Kidney vacuolar H+ -ATPase: physiology and regulation

The vacuolar H(+)-ATPase is a multisubunit protein consisting of a peripheral catalytic domain (V(1)) that binds and hydrolyzes adenosine triphosphate (ATP) and provides energy to pump H(+) through the transmembrane domain (V(0)) against a large gradient. This proton-translocating vacuolar H(+)-ATPase is present in both intracellular compartments and the plasma membrane of eukaryotic cells. Mutations in genes encoding kidney intercalated cell-specific V(0) a4 and V(1) B1 subunits of the vacuolar H(+)-ATPase cause the syndrome of distal tubular renal acidosis. This review focuses on the function, regulation, and the role of vacuolar H(+)-ATPases in renal physiology. The localization of vacuolar H(+)-ATPases in the kidney, and their role in intracellular pH (pHi) regulation, transepithelial proton transport, and acid-base homeostasis are discussed.     

The effect of kidney transplantation on distal tubular vacuolar H+-ATPase

BACKGROUND: The presence of vacuolar (v)H+-ATPase in distal tubule alpha-intercalated cells is essential for hydrogen excretion and maintenance of acid-base homeostasis. Loss of vH+-ATPase after kidney transplantation could cause posttransplant distal renal tubular acidosis (dRTA). METHOD: Immunostaining of the kidney specific vH+-ATPase of cortical collecting duct cells (CCT) was performed in 37 kidney biopsies taken immediately prior to transplantation and after engraftment (median [range]: 10 [1-181] months). Apical or intracytoplasmatic staining intensity was classified as grade 0 (absent), grade 1 (weak), or grade 2 (strong), and positive cells expressed as percentage of all CCT cells. In addition, kidney biopsies were scored for damage by the Banff schema. Serum and urinary pH, anion gap, and serum potassium were obtained for the diagnoses of dRTA. RESULTS: Fourteen transplant recipients had dRTA type I, 5 had rate-limited RTA, six had type IV dRTA, and 12 had no RTA. In pretransplant biopsies, 40% [3-77%] of CCT cells were positive for vH+-ATPase but only 17% [0-39%] after transplantation (P<0.0001). The loss of vH+-ATPase expression was similar in patients with dRTA type I (-21%), type IV (-25%), rate limited RTA (-21%), or no RTA (-29%). The decrease affected predominantly the apical proton pump expression. The individual loss of vH+-ATPase expression was not related to the time elapsed since transplantation, immunosuppressive drugs, acute transplant rejection, or tubulointerstitial changes. CONCLUSION: Kidney transplantation leads to a general decrease of distal tubular vH+-ATPase expression. Loss of proton pump activity occurs unrelated to immunosuppressive therapy or transplant related histologic changes.     

Na+ and K+ transport by the renal connecting tubule

PURPOSE OF REVIEW: The connecting tubule is emerging as a nephron segment critical to the regulation of Na+ and K+ excretion and the maintenance of homeostasis for these ions. The segment is difficult to study, however, and much of the available information we have concerning its functions is indirect. Here, we review the major transport mechanisms and transporters found in this segment and outline several unsolved problems in the field. RECENT FINDINGS: Recent electrophysiological and immunohistochemical measurements together with theoretical studies provide a more comprehensive view of ion transport in the connecting tubule. New signaling pathways governing Na+ and K+ transport have also been described. SUMMARY: Key questions about how Na+ and K+ transport are regulated remain unanswered. Is the connecting tubule the site of final regulation of both Na+ and K+ excretion? If so, how are the transport rates of these two ions independently controlled?     

Involvement of vacuolar H+ -ATPase in incorporation of risedronate into osteoclasts

Although osteoclasts incorporate bisphosphonates during bone resorption, the mechanism of this incorporation by osteoclasts is not known. We previously reported that bisphosphonates disrupt the actin rings (clear zones) formed in normal osteoclasts, but did not disrupt actin rings in osteoclasts derived from osteosclerotic oc/oc mice, which have a defect in the gene encoding vacuolar H(+)-ATPase (V-ATPase). The present study showed that V-ATPase is directly involved in the incorporation of risedronate, a nitrogen containing bisphosphonate, into osteoclasts. Treatment of osteoclasts with risedronate disrupted actin rings and inhibited pit formation by osteoclasts on dentine slices. Bafilomycin A(1), a V-ATPase inhibitor, inhibited the pit-forming activity of osteoclasts but did not disrupt actin rings. Risedronate failed to disrupt actin rings in the presence of bafilomycin A(1). E-64, a lysosomal cysteine proteinase inhibitor, showed no inhibitory effect on the demineralization of dentine by osteoclasts but inhibited the digestion of dentine matrix proteins without disrupting actin rings. Risedronate disrupted actin rings even in the presence of E-64. Treatment of osteoclasts placed on plastic plates with risedronate also disrupted actin rings. Bafilomycin A(1) but not E64 prevented the disruption of actin rings in osteoclasts treated with risedronate on plastic plates. Inhibition of V-ATPase with bafilomycin A(1) also prevented disruption of actin rings by etidronate, a non-nitrogen-containing bisphosphonate. These results suggest that V-ATPase induced acidification beneath the ruffled borders of osteoclasts and subsequent bone demineralization triggers the incorporation of both nitrogen-containing and non-nitrogen-containing bisphosphonates into osteoclasts.     

A new regulator of the vacuolar H(+)-ATPase in the kidney

Xu et al. identify Slc26a11, a novel member of the Slc26 anion exchanger family, as an electrogenic (Cl(-))(n)/HCO(3)(-) exchanger. Functional characterization of this transporter suggests that Slc26a11 mediates classical electroneutral Cl(-)/HCO(3)(-) exchange but also exhibits an electrogenic Cl(-) conductance. In the kidney, Slc26a11 colocalizes with the vacuolar H(+)-ATPase in intercalated cells, emphasizing the cooperation of the proton pump with chloride transporters.     

Actin binding activity of subunit B of vacuolar H+-ATPase is involved in its targeting to ruffled membranes of osteoclasts.

Adeno-associated virus was used to transduce primary mouse osteoclasts with the B1 isoform of vacuolar H(+)-ATPase. B1, which is not normally expressed in osteoclasts, was correctly targeted to ruffled membranes of resorbing osteoclasts. Mutant subunit B1 that lacked a functional actin-binding site did not accumulate in ruffled membranes. INTRODUCTION: The B1 "kidney" and B2 "brain" isoforms of subunit B of vacuolar H(+)-ATPase (V-ATPase) have actin binding sites that mediate interactions between the intact enzyme and filamentous-actin. Accumulating data support the hypothesis that the actin binding activity in subunit B is required for targeting of V-ATPases to the ruffled plasma membrane of osteoclasts. This study was designed to directly test this hypothesis. MATERIALS AND METHODS: Osteoclasts express B2, but not B1. Adeno-associated virus vectors were used to transduce mouse osteoclasts with wildtype B1 or B1(mut), a full-length B subunit that contained minor alterations that disrupted actin-binding activity. Immunofluorescence was performed using polyclonal antibodies specific for subunit E, B2, and B1 of V-ATPase. Immunoprecipitations were performed using an anti-E subunit antibody. Microfilaments were detected with phalloidin and actin rings were stained with phalloidin or anti-vinculin antibodies. Images were collected using a confocal microscope. RESULTS: Immunoprecipitations of transduced osteoclasts suggested that both B1 and B1(mut) assembled with endogenous V-ATPase subunits to form intact enzyme in osteoclasts. Both B1 and B1(mut) were localized like endogenous V-ATPase subunits in unactivated osteoclasts. Wildtype B1 associated with the detergent-insoluble cytoskeleton and was transported to ruffled membranes of resorbing osteoclasts. In contrast, B1(mut) failed to associate with the actin cytoskeleton and was not transported efficiently to ruffled membranes. CONCLUSIONS: The B1 isoform of B subunit contains the necessary information for targeting to the ruffled membranes of osteoclasts even though it is not normally expressed in osteoclasts. The actin binding activity of B1 is involved in proper ruffled membrane targeting.     

Angiotensin II stimulates vacuolar H+ -ATPase activity in renal acid-secretory intercalated cells from the outer medullary collecting duct

Final urinary acidification is mediated by the action of vacuolar H(+)-ATPases expressed in acid-secretory type A intercalated cells (A-IC) in the collecting duct. Angiotensin II (AngII) has profound effects on renal acid-base transport in the proximal tubule, distal tubule, and collecting duct. This study investigated the effects on vacuolar H(+)-ATPase activity in A-IC in freshly isolated mouse outer medullary collecting ducts. AngII (10 nM) stimulated concanamycin-sensitive vacuolar H(+)-ATPase activity in A-IC in freshly isolated mouse outer medullary collecting ducts via AT(1) receptors, which were also detected immunohistochemically in A-IC. AngII increased intracellular Ca(2+) levels transiently. Chelation of intracellular Ca(2+) with BAPTA and depletion of endoplasmic reticulum Ca(2+) stores prevented the stimulatory effect on H(+)-ATPase activity. The effect of AngII on H(+)-ATPase activity was abolished by inhibitors of small G proteins and phospholipase C, by blockers of Ca(2+)-dependent and -independent isoforms of protein kinase C and extracellular signal-regulated kinase 1/2. Disruption of the microtubular network and cleavage of cellubrevin attenuated the stimulation. Finally, AngII failed to stimulate residual vacuolar H(+)-ATPase activity in A-IC from mice that were deficient for the B1 subunit of the vacuolar H(+)-ATPase. Thus, AngII presents a potent stimulus for vacuolar H(+)-ATPase activity in outer medullary collecting duct IC and requires trafficking of stimulatory proteins or vacuolar H(+)-ATPases. The B1 subunit is indispensable for the stimulation by AngII, and its importance for stimulation of vacuolar H(+)-ATPase activity may contribute to the inappropriate urinary acidification that is seen in patients who have distal renal tubular acidosis and mutations in this subunit.     

Immunolocalization of NBC3 and NHE3 in the rat epididymis: colocalization of NBC3 and the vacuolar H+-ATPase

In the male reproductive tract, the epididymis plays an important role in mediating transepithelial bicarbonate transport and luminal acidification. In the proximal vas deferens, a significant component of luminal acidification is Na+-independent, and mediated by specific cells that possess apical vacuolar proton pumps. In contrast, luminal acidification in the cauda epididymidis is an Na+-dependent process. The specific apical Na+-dependent H+/base transport process(es) responsible for luminal acidification have not been identified. A potential clue as to the identity of these apical Na+-dependent H+/base transporter(s) is provided by similarities between the transport properties of the epididymis and the mammalian nephron. Specifically, the H+/base transport properties of caput epididymidis resemble the mammalian renal proximal tubule, whereas the distal epididymis and vas deferens have characteristics in common with renal collecting duct intercalated cells. Given the known expression of the Na+/H+ antiporter, NHE3, in the proximal tubule, and of the electroneutral sodium bicarbonate cotransporter, NBC3, in renal intercalated cells, we determined the localization of NHE3 and NBC3 in various regions of rat epididymis. NBC3 was highly expressed on the apical membrane of apical (narrow) cells in caput epididymidis, and light (clear) cells in corpus and cauda epididymidis. The number of cells expressing apical NBC3 was highest in cauda epididymidis. The localization of NBC3 in the epididymis was identical to the vacuolar H+-ATPase. The results indicate that colocalization of NBC3 and the vacuolar H+-ATPase is not restricted to kidney intercalated cells. Moreover, the close association of the two transporters appears to be a more generalized phenomenon in cells that express high levels of vacuolar H+-ATPase. Unlike NBC3, NHE3 was most highly expressed on the apical membrane of all epithelial cells in caput epididymidis, with less expression in the corpus, and no expression in the cauda. These results suggest that apical NBC3 and NHE3 potentially play an important role in mediating luminal H+/base transport in epididymis.     

Immunocytochemical localization of vacuolar H+-ATPase and Cl−-HCO 3 − anion exchanger (erythrocyte band-3 protein) in avian osteoclasts: Effect of calcium-deficient diet on polar expression of the H+-ATPase pump

Osteoclasts attach to the bone surface and resorb bone by secreting protons into an isolated subosteoclastic compartment. Previous studies have shown the presence of a vacuolar type H⁺-ATPase, and a functional Cl⁻-HCO ₃ ⁻ anion exchanger in the osteoclast. In the present studies, using a monoclonal antibody to the 31-kDa subunit of H⁺-ATPase and a rabbit antiserum to the erythrocyte band-3 protein (Cl⁻-HCO ₃ ⁻ anion exchanger) we have immunocytochemically localized the respective pumps in bone sections obtained from chickens fed a normal or a calcium-deficient diet for 4 weeks. Our results indicate that although H⁺-ATPase is either evenly distributed throughout the osteoclast or is more polarized at its ruffled membrane juxtaposed to the bone surface, the band-3 protein immunoreactivity is always localized to the plasma membrane which is not attached to the bone surface (basolateral membrane). Four weeks of a calcium-deficient diet resulted in a significant increase in the percentage of osteoclasts that were polarized for the H⁺-ATPase pump at their ruffled membrane, and a trend toward increased total number of osteoclasts, although the latter did not reach statistical significance (P =0.09). These changes were not accompanied by a significant increase in the intensity of staining for H⁺-ATPase. Band-3 protein immunoreactivity was always prominent, limited to the basolateral membrane, and did not alter with calcium-deficient diet or with changes in the degree of H⁺-ATPase polarization. Parts of this work have been presented in the American Society of Nephrology in November 1992     

The connecting tubule: a functional subdivision of the rabbit distal nephron segments

Studies on adenylate cyclase response of the nephron fragments to hormones and drugs have suggested that there is a functionally distinct segment tentatively called the connecting tubule (CNT), which is located between the distal (DT) and the cortical collecting tubule (CCT). The functional significance of these biochemical findings was examined with isolated rabbit renal tubules perfused in vitro. The transepithelial voltage (PDt) of the DT, CNT, and CCT were, respectively, -28.7 +/- 3.24 mV (24), -27.0 +/- 2.69 mV (24), and -3.5 +/- 2.14 mV (11) in the normal rabbits. The PDt of the CCT increased to -32.2 +/- 2.02 mV (33) when rabbits were pretreated for at least 3 days with deoxycorticosterone acetate, DOCA (1 mg/kg/day, i.m.), whereas the PDt of the DT and the CNT remained unchanged. The PDt of the CCT obtained from deoxycorticosterone acetate- (DOCA) treated animals decreased after addition of antidiuretic hormone (ADH) or isoproterenol (ISO) to the bath. The PDt of the CNT also responded to these agents, but the dose required to obtain the same response was quite different: The CNT was 100-fold more sensitive to ISO as compared to the CCT, whereas the CCT was 10-fold more sensitive to ADH than was the CNT. In contrast, the PDt of the DT did not respond to any of these agents, even at a higher concentration. After addition of ADH (200 microU/ml) to the bath, the osmotic water permeability (10(-8) cm2 . sec-1 . atm-1) of the CCT increased from 1.13 +/- 0.83 to 7.46 +/- 2.36, but that of the CNT remained low (0.36 +/- 0.78 in control vs. 0.48 + 0.64 after ADH). These observations support the view that the CNT is functionally distinct from either the DT or the CCT.     

The gamma-Na+,K+-ATPase subunit assembles selectively with alpha1/beta1-Na+,K+-ATPase but not with the colonic H+,K+-ATPase

BACKGROUND: The ubiquitous Na+-pump (Na+,K+-ATPase) assembles as a heterodimer of composition alpha/beta in some nephron segments, while in other segments it may exist as a heterotrimer of composition alpha/beta/gamma. The gamma-subunit has been reported to increase the affinity of the Na+-pump for adenosine 5'-triphosphate (ATP), and decrease affinity for both Na+ and K+. The alpha-subunit of the colonic H+,K+-ATPase (cHK) shares 75% sequence similarity with alpha1-Na+,K+-ATPase (alpha1) and assembles with beta1-Na+,K+-ATPase (beta1) in distal colon and renal medulla. Differences in pharmacological properties have been ascribed to when heterologously expressed function has been compared to function in vitro. The purpose of this study was to determine if cHK might associate with the gamma-subunit of the Na+,K+-ATPase (gamma) as a possible explanation for these variations in function. METHODS: An antibody specific for the gamma was used in coimmunoprecipitation experiments to determine if the gamma assembles stably in vitro with cHK and beta1 in rat renal medulla or distal colon. RESULTS: Our results demonstrate that the gamma-subunit assembles specifically with the Na+-pump, but not with cHK. Furthermore, the gamma-subunit assembly was specific for rat kidney and was not observed in distal colon. CONCLUSION: Since the gamma-subunit did not assemble with the cHK/beta1 complex, gamma-subunit assembly cannot explain those variations in ex vivo and in vitro pharmacologic properties ascribed to cHK.     

Regulation of vacuolar proton pumping ATPase-dependent luminal acidification in the epididymis

Luminal acidification in the epididymis is an important process for the regulation of male fertility. Low pH and low bicarbonate concentration are among key factors that keep spermatozoa in a dormant state while they mature and are stored in this organ. Although significant bicarbonate reabsorption is achieved by principal cells in the proximal regions of the epididymis, clear and narrow cells are specialized for net proton secretion. Clear cells express very high levels of the vacuolar proton pumping ATPase （V-ATPase） in their apical membrane and are responsible for the bulk of proton secretion. In the present paper, selected aspects of V-ATPase regulation in clear cells are described and potential pathologies associated with mutations of some of the V-ATPase subunits are discussed. （Asian J Androl 2007 July; 9： 476-482）     

囊泡型H+-ATPase B1 亚基的突变对大鼠内髓集合管细胞H+-ATPase结构和泵氢功能的影响

目的 研究致遗传性远端肾小管酸中毒（dRTA）的囊泡型H+-ATPase B1 亚基（ATP6V1B1）的点突变对大鼠内髓集合管（IMCD）细胞H+-ATPase结构和泵氢功能的影响。 方法 模拟致人类遗传性dRTA的 B1亚基点突变构建野生型（WT）和7种突变型（M)质粒,转染大鼠IMCD细胞并筛选稳定表达绿色荧光蛋白（GFP）-B1 M和GFP-B1 WT的IMCD细胞系。应用免疫荧光、免疫蛋白印迹法、ATP-NADH 耦合实验和快速酸负荷后不依赖钠的细胞内pH的变化,来观察GFP-B1 M和GFP-B1 WT在细胞内的分布,及其与H+-ATPase其他（E、H和c）亚基结合能力对ATP酶活性和H+-ATPase 泵氢功能的影响。 结果 GFP-B1 WT在转染细胞中呈囊泡样分布,与H+-ATPase的分布一致;而GFP-B1 M 则为弥散分布。免疫沉淀结果显示只有GFP-B1 WT融合蛋白能和其他的H+-ATPase 亚基（E、H和c）结合形成复合物,而 GFP-B1 M融合蛋白无此作用。ATP酶活性只有在GFP-B1 WT转染细胞株的免疫沉淀产物中存在,在GFP-B1 M转染细胞株的免疫沉淀产物中不存在。在GFP-B1 M 转染的IMCD细胞快速酸负荷后H+-ATPase介导的钠不依赖pHi 的恢复受到显著抑制[pHi的恢复率（pH U/min）在L81P、R124W、M174R、P275R、G316E、P346R、G364S GFP-B1M转染的IMCD细胞分别为0.007±0.002、0.004±0.002、0.002±0.002、0.003±0.002、0.006±0.004、0.009±0.004、0.015±0.006,P < 0.05,n = 5]。而GFP-B1 WT转染的IMCD细胞pHi的恢复率与未转染IMCD细胞相似[（0.040±0.006） pH U/min],且能被1 μmol/L巴弗洛霉素（H+-ATPase特异性抑制剂）所抑制。 结论 遗传性dRTA囊泡型H+-ATPase B1 亚基点突变影响GFP-B1融合蛋白与其他亚基正常结合组装形成完整的H+-ATPase,并抑制H+-ATPase 的泌酸功能。     

Calcium-sensing receptor (CaSR) modulates vacuolar H<Superscript>+</Superscript>-ATPase activity in a cell model of proximal tubule

Background

The calcium-sensing receptor (CaSR) is localized in the apical membrane of proximal tubules in close proximity to the transporters responsible for proton secretion. Therefore, the aim of the present study was to analyze the effects of CaSR stimulation on the biochemical activity of the vacuolar H⁺-ATPase in a cellular model of proximal tubule cells, OKP cells.

Methods

Biochemical activity of H⁺-ATPase was performed using cell homogenates, and the inorganic phosphate released was determined by a colorimetric method. Changes in cytosolic ionized calcium [Ca²⁺]_i were also determined using Fluo-4.

Results

A significant increase of vacuolar H⁺-ATPase activity was observed when the CaSR was stimulated with agonists such as Gd³⁺ (300 µM) and neomycin (200 µM). This activity was also stimulated in a dose-dependent fashion by changes in extracellular Ca²⁺ (Ca²⁺_o) between 10^?4 and 2 mM. Gd³⁺ and neomycin produced a sustained rise of [Ca²⁺]_i, an effect that disappears when extracellular calcium was removed in the presence of 0.1 µM thapsigargin. Inhibition of phospholipase C (PLC) activity with U73122 (5?×?10^?8 M) reduced the increase in [Ca²⁺]_i induced by neomycin.

Conclusion

CaSR stimulation induces an increase in the vacuolar H⁺-ATPase activity of OKP cells, an effect that involves an increase in [Ca²⁺]_i and require phospholipase C activity. The consequent decrease in intratubular pH could lead to increase ionization of luminal calcium, potentially enhancing its reabsorption in distal tubule segments and reducing the formation of calcium phosphate stones.

     

Slc26a11, a chloride transporter, localizes with the vacuolar H(+)-ATPase of A-intercalated cells of the kidney

Chloride has an important role in regulating vacuolar H(+)-ATPase activity across specialized cellular and intracellular membranes. In the kidney, vacuolar H(+)-ATPase is expressed on the apical membrane of acid-secreting A-type intercalated cells in the collecting duct where it has an essential role in acid secretion and systemic acid base homeostasis. Here, we report the identification of a chloride transporter, which co-localizes with and regulates the activity of plasma membrane H(+)-ATPase in the kidney collecting duct. Immunoblotting and immunofluorescent labeling identified Slc26a11 (～72?kDa), expressed in a subset of cells in the collecting duct. On the basis of double-immunofluorescent labeling with AQP2 and identical co-localization with H(+)-ATPase, cells expressing Slc26a11 were deemed to be distinct from principal cells and were found to be intercalated cells. Functional studies in transiently transfected COS7 cells indicated that Slc26a11 (designated as kidney brain anion transporter (KBAT)) can transport chloride and increase the rate of acid extrusion by means of H(+)-ATPase. Thus, Slc26a11 is a partner of vacuolar H(+)-ATPase facilitating acid secretion in the collecting duct.