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

Inoue BH, dos Santos L, Pessoa TD, Antonio EL, Pacheco BPM, Savignano FA, Carraro-Lacroix LR, Tucci PJF, Malnic G, Girardi ACC. Increased NHE3 abundance and transport activity in renal proximal tubule of rats with heart failure. Am J Physiol Regul Integr Comp Physiol 302: R166-R174, 2012. First published October 26, 2011; doi:10.1152/ajpregu.00127.2011.-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 underlying the inhibitory effects of uroguanylin on NHE3 transport activity in renal proximal tubule

Lessa LM, Carraro-Lacroix LR, Crajoinas RO, Bezerra CN, Dariolli R, Girardi AC, Fonteles MC, Malnic G. Mechanisms underlying the inhibitory effects of uroguanylin on NHE3 transport activity in renal proximal tubule. Am J Physiol Renal Physiol 303: F1399-F1408, 2012. First published September 5, 2012; doi: 10.1152/ajprenal.00385.2011.-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-PK1). 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 mu M) 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-PK1 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-PK1 cells. Finally, treatment of LLC-PK1 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

Dose-dependent effects of angiotensin-(1-7) on the NHE3 exchanger and [Ca(2+)](i) in in vivo proximal tubules

The acute direct action of angiotensin-(1-7) [ANG-(1-7)] on bicarbonate reabsorption (JHCO(3)(-)) was evaluated by stationary microperfusions on in vivo middle proximal tubules in rats using H ion-sensitive microelectrodes. The control JHCO(3)(-) is 2.82 ± 0.078 nmol·cm(-2)·s(-1) (50). ANG-(1-7) (10(-12) or 10(-9) M) in luminally perfused tubules decreases JHCO(3)(-) (36 or 60%, respectively), but ANG-(1-7) (10(-6) M) increases it (80%). A779 increases JHCO(3)(-) (30%) and prevents both the inhibitory and the stimulatory effects of ANG-(1-7) on it. S3226 decreases JHCO(3)(-) (45%) and changes the stimulatory effect of ANG-(1-7) to an inhibitory effect (30%) but does not affect the inhibitory effect of ANG-(1-7). Our results indicate that in the basal condition endogenous ANG-(1-7) inhibits JHCO(3)(-) and that the biphasic dose-dependent effect of ANG-(1-7) on JHCO(3)(-) is mediated by the Mas receptors via the Na(+)/H(+) exchanger 3 (NHE3). The control value of intracellular Ca(2+) concentration ([Ca(2+)](i)), as monitored using fura-2 AM, is 101 ± 2 nM (6), and ANG-(1-7) (10(-12), 10(-9), or 10(-6)M) transiently (3 min) increases it (by 151, 102, or 52%, respectively). A779 increases the [Ca(2+)](i) (25%) but impairs the stimulatory effect of all doses of ANG-(1-7) on it. The use of BAPTA or thapsigargin suggests a correlation between the ANG-(1-7) dose-dependent effects on [Ca(2+)](i) and JHCO(3)(-). Therefore, the interaction of the opposing dose-dependent effects of ANG II and ANG-(1-7) on [Ca(2+)](i) and JHCO(3)(-) may represent an physiological regulatory mechanism of extracellular volume and/or pH changes. However, whether [Ca(2+)](i) modification is an important direct mechanism for NHE3 activation by these peptides or is a side effect of other signaling pathways will require additional studies.

Distinct mechanisms underlie adaptation of proximal tubule Na+/H+ exchanger isoform 3 in response to chronic metabolic and respiratory acidosis

The Na+/H+ exchanger isoform 3 (NHE3) is essential for HCO3- reabsorption in renal proximal tubules. The expression and function of NHE3 must adapt to acid-base conditions. The goal of this study was to elucidate the mechanisms responsible for higher proton secretion in proximal tubules during acidosis and to evaluate whether there are differences between metabolic and respiratory acidosis with regard to NHE3 modulation and, if so, to identify the relevant parameters that may trigger these distinct adaptive responses. We achieved metabolic acidosis by lowering HCO3- concentration in the cell culture medium and respiratory acidosis by increasing CO2 tension in the incubator chamber. We found that cell-surface NHE3 expression was increased in response to both forms of acidosis. Mild (pH 7.21 +/- 0.02) and severe (6.95 +/- 0.07) metabolic acidosis increased mRNA levels, at least in part due to up-regulation of transcription, whilst mild (7.11 +/- 0.03) and severe (6.86 +/- 0.01) respiratory acidosis did not up-regulate NHE3 expression. Analyses of the Nhe3 promoter region suggested that the regulatory elements sensitive to metabolic acidosis are located between -466 and -153 bp, where two consensus binding sites for SP1, a transcription factor up-regulated in metabolic acidosis, were localised. We conclude that metabolic acidosis induces Nhe3 promoter activation, which results in higher mRNA and total protein level. At the plasma membrane surface, NHE3 expression was increased in metabolic and respiratory acidosis alike, suggesting that low pH is responsible for NHE3 displacement to the cell surface.

Sildenafil reduces polyuria in rats with lithium-induced NDI

Sanches TR, Volpini RA, Massola Shimizu MH, de Bragan a AC, Oshiro-Monreal F, Seguro AC, Andrade L. Sildenafil reduces polyuria in rats with lithium-induced NDI. Am J Physiol Renal Physiol 302: F216-F225, 2012. First published October 12, 2011; doi:10.1152/ajprenal.00439.2010.-Lithium (Li)-treated patients often develop urinary concentrating defect and polyuria, a condition known as nephrogenic diabetes insipidus (NDI). In a rat model of Li-induced NDI, we studied the effect that sildenafil (Sil), a phosphodiesterase 5 (PDE5) inhibitor, has on renal expression of aquaporin-2 (AQP2), urea transporter UT-A1, Na(+)/H(+) exchanger 3 (NHE3), Na(+)-K(+)-2Cl(-) cotransporter (NKCC2), epithelial Na channel (ENaC; alpha-, beta-, and gamma-subunits), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase. We also evaluated cGMP levels in medullary collecting duct cells in suspension. For 4 wk, Wistar rats received Li (40 mmol/kg food) or no treatment (control), some receiving, in weeks 2-4, Sil (200 mg/kg food) or Li and Sil (Li+Sil). In Li+Sil rats, urine output and free water clearance were markedly lower, whereas urinary osmolality was higher, than in Li rats. The cGMP levels in the suspensions of medullary collecting duct cells were markedly higher in the Li+Sil and Sil groups than in the control and Li groups. Semiquantitative immunoblotting revealed the following: in Li+Sil rats, AQP2 expression was partially normalized, whereas that of UT-A1, gamma-ENaC, and eNOS was completely normalized; and expression of NKCC2 and NHE3 was significantly higher in Li rats than in controls. Inulin clearance was normal in all groups. Mean arterial pressure and plasma arginine vasopressin did not differ among the groups. Sil completely reversed the Li-induced increase in renal vascular resistance. We conclude that, in experimental Li-induced NDI, Sil reduces polyuria, increases urinary osmolality, and decreases free water clearance via upregulation of renal AQP2 and UT-A1.

The regulation of NHE₁ and NHE₃ activity by angiotensin II is mediated by the activation of the angiotensin II type I receptor/phospholipase C/calcium/calmodulin pathway in distal nephron cells

Angiotensin II (Ang II), acting via the AT1 receptor, induces an increase in intracellular calcium [Ca(2+)]i that then interacts with calmodulin (CaM). The Ca(2+)/CaM complex directly or indirectly activates sodium hydrogen exchanger 1 (NHE1) and phosphorylates calmodulin kinase II (CaMKII), which then regulates sodium hydrogen exchanger 3 (NHE3) activity. In this study, we investigated the cellular signaling pathways responsible for Ang II-mediated regulation of NHE1 and NHE3 in Madin-Darby canine kidney (MDCK) cells. The NHE1- and NHE3-dependent pHi recovery rates were evaluated by fluorescence microscopy using the fluorescent probe BCECF/AM, messenger RNA was evaluated with the reverse transcription polymerase chain reaction (RT-PCR), and protein expression was evaluated by immunoblot. We demonstrated that treatment with Ang II (1pM or 1 nM) for 30 min induced, via the AT1 but not the AT2 receptor, an equal increase in NHE1 and NHE3 activity that was reduced by the specific inhibitors HOE 694 and S3226, respectively. Ang II (1 nM) did not change the total expression of NHE1, NHE3 or calmodulin, but it induced CaMKII, cRaf-1, Erk1/2 and p90(RSK) phosphorylation. The stimulatory effects of Ang II (1 nM) on NHE1 or NHE3 activity or protein abundance was reduced by ophiobolin-A (CaM inhibitor), KN93 (CaMKII inhibitor) or PD98059 (Mek inhibitor). These results indicate that after 30 min, Ang II treatment may activate G protein-dependent pathways, including the AT1/PLC/Ca(2+)/CaM pathway, which induces CaMKII phosphorylation to stimulate NHE3 and induces cRaf-1/Mek/Erk1/2/p90(RSK) activity to stimulate NHE1