Urotensin II (UII), an 11-amino acid vasoconstrictive peptide, was identified as the ligand for a novel G protein-coupled receptor, GPR-14. In addition to its potent vasoconstrictive action, UII was found to have pleiotrophic and profibrotic effects in the heart. The present study sought to define whether expression of UII and GPR-14 also plays a critical role in diabetes-induced renal fibrosis and dysfunction. Diabetic rats were induced using streptozotocin by intraperitoneal injection and the rat proximal tubular epithelial cells (NRK-52E) were utilized for in vitro mechanisms by manipulating gene expression with pharmacological and small inference RNA approaches. Results showed that expression of UII and GPR14 were significantly up-regulated in both mRNA and protein levels in the diabetic kidneys compared to controls. The up-regulated expressions of UII and GPR14 in the kidney were accompanied by significant increases in the renal pro-fibrotic factor TGF-1 expression, the renal extracellular matrix (fibronetin and collagen IV) accumulation, and the renal dysfunction (increases in urinal N-acetyl--D-glucosaminidase contents, 24-hour urinary retinol binding protein excretion rates, and creatinine clearance rate). Exposure of NRK-52E cells to 10-8 mol/L UII for 48 hours caused a significant increase of TGF-1, but not angiotensin II (Ang II), production that was GPR14- and calcium-dependent since GPR14 siRNA and calcium channel blocker nimodipine or calcium chelator EDTA all could abolish the induction of TGF- 1 by UII. Furthermore, exposure of NRK-52E cells to TGF-1 or Ang II also increased UII and GPR14 mRNA expressions. These results indicated that diabetes-induced up-regulation of UII and its receptor that is mediated by autocrine and/or paracrine mechanisms plays an important role in TGF-1-mediated renal fibrosis and dysfunction.