Monoclonal antibody to Na,K-ATPase: immunocytochemical localization along nephron segments

To obtain a highly specific reagent that could be utilized for ultrastructural localization of Na,K-ATPase, monoclonal antibodies were produced using microsomal preparations of outer renal medulla of dog and rat enriched for Na,K-ATPase. The monoclonal antibody (C62.4) raised against dog antigen, immunoprecipitated a 96,000 Dalton protein from membranes labeled either with 35S methionine or 3H NAB ouabain. Na,K-ATPase, Na-ATPase, and KpNPPase activity were 25, 60, and 100% maximal after reaction with C62.4. Na,K-ATPase activated with SDS was inhibited, but Na,K-ATPase in tight right-side-out membrane vesicles was not. C62.4 inhibited ouabain binding in the presence of Na,K, and Mg, but did not inhibit ouabain binding in the presence of Mg and Pi. Labeling of broken membranes was readily seen using C62.4 labeled with colloidal gold. Intact right-side-out vesicles showed no evidence of labeling, demonstrating that the antibody is directed to an epitope of the cytoplasmic domain of the enzyme. Differential localization of C62.4 along the nephron was identified. Glomeruli showed no significant antibody binding except by occasional cells in the mesangial regions. Only basal lateral membranes of cells from all tubule segments labeled with C62.4. There was no evidence of specific apical labeling. The thick ascending limb of Henle's loop demonstrated the greatest concentration of antibody binding. In the cortical and outer medullary collecting duct, only principal cells showed abundant antibody binding. Intercalated cells showed no detectable evidence of antibody binding on any surface. These studies demonstrate that Na,K-ATPase is localized exclusively to the basal lateral membrane of renal tubular epithelial cells and varies in density and distribution in different nephron segments.     

Salt loading induces redistribution of the plasmalemmal Na/K-ATPase in proximal tubule cells

BACKGROUND: We have reported that digitalis-like substances (cardiotonic steroids), including marinobufagenin (MBG), induce endocytosis of the plasmalemmal Na/K-ATPase in LLC-PK1 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 (86)Rb uptake, Na/K-ATPase enzymatic activity, and Na/K-ATPase alpha1 subunit density were determined. RESULTS: The high salt diet increased urinary sodium (17.8 +/- 1.8 vs. 2.5 +/- 0.3 mEq/day, P < 0.01) and MBG excretion (104 +/- 12 vs. 26 +/- 4 pmol/day), and decreased proximal tubular (86)Rb uptake (0.44 +/- 0.07 vs. 1.00 +/- 0.10, P < 0.01) and Na/K-ATPase enzymatic activity (5.1 +/- 1.1 vs. 9.9 +/- 1.6 micromol/mg pr/hr, P < 0.01) relative to the normal diet. Proximal tubular Na/K-ATPase alpha1 protein density was decreased in the plasmalemma fraction but increased in both early and late endosomes following the high salt diet. In rats fed a high salt diet, anti-MBG antibody caused a 60% reduction in urinary sodium excretion, substantial increases in proximal tubule (86)Rb uptake, and Na/K-ATPase enzymatic activity, as well as significant decreases in the early and late endosomal Na/K-ATPase alpha1 protein content. CONCLUSION: These data suggest that redistribution of the proximal tubule Na/K-ATPase in response to endogenous cardiotonic steroids plays an important role in renal adaptation to salt loading.     

Effects of hydrochlorothiazide on Na-K-ATPase activity along the rat nephron

Na-K-ATPase activity was determined in seven nephron segments of five-week-old, spontaneously hypertensive rats (SHR) with or without continuous hydrochlorothiazide (HCTZ) treatment for seven days. For comparison, the effects of HCTZ treatment on Na-K-ATPase activity in the nephron segments of age-matched normotensive Wistar-Kyoto rats (WKY) were also determined. Na-K-ATPase activity in proximal convoluted tubule (PCT), medullary thick ascending limb (MTAL), cortical thick ascending limb (CTAL), distal convoluted tubule (DCT) and cortical collecting duct (CCD) was significantly lower in HCTZ-treated SHR compared to control (untreated) SHR. However, there was no significant difference in Na-K-ATPase activity in proximal straight tubule (PST) and medullary collecting duct (MCD) between HCTZ-treated and control SHR. HCTZ treatment also produced a significant decrease in blood pressure (BP) and creatinine clearance (CCr) in SHR. On the other hand, HCTZ treatment did not produce a significant change in Na-K-ATPase activity in PCT, PST, MTAL, CTAL and MCD, in BP or in CCr in WKY. However, HCTZ treatment produced a decrease in the enzyme activity in the DCT and an increase in the enzyme activity in the CCD in WKY. The decrease in Na-K-ATPase activity in almost all nephron segments from SHR may be due to a significant decrease in CCr produced by HCTZ. On the other hand, a decrease in Na-K-ATPase activity in the DCT with an increase in the enzyme activity in the CCD from WKY suggest that renal compensation to the natriuretic effect of HCTZ occurs by an increase in Na+ reabsorption in the CCD.     

Branched-chain amino acid aminotransferase along the rabbit and rat nephron

The activity of branched-chain amino acid aminotransferase (EC 2.6.1.42) is reported for four or five different segments of the rat and rabbit nephron as well as for patches from the papilla. In the rat the levels ranged 40-fold, from a high in the thick ascending limb of Henle to a low in the proximal convoluted tubule. The peak activity is far above that reported for most other parts of the body. Maximum activity was located also in the thick ascending limb in the rabbit, but the level was only one-third as high as in the rat. It is postulated that ammonia liberated by this amino transferase, in cooperation with glutamate dehydrogenase, could diffuse readily into the adjacent proximal straight tubule where all of the renal glutamine synthase and the highest level of alanine aminotransferase are located. Thus alanine and glutamine could be produced when the ammonia was not needed to neutralize excess acidity.     

Gene expression of prostanoid forming enzymes along the rat nephron

BACKGROUND: To obtain information about the general capability of nephron segments to elaborate prostanoids, we determined the gene expression of key enzymes for prostanoid formation. METHODS: For this goal mRNAs were assayed for cyclooxygenases-1 and -2 as well as for the synthases of prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostacyclin (PGI2) and thromboxane A2 (TXA2) in microdissected rat nephron segments by RT-PCR. RESULTS: Cyclooxygenase-1 (COX-1) mRNA was strongly expressed in all segments of the collecting ducts and to a lesser extent in glomeruli. COX-2 mRNA was found in the cortical thick ascending limb of Henle, and weaker expression also was detected in glomeruli. The lipocalin-type PGD synthase mRNA displayed a broad expression pattern in the cortex and outer medulla, including proximal convoluted tubule, thick ascending limb of Henle, distal convoluted tubule, and cortical and outer medullary collecting duct. The hematopoietic PGD synthase mRNA was restricted to the outer medullary collecting duct, and the membrane-associated PGE-synthase mRNA was exclusively expressed in the whole collecting duct system. Prostacylin-synthase mRNA was found in the whole kidney, but not in any microdissected nephron segment analyzed in this study. TXA-synthase mRNA was expressed in glomeruli. CONCLUSION: Given that the existence of cyclooxygenase in combination with the different PG-synthases is a prerequisite for the formation of prostanoids, our data suggest that PGD2 is mainly formed in the thick ascending limb and in the collecting duct, while PGE2 appears to be mainly generated by the collecting ducts. Probably no formation of PGI2 occurs within the nephron. Whether TXA2 can be formed by nephron segments remains questionable.     

Ouabain induces endocytosis of plasmalemmal Na/K-ATPase in LLC-PK1 cells by a clathrin-dependent mechanism

BACKGROUND: We have demonstrated that ouabain causes dose- and time-dependent decreases in (86)Rb 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 3-kinase (PI(3)K), and also caused a translocation of some Na/K-ATPase to the nucleus. Immunofluorescence demonstrated that the Na/K-ATPase colocalized with clathrin both before and after exposure to ouabain, and immunoprecipitation experiments confirmed that ouabain stimulated interactions among the Na/K-ATPase, adaptor protein-2 (AP-2), and clathrin. Potassium (K) depletion, chlorpromazine, or PI(3)K inhibition all significantly attenuated this ouabain-induced endocytosis. Inhibition of the ouabain-activated signaling process through Src by 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) significantly attenuated ouabain-induced endocytosis. Moreover, experiments performed in SYF cells demonstrated that ouabain induced increases in the endocytosis of the Na/K-ATPase when Src was reconstituted (SYF+), but not in the Src-deficient (SYF-) cells. CONCLUSION: These data demonstrate that ouabain stimulates a clathrin-dependent endocytosis pathway that translocates the Na/K-ATPase to intracellular compartments, thus suggesting a potential role of endocytosis in ouabain-induced signal transduction as well as proximal tubule sodium handling.     

Inhibition of Na,K-ATPase by dopamine in proximal tubule epithelial cells

In the current report we review the results that lay grounds for the model of intracellular sodium-mediated dopamine-induced endocytosis of Na,K-ATPase. Under conditions of a high salt diet, dopamine activates PKCzeta, which phosphorylates NKA alpha1 Ser-18. The phosphorylation produces a conformational change of alpha1 NH2-terminus, which through interaction with other domains of alpha1 exposes PI3K- and AP-2-binding domains. PI3K bound to the NKA alpha1 induces the recruitment and activation of other proteins involved in endocytosis, and PI3K-generated 3-phosphoinositides affect the local cytoskeleton and modify the biophysical conditions of the membrane for development of clathrin-coated pits. Plasma membrane phosphorylated NKA is internalized to specialized intracellular compartments where the NKA will be dephosphorylated. The NKA internalization results in a reduced Na+ transport by proximal tubule epithelial cells.     

Multiple functions of Na,K-ATPase in epithelial cells

The Na,K-adenosine triphosphatase (ATPase), or sodium pump, has been well studied for its role in the regulation of ion homeostasis in mammalian cells. Recent studies suggest that Na,K-ATPase might have multiple functions such as a role in the regulation of tight junction structure and function, induction of polarity, regulation of actin dynamics, control of cell movement, and cell signaling. These functions appear to be modulated by Na,K-ATPase enzyme activity as well as protein-protein interactions of the alpha and beta subunits. In this review we attempt to differentiate functions associated with enzyme activity and subunit interactions. In addition, the consequence of impaired Na,K-ATPase function or reduced subunit expression levels in kidney diseases such as cancer, tubulointerstitial fibrosis, and ischemic nephropathy are discussed.     

Ouabain-induced endocytosis of the plasmalemmal Na/K-ATPase in LLC-PK1 cells requires caveolin-1

BACKGROUND: We have demonstrated that ouabain causes dose- and time-dependent decreases in (86)Rb 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 beta-cyclodextrin reduced ouabain-induced accumulation of Na/K-ATPase alpha-1 subunit, EGFR, Src, and MAPKs in clathrin-coated vesicles, as well as in endosomes. Depletion of cholesterol also significantly reduced the protein-protein interaction among alpha-1 subunit, AP2, PI-3K, and clathrin heavy chain. In LLC-PK1 cells expressing mock-vehicle and caveolin-1 siRNA, depletion of caveolin-1 abolished ouabain-induced decrease in Rb uptake and decrease in the plasmalemmal Na/K-ATPase content. Depletion of caveolin-1 also significantly reduced the ouabain-induced accumulation of Na/K-ATPase alpha-1 subunit, EGFR, Src, and MAPKs in clathrin-coat vesicles, as well as early and late endosomes. In addition, depletion of caveolin-1 also significantly reduced the protein-protein interaction among alpha-1 subunit, AP2, PI-3K, and clathrin heavy chain. These data suggest that caveolae are involved in ouabain-induced endocytosis and signal transduction by initiating assembly of signaling cascades through the caveolar Na/K-ATPase and/or the interaction with clathrin-mediated endocytosis of the Na/K-ATPase.     

Adaptation of intercalated cells along the collecting duct to systemic acid/base changes

Collecting duct intercalated cells respond to short-term acid/base perturbations by rapidly shuttling H(+)-ATPase to and from the plasma membrane. Purkerson et al. provide information on the regulation of the anion transporters during chronic acidosis and acute recovery (alkalosis). They found that the major mechanism for both acute and chronic states is regulation of both the H(+)-ATPase and the anion exchangers plus changes in the overall expression level of these anion transporters in chronic adaptation.     

Expression and cellular localization of Na,K-ATPase isoforms in the rat ventral prostate

OBJECTIVE: To determine the expression and plasma membrane domain location of isoforms of Na,K-ATPase in the rat ventral prostate. MATERIALS AND METHODS: Ventral prostate glands from adult male rats were dissected, cryosectioned (7 micro m) and attached to poly-l-lysine coated glass slides. The sections were then fixed in methanol and subjected to indirect immunofluorescence and immunoperoxidase procedures using a panel of well-characterized monoclonal and polyclonal antibodies raised against known Na,K-ATPase subunit isoforms. Immunofluorescence micrographs were digitally captured and analysed by image analysis software. RESULTS: There was expression of Na,K-ATPase alpha1, beta1, beta2 and beta3 subunit isoforms in the lateral and basolateral plasma membrane domains of prostatic epithelial cells. The alpha1 isoform was abundant but there was no evidence of alpha2, alpha3 or gamma isoform expression in epithelial cells. The alpha3 isoform was not detected, but there was a relatively low level of alpha2 isoform expression in the smooth muscle and stroma. CONCLUSION: Rat prostate Na,K-ATPase consists of alpha1/beta1, alpha1/beta2 and alpha1/beta3 isoenzymes. These isoform proteins were located in the lateral and basolateral plasma membrane domains of ventral prostatic epithelial cells. The distribution and subcellular localization of Na,K-ATPase is different in rodent and human prostate. Basolateral Na,K-ATPase probably contributes to the establishment of transepithelial ionic gradients that are a prerequisite for the uptake of metabolites by secondary active transport mechanisms and active citrate secretion.     

Distribution of pyruvate carboxylase along the rat nephron: an immunological and enzymatic study

Antibodies against purified rat-kidney-cortex pyruvate carboxylase were raised in rabbits. These polyspecific antibodies recognize pyruvate carboxylase enzymes alone without cross reactivity with other carboxylases as detected by immunoblotting. The abundance of pyruvate carboxylase in the various renal fractions was measured by ELISA and its activity by the fixation of [14C]CO3H-. The results were corroborated by a combination of immunocytochemistry and transmission electron microscopy. A good correlation was found between the enzymatic activity and the quantity of enzyme contained in each fraction. The kidney-cortex pyruvate carboxylase was primarily located in proximal tubules, in accord with its important role in gluconeogenesis.     

Expression of the alpha-subunit of Na/K-ATPase in renal collecting duct epithelium during development

Aldosterone enhances synthesis and enzyme activity of Na/K-ATPase and has been found to stimulate sodium reabsorption by the renal cortical collecting duct. Moreover, chronic exposure to aldosterone is associated with a remarkable morphological-functional adaptation. This is seen as a magnification in basolateral membrane area of principal cells. In the present paper we investigated the acquistion of Na/K-ATPase alpha-subunit in renal tissue and examined whether aldosterone initiates functional and adaptive changes in cultured collecting duct cells similar to those observed in vivo. Using a monoclonal antibody, immunofluorescence microscopy demonstrated the acquisition of the alpha-subunit of Na/K-ATPase in the developing cortical collecting ducts of the kidney of neonatal rabbits. The mature collecting ducts in the medulla and papilla of the developing kidney were strongly labelled at the basolateral side, while in the cortical portion of the fetal collecting duct adjacent to the embryonic ampullae the immunolabel was found at the apical and the basolateral aspect of the epithelium. However, the embryonic collecting duct ampullae in the outer cortex did not show any reaction with the antibody. In the collecting duct cells cultured for 24 hours the alpha-subunit of Na/K-ATPase was found to be distributed at both the apical and basolateral aspect of the epithelium. After two to 16 days, the immunolabel was strictly found distributed at the basolateral side. Culturing collecting duct cells in the presence of aldosterone (10(-6) M), the hormone modulated the cellular shape of epithelia after five days by infolding the lateral plasma membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin regulation of Na/K pump activity in rat hepatoma cells

Insulin rapidly increases Na/K pump activity in HTC rat hepatoma cells in tissue culture, as measured by the ouabain-sensitive influx of the potassium analogue 86Rb+. Increased influx is observed within minutes and is maximal (70% above control) within 1-2 h. The effect appears to be mediated by the insulin receptors, as: the concentration dependence on insulin is identical to that for insulin induction of tyrosine aminotransferase and stimulation of 2-aminoisobutyric acid transport, proinsulin is 6% as potent as insulin, and the effect is blocked by anti-receptor antibodies. The early stimulation of potassium influx is not blocked by cycloheximide and is not associated with an increased number of pump sites as measured by 3H-ouabain binding. The insulin effect is blocked by amiloride, which blocks sodium influx, and is mimicked by the sodium ionophore monensin, which increases sodium influx and intracellular accumulation. Insulin also rapidly increases the initial rate of 22Na+ influx, suggesting that insulin may enhance Na/K pump activity, in part, by increasing intracellular sodium concentration. Incubation of HTC cells with insulin for 24 h causes complete unresponsiveness to the insulin induction of transaminase and stimulation of amino acid transport, a phenomenon mediated by postbinding mechanisms. In contrast, similar incubation with insulin does not cause unresponsiveness to the insulin stimulation of Na/K pump activity. Therefore, the site of regulation of responsiveness to insulin must be distal to, or separate from, those events causing stimulation of ion fluxes.     

Calbindin-D9k and parvalbumin are exclusively located along basolateral membranes in rat distal nephron.

There is strong evidence that vitamin D-dependent Ca(2+)-binding proteins, i.e., calbindin-D9k and calbindin-D28k, facilitate diffusion of Ca2+ through the cytosolic compartment of renal and intestinal cells, which transport Ca2+ transcellularly. In the study presented here, parvalbumin, calbindin-D9k, and calbindin-D28k were localized precisely by immunocytochemistry in rat kidney. Antisera recognizing specifically the thick ascending loop of Henle, the connecting tubules and collecting ducts, and the intercalated cells of the collecting ducts were used to identify different cell types. In rat kidney cortex, parvalbumin and calbindin-D9k colocalized in the thick ascending loop of Henle, the distal convoluted tubule, the connecting tubule, and the intercalated cells of the collecting duct. Strikingly, in all responsive cells, parvalbumin and calbindin-D9k were exclusively present in a thin layer along the basolateral membrane. In contrast, calbindin-D28k was only present in the distal convoluted and connecting tubule, where it was evenly distributed through the cytosol. In conclusion, the exclusive localization of parvalbumin and calbindin-D9k at the basolateral membrane of immunopositive renal cells implies their involvement in the regulation of transport processes located in these membranes rather than a role as intracellular Ca2+ buffer and Ca2+ shuttle between the two opposing membranes.     

Extracellular hypotonicity increases Na,K-ATPase cell surface expression via enhanced Na+ influx in cultured renal collecting duct cells

In the renal collecting duct (CD), the Na,K-ATPase, which provides the driving force for Na+ absorption, is under tight multifactorial control. Because CD cells are physiologically exposed to variations of interstitial and tubular fluid osmolarities, the effects of extracellular anisotonicity on Na,K-ATPase cell surface expression were studied. Results show that hypotonic conditions increased, whereas hypertonic conditions had no effect on Na,K-ATPase cell surface expression in confluent mpkCCDcl4 cells. Incubating cells with amphotericin B, which increases [Na+]i, under isotonic or anisotonic conditions, revealed that Na,K-ATPase recruitment to the cell surface was not directly related to variations of cell volume and osmolarity. The effects of amphotericin B and extracellular hypotonicity were not additive, and both were prevented by protein kinase A and proteasome inhibitors, suggesting a common mechanism of action. In line with this hypothesis, extracellular hypotonicity induced a sustained stimulation of the amiloride-sensitive short-circuit current, indicating increased Na+ influx through the apical epithelial Na+ channel. Moreover, inhibiting apical Na+ entry by amiloride, a blocker of epithelial Na+ channel, or incubating cells in Na+ -free medium prevented the cell surface recruitment of Na,K-ATPase in response to extracellular hypotonicity. Altogether, these findings strongly suggest that extracellular hypotonicity stimulates apical Na+ influx leading to increased [Na+]i, protein kinase A activation, and recruitment of Na,K-ATPase units to the cell surface of mpkCCDcl4 cells.     

Heme oxygenase-1 localization in the rat nephron

BACKGROUND/AIMS: Renal tubules undergo oxidative injury in various nephropathies. It is unknown whether tubular cells possess mechanisms to attenuate this form of injury. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, may provide such a mechanism by reducing levels of free heme, a prooxidant molecule, and by limiting activity of heme-containing prooxidant enzymes. Determination of the distribution of HO-1 in the nephron may identify those segments where HO-1 can afford protection against oxidative injury. METHODS: Rats were injected subcutaneously with two different inducers of HO-1: Stannous chloride and cobalt protoporphyrin. At completion of injections, frozen sections of kidneys were stained for HO-1 using a biotin-conjugated monoclonal anti-HO-1 antibody. To identify the origin of tubules staining positive for HO-1, Tetragonolobus purpureas (TP)-derived lectin and Arachnis hypogaea (AH)-derived lectin were applied to sequential sections of the kidney cortex. RESULTS: In rats injected with either HO-1 inducer, HO-1 was immunolocalized in tubules but not in glomeruli. Staining of sequential sections with TP-derived lectin, which binds mainly to proximal tubular cells, was negative in the tubules that stained positive for HO-1. Staining of sequential sections with AH-derived lectin, which binds mainly to distal and collecting tubular cells, was positive in those tubules that were also positive for HO-1. CONCLUSIONS: In kidneys of rats injected with inducers of HO-1, distal and collecting tubular cells were identified as the main segments of the nephron that express HO-1. We suggest that the distal nephron, by expressing HO-1, may be less vulnerable to oxidative injury.