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.