| OBJECTIVE: The adriamycin nephropathy model was established by BALB/C mice through caudal vein injection.The effect of Nephropathy Prescription I on biochemical parameters and pathomorphology in BALB/c mice with adriamycin-induced nephropathy were investigated.To explorer the protective effect for the herbs on the podocytes in mice models,we have also measured Angptl3 and podocyte-associated molecules,such as Nephrin,Podocin,and CD2 AP.METHODS: Mice model establishments and drug interventions: 8 weeks old balb/c mice,20-25 g,male,were randomly divided into 5 groups,10 in each group.They were saline control group(C),model group(M),TCM intervention group(M+Z),steriod intervention group(M+S),Combined TCM and steriod group(M+Z+S).Excepted for C group,other groups were injected adriamycin(10mg/kg)via caudal vein to make nephropathy models,with equal dose saline for group C as a control.The corresponding groups were given Nephropathy Prescription I(26.1g /(kg.d)),and prednisone(18mg /(kg.d))through intragastric gavage respectively,from Day 1 after modeling,once a day for 28 consecutive days.The senarios for mice were observed with urine for 24 hours collected in the metabolic cage on the 7th,14 th,21st,and 28 th day of modeling.All mice were sacrificed on the 29 th day for blood sample and kidney tissue specimen.Urine biochemical indicators: urine albumin creatinine ratio;serum biochemical indicators: serum albumin,total cholesterol,triglycerides,serum creatinine,urea nitrogen;The renal morphological structure and podocyte ultrastructure were observed under light and electron microscope.The podocyte related albumins as Angptl3,Nephrin,Podocin and CD2 AP molecules were measured by Western blottong.RESULTS:(1)urine biochemical indicators(urine albumin / creatinine)1)After the modeling with doxorubicin,albuminuria appeared in the first week.The difference between the model group(2.13±0.25)and the control group(1.63±0.49)was statistically significant(P<0.05).The creatinine ratio reached the maximum(2.54±0.39)and the difference was statistically significant at the same time point(1.64±0.67)(P<0.05).2)There was no significant difference for urinary albumin creatinine ratio between the first week and the second week in the TCM intervention group(P>0.05).Compared with the models at the third week(2.02±0.09)and the fourth week(2.06±0.21)urinary albumin,the difference for creatinine ratio was significant(P<0.05).3)Compared with the model group the urinary albumin creatinine ratio in steroid intervention group decreased in the first week(1.69±0.38,2.13±0.25),the difference was statistically significant(P<0.05).At the second week compared with the model group(1.87±0.12,2.19±0.33),the third week compared with the model group(1.97±0.04,2.30±0.28),the fourth week compared with the model group(2.04±0.07,2.54±0.39),the ratio of urinary albumin creatinine all droped,the difference was statistically significant(P<0.05).4)In the first week compared with the model group(1.64±0.23,2.13±0.25),the second week compared with the model group(1.82±0.06,2.19±0.33),the ratio of urinary albumin creatinine in the combined TCM and steriod group was lower,and the difference was statistically significant(P<0.05).in the third week compared with the model group(1.86±0.21,2.30±0.28),the fourth week compared with the model group(1.93±0.09,2.54±0.39),the ratio of urinary albumin creatinine all droped,the difference was statistically significant(P<0.01).(2)Serum biochemical indicators 1)After four weeks of doxorubicin modeling,the serum albumin content(g/l)of the model group was lower than that of the control group(25.38±5.53,31.14±3.66).The difference was statistically significant(P<0.05).There was no significant difference(P>0.05)in serum albumin level between TCM intervention group and model group(25.32±2.89,25.38±5.53).The serum albumin level of the steriod intervention group was higher than the model group(31.53±4.12,25.38±5.53),differences have a statistically significant(P<0.05);the serum albumin level in the Combined TCM and steriod group was higher than that in the model group(32.68±4.38,25.38±5.53),differences have a statistically significant(P <0.01).2)The mice in the model group had higher levels of triglyceride(mmol/l)than the control group(3.76±0.94,1.73±0.25),and differences have a statistically significant(P<0.05).In the TCM intervention group(2.72±0.81),the steroid intervention group(2.34±0.59),and combined TCM and steriod group(1.84±0.68)the levels of triglyceride were lower than the model group(3.76±0.94),differences have a statistically significant(P<0.05);in the combined TCM and steriod group the triglyceride level was significantly reduced.3)Compared with the control group(2.84±0.44),the total cholesterol(mmol/l)content of the model group(6.42±1.39)was higher,differences have a statistically significant(P<0.05);compared with the model group,total cholesterol decreased in the steriod intervention group(3.23±0.44)and the combined TCM and steriod group(3.02±0.37),differences have a statistically significant(P<0.05).4)Serum creatinine and urea nitrogen level in each group was no significant change,differences have a statistically significant(P>0.05)(3)Changes in renal pathology After 4 weeks of modeling,there was no obvious pathological change in the glomerular structure of the mice under the light microscope.The glomerular foot process fusion disappeared in the mice of the model group under the electron microscope.The TCM intervention group,the steroid intervention group and combined TCM and steriod group all showed different degrees of remission.Among them,Combined TCM and steriod group was more effective in alleviating the fusion of glomerular foot processes in mice with adriamycin-induced nephropathy.(4)Expression of Angptl3 and podocyte-associated albumins(comparison of gray value of each albumin molecule)1)Nephrin The expression level of nephrin in the model group(1.02±0.13,0.49±0.09)dropped compared with the control group,differences have a statistically significant(P<0.05).The expression of nephrin was decreased in the TCM intervention group(0.84±0.26),steroid intervention group(0.80±0.33)and combined TCM and steriod group(0.99±0.26),compared with the model group(0.49±0.09),differences have a statistically significant(P<0.05).In combined TCM and steriod group the expression level of nephrin was significantly reduced,differences have a statistically significant(P<0.01).2)Podocin The expression level of Podocin in the model group(0.44±0.12,0.72±0.15)was decreased compared with the control group,differences have a statistically significant(P<0.05);Compared with the model group(0.44±0.12),the expression of Podocin was decreased in the TCM intervention group(0.62±0.05),the steriod intervention group(0.67±0.19)and combined TCM and steriod group(0.69±0.17),compared with the model group(0.44±0.12),differences have a statistically significant(P<0.05),in combined TCM and steriod group the expression level of Podocin was significantly decreased,differences have a statistically significant(P<0.01).3)CD2AP The expression level of CD2 AP in the model group(0.53±0.24,1.39±0.47)was decreased,compared with the control group,differences have a statistically significant(P<0.05).In the TCM intervention group(1.03±0.20),the steroid intervention group(1.17±0.55)and combined TCM and steriod group(1.36±0.46),the expression of CD2 AP was decreased,compared with the model group(0.53±0.24),differences have a statistically significant(P<0.05),in combined TCM and steriod group the expression of CD2 AP was significantly reduced,differences have a statistically significant(P<0.01).4)Angptl3 The expression level of Angptl3 n the model group(0.04±0.02,0.18±0.05)i was increased,compared with the control group,differences have a statistically significant(P<0.05.In the TCM intervention group(0.11±0.06),the steriod intervention group(0.10±0.07)and combined TCM and steriod group(0.09±0.03),the expression of Angptl3 was decreased,compared with the model group(0.18±0.05),differences have a statistically significant(P<0.05),in the combined TCM and steriod group the expression level of Angptl3 was significantly reduced,differences have a statistically significant(P<0.01).CONCLUSION: Nephropathy Prescription I can alleviate the albuminuria in mice with adriamycin-induced nephropathy with increased serum albumin,lowered blood lipids and reduced foot process fusion.The protective effection of the herbs for reducing podocyte damage is through regulating the level of some podocyte related albumins by decreased Angptl3 and the increased Nephrin,Podocin and CD2 AP expression. |
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