Cytokines by Epac1-rap1a-nhe3 Pathway: Implications

38 39 Besides glomerulus, tubulo-interstitium is often concomitantly affected in certain diseases, 40 e.g., diabetic nephropathy, and activation of renin-angiotensin system, to a certain extent, 41 worsens its outcome because of perturbations in hemodynamics and possibly tubulo-glomerular 42 feedback. Certain studies suggest that pathobiology of tubulo-interstitium is influenced by small 43 GTPases, e.g., Rap1. We investigated effect of angiotensin II (Ang II) on inflammatory cytokines, 44 while at the same time focusing on upstream effector of Rap1, i.e., Epac1, and some of the 45 downstream tubular transport molecules, i.e., NHE3. Ang II treatment of LLC-PK1 cells 46 decreased Rap1a GTPase activity in a timeand dose-dependent manner. Ang II treatment led 47 to an increased membrane translocation of NHE3, which was reduced with Epac1 and PKA 48 activators. Ang II-induced NHE3 translocation was notably reduced with the transfection of 49 Rap1a dominant positive mutants, i.e., Rap1a-G12V or Rap1a-T35A. Transfection of cells with 50 dominant negative Rap1a mutants, i.e., Rap1a-S17A, or Epac1 mutant, i.e., EPAC-ΔcAMP, 51 normalized Ang II-induced translocation of NHE3. In addition, Ang II treatment led to an 52 increased expression of inflammatory cytokines, i.e., IL-1β, IL-6, IL-8 and TNF-α, which was 53 reduced with Rap1a-G12V or Rap1a-T35A transfection; while it reverted to previous 54 comparable levels following transfection of Rap1a-S17A or EPAC-ΔcAMP. Ang II-induced 55 expression of cytokines was reduced with the treatment of NHE3 inhibitor, S3226, or with Epac1 56 and PKA activators. These data suggest that this novel Epac1-Rap1a-NHE3 pathway 57 conceivably modulates Ang II-induced expression of inflammatory cytokines, and this 58 information may yield impetus for developing strategies to reduce tubulo-intertstitial 59 inflammation in various renal diseases. 60