The Effect And Mechanism Study Of Macrophage On Podocyte Apoptosis In Diabetic Nephropathy

Objective:Podocyte apoptosis is a key event in podocyte impairment of DN.Macrophage accumulation within glomeruli and interstitium is a vital feature in DN.However,the effect of macrophages on podocytes apoptosis remains poorly elucidated.The present study aimed to explore the effect and mechanism of macrophage on podocyte apoptosis in diabetic nephropathy via STZ-induced DN rats and cell co-culture of macrophage and podocyte in vitro.Methods:In vivo:Rats were randomly divided into two groups:control and DN group.DN was induced by a single intraperitoneal injection of streptozotocin(STZ).Rats were sacrificed at 18 weeks after DN establishment.Blood,urine and kidney samples were collected.Blood and urine samples were taken to detect blood glucose,serum creatinine(Scr)and 24h-urinary proteinuria.Tunel staining was performed to determine podocytes apoptosis.Immunohistochemical staining was used to detect number of CD68~+macrophage within glomeruli.Immunohistochemical staining of8-OHdG was used to detect ROS in kidney.Protein expression of cleaved casepase-3,p38MAPK and p-p38MAPK in kidney was performed by Western Blot.In vitro:1.In a transwell co-culture system of macrophage and podocyte,Raw 264.7 cells(2×10~5,4×10~5,8×10~5)seeded on a 0.4μm Transwell insert were co-cultured with mouse conditionally immortalized podocyte cells(MPC)(4×10~5)in the absence or presence of 25 mM high glucose treatment.2.Firstly,Raw 264.7 cells were treated with normal RPMI 1640 media(NC,containing 11.1mM glucose),25mM mannitol(Man)or25mM glucose(HG)for 24h.Then cells were wash with PBS and cultured within normal media for another 24h.The supernatants were collected and referred to as conditioned media(CM)from normal glucose-cultured macrophages(NC-CM)or CM from high glucose-activated macrophages(HG-CM),respectively.The classical marker of M1(iNOS and TNF-α)and specific marker of M2 macrophages(MR)were examined by Western Blot and immunofluorescent staining.The TNF-αlevel in the CM of macrophage was detected by ELISA.Secondly,the MPC cells were treated by normal PRMI 1640 media(control),NC-CM,HG-CM,HG-CM+ROS inhibitors(Tempo),HG-CM+p38MAPK inhibitor(SB203580),HG-CM+anti-TNF-αneutralizing antibody,HG-CM+IgG1 Isotype control or recombinant mouse TNF-αalone.Apoptosis in podocyte was detected by Annexin V-FITC/PI staining and Hoechst-33342staining.ROSinpodocytewasmeasuredusing2’,-7’-dichlorodihydrofluorescein diacetate(DCFHDA)staining.Western Blot was used to examine the protein expression of cleaved casepase-3,p38MAPK and p-p38MAPK.Results:In vivo:DN rats developed overt diabetes with higher blood glucose concentration and lower body weight compared with the non-diabetic group.Scr and24h-urinary proteinuria were markedly increased in diabetic rats than in non-diabetic rats.Apoptosis in podocytes and the number of infiltrated CD68~+macrophages were significantly increased in the diabetic group compared with the non-diabetic group.In vitro:1.In order to explore the effect of macrophage on podocyte apoptosis in DN,we established the Transwell co-culture system of macrophage and podocyte.Results showed that both podocytes apoptosis and cleaved caspase 3 protein expression were increased after high glucose stimulation.podocytes apoptosis and cleaved caspase 3protein expression were further enhanced when podocytes were co-cultured with macrophages in the presence of high glucose in the Transwell co-culture system,which displayed in a cell number dependent manner.However,podocyte apoptosis was not increased when podocytes were co-cultured with macrophages in the absence of high glucose in the Transwell co-culture system.There results indicated that macrophage activated in the condition of high glucose aggravate podocyte apoptosis.Macrophages displayed pro-inflammatory M1 activation with higher expression of iNOS and lower expression of MR after high glucose treatment.Furthermore,using conditioned media(NC-CM or HG-CM)to treat podocyte,we found that there was no difference in podocytes apoptosis between control group and NC-CM group.Apoptosis in podocytes was significantly increased in a time-dependent manner when podocytes were exposed to HG-CM compared with either control or NC-CM group.2.ROS generaion in kidney tissue was significantly increased in the diabetic rats compared with the control rats.p38MAPK activation characterized by increased protein expression of p-p38MAPK and ratio of p-p38MAPK/p38MAPK in kidney tissue was significantly raised in the diabetic rats compared with the control rats.In vitro,the level of ROS in podocytes was significantly increased when podocytes were exposed to HG-CM compared with either control or NC-CM group.p38MAPK activation in podocytes was significantly increased when podocytes were exposed to HG-CM compared with either control or NC-CM group,displaying increased protein expression of p-p38MAPK and ratio of p-p38MAPK/p38MAPK.Tempo,an inhibitor of ROS,markedly reduced the proportion of apoptotic podocytes,cleaved caspase 3expression and p-p38MAPK activation in podocyte elevated by HG-CM.SB203580,a specific p38MAPK inhibitor,effectively decreased podocytes apoptosis and cleaved caspase 3 expression induced by HG-CM.3.The level of TNF-αin the HG-CM was evidently increased compared with NC-CM.Meanwhile,TNF-αprotein expression was also increased when macrophages were exposed to high glucose compared with those cultured in normal glucose.Anti-TNF-αneutralizing antibody blocked the apoptotic rate and cleaved caspase 3 expression in podocytes and blunted the increasedROSproduction,p-p38MAPKexpressionandratioof p-p38MAPK/p38MAPK in podocytes elicited by HG-CM.In addition,podocyte apoptosis and cleaved caspase 3 expression was significantly increased when podocytes were exposed to TNF-αalone.The level of ROS and p38MAPK activation characterized by increased protein expression of p-p38MAPK and ratio of p-p38MAPK/p38MAPK in podocytes were also significantly raised when podocytes were exposed to TNF-αalone.Conclusions:1.Podocyte apoptosis was increased in STZ-induced DN rats,accompanying by increased macrophage infiltration in glomeruli.2.Macrophages exhibited proinflammatory M1 phenotype in the condition of high glucose.M1 macrophages activated by high glucose promoted podocytes apoptosis via ROS-p38MAPK pathway.3.TNF-αthat was released by high glucose-activated M1 macrophages promoted podocytes apoptosis via ROS-p38MAPK pathway.