| ObjectiveTo analyse the expression level of multiple components in complement system which including the classic pathway,alternative pathway,mannose binding lectin pathway in type 2 diabetic nephropathy kidney of rat and explore the pathogenic role of the complement components in type 2 diabetic nephropathy.Methods1.Established the model of type 2 diabetes and type 2 diabetic nephropathy:5 week-old SPF male SD rats were randomly divided into 3 groups includ control group,type 2diabetes group(DM group)and type 2 diabetic nephropathy group(DN group).The control group was given normal feed,and the DM group and DN group were given high-fat feed.After 8 weeks,the rats were fasting for 12 h,the DM group and DN group were intraperitoneally injected 1%STZ in 30mg/kg dose,and the control group was injected with the same dose of citric acid buffer.After rats developed diabetes,the DN group continued to prepare diabetic nephropathy model with high-fat feed for 8 weeks.The 24h urine albumin excretion which more than 30mg/24h was the standard for early renal lesion.2.Measure the body weight,kidney weight and measure blood glucose by Roche glucometer(photochemical method).Using ELISA method to measure fasting insulin,24h urine albumin concentration,then calculate the insulin resistance index(HOMA-IR=FINS×FPG/22.5).3.HE staining and PAS staining was applied to observe the morphological changes of the kidney,and calculated the mesangial index(mesangial index=mesangial area/glomerular area×100%),arteriosclerosis index(arteriosclerosis index=wall area/(wall area+lumen area)),and percentage of the cast area(percentage of the cast area=cast area/total area×100%).4.Immunohistochemistry and western blot were used to detect the expression of the complement components and nephrin which was the marker protein of podocyte in rat kidney.Results1.Before STZ injection,the weight of the DM group and the DN group was higher than that of the control group(525.6±35.0g vs.477.6±25.6g,P=0.010;541.4±39.7g vs.477.6±25.6g,P=0.001).In the DM group and DN group,HOMA-IR was higher than that of the control group(2.76±1.20 vs.1.50±0.13,P=0.006;2.40±1.27 vs.1.50±0.13,P=0.036),indicating insulin resistance have been induced in the DM group and DN group.After one weeks of STZ injection,the blood glucose of the DM group and DN group was higher than that of the control group(23.11±2.78mmol/L vs.6.06±0.92mmol/L,P<0.001;24.25±2.80mmol/L vs.6.06±0.92mmol/L,P<0.001).The HOMA-IR of both the DM group and the DN group were higher than the control group(5.88±3.09 vs.1.59±0.13,P<0.001;5.83±2.07 vs.1.59±0.13,P<0.001).After 17weeks,the amount of urine albumin in the DN group was higher than that in the control group and the DM group(111.64±25.61mg vs.9.74±2.08mg,P<0.001;111.64±25.61mg vs.18.09±6.05mg,P<0.001).In addition,the renal weight/body weight(mg/g)of the DN group was higher than that of the control group and the DM group(4.94±1.00vs.2.79±0.24,P<0.001;4.94±1.00 vs.3.02±0.20,P<0.001).2.HE staining and PAS staining showed that the mesangial index in DN group was higher than the control group and the DM group(16.25±1.67%vs.13.62±1.41%,P=0.003;16.25±1.67%vs.14.00±1.69%,P=0.010).The percentage of the cast area of the DN group was higher than that of the control group and the DM group(1.66±1.83%vs.0,P=0.005;1.66±1.83%vs.0,P=0.005).The renal arteriosclerosis index in DN group was higher than that in the control group and DM group(0.79±0.03 vs.0.76±0.03,P=0.024;0.79±0.03 vs.0.75±0.03,P=0.011).The above indexes showed no significant difference in DM group and DN group(P>0.05).3.The IOD of positive expression in Immunohistochemistry(IHC)(×10~4)and the relative grey value of western blot stripe shows:The IOD of C1q increased in DN group than the control group and DM group(10.38±4.15 vs.5.69±3.86,P=0.010;10.38±4.15 vs.3.37±9.76,P<0.001).the IOD of MBL in the DN group was increased than the control group and DM group(6.79±4.56 vs.2.39±0.94,P=0.004;6.79±4.56 vs.2.92±0.8,P=0.010),and the relative grey value of western blot stripe was increased than the control group and DM group(3.91±1.13 vs.0.83±0.39,P=0.000;3.91±1.13 vs.1.34±1.05,P=0.004).the IOD of MASP-2 in the DN group was increased than the control group and DM group(12.50±3.10 vs.3.27±2.78,P<0.001;12.50±3.10 vs.4.72±5.16,P<0.001),and the relative grey value of western blot stripe was increased than the control group and DM group(3.91±1.13 vs.0.83±0.39,P<0.001;3.91±1.13 vs.1.34±1.05,P<0.001).The IOD of factor B in DN group was increased than the control group and the DM group(8.35±5.05 vs.1.84±1.45,P=0.001;8.35±5.05 vs.2.67±1.91,P=0.002).the IOD of C3 in the DN group was increased than the control group and DM group(2.22±1.23 vs.1.13±0.86,P=0.031;2.22±1.23 vs.1.02±0.62,P=0.018),and the relative grey value of western blot stripe was increased than the control group and DM group(1.77±0.94 vs.0.66±0.33,P=0.002;1.77±0.94 vs.0.96±0.77,P=0.036).The IOD of C5b-9 was higher in the DN group than the control group and the DM group(4.29±1.80 vs.1.64±0.79,P<0.001;4.29±1.80 vs.1.69±0.68,P<0.001).There was no significant difference in the expression of complement components in the control group and DM group(P>0.05)?4.The immunohistochemical results showed that C1q,MBL,MASP-2,factor B and C5b-9 were mainly expressed in renal tubular,and C3 was mainly expressed in glomerulus.5.IHC showed that the expression of nephrin have no significant difference in each group(P>0.05).6.correlation analysis results show that 24h urine albumin was positively correlated with C1q(r=0.580,P=0.003),MBL(r=0.640,P=0.001),MASP-2(r=0.751,P<0.001),factor B(r=0.728,P<0.001)and C5b-9(r=0.718,P<0.001).The mesangial index was positively correlated with MBL(r=0.457,P=0.025),factor B(r=0.429,P=0.036),C3(r=0.410,P=0.046),C5b-9(r=0.498,P=0.013).The percentage of cast area was positively correlated with C1q(r=0.431,P=0.035),MBL(r=0.548,P=0.006),and factor B(r=0.553,P=0.005).The arteriosclerosis index was positively correlated with C1q(r=0.481,P=0.017),MBL(r=0.465,P=0.022),MASP-2(r=0.494,P=0.014),and factor B(r=0.486,P=0.016).Conclusions1.The high expression levels of complement components of the three pathway in type 2diabetic nephropathy was associated with the lesion of DN.2.Different complement pathways may play different roles in glomerulus and renal tubular injury respectively. |
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