6.2 Involvement of PAR2 in progression of diabetic nephropathy
After PAR1, the expression of PAR2 was found to be abundant in kidneys [2, 28-30]. The activity of PAR2 is tissue factor (FXa) dependent [36, 27, 59, 60] and its activation exacerbates the renal tissue injury [61, 62]. Furthermore, a study has demonstrated the involvement of tissue factor-mediated inhibition of eNOS signaling as the pathological mechanism responsible for the progression of diabetic nephropathy [63]. In this study both eNOS-/- and WT mice were made diabetic by 5 consecutive i.p injections of low-dose STZ (40 mg/kg). It was observed that after 5 weeks of diabetes induction, the kidneys of eNOS-/- mice showed higher tissue factor (TF) mRNA expression than the kidneys of WT diabetic mice, thereby suggesting the eNOS deficiency mediated up-regulation of TF expression. Additionally, immunohistochemical analysis revealed that in kidneys of eNOS-/- mice, tissue factor is mainly expressed in the macrophages which involved the kidney glomeruli. Moreover, the mouse mesangial cell line (CRL-1927) showed increased inflammation upon treatment with mouse factor VIIa as depicted by an increase in monocyte chemoattractant protein-1(MCP-1) levels, which suggests the TF dependent activation of inflammation. Notably, in this study renal disease was exacerbated in high fat (HF) diet-fed eNOS-/- diabetic mice which lead to TF up-regulation, and development of functional and morphological changes in the kidneys as marked by an increase in urinary albumin, thickening of glomerular basement membrane (GBM), glomerulosclerosis, fibrin deposition, and also increase in various inflammatory and fibrogenic genes such as IL-6, TNF-α, MCP-1, TGF-β and type IV collagen expression. However, the administration of the anti-TF antibody, AF3178 has significantly reduced these manifestations in the kidney of HF-fed eNOS-/-diabetic mice. Therefore, these findings suggest that lack of eNOS and subsequent TF up-regulation is responsible for the development and progression of DN in additional fat-fed eNOS-/-diabetic mice by promoting inflammation. However, it needs to be determined whether direct inhibition of PAR2 could exhibit similar renoprotective effects in eNOS-/- diabetic mice.
More studies have been performed to find out the pathological role of coagulation factor Xa and PAR2 interaction in experimental type-2 diabetic nephropathy model using Akita mice [59]. In diabetic Akita mice, mutation of insulin 2 gene results in pancreatic β-cell destruction and hyperglycemia, followed by insulin resistance and metabolic disturbance along with the development of albuminuria. Notably in this study, the eNOS-/- mice showed higher plasma FXa levels as compared to eNOS+/+ mice, regardless of the status of DM. Furthermore, this study also showed that in diabetic conditions, carbonyl stress was elevated in the peritoneal macrophages, which further up-regulated the expression of FXa in the peritoneal macrophage. Moreover, in eNOS-/- DM mice macrophage infiltration increased rapidly in the glomeruli and co-localized with FXa that lead to glomerular damage and nephropathy. Further inhibition of FXa by edoxaban retarded the invasion of macrophages in glomeruli of both eNOS+/- and eNOS-/- mice and resulted in amelioration of diabetic nephropathy as observed by a reduction in urinary albumin excretion, mesangial matrix production along with a reduction in PAR2 mediated inflammation evidenced by decreased expression of pro-inflammatory cytokines (Tgfb, Tnf-α), and profibrotic genes (Pai1, Col1). Similarly, knockout of PAR2-/- gene in AKITA mice with reduced FXa expression attenuated diabetic nephropathy by suppressing the release of inflammation. This finding suggested that clotting FXa dependent PAR2 activation promotes DN by inducing glomerular macrophage infiltration and inflammation.
Similarly, the impact of PAR2-/- knockout on the development of renal injury has been evaluated in streptozotocin-induced type-1 diabetic mice [64]. In this study, type-1 diabetes was induced by five consecutive injections of STZ at a dose of 50 mg/kg body weight. Six months after diabetes induction in PAR2 deficient mice reduced albuminuria was observed, but it was accompanied by glomerular injury as marked by increased mesangial expansion and collagen deposition in the glomeruli. In this case, these findings are opposite to the glomerular outcomes observed in PAR2 deficient type-2 diabetic Akita mice [59]. Further, histological analysis showed that PAR2 deficiency did not influence the podocyte number, and strikingly multiplex ligation-dependent probe amplification (MLPA) analysis using apoptosis, inflammation, and coagulation gene panels, showed increased expression of other subtypes PAR1 and SERPINE1 genes in PAR2 deficient diabetic mice as compared to wild type diabetic mice. SERPINE1 is a gene, encoding for plasminogen activator inhibitor 1(PAI-1), which belongs to the family of serine protease inhibitors, and regulates controlled blood clot resolution. Notably, the increasing compensatory level of PAR1 transcription in PAR2 deficient type-1 diabetic mice might be one of the possible reasons for the increased mesangial expansion in these d/iabetic mice. Certainly, it could be concluded from these findings that the PAR2 subtype is not involved in mediating the pathophysiology of type-1 diabetic nephropathy. These discrepancies could partly be explained based on different expression profiles of PAR1 subtype in PAR2 knockout AKITA mice and STZ induced type 1 diabetic mice.  However, the direct cross-talk between PAR1 and PAR2 receptors in the pathogenesis of type-1 DN needs further exploration.