Role Of ROCK1 In The Podocyte Injury Induced By Oxidized Low-density Lipoprotein

BACKGROUDChronic kidney disease(CKD)in China in the general population prevalence rate is as high as 11.8%-13.0%,correspondingly,the number of patients with an annual increase of renal replacement therapy in our country,how to delay progress in CKD patients with kidney problems become to be solved urgently.In recent years,studies have shown that lipid abnormalities is recognized as independent risk factors promote the continuous progress of chronic kidney disease(CKD).With the increase of the incidence of hyperlipidemia and lipid epicuticular kidney effects,especially to merge kidney toxicity for patients with chronic kidney disease(CKD)is becoming more and more attention.Further study of the molecular mechanism of lipid kidney damage can not only deepen the theoretical knowledge of lipid nephrotoxicity and also helps to gain control and delay the new thought of continuous progress of chronic kidney disease(CKD).In 1982,Moorhead "lipid nephrotoxicity" hypothesis is proposed:glomerular injury with hyperlipidemia and proteinuria can lead to kidney disease progresses,and lipid abnormalities will also lead to atherosclerosis and glomerular sclerosis,glomerular sclerosis and atherosclerosis was part of a series of interrelated clinical disease.Our experiment in vitro cultured glomerular podocytes was first discovered in mice,the mice cells express ROCK1,ox-LDL stimulates cells can regulate the foot on the activity of ROCK1 and decrease in the expression of membrane protein nephrin.The results suggest the glomerular podocyte may be involved in lipid by increasing the activity of ROCK1 kidney damage.But the ox-LDL mediated cell damage of specific regulatory mechanism,has not been thorough research.Rho kinase(ROCK)is one of the most important effector molecule RhoA downstream,it is a kind of serine and threonine kinase,ROCK1 and ROCK2 two isomers,and can be GTP binding protein made RhoA activation(and GTP combination made is activated,when combined with GDP inactivation),activation of ROCK can make its substrates such as myosin light chain,LIM kinase,ades protein-root protein-membrane to protein,myosin phosphorylation subunits(MYPT)phosphorylation,such as in cell shrinkage,neutrophil migration,nerve cells in the process of growth and apoptosis play an extremely important role.Experiment indicated that by inhibiting the expression of ROCK can increase the volume of the autophagosome,then promote the occurrence of autophagy.Have other studies have shown that in vitro using Y-27632 ROCK inhibitors can significantly increase cell protein decomposition,decrease the accumulation of abnormal proteins,also can activate intracellular other protease such as ubiquitin proteasome system,increase the phagocytosis,promotes the increase in the number of abnormal cells in the digestive and decomposition of matter.In conclusion,this study through the establishment of Ox-LDL induced in vitro experiment of sertoli cell lipid damage model,to observe the Ox-LDL in mice podocyte ROCK1 activity and the influence of nephrin expression;And through the use of ROCK1 siRNA,discuss wtROCKl plasmid bidirectional regulation ROCK1 ROCK1 absorb lipids and cholesterol to the cells of the foot and the influence of nephrin,LC3-Ⅱ,P62,p-ULK1(Ser757)expression,the relationship between preliminary discussion ROCK1 involved in lipid may effect to the pathogenesis of kidney damage.METHODSPart one Mouse of podocytesculture,identification and treatment group1.Cultivation of mouse podocytesImmortalized cells under the condition of the foot(MPC)after recovery，use of RPMI-1640 medium containing 10%FBS and 20-100-U/mL restructuring of mice with gamma interferon in 5%CO2,in 33 ℃ under the condition of the induced proliferation and batches.Subsequent to 5%CO2,37℃ incubator for mature.2.Observation and identification of mouse podocytesFirst of all,The podocytes in the incubator at 33℃ and the incubator in the incubator at 37℃ were taken under inverted phase contrast microscope and the morphological differences between the two were observed.The podocyte was determined by immunofluorescence of podocyte-specific skeleton protein synaptopodin Has been divided into mature,mature podocytes that express and extend the cytoskeleton clearly.3.Intervention and treatment of differentiated Podocytes as follows(1)Effects of Ox-LDL stimulation on the activity of podocyte ROCK1.(2)To investigate the effect of ROCK1 expression on p-MYPT/MYPT,nephrin expression,podocyte uptake and cell cholesterolPart three DetectionsThe levels of p-MYPT/MYPT/MYPT,Nephrin,LC3-Ⅱ and LC3-Ⅱ were detected by Western blot.The changes of lipid uptake were detected by confocal immunofluorescence assay.Part four Statistical analysesThe data of the measurement data were expressed as mean ± standard deviation(SD),and SPSS 20.0 statistical software was used for statistical analysis.Multiple-group results were compared using one-way ANOVA method.If there was statistical differences in groups,the comparision between two groups would be uesd.Significance was difined as P<0.05.RESULTSCompared with normal control group,the ox-LDL stimulates cells raised foot ROCK activity increases,at the same time companion has nephrin,LC3-Ⅱexpression decreased(p<0.05),P62,p-ULK1 expression increased(p<0.05),intracellular cholesterol levels increased(p<0.05);Inhibition of ROCK1 expression can inhibit ox-LDL induced by p-MYPT level increases,at the same time,raised the expression of nephrin and LC3-Ⅱ cut P62(p<0.05),and the expression of p-ULK1(p<0.05),lower cholesterol levels in the cell(p<0.05);In contrast,increase ROCK1 express can fiirther increase the ox-LDL induced p-MYPT level,and further reduce the expression of nephrin and LC3-Ⅱ,increase the expression of P62 and p-ULK1(p<0.05)and cholesterol(p<0.05)in the cell.CONCLUSIONROCK1 mediated disfunction of lipophagy contributed to the ox-LDL induced podocyte injury.