6.3 Dual blockade of PAR1 and PAR2 in type-1 DKD
The initial studies have addressed the individual roles of PAR1 and PAR2 antagonism in an experimental model of DKD. So far, the beneficial effects of  PAR1 and PAR2 blockade alone have been conclusively known to abrogate the pathological events associated with experimental diabetic nephropathy by preventing structural alterations such as glomerulosclerosis, collagen deposition, mesangial expansion and attenuating the release of pro-inflammatory and pro-fibrotic mediators [52-54,59]. Recently, attention has also been focused on inhibiting both the receptor subtypes concurrently in the development of diabetic nephropathy (Figure-02). Interestingly, an experimental study conducted by Mitsui et al [65] investigated the impact of both PAR-1 and PAR-2 inactivation in eNOS deficient type I diabetic Akita mice that is a well-characterized animal model of renal complication underlying diabetes [63]. These eNOS depleted diabetic mice present distinct features of progressive DN, particularly elevated mean blood glucose and creatinine levels, pronounced albuminuria, mesangial expansion, CD68+ macrophage infiltration and collagen type IV deposition in glomeruli leading to deleterious nephropathic changes. This eventually resulted into critical upregulation of pro-inflammatory mediators such as TNF-α, MMP-1, EGF-like module-containing mucin-like hormone receptor-like 1 (Emr1) mainly expressed on murine macrophages and significantly enhanced the expressions of TGF-β, plasminogen activator inhibitor-1  ( Pai-1), Collagen type-1 (Col1) which contributes to the development of tissue fibrosis. In this study, diabetic mice were administered with PAR1 inhibitor E5555 (60mg/kg/p.o ) and PAR2 antagonist FSLLRY-NH2 (60mg/kg/i.p ) either alone or in combination continually for four weeks. Although, it was observed that co-administration of E5555 + FSLLRY-NH2 did not improve creatinine and glucose levels in diabetic rats but appeared to produce a marked decrease in albuminuria, collagen IV deposition that halted the fibrotic events as confirmed by downregulation of TGF-β, Pai-1, Col1 gene expressions. The upregulation of these genes has pathological implications in glomerulosclerosis and renal interstitial fibroma [65]. Aside from this, the combination E5555 + FSLLRY-NH2 also diminished pro-fibrotic biomarker Emr1 resulting in reduced infiltration of CD68 positive macrophage cells, TNF-α, MMP-1. It is worth highlighting that synergistic effects of dual blockade of PAR subtypes are more prominent as compared to their individual effects in DKD. Additionally, in consideration of the wide localization of PAR subtypes on endothelial cells [2, 28-30] authors examined the independent and additive effects of PAR1 agonist (TFLLR-NH2) and PAR2 agonist (2f-LIGRLO) in cultured human endothelial cells in vitro. This treatment resulted in a remarkable rise in mRNA expression of macrophage chemoattractant protein 1 (MCP-1) or plasminogen activator inhibitor (PAI-1). The role of MCP-1 and PAI-1 has been well documented in triggering the migration of leukocytes and acts as a fibrosis-promoting molecule collectively leading to degrading renal functions [66, 67]. Apparently, PAR1 agonist-mediated increase was found to be completely subsided when Bay11-708 the inhibitor of NF-kB was pre-added to the endothelial cells. Whilst PAR2 agonist triggered elevation in MCP-1 and PAI-1 levels was blocked by MAPK inhibitor U-0126. In summary, it can be presumed that PAR1 and PAR2 mediated up-regulation in MCP-1 and PAI-1 expressions follow distinct mechanistic pathways in producing injury. Nevertheless, findings from the present study suggest the synergistic effects of PAR1 & PAR2 antagonism in type-1 diabetes-induced kidney disease which is mediated by attenuating fibrosis, inflammatory cascades, and infiltration of macrophages [65]. However, the exploration of PAR1 and PAR2 additive renoprotective effects in type2 diabetes are still lacking and needs prompt investigation.