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Background: The kinase, mTOR complex-2 (mTORC2), is implicated in the regulation of renal K+ secretion and maintenance of renal sodium and potassium homeostasis. Recent evidences suggest differential regulation of epithelial sodium channel, ENaC, and ENaC-dependent K+ secretion along the distal nephron under various levels of dietary K perturbations. However, the role of mTORC2 in this nephron segment-specific control of K+ secretion is not fully understood yet. Here we have generated multiple mouse models with renal tubular segment specific mTORC2 KO, and studied its physiological consequences as well as the ability of these mice to respond to dietary potassium load to assess the role of mTORC2 in K+ secretion. Methods: mTORC2 was inactivated in either the entire nephron or selectively in different segments of the distal nephron using segment-specific Cre-Lox-mediated knockout (KO) of Rictor (a core component of mTORC2). WT and KO mice were subjected to a high K diet for 2 days. Urinary and blood parameters, and renal expression and activity of signaling molecules and transport proteins were assessed. Results: At 48 h on a high K+ diet, WT mice demonstrated increased activated ENaC and ROMK in the plasma membrane with normal plasma [K+] and moderately elevated aldosterone level. Downstream targets of mTORC2 implicated in ENaC regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated. At 24 h on HK diet, the entire tubule-specific Rictor knockout (TRKO) mice had significantly higher urinary Na/K ratio, greater UNa*V (net urine Na excretion) and reduced UK*V than WT, consistent with a functional defect in ENaC. By 48h, TRKO mice displayed severe pathophysiologic changes, including marked weight loss, severe hyperkalemia and volume depletion along with significantly higher aldosterone level and BUN than WT. Activation of ENaC and ROMK and phosphorylation of both SGK1 and Nedd4-2 were also severely impaired in TRKO mice. Knocking out of Rictor at the distal part of the DCT and at CNT, resulted in nearly similar pathophysiologic responses to the HK load, which included weight loss, reduced food intake, and urine output, significantly higher urinary Na/K ratio, BUN and plasma aldosterone level, hyperkalemia and abnormal urinary K+ excretion, as compared to the WT mice. Interestingly, deletion of Rictor in more distal part of the CNT and CCD, resulted in only milder abnormal phenotype than the entire nephron- or DCT2/CNT-specific Rictor KO mice on a high K diet. Conclusions: Overall, the data suggest that mTORC2 activity in the late DCT and CNT plays a major role in adjusting renal K+ secretion in response to an increased dietary K+ load. National Institutes of Health (R01- DK56695), James Hilton Manning and Emma Austin Manning Foundation This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.