Essential regulatory elements for NHE3 gene transcription in renal proximal tubule cells

The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5’-flanking sequence (5’FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5’FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5’FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5’FS are used as promoter, efficient transcription only occurs with 44 bp of 5’FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5’FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.

Long-term regulation of vacuolar H(+)-ATPase by angiotensin II in proximal tubule cells

Long-term effects of angiotensin II (Ang II) on vacuolar H(+)-ATPase were studied in a SV40-transformed cell line derived from rat proximal tubules (IRPTC). Using pH(i) measurements with the fluorescent dye BCECF, the hormone increased Na(+)-independent pH recovery rate from an NH(4)Cl pulse from 0.066 +/- 0.014 pH U/min (n = 7) to 0.14 +/- 0.021 pH U/min (n = 13; p < 0.05) in 10 h Ang II (10(-9) M)-treated cells. The increased activity of H(+)-ATPase did not involve changes in mRNA or protein abundance of the B2 subunit but increased cell surface expression of the V-ATPase. Inhibition of tyrosine kinase by genistein blocked Ang II-dependent stimulation of H(+)-ATPase. Inhibition of phosphatidylinositol-3-kinase (PI3K) by wortmannin and of p38 mitogen-activated protein kinase (MAPK) by SB 203580 also blocked this effect. Thus, long-term exposure of IRPTC cells to Ang II causes upregulation of H(+)-ATPase activity due, at least in part, to increased B2 cell surface expression. This regulatory pathway is dependent on mechanisms involving tyrosine kinase, p38 MAPK, and PI3K activation.

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

Carraro-Lacroix LR, Malnic G, Girardi AC. Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells. Am J Physiol Renal Physiol 297: F1647-F1655, 2009. First published September 23, 2009; doi:10.1152/ajprenal.00082.2009.-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 similar to 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 pHi 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.

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. Copyright (C) 2010 S. Karger AG, Basel

Direct action of aldosterone on bicarbonate reabsorption in in vivo cortical proximal tubule

Pergher PS, Leite-Dellova D, de Mello-Aires M. Direct action of aldosterone on bicarbonate reabsorption in in vivo cortical proximal tubule. Am J Physiol Renal Physiol 296: F1185-F1193, 2009. First published February 18, 2009; doi:10.1152/ajprenal.90217.2008.-The direct action of aldosterone (10(-12) M) on net bicarbonate reabsorption (J(HCO3)(-)) was evaluated by stationary microperfusion of an in vivo middle proximal tubule (S2) of rat kidney, using H ion-sensitive microelectrodes. Aldosterone in luminally perfused tubules caused a significant increase in J(HCO3)(-) from a mean control value of 2.84 +/- 0.08 [49/19 (n degrees of measurements/n degrees of tubules)] to 4.20 +/- 0.15 nmol.cm(-2).s(-1) (58/10). Aldosterone perfused into peritubular capillaries also increased J(HCO3)(-), compared with basal levels during intact capillary perfusion with blood. In addition, in isolated perfused tubules aldosterone causes a transient increase of cytosolic free calcium ([Ca(2+)](i)), monitored fluorometrically. In the presence of ethanol ( in similar concentration used to prepare the hormonal solution), spironolactone (10(-6) M, a mineralocorticoid receptor antagonist), actinomycin D (10(-6) M, an inhibitor of gene transcription), or cycloheximide (40 mM, an inhibitor of protein synthesis), the J(HCO3)(-) and the [Ca(2+)](i) were not different from the control value; these drugs also did not prevent the stimulatory effect of aldosterone on J(HCO3)(-) and on [Ca(2+)](i). However, in the presence of RU 486 alone [10(-6) M, a classic glucocorticoid receptor (GR) antagonist], a significant decrease on J(HCO3)(-) and on [Ca(2+)](i) was observed; this antagonist also inhibited the stimulatory effect of aldosterone on J(HCO3)(-) and on [Ca(2+)](i). These studies indicate that luminal or peritubular aldosterone (10(-12) M) has a direct nongenomic stimulatory effect on J(HCO3)(-) and on [Ca(2+)](i) in proximal tubule and that probably GR participates in this process. The data also indicate that endogenous aldosterone stimulates J(HCO3)(-) in middle proximal tubule.

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(NO(2))]-pyrrolidide (I40; 60 mg center dot kg(-1)center dot day(-1) ip) for 7 days. Rats injected with equal amounts of the noninhibitory compound Lys[ Z(NO(2))]-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 NaHCO(3) 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.

Treatment with a p38 MAPK inhibitor attenuates cisplatin nephrotoxicity starting after the beginning of renal damage

Aims: Cisplatin (CP) promotes increased production of reactive oxygen species, which can activate p38 mitogen activated protein kinases (p38 MAPKs) leading to apoptosis and increased expression of proinflammatory mediators that intensify the cytotoxic effects of CP. We investigated the effect of the treatment with S13203580, a p38 MAPKs inhibitor, on oxidative stress, on the oxidation-associated signal, p38 MAPK and on apoptosis in U-injected rats, starting after the beginning of the renal damage. Main methods: Rats (n = 21) were injected with CP (5 mg/kg, i.p.) and 3 and 4 days after some of them (n = 8) were treated with SB203580 (0.5 mg/kg, i.p.). Controls (n = 6) received saline (i.p.). Two or five days after saline or CP injections, plasma creatinine, urinary volume, sodium and potassium fractional excretions, blood urea nitrogen and urinary lipid peroxidation were measured. The kidneys were removed for histological, apoptosis, immunohistochemical and Western blot studies. Key findings: CP caused abnormalities in kidney functions and structure associated with raised urinary peroxidation levels and higher number of apoptotic cells in the outer medulla. The immunostaining studies showed increased numbers of macrophages/monocytes and p-p38 MAPKs positive cells in the renal outer medulla. The increase of p-p38 MAPKs expression was confirmed by Western blot analysis. All of these alterations were attenuated by treatment with S13203580. Significance: These data suggest that the beneficial effect of SB203580 on CP-induced renal damage might be related, in part, to the blockade of p38 MAPK activation with reduction of the inflammatory process, oxidative stress and apoptotic cell death. (C) 2009 Elsevier Inc. All rights reserved.

Mechanisms underlying the long-term regulation of NHE3 by parathyroid hormone

The activity of the Na(+)/H(+) exchanger NHE3 is regulated by a number of factors including parathyroid hormone (PTH). In the current study, we used a renal epithelial cell line, the opossum kidney (OKP) cell, to elucidate the mechanisms underlying the long-term effects of PTH on NHE3 transport activity and expression. We observed that NHE3 activity was reduced 6 h after addition of PTH, and this reduction persisted almost unaltered after 24 h. The decrease in activity was associated with diminished NHE3 cell surface expression at 6, 16, and 24 h after PTH addition, total cellular NHE3 protein at 16 and 24 h, and NHE3 mRNA abundance at 24 h. The lower levels of NHE3 mRNA were associated to a small, but significant, decrease in mRNA stability. Additionally, by analyzing the rat NHE3 gene promoter activity in OKP cells, we verified that the regulatory region spanning the segment -152 to +55 was mildly reduced under the influence of PTH. This effect was completely abolished by the presence of the PKA inhibitor KT 5720. In conclusion, long-term exposure to PTH results in reduction of NHE3 mRNA levels due to a PKA-dependent inhibitory effect on the NHE3 promoter and a small reduction of mRNA half-life, and decrease in the total amount of protein which is preceded by endocytosis of the apical surface NHE3. The decreased NHE3 expression is likely to be responsible for the reduction of sodium, bicarbonate, and fluid reabsorption in the proximal tubule consistently perceived in experimental models of PTH disorders.

Interactions of Diorganolead(IV) with 3-(2-Thienyl)-2-sulfanylpropenoic Acid and/or Thiamine: Chemical and in Vitro and in Vivo Toxicological Results

The reactions of PbR(2)(OAc)(2) (R=Me, Ph) with 3-(2-thienyl)-2-sulfanylpropenoic acid (H(2)tSpa) in methanol or ethanol afforded complexes [PbR(2)(tspa)] that electrospray ionization-mass spectrometry (ESI-MS) and IR data suggest are polymeric. X-ray studies showed that [PbPh(2)(tspa)(dmso)] center dot dmso, crystallized from a solution of [PbPh(2)(tspa)] in dmso, is dimeric, and that [HQ](2)[PbPh(2)(tspa)(2)] (Q=diisopropylamine), obtained after removal of [PbPh(2)(tspa)] from a reaction including Q, contains the monomeric anion [PbPh(2)(tSpa)(2)](2-). In the solid state the lead atoms are O,S-chelated by the tspa ligands in all these products, and in the latter two have distorted octahedral coordination environments. NMR data suggest that tspa(2-) remains coordinated to PbR(2)(2+) in solution in dmso. Neither thiamine nor thiamine diphosphate reacted with PbMe(2)(NO(3))(2) in D(2)O. Prior addition of H(2)tSpa protected LLC center dot PK1 renal proximal tubule cells against PbMe(2)(NO(3))(2); thiamine had no statistically significant effect by itself, but greatly potentiated the action of H(2)tSpa. Administration of either H(2)tspa or thiamine to male albino Sprague-Dawley rats dosed 30 min previously with PbMe(2)(NO(3))(2) was associated with reduced inhibition of delta-ALAD by the organolead compound, and with lower lead levels in kidney and brain, but joint administration of both H(2)tspa and thiamine only lowered lead concentration in the kidney.

Genomic and nongenomic stimulatory effect of aldosterone on H(+)-ATPase in proximal S3 segments

Leite-Dellova DC, Malnic G, Mello-Aires M. Genomic and non-genomic stimulatory effect of aldosterone on H(+)-ATPase in proximal S3 segments. Am J Physiol Renal Physiol 300: F682-F691, 2011. First published December 29, 2010; doi:10.1152/ajprenal.00172.2010.-The genomic and nongenomic effects of aldosterone on the intracellular pH recovery rate (pHirr) via H(-)(+)ATPase and on cytosolic free calcium concentration ([Ca(2+)](i)) were investigated in isolated proximal S3 segments of rats during superfusion with an Na(+)-free solution, by using the fluorescent probes BCECF-AM and FLUO-4-AM, respectively. The pHirr, after cellular acidification with a NH(4)Cl pulse, was 0.064 +/- 0.003 pH units/min (n = 17/74) and was abolished with concanamycin. Aldosterone (10(-12), 10(-10),10(-8), or 10(-6) M with 1-h or 15- or 2-min preincubation) increased the pHirr. The baseline [Ca(2+)](i) was 103 +/- 2 nM (n = 58). After 1 min of aldosterone preincubation, there was a transient and dose-dependent increase in [Ca(2+)](i) and after 6-min preincubation there was a new increase in [Ca(2+)](i) that persisted after 1 h. Spironolactone [mineralocorticoid (MR) antagonist], actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min preincubation) on pHirr and on [Ca(2+)](i) but inhibited the effects of aldosterone (1-h preincubation) on these parameters. RU 486 [glucocorticoid (GR) antagonist] and dimethyl-BAPTA (Ca(2+) chelator) prevented the effect of aldosterone on both parameters. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on the H(+)-ATPase and on [Ca(2+)](i). The results are compatible with stimulation of the H(+)-ATPase by increases in [Ca(2+)](i) (at 10(-12)-10(-6) M aldosterone) and inhibition of the H(+)-ATPase by decreases in [Ca(2+)](i) (at 10(-12) or 10(-6) M aldosterone plus RU 486).

Genomic and nongenomic dose-dependent biphasic effect of aldosterone on Na(+)/H(+) exchanger in proximal S3 segment: role of cytosolic calcium

Leite-Dellova DC, Oliveira-Souza M, Malnic G, Mello-Aires M. Genomic and nongenomic dose-dependent biphasic effect of aldosterone on Na(+)/H(+) exchanger in proximal S3 segment: role of cytosolic calcium. Am J Physiol Renal Physiol 295: F1342-F1352, 2008. First published August 20, 2008; doi:10.1152/ajprenal.00048.2008.-The effects of aldosterone on the intracellular pH recovery rate (pHirr) via Na(+)/H(+) exchanger and on the [Ca(2+)](i) were investigated in isolated rat S3 segment. Aldosterone [10(-12), 10(-10), or 10(-8) M with 1-h, 15- or 2-min preincubation (pi)] caused a dose-dependent increase in the pHirr, but aldosterone (10(-6) M with 1-h, 15- or 2-min pi) decreased it (these effects were prevented by HOE694 but not by S3226). After 1 min of aldosterone pi, there was a transient and dose-dependent increase of the [Ca(2+)](i) and after 6-min pi there was a new increase of [Ca(2+)](i) that persisted after 1 h. Spironolactone, actinomycin D, or cycloheximide did not affect the effects of aldosterone (15 -or 2-min pi) but inhibited the effects of aldosterone (1-h pi) on pHirr and on [Ca(2+)](i). RU 486 prevented the stimulatory effect of aldosterone (10(-12) M, 15 -or 2-min pi) on both parameters and maintained the inhibitory effect of aldosterone (10(-6) M, 15- or 2-min pi) on the pHirr but reversed its stimulatory effect on the [Ca(2+)](i) to an inhibitory effect. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on [Ca(2+)](i) and on the basolateral NHE1 and are compatible with stimulation of the NHE1 by increases in [Ca(2+)](i) in the lower range (at 10(-12) M aldosterone) and inhibition by increases at high levels (at 10(-6) M aldosterone) or decreases in [Ca(2+)](i) (at 10(-6) M aldosterone plus RU 486).

Transcriptional regulation of the Na(+)/H(+) exchanger NHE3 by chronic exposure to angiotensin II in renal epithelial cells

Angiotensin II (Ang II) exerts an acute bimodal effect on proximal tubule NHE3: while low doses stimulate the exchanger, high doses inhibit it. In the present study, we have investigated the chronic effects of Ang II on NHE3 expression and transcriptional regulation. Treatment of a tubular epithelial cell line, OKP, with Ang II 10(-11) M significantly increased NHE protein expression and mRNA levels, without evidence of bimodal effect. No change in mRNA half-life was detected, but transient transfection studies showed a significant increase in NHE3 promoter activity. Binding sites for Sp1/Egr-1 and AP2 transcription factors of the NHE3 proximal promoter were mutated and we observed that the Sp1/Egr-1 binding site integrity is necessary for Ang II stimulatory effects. Inhibition of cytochrome P450, PI3K, PKA and MAPK pathways prevented the Ang II stimulatory effect on the NHE3 promoter activity. Taking all the results together, our data reveal that chronic Ang II treatment exerts a stimulatory effect on NHE3 expression and promoter activity. The Ang II up-regulation of the NHE3 promoter activity appears to involve the Sp1/Egr-1 binding site and the interplay of several intracellular signaling pathways. (C) 2011 Elsevier Inc. All rights reserved.

Chemical and in vitro study of the potential of 3-(aryl)-2-sulfanylpropenoic acids and their Zn(II) complexes as protective agents against cadmium toxicity

The free H(2)xspa ligands [xspa = pspa, Clpspa, tspa or fspa where p = 3-(phenyl), Clp = 3-(2-chlorophenyl), t = 3-(2-thienyl), f = 3-(2-furyl) and spa = 2-sulfanylpropenoato], their Zn(II) complexes of formula [HQ](2)[Zn(xspa)(2)] (HQ=diisopropylammonium) and the Cd(II) equivalents were prepared and characterized by elemental analysis and by IR, Raman and NMR ((1)H, (13)C) spectroscopy. X-Ray studies of the crystal structures of [HQ](2)[Zn(pspa)(2)], [HQ](2)[Zn(Clpspa)2], [HQ](2)[Zn(tspa)(2)] and [HQ](2)[Zn(fspa)(2)] show that the zinc atom is coordinated to two O atoms and two S atoms of the ligands in a distorted tetrahedral ZnO(2)S(2) environment. In the structures of [HQ](2)[Cd(pspa)(2)] and [HQ](2)[Cd(Clpspa)(2)] the cadmium atom is coordinated to three S atoms and two carboxylato O atoms of the ligands in a distorted trigonal bipyramidal environment. The interchange of ligands between Zn( II) and Cd( II) was studied by (113)Cd NMR spectroscopy. The in vitro protective effect of H(2)xspa and their Zn( II) complexes against Cd toxicity was investigated using the human hepatocarcinoma HepG2 cell line and the pig renal proximal tubule LLC-PK1 cell line. The incorporation of Zn( II) was found to be relevant in the case of H(2)pspa, with an increase observed in the cell viability of the LCC-PK1 cells with respect to the value for the free ligand.

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

Crajoinas RO, Lessa LMA, Carraro-Lacroix LR, Davel APC, Pacheco BPM, Rossoni LV, Malnic G, Girardi ACC. Posttranslational mechanisms associated with reduced NHE3 activity in adult vs. young prehypertensive SHR. Am J Physiol Renal Physiol 299:F872-F881, 2010. First published July 14, 2010; doi:10.1152/ajprenal.00654.2009.-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)xs), 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)xs). 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.

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

Crajoinas RO, Oricchio FT, Pessoa TD, Pacheco BP, Lessa LM, Malnic G, Girardi AC. Mechanisms mediating the diuretic and natriuretic actions of the incretin hormone glucagon-like peptide-1. Am J Physiol Renal Physiol 301: F355-F363, 2011. First published May 18, 2011; doi: 10.1152/ajprenal.00729.2010.-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 mu g.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.