Osmotic Diuresis and Impaired Urinary Concentrating Ability in Epac Knockouts

Abstract

Cyclic adenosine monophosphate (cAMP) is a universal second messenger regulating a plethora of processes in the kidney, such as water‐electrolyte transport and epithelial cell proliferation, to name a few. Downstream effectors of cAMP include protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac) – Epac1 and Epac2. The role of PKA as a classical cAMP acceptor in renal cells has been widely established. While both Epac isoforms are abundantly expressed in various segments of the nephron, their physiological relevance in the kidney remains obscure. Recent studies suggest that Epac is pivotal for arginine‐vasopressin (AVP) dependent water reabsorption in the collecting duct (CD) cells. Activation of Epac triggers intracellular Ca2+ ([Ca2+]i) mobilization in response to AVP, facilitating apical exocytosis of Aquaporin 2 water channels (AQP2) in inner medullary collecting ducts. AVPinduced stimulation of Epac activity may also contribute to long‐term regulation of AQP2 expression in CD cells.Here, we combined ratiometric calcium imaging with quantitative immunoblotting and balance studies in mice with targeted deletion of Epac1 or Epac2 to determine the role of Epac in renal water handling.Both Epac1 and Epac2 knockouts exhibit pronounced polyuria, with urine volumes 30% (Epac1‐/−) to 50% (Epac2−/−) larger than in wild‐type controls. Ablation of either Epac isoform results in moderate (10–15%, statistically significant) decrease in urine osmolality. Mice lacking Epac1 exhibit a prominent increase in total AQP2 protein expression, but largely unaltered plasma membrane resident AQP2 levels and AVP‐dependent [Ca2+]i signal in the CD principal cells. Deletion of Epac2 decreases the sustained AVP‐induced [Ca2+]i signal, markedly reduces total AQP2 abundance, but only mildly diminishes AQP2 levels on the plasma membrane of CD cells. Sodium and potassium concentration was not different in urine samples from Epac1−/−, Epac2−/− and wild‐type mice. Thus, the observed polyuria is, at large, the result of osmotic diuresis, which was attributed to the decreased abundance and activity of sodium‐hydrogen exchanger 3 (NHE3) in the proximal tubule. The ability to concentrate urine is modestly impaired in both knockouts, as evidenced by excessive production of diluted urine after 24h water deprivation. Interestingly, water deprivation markedly increased urea excretion in both Epac knockouts. In mice lacking Epac2−/− this loss of urea was attributed to decreased abundance of UT‐A2 transporter. Expression and activity of NHE3 in both knockouts remained significantly lower than in wild‐type controls, similarly pointing to a major contribution of solute diuresis into the observed increase in urine production.In summary, deletion of either Epac1 or Epac2 impairs water and electrolyte reabsorption in the proximal tubule, resulting in polyuria. The latter is exacerbated in Epac2−/− by defective urea and water handling in the distal nephron segments.Support or Funding InformationASN Ben J. Lipps Research Fellowship (to V. Tomilin), NIH‐NIAID 5R01AI111464‐04, NIH‐NIGMS 1R35GM122536‐01 (to X. Cheng), AHA‐15SDG25550150 (to M. Mamenko), NIH‐NIDDK DK095029, AHA 17GRNT33660488 (to O. Pochynyuk),This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.