| Objective:To analyze the number of renal tubular epithelial cells with necroptosis in stages1-4 of chronic kidney disease（CKD）and its correlation with clinical and pathological indexes,and to explore the role of necroptosis of renal tubular epithelial cells in the continuous progression of chronic kidney injury of patients with CKD.Meanwhile,in order to further understand the trigger factors of the necroptosis of renal injury in CKD patients,this experiment confirmed that AngⅡcan induce the necroptosis of renal tubular epithelial cells in vitro,and explored its possible mechanism.Methods:Collect the clinicopathological data and renal tissue specimen of CKD patients by renal specimen.CKD patients with e GFR≥15 ml·min-1·（1.73m2）-1from June 2017 to June 2019 in the First Affiliated Hospital of Hainan Medical College.CKD patients were divided into1-4 of CKD according to K/DOQI guidelines,（20 patients in each stage）.HE staining and PASM staining were used to observe the pathological changes of renal tissue and the degree of renal fibrosis in patients with stages1-4 CKD.TUNEL+RIP3 immunofluorescence staining and immunohistochemical techniques were used to detect the number of renal tubular epithelial cells and the expressions of necroptosis marker proteins RIP3 and MLKL in the renal tissues of CKD patients at different periods,as well as the expressions of AT1R and AT2R proteins,and to comparet their differences.Spearman correlation test was used to analyze the correlation between the necroptosis rate of renal tubular epithelial cells and clinicopathological indicators,AT1R and AT2R expression in renal tissue.HK-2 cells were treated with normal medium and Ang II with 10-10-10-5M concentration respectively,and collected 24 hours later;HK-2 cells were cultured with DMEM/F12 complete medium containing 10-9 M concentration Ang II,and samples were collected at 12,24,36,48 and 72 hours respectively;cells were divided into normal control group,Ang II group,Ang II+nec-1 group,Ang II+GSK’872group,Ang II group+NSA group.Detection of necrotic HK-2 cells by flow cytometry.The effects of vehicle,nec-1,GSK’872 and NSA on renal tubular epithelial cells were observed by electron microscopy and TUNEL staining.Western blot was used to detect the expression of RIP1,RIP3,MLKL protein and their phosphorylated proteins in different groups.Finally,the data were collected to analyze whether there was necroptosis of renal tubular epithelial cells in CKD patients and whether Ang II was an effective mediator of tubular cell necrosis.Results:In the general clinical data of CKD patients,according to the sequence of control group,CKD1,CKD2,CKD3,and CKD4,the EGFR of CKD patients decreased gradually in each period（P<0.01）;patients in age,SCR,BUN,SUA、the levels of Cys and 24-hour urinary protein and systolic blood pressure increased gradually,and the CKD stage 2,3,and 4 were statistically different from the control group（P<0.01）.CKD patients were divided into stages 1-4 of CKD staging according to K/DOQI guidelines.HE staining and PASM staining results show that as CKD progresses,the structural damage of the renal tubules shrink in CKD patients gradually worsens,the renal tubules in the corresponding area shrink.Associated with interstitial fibrosis.The renal tubules in the adjacent area were focally dilated and a large number of protein casts were visible in the tubules.The renal tubular injury score in patients with CKD stage 2 and 3 was higher than that in the control group（P<0.01）,and decreased in stage 4 CKD.TUNEL+immunofluorescence staining results showed that the percentage of TUNEL/RIP3double positive renal tubular epithelial cells（necroptotic cells）in CKDstage2and3 patients was significantly increased（P<0.01）.Immunohistochemical results showed that the renal tissue RIP3,MLKL and AT2R proteins in CKD patients were mainly expressed in the cytoplasm of renal tubular epithelial cells.In CKD2,stage 3 patients,the cytoplasmic expression of RIP3,MLKL in renal tubular epithelial cells was significantly increased（P<0.01）,while AT1R the protein expression results were not statistically significant（P>0.05）.The results of Spearman correlation analysis showed that the rate of necroptotic renal tubular epithelial cells was positively correlated with urea nitrogen,creatinine,cystatin C,uric acid and renal tubular injury score,renal interstitial fibrosis index,and AT2R（P<0.01）,and negatively correlated with e GFR（P<0.05）.HK-2 cells were induced by 10-10-10-5M Ang II at different concentrations.The necrosis rate of HK-2cells was determined by flow cytometry in 24 hours.The results showed that the necrosis rate of HK-2 cells was significantly higher in the 10-9M Ang II group than in the normal control group;HK-2 cells were induced by Ang II at the concentration of10-9M.,samples were collected at 12,24,36,48 and72 hours respectively.The necrosis rate of HK-2 cells was detected by flow cytometry.The results showed that the necrosis rate of HK-2 cells in 24 hours group was significantly higher than that in normal control group（P<0.01）.The morphological changes of HK-2 cells were observed under the electron microscope.It was found that HK-2 cells were obvious necrotic morphological characteristics in the 10-9m Ang II group.In the Ang II+Nec-1 group,Ang II+GSK’872 and Ang II+NSA groups,some cells were vacuolated,and the necroptotic cells was significantly lower than that of Ang I I group.The application of necroptosis-specific protein RIP3 and TUNEL Immunofluorescence staining to evaluate the necroptosis of renal tubular epithelial cells.The results showed that the percentage of TUNEL+RIP3 positive cells in Ang II+vehicle group was significantly higher than that in control group,and nec-1,GSK’872 and NSA could significantly reduce the percentage of TUNEL/RIP3 double positive cells compared with Ang II+vehicle group（P<0.01）.Western blot was used to detect the expression of the key proteins of necroptosis RIP1,RIP3,MLKL and their phosphorylated proteins.The results showed that the expression of RIP 1,RIP3,MLKL and their phosphorylated proteins in Ang II group was significantly higher than that in the control group（P<0.01）.Nec-1 can effectively inhibit the expression of RIP 1,RIP3,MLKL and their phosphorylated proteins.GSK’872 can inhibit the expression of RIP3,MLKL and their phosphorylated proteins.NSA can effectively reduce the expression of MLKL and p-MLKL protein（P<0.01）.Conclusions:In the progression of CKD,the tubular epithelial cells of CKD patients do have necroptosis,which may play an important role in the progression of renal injury,and the cytotoxicity of Ang II can trigger the programmed necrosis of tubular epithelial cells in vivo and in vitro.Nec-1,GSK’872 and NSA blockers can block the development of necroptosis. |
