Aim: Sexual dimorphisms are evident along the nephron: Females (F) exhibit higher ratios of renal distal to proximal Na + transporters' abundance, greater lithium clearance (C Li ) more rapid natriuresis in response to saline infusion and lower plasma [K + ] vs. males (M). During angiotensin II infusion hypertension (AngII-HTN) M exhibit distal Na + transporter activation, lower proximal and medullary loop transporters, blunted natriuresis in response to saline load, and reduced plasma [K + ]. This study aimed to determine whether responses of F to AngII-HTN mimicked those in M or were impacted by sexual dimorphisms evident at baseline. Methods: Sprague Dawley rats and C57BL/6 mice were AngII infused via osmotic minipumps 2 and 3 weeks, respectively, and assessed by metabolic cage collections, tail-cuff sphygmomanometer, semi-quantitative immunoblotting of kidney and patch-clamp electrophysiology. Results: In F rats, AngII-infusion increased BP to 190 mm Hg, increased phosphorylation of cortical NKCC2, NCC and cleavage of ENaC two to threefold, increased ENaC channel activity threefold and aldosterone 10-fold. K + excretion increased and plasma [K + ] decreased. Evidence of natriuresis in F included increased urine Na + excretion and C Li , and decreased medullary NHE3, NKCC2 and Na,K-ATPase abundance. In C57BL/6 mice, AngII-HTN increased abundance of distal Na + transporters, suppressed proximal-medullary transporters and reduced plasma [K + ] in both F and M. Conclusion: Despite baseline sexual dimorphisms, AngII-HTN provokes similar increases in BP, aldosterone, distal transporters, ENaC channel activation and K + loss accompanied by similar suppression of proximal and loop Na + transporters, natriuresis and diuresis in females and males. K E Y W O R D S angiotensin II, ENaC, female, potassium, proteinuria, sodium transport 2 of 12 | VEIRAS Et Al.

cAMP is a universal second messenger regulating a plethora of processes in the kidney. Two downstream effectors of cAMP are PKA and exchange protein directly activated by cAMP (Epac), which, unlike PKA, is often linked to elevation of [Ca2+]i. While both Epac isoforms (Epac1 and Epac2) are expressed along the nephron, their relevance in the kidney remains obscure. We combined ratiometric calcium imaging with quantitative immunoblotting, immunofluorescent confocal microscopy, and balance studies in mice lacking Epac1 or Epac2 to determine the role of Epac in renal water‐solute handling. Epac1−/− and Epac2−/− mice developed polyuria despite elevated arginine vasopressin levels. We did not detect major deficiencies in arginine vasopressin [Ca2+]i signaling in split‐opened collecting ducts or decreases in aquaporin water channel type 2 levels. Instead, sodium‐hydrogen exchanger type 3 levels in the proximal tubule were dramatically reduced in Epac1−/− and Epac2−/− mice. Water deprivation revealed persisting polyuria, impaired urinary concentration ability, and augmented urinary excretion of Na+ and urea in both mutant mice. In summary, we report a nonredundant contribution of Epac isoforms to renal function. Deletion of Epac1 and Epac2 decreases sodium–hydrogen exchanger type 3 expression in the proximal tubule, leading to polyuria and osmotic diuresis.—Cherezova, A., Tomilin, V., Buncha, V., Zaika, O., Ortiz, P. A., Mei, F., Cheng, X., Mamenko, M., Pochynyuk, O. Urinary concentrating defect in mice lacking Epac1 or Epac2. FASEB J. 33, 2156–2170 (2019). http://www.fasebj.org

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Hypertension is a major modifiable risk factor for cardiovascular disease that remains suboptimally controlled despite all recent advances in biomedical research. Sex differences pertaining to development of hypertension and its pathophysiological outcomes are becoming universally recognized. Mechanistic insights into those differences can be utilized to improve the existing or develop novel therapeutic interventions for both sexes. We have previously demonstrated that excessive stimulation of Epithelial Na+ Channel (ENaC), resistant to mineralocorticoid receptor (MR) inhibition, contributes to Ang II‐induced hypertension in male mice. Given the interaction between aldosterone and estrogen signaling pathways, herein, we hypothesize that resistance to MR blockade in Ang II hypertensive subjects is sex‐specific. We combined physiological, pharmacological, biochemical and biophysical approaches to compare how MR antagonism affects renal sodium handling and blood pressure in Ang II‐infused male and female Sprague Dawley rats. Patch‐clamp electrophysiology in split‐opened collecting ducts demonstrated that systemic spironolactone administration (30 mg/kgBW·day for 14 days) only partially attenuated the stimulatory effect of Ang II infusion on ENaC in males. The activity of the channel remained at least twofold higher than in untreated normotensive controls. MR inhibition was remarkably more efficient in Ang II‐infused females, suppressing renal ENaC activity to its basal levels observed in non‐hypertensive rats. In both sexes, the reduction of ENaC activity driven by spironolactone was primarily attributed to decreased functional expression of the channel. This effect was complemented by a moderate decrease of ENaC open probability in spironolactone‐treated hypertensive females. Circulating aldosterone levels were lower in Ang II‐infused females when compared to males. This difference was further accentuated by spironolactone administration. Quantitative western‐blotting revealed that only in hypertensive females MR antagonism significantly increased renal expression of Nedd4‐2, facilitating ENaC degradation and reducing Na+ reabsorption. MR blockade did not alter the abundance of active phosphorylated forms of other renal Na+ transporters – NCC and NKCC2, in both sexes. In line with our observations on renal Na+ handling, we found that spironolactone markedly attenuated hypertension in Ang II‐infused females, while MR inhibition failed to reduce blood pressure in hypertensive males. Aldosterone antagonism did not elevate plasma K+ in all animal groups. Flow cytometry showed significantly lower levels of Th17 cells in the kidneys of spironolactone‐treated females, indicating reduced inflammatory response. Overall, our findings suggest that the action of spironolactone on ENaC‐mediated renal sodium reabsorption is sex‐specific and MR antagonism can be an effective strategy to reduce blood pressure and kidney damage in females with Ang‐II induced hypertension.Support or Funding InformationAHA 15SDG25550150This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Spontaneously hypertensive rat (SHR) has been widely used as a model to interrogate the contribution of the kidney to the pathogenesis of essential hypertension in humans. Elevation of blood pressure in SHR has been associated with altered renal function, since predisposition to hypertension can be transferred with a transplanted kidney into a normotensive Wistar‐Kyoto (WKY) strain. Renal defects result in excessive water and sodium retention leading to increased circulating volume and blood pressure. Abnormally elevated Na+ reabsorption in SHR is associated with the overactive renin‐angiotensin‐aldosterone system (RAAS) and with the increased abundance of the renal epithelial Na+ channel (ENaC) responsible for final regulation of sodium reuptake in the kidney and pivotal for long‐term blood pressure control. The existing data on regulation of ENaC by RAAS components in SHR is inconsistent and molecular determinants of excessive ENaC activation remain largely obscure. In the present study, we tested the efficacy of aldosterone antagonism to inhibit ENaC activity, decrease renal Na+ reabsorption and reduce blood pressure in SHR of both sexes. Where necessary, age‐matched (12–15 week‐old) WKY rats were used as controls. Consistent with previously published data, enzyme immunoassay analysis revealed elevated circulating aldosterone levels and semi‐quantitative immunoblotting – higher expression of renal ENaC protein in SHR when compared to WKY controls. Patch‐clamp experiments in split‐opened collecting ducts showed that ENaC activity was moderately increased in SHR when compared to WKY. Systemic administration of aldosterone antagonist, spironolactone (30 mg/kgBW·day) did not affect renal ENaC activity or protein abundance in SHR. Aldosterone antagonism did not alter the expression of sodium‐chloride co‐transporter (NCC) in the kidneys of SHR. Tail‐cuff plethysmography showed that treatment with spironolactone at doses 30–200 mg/kgBW·day for 2 weeks failed to decrease blood pressure in SHR and did not result in any detectable changes in urinary sodium excretion. We did not observe any sex‐specific differences pertaining to ENaC activity or responsiveness to spironolactone in SHR males and females. Thus, we conclude that aldosterone is not a primary driver of excessive ENaC‐dependent renal Na+ reabsorption in SHR.