7. Potential therapeutic effects of PAR2 in glomerular and podocyte injury
A study has demonstrated the role of PARs in mediating podocyte injury in a puromycin aminonucleoside (PAN) rat model of nephrotic syndrome (NS) [68] Pathologically, NS contributes to podocyte damage associated with high proteinuria. The persistent rise in proteinuria is one of the common causes of ESRD [69-72]. In PAN-induced nephrotic rats, the immunofluorescence histology of glomeruli showed co-localization of thrombin in podocytes, which is parallel with the proteinuria development during the disease progression. Moreover, this study revealed that inhibition of thrombin by hirudin significantly reduced the level of proteinuria as compared to control-treated nephrotic rats, which is probably by inhibition of ERK1/2 phosphorylation. From previous studies, it is evident that extracellular signal-regulated kinase (ERK) is an important sub-family of the MAPK signaling pathway, and it is mainly stimulated by growth factors and hormones. In disease conditions such as nephropathic conditions, the activated ERK is responsible for the induction of mitochondrial dysfunction, renal inflammation, and tubular apoptosis [73]. Notably, more studies have confirmed the expression of all PAR receptors in podocytes via RT-PCR and western blot techniques [74,75]. In addition, the study by Sharma et al [68] also revealed that in PAN induced nephrotic rats thrombin induced podocyte injury is by PAR1/PAR4 dependent manner, which is different in human podocytes (PAR3/PAR4 dependent). These findings were obtained by proximity ligation assays (PLAs), which showed cross-talk between PAR1/PAR4 and PAR3/PAR4 in cultured rat podocyte cells in presence of thrombin. Thrombin-induced podocyte injury was reduced upon pre-incubation with anti-PAR1 or anti-PAR4 antibody and additionally, thrombin-dependent ERK signaling was also inhibited. On the other hand, in cultured human podocyte cells, PLA assay revealed reduced podocyte injury on pre-incubation with anti-PAR3 or anti-PAR4 antibodies. Thus, these findings suggested the interactions between PAR1 and PAR4 in rat podocytes, whereas PAR3 and PAR4 in human podocytes [68].
Another study has investigated the role of PAR1 in the development of podocyte injury in a mouse model of drug-induced nephropathy (DIN) [49]. Gene expression studies showed that doxorubicin increased the expression of PAR1 mRNA in the isolated glomeruli, which was accompanied by increased albuminuria and also increased plasma creatinine levels. However, the administration of PAR1 antagonist, Q94 retarded the development of albuminuria and elevation of plasma creatinine levels in the DIN mice model. Additionally, Q94 treatment also prevented doxorubicin-induced glomerulosclerosis, renal oxidative stress, and also increased the immunostaining for podocin and nephrin (surface marker proteins of podocytes). Previously, studies have reported the involvement of calcium dyshomeostasis, such as persistent Ca2+ influx in the development of podocyte injury [2,76-79]. Moreover, thrombin is reported to activate PAR1 dependent calcium influx in the endothelial cells [80]. Furthermore, the addition of PAR1 antagonist (Q94) in cultured podocytes attenuated the doxorubicin-induced caspase 9/3 activation and increased intracellular calcium levels in the podocytes, thus suggesting the role of PAR1 in regulating intracellular calcium signaling and apoptosis in the podocytes [49]. Thus it could be concluded that at least PAR1 might be a novel target for the protection of podocyte damage in drug-induced renal injury, however further studies are required in other drug-induced nephrotoxicity models.
The previous study has demonstrated that inhibition of PAR1 is protective against podocyte injury in drug-induced nephrotoxicity models [49]. Another study has investigated the beneficial effect of PAR2 in VEGF inhibitor-induced glomerular and podocyte injury in mice [81]. Vascular endothelial growth factor  (VEGF)  is a glycoprotein and is mainly expressed in the cells such as endothelial cells, podocytes, fibroblasts, macrophages, and certain tumor cells, where it is mainly responsible for maintaining homeostasis, cell proliferation, and differentiation, cell growth survival, regulation of angiogenesis and prevention of apoptosis [82,83]. VEGF inhibitors are used together with chemotherapy for the treatment of various metastatic cancers. However, the increased use of VEGF inhibitors in cancer patients has led to the development of renal injuries as the most common side effect. These renal injuries include proteinuria, thrombotic microangiopathy, and glomerular injury, and podocyturia [84]. Additionally, inhibition of VEGF is accompanied by hypercoagulability, and fibrin deposition in the glomeruli [85]. It was observed that inhibition of VEGF by anti-VEGF antibody at a dose of 58.6±16.4 μg/mg creatinine has shown no effect on urinary albumin excretion, as well as in glomerular histology in wild type mice. However, in the eNOS-/- mice anti-VEGF administration elevated the urinary albumin excretion and histological damage depicted by open capillary area. The endothelial nitric oxide synthase (eNOS) is known to mediate the proliferation and migration of endothelial cells during injury. Moreover, deletion of PAR2 subtype in eNOS-/-mice leads to further worsening of renal dysfunction and structural changes upon anti-VEGF treatment. The immunohistochemical analysis showed that deletion of PAR2 resulted in reduced immunopositive CD31 area (an endothelial marker of differentiation) in the glomeruli of VEGF inhibitor administered eNOS-/- mice, thus indicating impaired glomerular endothelial cells repair which resulted in albuminuria. Moreover, deletion of PAR2 also reduced the level of podocyte-specific protein called nephrin, podocyte foot process effacement in the VEGF inhibitor administered eNOS-/-mice, which indicated the development of podocyte cell damage. Notably, it was found out in the study that deletion of PAR2 has reduced the expression of glomerular VEGF protein expression and other pro-angiogenic factors such as VEGF-A, and Tie2 as compared to PAR2 positive VEGF inhibitor administered eNOS-/- mice. In addition, administration of VEGF inhibitor leads to a reduction in pro-angiogenic cytokines Ccl2, Ccr2, Cxcl1, and Cxcr2 release in PAR2 deficient eNOS-/- mice. Moreover, cell line study in human endothelial cells (EA.hy926) also showed that the addition of PAR2 agonist, 2f-LIGRLO has significantly elevated the expressions of pro-angiogenic factors such as VEGF-A via MAPK and PI3K signaling pathways, thus indicating the crosstalk between PAR and growth factors. Thus, PAR2 regulates the expression of growth-promoting cytokines which induces repair and attenuates damage, thus PAR2 deficiency is partially responsible for inducing renal damage during anti-angiogenic inhibitor therapy and thus PAR levels should be carefully monitored in cancer patients undergoing anti-VEGF therapy.