ABSTRACT
Objective To investigate the potential role of CXC chemokine ligand 16 (CXCL16)/CXC chemokine receptor 6 (CXCR6) pathway in the progression of diabetic nephropathy (DN). Methods 8?week old male db/db mice were randomly divided into DN group and DN inflamed group. 10% casein was subcutaneously injected to induce the DN mouse model with inflammation. In vitro, HK?2 cells were treated with high glucose (HG), and IL?1β+HG to investigate the effect of inflammatory stress on HK?2 cells. Further knockdown CXCL16 was mediated by RNA interference to determine the effects of CXCl16, then cells were divided into HG+IL?1βgroup, HG+IL?1β + siCXCL16 group and HG + IL?1β + vehicle group. Changes of renal function in mice were assessed by 24 h proteinuria and N?acetyl?β?D?glucosaminidase (NAG) during 8 weeks. The ultra?microstructure was checked by electron microscopy at 8th week. Lipid accumulation in kidneys and HK?2 were observed by Filipin staining and quantitative assay of intracellular free cholesterol. The protein expressions of CXCl16, CXCR6, a disintegrin and metalloproteinase?10 (ADAM10), fibronectin and α smooth muscle actin (α?SMA) in renal tissue were detected by immunohistochemistry and Western blotting. The mRNA and protein expressions of CXCl16, CXCR6, ADAM10, fibronectin andα?SMA in HK?2 cells were detected by real?time PCR and Western blotting, and protein expressions of CXCl16, CXCR6 and ADAM10 in HK?2 cells were also tested by cell immunofluorescence. Results Mice in DN inflamed group had higher 24 h proteinuria and NAG than those in DN group, and the differences between two groups shown statistical significance at 8th week (all P<0.05). Compared with DN mice, DN inflamed mice had more vacuoles within renal tubular cells, with mitochondrial swelling, deformation and decrease. Lipid accumulation and protein expressions of fibronectin and α?SMA were increased in DN inflamed group when compared with DN group (all P<0.05). Further, the expressions of CXCL16, CXCR6, ADAM10 were significantly increased in DN inflamed group (all P<0.05). In vitro, the mRNA and protein expressions of CXCL16, CXCR6, ADAM10, fibronectin and α?SMA, and lipid accumulation were increased in high glucose plus IL?1βgroup when compared with high glucose group (all P<0.05). However, after siRNA of CXCL16 transfection, the mRNA and protein expressions of CXCL16, CXCR6, ADAM10, fibronectin andα?SMA were down?regulated in HG+IL?1β+siCXCL16 group as compared with high glucose+IL?1βgroup (all P<0.05). Furthermore, lipid accumulation was decreased (P<0.05). Conclusion Inflammation accelerates tubulointerstitial injury in DN partly through the activation of CXCL16 pathway, which may facilitate the lipid accumulation in tubular epithelial cells.
ABSTRACT
Objective To investigate the effects of low density lipoprotein receptor (LDLr) pathway on podocyte injury in diabetic nephropathy (DN) under inflammatory stress.Methods Male db/db mice and db/m mice were randomly divided into four groups (8 mice in each group):db/m group (control),casein injected db/m group (db/m + casein),db/db group (db/db),and casein injected db/db group (db/db + casein).An inflamed model of DN was established according to our previous study.24-hour urinary protein was measured every week.The plasma lipid profile was detected by clinical biochemistry assay.Podocyte changes were evaluated by electron microscope and immunofluorescent staining.Lipid accumulation in the kidney was evaluated by oil red O staining and intracellular cholesterol quantitative assay.The protein expression of Wilm's tumor-1 (WT-1),nephrin,α-smooth muscle actin (t-SMA),and molecules correlated with LDLr pathway were examined by immunohistochemical staining or Western blotting.The colocalized protein expression of LDLr with WT-1 was examined by immunofluorescent staining and laser confocal microscopy.Results There were no differences in plasma levels of LDL and HDL among four groups.Compared with db/db group,the db/db+ casein group showed markedly increased 24-hour urinary protein,more significant podocyte foot process effacement and podocyte damage,increased lipid droplet accumulation in kidneys,increased protein expressions of LDLr,SCAP and SREBP-2 in kidneys (all P ＜ 0.05).Interestingly,increased LDLr protein expression in kidneys of db/db mice was negatively correlated with decreased nephrin protein expression (r =-0.855,P ＜ 0.01) and positively correlated with increased α-SMA protein expression (r=0.768,P ＜ 0.01).Conclusions The disruption of LDLr pathway induced by inflammation contributes to podocyte injuries in diabetic nephropathy.