| Diabetic nephropathy(DN)is one of the common microvascular complication of diabetes mellitus.It was demonstrated that about 33 percentage of patients with diabetes will develop into diabetic nephropathy.According to clinical studies,diabetic nephropathy is characterized by microalbuminuria at early stage.Accumulating evidences suggest podocytes damage will surely impact on the integrity of glomerular filtration barrier,resulting in proteinuria which essentially promotes the progression of DN.However,the underlying molecular pathogenesis remains unclear.Swiprosin-1/EFhd2 is a new kind of protein with molecular weight of 27 kD,which was firstly found and reported by a Swiss scientist named Vuadens F.in 2004.So far,there have been only 25 articles on PubMed related to swiprosin-1/EFhd2.These reports mainly elaborated its distribution,function and features in immune system.Our previous study showed that swiprosin-1/EFhd2 was expressed in renal cortex of mice,and mainly distributed in glomerular podocytes.Further more,our studies showed that swiprosin-1/EFhd2 was increased in renal cortex of 8-week diabetic mice.There has been no report about the role of swiprosin-1/EFhd2 in diabetic nephropathy heretofore.Our previous study demonstrated that deficiency of swiprosin-1/EFhd2 attenuated glomerular hypertrophy,reduced the thickening of the basement membrane and podocytes apoptosis.Therefore,we hypothesized that swiprosin-1/EFhd2 in podocyte played an important role in the progression of diabetic nephropathy.Thus,we aim to investigate the effect of swiprosin-1/EFhd2 and its mechanism at the early stage of diabetic nephropathy both in vivo and in vitro and to provide new target for the treatment of diabetic nephropathy.Methods and Results:Firstly,the expression and location of swiprosin-1/EFhd2 in mouse kidney cortex and MPC-5 cells1.The expression of swiprosin-1/EFhd2 in the mouse kidney cortex was detected by immunoblotting,and swiprosin-1/EFhd2 was co-located with podocyte-specific marker called nephrin by immunofluorescence.2.The expression and distribution of swiprosin-1/EFhd2 in MPC-5 cells were detected by immunoblotting and immunofluorescence,and the results showed that swiprosin-1/EFhd2 and nephrin which is the podocyte-specific marker were co-located mainly at the cytoplasm and cell membrane of MPC-5.Secondly,the expression of swiprosin-1/EFhd2 in renal cortex of diabetic miceC57 mice in diabetic group were induced by a single injection of 150 mg/kg STZ intraperitoneally and control group mice were received sodium-citrate vehicle correspondingly.After 72 h,diabetes were confirmed in STZ-treated mice by measuring tail vein plasma glucose.All mice were killed and kidneys were harvested after 8 weeks.1.Compared with the control group,the expression of swiprosin-1/EFhd2 in renal cortex of diabetic mice was significantly increased detected by immunofluorescence and immunoblotting.Thirdly,the expression of swiprosin-1/EFhd2 in MPC-5 cells induced by high glucose1.MPC-5 cells were induced by the different concentration of glucose(5.5,11,22,33,44 m M)for 48 h and the expression of swiprosin-1/EFhd2 detected by immunoblotting.The results showed the level of swiprosin-1/EFhd2 was increased in podocytes after stimulation with medium containing glucose at 22 mM for 48 h compared with that of control group,continuously increased at 33 mM and 44 mM.2.MPC-5 cells were stimulated by 33 mM glucose for 12,24,48,72,96 h and the expression of swiprosin-1/EFhd2 was detected by immunoblotting.The results showed that the expression of swiprosin-1/EFhd2 was increased at 48 h,72h and 96 h compared with control group.Fourth,the effect of swiprosin-1/EFhd2 deficiency in the progression of early stage of diabetic nephropathySwiprosin-1-/-and swiprosin-1+/+ mice were treated with intraperitoneal injection of STZ,and control group mice were received sodium-citrate vehicle correspondingly.After 72 h,diabetes were confirmed in STZ-treated mice by measuring tail vein plasma glucose(random blood glucose value >16.7 mM).Swiprosin-1-/-diabetic mice namely KO model and swiprosin-1+/+ diabetic mice namely WT model group.Appropriate control group were named KO control group and WT control group,respectively.1.Morphology of renal cortex of mice were detected by PAS.Our results demonstrated that compared with mice in WT model group,there were some changes in general pathological characteristics,such as glomerular hypertrophy and extracellular matrix deposition.2.The results showed that the weight ratio of kidney/body of mice in KO model group was decreased than that in WT model group.3.Mice blood glucose in four group was measured,and we found that the blood glucose level was decreased in KO model group compare to that in the WT models.4.We collected mouse urine,and detected urinary protein and cystatin C levels by ELISA Kits.The results demonstrated that compared with diabetic swiprosin-1+/+ mice,urine protein level was decreased,and the level of cystatin C was increased in diabetic swiprosin-1-/-mice.5.The mRNA levels of collagen IV,TGF-? and fibronectin were detected by real time-PCR.The results demonstrated that compared with diabetic swiprosin-1+/+ mice,collagen IV,TGF-? and fibronectin mRNA levels were decreased in diabetic swiprosin-1-/-mice.Fifthly,the effect of interfered or over-expressed swiprosin-1/EFhd2 in MPC-5 cells induced by high glucose1.Swiprosin-1/EFhd2 interference small interfering RNA fragment and control fragments were constructed to transfect MPC-5 cells,respectively.The results of flow cytometry and JC-1 mitochondrial membrane potential assay exhibited that podocytes deficient of swiprosin-1/EFhd2 exhibited lower level of apoptosis rate compared with podocytes transfected with control siRNA and treated with HG.2.We constructed full-length swiprosin-1(LV-Swi)to over-expressed swiprosin-1/EFhd2 and LV-GFP as control,respectively.The results of flow cytometry exhibited that the apoptosis level of podocytes over-expressed swiprosin-1/EFhd2 was increased than that in the podocytes transfected with LV-GFP.Sixthly,the mechanism of swiprosin-1/EFhd2 in the progression of diabetic nephropathy1.Electron microscopy of mice kidney showed that thickened glomerular basement membrane(GBM)and degradation or fusion of the podocyte foot process(FP)were increased in WT model group compared with those in KO model group.The results of TUNEL staining in nephrin positive cells revealed that the apoptotic rate of podocytes in diabetic swiprosin-1+/+ mice was increased compared with that in diabetic swiprosin-1-/-mice.2.We detected the expression of pro-apoptosis proteins such as Bax,caspase3,caspase9,and anti-apoptosis protein Bcl-2 in vivo and vitro.The results revealed that the level of pro-apoptosis proteins in renal cortex of swiprosin-1-/-diabetic mice were lower than those in swiprosin-1+/+ diabetic mice.While Bcl-2 protein level was higher in swiprosin-1+/+ diabetic mice.The results in vivo was similar with those in vitro.These results indicated that swiprosin-1/EFhd2 may be involved in the mitochondria-dependent apoptosis in podocytes.3.Diabetic swiprosin-1-/-mice showed attenuated phosphorylation level of p38 and ERK in renal cortex than that in swiprosin-1+/+ diabetic mice which was detected by immunoblotting.Furthermore,we found that knockdown of swiprosin-1 by siRNA down-regulated the phosphorylation of p38 and ERK in podocytes stimulated by HG compared with those of control siRNA transfected cells stimulated by HG.These results suggested that swiprosin-1/EFhd2 could promote the activation of p38 and ERK MAPK signaling pathways.4.P38 and ERK was knocked down respectively in podocytes using si RNA and we constructed full-length swiprosin-1(LV-Swi)to over-express swiprosin-1.The LV-GFP was used as control.Apoptosis levels in p38 or ERK knockdown podocytes was decreased compared with control siRNA transfected cells after HG stimulation.Targeted knockdown p38 or ERK attenuated the increase of apoptosis level in podocytes over-expressed swiprosin-1.Conclusion:1.The expression of swiprosin-1/EFhd2 was increased in MPC-5 cells stimulated by high glucose and in the kidney of diabtic mice.2.Deficiency of swiprosin-1/EFhd2 could reduce podocyte apoptosis at early stage of diabetic nephropathy.3.Swiprosin-1/EFhd2 contributes to the mitochondria-dependent podocyte apoptosis stimulated by high glucose or in the kidney of diabtic mice.4.Swiprosin-1/EFhd2 mediates mitochondria-dependent podocyte apoptosis by activating p38 and ERK signaling pathways. |
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