Effect Of Endothelin-1 On The Interaction Between Human Mesangial Cells And Endothelial Cells On The Condition Of High Glucose And LPC

Part 1.The effect of high glucose and lysophosphatidylcholine on the role of extracellular matrix accumulation via the interaction of mesangial cells and endothelial cellsObjective:To investigate the interaction between mesangial cells and endothelial cells under different concentrations of glucose and lysophosphatidylcholine(LPC) at different time points,further explored the mechanism of diabetic nephropathy through examining concentration of fibronectin(Fn) and collagen typeⅣ(ColⅣ) in culture media.Methods:The model of intercellular interaction between mesangial cells and endothelial cells was established using Millicell membrane culture dish.The mesangial cells were cultured separately or co-cultured with endothelial cells and divided into three guoups:control,high glucose (10,20,30mM glucose) and LPC(5,10,20mg/L LPC),each group was cultured for 6,12,24,48 hours respectively.The concentration of Fn and ColⅣin culture media was determined by enzyme-linked immunosorbent assay(ELISA).Results:(1) Both in monolayer and co-culture cell culture,the concentration of Fn and ColⅣhad no significant change in control group at different time points(P＞0.05).With the same cultured time,the level of Fn and ColⅣof high glucose was higher than that of control group in co-culture and monolayer cell culture(P＜0.05),and the expression intensity increased in a concentration-dependent manner;The concentration of Fn and ColⅣin culture media treated with high glucose for 6,12,24,48 hours respectively increased in a time-dependent manner(P＜0.05), arrived peak level at 24h,then decreased slightly at 48 h.(2) Compared with control group,the concentration of Fn and ColⅣincreased in LPC group from 6h time point,the expression intensity enhanced in a time-dependent manner(P＜0.05),arrived peak level at 24 h; 5,10,20mg/L LPC could increase the secrection of Fn and ColⅣin culture media in a concentration-dependent manner(P＜0.05).(3) In control group,the level of Fn and ColⅣbetween co-culture and monolayer cells culture had no significant difference(P＞0.05). Exposed to high glucose or LPC,the concentration of Fn and ColⅣin co-cultured cells was higher than that in monolayer cells culture at 6,12, 24,48h time points(P＜0.05).Conclusions:(1) Both in co-culture and monolayer cell culture,high glucose and LPC increases the concentration of Fn and ColⅣin a concentration and time-dependent manner.(2) On the condition of high glucose and LPC,there is some kind of interaction between mesangial cells and endothelial cells which can increase the excretion of extracellular matrix.Part 2.Effect of endothelin-1 on the interaction between human mesangial cells and endothelial cells on the condition of high glucose and LPCObjective:To investigate the effect of endothelin-1 on the role of extracellular matrix accumulation via the interaction between mesangial cells and endothelial cells on the condition of high glucose and LPC through examining expression of endothelin-1(ET-1),Fn and ColⅣin co-cultured cell culture.Methods:The model of intercellular interaction between mesangial cells and endothelial cells was established and divided into six groups: control,mannitol,high glucose,LPC,high glucose and LPC, BQ-123(0.01μM,0.1μM,1μM),each group was cultured for 24 hours. The concentration of Fn and ColⅣin culture media was determined by ELISA,ET-1 in supernatant was detected by radio-immunoassay,ET-1 mRNA of endothelial cells and ETRA mRNA of mesangial cells was measured by reverse transcription polymerase chain response(RT-PCR).Results:(1) When mesangial cells were co-cultured with endothelial cells cultured for 24 hours,the concentration of Fn and ColⅣin culture media increased in high glucose group and LPC group(P＜0.05).Compare with high glucose group and LPC group,the expression of Fn and ColⅣincreased significantly in high glucose and LPC group(P＜0.05),but there was no significant difference between mannitol group and control group(P＞0.05).(2) Compared with control group,ET-1 increased remarkably in high glucose group and LPC group(P＜0.05),the stimulating effect was more pronounced in high glucose and LPC group(P＜0.05).(3) In co-culture cell culture,compared with control group,ETRA mRNA expression of mesangial cells in high glucose group and LPC group increased remarkably(P＜0.05),the stimulating effect was more pronounced in high glucose and LPC group(P＜0.05).(4) Compared with high glucose and LPC group,ET-1 receptor antagonist BQ-123 could inhibit Fn and ColⅣsecretion in a dosedependent manner in co-cultured model(P＜0.05),but BQ-123 had no effect on the expression of ET-1 and ETRA(P＞0.05).Conclusions:(1) The expression of Fn,ColⅣand ET-1 is found to be remarkably increased on the condition of high glucose and LPC in co-cultured mode.(2) ET-1 receptor antagonist BQ-123 can inhibit the secretion of Fn and ColⅣin a dose-dependent manner.ET-1 may play an important role in extracellular matrix accumulation on the condition of high glucose and LPC.(3) The expression of ETRA in mesangial cells is promoted by high glucose and LPC,which can enlarge the biological effect of ET-1.(4) ET-1 produced by endothelial cells may have an important impact on mesangial cells,which can improve the synthesis of extracellular matrix.Part 3.The mechanism of extracellular matrix accumulation induced by endothelin-1 on the condition of high glucose and LPC and the intervention effect of losartanObjective:To define whether ET-1 activated protein kinase CβI/ platelet- derived growth factor-B(PKCβI/PDGF-B) signaling pathway to mediate extracellular matrix accumulation,further explored the molecular mechanism of diabetic nephropathy and the intervention effect of losartan. Methods:(1) Mesangial cells were cultured seperately and divided into eight groups:control,ET-1,ET-1 + RG50864,ET-1 + LY333531,high glucose and LPC+ ET-1,high glucose and LPC + ET-1 + RG50864,high glucose and LPC + ET-1 + LY333531,high glucose and LPC + ET-1 + losartan, and each group was cultured for 24 hours.Fn and ColⅣin supernatant was detected by ELISA,the expression of PDGF-B mRNA was measured by RT-PCR and PDGF-B protein expression was detected by Westernblot.(2) The monolayer mesangial cells were divided into six groups: control,ET-1,ET-1 + LY333531,high glucose and LPC + ET-1,high glucose and LPC + ET-1 + LY333531,high glucose and LPC + ET-1 + losartan,and each group was cultured for 24 hours.Fn and ColⅣin supernatant was detected by ELISA,the expression of PKCβ1 mRNA was measured by RT-PCR,the protein expression of PKCβI was detected by Western-blot and immunofluorescence.Results:(1)The expression and secretion of ColⅣ,Fn and PDGF-B increased remarkably at 24h after ET-1 stimulation(P＜0.05),the stimulating effect could be enlarged by high glucose and LPC(P＜0.05). Addition of a tyrosine kinase inhibitor RG50864 to cells could prevent ET-1-induced Fn and ColⅣprotein up-secretion(P＜0.05),but RG50864 had no effect on the PDGF-B mRNA and protein expression of mesangial cells(P＞0.05).(2)After ET-1 stimulation,HMC PKCβ1 mRNA and protein expression was up-regulated compared with control(P＜0.05),and immunofluorescence demonstrated that ET-1-induced PKCβ1 transmitting from cytoplasm to nucleus and membrane,and the stimulating effect could be enlarged by high glucose and LPC(P＜0.05). Further more,LY333531 could prevent ET-1-induced Fn and ColⅣprotein up-secretion through attenuating PKCβI and PDGF-B expression of mesangial cells(P＜0.05).(3)Compared with high glucose and LPC + ET-1 group,losartan could decrease PKCβI and PDGF-B expression(P＜0.05),partly prevented ET-1-induced extracellular matrix secretion on the condition of high glucose and LPC(P＜0.05).Conclusions:(1) ET-1 stimulates human glomerular mesangial cells extracellular matrix secretion may by activating PKCβI/PDGF-B signaling on the condition of high glucose and LPC.(2) Losartan can protect kidney by inhibitting PKCβI/PDGF-B signaling and decreasing the extracellular matrix secretion.