Kidney denervation prevents the development of tubulointerstitial fibrosis, but the neuropeptide calcitonin gene-related peptide (CGRP) in the denervated kidneys restores the fibrotic feature through the upregulation of profibrogenic growth factors. CGRP is involved in aggravation of inflammation by increasing the number of circulating cells and chemotactic factors. However, it is not clear how CGRP contributes to the upregulation of profibrogenic factors during fibrogenesis. In both human and pig kidney proximal tubular cell lines, administration of 1 nM CGRP significantly increased the levels of transforming growth factor-β1 (TGF-β1) production and connective tissue growth factor (CTGF) expression at 6 and 24 h after the administration. Exogenous CGRP also increased the TGF-β1 and CTGF protein levels in the incubation media, indicating release of these proteins from the cells. Treatment with 100 nM CGRP receptor antagonist (CGRP8-37) for 24 h significantly inhibited the increase in intracellular levels and released levels of TGF-β1 and CTGF in CGRP-treated cells. Genetic inhibition of CGRP receptor using siRNA transfection also suppressed the increase in TGF-β1 production and release at 24 h after CGRP stimulation. Furthermore, treatment with a specific protein kinase C (PKC) inhibitor chelerythrine (1 thru 10 μM) markedly reduced the upregulation and release of TGF-β1 and CTGF 6 h after CGRP administration. Finally, inhibition of c-Jun N-terminal protein kinase (JNK) phosphorylation using 1 μM SP600125 prevented the increase in TGF-β1 and CTGF upregulation and release 6 h after CGRP administration. Consistent with the in vitro data, exogenous CGRP in denervated UUO kidneys upregulated and secreted TGF-β1 and CTGF in dependence on PKC activation and JNK phosphorylation. In conclusion, these data suggest that exogenous CGRP induces the upregulation and secretion of profibrogenic TGF-β1 and CTGF proteins through the CGRP receptor/PKC/JNK signaling pathway in kidney proximal tubular cells.
Keywords: C-Jun N-terminal protein kinase (JNK); Calcitonin gene-related peptide (CGRP); Connective tissue growth factor (CTGF); Kidney proximal tubular cell; Protein kinase C (PKC); Transforming growth factor-β1 (TGF-β1).