Inhibition Of P2Y12 Alleviates Angiotensin ?-induced Renal Inflammation And Fibrosis By Inactivating TGF-beta/Smad3 Signaling

Hypertensive nephropathy is a leading cause of death in chronic kidney disease(CKD)around the world.Fibrosis is a hallmark and common pathway leading to the end-stage organ disease including chronic kidney disease(CKD).Recent studies identified that macrophage-to-myofibroblast transition(MMT)is a new and important pathway contributing to 60-85% of ?-SMA+ myofibroblasts in both human and experimental mouse models of CKD including chronic renal allograft rejection.TGF-?/Smad3 signaling also is the master regulator of tissue fibrosis and the process of MMT.The investigators of our group have successfully obtained the unique profile of Smad3-dependent MMT transcriptomes by using single-cell RNA-seq(sc RNA-seq)and identified that P2Y12,a G protein-coupled(GPCR)purinergic receptors as a chemoreceptor for adenosine diphosphate(ADP),was detected in MMT cells of the fibrotic kidney of both human and Ang ?-induced animals.It was also identified that P2Y12,which predicted Smad3 binding its promoter by ECR browser,can activate TGF-?1/Smad3 signaling to mediate renal inflammation and fibrosis in Ang ?-induced mice.Surprisingly,inhibition of P2Y12 with specific inhibitor Clopidogrel effectively blocked Ang ?/TGF-?1 induced MMT and renal fibrosis in vitro and in vivo.The present study tested a novel hypothesis that P2Y12 may function beyond platelet activation to mediate renal inflammation and fibrosis in Ang ?-induced hypertensive nephropathy.A mouse model of hypertension was induced in mice by subcutaneous infusion of Ang ? via Alzet osmotic mini-pumps at the delivery rate of 1.44?mg/kg per day for 2 or 4 weeks.Mice were administered orally with saline or clopidogrel,P2Y12 inhibitor,at 20,40,60mg/kg daily until sacrificed.Blood pressure and urinary protein were measured weekly and kidney tissues were collected for analysis.1.To investigate the expression of P2Y12 on macrophages in hypertensive patients and mouse models induced by Ang ?Immunohistochemistry revealed that P2Y12 has upregulated on CD68+ ?-SMA+ cells in fibrotic kidneys of both patients with hypertensive nephropathy and Ang ?-induced mice,suggesting P2Y12 may contribute to macrophage fibrotic phenotype alteration and renal fibrosis in hypertensive nephropathy.2.To investigate the preventive and therapeutic effects of different doses of P2Y12 specific antagonist clopidogrel on renal inflammation and fibrosis in Ang ?-induced hypertensive nephropathy.Compared to the control group,systolic blood pressures of groups infused Ang ? were significantly higher and do be not affected by clopidogrel.Meanwhile,clotting times were no significant among groups administrated with different dosages of clopidogrel but were significantly prolonged compared to the groups without clopidogrel.In Ang ?-induced mice,Clopidogrel,P2Y12 antagonist,alleviated cardiac injury and renal injury,including reducing the level in serum of Cardiac Troponin I(CTN-I),Aspartate Aminotransferase(AST),Creatinine(SCr),Lactate Dehydrogenase(LDH),and ameliorating urinary protein.Although,the indicator of liver injury,alanine aminotransferase(ALT),shown no difference in all groups.This was confirmed by blocking P2Y12 with clopidogrel to inhibit renal inflammation and fibrosis by blocking the fibrotic macrophage transition,IL-1? and MCP-1 expression,and collagen matrix accumulation by Western blot and real-time PCR.Histology results have shown that Ang ?-induced kidney injury was attenuated in groups with clopidogrel,improving the morphology of hypertensive nephropathy,reducing MMT(F4/80 or CD68 and ?-SMA positive)cells.3.To explore the mechanism of P2Y12 on renal inflammation and fibrosis in Ang?-induced hypertensive nephropathy and in vitro.P2Y12 contributed to the Ang ?-induced renal inflammation and macrophage fibrotic phenotype alteration and renal fibrosis in hypertensive nephropathy.It also is pathogenic and mediates Ang ?-induced renal inflammation and fibrosis in hypertensive nephropathy independently of blood pressure and platelet activation by activating and TGF-?/Smad3 signaling In summary,we firstly identified that P2Y12 expresses on macrophages and mediates renal inflammation and fibrosis in Ang ?-induced hypertensive nephropathy by activating TGF-1/Smad3 signaling.And P2Y12 antagonist,clopidogrel,can attenuate the renal inflammation and fibrosis in Ang ?-induced mice.Thus,targeting P2Y12 may be a novel therapy for hypertensive nephropathy.