Total NHE3 Research Articles

Empagliflozin Downregulates Renal NHE3 Activity and NaPi‐2 Expression and Reduces Blood Pressure in Hypertensive Rats

Sodium-glucose cotransporter 2 (SGLT2) inhibitors, also known as gliflozins, can lower blood pressure (BP) and reduce cardiovascular mortality in hypertensive patients with type 2 diabetes. However, the molecular mechanisms underlying the antihypertensive effects of the SGLT2 inhibitors have not been elucidated. This study tested the hypothesis that empagliflozin, an SGLT2 inhibitor, reduces BP of nondiabetic hypertensive rats and that this effect may be associated with altered expression and/or covalent modification of apical sodium transporters in the renal proximal tubule. To this end, spontaneously hypertensive rats (SHR) and normotensive Wistar rats (12-14 weeks of age) were treated for 2 weeks with empagliflozin 10 mg/kg/day or vehicle (water) orally. BP was measured non-invasively (tail-cuff plethysmography) and invasively (arterial catheterization). Proximal tubule Na+/H+ exchanger isoform 3 (NHE3) activity was assessed by in vivo stationary microperfusion. NHE3 phosphorylated at serine 552 (pNHE3-552), total NHE3 and Na+-dependent phosphate cotransporter type II (NaPi-2) expression in the renal cortex were evaluated by immunoblotting. We found that vehicle-treated SHR exhibited a small increase in BP (9±2 mmHg), whereas empagliflozin reduced BP of hypertensive rats (-10±2 mmHg). No changes in BP were observed in Wistar rats. The in vivo stationary microperfusion analyses showed that empagliflozin inhibited the activity of NHE3 in both hypertensive and normotensive rats. Inhibition of NHE3 activity by empagliflozin was not accompanied by differences in the levels of pNHE3-552 or total NHE3 expression in the renal cortex. On the other hand, NaPi-2 expression was upregulated in the renal cortex of vehicle-treated SHR (226±18 vs. 100±5% in vehicle-treated Wistar rats, p< 0.0001), and treatment with empagliflozin significantly reduced it (163±11 vs. 226±18%, p< 0.01). Renal cortical NaPi-2 abundance was not affected by empagliflozin in normotensive rats. These findings demonstrate empagliflozin lowers blood pressure in nondiabetic hypertensive rats. Moreover, it shows that SGLT2 inhibition may reduce proximal tubule sodium reabsorption via inhibition of NHE3 and downregulation of NaPi-2. Future studies are necessary to evaluate the impact of SGLT2 inhibitors in the distal nephron and its association with the antihypertensive effects of the gliflozins.

Renal Proximal Tubule‐Specific Ablation of Atp1a1Reveals a Novel Tonic Inhibitory Mechanism of Sodium Reabsorption

Systemic levels of endogenous cardiotonic steroids (CTS) increase markedly during salt loading, volume expansion, and renal insufficiency, suggesting a physiological role in the regulation of renal Na+ handling. Rather than the classic CTS‐mediated inhibition of Na+/K+‐ATPase (NKA)‐mediated ion transport in the renal proximal tubule (RPT), in vitro pharmacological approaches have suggested that low concentrations of CTS (in the range of those reported in the blood) may initiate NKA/Src‐mediated signaling to reduce apical Na+/H+ Exchanger‐3 (NHE3) and transepithelial Na+ flux in the RPT.To obtain genetic evidence of this putative NKA/Src mechanism in the RPT and asses its physiological impact, we used a knockdown and rescue approach in pig renal epithelial cells (LLC‐PK1) and generated a PT‐specific NKA α1 knockout mouse (RPTα1−/−) by crossing SGLT2 (sodium glucose co‐transporter 2)‐Cre mice with Floxed Atp1a1 mice. A SGLT2‐Cre/Rosa 26 system was then used to re‐introduce expression of wild‐type NKA α1 (RPT α1WT) or a Src‐null mutant NKA α1Y260A (RPTα1Y260A) with intact ion‐pumping.In cells with 90% NKA α1 knockdown compared to the parent LLC‐PK1 cell line, we observed a 50% decrease in phosphorylated NHE3 (inactive form) without change in total NHE3, and a 50% increase in total Sodium‐Bicarbonate cotransporter‐1A (NBCe1A) expression. Comparable NHE3 activation with NBCe1A increase was observed when NKA α1 knockdown cells were rescued with a Src‐binding NKA α1 null‐mutant or non‐src binding NKA α2, but not with Src‐binding gain‐of‐function α2 mutant or the WT NKA α1, suggesting a role for NKA/Src receptor function in the tonic inhibition of NHE3.RPT‐specific KO and rescue confirmed by immunohistochemistry in kidney cross‐section from RPTα1−/−, RPTα1WT, and RPTα1Y260A mice did not alter kidney size, morphology or overall structure as assessed by periodic acid shift (PAS) and Masson’s trichrome staining. Western blot analyses of RPTα1−/− cortex indicated a decrease in phosphorylated NHE3 with no change in total NHE3 and an increase in NBCe1A expression comparable to those observed in vitro. Functionally, a 65% decrease in daily urine output and absolute Na+ excretion was observed in RPTα1−/− mice (n=6–10). Consistent with a NKA‐dependent tonic inhibition of NHE3, increased PT Na+ reabsorption was indicated by a 65% decrease in urinary lithium clearance in RPTα1−/−, with no change in glomerular filtration rate measured by FITC‐sinistrin clearance (n=10–12). The absolute Na+ excretion was rescued in the RPTα1WT but not in the RPTα1Y260A mouse, which supported the role of NKA α1/Src signaling in the PT Na+ reabsorption (n=5).These studies reveal a novel mechanism of tonic inhibition of NHE3 and NBCe1A by Atp1a1. In vitro results provide genetic evidence that NKA/Src receptor function is critical to this mechanism, which was corroborated in vivo. Animal studies further indicate a significant physiologically impact of this hitherto unrecognized regulation of Na+ reabsorption in the PT, which may be regulated by endogenous CTS in health and disease.Support or Funding InformationAHA fellowship #19PRE34450095

Expression of lysophosphatidic acid receptor 5 is necessary for the regulation of intestinal Na+/H+ exchanger 3 by lysophosphatidic acid in vivo.

Lysophosphatidic acid (LPA) is a bioactive lipid molecule, which regulates a broad range of pathophysiological processes. Recent studies have demonstrated that LPA modulates electrolyte flux in the intestine, and its potential as an antidiarrheal agent has been suggested. Of six LPA receptors, LPA5 is highly expressed in the intestine. Recent studies by our group have demonstrated activation of Na+/H+ exchanger 3 (NHE3) by LPA5. However, much of what has been elucidated was achieved using colonic cell lines that were transfected to express LPA5. In the current study, we engineered a mouse that lacks LPA5 in intestinal epithelial cells, Lpar5ΔIEC, and investigated the role of LPA5 in NHE3 regulation and fluid absorption in vivo. The intestine of Lpar5ΔIEC mice appeared morphologically normal, and the stool frequency and fecal water content were unchanged compared with wild-type mice. Basal rates of NHE3 activity and fluid absorption and total NHE3 expression were not changed in Lpar5ΔIEC mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5ΔIEC mice, providing direct evidence for the regulatory role of LPA5. NHE3 activation involves trafficking of NHE3 from the terminal web to microvilli, and this mobilization of NHE3 by LPA was abolished in Lpar5ΔIEC mice. Dysregulation of NHE3 was specific to LPA, and insulin and cholera toxin were able to stimulate and inhibit NHE3, respectively, in both wild-type and Lpar5ΔIEC mice. The current study for the first time demonstrates the necessity of LPA5 in LPA-mediated stimulation of NHE3 in vivo. NEW & NOTEWORTHY This study is the first to assess the role of LPA5 in NHE3 regulation and fluid absorption in vivo using a mouse that lacks LPA5 in intestinal epithelial cells, Lpar5ΔIEC. Basal rates of NHE3 activity and fluid absorption, and total NHE3 expression were not changed in Lpar5ΔIEC mice. However, LPA did not activate NHE3 activity or fluid absorption in Lpar5ΔIEC mice, providing direct evidence for the regulatory role of LPA5.

Dietary sodium induces a redistribution of the tubular metabolic workload.

Key points Body Na+ content is tightly controlled by regulated urinary Na+ excretion.The intrarenal mechanisms mediating adaptation to variations in dietary Na+ intake are incompletely characterized.We confirmed and expanded observations in mice that variations in dietary Na+ intake do not alter the glomerular filtration rate but alter the total and cell‐surface expression of major Na+ transporters all along the kidney tubule.Low dietary Na+ intake increased Na+ reabsorption in the proximal tubule and decreased it in more distal kidney tubule segments.High dietary Na+ intake decreased Na+ reabsorption in the proximal tubule and increased it in distal segments with lower energetic efficiency.The abundance of apical transporters and Na+ delivery are the main determinants of Na+ reabsorption along the kidney tubule.Tubular O2 consumption and the efficiency of sodium reabsorption are dependent on sodium diet. Na+ excretion by the kidney varies according to dietary Na+ intake. We undertook a systematic study of the effects of dietary salt intake on glomerular filtration rate (GFR) and tubular Na+ reabsorption. We examined the renal adaptive response in mice subjected to 7 days of a low sodium diet (LSD) containing 0.01% Na+, a normal sodium diet (NSD) containing 0.18% Na+ and a moderately high sodium diet (HSD) containing 1.25% Na+. As expected, LSD did not alter measured GFR and increased the abundance of total and cell‐surface NHE3, NKCC2, NCC, α‐ENaC and cleaved γ‐ENaC compared to NSD. Mathematical modelling predicted that tubular Na+ reabsorption increased in the proximal tubule but decreased in the distal nephron because of diminished Na+ delivery. This prediction was confirmed by the natriuretic response to diuretics targeting the thick ascending limb, the distal convoluted tubule or the collecting system. On the other hand, HSD did not alter measured GFR but decreased the abundance of the aforementioned transporters compared to NSD. Mathematical modelling predicted that tubular Na+ reabsorption decreased in the proximal tubule but increased in distal segments with lower transport efficiency with respect to O2 consumption. This prediction was confirmed by the natriuretic response to diuretics. The activity of the metabolic sensor adenosine monophosphate‐activated protein kinase (AMPK) was related to the changes in tubular Na+ reabsorption. Our data show that fractional Na+ reabsorption is distributed differently according to dietary Na+ intake and induces changes in tubular O2 consumption and sodium transport efficiency.

PDZ domain-dependent regulation of NHE3 protein by both internal Class II and C-terminal Class I PDZ-binding motifs

NHE3 directly binds Na+/H+ exchanger regulatory factor (NHERF) family scaffolding proteins that are required for many aspects of NHE3 regulation. The NHERFs bind both to an internal region (amino acids 586-660) of the NHE3 C terminus and to the NHE3 C-terminal four amino acids. The internal NHERF-binding region contains both putative Class I (-592SAV-) and Class II (-595CLDM-) PDZ-binding motifs (PBMs). Point mutagenesis showed that only the Class II motif contributes to NHERF binding. In this study, the roles in regulation of NHE3 activity of these two PBMs were investigated, revealing the following findings. 1) Interaction occurred between these binding sites because mutation of either removed nearly all NHERF binding. 2) Mutations in either significantly reduced basal NHE3 activity. Total and percent plasma membrane (PM) NHE3 protein expression was reduced in the C-terminal but not in the internal PBD mutation. 3) cGMP- and Ca2+-mediated inhibition of NHE3 was impaired in both the internal and the C-terminal PBM mutations. 4) There was a significant reduction in half-life of the PM pool of NHE3 in only the internal PBM mutation but no change in total NHE3 half-life in either. 5) There were some differences in NHE3-associating proteins in the two PBM mutations. In conclusion, NHE3 binds to NHERF proteins via both an internal Class II PBM and C-terminal Class I PBM, which interact. The former determines NHE3 stability in the PM, and the latter determines total expression and percent PM expression.

Phosphorylation and subcellular localization of Na+/H+ exchanger isoform 3 (NHE3) are associated with altered gallbladder absorptive function after formation of cholesterol gallstones.

Na+/H+ exchanger isoform 3 (NHE3) dysfunction is thought to contribute to the altered gallbladder absorption that occurs in cholesterol gallstone disease, but the mechanism is unknown. The current study was undertaken to examine the expression, phosphorylation, and subcellular localization of NHE3 in gallbladder epithelium cells (GBECs) of male C57BL/6 mice on a control or lithogenic diet. Thirty-six 8-week-old male C57BL/6 mice were randomly assigned to receive a high cholesterol diet or a regular diet for 8weeks. Gallstone formation was recorded. Gallbladder bile cholesterol, phospholipid, and total bile acids were examined. RT-PCR was used to measure NHE3 mRNA expression. NHE3 protein expression and subcellular localization were examined by Western blotting and immunofluorescence microscopy, respectively. Gallstones were formed in all mice fed the lithogenic diet. Despite higher NHE3 mRNA expression in gallbladders of the mice on the lithogenic diet than in those on the control diet, there was no significant difference in expression of total NHE3 protein. However, a higher level of NHE3 phosphorylated at serine-552 (P-NHE3) was seen on the lithogenic diet. In immunofluorescence studies, NHE3 protein was expressed both on the apical membrane and in the cytoplasm of mouse GBEC. This pattern of subcellular distribution of NHE3 strongly corroborates an exchanger trafficking mechanism in NHE3 activity regulation in mouse GBEC. We conclude that increased phosphorylation of NHE3 following gallstone formation leads to turnover of the exchanger, resulting in decreased gallbladder concentrating function.

Caffeine-induced diuresis and natriuresis is independent of renal tubular NHE3.

Caffeine is one of the most widely consumed behavioral substances. We have previously shown that caffeine- and theophylline-induced inhibition of renal reabsorption causes diuresis and natriuresis, an effect that requires functional adenosine A1 receptors. In this study, we tested the hypothesis that blocking the Gi protein-coupled adenosine A1 receptor via the nonselective adenosine receptor antagonist caffeine changes Na(+)/H(+) exchanger isoform 3 (NHE3) localization and phosphorylation, resulting in diuresis and natriuresis. We generated tubulus-specific NHE3 knockout mice (Pax8-Cre), where NHE3 abundance in the S1, S2, and S3 segments of the proximal tubule was completely absent or severely reduced (>85%) in the thick ascending limb. Consumption of fluid and food, as well as glomerular filtration rate, were comparable in control or tubulus-specific NHE3 knockout mice under basal conditions, while urinary pH was significantly more alkaline without evidence for metabolic acidosis. Caffeine self-administration increased total fluid and food intake comparably between genotypes, without significant differences in consumption of caffeinated solution. Acute caffeine application via oral gavage elicited a diuresis and natriuresis that was comparable between control and tubulus-specific NHE3 knockout mice. The diuretic and natriuretic response was independent of changes in total NHE3 expression, phosphorylation of serine-552 and serine-605, or apical plasma membrane NHE3 localization. Although caffeine had no clear effect on localization of the basolateral Na(+)/bicarbonate cotransporter NBCe1, pretreatment with DIDS inhibited caffeine-induced diuresis and natriuresis. In summary, NHE3 is not required for caffeine-induced diuresis and natriuresis.

Rapamycin Inhibition of mTOR Reduces Levels of the Na+/H+ Exchanger 3 in Intestines of Mice and Humans, Leading to Diarrhea

The immunosuppressant rapamycin frequently causes noninfectious diarrhea in organ transplant recipients. We investigated the mechanisms of this process. We performed a retrospective analysis of renal transplant recipients treated with rapamycin from 2003 through 2010 at Albany Medical College, collecting data on serum levels of rapamycin. Levels of the Na+/H+ exchanger 3 (NHE3) were measured in human ileal biopsy specimens from patients who did and did not receive rapamycin (controls), in ileum tissues from rats or mice given rapamycin, and in mice with intestine-specific disruption of mammalian target of rapamycin (Mtor) (mTOR(f/f):Villin-cre mice) or Atg7 (Atg7(flox/flox); Villin-Cre). Exchange activity and intestinal water absorption were measured using a pH-sensitive dye and small intestine perfusion, respectively. Episodes of noninfectious diarrhea occurred in organ recipients after increases in serum levels of rapamycin. The expression of NHE3 was reduced in the ileal brush border of patients with diarrhea. In rats and mice, continuous administration of low doses of rapamycin reduced levels of NHE3 in intestinal tissues; this effect was not observed in mice with intestinal deletion of ATG7, indicating that autophagy is required for the reduction. Administration of single high doses of rapamycin to mice, to model the spikes in rapamycin levels that occur in patients with severe diarrheal episodes, resulted in reduced phosphorylation of S6 and AKT in ileal tissues, indicating inhibition of the mTOR complex (mTORC1 and mTORC2). The intestines of mice with intestine-specific deletion of mTOR were dilated and contained large amounts of liquid stools; they also had reduced levels of total NHE3 and NHERF1 compared with control mice. We observed a significant reduction in Na(+)/H(+) exchange activity in ileum tissues from these mice. Rapamycin inhibition of mTOR reduces levels of NHE3 and Na(+)/H(+) exchange activity in intestinal tissues of patients and rodents. This process appears to require the autophagic activity mediated by ATG7. Loss of mTOR regulation of NHE3 could mediate the development of diarrhea in patients undergoing rapamycin therapy.

Renal nerve stimulation leads to the activation of the Na+/H+exchanger isoform 3 via angiotensin II type I receptor

Renal nerve stimulation at a low frequency (below 2 Hz) causes water and sodium reabsorption via α1-adrenoreceptor tubular activation, a process independent of changes in systemic blood pressure, renal blood flow, or glomerular filtration rate. However, the underlying mechanism of the reabsorption of sodium is not fully understood. Since the sympathetic nervous system and intrarenal ANG II appear to act synergistically to mediate the process of sodium reabsorption, we hypothesized that low-frequency acute electrical stimulation of the renal nerve (ESRN) activates NHE3-mediated sodium reabsorption via ANG II AT1 receptor activation in Wistar rats. We found that ESRN significantly increased urinary angiotensinogen excretion and renal cortical ANG II content, but not the circulating angiotensinogen levels, and also decreased urinary flow and pH and sodium excretion via mechanisms independent of alterations in creatinine clearance. Urinary cAMP excretion was reduced, as was renal cortical PKA activity. ESRN significantly increased NHE3 activity and abundance in the apical microvillar domain of the proximal tubule, decreased the ratio of phosphorylated NHE3 at serine 552/total NHE3, but did not alter total cortical NHE3 abundance. All responses mediated by ESRN were completely abolished by a losartan-mediated AT1 receptor blockade. Taken together, our results demonstrate that higher NHE3-mediated proximal tubular sodium reabsorption induced by ESRN occurs via intrarenal renin angiotensin system activation and triggering of the AT1 receptor/inhibitory G-protein signaling pathway, which leads to inhibition of cAMP formation and reduction of PKA activity.

Abstract 405: Novel Signaling Mechanisms by Which Intracellular Angiotensin II Induces Na + /HCO 3 - CotransporterExpression In The Proximal Tubule of The Kidney

Previous studies have shown that endocrine and/or paracrine angiotensin II (ANG II) plays an important role in the regulation of sodium and bicarbonate reabsorption in the proximal tubule of the kidney. However, it is not known whether intracellular (or intracrine) ANG II also plays a role in these responses in the proximal tubule. The present study tested the hypothesis that overexpression of an intracellular cyan fluorescent fusion protein of ANG II (ECFP/ANG II) in the proximal tubule of the kidney induces the expression of the Na + /HCO 3 - cotransporter via MAPK- and NF-kB signaling pathways. To test the hypothesis, transport-competent mPCTs from wild-type and type 1a ANG II receptor-deficient mice (AT 1a -KO) were transfected with ECFP/ANG II, and treated with the AT 1 receptor blocker losartan, the MEK1/MEK2 inhibitor U0126, or the NF-κB inhibitor RO 106-9920. In wild-type mPCT cells, the expression of ECFP/ANG II more than doubled total and/or phosphorylated NHE3 antiporter and Na + /HCO 3 - cotransporter proteins (p&lt;0.01). These response were accompanied by more than threefold increases in phospho-ERK 1/2, p65 subunit of NF-κB, and phospho-IKKα/β (Ser 176/180) proteins (p&lt;0.01). Pretreatment of mPCT cells with losartan, U0126, or RO 106-9920 significantly blocked the effects of ECFP/ANG II (p&lt;0.01). Furthermore, the effects of ECFP/ANG II were significantly attenuated in mPCT cells of AT 1a -KO mice (p&lt;0.01),. In wild-type C57BL/6J mice, adenovirus-mediated overexpression of ECFP/ANG II selectively in the proximal tubule of the kidney, driven by the sodium and glucose cotransporter 2 (sglt2) promoter, significantly increased blood pressure, total and/or phosphorylated NHE3 and Na + /HCO 3 - proteins, and proximal tubular lithium reabsorption (p&lt;0.01). These responses to ECFP/ANG II as observed in C57BL/6J mice were also attenuated in AT 1a -KO mice (p&lt;0.01). Our results strongly suggest that intracellular ANG II may induce NHE3 and Na + /HCO 3 - expression, and increase proximal tubular sodium and bicarbonate reabsorption via AT 1a receptor-mediated activation of MAP kinases ERK 1/2 and NF-κB signaling pathways.

Inhibition of central Na+/H+ exchanger type 3 can alleviate sleep apnea in Sprague-Dawley rats

Background Recent studies showed the central Na+/H+ exchanger type 3 (NHE3) has a close relationship with ventilation control. The objective of the study is to investigate the role of NHE3 in sleep apnea in Sprague-Dawley (SD) rats. Methods A sleep study was performed on 20 male SD rats to analyze the correlation between the sleep apneic events and total NHE3 protein content and inactive NHE3(pS552) in the brainstem measured by Western blotting. Another 20 adult male SD rats received 3 days of sleep and respiration monitoring for 6 hours a day, with adaption on the first day, 0.5% DMSO microinjection into the fourth ventricle on the second day, and AVE0657 (specific inhibitor of NHE3) microinjection on the third day. Rats were divided into two groups with injection of 5 μmol/L or 8 μmol/L AVE0657 before the sleep study. The effects of AVE0657 on sleep apnea and sleep structure of rats were analyzed through self-control. Results The total post-sigh apnea index (TPSAI) and post-sigh apnea index in non-rapid eye movement (NREM) sleep (NPSAI) and total apnea index (AI) in NREM sleep (NAI) were negatively correlated with NHE3(pS552) protein contents in the brainstem (r=-0.534, -0.547 and -0.505, respectively, P&lt;0.05). The spontaneous apnea index in REM sleep (RSPAI) was positively correlated with the level of NHE3(pS552) protein expression in the brainstem (r=0.556, P&lt;0.05). However, the sleep AI had no relationship with total NHE3 protein. Compared with the blank control and microinjection of 0.5% DMSO, 5 μmol/L AVE0657 significantly reduced the total AI and NPSAI (both P&lt;0.05) without a significant effect on sleep architecture. In contrast to blank control and microinjection of 0.5% DMSO, injection of 8 μmol/L AVE0657 significantly reduced the AI and PSAI in NREM and REM sleep (all P&lt;0.05). Conclusions The severity of sleep apnea was negatively correlated with central inactive NHE3. A specific inhibitor of NHE3 decreased the sleep AI. Thus, our results indicate that central NHE3 might be a molecular target for sleep apnea treatment, whose inhibitors may be potential therapeutic drugs for sleep apnea.

Dopamine D3 receptor inhibits the ubiquitin‐specific peptidase 48 to promote NHE3 degradation

The dopamine D3 receptor (D3R) is crucial in the regulation of blood pressure and sodium balance, in that Drd3 gene ablation in mice results in hypertension and failure to excrete a dietary salt load. The mechanism responsible for the renal sodium retention in these mice is largely unknown. We now offer and describe a novel mechanism by which D3R decreases sodium transport in the long term by inhibiting the deubiquitinylating activity of ubiquitin-specific peptidase 48 (USP48), thereby promoting Na(+)-H(+) exchanger (NHE)-3 degradation. We found that stimulation with the D3R-specific agonist PD128907 (1 μM, 30 min) promoted the interaction and colocalization among D3R, NHE3, and USP48; inhibited USP48 activity (-35±6%, vs. vehicle), resulting in increased ubiquitinylated NHE3 (+140±10%); and decreased NHE3 expression (-50±9%) in human renal proximal tubule cells (hRPTCs). USP48 silencing decreased NHE3's half-life (USP48 siRNA t1/2=6.1 h vs. vehicle t1/2=12.9 h), whereas overexpression of USP48 increased NHE3 half-life (t1/2=21.8 h), indicating that USP48 protects NHE3 from degradation via deubiquitinylation. USP48 accounted for ∼30% of the total deubiquitinylating activity in these cells. Extending our studies in vivo, we found that pharmacologic blockade of D3R via the D3R-specific antagonist GR103691 (1 μg/kg/min, 4 d) in C57Bl/6J mice increased renal NHE3 expression (+310±15%, vs. vehicle), whereas an innovative kidney-restricted Usp48 silencing via siRNA (3 μg/d, 7 d) increased ubiquitinylated NHE3 (+250±30%, vs. controls), decreased total NHE3 (-23±2%), and lowered blood pressure (-24±2 mm Hg), compared with that in control mice that received either the vehicle or nonsilencing siRNA. Our data demonstrate a crucial role for the dynamic interaction between D3R and USP48 in the regulation of NHE3 expression and function.

Altered protein phosphatase 1 (PP1) activity and expression contribute to the differential regulation of proximal tubule NHE3 before and after onset of hypertension in SHR

We have previously demonstrated that NHE3 activity is higher in the proximal tubule of young pre‐hypertensive SHR (Y‐SHR) compared to adult hypertensive SHR (A‐SHR). This fall in NHE3 activity with blood pressure increase was associated with a higher ratio of phosphorylated NHE3 at serine 552 to total NHE3 (P‐NHE3/ total). The present study was undertaken to test the hypothesis that the increased levels of phosphorylated NHE3 at the PKA consensus site serine 552 was due to an increase of PKA activity, or a decrease of PP1 activity, or both. To confirm that PKA‐mediated phosphorylation of NHE3 regulation is altered in SHR, we examined the effect of 6MB‐cAMP (an cAMP analog that specifically activates PKA) on both phosphorylation levels and activity of NHE3 in young and adult SHR. We found that the 6MB‐cAMP‐induced increase in the levels of NHE3 phosphorylation at the serine 552 was much higher in the renal cortex from Y‐SHR than from A‐SHR (179 ± 14 vs. 36 ± 4%, P &lt; 0.001). Likewise, the inhibitory effect of 6MB‐cAMP on NHE3‐mediated bicarbonate reabsorption was much more pronounced in proximal tubule from Y‐SHR than from A‐SHR (65 ± 10 vs. 23 ± 9%, P &lt; 0.01). PKA activity remained unchanged between Y‐SHR and A‐SHR. On the other hand, Y‐SHR exhibited higher PP1 activity than A‐SHR (1640 ± 107 vs. 940 ± 119 pM/μg, P &lt; 0.01). Furthermore, PP1α expression was decreased in the renal cortex of A‐SHR compared to Y‐SHR (32 ± 8 %, P &lt; 0.01). We conclude that the differential regulation of proximal tubule NHE3 activity and phosphorylation levels before and after onset of hypertension in SHR is, at least in part, due to changes in the activity and expression of PP1.Supported by FAPESP

Chronic regulation of the renal Na+/H+exchanger NHE3 by dopamine: translational and posttranslational mechanisms

The intrarenal autocrine/paracrine dopamine (DA) system contributes to natriuresis in response to both acute and chronic Na(+) loads. While the acute DA effect is well described, how DA induces natriuresis chronically is not known. We used an animal and a cell culture model to study the chronic effect of DA on a principal renal Na(+) transporter, Na(+)/H(+) exchanger-3 (NHE3). Intraperitoneal injection of Gludopa in rats for 2 days elevated DA excretion and decreased total renal cortical and apical brush-border NHE3 antigen. Chronic treatment of an opossum renal proximal cell line with DA decreased NHE3 activity, cell surface and total cellular NHE3 antigen, but not NHE3 transcript. The decrease in NHE3 antigen was dose and time dependent with maximal inhibition at 16-24 h and half maximal effect at 3 × 10(-7) M. This is in contradistinction to the acute effect of DA on NHE3 (half maximal at 2 × 10(-6) M), which was not associated with changes in total cellular NHE3 protein. The DA-induced decrease in total NHE3 protein was associated with decrease in NHE3 translation and mediated by cis-sequences in the NHE3 5'-untranslated region. DA also decreased cell surface and total cellular NHE3 protein half-life. The DA-induced decrease in total cellular NHE3 was partially blocked by proteasome inhibition but not by lysosome inhibition, and DA increased ubiquitylation of total and surface NHE3. In summary, chronic DA inhibits NHE3 with mechanisms distinct from its acute action and involves decreased NHE3 translation and increased NHE3 degradation, which are novel mechanisms for NHE3 regulation.

Mechanisms of Na+/H+ Exchanger Isoform 3 Gene Proximal Promoter Modulation by Parathyroid Hormone

Previous studies have shown that parathyroid hormone (PTH) chronically inhibits NHE3 in opossum kidney cells (OKP) by reducing both, total NHE3 protein and mRNA levels due to transcription modulation. The purpose of this study was to elucidate the inhibitory mechanisms of PTH on the NHE3 gene promoter. Different segments of the proximal promoter (−157/+31; −85/+31; −65/+31 and −44/+31) were inserted in the pGL3-basic luciferase reporter vector. OKP cells were transiently transfect with the vector constructs and kept in serum-free media for 24 h. Cells were then treated with 10-7M PTH for 24 h and promoter activity was determined. These segments were also evaluated by gel shift assays (GMSA). Nuclear expression of the Sp3/EGR-1 transcription factors was also assayed after treatment with 50 μM 1, 2, 3, 4, 5, 6-hexabromocyclohexane (JAK2 autophosphorylation inhibitor), 10 μM Static (STAT3 inhibitor), 1 μM KT5720 (PKA inhibitor) for 24 h.PTH decreased the promoter activity of the fragments −65 and −44 (∼24% and 29% respectively). Inhibition of JAK/STAT and PKA pathways abolished this suppressor effect of PTH. In GMSA, PTH reduced the protein-DNA affinity of the segment −44/+31. Western blot analysis of the transcription factors (Sp3/EGR-1) after treatment with PKA and JAK/STAT signaling pathways inhibitors showed that alone, these inhibitors did not affect their nuclear expression. In conclusion, these data suggest that the cis-element(s) required for PTH responsiveness must be localized in the proximal promoter; the JAK/STAT and PKA pathways may be involved in this inhibitory response; and this effect of PTH on promoter transcription is probably due to changes in protein-DNA affinity without altering transcription factors nuclear expression.

Chronic effect of parathyroid hormone on Na+/H+ exchanger isoform 3 gene proximal promoter

Previous studies have shown that parathyroid hormone (PTH) chronically inhibits NHE3 in opossum kidney cells (OKP) by reducing total NHE3 protein and mRNA levels by modulation of the transcription. The purpose of this study was to elucidate the inhibitory mechanisms of PTH on the NHE3 gene promoter. Different segments of the proximal promoter (−157/+31; −85/+31; −65/+31 and −44/+31) were inserted in the pGL3‐ basic luciferase reporter vector, and point mutations were introduced in some regulatory elements, which were also evaluated by gel shift assays (GMSA). OKP cells were transiently transfect with the vector constructs and kept in serum‐free media for 24 h, then treated with 10−7M PTH for 24 h. PTH decreased the promoter activity of the fragments −65 and −44 (~21% and 31% respectively). Mutations on Sp1/Egr‐1 and AP2 elements completely abolished the PTH inhibitory effect. Inhibition of JAK/STAT pathway abolished the suppressor effect of PTH on fragment −65. In GMSA, PTH reduced the protein‐DNA affinity of the segment −44/+31.In conclusion, these data suggest that the cis‐element(s) required for PTH responsiveness must be localized in the proximal promoter and the JAK/STAT pathway is involved in this inhibitory response.Financial Support: FAPESP and CAPES

Membrane surface charge dictates the structure and function of the epithelial Na+/H+exchanger

The Na(+)/H(+) exchanger NHE3 plays a central role in intravascular volume and acid-base homeostasis. Ion exchange activity is conferred by its transmembrane domain, while regulation of the rate of transport by a variety of stimuli is dependent on its cytosolic C-terminal region. Liposome- and cell-based assays employing synthetic or recombinant segments of the cytosolic tail demonstrated preferential association with anionic membranes, which was abrogated by perturbations that interfere with electrostatic interactions. Resonance energy transfer measurements indicated that segments of the C-terminal domain approach the bilayer. In intact cells, neutralization of basic residues in the cytosolic tail by mutagenesis or disruption of electrostatic interactions inhibited Na(+)/H(+) exchange activity. An electrostatic switch model is proposed to account for multiple aspects of the regulation of NHE3 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.

α2-Adrenergic Receptors Attenuate Secretagogue-Induced Endocytosis and Promote Exocytosis of Intestinal NHE2 and NHE3

Adrenergic agonists, through activation of intestinal epithelial alpha2-adrenergic receptors (alpha2AR), inhibit electrolyte secretion and promote absorption. The mechanisms of action to promote basal Na(+) absorption and inhibit stimulated secretion are not understood completely. The effects of alpha2-agonists on Na(+) transport were studied in a cell line, Caco2-3B, derived from the Caco2 cell line engineered to permanently express human alpha2A-adrenergic receptors. Serosal, but not mucosal, addition of the alpha2AR agonist N-(2,6-dichlorophenyl)-4,5-dihydro-1H-imidazol-2-amine (clonidine) increased Caco2-3B apical (22)Na(+) uptake, an effect not seen in the Caco2 parent line that lacks alpha2AR expression. This effect was blocked by the alpha2AR antagonist 17alpha-yohmban-16alpha-carboxylic acid methyl ester (yohimbine). Increased Na(+) uptake was paralleled by increased apical surface abundance of the sodium/hydrogen exchangers NHE2 and NHE3. No changes in total cell NHE2 and NHE3 expression were observed. Clonidine also inhibited both cAMP and Ca(2+)-induced decreases in apical Na(+) uptake and apical membrane NHE2 and NHE3 endocytosis stimulated by these agents. alpha2AR actions were mediated via stimulation of phospholipase C, and metabolism of arachidonic acid by an epoxygenase activity followed epidermal growth factor release and activation of the epidermal growth factor receptor, resulting in phosphatidylinositol-3-kinase and Akt stimulation. In summary, activation of intestinal epithelial alpha2AR significantly blocks the inhibition of apical Na(+) transporters by cAMP- and Ca(2+)-mediated pathways and also directly increases apical sodium/hydrogen exchange activities. By both blocking electrolyte secretion and promoting absorption, alpha2-agonists could be potent antidiarrheal agents that could directly counteract the actions of toxigenic pathogens and other secretagogues causing secretory diarrhea.

Role of the proximal tubule AT1A receptor in sodium transport regulation

Chronic AngII infusion raises blood pressure (BP) in mice which depends on renal AngII receptors (AT1R). AngII is anti‐natriuretic while high BP is natriuretic.Aim 1To determine how AngII infusion affects Na+ transporter abundance and phosphorylation (by immunoblot of homogenates). AngII infusion (1 μg/kg/min x 14d) increases systolic BP ~30 mmHg. Versus sham infused (=1.0), AngII decreased total NHE3 to 0.7±0.1, NHE3‐P (at Ser552) to 0.8±0.1 without altering NHE3 ‐P/total ratio. NaPi2 and myosin VI were unchanged. Distal convoluted tubule (DCT) total NCC increased to 2.2±0.3, NCC‐P (at Thr60) to 5.4 ± 1.6 and NCC‐P/total ratio to 2.2±0.5 fold.Aim 2To determine the role of proximal tubule (PT) AT1R in regulation of Na+ transporters in mice lacking PT AT1R (KO). BP decreased 10mmHg in KO vs. WT and baseline expression of NHE3, NaPi2, and NCC were unchanged in KO vs. WT. AngII infusion increased BP 20mmHg less than in WT. AngII infusion in KO decreased: total NHE3 to 0.55±0.1, NHE3‐P to 0.66±0.1 (no change in ‐P/total NHE3 ratio), myosin VI to 0.56±0.1, NaPi2 to 0.64±0.1. NCC total and NCC‐P were unchanged in the AngII infused KO.Conclusions1) increased NCC contributes to AngII induced high BP while decreased PT NHE3 compensates for high BP in WT, 2) AngII dependent increase in NCC depends on PT AT1R, and 3) the removal of PT AT1R facilitates pressure natriuresis by further decreasing PT transporters during AngII infusion.