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Reactive Oxygen Species Modulation Of Na/K-Atpase Regulates Fibrosis And Renal Proximal Tubular Sodium Handling, Jiang Liu, David J. Kennedy, Yanling Yan, Joseph I. Shapiro Md

Jiang Liu

The Na/K-ATPase is the primary force regulating renal sodium handling and plays a key role in both ion homeostasis and blood pressure regulation. Recently, cardiotonic steroids (CTS)-mediated Na/K-ATPase signaling has been shown to regulate fibrosis, renal proximal tubule (RPT) sodium reabsorption, and experimental Dahl salt-sensitive hypertension in response to a high-salt diet. Reactive oxygen species (ROS) are an important modulator of nephron ion transport. As there is limited knowledge regarding the role of ROS-mediated fibrosis and RPT sodium reabsorption through the Na/K-ATPase, the focus of this review is to examine the possible role of ROS in the regulation of Na/K-ATPase …

Cd36 And Na/K-Atpase- Α 1 Form A Proinflammatory Signaling Loop In Kidney, David Kennedy, Yiliang Chen, Wenxin Huang, Jamie Viterna, Jiang Liu, Kristen Westfall, Jian Tian, David Bartlett, W.H. Tang, Zijian Xie, Joseph Shapiro, Roy Silverstein

Jiang Liu

Proatherogenic, hyperlipidemic states demonstrate increases in circulating ligands for scavenger receptor CD36 (eg, oxidized low-density lipoprotein [oxLDL]) and the Na/K-ATPase (eg, cardiotonic steroids). These factors increase inflammation, oxidative stress, and progression of chronic kidney disease. We hypothesized that diet-induced obesity and hyperlipidemia potentiate a CD36/Na/K-ATPase–dependent inflammatory paracrine loop between proximal tubule cells (PTCs) and their associated macrophages and thereby facilitate development of chronic inflammation and tubulointerstitial fibrosis. ApoE^-/- and apoE^-/-/cd36^-/- mice were fed a high-fat diet for ≤32 weeks and examined for physiologic and histologic changes in renal function. Compared with apoE^-/-, apoE^{-/- …}

Initial Evidence Of Endothelial Cell Apoptosis As A Mechanism Of Systemic Capillary Leak Syndrome, Ragheb Assaly, Dan Olson, Jeffrey Hammersley, Pan-Sheng Fan, Jiang Liu, Joseph Shapiro, M. Kahaleh

Jiang Liu

Background: Systemic capillary leak syndrome (SCLS) is a rare disorder of unknown etiology that is characterized by acute recurrent attacks of hypovolemic shock commonly following an inflammatory stimulus such as a viral illness. Prophylactic therapy is generally ineffective, and the outcome is frequently fatal. Methods: In order to investigate the cellular mechanisms leading to SCLS, we examined the effects of sera from two patients with active SCLS on microvascular endothelial cell apoptosis in vitro. Apoptosis was determined by morphologic criteria, DNA fragmentation, annexin V stain, and by a quantitative photometric assay. The apoptotic pathway was investigated by Western blot of …

Grp78/Bip Is Involved In Ouabain-Induced Endocytosis Of The Na/K-Atpase In Llc-Pk1 Cells, Riad Kesiry, Jiang Liu

Jiang Liu

We have demonstrated that ouabain causes dose- and time-dependent decreases both in 86Rb+ uptake and plasmalemmal Na/K-ATPase content of LLC-PK1 cells, which is related to ouabain-induced endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells through a clathrin-dependent mechanism. GRP78/BiP is a resident protein of the endoplasmic reticulum (ER) and acts as a molecular chaperone. Recently, several studies have shown that GRP78/BiP is also expressed on the cell surface and forms heterogeneous, high molecular weight complexes with other proteins. To identify the proteins that are possibly involved in ouabain-induced endocytosis of the Na/K-ATPase in LLC-PK1 cells, we separated and identified endosomal proteins …

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

Jiang Liu

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 …

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

Jiang Liu

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, ouabain stimulated direct …

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

Jiang Liu

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 …

Ouabain-Induced Endocytosis And Signal Transduction Of The Na/K-Atpase, Jiang Liu

Jiang Liu

Na/K-ATPase can function as a signal transducer as well as an energy transducing ion pump. Cardiac glycosides (including ouabain and marinobufagin, MBG) are a new class of steroid hormones. Ouabain-activated signaling pathways lead to the induction of some early response proto-oncogenes, activation of transcription factors, and cardiac hypertrophy. Low concentration of ouabain also induced endocytosis of the Na/K-ATPase and compartmentalization of some signaling molecules (e.g. c-Src, EGFR, and p42/44 MAPKs) into clathrin-coated pits, early and late endosomes. Ouabain-induced endocytosis of the Na/K-ATPase depends on the activation of Src kinase, clathrin-coated pits formation, and caveolin-1 (the major component of caveolae). Moreover, …

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

Jiang Liu

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 …

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

Jiang Liu

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 …

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

Jiang Liu

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 …

The Sodium Pump And Cardiotonic Steroids-Induced Signal Transduction Protein Kinases And Calcium-Signaling Microdomain In Regulation Of Transporter Trafficking, Jiang Liu

Jiang Liu

The Na/K-ATPase was discovered as an energy transducing ion pump. A major difference between the Na/K-ATPase and other P-type ATPases is its ability to bind a group of chemicals called cardiotonic steroids (CTS). The plant-derived CTS such as digoxin are valuable drugs for the management of cardiac diseases, whereas ouabain and marinobufagenin (MBG) have been identified as a new class of endogenous hormones. Recent studies have demonstrated that the endogenous CTS are important regulators of renal Na+ excretion and blood pressure. The Na/K-ATPase is not only an ion pump, but also an important receptor that can transduce the ligand-like effect …

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

Jiang Liu

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

Jiang Liu

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

Jiang Liu

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

Jiang Liu

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-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

Jiang Liu

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 …

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

Jiang Liu

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 ± …