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DiaComp Funded Abstracts Pilot & Feasibility Funding Programs

Pilot & Feasibility Program Application Abstract
Protease-activated receptors in diabetic complications
Nobuyuki Takahashi   (Sendai, 0)
Diabetic nephropathy is a leading cause of renal failure and is a major life-threatening problem. Although pharmacological inhibition of the renin-angiotensin system slows progression of diabetic nephropathy, patient prognosis remains poor. We have clarified that lack or reduced expression of endothelial nitric oxide synthase (eNOS, Nos3) exacerbates diabetic nephropathy, which is associated with increased expression and activity of renal tissue factor (TF), an initiator of the coagulation cascade. Anti-TF neutralizing antibody reduced inflammation in diabetic nephropathy. Coagulation proteases contribute to tissue injury mediated by protease-activated receptors (PARs). PAR2 is activated by TF- coagulation factor VIIa (FVIIa) complex and coagulation factor Xa (FXa) and up-regulates inflammation and fibrosis. Most recently we reported that lack of PAR2 are protective against diabetic nephropathy. Interestingly, these mice have reduced PAR1 expression in the kidney. Both PAR1 and PAR2 are proinflammatory, and PAR1 is known to be increased in diabetic kidneys. Accordingly, PAR2 and possibly PAR1 are promising target for diabetic complications. Specific Aim 1 will clarify whether inhibiting both PAR1 and PAR2 is more effective in preventing diabetic nephropathy than inhibiting PAR2 alone. We generate male mice lacking PAR2 (F2rl1-/-) or having wild type PAR2 (F2rl1+/+) on Nos3+/- and Ins2Akita/+ background. These mice will be divided into two groups and treated or not treated with PAR1 specific antagonist E5555. Specific Aim 2 will identify chemical compounds with PAR2 inhibiting properties. PAR2 antagonists will be screened using several chemical compound libraries. We add library compounds and calcium sensitive dye Fluo4 to human endothelial cell line EA.hy926, and look for compounds that inhibit the increase in intracellular calcium by a PAR2 agonist peptide 2f-LIGRLO. Selectivity of the compounds for PAR2 and PAR1 will be evaluated by inhibition of the increase in intracellular calcium by a PAR1 agonist.