a variety of cations across the
1 membranes (Sachs and Munson 1991; Lutsenko and Kaplan 1995). P-ATPase is
designated due to catalyzing auto- or self- phosphorylation of a key conserved aspartate
residue within the pump. The Na/K-ATPase, the largest protein complex in the family of
P-type cation pump, is an oligomer consisting of two major polypeptides, α- and β- subunits, and a tissue-specific auxiliary regulatory subunit known as FXYD. A heterodimer of α–subunit and β–subunits is the minimum functional unit of Na/K-
ATPase (Jorgensen and Andersen 1988). In humans, there are four isoforms for the α– subunit (α1-α4), three for the β-subunit (β1-β3) (Lingrel, Orlowski et al. 1990; Axelsen
and Palmgren 1998) and seven for the FXYD (Kuster, Shainskaya et al. 2000; Sweadner
and Rael 2000), leading to different Na/K-ATPase variants.
1.1.1 α subunit
The α subunit is the catalytic unit with a molecular mass of 112 KDa, which
consists of ten transmembrane ((TM)) helices (Figure 1-1, M1-M10) (Blanco and Mercer
1998), three cytoplasmic conserved domains, actuator (A), nucleotide binding (N), and
phosphorylation (P) (Figure 1-2). ATP binds to the N domain and phosphrylates P
domain. The A domain is involved in this reaction to manipulate the TM segments to
open the occlusion for sodium or potassium binding, depending on which state, E1 or E2,
the protein is in. The lysine-rich N-terminal extension of around 40 residues
phosphorylated by protein kinase C might provide a platform for interacting with other
proteins such as IP3 receptor (Figure 1-2B). The fascinating observation by Toyoshima
team (Toyoshima, Kanai et al. 2011) (Xie and Cai 2003)is that the N-domain is less
(~20°) inclined toward the A-domain, and a salt bridge between the positively charged
residue Arg551 in the N-domain and the negatively charged residue Glu223 in the A-
2 domain is one crucial link between them (Figure 1-2A). Since Arg551 interacts with the
β-phosphate of ATP, the salt bridge will be broken by ATP/ADP binding, which speed
up the opening of α subunit headpiece and then transition into E1 (Shinoda, Ogawa et al.
2009).
Figure 1-1: Scheme of the membrane topology of the α and β isoforms of the Na/ K-ATPase (Blanco and Mercer 1998). Sequences of rat α and β isoforms are shown. Residues are colored to indicate the amino acid homology among the different α isoform or β isoforms.
In addition, the α-subunit contains the binding sites for the cations, ATP and the inhibitors such as ouabain (Pedemonte and Kaplan 1990; Mercer 1993; Lingrel and
Kuntzweiler 1994; Pressley 1996). The binding sites for Na+ and cardiotonic steroids
(CTS) are on the extracellular segments and the binding sites for K+ and ATP are on the
intracellular loops. Specifically, the biding sites for the inhibitors CTS include TM1-TM2,
3 TM5-TM6, and TM7-TM8 loops and several amino acids from the transmembane regions M4, M6, and M10. The biding site for ATP is located in the pocket formed by the intracellular loop TM4-TM5. The proximal and distal regions of intracellular loop TM4-
TM5 are the phosphorylation domains. Besides, of the ten TM helices, M4-M6 and M8 contain the binding sites for the cations. The M7-M10 helices have special function and might undergo large-scale rearrangement in Na/K-ATPase. The cytoplasmic half of M7 is distinctly kinked in Na/K-ATPase. The outside of M9 interacts with FXYD, whereas
M10 associates with TM helix of the β subunit (Figure 1-2) (Bagrov, Shapiro et al. 2009;
Toyoshima, Kanai et al. 2011).
1.1.2 β subunit
The β subunit is a heavily glycosylated polypeptide that crosses the membrane once (Blanco and Mercer 1998) and has a molecular weight between 40 and 60 KDa depending on the degree of glycosylation in different tissues. The β subunit is composed of a short N-terminal cytoplasmic domain, one transmembrane helix located in the groove between the M7 and M10 helices of the α-subunit and a large extracellular domain that almost covers the entire extracellular surface of the α-subunit (Figure 1-1.
Figure 1-2A).
The primary role of the β subunit is to function as a chaperone, facilitating routing of the α-subunit to the plasma membrane and stabilizing its conformation (Chow and
Forte 1995; Geering 2001; Geering 2008). In addition, the β subunit is also involved in the occlusion of K+ and the modulation of the K+ and Na+ affinity of the enzyme (Jaisser,
4 Horisberger et al. 1992; Lutsenko and Kaplan 1993; Blanco, Koster et al. 1995; Eakle,
Lyu et al. 1995; Geering 2001).
1.1.3 FXYD (γ subunit)
The FXYD protein, composing of one transmembrane helix, one N-terminal
extracellular region and a cytoplasmic part, is a small polypeptide of 8-14KDa (Blanco
and Mercer 1998). γ subunit is termed for FXYD in kidney outer medullar (Morth,
Pedersen et al. 2007). It interacts with the αβ-complex as a third subunit and modulates
the affinities of Na+, K+ and ATP, and then regulates Na/K-ATPase in a tissue- and
isoform-specific manner (Blanco and Mercer 1998; Cornelius and Mahmmoud 2003;
Geering 2006). However, it is unclear about how various FXYD proteins affect binding
of Na+/K+, and the kinetic properties of Na/K-ATPase (Blanco and Mercer 1998;
Toyoshima, Kanai et al. 2011).
1.2 Na/K-ATPase function (Post-Albers Model)
The Na/K-ATPase belongs to P-ATPases (known as E1-E2 ATPases) that is composed of two main conformations called E1 and E2, which are responsible for the
active transport of a variety of cations across cell membranes. In the E1 state, the TM cation binding sites have high affinity for Na+ and face the cytoplasm. In E2, the binding
sites have low affinity for Na+ but high affinity for K+ and face the extracellular side. In
E1 state, three sodium ions bind to the pump from the cytoplasmic side of the membrane.
ATP phosphorylates the enzyme (as indicated by the P attached to the cytoplasmic side of the pump), and the remaining ADP is released. The conformation then changes to E2, and the three sodium ions are released into the extracellular solution.
5
Figure 1-2: Architecture of Na/K-ATPase from Shark Rectal Gland with Bound 2− + + MgF4 and K , a Stable Analog of the E2·Pi·2K State (Toyoshima, Kanai et al. 2011). (A) A ribbon diagram of Na/K-ATPase with ouabain (shown in space fill) bound at low affinity (PDB ID: 3A3Y). Color changes gradually from the N-terminal (blue) to the C- terminal (red). ATP is taken from the E2(TG)·ATP crystal structure of Ca2+-ATPase (SERCA1a) (PDB ID: 3AR4) and docked in the corresponding position. Bound K+ ions are marked (I, II, and C) and circled. Inset shows a simplified diagram of the Post-Albers scheme. CLR, cholesterol; OBN, ouabain. (B) Digestion sites (proteinase K, black “x”; chymotrypsin, magenta “x”) and interaction sites with other proteins (dotted circles). Residue numbers in parentheses refer to corresponding residues in SERCA1a. AP-2, adaptor protein 2; IP3R, inositol 1,4,5-triphosphate receptor; PI3K, phophoinositide-3 kinase.(C) Disposition of transmembrane helices viewed approximately perpendicular to the membrane from the cytoplasmic side (Na/K-ATPase, yellow; SERCA1a, cyan). CLR, cholesterol.
After two potassium ions bind to the pump from the extracellular side, the pump is dephosphorylated and returns to E1 state. ATP binding occurs and the potassium is released into the intracellular solution (Figure 1-2, inset) (Albers 1967; Post, Hegyvary et al. 1972).
6 2. Na/K-ATPase ligand – Cardiotonic steroids
Cardiotonic steroids (CTS), also referred to as digitalis-like factors, are inhibitors of
Na/K-ATPase. CTS are composed of plant-derived ouabain, digoxin, and vertebrate- derived marinobufagenin (MBG). First and perhaps foremost, many endogenous CTS have been purified and characterized in animals and human (Hamlyn, Blaustein et al.
1991; Lichtstein, Gati et al. 1993; Bagrov and Fedorova 1998; Komiyama, Dong et al.
2005). Both endogenous ouabain and MBG synthesized in and released from hypothalamus and adrenal contex could be regulated by ACTH, angiotensin, vasopressin, and phenylephrine (Laredo, Shah et al. 1997). Generally, endogenous CTS circulate in a protein-bound form with plasma levels from subnanomolar to nanomolar concentration in different condition (Bagrov, Shapiro et al. 2009).
The α subunit of Na/K-ATPaseh as a specific binding site for CTS on the extracellular loops (TM1-TM2, TM5-TM6, and TM7-TM8). Extracellular TM1-TM2 loop (amino acids from 111-122 sequences) form the most important part of the putative
CTS binding site.
The sodium pump has a different sensitivity to CTS due to the different amino acid sequence. CTS, on the other hand, display different affinity to different isoforms. For instance, a nanomolar concentration of bufadienolide marinobufagenin can inhibit Na/K-
ATPase in rodent renal epithelial cells consisting of nearly exclusively the α1 isoform, whereas ouabain is much less active with renal Na/K-ATPase (Bagrov, Shapiro et al.
2009).
3. Na/K-ATPase signaling
7 Na/K-ATPase has three classic features that are the pump, the enzyme, and the receptor to CTS. Quite importantly, low concentrations of CTS, for instance, circulating
levels from subnanomolar to nanomolar in humans that do not inhibit the enzymatic
function of sodium pump, are able to initiate its signaling pathway (Liu, Periyasamy et al.
2002), which plays a key role in genomic and nongenomic functions. Moreover, the
following studies provide the positive evidence supporting the signaling function for the
Na/K-ATPase. First, no change in cytosolic [Na+] is measured in mammalian cells
exposed to those concentrations of CTS (Liu, Tian et al. 2000). Second, the caveolar
Na/K-ATPase is closely related to the key signaling proteins such as c-Src and EGFR
instead of pumping sodium (Liang, Cai et al. 2006). Third, the phosphorylation of key
signaling proteins caused by CTS can also be observed in cell-free systems, in which no
[Na+] is changed (Wang, Haas et al. 2004). Although increasing evidence demonstrates
that Na/K-ATPase signaling effects might be independent of its inhibition of transmembrane sodium transport (Xie and Askari 2002; Schoner and Scheiner-Bobis
2007; Tian and Xie 2008), we propose that the classic and signaling pathway may work both in parallel and synergistically to affect physiological consequence of Na/K-ATPase
(Bagrov, Agalakova et al. 2009).
The Na/K-ATPase signaling pathway involves the association of c-Src with the
Na/K-ATPase in a caveolar domain. Binding of CTS to the Na/K-ATPase stimulates c-
Src activation, which, in turn, transactivates the EGFR and phospholipase C (PLC), leading to a sequential cascade that involves generation of reactive oxygen species (ROS), activation of mitogen-activated protein kinase (MAPK) through activation of its mitogen- activated protein kinase kinase (MEK), activation of PI3K, and activation of protein
8 kinase C (PKC) (Figure 1-3). The activated pathways that exist in different cells including
renal proximal tubular cells, cardiac myocytes and whole animal models, will generate the
genomic and nongenomic effects (Xie and Cai 2003; Bagrov, Shapiro et al. 2009).
Figure 1-3: Schematic presentation of Na/K-ATPase signalosome (Xie and Cai 2003). The Na+/K+-ATPase (pump) is preassembled with its partners in caveolae; ouabain binding to the pump activates the signalosome, and transduces signals via multiple pathways. EGFR, epithelial growth factor receptor; PKC, protein kinase C; PI3K, phosphoinositide 3’ kinase; Grb2, growth factor receptor-bound protein 2; SOS, son of sevenless; Shc, src homology collagenlike protein; PLC, phospholipase C; MAPK, mitogen-activated protein kinase; MEK, MAPK-ERK activating kinase; ROS, reactive oxygen species.
It is noteworthy that Src activation is the critical step for Na/K-ATPase signaling.
Under basal circumstance, the caveolar Na/K-ATPase binds to Src and maintains Src
inactivated. The binding of Na/K-ATPase to CTS will cause an alternation of
conformation, which allows the Src to be activated (Figure 1-4) (Tian, Cai et al. 2006).
4. Na/K-ATPase signaling and renal sodium handling
4.1 Sodium proton exchanger 3 (NHE3)
9
Figure 1-4: Schematic presentation shows how ouabain regulates the Na/K- ATPase/Src receptor complex (Tian, Cai et al. 2006).
There are nine NHE isoforms in the mammalian genome. NHE 1-5 reside predominantly in the plasma membrane and NHE 6-9 are expressed largely in
endomembrane organelles (Alexander and Grinstein 2009; Donowitz, Mohan et al. 2009).
They have various tissue distribution and function. In the renal proximal tubules, NHE3
(SLC9A3) presented along apical/luminal brush border membrane is responsible for two-
third of filtered sodium and fluid reabsorption as well as maintenance and regulation of intravascular volume and BP (Biemesderfer, Pizzonia et al. 1993; Amemiya, Loffing et al.
1995; Alexander and Grinstein 2006). It is widely accepted that apical NHE3 is the rate- limiting step of apical Na+ entry and sodium reabsorption. In other words, NHE3 is a
major Na+ uptake mechanism in the proximal tubules, and increased activity of NHE3 in the renal brush border membrane is involved in the pathogenesis of hypertension
(Morduchowicz, Sheikh-Hamad et al. 1989). Conversely, NHE3-deficient mice are hypotensive and develop acidosis (Schultheis, Clarke et al. 1998; Lorenz, Schultheis et al.
1999; Ledoussal, Lorenz et al. 2001) because of reduced Na+ reabsorption and increased
Na+ excretion in the proximal tubule (Liu and Xie 2010).
10 Rats on low salt exhibit a higher NHE activity compared with rats on high salt diet,
which may contribute to decreased NaCl reabosorption in extracellular fluid volume
expansion, enhanced NaCl reabosorption in extracellular fluid volume depletion (Moe,
Tejedor et al. 1991; Fisher, Lee et al. 2001; Yang, Sandberg et al. 2008). The possible
molecular mechanisms underlying the decreased NHE3 activity in response to high salt is
attributed to the redistribution of NHE3 (Liu, Yan et al. ; McDonough ; Oweis, Wu et al.
2006; Yang, Sandberg et al. 2008). In this scenario, NHE3 plays a protective role
counterbalancing blood pressure rising. For instance, the NHE3 activity decreases with
age in SHR rats (Crajoinas, Lessa et al. ; Yang, Leong et al. 2007).
NHE3 activity is regulated by many factors such as NHE regulatory factor-1
(NHERF-1), angiotension II, sympathetic nervous system, thyroid hormone, insulin and
other humoral agents (Wang, Armando et al. 2009). Importantly, the total number of
NHE3 in the apical membrane and its activity are involved in acute regulation of its
function. The surface expression of NHE3 is mainly regulated by alterations in
endocytosis/exocytosis, and is considered to be the primary regulatory mechanism of
NHE3 activity. NHE3 traffics between the plasma membrane and early/recycling endosomes via a clathrin- and PI3K-dependent pathway (D'Souza, Garcia-Cabado et al.
1998; Kurashima, Szabo et al. 1998; Chow, Khurana et al. 1999; Janecki, Janecki et al.
2000). The NHE3 activity can be stimulated by exocytosis (Collazo, Fan et al. 2000;
Yang, Amemiya et al. 2000; Hu, Fan et al. 2001) and inhibited by endocytosis. Moreover,
activation of Src, PKA, PKC and an increase in intracellular Ca2+ are involved in the
regulation of NHE3 trafficking (Liu and Xie 2010).
11 Our lab has reported that CTS can induce the endocytosis of basolateral Na/K-
ATPase and apical NHE3 coordinately in the renal proximal tubules through Na/K-
ATPase signaling pathway, leading to a decrease in transepithelial sodium reabsorption
(Figure 1-5) (Oweis, Wu et al. 2006; Liu and Shapiro 2007; Cai, Wu et al. 2008). This mechanism contributes to renal proximal tubular sodium handling in response to volume expansion and high blood pressure (see below for details).
4.2 CTS as a 3rd factor and Na/K-ATPase signaling
The “3rd factor”, first described by De Wardener (De Wardener, Mills et al. 1961), is called because it was a factor in addition to aldosterone and glomerular filtration rate
(GFR) in determining renal sodium handling. Bricker postulated that this 3rd factor might be an inhibitor of the Na/K-ATPase (Bricker 1967). Buckalew, et al demonstrated inhibitor of Na/K-ATPase (Buckalew, Martinez et al. 1970) in plasma and plasma ultrafiltrate of volume expanded dogs . Yates et al reported that either ouabain or plasma volume expansion produces modest increases in sodium excretion and urine flow, whereas ouabain combined with plasma volume expansion increases sodium excretion in a synergistic rather than additive manner (Yates and McDougall 1995). Interestingly, high salt diet stimulates an increase in plasma ouabain/MBG and urinary ouabain/MBG excretion (Manunta, Hamilton et al. 2006). In addition, mounting evidence favors the view that a 3rd factor, which is independent of both glomerular filtration rate and aldosterone levels, modulates sodium excretion (Bricker 1967).
Our lab has been focusing on CTS and renal sodium handling for more than ten years. The following in vitro and in vivo studies from our lab suggest that CTS may function as a natriuretic factor through Na/K-ATPase signaling (Liu and Shapiro 2007).
12 (1) Administration of CTS induces a time- and dose-dependent endocytosis of Na/K-
ATPase in LLC-PK1 cells (porcine renal proximal tubular cells) (Liu, Periyasamy et al.
2002). Ouabain, digoxin and MBG have a different efficacy. Specifically, digoxin seems to be the least effective, whereas the others are more effective. (2) CTS inhibit transcellular 22Na+ transport in LLC-PK1 cells. The endocytosis of Na/K-ATPase is
mediated through clathrin-coated pits and activation of Na/K-ATPase signaling cascades
containing Na/ K-ATPase/Src/EGFR (Liu, Kesiry et al. 2004; Liu, Liang et al. 2005). (3)
In vivo studies provide evidence showing CTS-induced endocytosis involving decreased
sodium reabsorption in RPTs after salt loading (Periyasamy, Liu et al. 2005). (4) CTS can
mediate decreases in the apical cell surface NHE3 due to its endocytosis (Oweis, Wu et al.
2006; Cai, Wu et al. 2008).
Much interestingly, several fascinating reports further confirm this concept -CTS
as a natriuretic factor through Na/K-ATPase signaling. Genetically engineered mice with
ouabain-sensitive α1 subunit of Na/K-ATPase display greater natriuretic responses to salt
loading than mice with ouabain-resistant α1 subunit of Na/K-ATPase, confirming that α1
subunit of Na/K-ATPase as ouabain receptor participates in natriuretic response to salt
loading (Dostanic-Larson, Van Huysse et al. 2005; Loreaux, Kaul et al. 2008). In addition,
Nesher and Dvela et al, for the first time, show that endogenous circulatory ouabain
originating in the adrenal has physiological roles controlling vasculature tone and sodium
homeostasis in normal male Wistar rats (Nesher, Dvela et al. 2009). Likewise, another
endogenous MBG has similar natriuretic and vasoconstrictor function (Fedorova,
Kolodkin et al. 2001; Fedorova, Talan et al. 2002; Bagrov and Shapiro 2008).
13
Figure 1-5: Schematic illustration of the effect of CTS-mediated Na/K-ATPase signaling on renal proximal tubular sodium reabsorption (Liu and Xie 2010). NKA, Na+, K+-ATPase.
In summary, studies from our laboratory has demonstrated CTS-mediated redistribution of Na/K-ATPase and NHE3 via activation of Na/K-ATPase signaling cascade might be one of mechanisms linking CTS to a natriuretic factor. Specifically, binding of CTS to Na/K-ATPase stimulate Na/K-ATPase-Src signaling pathway and induce the redistribution of basalateral α1 subunit of Na/K-ATPase and apical NHE3, which mediates sodium and water reabsorption in renal proximal tubules. The downregulation of NHE3 will decrease transepithelial sodium transportation from apical membrane into basalateral membrane, leading to a net increase in urinary sodium excretion (Figure 1-5) (Liu and Shapiro 2007; Liu and Xie 2010).
5. Na/K-ATPase signaling and blood pressure regulation
Accumulating evidence suggests that CTS are important modulators of blood pressure through Na/K-ATPase. Approximately 50% of uncomplicated essential
14 hypertensive patients have an increase in plasma ouabain level (Rossi, Manunta et al.
1995; Manunta, Stella et al. 1999; McKinnon, Lord et al. 2003). Interestingly, some
hypertensive patients with elevated circulating levels of endogenous ouabain do not
appear to be related to plasma renin activity, and may not involve adrenal type-2
dopaminergic receptors (Rossi, Manunta et al. 1995). On the one hand, elevated
endogenous ouabain would lead to vasoconstriction and high blood pressure by inhibition
α2 subunit of Na/K-ATPase, followed by Ca2+ entry via the Na+/Ca2+ exchanger in
vascular smooth muscle cells (Bagrov, Fedorova et al. 1996; Fedorova, Doris et al. 1998;
Manunta, Hamilton et al. 2006; Krzesinski and Cohen 2007; Bagrov and Shapiro 2008;
Bagrov, Shapiro et al. 2009). In kidney, on the other hand, elevated endogenous ouabain
functions as a natriuretic substance by binding to α1 subunit of Na/K-ATPase, followed
by endocytosis of α1 subunit of Na/K-ATPase and NHE3, leading to an increase in urinary sodium excretion (see 4.2 for detail). The latter could counterbalance the
conditions of volume expansion and salt loading, which is consistent with Arthur
Guyton’s following quantitative mathematical explanation.
Based on Arthur Guyton’s quantitative mathematical model for the relation
between blood pressure an natriuresis (pressure-natriuresis relationship) (Guyton 1961;
Guyton and Coleman 1999), salt loading would transiently increase blood pressure,
which in turn would raise sodium excretion until the baseline steady state pressure is
reached. When the blood pressure/nariuresis relationship is shifted to the right, higher
blood pressure values are required to enable the kidney to excrete sodium loads (Figure
1-6).
15
Figure 1-6: Different renal set points for natriuresis in normotensive and hypertensive patients. When the curve shifts to the right in impaired pressure natriuresis, the equilibrium point shifts and the mean arterial pressure is elevated.
Taken together, Na/K-ATPase appears to have a significant effect on the relationship between blood pressure and natriuresis.
When it comes to the relationship between ouabain and MBG in the regulation of blood pressure, our co-laboratory postulated that brain endogenous ouabain caused by salt loading activates the central renin-angiotensin system (RAS) in the hypothalamus and pituitary, which in turn stimulates the RAS in the adrenal cortex via sympathetic nervous system (SNS) activation. Activation of angiotensin II facilitates the production
and release of MBG in adrenal cortex with a primary adaptive aim to induce natriuresis
through inhibition of renotubular Na/K-ATPase. Excessive MBG production, however,
induces vasoconstriction by inhibition of α2 subunit of Na/K-ATPase in vascular smooth
muscle cells (Fedorova, Agalakova et al. 2005; Bagrov, Shapiro et al. 2009). The above
postulation is supported by the following observations: (1) a transient increase in
circulating endogenous ouabain precedes a sustained increase in circulating MBG in Dahl
salt-sensitive rats after both acute and chronic salt loading (Fedorova, Lakatta et al. 2000;
16 Fedorova, Talan et al. 2002); (2) peak transient responses is observed in the amygdala,
hippocampus, and supraoptic nucleus of hypothalamus in response to salt loading in Dahl
salt-sensitive rats (Fedorova, Zhuravin et al. 2007); (3) intrahippocampal microinjection of low dose of ouabain (30pg in 0.5µl in each hemisphere) mimicked the above events completely (Fedorova, Zhuravin et al. 2007); (4) normotensive humans have a transient increase in urinary endogenous ouabain after salt loading, followed by a more sustained
increase in renal marinobufagenin excretion (Anderson, Fedorova et al. 2008; Bagrov and
Shapiro 2008).
6. Reactive oxygen species, Na/K-ATPase signaling and renal sodium handling
6.1 Reactive oxygen species (ROS) and Na/K-ATPase
- ROS, including superoxide anions (O2 ), hydroxyl radicals (·OH) and hydrogen
peroxide (H2O2), are generated as a natural byproduct of normal metabolism from
mitochondria and peroxisomes, as well as from various cytosolic enzyme systems (i.e. xanthine-xanthine oxidase and NADPH oxidase). Antioxidant systems that remove ROS
− encompasse superoxide dismutases (SOD), which converts O2 to H2O2, and catalase that
convert to water, as well as glutathione peroxidase (GPx), which can reduce lipid
hydroperoxides to their corresponding alcohols and to reduce to water. Besides, free radical scavengers such as vitamin C and E also limit ROS. In a word, an antioxidant system can regulate overall ROS levels to maintain physiological homeostasis (Halliwell and Gutteridge 1990).
- Particularly, ·O2 and H2O2 appear to be more important in cardiovascular cells
- (Paravicini and Touyz 2008). ·O2 is short-lived. It can be produced by the reduction of
17 molecular oxygen by NADPH oxidase. Further reduction of superoxide, by superoxide
dismutase, results in the formation of H2O2 (Curtin, Donovan et al. 2002; England,
O'Driscoll et al. 2004). Therefore, the main source of H2O2 in vascular tissue is the
- - dismutation of ·O2 either spontaneously or by SOD. ·O2 is unable to cross cellular
membranes owing to its charge, except possibly through ion channels. In contrast, H2O2
- has a longer biological lifespan than ·O2 . It is relatively stable and is easily diffusible
within and between cells (Paravicini and Touyz 2008).
It is worth to note that ROS can induce a number of nonenzymatic modifications of proteins such as carbonylation, nitrotyrosin, o-tyrosine, and dityrosine. Protein
carbonylation is widely used a marker for oxidative stress (Chevion, Berenshtein et al.
2000). ROS cause the oxidation of amino acid side chains on proteins, leading to exact
conformation and pattern of folding transition, which are tightly connected to their activity and function (Berlett and Stadtman 1997; Chevion, Berenshtein et al. 2000;
Stadtman and Levine 2000; Wondrak, Cervantes-Laurean et al. 2000).
Although excessive generation of ROS is related to cell injury in a variety of pathological conditions including those of the cardiovascular system, mounting evidence indicates that ROS also serve as second messengers within several signal pathways involved in the control of gene transcription (Sen and Packer 1996; Lander 1997). To our interest, an increase in ROS by hypoxia can induce the endocytosis of Na/K-ATPase through reducing Na/K-ATPase activity at the cell surface (Dada and Sznajder 2003).
In a word, an increase in ROS levels may also activate diverse signaling pathways, which may have either damaging or potentially protective functions (Miller, Suzuki et al. ;
Finkel and Holbrook 2000).
18 6.1.1 Na/K-ATPase sensitivity to ROS
Na/K-ATPase is one of the targets for ROS (Lees 1991; Boldyrev, Bulygina et al.
1995). ROS interact with Na/K-ATPase and induce its conformational change, which is
associated with oxidant sensitivity (Xie, Wang et al. 1990; Huang, Wang et al. 1994).
The sensitivity of Na/K-ATPase is dependent on its different isoforms in various tissues
(Xie, Jack-Hays et al. 1995). The α2 and α3 subunit of Na/K-ATPase appear to be more sensitive to H2O2 than the α1 subunit. More sensitive to oxidants the Na/K-ATPase
isoform is, more susceptible to degradation it is (Xie, Jack-Hays et al. 1995). In addition,
brain Na/K-ATPase has more sensitivity than kidney Na/K-ATPase to inhibition by H2O2
(Kurella, Tyulina et al. 1999), while similar sensitivity to oxidants has been found
between ouabain-sensitive α1 (canine) and insensitive α1 (rat), indicating that the
sensitivity of Na/K-ATPase isoform seems not to be species specific, but to be tissue-
dependent.
Taken together, oxidant sensitivity of Na/K-ATPase is related to structural features,
rather than to features that control ouabain sensitivity. Different sensitivity of the Na/K-
ATPase isoform to oxidants appear to depend on the primary sequences of different
subunits and different compositions of subunits in various tissues (Huang, Wang et al.
1994).
6.1.2 Oxidative modification of Na/K-ATPase and its functions
Na/K-ATPase is redox-sensitive (redox, short for reduction/oxidation). H2O2
induces oxidation of Na/K-ATPase, which could be prevented by antioxidants including
the hydrophilic natural antioxidant carnosine, the hydrophobic natural antioxidant alpha-
tocopherol, and the synthetic antioxidant ionol as well as the ferrous ion chelating agent
19 deferoxamine (Kurella, Tyulina et al. 1999). Besides, studies in cerebellar granule cells
(Petrushanko, Bogdanov et al. 2006) suggest that redox state is a potent regulator of the
Na/K-ATPase function. (1) Transport activity of the enzyme is maximal within in a narrow range of redox potentials. Shifts from an "optimal redox potential range" to higher or lower levels cause suppression of the sodium pump activity. (2) Oxidized Na/K-
ATPase (α/β) and FXYD proteins induced by ROS not only inhibit the Na/K-ATPase activity, but also promote its susceptibility to degradation by proteasomal and endosomal/lysosomal proteolytic pathways in different types of cells containing cardiac myocytes, vascular smooth muscle cells, and renal proximal tubular cells (Huang, Wang et al. 1992; Zolotarjova, Ho et al. 1994; Thevenod and Friedmann 1999).
Recently, Rasmussen and co-workers demonstrated that redox signaling mediated by angiotensin receptors and β adrenergic receptors induces oxidative modification
(glutathionylation) of cysteine residue in the β1 subunit of Na/K-ATPase, which could be a physiologic means of regulating protein function. Interestingly, oxidative modification is reversible as phosphorylation does (Figtree, Liu et al. 2009; Rasmussen, Hamilton et al.
2010). However, some studies show that purified enzyme has also been shown to be irreversibly inhibited in response to hydrogen peroxide, the superoxide anion, and the hydroxyl radical (Huang, Wang et al. 1992).
6.1.3 Oxidative modification of Na/K-ATPase and Na/K-ATPase structural regulation
In dog kidney, oxidative modification of Na/K-ATPase by H2O2 induces a decrease in the amount of sulfhydryl (SH) groups, followed by oligomeric structure formation of
Na/K-ATPase, resulting in 50% inhibition of its activity (Boldyrev and Kurella 1996;
20 Dobrota, Matejovicova et al. 1999). Furthermore, the oxidative stability of Na/K-ATPase
is depend on the differences in the number, location, and accessibility of SH groups in
Na/K-ATPase isozymes (Kurella, Tyulina et al. 1999). Besides, the cysteine residues
located in α subunit cytosolic loops is associated with its redox sensitivity.
In the future, studies will be investigated to help characterize changes in the Na/K-
ATPase structure and its possible chemical modification that occurs in response to redox stress.
6.2 ROS involved in Na/K-ATPase signaling
Binding of CTS to the Na/K-ATPase causes cellular ROS production and its downstream effects according to the following observations (Liu, Kennedy et al. 2012).
(1) Ouabain, an inhibitor of Na/K-ATPase, stimulates ROS generation in isolated rat myocytes, pancreatic islets and other cells (Liu, Tian et al. 2000; Kajikawa, Fujimoto et al. 2002; Boldyrev, Bulygina et al. 2003; Huang, Chueh et al. 2004). Moreover, infusion of CTS (MBG) induces ROS generation in experimental animals (Kennedy, Vetteth et al.
2006). (2) ROS induced by ouabain are essential second messengers in ouabain-initiated pathway (Xie, Kometiani et al. 1999). (3) In Na/K-ATPase signaling cascade (Figure 1-
2+ 3), Ras-Raf-MEK-ERK activation increases [Ca ]i, resulting in opening of mitochondrial
ATP-sensitive K+ channels (Tian, Liu et al. 2003) and generation of mitochondrial ROS
(Xie, Kometiani et al. 1999; Liu, Tian et al. 2000). Conversely, ROS stimulate the
signaling function of Na/K-ATPase, followed by a positive amplification loop due to the
mitochondrial production of ROS caused by activation of signaling function of Na/K-
ATPase (Xie, Jack-Hays et al. 1995; Xie and Cai 2003).
21 There is growing convincing data showing the key role of Na/K-ATPase signaling in regulating ROS production. (1) c-Src as a critical initiating step of Na/K-ATPase signaling, is also redox-sensitive and required for ROS generation (derived from NADPH oxidases) (Seshiah, Weber et al. 2002; Touyz, Yao et al. 2003). (2) Lipid rafts are necessary in Na/K-ATPase signaling. In coronary endothelial cells, clustering of lipid rafts on the membrane promotes aggregation and activation of NADPH oxidase, forming a platform for redox signaling through ROS (Touyz 2006; Zhang, Yi et al. 2006). (3) The
Na/K-ATPase regulates caveolin-1 endocytic trafficking and stabilizes the caveolin-1 plasma membrane pool (Cai, Wu et al. 2008). Caveolin 1 is also required in ROS- dependent Angiotensin II signaling (Zuo, Ushio-Fukai et al. 2005).
6.3 ROS in regulation of renal sodium handling and blood pressure
Oxidative stress has been shown to regulate blood pressure and renal sodium handling in various animal models. Physiologically, ROS are necessary in normal redox signaling, while pathological levels of ROS are responsible for renal and vascular dysfunction and remodeling due to oxidative damage (Touyz 2004; Wilcox and
Gutterman 2005; Paravicini and Touyz 2006). Genetic factor(s) partially contribute to high basal ROS levels and the development of hypertension (Lacy, O'Connor et al. 1998;
Lacy, Kailasam et al. 2000).
As mentioned above, CTS-stimulated Na/K-ATPase signaling contributes to RPT sodium handling. ROS serve as important intracellular and extracellular second messengers to regulate many signaling molecules containing Na/K-ATPase signaling.
22 Here we review the possible role of ROS in renal sodium handling and blood pressure regulation.
6.3.1 ROS and renal salt handling
6.3.1.1 ROS and renal NADPH oxidases
A major source of ROS in vascular tissues and kidney is NADPH oxidase (Nox).
Nox originally found in neutrophils, is an enzymatic complex consisting of seven subunits: membrane subunits p22phox and gp91phox (also termed “Nox2”) (Babior 2004),
cytosolic components p40phox, p47phox, and p67phox, and a low-molecular-weight G protein
Rac1 or Rac2 (“phox” stands for phagocyte oxidase) (Burritt, Quinn et al. 1995;
Fontayne, Dang et al. 2002; Vignais 2002; Paravicini and Touyz 2008). Generally, in
unstimulated cells, p22phox and gp91phox occur as a heterodimeric flavoprotein, cyto- chrome b558. On stimulation, p47phox becomes phosphorylated and the cytosolic
subsunits form a complex that translocates to the membrane, where it associates with
cyto-chrome b558 to assemble active oxidase, transferring electrons from the substrate to
O2, forming superoxide (Touyz, Yao et al. 2003; Paravicini and Touyz 2008). This
process is also regulated by Rac2 and Rac1(Bokoch and Zhao 2006).
Seven members in the Nox family include Nox1, Nox2, Nox3, Nox4, Nox5, Duox1,
and Duox2 (Griendling 2006). They are expressed in all tissues and mediate a variety of
biological functions. Of the Nox family, Nox 4 (originally termed Renox due to its high
− expression in kidney tissue) has been shown to act as an O2 producing NADPH oxidase in vascular smooth muscle and kidney (Geiszt, Kopp et al. 2000; Touyz, Chen et al.
2002). Interestingly, Nox4 requires p22phox for activity, but does not require the assembly
23 of cytosolic subunits (Rivera, Sobey et al. 2010). Nox4 might be located in a number of
cellular components, containing the mitochondria (Graham, Kulawiec et al. 2010),
endoplasmic reticulum in addition to the plasma membrane (Yang, Lane et al. 2009;
Zhang, Zhang et al. 2011). Nox4 and p22phox co-localize to internal membranes where
superoxide generation occurs. This Nox4 and /or p22phox complex would constitute
significant levels of hydrogen peroxide involved in intra- and intercellular signaling
pathways affecting basic cellular functions (Martyn, Frederick et al. 2006). That is why
Nox4 does not require the assembly of cytosolic subunits. In the macula densa cells,
Nox2 is primarily responsible for NaCl-induced superoxide production, whereas Nox4 is major source of basal superoxide (Zhang, Harding et al. 2009).
6.3.1.2 ROS and renal proximal tubular sodium transport
Renal Na+ excretion must be tightly regulated to maintain body fluid balance. In
kidney, the amount of Na+ filtered is several times higher than Na+ intake. More than
99% of filtered Na+ must be reabsorbed and approximately 60–70% of this reabsorption occurs in the proximal tubules. Importantly, the NADPH oxidases are the main resource of ROS and present in the renal cortex, medulla, and vasculature (Shiose, Kuroda et al.
2001). Especially, NADPH oxidases are highly expressed in renal proximal tubules.
(Geiszt, Kopp et al. 2000) and play a critical role in sodium reabsorption.
In general, the Nox family NADPH oxidases transfer electrons from
− NADPH/NADH to oxygen across biological membranes, forming superoxide. O2 can then react with both enzymatic and nonenzymatic cellular constituents to form other
− radicals with biological activity such as H2O2 or OH , or with NO to produce
24 OONO−(Traylor and Mayeux 1997). As shown in Fig. 1-7 (Schreck and O'Connor 2011),
− + superoxide (O2· ) generated by NAD(P)H oxidase inhibits Na uptake into the proximal
tubular cell through inhibiting NHE3 (Na+/H+) and Na+-glucose cotransporters
+ − + + (Na :Glucose). The actions of O2· on other apical Na transporters including Na -
ammino acid coransporters (Na+:AA) and Na+-phospate cotransporters (Na+:Pi) remains
− unknown. O2· reacts with nitric oxide (NO) to produce peroxynitrite (OONO·−), which
inhibits basolateral Na/K-ATPase (Schreck and O'Connor ; Zhang, Imam et al. 2002;
Schreck and O'Connor 2011).
Figure 1-7: Diagramatic sketch indicating the actions of NAD(P)H oxidase on sodium transport in a proximal tubule cell (Schreck and O'Connor 2011). Black arrows represent direction of action/reaction only; thin dashed arrows indicate direction of Na+ transport; red arrows indicate an inhibitory action. Panico et al have also demonstrated that superoxide produced by NADPH oxidase
inhibits proximal tubular fluid reabsorption through reduced NHE3 activity in
spontaneously hypertensive rat (SHR) (Panico, Luo et al. 2009). But inhibition of
NADPH oxidase does not have any effect on fluid absorption in renal proximal tubules
25 from normotensive WKY rats (Panico, Luo et al. 2009), suggesting the effects of ROS might be associated with pathological conditions, such as hypertension. Interestingly,
Ortiz and Garvin have reported that ROS (superoxide) stimulate sodium reabsorption in the thick ascending limb of loop of Henle (THAL) and the collecting duct (Ortiz and
Garvin 2002; Silva, Ortiz et al. 2006). The above experiment evidence indicates that elevated ROS levels within proximal tubules might serve an important negative feedback function, promoting excretion of excess sodium and fluid for maintaining body fluid
− balance. Besides, in the outer medullar, O2· production in medullary thick ascending
− limb (mTAL) via the NAD(P)H oxidase pathway and interactions of O2· and nitric oxide
(NO) determine the effectiveness of in situ free radical cross-talk between the mTAL and the vasa recta in terms of regulation of medullar blood flow (Mori and Cowley 2003).
In a word, fluid reabsorption is regulated by redox balance in renal proximal tubules. ROS generation from either high salt or high levels of glucose or ANG II inhibit the Na+, K+-ATPase, apical NHE3 and sodium/glucose transporter and then promote renal proximal tubular sodium and fluid excretion under certain circumstances (Johns,
O'Shaughnessy et al. ; Zhang, Imam et al. 2002; Han, Lee et al. 2005).
Taken together, there might be some cross-talk between Na/K-ATPase/c-Src signaling and ROS generation in terms of renal sodium handling (Liu, Kennedy et al.) based on the following evidence : (1) we have demonstrated that cardiotonic steroids such as ouabain decrease renal proximal tubular Na+ reabsorption by the redistribution of
Na/K-ATPase and NHE3 through Na/K-ATPase/c-Src signaling (Liu, Kesiry et al. 2004;
Liu, Liang et al. 2005; Liu and Shapiro 2007). (2) Activation of Na/K-ATPase/c-Src signaling stimulates mitochondrial ROS production. (3) c-Src as initiating step of Na/K-
26 ATPase signaling, is also redox-sensitive and required for ROS generation (derived from
NADPH oxidases) (Seshiah, Weber et al. 2002; Touyz, Yao et al. 2003). (4) NADPH oxidase-derived ROS production reduces renal proximal tubular sodium reabsorption
(Panico, Luo et al. 2009).
Figure 1-8: Schematic illustration of the effects of ROS and Na/K-ATPase signaling on RPT sodium reabsorption (Liu, Kennedy et al. 2012). CTS, cardiotonic steroids; RPT, renal proximal tubules.
6.3.2 ROS and blood pressure regulation Oxidative stress is both a cause and consequence of hypertension (Kitiyakara,
Chabrashvili et al. 2003; Touyz 2004; Wilcox 2005; Vaziri and Rodriguez-Iturbe 2006;
Welch 2006).
A large body of evidence supports a role for ROS in the pathogenesis of hypertension. Increased ROS generation precedes development of hypertension, suggesting that ROS participate in the development and maintenance of hypertension
(Kitiyakara and Wilcox 1998; Houston 2005). In experimental hypertension including
- Dahl salt-sensitive hypertension, oxidative stress markers such as concentrations of O2
27 and H2O2, and activation of NADPH oxidase and xanthine oxidase are increased, whereas
levels of NO and antioxidant enzymes (SOD) are reduced (Kitiyakara, Chabrashvili et al.
2003; Touyz and Schiffrin 2004). In human, clinical studies have demonstrated that hypertensive patients produce excessive amounts of ROS (Lacy, Kailasam et al. 2000;
Fortuno, Olivan et al. 2004) and have reduced levels of antioxidant enzymes (SOD, glutathione peroxidase and catalase) (Saez, Tormos et al. 2004; Simic, Mimic-Oka et al.
2006). Based on the above experimental evidence and clinical studies, current therapeutic approaches that target ROS in the treatment of hypertension include the following mechanisms: (1) to increase antioxidant capacity through diet or supplementation, (2) to
- reduce ROS generation by decreasing activity of O2 generating enzymes, (3) and to
increase NO bioavailability(Hamilton, Miller et al. 2004; Touyz and Schiffrin 2004).
Antioxidant heme oxygenase (HO, encoded by HMOX genes) is the rate limiting
enzyme in heme degradation. Heme is a stable prosthetic group of a variety of enzymes.
A Fe2+ atom in its center of a protoporphyrin IX ring can be oxidized by hydrogen
peroxide to produce highly toxic hydroxyl radicals (HJH 1894). So it might be a
ubiquitous cytotoxic molecule involved in the pathogenesis of a broad spectrum of
diseases. HO can catalyze free heme to produce equimolar amounts of liable Fe (stored
by the ferritin H chain, FtH), carbon monoxide (CO), and biliverdin. Biliverdin is
subsequently converted to endogenous antioxidant bilirubin by biliverdin reductase
(Ndisang, Tabien et al. 2004; Abraham and Kappas 2005). CO like NO, signals and
augment NO levels to favor vasodilation and saliuresis. Bilirubin, which may enhance
- NO levels by reducing its rate of consumption by O2 , is an effective ROS scavenger
(Pallone 2007). All three end products of heme catabolism are cytoprotective due to their
28 potent antioxidant properties and antiapoptotic effects (Stocker, Yamamoto et al. 1987;
Abraham, Cao et al. 2009). Humans and rodents have two HO isoenzymes, namely HO-1
(32kDa) and HO-2 (36kDa) encoded by the HMOX1 and HMOX2 genes respectively.
HO-1 expression is induced ubiquitously in response to oxidative stress, whereas HO-2 is constitutively expressed and not inducible.
It has been shown that induction of HO-1 improves vascular function and ameliorates both genetic and experimental hypertension (Sacerdoti, Escalante et al. 1989;
Levere, Martasek et al. 1990; Sabaawy, Zhang et al. 2001; Yang, Quan et al. 2004;
Abraham and Kappas 2005; Botros, Schwartzman et al. 2005; Wang, Shamloul et al.
2006). Inhibition of HO-1 increases mean arterial pressure in Sprague-Dawley rats
(Johnson, Lavesa et al. 1995; Hirakawa and Hayashida 2006) and SHR rats (Johnson,
Lavesa et al. 1996). However, most current studies focus on vasculature. Given the crucial role of the kidneys in the long-term control of arterial blood pressure, it is imperative to determine whether and how local HOs in the kidneys, especial renal proximal tubules, participate in the regulation of the renal sodium excretion and arterial blood pressure. Li et al demonstrate that inhibition of renal medullary HO induces hypertension and increased salt sensitivity of arterial blood pressure through blunted natriuretic response to the elevation of renal perfusion pressure (Zou, Billington et al.
2000; Li, Yi et al. 2007; Pallone 2007). Increasingly, considerable attention has been paid mechanisms of pressure natriuresis because it favors amelioration of high blood pressure through reduction of extracellular fluid volume.
As discussed above (6.3.1.2), there might be some cross-talk between Na/K-
ATPase/c-Src signaling and ROS generation in terms of renal sodium handling (Liu,
29 Kennedy et al.). Binding of cardiotonic steroids such as ouabain to Na/K-ATPase decreases renal proximal tubular Na+ reabsorption by the redistribution of Na/K-ATPase and NHE3 through Na/K-ATPase/c-Src signaling (Liu, Kesiry et al. 2004; Liu, Liang et al. 2005; Liu and Shapiro 2007). Activation of Na/K-ATPase/c-Src signaling stimulates mitochondrial ROS production. c-Src as initiating step of Na/K-ATPase signaling, is also redox-sensitive and required for ROS generation (derived from NADPH oxidases)
(Seshiah, Weber et al. 2002; Touyz, Yao et al. 2003). NADPH oxidase-derived ROS production reduces renal proximal tubular sodium reabsorptiotn (Panico, Luo et al. 2009).
Together with the roles of renal medullar HO in regulation of salt and water excretion by the kidney, we propose a network among the Na/K-ATPase/c-Src signaling, NADPH oxidase-derived ROS generation and HO-1 that will favor understanding the ROS functional roles in renal proximal tubular sodium handling.
Although antioxidants exhibit a beneficial and protective effect on blood pressure in various animal models of hypertension, findings from clinical trials have been conflicting (reviewed in (Touyz 2004; Munzel, Gori et al. 2010). Some large trials including the Dietary Approaches to Stop Hypertension (DASH) have demonstrated that lower blood pressure associated with reduced dietary salt intake may be related to reductions in oxidative stress (Appel, Moore et al. 1997; Miller, Appel et al. 1998;
Sacks, Svetkey et al. 2001; John, Ziebland et al. 2002). However, antioxidant supplementation presently might not be recommended for the prevention and treatment of hypertension due to less evidence to prove the benefits from the routine use of antioxidants. Moreover, excessive antioxidant supplementation might be even dangerous (Huang, Caballero et al. 2006) due to the possible ‘over-antioxidant-
30 buffering’ effect. In this setting, excess antioxidants might become pro-oxidants (by
providing H+) if they cannot promptly be removed by the following anti-oxidant in the
biological anti-oxidant chain. Thus, it appears that the balance of the ROS status, within
a physiological range, may be more important to maintain a beneficial ROS signaling
(Liu, Kennedy et al.).
ROS is an appealing candidate target due to representing a common mechanism
leading from multiple risk factors to disease. Given the pivotal role of kidney in the
regulation of blood pressure, further studies, both in animal and humans, are needed in
order to achieve a better understanding of the role of ROS in the pathophysiology of
hypertension. Based on the literatures and our studies, this dissertation proposes: (1) ouabain stimulates trafficking regulation of the Na/K-ATPase and NHE3 in renal proximal tubule cells, (2) impairment of Na/K-ATPase signaling in renal proximal tubule contributes to Dahl salt-sensitive hypertension, (3) ROS are implicated in the modulation of the Na/K-ATPAse signaling and renal proximal tubular sodium handling. With greater insights and understanding of these processes regulating renal sodium handling and identification of molecular pathway that tip the equilibrium to states of oxidative stress which cause high blood pressure, the target therapies might be more effective in the prevention and treatment of hypertension.
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54 Chapter 2 – Manuscript 1
Title:
Ouabain-Stimulated Trafficking Regulation of the Na/K-ATPase and NHE3 in
Renal Proximal Tubule Cells
Authors:
Yanling Yan1,3, Steven Haller1, Anna Shapiro1, Nathan Malhotra1, Jiang Tian1,
Zijian Xie2,1, Deepak Malhotra1, and Joseph I. Shapiro1,2, Jiang Liu1
From the Departments of Medicine1 and Physiology and Pharmacology2, University
of Toledo College of Medicine, Toledo, OH; and Institute of Biomedical
Engineering3, Yanshan University, China.
Corresponding Author:
Jiang Liu, M.D., Ph.D. Department of Medicine, University of Toledo College of
Medicine, Health Science Campus, 3000 Arlington Avenue, Toledo, OH, 43614
Phone: (419) 383-3923
Email: [email protected]
FAX: (419) 383-6244
Published in Molecular and Cellular Biochemistry. 2012, # : #-#. (mcbi-5426)
55 2.1 Abstract
We have demonstrated that ouabain regulates protein trafficking of the
Na/K-ATPase α1 subunit and NHE3 (Na/H exchanger, isoform 3) via ouabain-activated Na/K-ATPase signaling in porcine LLC-PK1 cells. To investigate
whether this mechanism is species-specific, ouabain-induced regulation of the α1
subunit and NHE3 as well as transcellular 22Na+ transport were compared in three
renal proximal tubular cell lines (human HK-2, porcine LLC-PK1, and AAC-19
originated from LLC-PK1 in which the pig α1 was replaced by ouabain-resistant rat
α1). Ouabain inhibited transcellular 22Na+ transport due to an ouabain-induced
redistribution of the α1 subunit and NHE3. In LLC-PK1 cells, ouabain also inhibited
the endocytic recycling of internalized NHE3, but has no significant effect on
recycling of endocytosed α1 subunit. These data indicated that the ouabain-induced
redistribution of the α1 subunit and NHE3 is not a species-specific phenomenon, and
ouabain-activated Na/K-ATPase signaling influences NHE3 regulation.
Keywords: Ouabain, Na/K-ATPase signaling, Na/K-ATPase, NHE3, redistribution
56 2.2 Introduction
Renal sodium handling is a key determinant of long term regulation of blood
pressure (Guyton 1991; Stamler, Rose et al. 1991; Meneton, Jeunemaitre et al. 2005;
Haddy 2006). In the kidney, the renal proximal tubules (RPTs) are responsible for
more than 60% of the net tubular Na+ reabsorption, mainly through basolateral
Na/K-ATPase and apical NHE3. Endogenous CTS (cardiotonic steroids, also known
as digitalis-like substances), which were initially classified as specific inhibitors of
the Na/K-ATPase and now classified as a new family of steroid hormones, are
involved in regulation of blood pressure and renal sodium handling (Schoner and
Scheiner-Bobis 2008; Bagrov, Shapiro et al. 2009; Nesher, Dvela et al. 2009).
Circulating CTS are markedly increased under certain conditions such as salt
loading, volume expansion, renal insufficiency, and congestive heart failure (Lloyd,
Sandberg et al. 1992; Fedorova, Doris et al. 1998; Manunta, Hamilton et al. 2006).
The pathophysiological significance of endogenous CTS has been a subject of
debate since it was first proposed (Haddy and Overbeck 1976; Blaustein 1977; De
Wardener 1977). In essence, the Na/K-ATPase inhibitor (endogenous CTS) will rise
in response to either a defect in renal Na+ excretion or high salt intake. This increase,
while returning Na+ balance toward normal by increasing renal Na+ excretion, also
cause increases in blood pressure through acting on vascular Na/K-ATPase
(Blaustein, Zhang et al. 2009). Increases in endogenous CTS regulate both renal Na+ excretion and blood pressure through the Na/K-ATPase (Dostanic-Larson, Van
57 Huysse et al. 2005; Loreaux, Kaul et al. 2008; Blaustein, Zhang et al. 2009; Nesher,
Dvela et al. 2009; Liu, Yan et al. 2011).
In porcine RPT LLC-PK1 cells, we have shown that ouabain inhibits active
transepithelial 22Na+ transport (from apical to basolateral aspect) via protein trafficking regulation of the Na/K-ATPase and NHE3 (Liu, Kesiry et al. 2004; Cai
2008; Liu and Xie 2010), a process requiring ouabain-activated Na/K-ATPase signaling. This novel regulatory mechanism may contribute to CTS-induced natriuresis, especially in rodents expressing ouabain-resistant Na/K-ATPase α1 subunit (Liu, Yan et al. 2011). In the present study, we investigated if this regulatory mechanism is species-specific by characterizing the effect of ouabain on transcellular 22Na+ flux and redistribution of the Na/K-ATPase and NHE3.
Furthermore, we also investigate the endocytic recycling (reinsertion of endocytosed
protein back to plasma membrane) of internalized Na/K-ATPase and NHE3 in
LLC-PK1 cells.
2.3 Experimental Methods
2.3.1 Chemicals and Antibodies
All reagents, unless otherwise mentioned, were obtained from Sigma (St.
Louis, MO). Src kinase inhibitor PP2 was from CalBiochem (San Diego, CA).
EZ-Kink sulfo-NHS-ss-Biotin and ImmunoPure immobilized streptavidin-agarose
beads were obtained from Pierce Biotechnology (Rockford, IL). A rabbit polyclonal
antibody against a mixture of peptides from porcine NHE3 was prepared and affinity
58 purified (Oweis, Wu et al. 2006). Antibodies against Rab7, integrin-β1 and human
NHE3 were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody against the Na/K-ATPase α1-subunit (clone α6F) was from the
Developmental Studies Hybridoma Bank at the University of Iowa (Iowa City, IA).
Monoclonal antibodies against NHE3 (clone 4F5) and early endosome antigen-1
(EEA-1) were from Millipore Chemicon (Temecula, CA). Radioactive rubidium
(86Rb+) and sodium (22Na+) were from DuPont NEN Life Science (Boston, MA).
2.3.2 Cell cultures
Human HK-2 cells and pig LLC-PK1 cells were obtained from the American
Type Culture Collection (Manassas, VA). AAC-19 cells were generated from
LLC-PK1 cells as we described previously (Liang, Cai et al. 2006). Briefly, the
ouabain-sensitive pig α1 in LLC-PK1 cells was knock-down by siRNA method. To
rescue the α1 knock-down cell with ouabain-resistant rat α1 (AAC-19 cells), the α1
siRNA targeted sequence was silently mutated to introduce rat α1 with rat α1
pRc/CMV-α1AAC expression vector. The expression of ouabain-insensitive rat α1
was selected with 3µM ouabain in culture medium since untransfected LLC-PK1
cells are very sensitive to ouabain. Cells were cultured in DMEM (Dulbecco's
modified Eagle's medium)/F-12 mixed medium (1:1, vol/vol) for HK-2 or DMEM
for LLC-PK1 and AAC-19, with 10% fetal bovine serum (FBS), 100 U/ml penicillin,
and 100 µg/ml streptomycin) in a 5% CO2-humidified incubator. Culture medium
was changed daily until confluency. LLC-PK1 cells and AAC-19 cells were
serum-starved (in serum-free DMEM medium) for 16-18 h before treatment, and
59 HK-2 cells were changed to medium containing 1% FBS for 16-18 h before treatment. In assays for active transcellular 22Na+ flux, cells were grown on
Transwell membrane support to form monolayer, and then treated with ouabain either in the basolateral or apical compartment. Both LLC-PK1 and AAC-19 cells can be easily grown to monolayers in DMEM medium with 10% FBS. While HK-2 cells are hard to form monolayer with ATCC-recommended Keratinocyte Serum
Free Medium, we found that HK-2 cells could be easily grown to monolayer in
DMEM/F-12 medium (with 10% FBS) without losing its ouabain sensitivity.
2.3.3 Isolation of early endosome (EE) and late endosome (LE) fractions
EE and LE fractions were fractionated by a sucrose flotation gradient technique as we previously described (Liu, Kesiry et al. 2004; Cai 2008). EE and LE fractions were identified with antibodies against EE marker protein EEA-1 and LE marker protein Rab7, respectively (Liu, Kesiry et al. 2004; Cai 2008). In comparison with whole cell lysates, more than a 10-fold enrichment of these marker proteins was observed in representative endosome fractions as we have previously shown (Liu,
Kesiry et al. 2004).
2.3.4 Cell surface biotinylation
Cell surface biotinylation was conducted as we described before (Liu, Kesiry et al. 2004; Cai 2008). Biotinylated proteins were pulled down with streptavidin-agarose beads, eluted with 2x Laemmli buffer (125 mM Tris-HCl, 20%
Glycerol, 4% SDS, 0.025% Bromophenol Blue, 10% 2-mercaptoethanol, pH 6.8) at
60 55°C waterbath for 30 minutes, resolved by 10% SDS-PAGE, and then
immunoblotted for the Na/K-ATPase α1 and NHE3. The same membrane was also
imunoblotted with antibody against integrin-β1 to serve as loading control as
described previously (Liu, Yan et al. 2011).
2.3.5 Ouabain-sensitive Na/K-ATPase activity assay (86Rb+ uptake)
For 86Rb+ uptake assay, cells were cultured in 12-well plates and treated with or without different concentrations of ouabain for 15min. Monensin (20 µM), a
Na+-clamping agent, was added to the medium prior to the assay to assure that the
maximal capacity of active uptake was measured (Haber, Pressley et al. 1987). 86Rb+ uptake was initiated by the addition of 1 µCi of 86Rb+ as tracer of K+ to each well,
and the reaction was stopped after 15 minutes by washing four times with ice-cold
86 + 0.1 M MgCl2. Trichloroacetic acid (TCA)-soluble Rb was extracted with 10%
TCA and counted. TCA-precipitated cellular protein content was determined and used to calibrate the 86Rb+ uptake. Data were expressed as the percentage of control
86Rb+ uptake.
2.3.6 NHE3 mediated active transepithelial 22Na+ flux and 22Na+ uptake
Active transepithelial 22Na+ flux (from apical to basolateral aspect of the
Transwell membrane supports) was performed on monolayers (grown on Costar
Transwell culture filter inserts, filter pore size: 0.4 µm, Costar, Cambridge, MA) as
described by Haggerty and colleagues (Haggerty, Agarwal et al. 1988). Briefly, after
ouabain treatment for 1h at the concentration indicated, both apical (upper) and
61 basolateral (lower) compartments were rinsed with ouabain-free DMEM (0% FBS
for LLC-PK1 and AAC-19 cells, and 1% FBS for HK-2 cells). 1 ml DMEM
containing 22Na+ (1 µCi/ml) was added to the apical compartment of a filter insert, and the basolateral compartment was filled with 1 ml of DMEM. After 1h, aliquots
were removed from the basolateral compartments for scintillation counting.
H+-driven 22Na+ uptake were determined as described by Soleimani and colleagues
(Soleimani, Watts et al. 1998). Briefly,the cells grown on 12-well plate were treated
with ouabain at the concentration indicated and then washed three times with the
Na+-free buffer (in mM, 140 N-methyl-D-glucammonium (NMDG+) Cl, 4 KCl, 2
MgCl2, 1 CaCl2, 10 HEPES, pH 7.4). The cells were then incubated for 10 min in the
+ + + same Na -free buffer in which 20 mM NMDG was replaced with 20 mM NH4 . The
+ + uptake was initiated by replacing the NH4 -containing buffer with Na -free buffer
containing 1 µCi/ml 22NaCl+. 22Na+ uptake was stopped after 30 min by washing four
times with ice-cold saline. Cell-associated radioactivity was extracted with 1 ml of 1
N sodium hydroxide, quantified by scintillation counting, and calibrated with protein
content. In both experimental settings, cells were pretreated with 50 µM amiloride to
inhibit amiloride-sensitive NHE1 activity.
2.3.7 Assessment of endocytic recycling of NHE3 and Na/K-ATPase α1 subunit
in LLC-PK1 cells
Endocytic recycling of α1 and NHE3 were assessed by the method described
by the Moe Laboratory (Klisic, Zhang et al. 2003). Briefly, two sets of LLC-PK1
cells were biotinylated and quenched at 4°C, and then treated with ouabain (100nM)
62 or vehicle (as control) for 1h at 37°C to induce redistribution of the Na/K-ATPase α1
and NHE3. After rinsing with ice-cold PBS-Ca-Mg (1x PBS with 0.1mM CaCl2 and
1mM MgCl2), un-internalized surface biotinylated proteins were cleaved with 50
mM glutathione-SH (GSH-SH, a reducing agent) at 4°C, and un-reacted free
GSH-SH was oxidized by incubation with 30mM iodoacetamide for 10min. At this
point, one set of cells was lysed with RIPA buffer to retrieve total internalized
intracellular biotinylated α1 and NHE3 with streptavidin-agarose beads. Other set of
cells was changed to serum-free DMEM medium and cultured at 37°C to permit
further trafficking for 2h with or without 100nM ouabain. Biotinylated proteins that
recycled back to cell surface (reinsertion) were cleaved by GSH-SH method again.
The remaining intracellular biotinylated proteins after reinsertion were retrieved
with streptavidin-agarose beads, which represent the internalized biotinylated α1 and NHE3 that were not reinserted. The difference of the total intracellular α1 and
NHE3, before and after reinsertion, represents the α1 and NHE3 that was
internalized and then reinserted (endocytic recycling).
2.3.8 Western blot
Equal amounts of total protein were resolved by 10% SDS-PAGE and
immunoblotted with indicated antibodies (with dilution of 1:2000 for the
Na/K-ATPase α1 subunit and 1:1000 dilution for NHE3, in 4% non-fat dry milk in
1x Tris-buffered saline with 0.1% Tween-20). The same membrane was also
imunoblotted with antibodies against integrin-β1 (for surface biotinylation), EEA-1
(for EE fraction) and Rab7 (for LE fraction) to serve as loading controls (data not
63 shown), respectively, as we previously described (Liu, Kesiry et al. 2004; Cai 2008;
Liu, Yan et al. 2011). Signal detection was performed with an enhanced
chemiluminescence super signal kit (Pierce, Rockford, IL). Multiple exposures were
analyzed to assure that the signals were within the linear range of the film. The signal density was determined using Molecular Analyst software (Bio-Rad, Hercules, CA).
2.3.9 Statistical analysis
Data were tested for normality (all data passed) and then subjected to parametric analysis. When more than two groups were compared, one-way ANOVA
was performed prior to the comparison of individual groups with an unpaired t-test.
Statistical significance was reported at the P < 0.05 and P < 0.01 levels. SPSS
software was used for all analysis (SPSS, Chicago, IL). Values are given as
mean±S.E.
2.4 Results
2.4.1 Ouabain-mediated inhibition of the Na/K-ATPase
Ouabain-induced inhibition of the Na/K-ATPase “ion-pumping” activity
(ouabain-sensitive 86Rb+ uptake) in these RPT cell lines is summarized in Fig. 2-1.
The IC50 values are consistent with the established differences of α1 ouabain
sensitivity amongst these species (see Discussion). In LLC-PK1 cells with IC50 at
1µM, 100nM ouabain is sufficient to activate the Na/K-ATPase signaling and consequent regulation of the Na/K-ATPase and NHE3 (Liu and Xie 2010).
64 According to the Na/K-ATPase α1 sensitivity to ouabain, we chose ouabain concentrations that are able to activate the Na/K-ATPase signaling for these three cell lines (10nM for HK-2, 100nM for LLC-PK1, and 10μM for AAC-19 cells) without significant inhibition of Na/K-ATPase activity. No significant effect on cell viability was observed when these cells were treated for 1h with ouabain concentrations used that was evaluated by Trypan blue exclusion.
2.4.2 Ouabain-mediated inhibition of transepithelial 22Na+ flux and
22Na+uptake
We have shown that ouabain inhibits transepithelial 22Na+ flux by activating
Na/K-ATPase signaling in LLC-PK1 cells (Liu, Kesiry et al. 2004; Cai 2008). To
assess if this effect is species-specific, we measured H+-driven 22Na+ uptake and
transepithelial 22Na+ flux in these three RPT cell lines. As shown in Fig. 2-2 and 2-3,
when ouabain was added in the basolateral aspect, ouabain inhibited 22Na+ uptake
(Fig. 2-2) and active transepithelial 22Na+ flux (Fig. 2-3) in both HK-2 and AAC-19 cells in the same manner as in LLC-PK1 cells. The effect of ouabain on 22Na+ flux
and NHE3 activity was largely blunted when these cells were pretreated with the Src
kinase inhibitor PP2 (1µM for 30 min, at 37 °C). PP2 alone did not show significant effect. No significant inhibition of NHE3 activity was observed in all three cell lines
when ouabain was added in the apical aspect (data not shown), suggesting that
ouabain-induced regulation of 22Na+ flux and NHE3 activity requires
ouabain-activated Na/K-ATPase signaling.
65 2.4.3 Ouabain-induced protein trafficking of Na/K-ATPase and NHE3
In LLC-PK1 cells, ouabain-induced inhibition of 22Na+ flux is largely due to
an ouabain-mediated redistribution of the Na/K-ATPase and NHE3 via
Na/K-ATPase signaling. To further test this regulatory mechanism, these three cell
lines were treated with or without ouabain to assess the ouabain-induced
redistribution. As shown in Fig. 2-4, the ouabain induced redistribution of the α1
subunit and NHE3 was caused by a decrease in cell surface α1 subunit and NHE3 in
both HK-2 and AAC-19 cells (as we previously reported in LLC-PK1 cells (Liu,
Kesiry et al. 2004; Cai 2008)). Pretreatment with PP2 abolished ouabain-induced
redistribution of the α1 subunit and NHE3 (data not shown). As shown in Table 2.1,
ouabain (1h) caused a dose-dependent reduction of cell surface α1 subunit and
NHE3. Furthermore, the ouabain-induced reduction of cell surface α1 subunit and
NHE3 was closely correlated to ouabain-induced inhibition of transcellular 22Na+ flux and NHE3 activity (Fig. 2-2 and 2-3).
2.4.4 Ouabain-mediated regulation of endocytic recycling of the Na/K-ATPase
α1 subunit and NHE3 in LLC-PK1 cells
Endocytic recycling is essential for maintaining the distinction between apical and basolateral membranes in polarized cells, even though the recycling pathways may be redundant (Maxfield and McGraw 2004). It has been shown that ouabain redistributes the Na/K-ATPase into both late endosomes and lysosomes
(Algharably, Owler et al. 1986), presumably for degradation. To explore the underlying mechanism, we used LLC-PK1 cells to assess endocytic recycling of the
66 α1 subunit and NHE3. As shown in Fig. 2-5A, GSH-SH (glutathione-SH, a reducing agent) was able to cleave over 90% of protein-bound biotin. Ouabain (100nM, 1h)
accumulated the α1 subunit (control 100±9.6% vs. ouabain 198.3±18.2, n=3, p<0.01)
and NHE3 (control 100±8.9% vs. ouabain 187.4±16.5, n=3, p<0.01) in intracellular
compartments (Fig. 2-5B) after cleavage of surface protein-bound biotin with
GSH-SH method. After 2h-period of recycling of internalized biotinylated proteins,
recycled biotinylated proteins were cleaved again with GSH-SH. The total
intracellular biotinylated α1 and NHE3 after recycling, which represent un-recycled
biotinylated α1 and NHE3, are shown in Fig. 2-5C. The reinsertion (endocytic
recycling) was presented as the difference of the total intracellular α1 and NHE3
before and after reinsertion (Fig. 2-5D). The present data indicate that ouabain
treatment not only induced NHE3 redistribution, but it also inhibited the endocytic
recycling of NHE3. Interestingly, the endocytic recycling of the α1 subunit was not
significantly affected by ouabain. These observations suggested that, while most of
the internalized α1 was destined for degradation, at least part of internalized NHE3
was recycled back to cell membrane surface. Despite the lack of microvilli in
cultured renal proximal tubular cells (McDonough and Biemesderfer 2003), our
observation is reminiscent of the model of NHE3 moving along the microvilli
structure (Yang, Maunsbach et al. 2004).
To further explore the destination of the α1 subunit and NHE3, we determined
the protein content of these two transporters in EE and LE fractions in response to
ouabain. As shown in Fig. 2-6, ouabain treatment (100nM, 1h) stimulated
accumulation of both α1 and NHE3 in EE fractions. However, ouabain significantly
67 accumulated α1, but not NHE3 in LE fractions. The NHE3 protein content in LE fractions was not significantly increased after ouabain treatment, even in the presence of the lysosomotropic weak base agent chloroquine (0.2 mM, pretreated for
2h) which inhibits degradation by the lysosomal pathway. On the other hand, pretreatment with chloroquine caused a further accumulation of the α1 subunit in LE fractions in response to ouabain, suggesting that the endocytosed α1 subunit, but not
NHE3, is degraded through the LE-lysosome pathway.
2.5 Discussion
The renal tubular Na/K-ATPase comprises a final site for the regulation of renal sodium transport by many factors (Aperia 2001). Our recent work indicates that ouabain is one of these factors that act via a coordinated regulation of the
Na/K-ATPase and NHE3 through Na/K-ATPase signaling (Liu, Kesiry et al. 2004;
Cai 2008; Liu and Xie 2010; Liu, Yan et al. 2011). In the present study, we have investigated whether this ouabain mediated regulatory model is species-specific.
Our present study indicated that ouabain-induced regulation of the Na/K-ATPase and NHE3 is not species-specific. First, ouabain was able to inhibit the active transepithelial 22Na+ flux in these three cell lines. This effect was largely prevented by blocking c-Src activation with PP2 pretreatment. Secondly, ouabain-induced redistribution and reduction of the cell surface Na/K-ATPase and NHE3 contributed to the inhibition of active 22Na+ flux. Third, the effect of ouabain on active 22Na+ flux was only observed when ouabain was applied in the basolateral, but not in the apical
68 aspect. Fourth, the ouabain-induced reduction of the cell surface α1 subunit
contributed to the ouabain-induced inhibition of 22Na+ flux and 22Na+ uptake. Taken
together, these observations suggest that the ouabain-induced regulation of the
Na/K-ATPase and NHE3 is not species-specific, and may be explained if the
Na/K-ATPase is the functional receptor for ouabain-induced regulation.
Activation of c-Src is critical in the ouabain-activated Na/K-ATPase
signaling and redistribution of the Na/K-ATPase and NHE3 (Liu, Kesiry et al. 2004;
Liu, Liang et al. 2005; Oweis, Wu et al. 2006; Cai 2008; Liu and Xie 2010). When
compared with HK-2 and LLC-PK1 cells, a higher concentration of ouabain was
needed to regulate activity and redistribution of the Na/K-ATPase and NHE3 in
AAC-19 cells expressing native pig NHE3 and rat α1. This is consistent with the established differences in ouabain sensitivity of the α1 subunit as well as ouabain-stimulated c-Src activation between these two cell lines (Liang, Cai et al.
2006). It is well known that there are large differences in sensitivity of the
Na/K-ATPase to ouabain based on α isoforms and species (Sweadner 1989; Lingrel and Kuntzweiler 1994; Blanco and Mercer 1998). Specifically, the rodent α1 is far less sensitive than pig, dog, or human α1. Higher concentrations of ouabain were required to activate Na/K-ATPase signaling in rodents, compared to other species
(Liu, Tian et al. 2000; Aizman, Uhlen et al. 2001; Aydemir-Koksoy, Abramowitz et al. 2001; Haas, Wang et al. 2002; Liang, Cai et al. 2006). We have also shown that a higher concentration of ouabain (10µM) is needed to activate c-Src in isolated renal proximal tubules of Dahl salt-resistant rats (Liu, Yan et al. 2011). Most interestingly, different natriuretic responses were observed between transgenic mice expressing
69 ouabain-sensitive α1 and wild-type mice expressing ouabain-resistant α1(Loreaux,
Kaul et al. 2008) in which the ouabain binding site of the α1 subunit plays a critical
role (Lingrel 2010). As shown in Table 2.1, the ouabain sensitivity of the α1 subunit
influenced the ouabain-induced inhibition of 22Na+ flux as well as surface reduction of the α1 subunit and NHE3. The present study further suggests that species-specific
α1 sensitivity to ouabain might explain the species differences in ouabain-induced
natriuresis in vivo (Lloyd, Sandberg et al. 1992; Yates and McDougall 1993;
Loreaux, Kaul et al. 2008; Nesher, Dvela et al. 2009). Considering the high ouabain
sensitivity of human α1 subunit, this could be a explanation how pathophysiological
circulating CTS might affect renal sodium handling, especially in the view that
endogenous ouabain is a natriuretic hormone and has a physiological role in
controlling sodium homeostasis in normal rats (Nesher, Dvela et al. 2009).
After the redistribution of membrane proteins, subsequent intracellular trafficking differs among different endocytosed proteins. Receptor-mediated redistribution is believed to be an effective pathway to reduce cell surface signaling receptors. In our experimental settings, ouabain caused accumulation of the α1 subunit in LE fractions, but failed to affect its endocytic recycling (Fig. 2-5 and 6).
This is a reminiscence of the early observation that ouabain caused the redistribution of the Na/K-ATPase into late endosomes and lysosomes (Algharably, Owler et al.
1986), suggesting that the endocytosed Na/K-ATPase is most likely degraded in
LE/lysosome pathway. On the other hand, the cell surface expression of NHE3 is likely reduced by inhibition of the recycling of internalized NHE3 (Fig. 2-5 and 6).
However, the mechanism is not clear. In both cases, we cannot exclude the
70 possibility that both recycling and degradation were affected by ouabain, but the
overall observed effect was a balance between these two processes.
Renal Na+ reabsorption through NHE3 plays an important role in
salt-sensitivity, as well as in the development and control of sodium homeostasis and
blood pressure (Harris, Brenner et al. 1986; Kelly, Quinn et al. 1997; Schultheis,
Clarke et al. 1998; Lorenz, Schultheis et al. 1999). Recently, we have demonstrated
that impaired Na/K-ATPase-Src signaling contributes to salt sensitivity in Dahl rats
(Liu, Yan et al. 2011). This is consistent with the observation that a high salt diet
stimulates redistribution of RPT Na/K-ATPase and NHE3 (McDonough 2010).
Although the mechanisms are still being elucidated, accumulating evidence supports
the notion of coordinated regulation of the Na/K-ATPase and NHE3 (Liu and
Shapiro 2007). It appears that the pathways regulating the Na/K-ATPase and NHE3
are numerous and redundant, and CTS-induced coordinated regulation is one of the
pathways that occur in response to conditions that cause an increase in endogenous
CTS.
In summary, our data indicate that the Na/K-ATPase is the functional receptor for ouabain-induced regulation of the Na/K-ATPase and NHE3 (and thus transcellular Na+ transport), as we previously proposed (Liu and Xie 2010). This regulation is not species-specific, but the species-specific α1 ouabain sensitivity may
partially account for the species differences observed in ouabain-induced natriuresis
(Lloyd, Sandberg et al. 1992; Yates and McDougall 1993; Loreaux, Kaul et al. 2008;
Nesher, Dvela et al. 2009). In ouabain-induced trafficking regulation, endocytic recycling of internalized NHE3, but not the α1 subunit, was inhibited by ouabain.
71
Acknowledgments
The authors would like to thank Ms. Carol Woods her excellent help. Portions of this work were supported by National Institutes of Health Grants HL-105649 (to
J.T.), HL-109015 (to Z.X. and J.I.S.) and GM-78565 (to Z.X.).
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78 2.7 Figure Legends
Figure 2-1: Dose-dependent effects of ouabain (Oua) on Na/K-ATPase activity.
The HK-2, LLC-PK1 and AAC-19 cells were grown in 12-well plates with
Transwell membrane support to form monolayer. The Na/K-ATPase activity
(ouabain-sensitive 86Rb+ uptake) was assayed as described in Experimental Methods.
Data were shown as percentage of control, and each point is presented as mean ± S.E. of four sets of independent experiments. Curve fit analysis was performed by
GraphPad software.
Figure 2-2: Ouabain (Oua) inhibits H+-driven 22Na+ uptakes.
The HK-2, LLC-PK1 and AAC-19 cells were grown in 12-well plates to form
a monolayer. After treatment with ouabain (1h) and/or PP2 (1µM for 30min), 22Na+ was added and assayed for H+-driven 22Na+ uptake. To determine H+-driven Na+
+ uptake, cells were first acid loaded in Na -free buffer with 20 mM NH4Cl and then
assayed for 22Na+ uptake. 50 µM amiloride was used to inhibit amiloride-sensitive
NHE1 activity. Data are shown as mean ± S.E., percentage of control. n=4. ** p<0.01 compared to Control.
Figure 2-3: Ouabain (Oua) inhibits transcellular 22Na+ flux.
The HK-2, LLC-PK1 and AAC-19 cells were grown in 12-well plates with
Transwell membrane support to form a monolayer. The cells were treated with
ouabain (1h) and/or PP2 (1µM for 30min) in the basolateral or apical aspect. Active
79 transepithelial 22Na+ flux (apical to basolateral) was determined by counting radioactivity in the basolateral aspect at 1 h after 22Na+ addition. 50 µM amiloride
was added in the basolateral aspect to inhibit amiloride-sensitive NHE1 activity.
Data are shown as mean ± S.E., percentage of control. n=4. ** p<0.01 compared to
Control.
Figure 2-4: Ouabain (Oua) reduces cell surface expression of the α1 and NHE3.
The HK-2, LLC-PK1 and AAC-19 cells were treated with indicated
concentrations of ouabain (1h). Biotinylation of cell surface proteins was performed
to assess cell surface protein contents. Data are shown as mean ± S.E., percentage of control (Con). n=4. ** p<0.01 compared to control. Insert shows a representative western blot of four separate experiments. Immunoblotting with antibody against integrin-β1 served as loading controls (data not shown).
Figure 2-5: Ouabain (Oua) inhibits endocytic recycling of NHE3 in LLC-PK1 cells.
The experiments were performed as described in Experimental Methods. (A)
Determination of GSH-cleavage efficiency. Cell surface proteins were biotinylated and applied with or without GSH cleavage procedure. (B) Retrieval of total internalized biotinylated α1 and NHE3 after treatment with or without ouabain
(100nM, 1h) and GSH cleavage. (C) Assessment of the effect of ouabain on reinsertion. (D) The graph bars represented reinsertion of endocytosed α1 and NHE3,
80 the difference of total endocytosed α1 and NHE3 before and after resinsertion procedure. n=3. ** , p<0.01.
Figure 2-6: Ouabain (Oua) accumulates the Na/K-ATPase α1 subunit, but not
NHE3 in late endosome in LLC-PK1 cells.
LLC-PK1 cells were treated with or without ouabain (100nM for 1h), with or without pretreatment of chloroquine (Chlor, 0.2 mM, pretreated for 2h). Early endosome (EE) and late endosome (LE) fractions were isolated at the end of ouabain treatment. Equal amount of proteins (25 μg) was used to determine protein contents of the α1 and NHE3 by western blot analysis. n=4. ** p<0.01 compared to controls from EE and LE, respectively. # p<0.01, comparison of α1 subunit in LE fraction with or without chloroquine pretreatment. Insert shows a representative western blot of four separate experiments. Immunoblotting with antibodies against EEA-1 (for
EE fractionation) and Rab7 (for LE fractionation) served as loading controls, respectively (data not shown).
81 2.8 Tables and figures
Table 2.1: Ouabain causes a dose-dependent inhibition of transcellular 22Na+ flux, 22Na+ uptake, and surface α1 and NHE3. 22Na+ flux and 22Na+ uptake were measured as described in Experimental Methods. Surface α1 and NHE3 were determined by cell surface biotinylation. N=3 for each treatment. a, p<0.05 compared to controls; b, p<0.01 compared to controls.
HK-2 cells
Ouabain (nM) for 1h
0 1 5 10 50
22Na+ flux 100±4.5 102.1±3.8 81.2±5.2a 68.5±4.8b 61.8±5.9 b
22Na+ uptake 100±5.1 98.4±4.2 83.6±5.6a 76.6±6.2 b 67.8±5.3 b
Surface α1 100±6.1 96.5±6.7 84.2±6.2 a 63.5±7.1 b 59.7±7.4 b
Surface NHE3 100±4.5 99.2±6.1 86.2±6.8 a 67.3±7.1 b 69.1±7.9 b
LLC-PK1 cells
Ouabain (nM) for 1h
0 10 25 100 1,000
22Na+ flux 100±5.2 99.1±4.8 82.4±7.4 a 65.5±6.8 b 60.2±8.6 b
22Na+ uptake 100±5.8 95.7±6.7 88.3±8.1 a 64.8±7.2 b 58.5±7.8 b
Surface α1 100±7.9 97.3±8.1 85.6±7.2 a 65.2±7.7 b 67.6±8.4 b
Surface NHE3 100±6.5 98.6±8.8 83.2±7.4 a 72.7±6.1 b 69.3±7.6 b
82
AAC-19 cells
Ouabain (nM) for 1h
0 100 1,000 10,000 25,000
22Na+ flux 100±6.2 96.3±3.8 86.4±7.3 a 75.5±6.9 b 68.8±7.6 b
22Na+ uptake 100±7.2 97.8±7.1 85.1±8.5 a 71.6±8.1 b 65.2±9.6 b
Surface α1 100±8.2 96.5±7.6 87.4±8.1 a 75.2±7.9 b 67.4±9.1 b
Surface NHE3 100±9.1 94.7±8.7 84.8±9.3 a 69.5±8.8 b 61.8±9.9 b
83 Figure 2-1
84 Figure 2-2
85 Figure 2-3
86 Figure 2-4
87 Figure 2-5
88 Figure 2-6
89 Chapter 3 – Manuscript 2
Title:
Impairment of Na/K-ATPase Signaling in Renal Proximal Tubule Contributes to
Dahl Salt-Sensitive Hypertension
Authors:
Jiang Liu1, Yanling Yang1,3, Lijun Liu2, Zijian Xie2,1, Deepak Malhotra1, Bina Joe1, and
Joseph I. Shapiro1,2
From the Department of Medicine1, Physiology and Pharmacology2, University of Toledo
College of Medicine, Toledo, OH; and Institute of Biomedical Engineering3, Yanshan
University, China.
Running head : Na/K-ATPase signaling in salt sensitivity
Corresponding author:
Jiang Liu, Department of Medicine, University of Toledo College of Medicine, Health
Science Campus, 3000 Arlington Avenue, Toledo, Ohio 43614-2598.
Tel : 419-383-3923;
Email: [email protected]
Published in the Journal of Biological Chemistry. 2011, 286: 22806-22813.
90 3.1 Abstract
We have observed that, in renal proximal tubular cells, cardiotonic steroids such as ouabain in vitro signal through Na/K-ATPase which results in inhibition of transepithelial
22Na+ transport by redistributing Na/K-ATPase and NHE3. In the present study, we
investigate the role of Na/K-ATPase signaling in renal sodium excretion and blood
pressure regulation in vivo.
In Sprague Dawley rats, high salt diet activated c-Src and induced redistribution of
Na/K-ATPase and NHE3 in renal proximal tubules. In Dahl salt sensitive (S) and resistant
(R) rats given high dietary salt, we found different effects on blood pressure but, more
interestingly, different effects on renal salt handling. These differences could be explained by different signaling through the proximal tubular Na/K-ATPase. Specifically, in Dahl R
rats, high salt diet significantly stimulated phosphorylation of c-Src and ERK1/2, reduced
Na/K-ATPase activity and NHE3 activity, and caused redistribution of Na/K-ATPase and
NHE3. In contrast, these adaptations were either much less effective or not seen in the Dahl
S rats. To correct for the differences to high dietary salt, we also studied the primary culture
of renal proximal tubule isolated from Dahl S and R rats fed a low salt diet. In this system,
ouabain induced Na/K-ATPase/c-Src signaling and redistribution of Na/K-ATPase and
NHE3 in the Dahl R rats, but not in the Dahl S rats. Our data suggested that impairment of
Na/K-ATPase signaling and consequent regulation of Na/K-ATPase and NHE3 in renal proximal tubule may contribute to salt induced hypertension in the Dahl S rat.
91 3.2 Introduction
The Dahl salt resistant (R) and sensitive (S) strains were developed by selective
breeding of the outbred Sprague Dawley rat strain for resistance or susceptibility to the
hypertensive effects of high dietary sodium (Dahl, Heine et al. 1962). While the blood pressure (BP) response to salt-loading in Dahl R and S rats involves many regulatory factors (Rapp 1982), it has been proposed that renal proximal tubule (RPT) Na+ handling may be a critical determinant of the different BP responses in these strains (Dahl, Knudsen et al. 1969; Dahl, Heine et al. 1974).
Cardiotonic steroids (CTS) such as ouabain and marinobufagenin (MBG) appear to be involved in the regulation of BP and renal Na+ handling in vivo (Fedorova, Lakatta et al.
2000; Fedorova, Talan et al. 2002; Periyasamy, Liu et al. 2005) as well as the ion handling
of both primary cultures and proximal tubular cell lines in vitro (Blaustein and Hamlyn
1991; Yuan, Manunta et al. 1993; Liu, Periyasamy et al. 2002; Liu, Kesiry et al. 2004;
Periyasamy, Liu et al. 2005; Blaustein and Hamlyn 2010). Based on this background, we
performed the following studies to examine whether the proximal tubules of Dahl R and S
rats had different responses to CTS.
3.3 Materials and Methods
3.3.1 Animals:
Male Sprague Dawley rats (290-310 g body weight) were purchased from Charles
River. Male, age-matched Dahl R (SS/JrHsd) and S (SR/JrHsd) rats were bred and
maintained in-house and used at the age of 12-14 weeks. Rats were maintained with 12hr
92 dark/light cycle and had ad libitum access to the food (Teklad lab animal diets from Harlan
Laboratories) and water. All animal experimentation was conducted in accordance with the
National Institutes of Health Guide for the Care and Use of Laboratory Animals using
protocols approved by the University of Toledo Institutional Animal Use and Care
Committee.
3.3.2 Chemicals and Antibodies:
All chemicals, except otherwise mentioned, were obtained from Sigma-Aldrich (St.
Louis, MO). EZ-Link sulfo-NHS-ss-biotin and ImmunoPure immobilized
streptavidin-agarose beads were from Pierce Biotechnology (Rockford, IL). Antibodies
against Rab7, integrin-β1 and c-Src were from Santa Cruz Biotechnology (Santa Cruz,
CA). Monoclonal antibody against the Na/K-ATPase α1 subunit (clone α6F) was from the
Hybridoma Bank of the University of Iowa (Iowa City, IA). Monoclonal antibodies against
NHE3 (clone 4F5) and early endosome antigen-1 (EEA1) were from Millipore Chemicon
(Temecula, CA). Polyclonal anti-Src [pY418] phosphospecific antibody was from
Invitrogen (Camarillo, CA). Antibodies against phosphor-p44/42 ERK1/2 (Thr202/Tyr204)
and p44/42 were from Cell Signaling (Danvers, MA). Type 2 collagenase (activity of
323U/mg) was from Worthington Biochemical Corp (Lakewood, NJ). Sterile Percoll was
from GE Healthcare (Piscataway, NJ). Biomol Green was from BIOMOL Research
Laboratories (Plymouth Meeting, PA). Radioactive 22Na+ was from DuPont NEN Life
Science (Boston, MA).
3.3.3 Experimental groups and treatments:
93 For salt-loading studies in Sprague Dawley rats, the rats were randomly divided into two groups (10 rats per group) after adjustment to the new environment. The rats were then fed with 0.3% NaCl (as low salt diet) or 4% NaCl chow for 7 days. For
salt-loading studies in Dahl rats, age-matched, male R and S rats (total 48 rats) were given
either 0.3% NaCl (low salt diet, n=12 per strain) or 2% NaCl (n=12 per strain) diet for 7
days. Only 2% NaCl loading was used with the Dahl rats because of the profound effects
on BP and food consumption (inferred from urinary Na+ excretion data, see Table 2)
salt-loading has on the S animals.
3.3.4 Tail-cuff measurement of BP:
At day 0 and 7, BP was measured in conscious rats by the tail-cuff plethysmography with the aid of a computerized system (Amplifier model 229, Monitor model 31, Test
chamber Model 306; IITC Life Science). All rats were first trained for BP measurement
over a period of 2 days before day 0 in order to accustom them to the apparatus and restraint (Kennedy, Vetteth et al. 2006). After that, the actual BP (average of at least three
measurements) was recorded.
3.3.5 Isolation and primary culture of RPTs:
RPTs were isolated from the outer cortices as described by Vinay et al (Vinay,
Gougoux et al. 1981) with minor modifications. Briefly, harvested kidneys were
decapsulated and rinsed with ice-cold oxygenized PBS. Outer cortices were dissected,
minced, and digested in digesting solution (oxygenized DMEM with 1mg/ml collagenase
Type 2, 0.5% filtered BSA Fraction V) four times at 37 ºC, 15min each. Pooled tubular
94 segments were further separated with 42% Percoll gradient (pH7.4) and the RPT segments
were collected from the lowest two bands. The enrichment and purity of RPT isolation was
determined by measuring RPT brush-border membrane vehicles (BBMVs) marker enzyme
alkaline phosphatase activity (~10 fold increases) and by microscopic examination.
For in vitro primary culture, RPTs were isolated from R and S rats fed with low salt
diet. Isolated RPTs were cultured in collagen I-coated culture dishes in DMEM/F-12
medium (1:1 mixture, with 10% FBS and Insulin-transferrin-sodium selenite media
supplement, Sigma) until 90-100% confluency in a 5% CO2-humidified incubator. Before
ouabain treatment, cells were serum-starved (cultured in medium with 0.5% FBS) for
16-18 h.
3.3.6 Cell fractionation and biochemical studies:
Early endosome (EE, EEA-1- and Rab5-pisitive) and late endosome (LE,
Rab-7-positive) fractions were isolated by sucrose flotation centrifugation as previously
reported (Periyasamy, Liu et al. 2005; Cai 2008). The enrichment of EE and LE fractions
was assessed by the EE marker EEA1 and LE marker Rab7, respectively; Equal amounts of
total proteins from EE or LE fraction of each sample were precipitated with trichloroacetic
acid for subsequent Western blot. Surface biotinylation studies were conducted as
previously described (Liu, Kesiry et al. 2004; Cai 2008). Crude membrane isolation and
Na/K-ATPase enzymatic activity determinations (each performed in triplicate) were performed as previously described (Liu, Periyasamy et al. 2002).
3.3.7 RPT BBMVs Preparation and NHE3 activity assay (22Na+ uptake):
95 RPT BBMVs were prepared from isolated RPTs using the protocol described by
Murer’s Laboratory (Biber, Stieger et al. 2007). After homogenization, BBMVs were
2+ isolated with Mg precipitation method (final concentration of MgCl2 is 12 mM, 15 min
on ice) and differential centrifugation. Alkaline phosphotase activity was enriched ~10 fold
by this procedure. NHE3 activity (H+-driven 22Na+ uptake) in RPT BBMVs was
determined in triplicate using the filtration protocol described by Moe’s Laboratory (Bacic,
Kaissling et al. 2003).
3.3.8 Measurement of c-Src phosphorylation:
RPT whole cell lysates were prepared with Nonidet P-40 buffer containing 1%
Nonidet P-40, 0.25% sodium deoxycholate, 50 mM NaCl, 50 mM HEPES, 10% Glycerol
(pH 7.4), 1 mM sodium vanadate, 0.5 mM sodium fluoride, 1 mM Phenylmethanesulfonyl fluoride, and protease inhibitor cocktail for general use (Sigma). After clarification, 300μg of total protein was immunoprecipitated with antibody against c-Src and protein G-agrose beads (Millipore), and then eluted with 2x Laemmli buffer. After immunoblotting for phospho-c-Src (p-Src), the same membrane was stripped and immunoblotted for total c-Src (t-Src). The activation of c-Src was expressed as ratio of p-Src/ t-Src with both measurements normalized to 1.0 for the control samples.
3.3.9 Western blotting:
For Western blot analysis, equal amounts of total protein were resolved by 10%
SDS-PAGE and immunoblotted with antibodies against interested proteins. The same membrane was also immunoblotted with antibodies against integrin-β1 (for surface
96 biotinylation) or EEA-1 (for EE fractionation) to serve as loading controls, respectively.
Signal detection was performed with an enhanced chemiluminescence super signal kit
(Pierce, Rockford, IL). Multiple exposures were analyzed to assure that the signals were
within the linear range of the film. The signal density was determined using Molecular
Analyst software (Bio-Rad, Hercules, CA).
3.3.10 Statistical analysis:
Data were tested for normality (all data passed) and then subjected to parametric
analysis. When more than two groups were compared, one-way ANOVA was performed
prior to comparison of individual groups, and the post-hoc t-tests were adjusted for
multiple comparisons using Bonferroni’s correction. Statistical significance was reported
at the P < 0.05 and P < 0.01 levels. SPSS software was used for all analysis. Values are given as mean±SEM (Wallenstein, Zucker et al. 1980).
3.4 Results
3.4.1 Effect of high salt diet on RPT Na/K-ATPase and NHE3 in Sprague Dawley
rats:
As shown in Table 1, the high salt diet significantly increased absolute urinary Na+ excretion compared with control rats. The high salt diet also had a small effect on systolic
BP. The high salt diet reduced the Na/K-ATPase activity in RPT crude plasma membrane fractions and NHE3 activity in RPT BBMVs (Table 1). In addition, the high salt diet not only decreased RPT surface contents of both transporters (Fig. 3-1a), but also caused both
97 transporters in RPTs to accumulate in the EE fraction (Fig. 3-1b). The inhibition of the
Na/k-ATPase activity and NHE3 activity was well correlated with reduced protein content
of the α1 subunit and NHE3 on the cell surface. Furthermore, the high salt diet stimulated
c-Src phosphorylation in RPT whole cell lysates (Fig.3-1c).
RPT primary cultures isolated from control rats fed with low salt diet were treated
with ouabain or vehicle. Ouabain treatment (10 µmol/L for 1 h) accumulated the α1 subunit
and NHE3 in the EE fractions, reduced the surface protein content of these two ion
transporters (Fig. 3-2a), and caused an inhibition of both Na/K-ATPase activity (in
µmol/mg protein/hour, control 58.9±3.6 vs. ouabain 48.1±3.1, n=4, p<0.01 compared to
control) and NHE3 activities by measuring H+-driven 22Na+ uptake (in relative value,
control 100±4.5 vs. ouabain-treated 76.5±6.8, n=4, p<0.01 compared to control). Moreover,
ouabain (10 or 25 µmol/L for 5 min) stimulated c-Src phosphorylation (Fig. 3-2b).
3.4.2 Effect of high salt diet on RPT Na/K-ATPase and NHE3 from Dahl R and S
rats:
Male age-matched Dahl R and S rats were given a low salt (0.3% NaCl, n=12 per
strain) or high salt (2% NaCl, n=12 per strain) diet for 7 days. As briefly mentioned earlier,
we used only a 2% high salt diet as the Dahl S (but not R) rats showed profound increases
in blood pressure as well as very substantial decreases in food consumed as evidenced by
the relative decrease in UNa+V compared with the R rats (Table 2). There were no
significant differences amongst the groups in the plasma concentrations of Na+ and K+ at the end of the experiments. These and additional data are summarized in Table 2.
98 In the isolated RPTs from Dahl R and S rats fed a low salt diet, there were no differences in the expression of Na/K-ATPase α1 subunit and NHE3 or Na/K-ATPase and
NHE3 activities. High salt diet has no significant effect on total expression of
Na/K-ATPase α1 and NHE3 in isolated RPTs from the R and S rats (data not shown).
However, the high salt diet significantly decreased both Na/K-ATPase and NHE3 activities in the R but not the S rats (Fig.3-3).
In the isolated RPTs from the R rats, the high salt diet not only decreased the RPT surface content of both Na/K-ATPase α1 and NHE3 (Fig.3-4a) but also caused the accumulation of both transporters in the EE fraction (Fig.3-4b). Interestingly, the high salt diet did not cause redistribution of the α1 subunit and NHE3 in the S rat RPTs (Fig. 3-4a and 4b). While the high salt diet caused the α1 subunit to also accumulate in the LE fractions in the R rats (relative to control 100±3.5, high salt diet 246.5±7.6, n=3, p<0.01), the NHE3 did not accumulate in the LE fraction (Fig.3-4c). This suggests that the NHE3 which moves to the EE fraction remains available for recycling, but in any case doesn’t accumulate in the LE fraction.
In the RPTs isolated from the R but not the S rats, high salt diet stimulated activation of c-Src (Fig. 3-5a) and ERK1/2 (Fig. 3-5b). In the same immunoprecipitation studies shown in Fig. 3-5a, high salt diet also significantly enhanced association between c-Src and
Na/K-ATPase α1 (relative to the low salt control diet 100±3.7, high salt diet 184.6±9.3, n=3, p<0.01) in the R rats, but not in the S rats.
3.4.3 Effects of Ouabain on RPT primary cultures derived from Dahl R and S rats:
99 RPTs were isolated from age-matched male R and S rats (20 R rats and 20 S rats) fed
with a low salt diet (0.3% NaCl), and cultured to reach confluence. As observed in
salt-loading experiment with intact animals, ouabain treatment also caused differential
regulations in the RPT primary culture derived from the R and S rats. While ouabain
demonstrated no regulatory effect in the RPTs derived from the S rats, ouabain had
dramatic consequences in the RPTs derived from the R rats. Specifically, ouabain treatment (10 µmol/L for 1 h) caused the accumulation of the α1 subunit and NHE3 in the
EE fractions (Fig. 3-6a); reduced the surface protein content of these two ion transporters
(Fig. 3-6b); and inhibited both the membrane Na/K-ATPase and NHE3 activities in RPT
crude membrane fraction and BBMVs, respectively (Table 3). Moreover, ouabain
treatment (1 and 10 µmol/L for 5 min) stimulated phosphorylation of c-Src and ERK1/2
(Fig. 3-7a and 7b). In the same immunoprecipitation studies shown in Fig. 3-7a, ouabain
also enhanced the association between c-Src and Na/K-ATPase α1 (in values relative to
control 100±4.2, ouabain 1 µmol/L 167.6±8.2 and ouabain 10 µmol/L 192.8±6.3, n=4,
both p<0.01 compared to control) in the R rats. No significant association between c-Src
and Na/K-ATPase α1 was observed in the S rats in response to either dose of ouabain.
3.5 Discussion
The significance of endogenous CTS functioning as natriuretic hormones by
inhibiting the renal Na/K-ATPase has been a subject of debate for some time (Bagrov,
Shapiro et al. 2009). While volume expansion is considered to be the main cause of the
100 salt-sensitive hypertension in the S rats (Rapp 1982; Roman and Osborn 1987), accumulating evidence, some from our laboratories, suggest that endogenous CTS regulate renal Na+ handling and BP through the Na/K-ATPase signaling (Fedorova, Lakatta et al.
2000; Fedorova, Talan et al. 2002; Dostanic, Paul et al. 2005; Zhang, Lee et al. 2005;
Loreaux, Kaul et al. 2008; Nesher, Dvela et al. 2009; Fedorova OV 2010; Liu and Xie
2010). A role for endogenous CTS in salt sensitive hypertension has also been suggested by a number of authors (Schoner and Scheiner-Bobis 2008; Blaustein and Hamlyn 2010;
Jaitovich and Bertorello 2010; Ritz 2010). On this background, the Dahl R and S rats have been extensively studied for insight into the pathogenesis of salt sensitive hypertension.
Fedorova and coworkers have established that increases in circulating MBG are, in fact, greater during acute and chronic salt-loading in Dahl S as compared with R rats (Fedorova,
Lakatta et al. 2000; Fedorova, Talan et al. 2002).
In the current study, we observed that the outbred Sprague Dawley rats developed redistribution of the Na/K-ATPase in RPTs in response to a shift to a high salt diet. As we have previously observed (Periyasamy, Liu et al. 2005), this redistribution of the
Na/K-ATPase appears to depend on increases in the production of the CTS, MBG. In addition to what we had previously seen, we also noted what appeared to be a coordinated redistribution of the apical NHE3 into the EE but not LE fraction, a phenomenon which we have documented to be a consequence of Na/K-ATPase-c-Src signaling in LLC-PK1 cells
(Cai 2008). Interestingly, works from the laboratory of McDonough using immunofluorescence have shown that a high salt diet causes the redistribution of NHE3 to a deeper portion of microvilli whereas this high salt diet causes the sodium-phosphate
101 cotransporter to move completely into cells (Yang, Meng et al. 2008). We believe that our
data with the NHE3 are consistent with this report.
We further demonstrated that CTS signaling also appeared to occur in a similar fashion in Dahl R but not S rats. Specifically, we saw that high salt diet induced redistribution of the Na/K-ATPase and NHE3 along with evidence for c-Src and ERK1/2
activation in RPTs isolated from the Dahl R but not S rats. Impaired CTS signaling in the
RPTs of the S rats was further implicated by the failure of these RPTs isolated from the S
rats on a low salt diet to show ouabain signaling in vitro, which was readily apparent in
parallel studies performed in the RPTs isolated from the R rats. As the S rats developed
profound hypertension when dietary Na+ is increased to 2% whereas the R rat
demonstrated little change in BP with this dietary manipulation, these data implicated the
CTS-Na/K-ATPase-c-Src signal cascade in the control of BP in these animals.
Furthermore, since the S rats develop greater increases in the production of as well as
circulating levels of CTS following increases in dietary Na+ (Fedorova, Lakatta et al. 2000;
Fedorova, Talan et al. 2002), diminished sensitivity of the signaling cascade to CTS would
appear to be involved. However, as the α1 subunit sequence of the Na/K-ATPase does not
differ between Dahl S and R rats (Mokry and Cuppen 2008), one must implicate the
environment of the signaling Na/K-ATPase and/or the interactions between the
Na/K-ATPase and its signaling partners (Pierre and Xie 2006).
We believe that these data are interesting for several reasons. First, they implicate
CTS in the pathogenesis of salt sensitive hypertension by a renal mechanism, namely the
failure of the Dahl S to demonstrate the normal response to CTS seen with the Dahl R and
the outbred Sprague Dawley rats. This may give pause to implementing the therapeutic
102 strategy of antagonizing CTS-Na/K-ATPase signaling in vascular tissue which has been proposed by pioneering workers in this field (Yuan, Manunta et al. 1993; Blaustein and
Hamlyn 2010). Second, these data suggest that CTS may induce increases in renal sodium excretion by multiple mechanisms. Specifically, these agents may increase vascular resistance as others have demonstrated (Bagrov and Fedorova 2005; Fedorova, Kolodkin et al. 2005; Kashkin, Zvartau et al. 2007) while also working through the renal mechanism
discussed above. Although other hormonal systems no doubt interact with both of these
pathways (Fedorova, Agalakova et al. 2006), it does appear that the former mechanism is
much more intact in the Dahl S rats suggesting that the molecular mechanisms of CTS
signaling might be different in these different tissues. Whether this is explained by
Na/K-ATPase isoform differences, differences in specific CTS and/or mechanistic
differences in the way that CTS signal in vascular as compared to renal tissues is still quite
unclear. On this latter point, we might speculate that the classic ionic signal mechanism
involving actual pump inhibition might dominate in vascular tissue as proposed by
Blaustein and Hamlyn (Blaustein and Hamlyn 2010) whereas the CTS-c-Src signaling
might be more important in the RPTs. Of course, further work must be done to address this
complex area. Last, these data might possibly provide the basis for developing a tool for
clinical practice. If the sensitivity of the Na/K-ATPase-c-Src signaling pathway to CTS is
something that is impaired either on a genetic or acquired basis, it might be possible to use
this phenomenon to develop a biomarker for salt sensitive hypertension.
103 Acknowledgments:
Portions of this work were supported by grants from National Institutes of Health
(GM78565, HL36573, HL020176 and HL076709) and American Heart Association Ohio
Valley Affiliate.
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108 3.7 Figure Legends
Figure 3-1: In Sprague Dawley rats, 7-day high salt diet (HS) activated c-Src and stimulated redistribution of RPT Na/K-ATPase α1 subunit and NHE3.
HS reduced RPT cell surface contents of the α1 subunit and NHE3, n=5 (a), accumulated the α1 subunit and NHE3 in RPT EE fractions, n=5 (b), and stimulated c-Src phosphorylation in RPT whole cell lysates, n=4 (c). ** p<0.01 compared to control. RPT isolation, surface biotinylation and EE fraction isolation from control and treated rats were conducted in pairs concurrently. Immunoblotting of integrin-β1 and EEA-1 was served as loading control for surface biotinylation and EE fraction, respectively.
Figure 3-2:
In RPT primary culture derived from Sprague Dawley rats, ouabain (Oua) not only redistributed the Na/K-ATPase α1 subunit and NHE3, n=5 (a), but it also stimulated c-Src phosphorylation within 5 minute in RPTs, n=4 (b). ** p<0.01 compared to control. The experiments in (a) were performed as in Figure 2-1, with integrin-β1 and EEA-1 as loading control.
Figure 3-3:
In the R rats, high salt diet (HS) inhibited both the Na/K-ATPase activity (a) and
NHE3 activity (b). Crude heavy plasma membrane fractions and RPT BBMVs were prepared from isolated RPTs at the end of experiments. The Na/K-ATPase enzymatic
109 activity (µmol/mg protein/hour) and relative NHE3 activity were calibrated by protein
content. n=6. ** p<0.01 compared to control.
Figure 3-4: In Dahl rats, high salt diet (HS) redistributed the Na/K-ATPase α1 and
NHE3 in RPTs isolated from the R rats, but not in the S rats.
Male, age-matched Dahl R and S rats were paired for salt-loading. Cell surface α1
subunit and NHE3 were determined by surface biotinylation (a) with integrin-β1 blotting
as loading control, and internalized α1 subunit and NHE3 were determined by the protein
content in EE fractions (b) with EEA-1 blotting as loading control. n=6. ** p<0.01
compared to control. In LE fractions, high salt diet accumulated α1 subunit only in the R
rats, but NHE3 protein content was not significantly affected by high salt diet in both R and
S rats (c). Rab7 blotting was served as loading control.
Figure 3-5: High salt diet (HS) activated c-Src and ERK1/2 and enhanced association
between c-Src and the α1 subunit in RPTs of the R rats, but not the S rats.
(a) HS stimulated c-Src phosphorylation and its association with the α1 subunit. (b)
HS activated ERK1/2. Values are expressed relative to a control value of 100. n=4. **, p
＜0.01 compared to control.
Figure 3-6: Ouabain (Oua, 10µM for 1 hour) redistributed the Na/K-ATPase α1
subunit and NHE3 in RPT primary culture of the R rats, but not the S rats.
RPTs were isolated from the R and S rats fed with low salt (0.3% NaCl) diet. Cell surface NHE3 and α1 subunit were determined by surface biotinylation (a), and
110 internalized NHE3 and α1 subunit were determined by the protein content in EE fractions
(b). n=4. ** p<0.01 compared to control. Immunoblotting of integrin-β1 and EEA-1 was served as loading control for surface biotinylation and EE fraction, respectively.
Figure 3-7: Ouabain (Oua, 1 and 10µM for 5 min) activated c-Src and ERK1/2 and enhanced association between c-Src and the α1 subunit in RPTs of the R rats, but not the S rats.
(a) ouabain stimulated c-Src phosphorylation and its association with the α1 subunit.
(b) ouabain activated ERK1/2. Values are expressed relative to a control value of 100. n=4.
**, p＜0.01.
111 3.8 Tables and figures
Table 3.1: The effect of high salt diet (4% NaCl for 7 days) on renal sodium excretion
of Sprague Dawley rats.
Absolute Na+ excretion (UNa+V) was calculated as urinary Na+ concentration x
24-h urine volume. The Na/K-ATPase activity was measured from isolated
RPTs and expressed as µmol/mg protein/hour from four independent
experiments (each performed in triplicate). NHE3 activity (22Na+ uptake) in
BBMVs was measured from isolated RPTs and expressed as relative values (%,
control was normalized as 100%) from four independent experiments (each
performed in triplicate). ** p<0.01 compared to control. n=rat number (for
UNa+V and BP) or experiment number (for Na/K-ATPase and NHE3 activity).
UNa+V (mEq/24h) BP (mmHg) Na/K-ATPase activity NHE3 activity (%)
Control 2.7 ± 0.4 (n=10) 102.6±2.5 (n=10) 56.4±4.2 (n=4) 100±4.8 (n=4)
High salt diet 18.1 ± 1.7(n=10)** 115.3±3.2(n=10)** 40.2±6.8 (n=4) ** 67.6±10.5 (n=4) **
112 Table 3.2: High salt diet (HS, 2% NaCl, 7 days) caused differential regulation
between the R and S rats. Absolute Na+ excretion (UNa+V) was calculated as
Urine Na+ x 24-h urine volume. Fractional Na+ excretion (FENa+) was
calculated as 100 x (Urine Na+ x Plasma creatinine)/(Plasma Na+ x Urine
creatinine). Creatinine clearance was calculated as (Urine creatinine x 24-h
urine volume)/(Plasma creatinine x 24 x 60min). ** p<0.01 compared to
control LS diet of the same strain. # p<0.01 the R rats with HS compared to the
S rats with HS. n=rat number, per treatment, per group.
Strain LS HS
BP R 133±2.2 (n=12) 134±2.6 # (n=12)
S 146±3.1 (n=12) 169±5.6** (n=12)
Plasma Na+ R 145±2.8 (n=12) 146±1.4 (n=12)
S 146±2.1 (n=12) 147±3.6 (n=12)
Plasma K+ R 8.7±1.8 (n=12) 8.6±1.3 (n=12)
S 8.8±1.4 (n=12) 8.7±1.5 (n=12)
UNa+V (mEq/24h) R 2.9±0.6(n=8) 20.1±1.6**,#(n=8)
S 2.7±0.25(n=8) 6.5±1.0**(n=8)
FENa+ R 0.073±0.008(n=8) 0.287±0.064**,#(n=8)
S 0.071±0.004(n=8) 0.132±0.021**(n=8)
Creatinine clearance R 1.99±0.14(n=8) 2.66±0.38(n=8)
(ml/min) S 2.06±0.32(n=8) 2.67±0.64(n=8)
113 Table 3.3: The effect of ouabain on the Na/K-ATPase activity and NHE3 activity in
RPT primary cultures derived from the R and S rats. The Na/K-ATPase
activity in crude heavy membrane fractions was expressed as µmol/mg
protein/hour from four independent experiments (each performed in triplicate).
NHE3 activity (22Na+ uptake) in BBMVs was expressed as relative values (%
of control as 100%) from four independent experiments (each performed in
triplicate). ** p<0.01 compared to control.
Strain Control Ouabain (10µM, 1h)
Na/K-ATPase activity R 61.2±3.6 (n=4) 46.8±3.5** (n=4)
S 60.8±4.1 (n=4) 57.9±4.5 (n=4)
NHE3 activity (%) R 100±5.2 (n=4) 71.2±5.5 **(n=4)
S 100±6.1 (n=4) 96.4±6.5 (n=4)
114 Figure 3-1
a. b.
c.
115 Figure 3-2 a.
b.
116 Figure 3-3 a.
75 LS HS
50 **
25 Enzymatyic activity (uMol/mg protein/hour) (uMol/mg
0 R rats S rats
b.
125 LS HS
100 uptake, %) + 75 ** Na 22
50
25 NHE3 activity ( 0 R rats S rats
117 Figure 3-4 a. b.
c.
118 Figur 3-5 a.
b.
119 Figure 3-6 a.
b.
120 Figure 3-7 a.
b.
121 Chapter 4 – Manuscript 3
Title:
Redox Modulation of the Na/K-ATPase Signaling and Renal Proximal Tubular
Sodium Handling
Authors:
Yanling Yan1,3, Vinai Katragadda1, Steven Haller1, Anna P. Shapiro1, Joe Xie1, Chiamaka
Mbaso1, Deepak Malhotra1, Zi-jian Xie2,1, Joseph I. Shapiro1,2, and Jiang Liu1
1Departments of Medicine and Physiology, University of Toledo College of
Medicine, Toledo, OH; and 3Institute of Biomedical Engineering, Yanshan
University, China.
In preparation.
122 4.1 Abstract
We have shown that cardiotonic steroids (CTS) signaling through the Na/K-
ATPase regulate renal proximal tubule (RPT) sodium reabsorption and impairment of the Na/K-ATPase signaling contributes to experimental Dahl salt-sensitive hypertension. Here we report that reactive oxygen species (ROS) is critical in modulation of ouabain-induced Na/K-ATPase signaling and RPT ion transport.
Antioxidant NAC (N-Acetyl-L-cysteine) prevented ouabain-stimulated Na/K-
ATPase signaling cascade, protein carbonylation, redistribution of the Na/K-
ATPase and NHE3, and inhibition of transepithelial 22Na+ transport. However, a basal physiological range of ROS is necessary for initiation and amplification of ouabain-Na/K-ATPase signaling and redistribution of Na/K-ATPase and NHE3.
Furthermore, both ouabain and glucose oxidase caused direct carbonylation of two amino acid residues in the actuator (A) domain of the Na/K-ATPase α1 subunit.
123 4.2 Introduction
Excessive dietary salt intake significantly contributes to the development of hypertension and tends to be more pronounced in typical salt-sensitive patients
(Calhoun, Jones et al. 2008). Renal proximal tubule (RPT) sodium handling, which
claims over 60% reabsorption of filtered sodium, is an independent determinant in
the pathogenesis of salt-sensitive hypertension. Accumulating data indicate that
CTS (also known as endogenous digitalis-like substances) signaling through the
Na/K-ATPase regulates RPT sodium handling and salt sensitivity (reviewed in
(Schoner and Scheiner-Bobis 2008; Fedorova, Shapiro et al. 2010; Liu and Xie
2010)).
It is well documented that a high salt diet stimulates an increase in
endogenous CTS release and ROS generation. Our recent observations indicate that
RPT sodium handling via ligand-mediated Na/K-ATPase/c-Src signaling
counterbalances the sodium retention mediated increases in blood pressure, such as
that seen in salt-sensitive hypertension (Liu, Kesiry et al. 2004; Liu, Liang et al.
2005; Periyasamy, Liu et al. 2005; Oweis, Wu et al. 2006; Cai, Wu et al. 2008; Liu,
Yan et al. 2011). Ouabain, a ligand of the Na/K-ATPase, activates the Na/K-
ATPase/c-Src signaling pathway and subsequently redistributes basolateral Na/K-
ATPase and apical sodium/hydrogen exchanger isoform 3 (NHE3) in RPTs, leading
to reduced RPT sodium reabsorption and increased urinary sodium excretion. We
have also demonstrated that impairment of the RPT Na/K-ATPase/c-Src signaling
contributes to experimental Dahl salt-sensitive hypertension (Liu, Yan et al. 2011).
Since it failed to confirm the possible difference of the Na/K-ATPase α1 gene
124 (Atp1a1) coding (Mokry and Cuppen 2008) and ouabain sensitivity of the Na/K-
ATPase (Nishi, Bertorello et al. 1993) between the Dahl salt-resistant (R) and salt-
sensitive (S) rats (Jr strains), other factor(s) must be present to prevent activation of
the Na/K-ATPase/c-Src signaling in the S rats. Normal physiological ROS signaling
is critical in systematic and cellular functions, and excessive oxidative stress
contributes to the development and maintenance of hypertension in animal models in which, the RPT sodium and fluids reabsorption through the Na/K-ATPase and
NHE3 activities are dysregulated (Zhang, Imam et al. 2002; Panico, Luo et al. 2009;
Banday and Lokhandwala 2011). Amongst ROS-induces many kinds of post-
translational modifications, protein direct carbonylation, which are mostly
happened in lysine, arginine, threonine, and proline residues, is widely used as a
biomarker of oxidative stress (Dalle-Donne, Rossi et al. 2003; Stadtman and Levine
2003). We present here that ouabain-stimulated Na/K-ATPase signaling and
subsequent redistribution are redox-sensitive, and ouabain induces direct
carbonylation of proline/threonine residues in the actuator (A) domain of the Na/K-
ATPase α1 subunit. The data indicate that a proper redox modulation of RPT Na/K-
ATPase signaling is critical in renal sodium handling and might play an important
role in counteracting volume expansion mediated high blood pressure.
4.3 Materials and Methods
4.3.1 Chemicals and Antibodies
125 All chemicals, except otherwise mentioned, were obtained from Sigma-
Aldrich (St. Louis, MO). EZ-Link sulfo-NHS-ss-biotin and ImmunoPure immobilized streptavidin-agarose beads were obtained from Pierce Biotechnology
(Rockford, IL). Monoclonal antibody against the Na/K-ATPase α1 subunit (clone
α6F) was from the Developmental Studies Hybridoma Bank at the University of
Iowa (Iowa City, IA). Monoclonal antibody against early endosome antigen-1
(EEA1) and OxyBlot Protein Oxidation Detection Kit were from Millipore
Chemicon (Temecula, CA). Polyclonal anti-Src [pY418] phosphospecific antibody was from Invitrogen (Camarillo, CA). Antibodies against phosphor-p44/42 ERK1/2
(Thr202/Tyr204) and p44/42 were from Cell Signaling Technology (Danvers, MA).
DNPH (2,4-Dinitrophenylhydrazine) and antibody against DNP was from Sigma.
DMPO (5,5-Dimethyl-1-Pyrroline-N-Oxide) and polyclonal anti-DMPO antiserum were from Cayman Chemicals (Ann Arbor, MI). Radioactive 22Na+ was from
DuPont NEN Life Science (Boston, MA).
4.3.2 Cell cultures
Porcine renal proximal tubule LLC-PK1 cells, mice SYF and SYF+c-Src cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured with DMEM (Dulbecco's modified Eagle's medium with 10% FBS,
100 U/ml penicillin, and 100 µg/ml streptomycin) in a 5% CO2-humidified incubator. Culture medium was changed daily until confluence. LLC-PK1 cells were serum-starved for 16-18 h before treatment. In assays for active transcellular
22Na+ flux, cells were grown on Transwell membrane support to form monolayer,
126 and then treated with ouabain either in basolateral or apical compartment. For SYF
and SYF+c-Src cells, medium was changed daily until the cells reached 80-90%
confluence, at which time the medium was changed to DMEM with 1% FBS for at
least 12 hours before experiments.
4.3.3 Isolation of early endosome (EE) fraction
EE (EEA-1- and Rab5-pisitive) fraction was isolated based on a flotation
gradient using the technique of Gorvel (Gorvel, Chavrier et al. 1991) and identified
as we previously described (Liu, Kesiry et al. 2004; Cai, Wu et al. 2008). The enrichment of EE fraction was assessed by the EE marker EEA-1. Equal amounts of
total proteins from EE fraction of each sample were precipitated with trichloroacetic
acid for subsequent Western blot.
4.3.4 Active transepithelial 22Na+ flux assa
LLC-PK1 cells were cultured on Transwell membrane support (Costar
Transwell culture filter inserts, filter pore size: 0.4µM, Costar, Cambridge, MA ) to
form monolayers. Active transepithelial 22Na+ flux (from apical to basolateral aspect)
was determined by counting radioactivity in the basolateral aspect at 1 hour after
addition 22Na+ as we previously described (Cai, Wu et al. 2008). Cells were
pretreated with 50 µM amiloride to inhibit amiloride-sensitive NHE1 activity.
4.3.5 Measurement of c-Src phosphorylation
127 Whole cell lysates were prepared with Nonidet P-40 buffer containing 1%
Nonidet P-40, 0.25% sodium deoxycholate, 50 mM NaCl, 50 mM HEPES, 10%
Glycerol (pH 7.4), 1 mM sodium vanadate, 0.5 mM sodium fluoride, 1 mM
Phenylmethanesulfonyl fluoride, and protease inhibitor cocktail for general use
(Sigma). After clarification, 300μg of total protein was immunoprecipitated with
antibody against c-Src and protein G-agrose beads (Millipore), and then eluted with
2x Laemmli buffer. After immunoblotting for phospho-c-Src (p-Src), the same
membrane was stripped and immunoblotted for total c-Src (t-Src). The activation
(phosphorylation) of c-Src was expressed as ratio of p-Src/ t-Src with both
measurements normalized to 1.0 for the control samples (Liu, Yan et al. 2011).
4.3.6 Dihydroethidium staining
Intracellular superoxide levels are determined qualitatively by
- dihydroethidium (DHE) fluorescence, a specific probe for intracellular O2 production (Beswick, Dorrance et al. 2001; Elmarakby, Loomis et al. 2005;
Robinson, Janes et al. 2008; Cossarizza, Ferraresi et al. 2009). DHE is a lipophilic cell-permeable dye that is rapidly oxidized to fluorescent product ethidium in the presence of free radical superoxide. Produced ethidium tends to intercalate into nuclear DNA and fluoresces strongly at around 600 nm when excited at 500–530 nm, giving an indication of oxidant stress. Cells were loaded with 5 μM dihydroethidium (DHE) for 20 min. After washing 2 times in phosphate- buffered saline (PBS), cells were scanned for DHE red fluorescence using an Olympus
128 FSX100 box type fluorescence imaging device (Olympus America, Center Valley,
PA). The DHE excitation wavelength is 488nm and emission wavelength is 585nm.
4.3.7 Assessment of formation of DMPO-nitrone adducts (Spin-trap
immunoassay or Immuno-spin trapping)
The nitrone spin trap 5,5-dimethyl-1-5 pyrroline N-oxide (DMPO) is widely used
to measure protein-centered radicals (Ramirez 2005). In theory, protein-centered radicals might be trapped with the nitrone spin trap DMPO forming DMPO-radical adducts, which can be detected by using an antibody. LLC-PK1 cells were pretreated with DMPO
(50mM) for 1 h and then with ouabain or glucose oxidase for 1h at concentration indicated. Whole cell lysates were prepared with Nonidet P-40 buffer as aforementioned, separated by SDS-PAGE, and immunoblotted with anti-DMPO antibody.
4.3.8 Assessment of protein carbonylation
Whole cell lysates were prepared as aforementioned. Equal amount of proteins from each sample was denatured with 6% SDS (final concentration), derivatized with 1x DNPH (diluted freshly with distilled water from 10x DNPH stock solution, 100mM in 100% Trifluoroacetic acid), and neutralized with neutralization Buffer (30% of Glycerol in 2M Tris). This was followed by either
Western blot for protein carbonylation in whole cell lysates or immunoprecipitation
(IP-DNP) studies. For IP-DNP, neutralized DNP-derivatives were pull-down with anti-DNP antibody (Sigma) and protein G-agrose beads, and then eluted with 2x
129 Laemmli buffer. Elutes were immunoblotted with antibodies against Na/K-ATPase
α1 subunit, NHE3 and c-Src.
4.3.9 Protein Identification by LC-MS/MS
Protein identification was performed in Mass Spectrometry-based Proteomics
Facility (Department of Pathology, University of Michigan), based on previously
described protocols (Todi, Scaglione et al.). LLC-PK1 cells were treated with
ouabain (100nM) or GO (3mU/ml) for 1 hr. The Na/K-ATPase α1 subunit was
immunoprecipitated with anti-α1 antibody and separated with SDS-PAGE.
Coomassie Brilliant Blue staining was performed using mass spectrometry-
compatible NOVEX Coomassie Blue colloidal staining (Invitrogen) as instructed by
the manufacturer. The α1 bands were exercised and processed for in-gel trypsin
digestion with sequencing grade modified trypsin (Promega). Resulting peptides
were resolved on a nano-capillary reverse phase column (Picofrit column, New
Objective) and directly introduced into a linear ion-trap mass spectrometer (LTQ
Orbitrap XL, Thermo Fisher). Data-dependent MS/MS spectra on the five most
intense ions from each full MS scan were collected (relative Collision Energy
~35%). Proteins were identified by searching the data against database appended
with decoy (reverse) sequences using the X!Tandem/Trans-Proteomic Pipeline
(TPP) software suite. All peptides and proteins with a PeptideProphet and
ProteinProphet probability score of >0.85 (false discovery rate <3%) were
considered positive identifications of direct protein carbonylation manually verified.
130 4.3.8 Western blotting
Equal amounts of total protein were resolved by 10% sodium dodecylsulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF
membrane. For protein carbonylation assay, the membrane was stained using
Ponceau S solution (Sigma P7171-1L) to verify uniform loading before
immunoblotting with indicated antibodies. Signal detection was performed with an
enhanced chemiluminescence (ECL) super signal kit (Pierce, Rockford, IL).
Multiple exposures were analyzed to assure that the signals were within the linear
range of the film. The signal density was determined using Molecular Analyst
software (Bio-Rad, Hercules, CA).
4.3.9 Statistical analysis
Data were tested for normality (all data passed) and then subjected to parametric analysis. When more than two groups were compared, one-way analysis
of variance (ANOVA) was performed prior to comparison of individual groups, and
the post-hoc t-tests were adjusted for multiple comparisons using Bonferroni’s
correction. Statistical significance was reported at the P < 0.05 and P < 0.01 levels.
SPSS software was used for all analysis. Values are given as mean±SEM.
4.4 Results
4.4.1 The effect of ROS on ouabain-initiated Na/K-ATPase/c-Src signaling
131 In LLC-PK1 cells, we have demonstrated that the Na/K-ATPase α1 subunit directly interacts with c-Src kinase via two pairs of domain interactions to form a functional receptor complex, and ouabain stimulated phosphorylation of c-Src bound to the α1 subunit and association between the α1 subunit and c-Src (Tian, Cai et al.
2006; Li and Xie 2009). We determined the role of ROS on ouabain-stimulated c-Src phosphorylation in LLC-PK1 cells. As shown in Fig. 4-1, ouabain (100nM, 15min) stimulated c-Src, ERK1/2 activation and interaction between the α1 subunit and c-
Src, which was abolished by pretreatment with NAC (N-Acetyl-L-cysteine, 10mM,
30min) (Fig. 4-1a-d). NAC alone has no effect on c-Src activation. Cobalt protoporphyrin (CoPP), heme oxygenase (HO-1) inducer (antioxidant enzyme), attenuated ouabain stimulated-c-Src activation (Fig. 4-1e-f). Binding of ouabain to the Na/K-ATPase not only recruits c-Src to the signaling complex (Haas, Wang et al.
2002), but it also activates c-Src that binds to the Na/K-ATPase α1 subunit (Tian,
Cai et al. 2006). Since NAC is capable of eliminating basal ROS before ouabain stimulation, it appears that a basal level of ROS is required, as a co-effector, in ouabain-induced Na/K-ATPase/c-Src signaling in which, a basal level of ROS might affect ouabain binding to the α1 subunit by induction of Na/K-ATPase conformation change, favoring ouabain binding and c-Src activation.
4.4.2 The effect of ROS on ouabain-induced redistribution of the α1 subunit and
NHE3, and inhibition of transcellular 22Na+ flux
Ouabain and a high salt diet redistribute the Na/K-ATPase and NHE3 to promote renal sodium excretion by signaling through the Na/K-ATPase (Cai, Wu et
132 al. 2008; Liu, Yan et al. 2011). Consistent with the observation that NAC prevents
ouabain-induced c-Src, ERK1/2 activation (Fig. 4-1a-d), pretreatment with NAC
(10mM, 30min) also prevented ouabain (100nM, 1h) stimulated accumulation of
Na/K-ATPase α1 and NHE3 in early endosome (EE) fractions (Fig. 4-2a-c). The
Na/K-ATPase in rat cerebellar granule cells is redox-sensitive with a "optimal redox
potential range" (Petrushanko, Bogdanov et al. 2006). Interestingly, ouabain-induced
c-Src activation was significantly attenuated by pretreatment (30min) with high
doses but not low dose of NAC in LLC-PK1 cells (Fig. 4-2e). Functionally, ouabain
(100nM, 1h)-induced inhibition of active transepithelial 22Na+ flux was blunted by
pretreatment of 10mM NAC (30min), but not 1mM NAC (Fig. 4-2f). Taken together, the above data further supported our hypothesis that a certain level of ROS is
required in ouabain-induced Na/K-ATPase/c-Src signaling and redistribution of the
Na/K-ATPase and NHE3, which lead to inhibition of sodium reabsorption in renal
proximal tubules.
4.4.3 Ouabain stimulated ROS production and protein oxidation
- DHE (dihydroethidine) is extensively used as a probe for O2 , being oxidized
to a fluorescent product to evaluate ROS production. As shown in Fig. 4-3a, ouabain
(100nM, 1h) induced ROS production was assessed by DHE staining. In evaluating
protein oxidation studies, we use GO (glucose oxidase), which causes a steady
increase in intracellular ROS in cultured cardiac myocytes for at least 12 h (Liu,
Tian et al. 2000; Liu, Li et al. 2006), as positive control. Both ouabain (50 and
100nM, 1h) and GO (1 and 3 mU/ml, 1h) stimulated the formation of DMPO (5,5-
133 Dimethyl-1-Pyrroline-N-Oxide)-nitrone adducts assessed by a spin trap reagent
DMPO-based Western blot assay (Fig. 4-3b). Cells with DMPO treatment alone served as system control. Ouabain (100nM, 1h) and GO (3 mU/ml, 1h) also stimulated protein carbonylation in whole cell lysates (Fig. 4-3c). When whole cell lysate proteins were derivatized with DNPH (2,4-Dinitrophenylhydrazine) and immunoprecipitated with anti-DNP antibody, it appeared that both ouabain (100nM,
1h) and GO (3mU/ml, 1h) caused protein carbonylation of Na/K-ATPase α1 subunit,
NHE3 and c-Src (Fig. 4-3d). When whole cell lysate proteins were immunoprecipitated with anti-α1 subunit antibody and then derivatized with DNPH, similar carbonylation of the α1 subunit stimulated by ouabain and GO was observed
(data not shown). In Fig. 4-3b-c, control immunoblots with DMPO or DNPH alone suggest the presence of basal ROS and protein oxidation.
These results led us to speculate that oxidative modification of the α1 subunit might promote its internalization. We compared the protein carbonylation profiles in whole cell lysate and EE fraction. Equal amount of total proteins (20µg) was used to determine the protein carbonylation profiles. As shown in Fig. 4-3e, the profile of whole cell lysate is significantly different from that of EE fractions. The most profound difference is the band closely located between 100-115 kDa that is presented as the α1 subunit, as indicated in Fig. 4-3d.
134 4.4.4 ROS stress stimulated Na/K-ATPase signaling and redistribution of Na/K-
ATPase and NHE3, and inhibited transepithelial 22Na+ flux
It has been shown that oxidative stress induces conformation change and
inhibits Na/K-ATPase activity (Huang, Wang et al. 1992; Figtree, Liu et al. 2009).
Like ouabain (100nM, 15min), GO (3mU/ml, 15min) alone stimulated
phosphorylation of c-Src and ERK1/2 (Fig. 4-4a-b). Moreover, co-treatment with
ouabain (100nM) and GO (3mU/ml) for 15min had a slight but significant
synergistic effects on c-Src activation compared to ouabain or GO alone (Fig. 4-4a),
further indicating that ROS is a co-effector of ouabain-induced Na/K-ATPase/c-Src
signaling. The data also suggest that ouabain-induced ROS generation, functioning
as a downstream effector of Na/K-ATPase/c-Src signaling, might further stimulate
Na/K-ATPase/c-Src cascade by a positive feedback mechanism. Increases in ROS
stress by GO (1 and 3mU/ml) alone activated c-Src (Fig. 4-4c, 15min treatment) and
stimulated Na/K-ATPase endocytosis (Fig. 4-4c-e, 1h treatments). In transepithelial
22Na+ flux assay, GO (3mU/ml, 60 min) alone, like ouabain (100nM, 60min),
inhibited the 22Na+ flux. However, unlike ouabain-induced inhibition only occurred when ouabain was applied in the basolateral aspect (Cai, Wu et al. 2008), GO inhibited the transepithelial 22Na+ flux when it was applied on both basolateral and
apical aspects (Fig. 4-4f). Taken together, the data indicate that GO alone can
activate c-Src (Fig. 4-4a-c), stimulate protein oxidative modification (Fig. 4-3b-d)
and the redistribution of the Na/K-ATPase (Fig. 4-4c, e) in the same manner as
ouabain. This is consistent with out early observations that ROS induced by GO
135 crosstalk with the Na/K-ATPase and induce the Na/K-ATPase endocytosis (Liu, Li
et al. 2006).
4.4.5 The role of c-Src in ouabain and glucose oxidase mediated protein
carbonylation.
Because c-Src activation is the initiating step in Na/K-ATPase signaling
cascade, to obtain additional evidence showing the interplay between ROS and
Na/K-ATPase signaling, we investigated whether ROS generation is influenced by
c-Src. LLC-PK1 cells were pretreated with c-Src specific inhibitor PP2, and then
exposed to 100nM ouabain or 3mU/ml GO to assess c-Src activation, ERK1/2
activation, as well as protein carbonylation. As depicted in Fig. 4-5a-c, PP2
abrogated c-Src and ERK1/2 phosphorylation induced by GO. Interestingly, PP2
abolished ouabain-induced protein carbonylation and attenuated GO-mediated
protein oxidation. These data support our previous propositions that Na/K-ATPase
signaling is implicated in ROS generation and c-Src plays a key role in ouabain-
induced protein oxidation. Given PP2 is non-specific kinase inhibitor, both SYF and
SYF+c-Src cells were employed to further demonstrate the hypothesis that c-Src is
necessary in ROS generation caused by ouabain. The SYF+c-Src cells are derived
from mouse embryos harboring functional null mutations in both alleles of the c-Src
family kinases Src, Yes, and Fyn. The SYF+c-Src cells are the stable transfectants of
SYF cells that express c-Src (Haas, Wang et al. 2002). As shown in Fig. 4-5d-e, in
SYF cells, protein carbonylation was not significantly altered by ouabain compared
with controls. However, in SYF+c-Src cells, ouabain significantly increased protein
136 carbonylation. Glucose oxidase caused protein oxidation in both SYF and SYF+c-
Src cells. The data suggest c-Src is required in ROS generation mediated by ouabain, while glucose oxidase-induced protein oxidation is partially dependent on c-Src, and
Na/K-ATPase might not be the only pathway for GO-mediated protein carbonylation.
4.4.6 Disruption of Na/K-ATPase/c-Src signaling attenuated ouabain-induced
ROS production
To test the role of Na/K-ATPase/c-Src complex in ouabain-induced ROS generation, we used two stable cell lines generated from LLC-PK1 by siRNA method, with disrupted Na/K-ATPase/c-Src signaling and signaling complex. PY-17 cells only express about 8-10% of α1 compared to the parent LLC-PK1, and C2-9 cells are caveolin-1 knock-out LLC-PK1 cells showing disrupted formation of caveolar Na/K-ATPase/c-Src complex (Liu, Liang et al. 2005; Liang, Cai et al. 2006;
Liang, Tian et al. 2007). The basal level of c-Src phosphorylation is higher in PY-17 cells than that in LLC-PK1 cells (Liang, Cai et al. 2006; Liang, Tian et al. 2007).
Both PY-17 and C2-9 do not respond to ouabain stimulation in terms of c-Src activation. Furthermore, depletion of caveolin-1 or disruption of lipid rafts abolishes ouabain-induced endocytosis of the Na/K-ATPase (Liu, Liang et al. 2005; Cai, Wu et al. 2008). The ouabain-induced c-Src activation (ouabain=100nM, 15min) (Fig. 4-
6a) and ROS production (ouabain=100nM, 1h) (Fig. 4-6b-c) were significantly attenuated by disruption of the Na/K-ATPase/c-Src signaling complex. Since ouabain-induced redistribution of the Na/K-ATPase and NHE3 requires caveolin-1 and lipid rafts (Liu, Liang et al. 2005; Cai, Wu et al. 2008), the data suggest that
137 ouabain-induced ROS generation requires Na/K-ATPase/c-Src signaling, and ouabain-induced protein oxidation is a downstream effector of Na/K-ATPase/c-Src signaling (as shown in Fig. 4-5c-e and Fig. 4-6b-c) to promote the redistribution of certain proteins in EE fractions (Fig. 4-3e).
4.4.7 Identification of carbonylation sites in the Na/K-ATPase α1 subunit
To determine the carbonylation site(s), LC-MS/MS was conducted with native trypsinized α1 subunit isolated from LLC-PK1 cells by immunoprecipitation, with or without treatment with ouabain (100nM, 1 hr) or GO (3mU/ml, 1hr). Direct carbonylation modification of two amino acid residues Pro234 (mass 113.0477 with mass diff 15.9949) and Thr237 (mass 99.0320 with mass diff -2.0156) in peptide
226SPDFTNENPLETR238 (numbered by UniProtKB/Swiss-Prot No P05024
(AT1A1_PIG)) were present in control ouabain- and GO- treated cells. Both Pro224 and Thr226 in peptide 211VDNSSLTGESEPQTR225 were shown stimulated direct carbonylation only in response to ouabain and GO treatments (Fig. 4-7). Both peptides have 2 tolerable (tryptic) termini (NTT=2). Comparing to peptide
226SPDFTNENPLETR238, peptide 211VDNSSLTGESEPQTR225 is highly conserved and present in Na/K-ATPase α1/α2/α3/α4 isoforms. Moreover, these two peptides are integrity linked (211VDNSSLTGESEPQTRSPDFTNENPLETR238) and located in the actuator (A) domain (cytosolic loop between transmembrane M2 and M3 domains) of the α1 subunit, and Pro222/Thr224 is located on the surface facing the nucleotide binding (N) domain. Upon ouabain binding, the Na/K-ATPase undergoes conformation changes, in which the A domain is rotated to the N domain and
138 transmembrane M1/M2 domain is shifted toward M3/M4 domain (Yatime, Laursen
et al.). These conformation changes form a high affinity binding pocket for ouabain
as well as might stabilize caveolin-1 binding motif and affect binding of signaling
molecules such as c-Src, PI3K and IP3R (Yatime, Laursen et al.), which are critical
in the Na/K-ATPase signaling. Except the two carbonylation sites, the peptide
211VDNSSLTGESEPQTR225 also includes 217TGES220 motif and Glu221 residue that
affect conformation change and function. The 217TGES220 motif not only is the
anchor motif of A domain rotated toward N domain (~10˚), but it also protects spontaneous hydrolysis of phosphorylated Asp374 residue in E2P:Ouabain state
(Yatime, Laursen et al.). Moreover, Glu221 and Arg549 form the only link between the
A and N domains (Toyoshima, Kanai et al.). However, it is not clear the effect of
Pro224/Thr226 carbonylation on the link between Glu221 and Arg549.
4.5 Discussion
We have shown that activation of the Na/K-ATPase/c-Src signaling by a high
salt diet or ouabain inhibits RPT sodium reabsorption to counterbalance salt-loading
mediated volume expansion and related BP increase. We reported here that in LLC-
PK1 cells, ROS and ouabain-Na/K-ATPase/c-Src signaling are inextricably linked
forming a positive feedback mechanism. Basal ROS is required to initiate and
propagate the Na/K-ATPase signaling. Neutralization of ROS with NAC prevents
ouabain-induced activation of Na/K-ATPase/c-Src signaling, redistribution of Na/K-
ATPase and NHE3, and inhibition of transcellular 22Na+ flux. Furthermore, ouabain-
139 stimulated ROS generation might link the Na/K-ATPase signaling to NHE3 regulation.
It was first proposed that an increase in endogenous CTS stimulates renal sodium excretion by inhibition of renal tubular Na/K-ATPase to correct volume expansion and its related BP increase (Haddy, Pamnani et al. 1979; de Wardener and Clarkson 1985), but the finding that ouabain mediated vascular tone shifts the focus away from renal function (Blaustein, Zhang et al. 2009). Gene replacement studies have unequivocally demonstrated an important role of endogenous CTS in regulation of renal sodium excretion and BP (Dostanic-Larson, Van Huysse et al.
2005; Loreaux, Kaul et al. 2008). Recently, evidences including ours support this concept under pathological conditions such as high salt intake, chronic renal failure and chronic heart failure in various animal models and human beings. We have demonstrated that the Na/K-ATPase α1 subunit directly interacts with c-Src kinase via two pairs of domain-domain interactions to form a functional receptor complex
(Tian, Cai et al. 2006; Li and Xie 2009), as well as that CTS activate this receptor
Na/K-ATPase/c-Src complex, resulting in the stimulation of multiple protein kinase cascades including c-Src, ERK1/2, PI3K and PKC, and increased ROS production in both in vitro and in vivo models (reviewed in (Schoner and Scheiner-Bobis 2008;
Bagrov, Shapiro et al. 2009; Li and Xie 2009; Liu and Xie 2010)). Ouabain- stimulated ROS generation functions as a second messenger in ouabain-activated
Na/K-ATPase signaling (Liu, Tian et al. 2000; Liu, Li et al. 2006). Infusion of CTS, like angiotensin II, causes ROS generation and protein oxidation in experimental animals (Kennedy, Vetteth et al. 2006; Kennedy, Vetteth et al. 2006). Oxidative
140 modification of the Na/K-ATPase inhibits its activity, promotes its susceptibility to
degradation, and stabilizes the enzyme in an E2-prone conformation (Xie, Wang et al. 1990; Huang, Wang et al. 1992; THÉVENOD and FRIEDMANN 1999; Figtree,
Liu et al. 2009). An increase in ROS stimulates interaction between the Na/K-
ATPase and signaling proteins which leads to trafficking regulation of the Na/K-
ATPase (Liu, Li et al. 2006; Dada, Novoa et al. 2007). Our present study indicated
that ouabain binding stimulated carbonylation modification of the Na/K-ATPase α1
subunit and NHE3 might trigger the trafficking regulation.
Increases in oxidative stress are both a cause and consequence of
hypertension (Touyz 2004; Wilcox 2005; Vaziri and Rodriguez-Iturbe 2006; Welch
2006). High salt diet, which is well-documented for its stimulation of systematic
oxidative stress, regulates the activity and distribution of the Na/K-ATPase and
NHE3 that contribute to the development and maintenance of hypertension in
different animal models (Moe, Tejedor et al. 1991; Silva and Soares-da-Silva 2007;
Panico, Luo et al. 2009; McDonough 2010; Banday and Lokhandwala 2011; Liu,
Yan et al. 2011). In the kidney, increased oxidative stress influences a number of
physiologic processes including renal sodium handling in the proximal tubule
(Zhang, Imam et al. 2002; Han, Lee et al. 2005; Panico, Luo et al. 2009; Schreck
and O'Connor 2011), and contributes to salt-sensitivity and salt-sensitive
hypertension (Kopkan and Majid 2005; Taylor, Glocka et al. 2006). Anti-oxidant
agents such as Tempol and enzymes such as heme oxygenase-1 exhibit a beneficial
and protective effect on BP in various animal models of hypertension (Abraham
and Kappas 2005). However, the beneficial effect of antioxidants is controversy and
141 not seen in most clinic trials with administration of antioxidant supplementation
(reviewed in (Touyz 2004; Munzel, Gori et al. 2010)). It is suggested that synthetic
antioxidant supplementation may be ineffective or even dangerous due to the
possible ‘over-antioxidant-buffering’ effect of excessive antioxidant
supplementation (Huang, Caballero et al. 2006), while natural antioxidants from
high dietary intakes of fruits, vegetables, nuts and grains might significantly lower
systolic BP (Kizhakekuttu and Widlansky ; John, Ziebland et al. 2002). It appears
that maintaining a proper range of ROS, systematically and locally, is more
important.
Carbonylation modifications, via metal-catalyzed activation of hydrogen
peroxide, are very stable products and chemically irreversible, and carbonylated
proteins are prone to be degradated (Stadtman and Levine 2000; Dalle-Donne,
Aldini et al. 2006). However, a recent study has demonstrated the possible role of
carbonylation/decarbonylation process in signal transduction in which the thiol
groups were responsible for decarbonylation via enzymatic processes in the
biologic system (Wong, Marcocci et al. 2010). Our observations suggest that
carbonylated proteins were gradually recovered after removal of un-bound ouabain
(data not shown). Although the task of identifying the causal factors behind time-
dependent decreased carbonylation has been proven difficult, several principle
possibilities can be envisioned, including (1) a de novo protein synthesis to dilute
carbonylated protein levels, (2) degradation for removal of oxidized proteins, (3) decarbonylation. To our knowledge, this is the first time to demonstrate that CTS and/or ROS cause direct carbonylation modification, in the A domain that has the
142 potential to significantly affect Na/K-ATPase α1 conformation change and known binding domains for partners of the Na/K-ATPase signaling. Nonetheless, more studies are needed to explore the underlying mechanism.
Our present data indicate a novel mechanism regulating RPT sodium handling via interaction of ROS with the Na/K-ATPase/c-Src signaling pathway, which may influence salt retention related volume expansion that is responsible for certain types of hypertension. However, the mechanisms remain largely to be elucidated since the available data is limited (Garvin and Ortiz 2003; Schreck and
O'Connor 2011). Some pertinent questions remain to be resolved, such as how ROS interacts with and influences the Na/K-ATPase/c-Src signaling cascade as well as whether ouabain-induced ROS acutely boosts the Na/K-ATPase signaling by a positive feedback mechanism and chronically desensitizes the signaling cascade by stimulating Na/K-ATPase/c-Src endocytosis.
Acknowledgments
The authors would like to thank Dr. Venkatesha Basrur (Department of
Pathology, University of Michigan) for performing MASS analysis and Ms. Carol
Woods for her excellent help. Portions of this work were supported by grants from
National Institutes of Health (HL-109015 to Z.X. and J.I.S., and GM-78565 to Z.X.).
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151 4.7 Figure Legends
Figure 4-1: A basal level of ROS is required in ouabain-induced activation of the Na/K-ATPase signaling.
LLC-PK1 cells were pretreated with NAC (10mM, 30min) or cobalt protoporphyrin (CoPP, 2µM) before ouabain (100nM, 15min) treatment.
Immunoprecipitation against total c-Src was performed to determine c-Src phosphorylation and its association with the Na/K-ATPase α1 subunit. Phospho-
ERK1/2 and total ERK1/2 were assessed with whole cell lysates. (a) A representative Western blot showing pretreatment with NAC prevented ouabain- induced pY418 c-Src and ERK1/2 phosphorylation. (b) and (c) Quantitative analysis of the effect of NAC pretreatment on ouabain-induced pY418 c-Src and ERK1/2 phosphorylation. The phosphorylation of c-Src and ERK1/2 was expressed as the ratio of phosphorylated c-Src (p-Src) and ERK1/2 (p-ERK) versus total c-Src (t-Src) and ERK1/2 (t-ERK), respectively. n=4. (d) The effect of NAC pretreatment on ouabain-stimulated association between total c-Src and the Na/K-ATPase α1 subunit. The association was expressed as ratio of the α1 versus total c-Src. n=4. **, p<0.01 vs con. #, p<0.01 vs ouabain treatment. (e) A representative Western blot showing that pretreatment with CoPP prevented ouabain-induced pY418 c-Src phosphorylation. (f) Quantitative analysis of the effect of CoPP pretreatment on ouabain-induced pY418 c-Src phosphorylation. n=4. **, p<0.01 vs con. #, p<0.01 vs ouabain treatment.
152 Figure 4-2: A basal level of ROS is required in ouabain-induced redistribution
of the Na/K-ATPase α1 subunit and NHE3, and inhibition of transcellular
22Na+ flux.
(a) A representative Western blot showing that pretreatment with NAC
(10mM, 30min) prevented ouabain (100nM, 1h) stimulated accumulation of the α1 subunit and NHE3 in early endosome (EE) fractions. Graph (b-d) show quantitative analysis of (a), n=4; ** p<0.01 vs control. (e) The effect of different concentrations
of NAC (30min) on ouabain (100nM, 15min)-induced pY418 c-Src phosphorylation,
n=3; ** p<0.01 vs control. (f) The effect of different concentrations of NAC
(30min) on ouabain (100nM, 1h)-induced inhibition of active transcellular 22Na+
flux from apical to basolateral compartment in LLC-PK1 monolayer grown on
Transwell® membrane support, n=3, ** p<0.01 vs control; * p<0.05 vs control.
Figure 4-3: Ouabain stimulated ROS generation and protein oxidative
modification.
Ouabain (100nM, 30min) stimulated ROS generation assessed by
Dihydroethidium (DHE) staining (a), formation of DMPO-nitrone adducts (b), and
protein carbonylation (c). Immunoprecipitation against DNP showed carbonylation modification of the α1 subunit, NHE3 and c-Src (d). Protein carbonylation might promote the accumulation of certain proteins in EE fractions (e).
153 Figure 4-4: Oxidative stress induced by GO caused similar effect as ouabain.
GO, like ouabain, activated c-Src (a) and ERK1/2 (b); n=3, ** p<0.01 vs
control; # p<0.05 vs ouabain or GO alone. GO (1 and 3 mU/ml, 1h) accumulated the α1 subunit and NHE3 in EE fractions in a dose-dependent manner (c-e); n=4, **
p<0.01 vs control. Unlike ouabain that only inhibits activate 22Na+ flux when added
to the basolateral aspect, GO (3 mU/ml, 1h) inhibits active 22Na+ flux in LLC-PK1
monolayer, effective on both apical and basolateral aspect (f); n=3, ** p<0.01 and*
p<0.05 vs control.
Figure 4-5: c-Src is required in ouabain-induced protein carbonylation, but
GO-mediated protein oxidation is partially dependent on c-Src.
A representative Western blot showing that pretreatment of c-Src inhibitor
PP2 (10µM, 30min) blocked phosphorylation of c-Src and ERK1/2 induced by GO
(3mU/ml, 1h) (a), abrogated ouabain (100nM, 1h)-mediated protein carbonylation and attenuated GO (3mU/ml, 1h)-induced protein oxidation (c). Ponceau S staining showing loading control. (b) Quantitative analysis of (a), n=4, ** p<0.01 vs control.
# p<0.05 vs GO. (d) A representative Western blot showing that ouabain (100nM,
1h) stimulated protein carbonylation in SYF+c-Src cells, but not in SYF cells. GO
mediated protein oxidation in both SYF+c-Src and SYF cells. (e) Quantitative
analysis of (d). n=4, ** p<0.01, # p<0.05 vs control.
Figure 4-6: Both Na/k-ATPase α1 subunit and caveolin-1 are necessary in
ouabain-mediated protein carbonylation.
154 Ouabain stimulated c-Src phosphorylation (a), an increase in superoxide
level (b) and protein carbonylation (c) in LLC-PK1 cells, but not in PY-17 cells and
C2-9 cells. Ponceau S staining showing loading control. n=3, ** p<0.01.
Figure 4-7: A ribbon diagram of the Na/K-ATPase with Pro222 and Thr224 highlighted (PDDB ID: 2ZXE).
155 4.8 Figures
Figure 4-1 a.
b.
# **
2
1 (relative)
p-Src/T-Src 0 n a o ua u C O NAC O +
NAC
156 Figure 4-1 c.
** # 2
1 (relative) p-ERK/t-ERK1/2 0
n C A Co Oua N
NAC+Oua
d.
# 2.5 **
2.0
1.5
1/T-Src 1.0 α (relative) 0.5
0.0
n C o ua A ua C O N +O
NAC
157 Figure 4-1 e.
f.
# ** 3
2
1 c-Src/ total c-Src
418 pY 0 con CoPP Oua CoPP+Oua
158 Figure 4-2 a.
b. # 2 **
1
1 inEE (relative) α 0
ua Con Oua NAC +O C NA
159 Figure 4-2 c.
3 # ** 2
(relative) 1 NHE3 EE in
0 a on AC C Oua N +Ou AC N
d.
2 # **
1 1 in LE 1 in α
(relative)
0
on C Oua NAC
NAC+Oua
160 Figure 4-2 e.
3 Control ** Oua ** 2
1
p-Src/T-Src (%) p-Src/T-Src 0
NAC= 0 1 5 10 mM
f. Control Oua 100 * ** Flux (%) + 50 Na 22
0
NAC 0 1 10 mM
161 Figure 4-3 a.
b.
162 Figure 4-3 c.
Ponceau S Staining
. d.
163 Figure 4-3 e.
Ponceau S Staining
164 Figure 4-4 a.
3 **, # 2 ** **
1 (relative%) P-Src/T-Src
0 l a u O O GO ontro a+G C u O
b.
165 Figure 4-4 c.
d.
4 ** ** 3 **
2 (relative)
p-Src/T-Src 1
0 n a 1 3 u O O Co O G G
166 Figure 4-4 e.
3 ** ** 2 ** 1 in1 EE α (relative) 1
0 n 1 Co Oua GO GO 3
f.
100 * ** ** Flux (%)
+ 50
Na 22 0 n ) ) o so so C a a (B (B a 3 O Ou G GO 3 (Apical)
167 Figure 4-5
a.
b. con PP2+GO PP2 GO 2.0 2.0 ** ** phospho-ERK1/2/T-ERK1/2 # # 1.5 1.5
1.0 1.0 c-Src/T--c-Src
418 0.5 0.5 PY
0.0 0.0 c-Src ERK1/2
168 Figure 4-5
c.
169 Figure 4-5
d.
e.
2.0 con oua # ** ** GO 1.5
1.0
0.5 Carbonyl contents
0.0 SYF + c-Src SYF
170 Figure 4-6
a.
Control 300 ** Oua 15min
200
100
(%) p-Src/T-Src 0 LLC-PK1 PY-17 C2-9
b.
300 ** con oua 100nM 1h 200
100
(%) DHE signal
0 LLC-PK1 PY-17 C2-9
171 Figure 4-6 c.
172 Figure 4-7
173 Chapter 5 – Summary and Conclusions
The overall aim of this dissertation is to improve our understanding of Na/K-
ATPase and NHE3 trafficking regulation and determine molecular mechanisms of
renal proximal tubular (RPT) sodium handling, which might contribute to the
impaired sodium excretion associated with hypertension.
In porcine RPT LLC-PK1 cells, we have shown that ouabain inhibits active
transepithelial 22Na+ transport (from apical to basolateral aspect) via protein trafficking regulation of the Na/K-ATPase and NHE3 (Liu, Kesiry et al. 2004; Cai
2008; Liu and Xie 2010), a process requiring ouabain-activated Na/K-ATPase signaling. This novel regulatory mechanism may contribute to CTS-induced renal sodium excretion. To investigate (1) if this regulatory mechanism is species- specific, (2) and if the internalized Na/K-ATPase and NHE3 is the endocytic recycling (reinsertion of endocytosed protein back to plasma membrane) in LLC-
PK1 cells, in manuscript 1 entitled “Ouabain-stimulated trafficking regulation of
the Na/K-ATPase and NHE3 in renal proximal tubule cells”, (1) three renal
proximal tubular cell lines (human HK-2, porcine LLC-PK1, and AAC-19
originated from LLC-PK1 cells in which the pig α1 was replaced by ouabain-
resistant rat α1) were employed to compare ouabain-induced regulation of the α1
subunit and NHE3 as well as transcellular 22Na+ transport, (2) LLC-PK1 cells were
used to assess endocytic recycling of NHE3 and Na/K-ATPase α1 subunit. Our
174 data indicate that the Na/K-ATPase is the functional receptor for ouabain-induced regulation of the Na/K-ATPase and NHE3 (and thus transcellular Na+ transport), as
we previously proposed (Liu and Xie 2010). This regulation is not species-specific,
but the species-specific α1 ouabain sensitivity may partially account for the species
differences observed in ouabain-induced natriuresis (Lloyd, Sandberg et al. 1992;
Yates and McDougall 1993; Loreaux, Kaul et al. 2008; Nesher, Dvela et al. 2009).
Specifically, the rodent α1 is far less sensitive response to ouabain than pig, dog, or
human α1. Higher concentrations of ouabain were required to activate Na/K-
ATPase signaling in rodents, compared to other species (Liu, Tian et al. 2000;
Aizman, Uhlen et al. 2001; Aydemir-Koksoy, Abramowitz et al. 2001; Haas, Wang et al. 2002; Liang, Cai et al. 2006). And the ouabain sensitivity of the α1 subunit influenced the ouabain-induced inhibition of 22Na+ flux as well as surface reduction of the α1 subunit and NHE3. Additionally, in ouabain-induced trafficking
regulation, endocytic recycling of internalized NHE3, but not the α1 subunit, was
inhibited by ouabain. The endocytosed α1 subunit, but not NHE3, is degraded through the LE- lysosome pathway.
In manuscript 2 entitled “Impairment of Na/K-ATPase signaling in renal proximal
tubule contributes to Dahl salt-sensitive hypertension”, we employed Sprague Dawley
rats, Dahl salt resistant (R) and sensitive (S) rats to investigate the role of Na/K-ATPase
signaling in renal sodium excretion and blood pressure regulation in vivo, further confirming our in vitro data: CTS function as natriuretic factors by CTS-induced redistribution of Na/K-ATPase and NHE3 and then RPT sodium transport regulation.
Our observations are as follows: (1) In Sprague Dawley rats, high salt diet activated c-Src
175 and induced redistribution of Na/K-ATPase and NHE3 in renal proximal tubules. (2) In
Dahl R rats, high salt diet significantly stimulated phosphorylation of c-Src and ERK1/2,
reduced Na/K-ATPase activity and NHE3 activity, and caused redistribution of Na/K-
ATPase and NHE3. In contrast, these adaptations were either much less effective or not seen in the Dahl S rats. (3) In primary culture of renal proximal tubule isolated from Dahl
S and R rats fed a low salt diet, ouabain induced Na/K-ATPase/c-Src signaling and
redistribution of Na/K-ATPase and NHE3 in the Dahl R rats, but not in the Dahl S rats.
These data indicate that impaired Na/K-ATPase signaling and consequent regulation of
Na/K-ATPase and NHE3 in renal proximal tubule may contribute to salt-induced
hypertension in the Dahl S rat.
Since there is no difference in the Na/K-ATPase α1 gene (Atp1a1) coding (Mokry and Cuppen 2008) and α1 sensitivity to ouabain (Nishi, Bertorello et al. 1993) between
Dahl salt-resistant and Dahl salt-sensitive rats, we propose that other factors must be present to affect the activation of Na/K-ATPase/c-Src signaling in the Dahl S rats.
It has been demonstrated that a high salt diet stimulates an increase in endogenous
CTS release and ROS generation. Our observations indicate that RPT sodium handling
via ouabain-mediated Na/K-ATPase/c-Src signaling appears to counterbalance blood
pressure rising caused by sodium retention, as shown in salt-sensitive hypertension (Liu,
Kesiry et al. 2004; Liu, Liang et al. 2005; Periyasamy, Liu et al. 2005; Oweis, Wu et al.
2006; Cai, Wu et al. 2008; Liu, Yan et al. 2011). Activation of Na/K-ATPase/c-Src
signaling, on the other hand, stimulates mitochondrial ROS production. In addition, c-Src
as initiating step of Na/K-ATPase signaling, is also redox-sensitive and required for ROS
generation (especially derived from NADPH oxidases) (Seshiah, Weber et al. 2002;
176 Touyz, Yao et al. 2003). Together with recent report that NADPH oxidase-derived ROS
production reduces renal proximal tubular sodium reabsorption (Panico, Luo et al. 2009),
we propose that there might be some cross-talk between Na/K-ATPase/c-Src signaling
and ROS generation in terms of renal sodium handling (Liu, Kennedy et al.)
In manuscript 3 entitled “Redox modulation of the Na/K-ATPase signaling and renal proximal tubular sodium handling”, we report that reactive oxygen species
(ROS) is critical in modulation of ouabain-Na/K-ATPase signaling and RPT ion transport based on the following observations: (1) Antioxidant NAC (N-Acetyl-L- cysteine) prevented ouabain-stimulated Na/K-ATPase signaling cascade, protein carbonylation, redistribution of the Na/K-ATPase and NHE3, and inhibition of transepithelial 22Na+ transport. (2) CoPP, HO-1 inducer (antioxidant enzyme),
attenuated ouabain stimulated-c-Src activation, which is an initiating step of Na/K-
ATPase signaling. (3) Protein carbonylation assay, spin-trap immunoassay, and DHE
staining all demonstrated that ouabain stimulated ROS generation and protein
oxidation. (4) c-Src is required in ouabain-induced protein carbonylation. (5)
Disruption of Na/K-ATPase/c-Src signaling attenuated ouabain-induced ROS
production. Interestingly, pretreatment with high doses but not low dose of NAC
attenuated ouabain-induced c-Src activation and blunted ouabain-inhibited active
transepithelial 22Na+ flux in LLC-PK1 cells, indicating that a basal physiological
range of ROS is necessary for initiation and amplification of ouabain-Na/K-ATPase
signaling and redistribution of Na/K-ATPase and NHE3. Furthermore, LC-MS/MS
analysis has identified that both ouabain and glucose oxidase caused direct
carbonylation of two amino acid residues in the actuator (A) domain of the Na/K-
177 ATPase α1 subunit. Taken together, our above findings unveil a novel mechanism
regulating RPT sodium handling via interaction of ROS with the Na/K-ATPase/c-
Src signaling pathway, which may influence salt retention related to volume
expansion that is responsible for hypertension. Moreover, direct protein
carbonylation in the actuator (A) domain of the Na/K-ATPase α1 subunit by ROS
might affect ouabain binding to the α1 subunit by induction of Na/K-ATPase
conformation change, favoring ouabain binding and c-Src activation, as well as
subsequently signaling cascade. However, the mechanisms remain largely to be
elucidated since the available data is limited (Garvin and Ortiz 2003; Schreck and
O'Connor 2011). Some pertinent questions remain to be resolved, such as how ROS
interacts with and influences the Na/K-ATPase/c-Src signaling cascade, as well as
whether ouabain-induced ROS generation acutely boosts the Na/K-ATPase signaling
by a positive feedback mechanism and chronically desensitizes the signaling cascade
by stimulating Na/K-ATPase/c-Src endocytosis.
Despite increasing evidence showing that antioxidants effectively lower
blood pressure in animal models, findings in human beings are less conclusive. A major clinical challenge is that routinely used antioxidants are ineffective in preventing or treating hypertension. The failure of antioxidant treatment prompts us to explore the underlying mechanisms that how ROS regulate blood pressure so that specific intervention can be used. We hope this dissertation and future in vitro and
in vivo studies on this topic would open up the possibility of translational clinical research and might improve the prognosis of hypertension and develop personalized patient management.
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181