Leite-Dellova DC, Oliveira-Souza M, Malnic G, Mello-Aires M. Genomic and nongenomic dose-dependent biphasic effect of aldosterone on Na

mTORC2 is a kinase complex that targets predominantly Akt, SGK1, and PKC, and has well characterized roles in mediating hormone and growth factor effects on a wide array of cellular processes. Recent evidence suggests that mTORC2 is also directly stimulated in renal tubule cells by increased extracellular potassium (K+) concentration, leading to activation of the Na+ channel, ENaC, and increasing the electrical driving force for K+ secretion. We identify here a signaling mechanism for this local effect of K+. We show that an increase in extracellular [K+] leads to a rise in intracellular chloride (Cl-), which stimulates a previously unknown scaffolding activity of With No Lysine-1 (WNK1) kinase. WNK1 interacts selectively with SGK1 and recruits it to mTORC2 resulting in enhanced SGK1 phosphorylation, and SGK1-dependent activation of ENaC. This scaffolding effect of WNK1 is independent of its own kinase activity and does not cause a generalized stimulation of mTORC2 kinase activity. These findings establish a novel WNK1- dependent regulatory mechanism, which harnesses mTORC2 kinase activity selectively toward SGK1 to control epithelial ion transport and electrolyte homeostasis.

The rapid (2 min) nongenomic effects of aldosterone (ALDO) and/or spironolactone (MR antagonist), RU 486 (GR antagonist), atrial natriuretic peptide (ANP) and dimethyl-BAPTA (BAPTA) on the intracellular pH recovery rate (pHirr) via NHE1 (basolateral Na⁺/H⁺ exchanger isoform), after the acid load induced by NH₄Cl, and on the cytosolic free calcium concentration ([Ca²⁺](i)) were investigated in the proximal S3 segment isolated from rats, by the probes BCECF-AM and FLUO-4-AM, respectively. The basal pHi was 7.15±0.008 and the basal pHirr was 0.195±0.012 pH units/min (number of tubules/number of tubular areas=16/96). Our results confirmed the rapid biphasic effect of ALDO on NHE1: ALDO (10⁻¹² M) increases the pHirr to approximately 59% of control value, and ALDO (10⁻⁶ M) decreases it to approximately 49%. Spironolactone did not change these effects, but RU 486 inhibited the stimulatory effect and maintained the inhibitory effect. ANP (10⁻⁶ M) or BAPTA (5×10⁻⁵ M) alone had no significant effect on NHE1 but prevented both effects of ALDO on this exchanger. The basal [Ca²⁺](i) was 104±3 nM (15), and ALDO (10⁻¹² or 10⁻⁶ M) increased the basal [Ca²⁺](i) to approximately 50% or 124%, respectively. RU 486, ANP and BAPTA decreased the [Ca²⁺](i) and inhibited the stimulatory effect of both doses of ALDO. The results suggest the involvement of GR on the nongenomic effects of ALDO and indicate a pHirr-regulating role for [Ca²⁺](i) that is mediated by NHE1, stimulated/impaired by ALDO, and affected by ANP or BAPTA with ALDO. The observed nongenomic hormonal interaction in the S3 segment may represent a rapid and physiologically relevant regulatory mechanism in the intact animal under conditions of volume alterations.

The direct action of aldosterone (10(-12) M) on net bicarbonate reabsorption (J(HCO(3)(-))) 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(HCO(3)(-)) 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(HCO(3)(-)), 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(HCO(3)(-)) 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(HCO(3)(-)) 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(HCO(3)(-)) and on [Ca(2+)](i) was observed; this antagonist also inhibited the stimulatory effect of aldosterone on J(HCO(3)(-)) and on [Ca(2+)](i). These studies indicate that luminal or peritubular aldosterone (10(-12) M) has a direct nongenomic stimulatory effect on J(HCO(3)(-)) 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(HCO(3)(-)) in middle proximal tubule.

The acute effects of angiotensin-1-7 [ANG-(1-7)] on the reabsorptive bicarbonate flow (J[Formula: see text]) were evaluated using stationary microperfusion in vivo in the proximal tubules of spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar-Kyoto (WKY) rats, using a microelectrode sensitive to H In WKY rats, the control J[Formula: see text] was 2.40 ± 0.10 nmol·cm·s ( = 120); losartan (10 M) or A779 (10 M, a specific Mas antagonist), alone or in combination with losartan, decreased the J[Formula: see text] ANG-(1-7) had biphasic effects on J[Formula: see text]: at 10 M, it inhibited, and at 10, it stimulated the flow. S3226 [10 M, a specific Na-H exchanger 3 (NHE3) antagonist] decreased J[Formula: see text] and changed the stimulatory effect of ANG-(1-7) to an inhibitory one but did not alter the inhibitory action of ANG-(1-7). In SHR, the control J[Formula: see text] was 2.04 ± 0.13 nmol·cm·s ( = 56), and A779 and/or losartan reduced the flow. ANG-(1-7) at 10 M increased J[Formula: see text], and ANG-(1-7) at 10 M reduced it. The effects of A779, losartan, and S3226 on the J[Formula: see text] were similar to those found in WKY rats, which indicated that in SHR, the ANG-(1-7) action on the NHE3 was via Mas and ANG II type 1. The cytosolic calcium in the WKY or SHR rats was ~100 nM and was increased by ANG-(1-7) at 10 or 10 M. In hypertensive animals, a high plasma level of ANG-(1-7) inhibited NHE3 in the proximal tubule, which mitigated the hypertension caused by the high plasma level of ANG II.