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Regulation of ENaC in the distal nephron is essential for sodium homeostasis and blood pressure control. A unique feature of ENaC is its complex proteolytic processing leading to channel activation due to release of inhibitory tracts from its α- and γ-subunits. In particular, fully cleaved γ-ENaC is critical for high channel activity. However, the physiologically relevant proteases involved remain elusive. Using Xenopus laevis oocytes and H441 airway epithelial cells, we recently demonstrated that TMPRSS2 proteolytically activates ENaC by cleaving the channel’s γ-subunit ( J Biol Chem 2022 Jun;298(6):102004). Here we investigate whether TMPRSS2 also contributes to proteolytic ENaC regulation in the kidney. Using mRNA sequencing, we demonstrated that mouse cortical collecting duct (mCCDcl1) cells express TMPRSS2 at a high level unaffected by aldosterone, an important ENaC stimulating hormone. TMPRSS2 knockdown by CRISPR/Cas9 technology significantly reduced fully cleaved γ-ENaC in membrane-enriched fractions of mCCDcl1 cell lysates (n=5). Apical application of chymotrypsin significantly stimulated ENaC-mediated short circuit current ( I SC) in TMPRSS2-knockdown cells, but not in control cells (2.0 ± 1.1 μA/cm² vs. 0.2 ± 0.6 μA/cm²; n=13-14; p<0.001; mean ± SD). Moreover, the stimulatory effect of aldosterone on I SC was impaired in TMPRSS2-knockdown cells compared to control cells (9.1 ± 4.0 μA/cm² vs. 18.3 ± 4.0 μA/cm²; n=7-8; p<0.01). After aldosterone treatment I SC stimulation by chymotrypsin was enhanced in TMPRSS2-knockdown cells (5.3 ± 1.9 μA/cm²; n=7) but undetectable in control cells (−0.8 ± 1.6 μA/cm²; n=8). These findings indicate that TMPRSS2 deficiency impairs proteolytic ENaC activation in mCCDcl1 cells. To confirm this in an in vivo model, we used constitutive TMPRSS2 knockout mice ( Tmprss2 -/-). Using a combination of RNAscope technology and immunofluorescence, we demonstrated high expression of Tmprss2 mRNA in renal tubular cells, including cells with β-ENaC protein staining. Importantly, the ratio of fully to partially cleaved γ-ENaC was significantly reduced in membrane-enriched fractions of whole-kidney lysates from Tmprss2 -/- mice (n=10). Maintained on a standard diet, Tmprss2 -/- mice had no apparent renal phenotype with plasma aldosterone levels similar to those of Tmprss2 +/+ mice (120 ± 43 pg/ml in Tmprss2 -/- vs. 92 ± 17 pg/mL in Tmprss2 +/+; n=7-10). Moreover, the ability of Tmprss2 -/- mice to reduce urinary sodium excretion in response to four days of low sodium diet was fully preserved. However, under low sodium diet plasma aldosterone increased to significantly higher levels in Tmprss2 -/- mice compared to controls (752 ± 146 pg/ml in Tmprss2 -/- vs. 268 ± 55 pg/ml in Tmprss2 +/+; n=7-11; p<0.01). This is most likely a compensatory response to compromised proteolytic ENaC activation due to TMPRSS2 deficiency. We conclude that TMPRSS2 plays a physiological role in proteolytic ENaC processing and activation in the kidney. This study was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - Project-ID 509149993, TRR 374 (subproject A4 to A.I. and C.K.) and the Bayerisches Staatsministerium für Wissenschaft und Kunst (Bavarian Ministry of Art and Science), project VI-Corona-Forschung (subproject 07 to M.B. and T.G). A.K. was supported by the Interdisziplinäres Zentrum für klinische Forschung (IZKF) Erlangen. S.W. was supported by the Bundesministerium für Bildung und Forschung (BMBF, Federal Ministry of Education and Research, Germany), project SENSE-CoV2, 01KI20172A. P.S. and F.A. were funded by the IZKF Tübingen. The authors declare no conflicts of interest. 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.