Role Of KLF2 In Cold Preservation Of Human Glomerular Endothelial Cells

Research background and purposeKidney transplants are the best treatment for end-stage kidney disease.The continuous development of transplantation technology and surgical methods has improved the success rate of kidney transplantation,and the improvement of cold preservation technology has also ensured that the survival rate of kidney transplantation is significantly improved.However,during static cold storage,the donor kidney will inevitably experience interruption of blood flow and long-term cold ischemia.The establishment of animal models found that the injury of glomerular endothelial cells was the earliest significant pathological change.This process leads to impaired filtering function of the kidney and is related to acute and chronic failure and functional recovery of the transplanted kidney,and also can affect the reperfusion process.The molecular mechanism of kidney cold storage injury is related to the decreased expression of blood flow-dependent mechanosensitive transcription factor KLF2(Krüppel-like factor 2).The expression of KLF2 is regulated by the tangential force of normal blood flow,which induces endothelial cells to produce nitric oxide synthase(e NOS),promotes the synthesis of vascular NO and exerts anti-inflammatory effects to maintain the normal function of endothelial cells.Due to the interruption of blood flow during cold storage,KLF2 expression decreased,endothelial protection was weakened,and endothelial cells appeared damaged.In addition to dynamic tangential force,KLF2 expression can also be up-regulated by statins.Based on the morphological observation of isolated kidney and the detection of related damage indicators,glomerular endothelial cells have been proven to be the initial damage site during cold storage,and microcirculation disorders caused by endothelial damage are a common feature of cold ischemic injury and other renal ischemic diseases.Therefore,in this study,by establishing a static cold preservation model of human glomerular endothelial cell lines and using Simvastatin to up-regulate KLF2 expression,the cold preservation injury mechanism of glomerular endothelial cells was studied.Through research to clarify the molecular mechanism of KLF2 in this process,it can not only provide new improved strategies and ideas for cold preservation of kidneys,but also help to generate new prevention and treatment methods for ischemic diseases.MethodHuman glomerular endothelial cells(HRGEC)were cultured and grouped according to different needs.Group one:(1)normal control group(Control,Ctrl);(2)cold storage 6 hours vehicle group(CS6h-Veh);(3)cold storage 6 hours simvastatin group(CS6h-Sim).Group two:(1)Normal control group(Control,Ctrl);(2)Interleukin-1? stimulated group(IL-1?);(3)Interleukin-1? stimulated cold storage for 6hours in vehicle group(IL-1? + CS6h-Veh);(4)Simvastatin group(IL-1? + CS6h-Sim)was stored cold for 6 hours after stimulation with interleukin-1?.Specific detection indicators:(1)Detect which time point of KLF2 expression changes significantly after cold storage or dosing,so as to determine the cold storage time point;(2)Observe the adherence of cells after cold storage;(3)Observation of cell ultrastructure;(4)Detection of related factors at the protein and gene levels: KLF2,NF-?B,Caspase-3,Cyt-C,Beclin-1,p62,e NOS.Result(1)In the cold-preserved model of human glomerular endothelial cells,KLF2 expression was significantly reduced at 6 hours,and KLF2 expression was significantly increased at 6 hours with the addition of simvastatin.(2)Morphological changes of human glomerular endothelial cells under cold microscope after 6h cold storage:Compared with the Ctrl group,the cells in the CS6h-Veh group became swollen and rounded,and the cytoplasmic protrusions became incomplete,with a large number of black spots appearing;Compared with the CS6h-Veh group,the swelling and round cells in the CS6h-Sim group were reduced.Ultrastructural changes: Compared with the Ctrl group,the nuclear chromatin of the CS6h-Veh group was uneven,and a large number of autophagosomes appeared in the cytoplasm;compared with the CS6h-Veh group,a large number of autophagy still existed in the cytoplasm of the CS6h-Sim group.(3)KLF2 and NF-KB protein expression in human glomerular endothelial cells after cold storage for 6 hours: Compared with the Ctrl group,KLF2 protein expression in the CS6h-Veh group was reduced and NF-KB expression was increased;compared with the CS6h-Veh group,CS6h-The expression of KLF2 protein increased and the expression of NF-KB decreased in Sim group.(4)m RNA expression of KLF2 and its major downstream e NOS in human glomerular endothelial cells after cold storage for 6 hours:Compared with the Ctrl group,the expression of KLF2 m RNA and the expression of e NOS m RNA in the CS6h-Veh group were reduced;Compared with CS6h-Veh group,CS6h-Sim group increased KLF2 m RNA expression and e NOS m RNA expression.(5)Expression of apoptosis-related proteins in human glomerular endothelial cells after cold storage for 6 hours: Compared with the Ctrl group,the expression of Caspase-3 and Cyt-C proteins in the CS6h-Veh group was increased;compared to the CS6h-Veh group,the Caspase in the CS6h-Sim group-3.Cyt-C protein expression is reduced.(6)Expression of autophagy-related proteins in human glomerular endothelial cells after cold storage for 6 hours: Compared with the Ctrl group,the expression of Beclin-1protein in the CS6h-Veh group was increased,and the expression of p62 protein was decreased.Compared to the CS6h-Veh group,Beclin-1 protein expression increased,p62 protein expression decreased in the CS6h-Sim group.(7)KLF2 and NF-KB protein expression after interleukin-stimulated cells were cold-preserved: Compared with the Ctrl group,the expression of KLF2 decreased and the expression of NF-?B increased in the interleukin-stimulated group,but it was not statistically significant.Compared with the interleukin-free cold storage group(IL-?),the expression of KLF2 decreased and the expression of NF-KB increased in the IL-1?+ CS6h-Veh group;Compared with the IL-1? + CS6h-Veh group,the expression of KLF2 increased and the expression of NF-KB decreased in the IL-1? + CS6h-Sim group.ConclusionIn the human glomerular endothelial cell cold preservation injury model,KLF2 can exert endothelial cell protection by increasing autophagy,inhibiting the key inflammatory factor NF-?B,and up-regulating endothelial protective factor e NOS.