Independent regulation of Piezo1 activity by principal and intercalated cells of the collecting duct.

The renal collecting duct is continuously exposed to a wide spectrum of fluid flow rates and osmotic gradients. Expression of a mechanoactivated Piezo1 channel is the most prominent in the collecting duct. However, the status and regulation of Piezo1 in functionally distinct principal and intercalated cells (PCs and ICs) of the collecting duct remain to be determined. We used pharmacological Piezo1 activation to quantify Piezo1-mediated [Ca2+]i influx and single-channel activity separately in PCs and ICs of freshly isolated collecting ducts with fluorescence imaging and electrophysiological tools. We also employed a variety of systemic treatments to examine their consequences on Piezo1 function in PCs and ICs. Piezo1 selective agonists, Yoda-1 or Jedi-2, induced a significantly greater Ca2+ influx in PCs than in ICs. Using patch clamp analysis, we recorded a Yoda-1-activated nonselective channel with 18.6 ± 0.7 pS conductance on both apical and basolateral membranes. Piezo1 activity in PCs but not ICs was stimulated by short-term diuresis (injections of furosemide) and reduced by antidiuresis (water restriction for 24 h). However, prolonged stimulation of flow by high K+ diet decreased Yoda-1-dependent Ca2+ influx without changes in Piezo1 levels. Water supplementation with NH4Cl to induce metabolic acidosis stimulated Piezo1 activity in ICs but not in PCs. Overall, our results demonstrate functional Piezo1 expression in collecting duct PCs (more) and ICs (less) on both apical and basolateral sides. We also show that acute changes in fluid flow regulate Piezo1-mediated [Ca2+]i influx in PCs, whereas channel activity in ICs responds to systemic acid-base stimuli.

Compromised regulation of the collecting duct ENaC activity in mice lacking AT1a receptor.

ENaC-mediated sodium reabsorption in the collecting duct (CD) is a critical determinant of urinary sodium excretion. Existing evidence suggest direct stimulatory actions of Angiotensin II (Ang II) on ENaC in the CD, independently of the aldosterone-mineralocorticoid receptor (MR) signaling. Deletion of the major renal AT1 receptor isoform, AT1a R, decreases blood pressure and reduces ENaC abundance despite elevated aldosterone levels. The mechanism of this insufficient compensation is not known. Here, we used patch clamp electrophysiology in freshly isolated split-opened CDs to investigate how AT1a R dysfunction compromises functional ENaC activity and its regulation by dietary salt intake. Ang II had no effect on ENaC activity in CDs from AT1a R -/- mice suggesting no complementary contribution of AT2 receptors. We next found that AT1a R deficient mice had lower ENaC activity when fed with low (<0.01% Na+ ) and regular (0.32% Na+ ) but not with high (∼2% Na+ ) salt diet, when compared to the respective values obtained in Wild type (WT) animals. Inhibition of AT1 R with losartan in wild-type animals reproduces the effects of genetic ablation of AT1a R on ENaC activity arguing against contribution of developmental factors. Interestingly, manipulation with aldosterone-MR signaling via deoxycosterone acetate (DOCA) and spironolactone had much reduced influence on ENaC activity upon AT1a R deletion. Consistently, AT1a R -/- mice have a markedly diminished MR abundance in cytosol. Overall, we conclude that AT1a R deficiency elicits a complex inhibitory effect on ENaC activity by attenuating ENaC Po and precluding adequate compensation via aldosterone cascade due to decreased MR availability.