Mechanism Of The Renal Tubular Epithelial Cell Necroptosis Promotes Tubulointerstitial Fibrosis In Chronic Kidney Disease

Part Ⅰ: Role of renal tubular epithelial cell necroptosis in tubulointerstitial fibrosis in patients with CKD and its relationship with TGF-β1/Smads signal molecular Objective:To identify the significance of the renal tubular epithelial cell necroptosis in the progression of CKD in patients with different stages of chronic kidney disease(CKD),To further investigate the correlation between renal tubular epithelial cell necroptosis with the activation of fibroblasts,epithelial-to-mesenchymal transition and the development of tubulointerstitial fibrosis in patients with CKD.Methods:1.The clinical data and renal tissue samples from 100 patients(18-65 years old)with CKD by kidney biopsy were collected in the First Affiliated Hospital of Hainan Medical University and The First Affiliated Hospital of Xinxiang Medical University from March 2020 to March 2021,and the estimate glomerular filtration rate(e GFR)≥15 ml·min-1·(1.73 m2)-1.2.According to internationally accepted K/DOQI guidelines,the patients were divided into 1-4 stages of CKD,with 20 cases in CKD 1,2,4 stage,and 40 patients in CKD3.15 patients with renal trauma without underlying renal disease were selected as the control group.3.The morphological structure of renal tubular epithelial cell necroptosis and surrounding interstitial fibroblasts in in patients with chronic kidney disease were observed by transmission electron microscope.4.HE staining and PASM staining were used to observe the pathological changes of renal tissue and the degree of renal fibrosis in patients with stages 1-4CKD.5.The expression of renal tubular epithelial cells necroptosis key signaling molecules(RIPK3,MLKL,p-MLKL),pro-fibrosis factors(TGF-β1,Smad2/3),mesenchymal fibroblast activation markers(FGF-2,FSP1,Collagen-I)and EMT marker molecules(E-cadherin,a-SMA)in patients with CKD at different stages by immunohistochemistry,and compares their differences.6.The expression of TUNEL+RIP3、TUNEL+MLKL、TUNEL+TGF-β1、TUNEL+FSP1、TUNEL+Collagen-I、TUNEL+E-cadherin and TUNEL+a-SMA in patients with CKD at different stages by immunofluorescence,and compares their differences.7.The expression of RIP3 and TGFβ1,RIP3 and Collagen-I in renal tissue of control group and CKD group by double label immunofluorescence staining,and compares their differences.8.Pearson analysis the correlation between renal tubular epithelial cell necroptosis and clinicopathological indexes in patients with CKD.Results:1.With the development of CKD,the e GFR of CKD patients decreased gradually in each period,the levels of Cys C,BUN,SCR,24-hour urinary protein and systolic blood pressure increased gradually,and the CKD stage 3 and 4 were statistically different from the control group(P <0.01).2.TEM showed the morphological changes in necroptosis of human renal tissue,including cell volume expansion,ruptured cell membranes,spillage of cytoplasmic contents,apparent vacuolization,the formation of extensive vesicles in cells.3.HE staining and PASM staining results show,with the development of CKD,the structural destruction of renal tubules in patients with CKD was gradually aggravated,and the renal tubules in the corresponding areas were atrophied,accompanied by worsening interstitial fibrosis,the adjacent renal tubules were focally dilated and numerous protein tubules were seen in the tubules.Importantly,renal tubular injury score in second and third stage of CKD was significantly higher than that in control group,and decreased in stage 4 CKD.4.Immunohistochemical results showed that the renal tissue RIP3,MLKL and phosphorylated proteins in CKD patients were mainly expressed in the cytoplasm of renal tubular epithelial cells.The cytoplasmic expression of renal tubular epithelial cells in patients with CKD 2 and 3a was significantly increased(P <0.01).Compared with the control group,the expression of pro-fibrosis factors(TGF-β1,Smad2/3),mesenchymal fibroblast activation markers(FGF-2,FSP1,Collagen-I)and EMT marker molecules(E-cadherin,a-SMA)were specifically upregulated in patients with CKD stage 2,3a(both P<0.01),whereas the expression of E-cadherin protein was significantly decreased.5.Immunofluorescence staining results showed that the percentage of TUNEL+/RIP3+ and TUNEL+/MLKL+ double positive renal tubular epithelial cells(necroptotic cells)in CKD stage 2 and 3a patients was significantly increased(P<0.01).Compared with the control group,the expression of TUNEL+TGF-β1,TUNEL+FSP1,TUNEL+Collagen-I and TUNEL+a-SMA were specifically upregulated in patients with CKD stage 2,3a(both P<0.01),whereas the expression of E-cadherin protein was significantly decreased.Compared with the control group,RIP3 and TGF β1,RIP3 and Collagen-I in CKD group were positive results(P < 0.01).6.Pearson correlation analysis showed that the percentage of necroptotic renal tubular epithelial cells was positively correlated with TGF-β1,Collagen-I,renal tubules injury score,renal interstitial fibrosis index(both P < 0.05),was negatively correlated with e GFR(P < 0.01).Conclusions:1.In the progression of CKD,the tubular epithelial cells of CKD patients had necroptosis,and the peak of necroptosis was in stages 2 and 3a CKD.2.Renal tubular epithelial cells necroptosis in patients with CKD may be related to expression of fibrosis-promoting factor TGF-β1 and signal molecules Smad2/3,activation of interstitial fibroblasts and the occurrence of EMT in renal tubular epithelial cells.Part Ⅱ: Mechanism of the renal tubular epithelial cell necroptosis activated interstitial fibroblasts in HK-2 of TNF-α-induced in vitro Objective:At the cellular level,it was elucidated that the renal tubular epithelial cell necroptosis mediates renal tubulointerstitial fibrosis in chronic kidney disease by increasing fibroblast activation and EMT(Epithelial mesenchymal transformation)via the TGF-β1/ Smad3 pathway.Methods:1.HK-2 cells were treated with normal medium and containing TNF-α(100ug/L)respectively,and collected 24 hours later.HK-2 cells were divided into control group,TNF-α group,TNF-α +Nec-1s group,TNF-α+GSK’872 group and TNF-α+NSA group.In the final three groups,Nec-1s,GSK,872 and NSA were pretreated for 30 min respectively.The protein levels of RIP3,MLKL,TGF-β1 and Smad3 in HK-2 cells were determined by Western blot,and compares their differences.2.To established a model of renal tubular epithelial cells necroptosis,HK-2cells were treated with containing TNF-α(100ug/L)for 24 hours.The necroptic of renal tubular epithelial cells were treated with normal medium and interstitial fibroblasts respectively,and collected 48 hours later.HK-2 cells were divided into control group,TNF-α group,TNF-α +Nec-1s group,TNF-α+GSK’872 group and TNF-α+NSA group.In the final three groups,Nec-1s,GSK,872 and NSA were pretreated for 30 min respectively.The protein levels of FGF-2 and FN in HK-2 cells were determined by Western blot,and compares their differences.Next,the cells were divided into control group,vehicle group,LY364947 group,SIS3 group and FGF-2antibodies group.In the final three groups,LY364947,SIS3 and FGF-2 antibodies were pretreated for 30 min respectively.The protein levels of FGF-2,FN and α-SMA were determined by Western blot,and compares their differences.To clarify the correlation between renal tubular epithelial cell necroptosis,activation of interstitial fibroblasts and EMT of renal tubular epithelial cells.Results:1.Cell experiment Western blot results showed that compared with control group,the number of RIP3 and MLKL proteins in HK-2 cells were increased significantly in TNF-α group(P<0.05).The levels of TGF-β1 and Smad3 were up-regulated in TNF-α group(P<0.05).In addition,Nec-1s,GSK’872 or NSA intervention significantly reduced the protein levels of RIP3,MLKL,TGF-β1 and Smad3 level in HK-2 cells induced by TNF-α.2.Western blot results showed that compared with control group,mesenchymal fibroblast activation markers FGF-2 and FN proteins were increased significantly in in vehicle group(P<0.01).The levels of α-SMA protein was up-regulated in vehicle group(P<0.05).In addition,LY364947,SIS3 and FGF-2 antibodies intervention significantly reduced the protein levels of FGF-2、FN and α-SMA(both P<0.01).Conclusions:In the progression of CKD,the tubular epithelial cells of CKD patients had necroptosis,the renal tubular epithelial cells necroptosis may promote the activation of interstitial fibroblasts and the occurrence of EMT in renal tubular epithelial cells,and promote the occurrence and development of TIF by mediating fibrosis-promoting factor and signal molecules TGF-β1 and Smad3.