Molecular Mechanism Of Inflammation Promoting Podocyte Injury Via Breaking The Cholesterol Balance Hold By NPC1 In Primary Nephrotic Syndrome

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THE RELATIONSHIP BETWEEN CHOLESTEROLIMBALANCE AND ENDOPLASMIC RETICULUMSTRESS IN ADRIAMYCIN NEPHROPATHY MICEMODELObjective: To investigate the relationship between cholesterol imbalance and primary nephrotic syndrome via observing endoplasmic reticulum stress response and cholesterol level of serum,kidney tissue and renal endoplasmic reticulum in Adriamyc in nephropathy mice.Methods: 6~8-week-old male mice were randomly divided into control group(CTL)and Adriamyc in group(ADR),both of which were sacrificed in the 4th,8th and 12 th week,respectively.24-hour urine protein was measured by Coomassie brilliant blue.Kidney tissues were observed by optical microscope and electron microscope.Triglyceride,low dens ity lipoprotein,creatinine and cholesterol level of serum,cholesterol level of kidney tissue and renal endoplasmic reticulum were measured by enzymatic analysis.m RNA expressions of GRP78 and CHOP were measured by q PCR.Results: 1.Compared with CTL group,mice in ADR group were inactive and lost weight.2.Compared with CTL group,mice in ARD group suffered proteinuria from the 2nd week,which peaked in the 8th week and alleviated from then on(P<0.05).3.In the 4th week,compared with CTL group,foot process fusion and lipid droplet were found in the kidney of ADR group by electron microscope,while nothing different was found between the two groups by light microscope;in the 8th week,inflammatory cell infiltration and glomerulosclerosis were found in the kidney of ADR group by light microscope,while extensive foot process fusion,mesangial cell proliferation,protein deposition and more lipid droplet and lysosome were found by electron microscope;in the 12 th week,more inflammatory cell infiltration and focal segmental glomerulosclerosis were found in the kidney of ADR group by light microscope,while more serious mesangial cell proliferation and extensive foot process fusion,even apoptosis of podocyte were found by electron microscope.4.Compared with CTL group,cholesterol level of renal endoplasmic reticulum in ARD group elevated from the 4th week(P<0.05).5.Compared with CTL group,serum total cholesterol and triglyceride in ARD group increased from the 4th week;serum low dens ity lipoprotein decreased in the 4th week and made no difference in the 8th and 12 th week;serum creatinine increased from the 8th week(P<0.05).6.Compared with CTL group,m RNA expression of GRP78 and CHOP in ADR group increased obviously from the 4th week(P<0.05).Conclusions: 1.renal pathological variation of adriamycin nephropathy mice model was minimal change nephrotic syndrome in the early phase,then progressed into focal segmental glomerulosclerosis with enal failure.2.Cholesterol imbalance and endoplasmic reticulum stress were found in adriamycin nephropathy mice model.PART ? NPC1 ABNORMAL EXPRESSION IN THEKIDNEY OF ADRIAMYCIN NEPHROPATHY MODELObjective: To investigate the relationship between NPC1 and primary nephrotic syndrome via observing NPC1 expression in the kidney of mice of adrimamycin nephropathy model.Methods: 6~8-week-old male mice were randomly divided into control group(CTL)and adriamycin group(ADR),both of which were sacrificed in the 4th,8th and 12 th week,respectively.NPC1 expression in the kidney of both groups was measured by q PCR,western blot,immunohistochemical and immunofluorescence.Results: Compared with CTL group,NPC1 expression in the kidney of ADR group increased obviously from the 4th week(P<0.05).Conclusions: NPC1 expression in the kidney of ADR group was increased,which might be related to primary nephrotic syndrome.PART ? INFLAMMATION PROMOTS PODOCYTE INJURY VIA BREAKING THE CHOLESTEROL BALANCEHOLD BY NPC1Objective: To investigate the possible role of NPC1 in podocyte,which was injured by inflammation,via observing NPC1 expression in podocyte and the changes of cell physiology,cholesterol balance and endoplasmic reticulum stress after blocking NPC1.Methods: Mouse podocytes(MPC5)was divided into 4 groups: control group(CTL),LDL group,LDL+IL-1?group,U18666 A group.NPC1 expression in podocyte was measured by q PCR,western blot and immunofluorescence;apoptosis of podocyte was measured by flow cytometry;m RNA expression of GRP78 and CHOP in podocyte were measured by q PCR;total cholesterol level of podocyte and its endoplasmic reticulum were measured by enzymatic analys is;lipid deposition in podocyte was detected by oil red stain.Results: 1.Compared with CTL group,NPC1 expression in podocyte was upregulated(P<0.05).2..Compared with CTL group,apoptosis rate of podocyte was increased in LDL group and more severe in LDL+IL-1?group,both of which were inhibited by co-treatment with an NPC1 inhibitor(P<0.05).3.Compared with CTL group,m RNA expression of GRP78 and CHOP was incresead in LDL group increased,which was more obviously in LDL+IL-1? group,both of which were inhibited by co-treatment with an NPC1 inhibitor(P<0.05).4.Compared with CTL group,total cholesterol level of podocyte was increased in LDL+IL-1? group;while cholesterol level of endoplasmic reticulum in podocyte was incresead in LDL group increased,which was more obviously in LDL+IL-1? group,both of which were inhibited by co-treatment with an NPC1 inhibitor(P<0.05)Conclusions: Inflammation might play an important role in podocyte apoptosis by disrupting the cholesterol balance keeping by NPC1 in primary nephrotic syndrome.PART ? CD36 PROMOTES PODOCYTE APOPTOSIS BYACTIVATING THE NLRP3 INFLAMMASOME INPRIMARY NEPHROTIC SYNDROMEObjective: To investigate the possible role of CD36 and NLRP3 inflammasome in podocyte apoptosis in primary nephrotic syndrome,via observing CD36 expression in the kidney of mice of adrimamyc in nephropathy model and podocyte,as well as the changes of NLRP3 expression after blocking CD36.Methods: The mouse podocyte cell line MPC5 was used as a model.m RNA and protein expression of CD36 and NLRP3 was quantified by real-time PCR and western blotting,respectively.Levels of caspase-1 activity and total cholesterol were determined us ing commercial kits.Intracellular lipid droplets were detected by Oil Red O staining.CD36 expression was also examined in nephrotic mouse kidney tissue by immunohistochemistry and immunofluorescence.Intracellular lipid droplet was examined by Oil Red O staining.Results: CD36 expression was increased in nephrotic mouse kidney tissue(P<0.05).Treatment with interleukin-1? increased expression of CD36 and total cholesterol in MPC5 cells(P<0.05).Moreover,this treatment increased expression of NLRP3 and the percentage of apoptotic cells,both of which were inhibited by co-treatment with an anti-CD36 antibody(P<0.05).Conclusions: CD36 might play an important role in podocyte apoptosis by activating the NLRP3 inflammasome in primary nephrotic syndrome.