Change of V-ATPase B subunit in transforming growth factor β1-induced epithelial-mesenchymal transition of rat renal proximal tubular cells / 中华肾脏病杂志

Objecfive To investigate the change of V-ATPase B subunits on epithelial to mesenchymal transition (EMT)in rat renal tubular epithelial cells (NRK52E) stimulated by transforming growth factor β1 (TGF-β1). Methods NRK52E cells were stimulated by TGF-β1 (10 μg/L)for O h(control),12 h,24 h,48 h,72 h after sefrum-free culture for 24 h.The mRNA and protein expression of E-cadherin,α-SMA,B2 and B1 subunits of V-ATPase were detected by real-time PCR,Western blotting and immunofluorescence. Results After stimulated by TGF-β1 (10 μg/L)for 48 h,the expression of α-SMA was markedly increased(P<0.05),but the expression of E-cadherin was dramatically decreased(P<0.05).Meanwhile,the expressions of V-ATPase subunit B2 was significantly increased (P<0.05).However,the B1 subunit distributed rarely in NRK 52E cells,and did not increase after TGF-β1 stimulation.Double-label immunofluoerscence staining also showed that the V-ATPase B2 subunit was increased in the cytosol.tending to accumulate to the cell membrane after TGF-β1 stimulation. Conclusions The main isoform of V-ATPase distributed in NRK52E cells is B2 subunit.B2 subunit is increased alone with TGF-β1-induced EMT.It may suggest that V-ATPase B2 subunit may play a potential role in TGF-β1-induced tubular EMT and renal fibrosis.

Physiological implications of the regulation of vacuolar H+-ATPase by chloride ions

Vacuolar H+-ATPase is a large multi-subunit protein that mediates ATP-driven vectorial H+ transport across the membranes. It is widely distributed and present in virtually all eukaryotic cells in intracellular membranes or in the plasma membrane of specialized cells. In subcellular organelles, ATPase is responsible for the acidification of the vesicular interior, which requires an intraorganellar acidic pH to maintain optimal enzyme activity. Control of vacuolar H+-ATPase depends on the potential difference across the membrane in which the proton ATPase is inserted. Since the transport performed by H+-ATPase is electrogenic, translocation of H+-ions across the membranes by the pump creates a lumen-positive voltage in the absence of a neutralizing current, generating an electrochemical potential gradient that limits the activity of H+-ATPase. In many intracellular organelles and cell plasma membranes, this potential difference established by the ATPase gradient is normally dissipated by a parallel and passive Cl- movement, which provides an electric shunt compensating for the positive charge transferred by the pump. The underlying mechanisms for the differences in the requirement for chloride by different tissues have not yet been adequately identified, and there is still some controversy as to the molecular identity of the associated Cl--conducting proteins. Several candidates have been identified: the ClC family members, which may or may not mediate nCl-/H+ exchange, and the cystic fibrosis transmembrane conductance regulator. In this review, we discuss some tissues where the association between H+-ATPase and chloride channels has been demonstrated and plays a relevant physiologic role.

Regulation of Vacuolar H+-ATPase c Gene Expression by Oxidative Stress

By using differential display, we identified one of the genes encoding the multi-subunit complex protein V-ATPase, c subunit gene (ATP6L), and showed alterations of the gene expression by oxidative stresses. Expression of the ATP6L gene in Neuro-2A cells was increased by the treatment with H2O2 and incubation in hypoxic chamber, implying that the expression of the ATP6L gene is regulated by oxidative stresses. To examine mechanisms involved in the regulation of the gene expression by oxidative stresses, the transcriptional activity of the rat ATP6L promoter was studied. Transcription initiation site was determined by primer extension analysis and DNA sequencing, and promoter of the rat ATP6L and its deletion clones were constructed in reporter assay vector. Significant changes of the promoter activities in Neuro-2A cells were observed in two regions within the proximal 1 kbp promoter, and one containing a suppressor was in -195 to -220, which contains GC box that is activated by binding of Sp1 protein. The suppression of promoter activity was lost in mutants of the GC box. We confirmed by electrophoretic mobility shift and supershift assays that Sp1 protein specifically binds to the GC box. The promoter activity was not changed by the H2O2 treatment and incubation in hypoxic chamber, however, H2O2 increased the stability of ATP6L mRNA. These data suggest that the expression of the ATP6L gene by oxidative stresses is regulated at posttranscriptional level, whereas the GC box is important in basal activities of the promoter.

A Case of Distal Renal Tubular Acidosis by Damage of H+-ATPase Pump after Paraquat Intoxication / 대한신장학회잡지

Distal Renal tubular acidosis is characterized by tubular dysfunction with a decrease in net H+-secretion in the collecting tubules regardless of normal glomerular filtration rate. It classified into primary and secondary form. The causes of secondary form could be many drugs such as amphotericin B, toluene, lithium carbonate, ifosfamide, but paraquat has not been reported. The mechanism of renal damage by paraquat has not be fully comprehensive but it is thought that paraquat causes damage to renal proximal tubules and clinically induces acute tubular necrosis. Our case demonstrated that immunohistochemical staining of renal biopsy specimen with anti H+-ATPase antibody showed absence of proton pump in collecting duct. Thus a case of distal renal tubular acidosis in association with paraquat intoxication is reported with a review of literatures.

Expression of H(+)-ATPase in Inner Medullary Collecting Duct Cells of Aquaporin-3-Deficient Mice / 대한해부학회지

It has been reported that the decrease in urinary pH observed in AQP1 null mice with a urinary concentrating defect is due to upregulation of H(+)-ATPase in the IMCD. This is thought to be caused by the chronically low interstitial osmolality in these animals. To explore whether increase of H(+)-ATPase expression in the IMCD is associated with changes in the prolonged decrease of interstitial osmolality, we examined the expression of H(+)-ATPase and Na(+)-H(+) exchanger (NHE3) using light and electron microscopic immunocytochemistry in the kidneys of AQP3 null mice which are polyuric and manifest a urinary concentrating defect because of an inability to create a hypertonic medullary interstitium. In both AQP3 (-/-) and AQP1 (-/-) mouse kidneys, type A intercalated cells in cortical and medullary collecting ducts are slightly activated, and strong H(+)-ATPase immunostaining was present in the apical plasma membrane of IMCD cells, whereas no H(+)-ATPase labeling was observed in IMCD cells in wild type mice. No differences of the immunoreactivity for NHE3 in the proximal tubule and thick ascending limb of loop of Henle were observed between AQP3 or AQP1 (-/-) mice and AQP3 (+/+) mouse. These results suggest that the induction of H(+)-ATPase expression in IMCD cells of AQP3 null mice, as well as AQP1 null mice, may be related to their chronically low interstitial osmolality.

H+ -ATPase Pump Defects of Distal Renal Tubular Acidosis as Initial Manifestation of Systemic Lupus Erythematosus / 대한신장학회잡지

Systemic lupus erythematosus (SLE) is a multisystem disease with marked variability in its manifestation. Tubulointerstitial involvement is well recognized in SLE. But usually the tubular dysfunction is latent and usually presents after diagnosis of SLE. We report a 20 years old female whose initial symptom of SLE was distal renal tubular acidosis (RTA). She presented with severe muscle weakness at emergency room with laboratory fingding consistent with distal RTA. After several months she developed fever, arthritis, serologic fingding which was compatible to diagnose SLE. We report a case whose initial symptom of SLE had been distal RTA.

Changes of Urinary Acidification and Collecting Duct H(+)-ATPase Abundance in Response to Chronic Diuretic Administration / 대한신장학회잡지

PURPOSE: Commonly used diuretics such as furosemide and hydrochlorothiazide may cause metabolic alkalosis by increasing proton secretion from distal nephron. We evaluated changes in urinary acidification and abundance of proton-secreting transporters in response to chronic subcutaneous infusion of diuretics. METHODS: Osmotic minipumps were implanted into Sprague-Dawley rats to deliver 12 mg/day furoemide or hydrochlorothiazide 7.5 mg/day for 7 days. All animals were offered tap water and a solution containing 0.8% NaCl and 0.1% KCl as drinking fluid. RESULTS: Compared with vehicle-infused controls, diuretic and natriuretic responses were evident from furosemide or hydrochlorothiazide infusion. However, there were no changes in body weight, serum aldosterone and creatinine clearance between diuretic- infused(n=6) and control(n=6) rats. In both furosemide-infused and hydrochlorothiazide-infused rats, urine pH was significantly lowered compared with controls. Furosemide-infused rats showed significantly larger excretion of urinary ammonium. Semiquantitative immunoblotting was carried out from rat kidneys to investigate abundance of proximal tubule or medullary thick ascending limb Na(+)/H(+) exchanger type 3(NHE3) and collecting duct H(+)- ATPase using specific polyclonal antibodies to NHE3 and H(+)-ATPase B1 subunit, respectively. The abundance of NHE3 from cortical homogenates was not changed by either furosemide or hydrochlorothiazide infusion. However, the abundance of NHE3 from outer medullary homogenates was increased by furosemide infusion. The H(+)-ATPase B1 subunit abundance was increased by furosemide or hydrochlorothiazide infusion in both cortical and outer medullary homogenates. CONCLUSION: These increases in the abundance of proton-secreting transporters may account for the enhanced distal urinary acidification in response to chronic diuretic administration.

Transport of Tetraethylammonium in Renal Cortical Endosomes of Cadmium-Intoxicated Rats

Effects of cadmium (Cd) intoxication on renal endosomal accumulation of organic cations (OC ) were studied in rats using 14C-tetraethylammnium (TEA) as a substrate. Cd intoxication was induced by s.c. injections of 2 mg Cd/kg/day for 2-3 weeks. Renal cortical endosomes were isolated and the endosomal acidification (acridine orange fluorescence change) and TEA uptake (Millipore filtration technique) were assessed. The TEA uptake was an uphill transport mediated by H /OC antiporter driven by the pH gradient established by H -ATPase. In endosomes of Cd-intoxicated rats, the ATP-dependent TEA uptake was markedly attenuated due to inhibition of endosomal acidification as well as H /TEA antiport. In kinetic analysis of H /TEA antiport, Vmax was reduced and Km was increased in the Cd group. Inhibition of H /TEA antiport was also observed in normal endosomes directly exposed to free Cd (but not Cd-metallothionein complex, CdMt) in vitro. These data suggest that during chronic Cd exposure, free Cd ions liberated by lysosomal degradation of CdMt in proximal tubule cells may impair the endosomal accumulation of OC by directly inhibiting the H /OC antiporter activity and indirectly by reducing the intravesicular acidification, the driving force for H /OC exchange.

Defect of Acid-base Transporters in Distal Renal Tubular Acidosis / 대한신장학회잡지

The purpose of this study was to elucidate whether the molecular defect of acid-base transporters in renal tubules is related to the functional defect of urinary acidification in distal renal tubular acidosis(RTA). We performed NH4Cl, furosemide, or bicarbonate loading test to evaluate renal acidification function, and immunohistochemistry using antibodies to H+- ATPase, Cl-/HCO3- exchanger(band-3 protein), and Na+/K+-ATPase in kidney tissue in 6 patients with RTA and renal cell carcinoma patients as normal controls. Kidney tissue was obtained either by percutaneous needle biopsy(RTA) or nephrectomy(NC). The results were as follows; 1) In all six RTA patients, proton secretory defect of distal acidification was shown by a failure to lower the urine pH after NH4Cl loading or furosemide test or abnormally low urine-blood pCO2 difference during bicarbonate loading. In two patients with RTA, proximal acidification defect was combined, which was demonstrated by increased fractional excretion of bicarbonate. 2) In normal control, intense H+-ATPase and band-3 protein staining was observed in collecting ducts. 3) In distal RTA patients, H+-ATPase and band- 3 protein staining was not demonstrable or markedly decreased in the intercalated cells of distal nephron. 4) In two patients who had both proximal and distal RTA, H+-ATPase staining was markedly decreased in the brush border of proximal tubules as well as the distal nephron. In conclusion, the defect of acid-base transporters in renal tubule was related with the functional defect of urinary acidification in distal RTA.

Cadmium inhibition of renal endosomal acidification

Chronic exposure to cadmium (Cd) results in an inhibition of protein endocytosis in the renal proximal tubule, leading to proteinuria. In order to gain insight into the mechanism by which Cd impairs the protein endocytosis, we investigated the effect of Cd on the acidification of renal cortical endocytotic vesicles (endosomes). The endosomal acidification was assessed by measuring the pH gradient-dependent fluorescence change, using acridine orange or FITC-dextran as a probe. In renal endosomes isolated from Cd-intoxicated rats, the Vmax of ATP-driven fluorescence quenching (H -ATPase dependent intravesicular acidification) was significantly attenuated with no substantial changes in the apparent Km, indicating that the capacity of acidification was reduced. When endosomes from normal animals were directly exposed to free Cd in vitro, the Vmax was slightly reduced, whereas the Km was markedly increased, implying that the biochemical property of the H -ATPase was altered by Cd. In endosomes exposed to free Cd in vitro, the rate of dissipation of the transmembrane pH gradient after H -ATPase inhibition appeared to be significantly faster compared to that in normal endosomes, indicating that the H -conductance of the membrane was increased by Cd. These results suggest that in long-term Cd-exposed animals, free Cd ions liberated in the proximal tubular cytoplasm by lysosomal degradation of cadmium-metallothionein complex (CdMT) may impair endosomal acidification 1) by reducing the H -ATPase density in the endosomal membrane, 2) by suppressing the intrinsic H -ATPase activity, and 3) possibly by increasing the membrane conductance to H+ ion. Such effects of Cd could be responsible for the alterations of proximal tubular endocytotic activities, protein reabsorption and various transporter distributions observed in Cd-exposed cells and animals.

Vesicular transport with emphasis on exocytosis

The eukaryotic cell is compartmentalized by a series of vesicular organelles which constitute the endocytic and exocytic transport pathways. Each vesicular compartment has distinct sets of membrane proteins, structures and functions. Despite continuous vesicular transport, each vesicular compartment maintains its structure and function by use of retention and retrieval signal for its own resident proteins. Proteins in transit along the endocytic and exocytic pathway are transported without admixing with cytoplasmic constituents by successive steps of budding from the donor vesicles, formation of intermediate transport vesicles, transport, targeting to and fusion with acceptor vesicles. Specificity and fidelity of the vesicular transport are conferred by vesicular membrane proteins and small molecular weight GTP-binding proteins of the Rab subfamily. Proteins for export are packaged into specific vesicles for their final destinations. Insertion into and retrieval from the plasma membrane of transport proteins in response to cellular stimulus are a new paradigm of cellular regulatory mechanism. Secretion of neurotransmitters, hormones and enzymes by exocytosis involves a complex set of cytosolic proteins, G-proteins, proteins on the secretory granule membrane and plasma membrane. Much progress has been recently made in identifying proteins and factors involved in the exocytosis. But the molecular interactions among identified proteins and regulatory factors are unknown and remain to be elucidated. Finally our chemiosmotic hypothesis which involves the H+ electrochemical gradient across the secretory granule membrane generated by an ATP-dependent electrogenic H(+)-ATPase as the potential driving force for fusion and release of granule contents will be discussed.