Epac-dependent regulation of NHE-3 abundance and activity in the proximal tubule determines the sexual dimorphism in urinary volume production

Background: Abundant basic and clinical evidence demonstrate a critical role of androgenic hormones in regulation of water-electrolyte transport in both proximal tubule and collecting duct system. This culminates in the notable differences in renal function with females producing lower urinary volume and having a greater urinary concentrating ability than males. At the same time, the key intermediate signaling pathways linking binding the androgenic hormones to their respective receptors with integrative water-solute handling by the kidney remain largely underexplored. Exchange proteins directly activated by cAMP isoform 1 and isoform 2 (Epac1 and Epac2, respectively) are known to mediate a plethora of intracellular signaling processes downstream of receptor-ligand triggered events. Epac isoforms are abundantly expressed in the kidney. We previously showed that deletion of Epac1 or Epac2 increases urinary volume and reduces urinary concentrating ability. Here, we aimed to explore the potential relevance of Epac signaling in the kidney in setting sex-dependent dimorphism in urinary production. Methods: Age-matched mice with global or kidney specific (KS) deletion of Epac1 and Epac2 underwent balance studies in metabolic cages and water deprivation tests with the goal to examine sex-dependent differences in urinary production. In addition, gonadectomy was performed in males and females to ablate the androgenic input. Isolated kidneys were subjected to biochemical and immunofluorescent microscopy assessment. Results: Western blot analysis of whole kidney lysates from wild type (WT) mice revealed over 2-fold greater expression of both Epac1 and Epac2 in females than in males pointing to a sex-dependent contribution of Epac signaling in regulation of urinary volume and osmolarity. Genetic deletion of Epac isoforms increased urinary volume and reduced urinary osmolarity in an additive manner. Importantly, females lacking both isoforms exhibited drastically larger polyuria than males (increases urinary volume by 320% versus 38%). In addition, urinary concentrating ability was significantly more compromised in Epac isoform deficient females than in males. The polyuric phenotype was also reproduced in mice with renal tubule specific deletion of Epac isoforms suggesting the central role of renal Epac expression in this pathology. At the molecular level, deletion of both Epac12 decreased NHE-3 expression by 90% in females, whereas only by 58% in males. The reduced NHE-3 expression was also associated with translocation to the base of brush border of proximal tubule cells and impaired Na + /H + transport suggesting an increased fluid flow to the loop of Henle with a greater extent in females. Consistently, we also observed a compensatory elevation of AQP2 water channel levels in the collecting duct, which was larger in females. Importantly, ovariectomy greatly reduced urinary volume and improved urinary concentrating ability in Epac1&2 -/- females, whilst castration led to only a mild antidiuresis in Epac1&2 -/- males. In fact, gonadectomy abolished sex-dependent differences in urinary production in the knockouts. Conclusions: Our findings demonstrate a novel role of Epac signaling cascade in establishing sex-dependent differences in urinary production. We propose that higher Epac isoform expression in females is instrumental to control NHE-3 expression and activity in the proximal tubule to a larger extent than in males. This allows more effective regulation of fluid flow to the medulla to preserve interstitial hypertonicity and reduce urinary volume at the baseline and in response to androgenic inputs especially in females. This research was supported by NIDDK DK117865, DK136462, AHA 20EIA35260097 (to O. Pochynyuk), AHA 24POST1191738 (to A. Atamanchuk) and AHA 24SCEFIA1259812 (to K. Pyrshev). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.