Abstract 2409 Transmembrane serine protease 2 (TMPRSS2) regulates the α subunit of the epithelial sodium channel (ENaC) and epithelial barrier function

Transcellular and paracellular transport are key routes for the transfer of substances between and through cells. Both transport routes are heavily regulated by a myriad of molecular pathways. The epithelial sodium channel (ENaC) transports sodium across the tight epithelia in many different tissues and is primarily composed of three subunits (α, β and γ). A complex array of intrinsic and extrinsic factors, including channel activation by protease cleavage, regulate the expression and function of this evolutionary conserved ion channel. It has been shown by Xenopus oocyte functional experiments that the serine protease TMPRSS2 activates ENaC by cleaving the γ subunit. Our work explores whether TMPRSS2 is also involved in the regulation of ENaC expression and function, with a particular focus on the α subunit of ENaC. TMPRSS2 and αENaC are similarly expressed in most organs and both are highly expressed in the kidney. ENaC is known to localise to the distal convoluted tubule and cortical collecting duct of the nephron in the kidney. When analysing mouse kidney by RNAscope we find that TMPRSS2 is highly expressed in αENaC positive cells. We utilise the mouse cortical collecting duct (mCCD) cell line and CRISPR-Cas9 gene editing to explore this functional relationship further in vitro. TMPRSS2 KO cells reveal that αENaC gene expression is significantly reduced, thus abolishing transepithelial sodium transport. Upon further investigation of these cells by RNA-sequencing, we discover that genes (Epcam, Cldn3 and Cldn7) involved in maintaining the paracellular barrier are downregulated in TMPRSS2 KO cells. Subsequent protein analysis by Western blotting and immunostaining shows that the protein expression of EpCAM, Claudin-3 and Claudin-7 are also reduced in TMPRSS2 KO cells. Taken together, our data indicates that TMPRSS2 is instrumental in regulating ENaC mediated transepithelial transport via αENaC and maintaining the paracellular barrier via the EpCAM/claudin-7 tight junction complex. Swiss NationalFoundation (Grant FNRS 31003A-182478/1) and the National Center of Competence in Research "Kidney.CH," Lausanne, Switzerland (NCCR, N-403-07-23).