Protease activated-receptor-2 induced inflammation and fibrosis is mediated in part through activation of transforming growth factor-beta signalling.

Abstract

Aim: To explore the pro-inflammatory and pro-fibrotic responses of primary human kidney tubular epithelial cells (HTECs) following activation of protease-activated receptor-2 (PAR-2) and/or the transforming growth factor-beta (TGF-beta) receptor. Background(s): Tubulointerstitial inflammation and fibrosis are observed in all progressive forms of chronic kidney disease (CKD) irrespective of the initiating insult. Recent evidence suggests that serine proteases, commonly produced following kidney injury, promote and exacerbate these responses by activating PAR-2 and TGF-beta receptors. Method(s): Confluent HTECs were treated with the synthetic PAR-2 agonist 2f-LIGRLO-NH2 (1 muM) and/or TGF-beta1 (5ng/ mL) in the presence or absence of the pan-protein kinase C (PKC) inhibitor Go6983, the TGF-beta receptor kinase inhibitor SB-431542, and PAR-2 antagonist GB88. Inductions of pro-inflammatory and pro-fibrotic molecules [e.g. CSF2, TNF-alpha, PAI- 1,MMP-1,MMP-9, and MMP-10] was measured at the mRNA and protein level by qPCR, western blot, and ELISA. Result(s): Alone, 2f-LIGRLO-NH2 significantly induced production range of molecules including CSF2, TNF-alpha, CTGF, PAI-1, MMP-1,MMP-9, and MMP-10 from HTEC. Smad2 was also activated by this treatment. TGF-beta1 treatment induced production CSF2, CTGF, and PAI-1 although to a lesser extent than with PAR-2 activation. Concurrent activation of PAR-2 and TGF-beta receptors gave a synergistic increase in the production of a number of these factors, particularly CSF2 and MMP-9. GB88, Go6983 and SB-431542 significantly reduced production of molecules CTGF, CSF2, PAI-1, and MMP-9. Conclusion(s): PAR-2 activation induces pro-inflammatory and pro-fibrotic responses in HTECs which involve PKC and TGF-beta signalling pathways. PAR-2 antagonists may be useful for pharmacotherapy of CKD, and are currently being tested for efficacy in animal models.