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.