High glucose promotes renal tubular CTGF expression via the activation of protease-activated receptors

Abstract

BACKGROUND: Protease-Activated Receptors (PARs) are potent mediators of inflammation and fibrosis after being cleaved and activated by serine proteases. Overexpression of PARs in several kidney diseases suggests a possible role in the progression of kidney damage. Based on our previous study in which high glucose stimulates pro-inflammatory and pro-fibrotic signals in cultured human proximal tubular epithelial cells (PTEC), we here investigate the role of PARs in high glucose induced expression of connective tissue growth factor (CTGF), the pro-fibrotic mediator for the accumulation of extracellular matrix protein and the pathogenesis of diabetic nephropathy. METHODS: Human PTEC were cultured in medium with normal glucose (5mM), high glucose (30mM) or high glucose with specific PAR antagonist (SCH79797 for PAR-1, ENMD-1068 for PAR-2 and trans-Cinnamoyl-YPGKF-NH2 for PAR-4) added 1h before stimulation. RNA were isolated after 6h incubation for gene expression analysis by quantitative real time PCR and culture supernatant were collected after 24h incubation for protein expression analysis by ELISA. RESULTS: We demonstrated the expression of PARs in PTEC. PAR-2 was the most abundant receptor in PTEC compared to PAR-1 and PAR-4, whereas PAR-4 had the lowest expression among the receptor family. However, high glucose (30mM) selectively enhanced PAR-4, but not PAR-1 and PAR-2 mRNA expression in PTEC. In addition, high glucose stimulated a time-dependent increase in CTGF mRNA and protein levels, and the expression was significantly inhibited by pre-incubation with PAR-1, PAR-2 and PAR-4 antagonists. CONCLUSIONS: These results suggest that PARs mediate the modulation of CTGF expression in renal tubular cells and the up-regulation of PAR-4 may further contribute to the progression of fibrosis in the diabetic kidney.