These findings indicate that the modulation of Ca2+ handling may be one of the molecular mechanisms underlying the effect of salt intake on myocardial function in hypertension.
BackgroundA growing body of evidence suggests an interplay between extracellular volume and glucose homeostasis. NHE3 is the main pathway for Na reabsorption in the proximal tubule (PT). The sodium glucose cotransporter SGLT2 reabsorbs most of the glucose filtered by the kidneys and colocalizes with NHE3 in the apical membrane of PT. Thus, we tested the hypothesis that SGLT2 and NHE3 functionally interact. Given that SGLT2 inhibitors confer blood‐pressure lowering effects in T2DM patients, we also tested the hypothesis that the antihypertensive effects of SGLT2 inhibitors may be associated with PT NHE3 inhibition.MethodsThe acute and long‐term effects of the SGLT2 inhibitor empagliflozin on PT NHE3 activity were determined by in vivo stationary microperfusion in fourteen‐week‐old male Wistar rats and SHR‐treated rats. Additionally, twelve‐week‐old SHRs were treated with empagliflozin (10 mg/kg/day) or vehicle (control) for two weeks. Tail‐cuff blood pressure and renal function were measured before (baseline) and after treatment (post‐treatment). NHE3 expression and phosphorylation levels were evaluated by immunoblotting.ResultsEmpagliflozin acutely inhibited PT NHE3 activity in both Wistar (1.73 ± 0.07 vs. 2.55 ± 0.14 nmol/cm2.s, P < 0.001) and SHR (0.82 ± 0.07 vs. 1.21 ± 0.10 nmol/cm2.s, P < 0.01). Blood pressure (BP) of empagliflozin‐treated SHR was lower than baseline levels (176 ± 4 vs. 194 ± 4 mm Hg, P < 0.01) whereas no change in BP was observed in vehicle‐treated SHR (194 ± 6 vs. 192 ± 5 mm Hg). Compared to baseline, empagliflozin‐treatment enhanced cumulative urinary flow, calcium, sodium and glucose excretion whereas in vehicle‐treated SHR these parameters remained unchanged. Lower BP in empagliflozin‐treated SHR was associated with lower PT NHE3 activity (0.67 ± 0.10 vs. 1.18 ± 0.10 nmol/cm2.s, P < 0.001). No differences were observed on renal cortical NHE3 protein expression or NHE3 phosphorylation levels between the two groups of rats.ConclusionThese results suggest that NHE3 and SGLT2 functionally interact in the PT. Inhibition of NHE3 by SGLT2 inhibitors may underlie, at least in part, the antihypertensive effect of empagliflozin.Support or Funding InformationFAPESPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The antidiabetic agent class of sodium-glucose cotransporter 2 (SGLT2) inhibitors confer unprecedented cardiovascular benefits beyond glycemic control, including reducing the risk of fatal ventricular arrhythmias. However, the impact of SGLT2 inhibitors on the electrophysiological properties of cardiomyocytes exposed to stimuli other than hyperglycemia remains elusive. This investigation tested the hypothesis that the SGLT2 inhibitor empagliflozin (EMPA) affects cardiomyocyte electrical activity under hypoxic conditions. Rat neonatal and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes incubated or not with the hypoxia-mimetic agent CoCl2 were treated with EMPA (1 μM) or vehicle for 24 h. Action potential records obtained using intracellular microelectrodes demonstrated that EMPA reduced the action potential duration at 30%, 50%, and 90% repolarization and arrhythmogenic events in rat and human cardiomyocytes under normoxia and hypoxia. Analysis of Ca2+ transients using Fura-2-AM and contractility kinetics showed that EMPA increased Ca2+ transient amplitude and decreased the half-time to recover Ca2+ transients and relaxation time in rat neonatal cardiomyocytes. We also observed that the combination of EMPA with the Na+/H+ exchanger isoform 1 (NHE1) inhibitor cariporide (10 µM) exerted a more pronounced effect on Ca2+ transients and contractility than either EMPA or cariporide alone. Besides, EMPA, but not cariporide, increased phospholamban phosphorylation at serine 16. Collectively, our data reveal that EMPA reduces arrhythmogenic events, decreases the action potential duration in rat neonatal and human cardiomyocytes under normoxic or hypoxic conditions, and improves cytosolic calcium handling at least partially independent of NHE1. Moreover, we provided further evidence that SGLT2 inhibitor-mediated cardioprotection may be partly attributed to its cardiomyocyte electrophysiological effects.
NHE3 transport function is inhibited by PKA. Binding of angiotensin II (ANG II) to AT1 receptor has been reported to activate Gi‐protein signaling pathway, resulting in the inhibition of adenylyl cyclase, and consequently, in decreased PKA activation. We therefore tested the hypothesis that decrement of PKA‐mediated NHE3 phosphorylation is one of the mechanisms by which ANG II acutely stimulates NHE3 activity in renal proximal tubule. To this end, OKP cells were treated with 100 μM Dopamine (DOP) for 30 min in the presence or absence of 10−10 M ANG II for 5, 15, and 30 min; or only with ANG II for 30 min. Extrusion of H+ from OKP cells was measured. DOP significantly inhibited NHE3 activity (0.2013 ± 0.009 vs. 0.3036 ± 0.0019 pH units/min in control). Simultaneous incubation of DOP with ANG II for 15 (0.2347 ± 0.044 pH units/min) and 30 min (0.3295 ± 0.032 pH units/min) completely blocked this inhibitory effect. As expected, ANG II stimulated NHE3 activity. DOP increased the levels of NHE3 phosphorylated at the PKA consensus phosphorylation sites [serines 552 and 605] by 64 ± 8% and 288 ± 37% respectively vs. control. ANG II treatment for 15 and 30 min prevented this increase. PKA activity was also measured in these groups of cells. We found that PKA activity was significantly increased in cells treated with DOP for 30 min and in cells treated with DOP plus ANG II for 5 min. Longer incubation times with ANG II (15 and 30 min) in the presence of DOP restored PKA activity near to control levels. Losartan blocked all the effects of ANG II here reported. Collectively, these data suggest that inhibition of PKA activity leading to lower levels of endogenous phosphorylation of NHE3 is one of the mechanisms by which ANG II stimulates NHE3 activity in the proximal tubule.Supported by FAPESP
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