Medicine and Health Sciences Commons^™

Rapamycin Attenuates Cardiac Fibrosis In Experimental Uremic Cardiomyopathy By Reducing Marinobufagenin Levels And Inhibiting Downstream Pro-Fibrotic Signaling, Steven T. Haller Phd, Yanling Yan Phd, Christopher A. Drummond Phd, Joe Xie Md, Jiang Tian Phd, David J. Kennedy Phd, Victoria Y. Shilova Phd, Zijian Xie Phd, Jiang Liu Phd, Christopher J. Cooper Md, Deepak Malhotra Md, Phd, Joseph I. Shapiro Md, Olga V. Fedorova Phd, Alexei Y. Bagrov Md, Phd

Zijian Xie

Background: Experimental uremic cardiomyopathy causes cardiac fibrosis and is causally related to the increased circulating levels of the cardiotonic steroid, marinobufagenin (MBG), which signals through Na/K‐ATPase. Rapamycin is an inhibitor of the serine/threonine kinase mammalian target of rapamycin (mTOR) implicated in the progression of many different forms of renal disease. Given that Na/K‐ATPase signaling is known to stimulate the mTOR system, we speculated that the ameliorative effects of rapamycin might influence this pathway.

Methods and Results: Biosynthesis of MBG by cultured human JEG‐3 cells is initiated by CYP27A1, which is also a target for rapamycin. It was demonstrated that 1 …

Protein Carbonylation Of An Amino Acid Residue Of The Na/K‐Atpase Α1 Subunit Determines Na/K‐Atpase Signaling And Sodium Transport In Renal Proximal Tubular Cells, Yanling Yan, Anna P. Shapiro, Brahma R. Mopidevi, Muhammad Chaudhry, Kyle Maxwell, Steven T. Haller, Christopher A. Drummond, David J. Keendey, Jiang Tian, Deepak Malhorta, Zijian Xie, Joseph I. Shapiro Md, Jiang Liu

Zijian Xie

Background We have demonstrated that cardiotonic steroids, such as ouabain, signaling through the Na/K‐ATPase, regulate sodium reabsorption in the renal proximal tubule. By direct carbonylation modification of the Pro222 residue in the actuator (A) domain of pig Na/K‐ATPase α1 subunit, reactive oxygen species are required for ouabain‐stimulated Na/K‐ATPase/c‐Src signaling and subsequent regulation of active transepithelial ²²Na⁺ transport. In the present study we sought to determine the functional role of Pro222 carbonylation in Na/K‐ATPase signaling and sodium handling.

Methods and Results Stable pig α1 knockdown LLC‐PK1‐originated PY‐17 cells were rescued by expressing wild‐type rat α1 and rat α1 with …

Sh2 Ligand-Like Effects Of Second Cytosolic Domain Of Na/K-Atpase Α1 Subunit On Src Kinase, Moumita Banerjee, Qiming Duan, Zijian Xie

Zijian Xie

Our previous studies have suggested that the α1 Na/K-ATPase interacts with Src to form a receptor complex. In vitro binding assays indicate an interaction between second cytosolic domain (CD2) of Na/K-ATPase α1 subunit and Src SH2 domain. Since SH2 domain targets Src to specific signaling complexes, we expressed CD2 as a cytosolic protein and studied whether it could act as a Src SH2 ligand in LLC-PK1 cells. Co-immunoprecipitation analyses indicated a direct binding of CD2 to Src, consistent with the in vitro binding data. Functionally, CD2 expression increased basal Src activity, suggesting a Src SH2 ligand-like property of CD2. Consistently, …

Involvement Of Reactive Oxygen Species In A Feed-Forward Mechanism Of Na/K-Atpase Mediated Signaling, Yanling Yan, Anna P. Shapiro, Steven Haller, Vinal Katragadda, Lijun Liu, Jiang Tian, Venkatesha Basrur, Deepak Malhotra, Zi-Jian Xie, Nader G. Abraham, Joseph I. Shapiro Md, Jiang Liu

Zijian Xie

Cardiotonic steroids (such as ouabain) signaling through Na/K-ATPase regulate sodium reabsorption in the renal proximal tubule. We report here that reactive oxygen species are required to initiate ouabain-stimulated Na/K-ATPase·c-Src signaling. Pretreatment with the antioxidant N-acetyl-L-cysteine prevented ouabain-stimulated Na/K-ATPase·c-Src signaling, protein carbonylation, redistribution of Na/K-ATPase and sodium/proton exchanger isoform 3, and inhibition of active transepithelial ²²Na⁺ transport. Disruption of the Na/K-ATPase·c-Src signaling complex attenuated ouabain-stimulated protein carbonylation. Ouabain-stimulated protein carbonylation is reversed after removal of ouabain, and this reversibility is largely independent of de novo protein synthesis and degradation by either the lysosome or the proteasome pathways. Furthermore, …

Pnaktide Inhibits Na/K-Atpase Reactive Oxygen Species Amplification And Attenuates Adipogenesis, Komal Sodhi, Kyle Maxwell, Yanling Yan, Jiang Liu, Muhammad Chaudhry, Morgan Getty, Zijian Xie, Nader G. Abraham, Joseph I. Shapiro Md

Zijian Xie

Obesity has become a worldwide epidemic and is a major risk factor for metabolic syndrome. Oxidative stress is known to play a role in the generation and maintenance of an obesity phenotype in both isolated adipocytes and intact animals. Because we had identified that the Na/K-ATPase can amplify oxidant signaling, we speculated that a peptide designed to inhibit this pathway, pNaKtide, might ameliorate an obesity phenotype. To test this hypothesis, we first performed studies in isolated murine preadipocytes (3T3L1 cells) and found that pNaKtide attenuated oxidant stress and lipid accumulation in a dose-dependent manner. Complementary experiments in C57Bl6 mice fed …

Identification Of Hydroxyxanthones As Na/K-Atpase Ligands, Zhongbing Zhang, Zhichuan Li, Jiang Tian, Wei Jiang, Yin Wang, Xiaojin Zhang, Zhuorong Li, Qidong You, Joseph Shapiro, Shuyi Si, Zijian Xie

Zijian Xie

We have screened a chemical library and identified several novel structures of Na/K-ATPase inhibitors. One group of these inhibitors belongs to polyphenolic xanthone derivatives. Functional characterization reveals the following properties of this group of inhibitors. First, like ouabain, they are potent inhibitors of the purified Na/K-ATPase. Second, their effects on the Na/K-ATPase depend on the number and position of phenolic groups. Methylation of these phenolic groups reduces the inhibitory effect. Third, further characterization of the most potent xanthone derivative, MB7 (3,4,5,6-tetrahydroxyxanthone), reveals that it does not change either Na⁺ or ATP affinity of the enzyme. Finally, unlike that of …

Central Role For The Cardiotonic Steroid Marinobufagenin In The Pathogenesis Of Experimental Uremic Cardiomyopathy, David Kennedy, Sandeep Vetteth, Sankaridrug Periyasamy, Mohamed Kanj, Larisa Fedorova, Samer Khouri, M. Kahaleh, Zijian Xie, Deepak Malhotra, Nikolai Kolodkin, Edward Lakatta, Olga Fedorova, Alexei Bagrov, Joseph Shapiro

Zijian Xie

Patients with chronic renal failure develop a “uremic” cardiomyopathy characterized by diastolic dysfunction, cardiac hypertrophy, and systemic oxidant stress. Patients with chronic renal failure are also known to have increases in the circulating concentrations of the cardiotonic steroid marinobufagenin (MBG). On this background, we hypothesized that elevations in circulating MBG may be involved in the cardiomyopathy. First, we observed that administration of MBG (10 g/kg per day) for 4 weeks caused comparable increases in plasma MBG as partial nephrectomy at 4 weeks. MBG infusion caused increases in conscious blood pressure, cardiac weight, and the time constant for left ventricular relaxation …

Marinobufagenin Stimulates Fibroblast Collagen Production And Causes Fibrosis In Experimental Uremic Cardiomyopathy, Jihad Elkareh, David Kennedy, Belvadi Yashaswi, Sandeep Vetteth, Amjad Shidyak, Eric Kim, Sleiman Smaili, Sankaridrug Periyasamy, Imad Hariri, Larisa Fedorova, Jiang Liu, Liang Wu, M. Kahaleh, Zijian Xie, Deepak Malhotra, Olga Fedorova, Vladimir Kashkin, Alexei Bagrov, Joseph Shapiro

Zijian Xie

We have observed recently that experimental renal failure in the rat is accompanied by increases in circulating concentrations of the cardiotonic steroid, marinobufagenin (MBG), and substantial cardiac fibrosis. We performed the following studies to examine whether MBG might directly stimulate cardiac fibroblast collagen production. In vivo studies were performed using the 5/6th nephrectomy model of experimental renal failure (PNx), MBG infusion (MBG), PNx after immunization against MBG, and concomitant PNx and adrenalectomy. Physiological measurements with a Millar catheter and immunohistochemistry were performed. In vitro studies were then pursued with cultured isolated cardiac fibroblasts. We observed that PNx and MBG increased …

Spironolactone Attenuates Experimental Uremic Cardiomyopathy By Antagonizing Marinobufagenin, Jiang Tian, Amjad Shidyak, Sankaridrug Periyasamy, Steven Haller, Mohamed Taleb, Nasser El-Okdi, Jihad Elkareh, Shalini Gupta, Sabry Gohara, Olga Fedorova, Christopher Cooper, Zijian Xie, Deepak Malhorta, Alexei Bagrov, Joseph Shapiro

Zijian Xie

Spironolactone has been noted to attenuate cardiac fibrosis. We have observed that the cardiotonic steroid marinobufagenin plays an important role in the diastolic dysfunction and cardiac fibrosis seen with experimental renal failure. We performed the following studies to determine whether and how spironolactone might ameliorate these changes. First, we studied rats subjected to partial nephrectomy or administration of exogenous marinobufagenin. We found that spironolactone (20 mg/kg per day) attenuated the diastolic dysfunction as assessed by ventricular pressure-volume loops and essentially eliminated cardiac fibrosis as assessed by trichrome staining and Western blot. Next, we examined the effects of spironolactone and its …

Marinobufagenin Induces Increases In Procollagen Expression In A Process Involving Protein Kinase C And Fli-1: Implications For Uremic Cardiomyopathy, Jihad Elkareh, Sankaridrug Periyasamy, Amjad Shidyak, Sandeep Vetteth, Jeremy Schroeder, Vanamala Raju, Imad Hariri, Nasser El-Okdi, Shalini Gupta, Larisa Fedorova, Jiang Liu, Olga Fedorova, M. Kahaleh, Zijian Xie, Deepak Malhotra, Dennis Watson, Alexei Bagrov, Joseph Shapiro

Zijian Xie

The cardiotonic steroid marinobufagenin (MBG) has been implicated in the pathogenesis of experimental uremic cardiomyopathy, which is characterized by progressive cardiac fibrosis. We examined whether the transcription factor Friend leukemia integration-1 (Fli-1) might be involved in this process. Fli-1-knockdown mice demonstrated greater cardiac collagen-1 expression and fibrosis compared with wild-type mice; both developed increased cardiac collagen expression and fibrosis after 5/6 nephrectomy. There was a strong inverse relationship between the expressions of Fli-1 and procollagen in primary culture of rat cardiac and human dermal fibroblasts as well as a cell line derived from renal fibroblasts and MBG-induced decreases in nuclear …

Effects Of Uremic Serum On Isolated Cardiac Myocyte Calcium Cycling And Contractile Function, Sankaridrug Periyasamy, Jie Chen, Derek Cooney, Patricia Carter, Eiad Omran, Jiang Tian, Snigdha Priyadarshi, Alexei Bagrov, Olga Fedorova, Deepak Malhotra, Zijian Xie, Joseph Shapiro

Zijian Xie

Background: Diastolic dysfunction occurs in patients with chronic renal failure. Moreover, serum from uremic patients contains one or more inhibitors of the plasmalemmal Na,K-ATPase (sodium pump). We hypothesized that a circulating substance present in uremic sera contributes to both sodium pump inhibition and diastolic dysfunction.

Methods: Serum samples were obtained from six patients with chronic renal failure and diastolic dysfunction.

Results: Their serum samples caused marked inhibition of Na,K-ATPase purified from dog kidney at all concentrations studied (all P < 0.01) and also impaired ouabain-sensitive rubidium uptake by myocytes isolated from Sprague-Dawley rats (P < 0.01). These cardiac myocytes were studied for their contractile function with video-edge detection and calcium metabolism with indo-1 fluorescence spectroscopy after exposure to these uremic sera. These uremic sera caused increases in myocyte fractional shortening (P < 0.01) as well as an increase in the time constant of relengthening (P < 0.01). Examining the calcium transient, the time constant for calcium recovery was also increased (P < 0.01). Exposure of these cells to sera from age- and sex-matched healthy subjects did not result in significant changes in contraction or calcium cycling. Extracts of uremic serum samples inhibited isolated Na,K-ATPase whereas extracts of normal serum samples did not. The effect of uremic serum extracts on contractile function and calcium cycling were quite similar to that of intact serum or the addition of ouabain. Co-incubation of uremic serum extract with an antibody fragment directed against digoxin markedly attenuated the inhibition of Na,K-ATPase activity and completely prevented any effects on calcium cycling or contractile function.

Conclusion: These data show that one or more substances are present in uremic sera that acutely cause increased force of contraction …

Reduction Of Na/K-Atpase Potentiates Marinobufagenin-Induced Cardiac Dysfunction And Myocyte Apoptosis, Changxuan Liu, Yan Bai, Yiliang Chen, Yu Wang, Yoann Sottejeau, Lijun Liu, Xiaomei Li, Jerry B. Lingrel, Deepak Malhorta, Christopher Cooper, Joseph I. Shapiro M.D., Zi-Jian Xie, Jiang Tian

Zijian Xie

Background: Na/K-ATPase decrease has been reported in patients with heart failure and is related to cardiac dysfunction. Results: Reducing Na/K-ATPase activates caspase 9 and induces cardiac dilation when treated with marinobufagenin. Conclusion: Reduction of Na/K-ATPase potentiates marinobufagenin-induced cardiac myocyte apoptosis. Significance: Decreased Na/K-ATPase content together with increased cardiotonic steroids levels is a novel mechanism that may account for cardiac dysfunction.

Effect Of Green Tea Extract On Cardiac Hypertrophy Following 5/6 Nephrectomy In The Rat, Snigdha Priyadarshi, Brandon Valentine, Chi Han, Olga Fedorova, Alexei Bagrov, Jiang Liu, Sankaridrug Periyasamy, David Kennedy, Deepak Malhorta, Zijian Xie, Joseph Shapiro

Zijian Xie

Background

Left ventricular hypertrophy commonly complicates chronic renal failure. We have observed that at least one pathway of left ventricular hypertrophy appears to involve signaling through reactive oxygen species (ROS). Green tea is a substance that appears to have substantial antioxidant activity, yet is safe and is currently widely used. We, therefore, studied whether green tea supplementation could attenuate the development of left ventricular hypertrophy in an animal model of chronic renal failure.

Methods

Male Sprague-Dawley rats were subjected to sham or remnant kidney surgery and given green tea extract (0.1% and 0.25%) or plain drinking water for the next …

Cardiac Performance In Inbred Rat Genetic Models Of Low And High Running Capacity, J. Chen, G. Feller, J. Barbato, S. Periyasamy, Zijian Xie, L. Koch, Joseph Shapiro, S. Britton

Zijian Xie

Previous work demonstrating that DA inbred rats are superior to COP inbred rats in aerobic treadmill running capacity has indicated their utility as genetic models to explore this trait. We tested the general hypothesis that intermediate phenotypes of cardiac function and calcium metabolism are responsible for the difference in capacity between these strains.

Logical cardiac trait differences were estimated at a tissue (isolated papillary muscle), cellular (isolated left ventricular cells), and biochemical level of organization.

DA hearts were found to give significantly higher values than COP hearts for: (1) maximal developed tension (38.3 % greater), and rates of tension change …

Identification Of A Pool Of Non-Pumping Na/K-Atpase, Man Liang, Jiang Tian, Lijun Liu, Sandrine Pierre Phd, Jiang Liu, Joseph I. Shapiro M.D., Zi-Jian Xie

Zijian Xie

Recent studies have ascribed many non-pumping functions to the Na/K-ATPase. Here, we present experimental evidence demonstrating that over half of the plasma membrane Na/KATPase in LLC-PK1 cells is performing cellular functions other than ion pumping. This “non-pumping” pool of Na/K-ATPase, like the pumping pump, binds ouabain. Depletion of either cholesterol or caveolin-1 moves some of the “non-pumping” Na/KATPase into the pumping pool. Graded knock-down of the 1 subunit of the Na/K-ATPase eventually results in loss of this “non-pumping” pool while preserving the pumping pool. Our prior studies indicate that a loss of the non-pumping pool is associated with a loss …

Na/K-Atpase Mimetic Pnaktide Peptide Inhibits The Growth Of Human Cancer Cells, Zhichuan Li, Zhongbing Zhang, Joe X. Xie, Xin Li, Jiang Tian, Ting Cai, Hongaun Cui, Hanfei Ding, Joseph I. Shapiro Md, Zijian Xie

Zijian Xie

Cells contain a large pool of non-pumping Na/K-ATPase that participates in signal transduction. Here, we show that the expression of α1 Na/K-ATPase is significantly reduced in human prostate carcinoma as well as in several human cancer cell lines. This down-regulation impairs the ability of Na/K-ATPase to regulate Src-related signaling processes. Supplement of pNaKtide, a peptide derived from α1 Na/K-ATPase, reduces activities of Src and Src effectors. Consequently, these treatments stimulate apoptosis and inhibit growth in cultures of human cancer cells. Moreover, administration of pNaKtide inhibits angiogenesis and growth of tumor xenograft. Thus, the new findings demonstrate the in vivo effectiveness …

Gender Differences In The Development Of Uremic Cardiomyopathy Following Partial Nephrectomy: Role Of Progesterone, Christopher Drummond, George Buddny, Steven Haller, Jiang Liu, Yanling Yan, Zijian Xie, Deepak Malhotra, Joseph Shapiro, Jiang Tian

Zijian Xie

Gender difference has been suggested as a risk factor for developing cardiovascular and renal diseases in humans and experimental animals. As a major sex hormone, progesterone was reported to compete with cardiotonic steroid binding to Na/K-ATPase. Our previous publication demonstrated that cardiotonic steroids (e.g., marinobufagenin) play an important role in the development of experimental uremic cardiomyopathy. We also observed that the putative mineralocorticoid antagonists, spironolactone and its major metabolite canrenone, antagonize binding of cardiotonic steroids to Na/K-ATPase in a competitive manner and also ameliorate experimental uremic cardiomyopathy induced by partial nephrectomy. In the following studies, we noted that progesterone displayed …

Effects Of Cardiac Glycosides On Sodium Pump Expression And Function In Llc-Pk1 And Mdck Cells, Jiang Liu, Sankaridrug Periyasamy, William Gunning, Olga Fedorova, Alexi Bagrov, Deepak Malhorta, Zijian Xie, Joseph Shapiro

Zijian Xie

Background: The decreases in proximal tubule sodium reabsorption seen with chronic renal failure and volume expansion have been ascribed to circulating digitalis-like substances (DLS). However, the circulating concentrations of DLS do not acutely inhibit the sodium pump to a degree consistent with the observed changes in proximal tubule sodium reabsorption.

Methods: We examined how cell lines that simulated proximal (LLC-PK1) and distal tubule (MDCK) cells responded to acute (30 min) and long-term (up to 12 hours) Na⁺,K⁺-ATPase inhibition with DLS.

Results: In LLC-PK1, but not MDCK cells, low concentrations of ouabain decreased ⁸⁶Rb uptake profoundly …

Ouabain Induces Endocytosis Of Plasmalemmal Na/K-Atpase In Llc-Pk1 Cells By A Clathrin-Dependent Mechanism, Jiang Liu, Riad Kesiry, Sankaridrug Periyasamy, Deppak Malhorta, Zijian Xie, Joseph Shapiro

Zijian Xie

Background: We have demonstrated that ouabain causes dose- and time-dependent decreases in ⁸⁶Rb uptake in porcine proximal tubular (LLC-PK1) cells. The present study addresses the molecular mechanisms involved in this process.

Methods: Studies were performed with cultured LLC-PK1 and Src family kinase deficient (SYF) cells.

Results: We found that 50 nmol/L ouabain applied to the basal, but not apical, aspect for 12 hours caused decreases in the plasmalemmal Na/K-ATPase. This loss of plasmalemmal Na/K-ATPase reverses completely within 12 to 24 hours after removal of ouabain. Ouabain also increased the Na/K-ATPase content in both early and late endosomes, activated phosphatidylinositol …

Regulation Of Apical Nhe3 Trafficking By Ouabain-Induced Activation Of Basolateral Na/K-Atpase Receptor Complex, Haiping Cai, Liang Wu, Weikai Qu, Deepak Malhotra, Zijian Xie, Joseph I. Shapiro, Jiang Liu

Zijian Xie

The long-term effects of ouabain on transepithelial Na+ transport involve transcriptional downregulation of apical Na+/H+ exchanger isoform 3 (NHE3). The aim of this study was to determine whether ouabain could acutely regulate NHE3 via a posttranscriptional mechanism in LLC-PK1 cells. We observed that the basolateral, but not apical, application of ouabain for 1 h significantly reduced transepithelial Na+ transport. This effect was not due to changes in the integrity of tight junctions or increases in the intracellular Na+ concentration. Ouabain regulated the trafficking of NHE3 and subsequently inhibited its activity, a process independent of intracellular Na+ concentration. Ouabain-induced NHE3 trafficking …

Cardiac Glycoside Downregulates Nhe3 Activity And Expression In Llc-Pk1 Cells, Shadi Oweis, Liang Wu, Pawel Kiela, Deepak Malhotra, Fayez Ghishan, Zijian Xie, Joseph Shapiro, Jiang Liu

Zijian Xie

Ouabain, a cardiotonic steroid and a specific inhibitor of the Na+-K+-ATPase, has been shown to significantly inhibit transcellular Na+ transport without altering the intracellular Na+ concentration ([Na+]i) in the epithelial cells derived from the renal proximal tubules. We therefore studied whether ouabain affects the activity and expression of Na_/H_ exchanger isoform 3 (NHE3) representing the major route of apical Na_ reabsorption in LLC-PK1 cells. Chronic basolateral, but not apical, exposure to low-concentration ouabain (50 and 100 nM) did not change [Na+]i but significantly reduced NHE3 activity, NHE3 protein, and mRNA wxpression. Inhibition of c-Src or phosphoinositide 3-kinase (PI3K) with PP2 …

Ouabain-Induced Endocytosis Of The Plasmalemmal Na/K-Atpase In Llc-Pk1 Cells Requires Caveolin-1, Jiang Liu, Man Liang, Lijun Liu, Deepak Malhotra, Zijian Xie, Joseph Shapiro

Zijian Xie

Background: We have demonstrated that ouabain causes dose- and time-dependent decreases in 86Rb uptake in pig renal proximal tubule cell line (LLC-PK1) cells; and ouabain induces endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells in a clathrin-dependent pathway. Our data also suggest a role of endocytosis in both ouabain-induced signal transduction and proximal tubule sodium handling. The present study addresses the molecular mechanisms involved in this process. Methods: Studies were performed with cultured LLC-PK1 and a stable-expressed caveolin-1 knockdown LLC-PK1 cell line by SiRNA method. Results: In wild-type LLC-PK1 cells, depletion of cholesterol by methyl -cyclodextrin reduced ouabain-induced accumulation of Na/K-ATPase …

Intracellular Reactive Oxygen Species Mediate The Linkage Of Na+/K+-Atpase To Hypertrophy And Its Marker Genes In Cardiac Myocytes, Joseph I. Shapiro, Amir Askari, Zijian Xie, Peter Kometiani, Jie Li, Jiang Liu

Zijian Xie

We showed before that in cardiac myocytes partial inhibition of Na+/K+-ATPase by nontoxic concentrations of ouabain causes hypertrophy and transcriptional regulations of growth-related marker genes through multiple Ca2+-dependent signal pathways many of which involve Ras and p42/44 mitogen-activated protein kinases. The aim of this work was to explore the roles of intracellular reactive oxygen species (ROS) in these ouabain-initiated pathways. Ouabain caused a rapid generation of ROS within the myocytes that was prevented by preexposure of cells to N-acetylcysteine (NAC) or vitamin E. These antioxidants also blocked or attenuated the following actions of ouabain: inductions of the genes of skeletal …

Ouabain And Insulin Induce Sodium Pump Endocytosis In Renal Epithelium, Shalini Gupta, Yanling Yan, Deepak Malhotra, Jiang Liu, Zijian Xie, Sonia Najjar, Joseph Shapiro

Zijian Xie

Cardiotonic steroids signaling through the basolateral sodium pump (Na/K-ATPase) have been shown to alter renal salt handling in intact animals. Because the relationship between renal salt handling and blood pressure is a key determinant of hypertension, and patients with insulin resistance are frequently hypertensive, we chose to examine whether there might be competition for resources necessary for receptor-mediated endocytosis. In LLC-PK1 cells, the Na/K-ATPase-α1 and carcinoembryonic antigen cell adhesion molecule 1, a plasma membrane protein that promotes receptor-mediated endocytosis, colocalized in the plasma membranes and translocated to the intracellular region in response to ouabain. Either ouabain or insulin alone caused …

Ouabain-Stimulated Trafficking Regulation Of The Na/K-Atpase And Nhe3 In Renal Proximal Tubule Cells, Yanling Yan, Steven Haller, Anna Shapiro, Nathan Malhotra, Jiang Tian, Zijian Xie, Deepak Malhotra, Joseph Shapiro, Jiang Liu

Zijian Xie

We have demonstrated that ouabain regulates protein trafficking of the Na/K-ATPase a1 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 a1 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 a1 was replaced by ouabain-resistant rat a1). Ouabain-induced inhibition of transcellular 22Na? transport is due to an ouabain-induced redistribution of the a1 subunit and NHE3. In LLC-PK1 cells, ouabain also …

Impairment Of Na/K-Atpase Signaling In Renal Proximal Tubule Contributes To Dahl Salt-Sensitive Hypertension, Jiang Liu, Yanling Yan, Lijun Liu, Zijian Xie, Deepak Malhotra, Bina Joe, Joseph I. Shapiro

Zijian Xie

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 …

Salt Loading Induces Redistribution Of The Plasmalemmal Na/K-Atpase In Proximal Tubule Cells, Sankaridrug Periyasamy, Jiang Liu, Feras Tanta, Besher Kabak, Brent Wakefield, Deepak Malhorta, David Kennedy, Alaa Nodoor, Olga Fedorova, William Gunning, Zijian Xie, Alexi Bagrov, Joseph Shapiro

Zijian Xie

Background: We have reported that digitalis-like substances (cardiotonic steroids), including marinobufagenin (MBG), induce endocytosis of the plasmalemmal Na/K-ATPase in LLCPK1 cells. The current report addresses the potential relevance of plasmalemmal Na/K-ATPase redistribution to in vivo salt handling. Methods: Male Sprague-Dawley rats were given 1 week of a high salt (4.0% NaCl) or normal salt (0.4% NaCl) diet. Urinary sodium excretion, as well as MBG excretion, was monitored, and proximal tubules were isolated using a Percoll gradient method. Tubular 86Rb uptake, Na/K-ATPase enzymatic activity, and Na/K-ATPase a1 subunit density were determined. Results: The high salt diet increased urinary sodium (17.8 ± …

Ouabain Interaction With Cardiac Na+/K+-Atpase Initiates Signal Cascades Independent Of Changes In Intracellular Na+ And Ca2+, Jiang Liu, Jiang Tian, Michael Haas, Joseph I. Shapiro, Amir Askari, Zijian Xie

Zijian Xie

We have shown previously that partial inhibition of the cardiac myocyte Na+/K+-ATPase activates signal pathways that regulate myocyte growth and growth-related genes and that increases in intracellular Ca2+ concentration ([Ca2+]i) and reactive oxygen species (ROS) are two essential second messengers within these pathways. The aim of this work was to explore the relation between [Ca2+]i and ROS. When myocytes were in a Ca2+-free medium, ouabain caused no change in [Ca2+]i, but it increased ROS as it did when the cells were in a Ca2+-containing medium. Ouabain-induced increase in ROS also occurred under conditions where there was little or no change …