Paracrine and endocrine regulation of renal K+ secretion.

The seminal studies conducted by Giebisch and coworkers in the 1960s paved the way for understanding the renal mechanisms involved in K+ homeostasis. It was demonstrated that differential handling of K+ in the distal segments of the nephron is crucial for proper K+ balance. Although aldosterone had been classically ascribed as the major ion transport regulator in the distal nephron, thereby contributing to K+ homeostasis, it became clear that aldosterone per se could not explain the ability of the kidney to modulate kaliuresis in both acute and chronic settings. The existence of alternative kaliuretic and antikaliuretic mechanisms was suggested by physiological studies in the 1980s but only gained form and shape with the advent of molecular biology. It is now established that the kidneys recruit several endocrine and paracrine mechanisms for adequate kaliuretic response. These mechanisms include the direct effects of peritubular K+, a gut-kidney regulatory axis sensing dietary K+ levels, the kidney secretion of kallikrein during postprandial periods, the upregulation of angiotensin II receptors in the distal nephron during chronic changes in K+ diet, and the local increase of prostaglandins by low-K+ diet. This review discusses recent advances in the understanding of endocrine and paracrine mechanisms underlying the modulation of K+ secretion and how these mechanisms impact kaliuresis and K+ balance. We also highlight important unknowns about the regulation of renal K+ excretion under physiological circumstances.

Empagliflozin Inhibits Proximal Tubule NHE3 Activity, Preserves GFR, and Restores Euvolemia in Nondiabetic Rats with Induced Heart Failure.

BACKGROUND: SGLT2 inhibitors reduce the risk of heart failure (HF) mortality and morbidity, regardless of the presence or absence of diabetes, but the mechanisms underlying this benefit remain unclear. Experiments with nondiabetic HF rats tested the hypothesis that the SGLT2 inhibitor empagliflozin (EMPA) inhibits proximal tubule (PT) NHE3 activity and improves renal salt and water handling. METHODS: Male Wistar rats were subjected to myocardial infarction or sham operation. After 4 weeks, rats that developed HF and sham rats were treated with EMPA or untreated for an additional 4 weeks. Immunoblotting and quantitative RT-PCR evaluated SGLT2 and NHE3 expression. Stationary in vivo microperfusion measured PT NHE3 activity. RESULTS: EMPA-treated HF rats displayed lower serum B-type natriuretic peptide levels and lower right ventricle and lung weight to tibia length than untreated HF rats. Upon saline challenge, the diuretic and natriuretic responses of EMPA-treated HF rats were similar to those of sham rats and were higher than those of untreated HF rats. Additionally, EMPA treatment prevented GFR decline and renal atrophy in HF rats. PT NHE3 activity was higher in HF rats than in sham rats, whereas treatment with EMPA markedly reduced NHE3 activity. Unexpectedly, SGLT2 protein and mRNA abundance were upregulated in the PT of HF rats. CONCLUSIONS: Prevention of HF progression by EMPA is associated with reduced PT NHE3 activity, restoration of euvolemia, and preservation of renal mass. Moreover, dysregulation of PT SGLT2 may be involved in the pathophysiology of nondiabetic HF.

The Role of ß-Adrenergic Overstimulation in the Early Stages of Renal Injury.

BACKGROUND/AIMS: To assess the possible contribution of the ß-adrenergic overstimulation in early stages of renal injury, the present study evaluated, in rats, the effects of the ß-adrenoceptor agonist isoproterenol (ISO) on renal function and morphology, as well as the renal mRNA and protein expression of the NADPH oxidase isoform 4 (Nox 4) and subunit p22phox, endoplasmic reticulum (ER) stress, pro-inflammatory, pro-apoptotic and renin-angiotensin system (RAS) components. METHODS: Wistar rats received ISO (0.3 mg.kg-1.day-1 s.c.) or vehicle (control) for eight days. At the end of the treatment, food and water intake, urine output and body weight gain were evaluated and renal function studies were performed. Renal tissue was used for the morphological, quantitative PCR and immunohistochemical studies. RESULTS: ISO did not change metabolic parameters or urine output. However it induced a decrease in renal blood flow and an increase in the filtration fraction. These changes were accompanied by increased cortical mRNA and protein expression for the renal oxidative stress components including Nox 4 and p22phox; ER stress, pro-inflamatory, pro-apoptotic as well as RAS components. ISO also induced a significant increase in medullar renin protein expression. CONCLUSION: These findings support relevant information regarding the contribution of specific ß-adrenergic hyperactivity in early stage of renal injury, indicating the reactive oxygen species, ER stress and intrarenal RAS as important factors in this process.

Escherichia coli Heat-Stable Enterotoxin Mediates Na+/H+ Exchanger 4 Inhibition Involving cAMP in T84 Human Intestinal Epithelial Cells.

The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl-release in intestinal cells, including the human colonic carcinoma T84 cell line, involving increased cGMP and membrane alkalization due to reduced Na+/H+ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pHi), and NHE1, NHE2, and NHE4 are expressed in T84 cells, we characterized the STa role as modulator of these exchangers. pHi was assayed by the NH4Cl pulse technique and measured by fluorescence microscopy in BCECF-preloaded cells. pHi recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 µmol/L STa (30 minutes), 25 µmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 µmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 µmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 µmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H+ efflux (JH+) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and JH+ (~63%), without altering basal pHi (range 7.144-7.172). STa did not alter ßi value in a range of 1.6 pHi units. The dpHi/dt and JH+ was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa-decreased dpHi/dt and JH+ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T84 cells with STa results in reduced NHE4 activity leading to a lower capacity of pHi recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human diarrhoea.

Fructose acutely stimulates NHE3 activity in kidney proximal tubule.

BACKGROUND/AIMS: Fructose causes a sodium-sensitive hypertension and acutely reduces the urinary Na+ excretion, suggesting that it may regulate the activity of renal tubular sodium transporters. NHE3 is highly expressed in proximal tubule (PT), along with proteins that mediate fructose transport and metabolism. The present work was outlined to investigate whether fructose modulates proximal NHE3 activity and to elucidate the molecular mechanisms underlying this modulation. METHODS/RESULTS: Using in vivo stationary microperfusion, we observed that fructose stimulates NHE3 mediated JHCO3- reabsorption. The MAPK pathway is not involved in this activation, as demonstrated by using of MEK/MAPK inhibitors, whereas experiments using a PKA inhibitor suggest that PKA inhibition plays a role in this response. These results were confirmed in vitro by measuring the cell pH recovery rate after NH4Cl pulse in LLC-PK1, a pig PT cell line, which showed reduced cAMP levels and NHE3 phosphorylation at serine-552 (PKA consensus site) after fructose treatment. CONCLUSIONS: NHE3 activity is stimulated by fructose, which increases proximal tubule Na+ reabsorption. The molecular mechanisms involved in this process are mediated, at least in part, by downregulation of the PKA signaling pathway. Future studies are needed to address whether fructose-stimulated NHE3 activity may contribute to renal injury and hypertension.

Mechanisms underlying the inhibitory effects of uroguanylin on NHE3 transport activity in renal proximal tubule.

We previously demonstrated that uroguanylin (UGN) significantly inhibits Na(+)/H(+) exchanger (NHE)3-mediated bicarbonate reabsorption. In the present study, we aimed to elucidate the molecular mechanisms underlying the action of UGN on NHE3 in rat renal proximal tubules and in a proximal tubule cell line (LLC-PK(1)). The in vivo studies were performed by the stationary microperfusion technique, in which we measured H(+) secretion in rat renal proximal segments, through a H(+)-sensitive microelectrode. UGN (1 µM) significantly inhibited the net of proximal bicarbonate reabsorption. The inhibitory effect of UGN was completely abolished by either the protein kinase G (PKG) inhibitor KT5823 or by the protein kinase A (PKA) inhibitor H-89. The effects of UGN in vitro were found to be similar to those obtained by microperfusion. Indeed, we observed that incubation of LLC-PK(1) cells with UGN induced an increase in the intracellular levels of cAMP and cGMP, as well as activation of both PKA and PKG. Furthermore, we found that UGN can increase the levels of NHE3 phosphorylation at the PKA consensus sites 552 and 605 in LLC-PK(1) cells. Finally, treatment of LLC-PK(1) cells with UGN reduced the amount of NHE3 at the cell surface. Overall, our data suggest that the inhibitory effect of UGN on NHE3 transport activity in proximal tubule is mediated by activation of both cGMP/PKG and cAMP/PKA signaling pathways which in turn leads to NHE3 phosphorylation and reduced NHE3 surface expression. Moreover, this study sheds light on mechanisms by which guanylin peptides are intricately involved in the maintenance of salt and water homeostasis.

Increased NHE3 abundance and transport activity in renal proximal tubule of rats with heart failure.

Heart failure (HF) is associated with a reduced effective circulating volume that drives sodium and water retention and extracellular volume expansion. We therefore hypothesized that Na(+)/H(+) exchanger isoform 3 (NHE3), the major apical transcellular pathway for sodium reabsorption in the proximal tubule, is upregulated in an experimental model of HF. HF was induced in male rats by left ventricle radiofrequency ablation. Sham-operated rats (sham) were used as controls. At 6 wk after surgery, HF rats exhibited cardiac dysfunction with a dramatic increase in left ventricular end-diastolic pressure. By means of stationary in vivo microperfusion and pH-dependent sodium uptake, we demonstrated that NHE3 transport activity was significantly higher in the proximal tubule of HF compared with sham rats. Increased NHE3 activity was paralleled by increased renal cortical NHE3 expression at both protein and mRNA levels. In addition, the baseline PKA-dependent NHE3 phosphorylation at serine 552 was reduced in renal cortical membranes of rats with HF. Collectively, these results suggest that NHE3 is upregulated in the proximal tubule of HF rats by transcriptional, translational, and posttranslational mechanisms. Enhanced NHE3-mediated sodium reabsorption in the proximal tubule may contribute to extracellular volume expansion and edema, the hallmark feature of HF. Moreover, our study emphasizes the importance of undertaking a cardiorenal approach to contain progression of cardiac disease.

Mechanisms mediating the diuretic and natriuretic actions of the incretin hormone glucagon-like peptide-1.

Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone considered a promising therapeutic agent for type 2 diabetes because it stimulates beta cell proliferation and insulin secretion in a glucose-dependent manner. Cumulative evidence supports a role for GLP-1 in modulating renal function; however, the mechanisms by which GLP-1 induces diuresis and natriuresis have not been completely established. This study aimed to define the cellular and molecular mechanisms mediating the renal effects of GLP-1. GLP-1 (1 µg·kg(-1)·min(-1)) was intravenously administered in rats for the period of 60 min. GLP-1-infused rats displayed increased urine flow, fractional excretion of sodium, potassium, and bicarbonate compared with those rats that received vehicle (1% BSA/saline). GLP-1-induced diuresis and natriuresis were also accompanied by increases in renal plasma flow and glomerular filtration rate. Real-time RT-PCR in microdissected rat nephron segments revealed that GLP-1 receptor-mRNA expression was restricted to glomerulus and proximal convoluted tubule. In rat renal proximal tubule, GLP-1 significantly reduced Na(+)/H(+) exchanger isoform 3 (NHE3)-mediated bicarbonate reabsorption via a protein kinase A (PKA)-dependent mechanism. Reduced proximal tubular bicarbonate flux rate was associated with a significant increase of NHE3 phosphorylation at the PKA consensus sites in microvillus membrane vesicles. Taken together, these data suggest that GLP-1 has diuretic and natriuretic effects that are mediated by changes in renal hemodynamics and by downregulation of NHE3 activity in the renal proximal tubule. Moreover, our findings support the view that GLP-1-based agents may have a potential therapeutic use not only as antidiabetic drugs but also in hypertension and other disorders of sodium retention.

Bases fisiológicas da nefrologia / Physiological bases of nephrology

O livro tem sua inspiração no Curso de Bases Fisiológicas de Prática Médica da Faculdade de Medicina da Universidade de São Paulo – USP, capítulo Nefrologia. Seu texto passou por sucessivas modificações didáticas e de conteúdo, ao longo dos 18 anos de existência do curso. Amadureceu, encorpou, ganhando aspecto interdisciplinar ao interagir a Fisiologia – centrada nos mecanismos de funcionamento de órgãos e sistemas, com a prática clínica dirigida à interpretação de dados clínico laboratoriais e a aplicação do raciocínio diagnóstico. É livro, pois, que bem reflete a excelência do ensino da Nefrologia integrado pela Fisiologia, Fisiopatologia Renal, Investigações clínicas e Exames Complementares. Sua equipe autorial é formada por 1 Editor, 2 Coeditores e 14 Colaboradores. Apresenta 15 capítulos e 3 Apêndices. É destinado aos estudantes de graduação de Medicina, Médicos Residentes e profissionais interessados em reciclar e atualizar seus conhecimentos em Nefrologia.

Bases fisiológicas da nefrologia

O livro tem sua inspiração no Curso de Bases Fisiológicas de Prática Médica da Faculdade de Medicina da Universidade de São Paulo – USP, capítulo Nefrologia. Seu texto passou por sucessivas modificações didáticas e de conteúdo, ao longo dos 18 anos de existência do curso. Amadureceu, encorpou, ganhando aspecto interdisciplinar ao interagir a Fisiologia – centrada nos mecanismos de funcionamento de órgãos e sistemas, com a prática clínica dirigida à interpretação de dados clínico laboratoriais e a aplicação do raciocínio diagnóstico. É livro, pois, que bem reflete a excelência do ensino da Nefrologia integrado pela Fisiologia, Fisiopatologia Renal, Investigações clínicas e Exames Complementares. Sua equipe autorial é formada por 1 Editor, 2 Coeditores e 14 Colaboradores. Apresenta 15 capítulos e 3 Apêndices. É destinado aos estudantes de graduação de Medicina, Médicos Residentes e profissionais interessados em reciclar e atualizar seus conhecimentos em Nefrologia

Bases fisiológicas da nefrologia / Physiological bases of Nephrology

O livro tem sua inspiração no Curso de Bases Fisiológicas de Prática Médica da Faculdade de Medicina da Universidade de São Paulo – USP, capítulo Nefrologia. Seu texto passou por sucessivas modificações didáticas e de conteúdo, ao longo dos 18 anos de existência do curso. Amadureceu, encorpou, ganhando aspecto interdisciplinar ao interagir a Fisiologia – centrada nos mecanismos de funcionamento de órgãos e sistemas, com a prática clínica dirigida à interpretação de dados clínico laboratoriais e a aplicação do raciocínio diagnóstico. É livro, pois, que bem reflete a excelência do ensino da Nefrologia integrado pela Fisiologia, Fisiopatologia Renal, Investigações clínicas e Exames Complementares. Sua equipe autorial é formada por 1 Editor, 2 Coeditores e 14 Colaboradores. Apresenta 15 capítulos e 3 Apêndices. É destinado aos estudantes de graduação de Medicina, Médicos Residentes e profissionais interessados em reciclar e atualizar seus conhecimentos em Nefrologia

Acid-base transport by the renal distal nephron.

The functional versatility of the distal nephron is mainly due to the large cytological heterogeneity of the segment. Part of Na+ uptake by distal tubules is dependent on Na+/H+ exchanger 2 (NHE2), implicating a role of distal convoluted cells also in acid-base homeostasis. In addition, intercalated (IC) cells expressed in distal convoluted tubules, connecting tubules and collecting ducts are involved in the final regulation of acid-base excretion. IC cells regulate acid-base handling by 2 main transport proteins, a V-type H+-ATPase and a Cl/HCO3- exchanger, localized at different membrane domains. Type A IC cells are characterized by a luminal H+-ATPase in series with a basolateral Cl/HCO3- exchanger, the anion exchanger AE1. Type B IC cells mediate HCO3- secretion through the apical Cl-/HCO3- exchanger pendrin in series with a H+-ATPase at the basolateral membrane. Alternatively, H+/K+-ATPases have also been found in several distal tubule cells, particularly in type A and B IC cells. All of these mechanisms are finely regulated, and mutations of 1 or more proteins ultimately lead to expressive disorders of acid-base balance.

Role of CFTR and ClC-5 in modulating vacuolar H+-ATPase activity in kidney proximal tubule.

BACKGROUND/AIMS: It has been widely accepted that chloride ions moving along chloride channels act to dissipate the electrical gradient established by the electrogenic transport of H(+) ions performed by H(+)-ATPase into subcellular vesicles. Largely known in intracellular compartments, this mechanism is also important at the plasma membrane of cells from various tissues, including kidney. The present work was performed to study the modulation of plasma membrane H(+)-ATPase by chloride channels, in particular, CFTR and ClC-5 in kidney proximal tubule. METHODS AND RESULTS: Using in vivo stationary microperfusion, it was observed that ATPase-mediated HCO(3)(-) reabsorption was significantly reduced in the presence of the Cl(-) channels inhibitor NPPB. This effect was confirmed in vitro by measuring the cell pH recovery rates after a NH(4)Cl pulse in immortalized rat renal proximal tubule cells, IRPTC. In these cells, even after abolishing the membrane potential with valinomycin, ATPase activity was seen to be still dependent on Cl(-). siRNA-mediated CFTR channels and ClC-5 chloride-proton exchanger knockdown significantly reduced H(+)-ATPase activity and V-ATPase B2 subunit expression. CONCLUSION: These results indicate a role of chloride in modulating plasma membrane H(+)-ATPase activity in proximal tubule and suggest that both CFTR and ClC-5 modulate ATPase activity.

Posttranslational mechanisms associated with reduced NHE3 activity in adult vs. young prehypertensive SHR.

Abnormalities in renal proximal tubular (PT) sodium transport play an important role in the pathophysiology of essential hypertension. The Na(+)/H(+) exchanger isoform 3 (NHE3) represents the major route for sodium entry across the apical membrane of renal PT cells. We therefore aimed to assess in vivo NHE3 transport activity and to define the molecular mechanisms underlying NHE3 regulation before and after development of hypertension in the spontaneously hypertensive rat (SHR). NHE3 function was measured as the rate of bicarbonate reabsorption by means of in vivo stationary microperfusion in PT from young prehypertensive SHR (Y-SHR; 5-wk-old), adult SHR (A-SHR; 14-wk-old), and age-matched Wistar Kyoto (WKY) rats. We found that NHE3-mediated PT bicarbonate reabsorption was reduced with age in the SHR (1.08 ± 0.10 vs. 0.41 ± 0.04 nmol/cm(2)×s), while it was increased in the transition from youth to adulthood in the WKY rat (0.59 ± 0.05 vs. 1.26 ± 0.11 nmol/cm(2)×s). Higher NHE3 activity in the Y-SHR compared with A-SHR was associated with a predominant microvilli confinement and a lower ratio of phosphorylated NHE3 at serine-552 to total NHE3 (P-NHE3/total). After development of hypertension, P-NHE3/total increased and NHE3 was retracted out of the microvillar microdomain along with the regulator dipeptidyl peptidase IV (DPPIV). Collectively, our data suggest that the PT is playing a role in adapting to the hypertension in the SHR. The molecular mechanisms of this adaptation possibly include an increase of P-NHE3/total and a redistribution of the NHE3-DPPIV complex from the body to the base of the PT microvilli, both predicted to decrease sodium reabsorption.

Regulation of Na+/H+ exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells.

The gut incretin hormone glucagon-like peptide 1 (GLP-1) is released in response to ingested nutrients and enhances insulin secretion. In addition to its insulinotropic properties, GLP-1 has been shown to have natriuretic actions paralleled by a diminished proton secretion. We therefore studied the role of the GLP-1 receptor agonist exendin-4 in modulating the activity of Na(+)/H(+) exchanger NHE3 in LLC-PK(1) cells. We found that NHE3-mediated Na(+)-dependent intracellular pH (pH(i)) recovery decreased approximately 50% after 30-min treatment with 1 nM exendin-4. Pharmacological inhibitors and cAMP analogs that selectively activate protein kinase A (PKA) or the exchange protein directly activated by cAMP (EPAC) demonstrated that regulation of NHE3 activity by exendin-4 requires activation of both cAMP downstream effectors. This conclusion was based on the following observations: 1) the PKA antagonist H-89 completely prevented the effect of the PKA activator but only partially blocked the exendin-4-induced NHE3 inhibition; 2) the MEK1/2 inhibitor U-0126 abolished the effect of the EPAC activator but only diminished the exendin-4-induced NHE3 inhibition; 3) combination of H-89 and U-0126 fully prevented the effect of exendin-4 on NHE3; 4) no additive effect in the inhibition of NHE3 activity was observed when exendin-4, PKA, and EPAC activators were used together. Mechanistically, the inhibitory effect of exendin-4 on pH(i) recovery was associated with an increase of NHE3 phosphorylation. Conversely, this inhibition took place without changes in the surface expression of the transporter. We conclude that GLP-1 receptor agonists modulate sodium homeostasis in the kidney, most likely by affecting NHE3 activity.

Dipeptidyl peptidase IV inhibition downregulates Na+ - H+ exchanger NHE3 in rat renal proximal tubule.

In the microvillar microdomain of the kidney brush border, sodium hydrogen exchanger type 3 (NHE3) exists in physical complexes with the serine protease dipeptidyl peptidase IV (DPPIV). The purpose of this study was to explore the functional relationship between NHE3 and DPPIV in the intact proximal tubule in vivo. To this end, male Wistar rats were treated with an injection of the reversible DPPIV inhibitor Lys [Z(NO2)]-pyrrolidide (I40; 60 mg.kg(-1).day(-1) ip) for 7 days. Rats injected with equal amounts of the noninhibitory compound Lys[Z(NO2)]-OH served as controls. Na(+) - H(+) exchange activity in isolated microvillar membrane vesicles was 45 +/- 5% decreased in rats treated with I40. Membrane fractionation studies using isopycnic centrifugation revealed that I40 provoked redistribution of NHE3 along with a small fraction of DPPIV from the apical enriched microvillar membranes to the intermicrovillar microdomain of the brush border. I40 significantly increased urine output (67 +/- 9%; P < 0.01), fractional sodium excretion (63 +/- 7%; P < 0.01), as well as lithium clearance (81 +/- 9%; P < 0.01), an index of end-proximal tubule delivery. Although not significant, a tendency toward decreased blood pressure and plasma pH/HCO(3)(-) was noted in I40-treated rats. These findings indicate that inhibition of DPPIV catalytic activity is associated with inhibition of NHE3-mediated NaHCO3 reabsorption in rat renal proximal tubule. Inhibition of apical Na(+) - H(+) exchange is due to reduced abundance of NHE3 protein in the microvillar microdomain of the kidney brush border. Moreover, this study demonstrates a physiologically significant interaction between NHE3 and DPPIV in the intact proximal tubule in vivo.

NHE1, NHE2, and NHE4 contribute to regulation of cell pH in T84 colon cancer cells.

The isoforms of the Na+/H+ exchanger present in T84 human colon cells were identified by functional and molecular methods. Cell pH was measured by fluorescence microscopy using the probe BCECF. Based on the pH recovery after an ammonium pulse and determination of buffering capacity of these cells, the rate of H+ extrusion (JH) was 3.68 mM/min. After the use of the amiloride derivative HOE-694 at 25 microM, which inhibits the isoforms NHE1 and NHE2, there remained 43% of the above transport rate, the nature of which was investigated. Evidence of the presence of NHE1, NHE2, and NHE4 was obtained by reverse transcriptase polymerase chain reaction (RT-PCR) (mRNA) and Western blot. There was no decrease of JH by the NHE3 inhibitor S3226 (1 microM) and no evidence of this isoform by RT-PCR was found. The following functional evidence for the presence of NHE4 was obtained: 25 microM EIPA abolished JH entirely, but NHE4 was not inhibited at 10 microM; substitution of Na by K increased the remainder, a property of NHE4; hypertonicity also increased this fraction of JH. Cl--dependent NHE was not detected: in 0 Cl- solutions JH was increased and not reduced. In 0 Cl- cell volume decreased significantly, which was abolished by the Cl- channel blocker NPPB, indicating that the 0 Cl- effect was because of reduction of cell volume. In conclusion, T84 human colon cells contain three isoforms of the Na+/H+ exchanger, NHE1, NHE2, and NHE4, but not the Cl-dependent NHE.

Signaling pathways involved with the stimulatory effect of angiotensin II on vacuolar H+-ATPase in proximal tubule cells.

It has been documented that angiotensin II (ANG II) (10(-9) M) stimulates proton extrusion via H(+)-adenosine triphosphatase (ATPase) in proximal tubule cells. In the present study, we investigated the signaling pathways involved in the effects of ANG II on H(+)-ATPase activity and on the cytosolic free calcium concentration in immortalized rat proximal tubule cells, a permanent cell line derived from rat proximal tubules. The effects of ANG on pH(i) and [Ca(+2)](i) were assessed by the fluorescent probes, 2',7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxy-methyl ester and fluo-4-acetoxy-methyl ester, in the absence of Na(+) to block the Na(+)/H(+) exchanger. In the control situation, the pH recovery rate following intracellular acidification with NH(4)Cl was 0.073+/-0.011 pH units/min (n=12). This recovery was significantly increased with ANG II (10(-9 )M), to 0.12+/-0.015 pH units/min, n=10. This last effect was also followed by a significant increase of Ca(+2) (i), from 99.72+/-1.704 nM (n=21) to 401.23+/-33.91 nM (n=39). The stimulatory effect of ANG II was blocked in the presence of losartan, an angiotensin II subtype 1 (AT(1)) receptor antagonist. H89 [protein kinase A (PKA) inhibitor] plus ANG II had no effect on the pH recovery. Staurosporine [protein kinase C (PKC) inhibitor] impaired the effect of ANG II. Phorbol myristate acetate (PKC activator) mimicked in part the stimulatory effect of ANG II, but reduced Ca(+2) (i). 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (intracellular calcium chelator) alone reduced the pH(i) recovery rate below control levels and impaired the effect of ANG II, in a way similar to that of trimethoxy benzoate (a blocker of Ca(+2) (i) mobilization). We conclude that ANG II regulates rat proximal tubule vacuolar H(+)-ATPase by a PKA-independent mechanism and that PKC and intracellular calcium play a critical role in this regulation.