The effects of angiotensin-(1-7) on the exchanger NHE3 and on [Ca2+]i in the proximal tubules of spontaneously hypertensive rats.

The acute effects of angiotensin-1-7 [ANG-(1-7)] on the reabsorptive bicarbonate flow (J[Formula: see text]) were evaluated using stationary microperfusion in vivo in the proximal tubules of spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar-Kyoto (WKY) rats, using a microelectrode sensitive to H+ In WKY rats, the control J[Formula: see text] was 2.40 ± 0.10 nmol·cm-2·s-1 (n = 120); losartan (10-7 M) or A779 (10-6 M, a specific Mas antagonist), alone or in combination with losartan, decreased the J[Formula: see text] ANG-(1-7) had biphasic effects on J[Formula: see text]: at 10-9 M, it inhibited, and at 10-6, it stimulated the flow. S3226 [10-6 M, a specific Na+-H+ exchanger 3 (NHE3) antagonist] decreased J[Formula: see text] and changed the stimulatory effect of ANG-(1-7) to an inhibitory one but did not alter the inhibitory action of ANG-(1-7). In SHR, the control J[Formula: see text] was 2.04 ± 0.13 nmol·cm-2·s-1 (n = 56), and A779 and/or losartan reduced the flow. ANG-(1-7) at 10-9 M increased J[Formula: see text], and ANG-(1-7) at 10-6 M reduced it. The effects of A779, losartan, and S3226 on the J[Formula: see text] were similar to those found in WKY rats, which indicated that in SHR, the ANG-(1-7) action on the NHE3 was via Mas and ANG II type 1. The cytosolic calcium in the WKY or SHR rats was ~100 nM and was increased by ANG-(1-7) at 10-9 or 10-6 M. In hypertensive animals, a high plasma level of ANG-(1-7) inhibited NHE3 in the proximal tubule, which mitigated the hypertension caused by the high plasma level of ANG II.

Uroguanylin modulates (Na++K+)ATPase in a proximal tubule cell line: Interactions among the cGMP/protein kinase G, cAMP/protein kinase A, and mTOR pathways.

BACKGROUND: The natriuretic effect of uroguanylin (UGN) involves reduction of proximal tubule (PT) sodium reabsorption. However, the target sodium transporters as well as the molecular mechanisms involved in these processes remain poorly understood. METHODS: To address the effects of UGN on PT (Na(+)+K(+))ATPase and the signal transduction pathways involved in this effect, we used LLC-PK1 cells. The effects of UGN were determined through ouabain-sensitive ATP hydrolysis and immunoblotting assays during different experimental conditions. RESULTS: We observed that UGN triggers cGMP/PKG and cAMP/PKA pathways in a sequential way. The activation of PKA leads to the inhibition of mTORC2 activity, PKB phosphorylation at S473, PKB activity and, consequently, a decrease in the mTORC1/S6K pathway. The final effects are decreased expression of the α1 subunit of (Na(+)+K(+))ATPase and inhibition of enzyme activity. CONCLUSIONS: These results suggest that the molecular mechanism of action of UGN on sodium reabsorption in PT cells is more complex than previously thought. We propose that PKG-dependent activation of PKA leads to the inhibition of the mTORC2/PKB/mTORC1/S6K pathway, an important signaling pathway involved in the maintenance of the PT sodium pump expression and activity. GENERAL SIGNIFICANCE: The current results expand our understanding of the signal transduction pathways involved in the overall effect of UGN on renal sodium excretion.

The physiological role of glucagon-like peptide-1 in the regulation of renal function.

Glucagon like peptide-1 (GLP-1) is an incretin hormone constantly secreted from the intestine at low basal levels in the fasted state; plasma concentrations rise rapidly after nutrient ingestion. Upon release, GLP-1 exerts insulinotropic effects via a G protein-coupled receptor, stimulation of adenylyl cyclase, and cAMP generation. Although primarily involved in glucose homeostasis, GLP-1 can induce diuresis and natriuresis when administered in pharmacological doses in humans and rodents. However, whether endogenous GLP-1 plays a role in regulating renal function remains an open question. This study aimed to test the hypothesis that blockade of GLP-1 receptor (GLP-1R) signaling at baseline influences renal salt and water handling. To this end, the GLP-1R antagonist exendin-9 (100 µg·kg(-1)·min(-1)) or vehicle was administered intravenously to overnight-fasted male Wistar rats for 30 min. This treatment reduced urinary cAMP excretion and renal cortical PKA activity, demonstrating blockade of renal GLP-1R signaling. Exendin-9-infused-rats exhibited reduced glomerular filtration rate, lithium clearance, urinary volume flow, and sodium excretion compared with vehicle-infused controls. Exendin-9 infusion also reduced renal cortical Na(+)/H(+) exchanger isotope 3 (NHE3) phosphorylation at serine 552 (NHE3pS552), a PKA consensus site that correlates with reduced transport activity. Collectively, these results provide novel evidence that GLP-1 is a physiologically relevant natriuretic factor that contributes to sodium balance, in part via tonic modulation of NHE3 activity in the proximal tubule.

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.

Uroguanylin inhibits H-ATPase activity and surface expression in renal distal tubules by a PKG-dependent pathway.

Cumulative evidence suggests that guanylin peptides play an important role on electrolyte homeostasis. We have previously reported that uroguanylin (UGN) inhibits bicarbonate reabsorption in a renal distal tubule. In the present study, we tested the hypothesis that the bicarbonaturic effect of UGN is at least in part attributable to inhibition of H(+)-ATPase-mediated hydrogen secretion in the distal nephron. By in vivo stationary microperfusion experiments, we were able to show that UGN inhibits H(+)-ATPase activity by a PKG-dependent pathway because KT5823 (PKG inhibitor) abolished the UGN effect on distal bicarbonate reabsorption and H89 (PKA inhibitor) was unable to prevent it. The in vivo results were confirmed by the in vitro experiments, where we used fluorescence microscopy to measure intracellular pH (pHi) recovery after an acid pulse with NH4Cl. By this technique, we observed that UGN and 8 bromoguanosine-cGMP (8Br-cGMP) inhibited H(+)-ATPase-dependent pHi recovery and that the UGN inhibitory effect was abolished in the presence of the PKG inhibitor. In addition, by using RT-PCR technique, we verified that Madin-Darby canine kidney (MDCK)-C11 cells express guanylate cyclase-C. Besides, UGN stimulated an increase of both cGMP content and PKG activity but was unable to increase the production of cellular cAMP content and PKA activity. Furthermore, we found that UGN reduced cell surface abundance of H+-ATPase B1 subunit in MDCK-C11 and that this effect was abolished by the PKG inhibitor. Taken together, our data suggest that UGN inhibits H(+)-ATPase activity and surface expression in renal distal cells by a cGMP/PKG-dependent pathway.

Functional role of glucose metabolism, osmotic stress, and sodium-glucose cotransporter isoform-mediated transport on Na+/H+ exchanger isoform 3 activity in the renal proximal tubule.

Na(+)-glucose cotransporter 1 (SGLT1)-mediated glucose uptake leads to activation of Na(+)-H(+) exchanger 3 (NHE3) in the intestine by a process that is not dependent on glucose metabolism. This coactivation may be important for postprandial nutrient uptake. However, it remains to be determined whether SGLT-mediated glucose uptake regulates NHE3-mediated NaHCO3 reabsorption in the renal proximal tubule. Considering that this nephron segment also expresses SGLT2 and that the kidneys and intestine show significant variations in daily glucose availability, the goal of this study was to determine the effect of SGLT-mediated glucose uptake on NHE3 activity in the renal proximal tubule. Stationary in vivo microperfusion experiments showed that luminal perfusion with 5 mM glucose stimulates NHE3-mediated bicarbonate reabsorption. This stimulatory effect was mediated by glycolytic metabolism but not through ATP production. Conversely, luminal perfusion with 40 mM glucose inhibited NHE3 because of cell swelling. Notably, pharmacologic inhibition of SGLT activity by Phlorizin produced a marked inhibition of NHE3, even in the absence of glucose. Furthermore, immunofluorescence experiments showed that NHE3 colocalizes with SGLT2 but not SGLT1 in the rat renal proximal tubule. Collectively, these findings show that glucose exerts a bimodal effect on NHE3. The physiologic metabolism of glucose stimulates NHE3 transport activity, whereas, supraphysiologic glucose concentrations inhibit this exchanger. Additionally, Phlorizin-sensitive SGLT transporters and NHE3 interact functionally in the proximal tubule.

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

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.

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.

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.

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.