505-P: ROCK2 Regulates TGF-Beta-Induced Expression of CTGF and Profibrotic Genes via NF-kappa B and Cytoskeleton Dynamics in the Mesangial Cells

The small GTPase Rho and its effector Rho-kinase are involved in the pathogenesis of diabetic kidney disease. Accumulating evidence shows profibrotic mediators, including connective tissue growth factor (CTGF), are key regulators of glomerulosclerosis under diabetic conditions. However, the interactions of Rho-kinase and these profibrotic mediators in the development of renal dysfunction remain unclear. Glomeruli isolated from type 2 diabetic db/db mice demonstrated increased gene expression of transforming growth factor β (TGF-β) and its downstream profibrotic mediators. Chemical inhibition of Rho-kinase suppressed the expression of profibrotic mediators in both isolated glomeruli and cultured mesangial cells. An investigation of mechanisms underlying this observation revealed activated Rho-kinase functions through the phosphorylation of JNK and Erk, and the nuclear translocation of nuclear factor-kappa B (NF-κB). Immunostaining studies demonstrated that a Rho-kinase inhibitor and Cytochalasin D, an inhibitor of actin polymerization, attenuated TGF-β-mediated nuclear translocation of NF-κB, indicating that Rho-kinase-mediated actin reorganization regulates NF-κB activation. Knockdown by small interfering RNA against two Rho-kinase isoforms, ROCK1 and ROCK2, showed that ROCK2 but not ROCK1 controls this fibrotic machinery. SLx-2119, a chemical ROCK2 inhibitor, also modulated the expression of TGF-β-induced profibrotic mediators via a similar mechanism by downregulating ROCK2 activity. A further in vivo study showed that ROCK2 activity in the renal cortex of db/db mice was elevated compared to control db/m mice. These observations indicate that ROCK2 is a key player in the development of diabetic renal injury. Glomerular ROCK2 may be a potential therapeutic target for the treatment of diabetic kidney disease.