Serum LPA and autotaxin levels are
Serum LPA and autotaxin levels are associated with proteinuria and kidney failure in the type 2 diabetic condition [27,36]. LPAR1-4 are expressed in the kidney tissue under normal conditions . LPAR1 activation promotes tubulointerstitial fibrosis in mice given unilateral ureteral obstruction . Our recent study and others have also demonstrated that LPARs-mediated signaling is activated in mesangial Amiloride HCL exposed to high glucose and in the renal cortex of a type 2 diabetic mouse model, whereas ki16425 or BMS002 treatment (antagonists for LPAR1/3) attenuated glomerular injury and recovered kidney dysfunction [32,35]. Consistent with these, our current study showed that LPAR1 expression is elevated in kidney of a STZ-induced diabetic mice. However, ki16425 and BMS002 have antagonistic effects for both LPAR1 and LPAR3 [32,35]. Thus, it is difficult to exclude the effects of antagonism on LPAR3 under pathogenic conditions, even though the expression of LPAR3 in renal cortex was not changed in the diabetic condition in our previous study . In contrast, Zhang et al. showed that both LPAR1 and 3 are significantly increased in endothelial nitric oxide synthase-knockout (eNOS−/−) db/db mice . AM095 is well known as an antagonist for LPAR1 . This characteristic of AM095 makes it more suitable for evaluating the role of LPAR1 on pathogenesis of DN, and thus, was used in the present study. Our data showed that AM095 treatment significantly decreased blood glucose levels, although mildly, in two different cohorts of STZ-induced diabetic mice. This result is inconsistent with our previous study using ki16425 in the db/db mouse model, in which there was no effect on blood glucose levels . However, Rancoule et al. reported that LPAR antagonism using ki16425 significantly reduced blood glucose by increasing the number of islets and insulin secretion, as well as insulin sensitivity in liver and muscle in high fat diet-induced obese mice . The discrepancy between these studies might be due to the different diabetic mouse models and the differences in the degree of affinity for LPAR1 between AM095 and ki16425. Histological analysis of the kidney showed that AM095 administration reduced the impairment of kidney structure as shown by a smaller glomerular surface area and tuft area in the AM095 treatment group compared to the STZ-vehicle group. The therapeutic effect at 30 mg AM095 was also comparable with losartan treatment group. These results are consistent with our previous reports and others using a type 2 DN mouse model [32,35]. Taken together, the results indicated that AM095 administration may effectively improve kidney function by recovering the damaged kidney structure by reducing inflammatory mediated fibrosis. Increasing evidence indicates that oxidative stress and ROS potentiate the development of DN . Our data showed that the increase of LPAR1 expression in the kidney of STZ-induced diabetic mice was correlated with the increase of the detection of 4-HNE, a marker of oxidative stress. In addition, LPA increased ROS production in mesangial cells, and AM095 reduced this elevated ROS production. These data strongly suggested that AM095 treatment may inhibit renal damage by reducing ROS production. Cellular ROS production is mediated by NADPH oxidase . Our data showed that the expression of NADPH oxidase expression was increased by LPA treatment in SV40 MES13 cells and in the kidney of diabetic mice. This increase may, in turn, induce ROS production. TLR4 is an innate immune receptor classically activated by lipopolysaccharide in immune cells or by endogenous ligands in nonimmune cells [18,19]. TLR4 can activate the NF-κB pathway leading to inflammatory cytokines production. In diabetic patients, TLR4 expression and activation of its downstream signaling is observed in various cell types and organs [16,20]. As it is known that TLR4 also activates NADPH oxidase, leading to ROS production, we examined the changes of TLR4 expression in LPA-treated SV40 MES13 cells. We found that LPA treatment increased TLR4 expression in SV40 MES13 cells as well as in the kidney of diabetic mice. Consistent with our result, it has been previously reported that LPA increases TLR4 expression in THP-1 cells . Furthermore, our study showed that LPA induced TLR4 expression was blocked by LPA antagonism using AM095 in SV40 MES13 cells and in the kidney of diabetic mice, suggesting that the TLR4/NF-κB pathway plays an important role in the development of DN.