Effects Of Danhong On Kidney Injury And Its Mechanisms In Rats With Early Chronic Kidney Disease

BackgroundChronic kidney Disease (CKD) is a kind of chronic diseases, which has become a serious threat to people’health, and consumed a large quantity of health resources. It is of great importance to diagnose and interve early and effectively for delaying the progression of CKD. The mechanisms of chronic kidney disease is so complex that it is involved with oxidative stress, endothelin disfunction, over-expression of angiotensin II,over-secretion of TGF-β,and dyslipidemia. We have already acknowledged that Salviamiltiorrhiza Bunge and Carthamus tinctoriusL are widely used in treatment of cardiovascular diseases, for it accelerating hemodynamics, ameliorating endothelial function, inhibiting oxidative stress. However, whether or not it affects the chronic kidney disease and its mechanisms are waiting to be understood and unveiled. So we planed to investigate the effects of Danhong injection on renal lesion in early CKD rats and its mechanisms, hoping to provide useful guidance for the treatment of chronic kidney disease.ObjectiveUsing the method of unilateral nephrectomy, we try to establish the early CKD animal model to study the effects of Danhong on kidney injury and its mechanisms in rats with early chronic kidney disease.MethodsBy right-side nephrectomy for12weeks, we tried to establish the animal model of early CKD. The40SD rats were divided into four groups randomly, including Group A(sham operation group),Group B(CKD group), Group C (danhong-treated CKD group),and Group D(losartan-treated CKD group). Each group had10rats.Group A and B were given8ml/kg saline gastric lavage and saline intraperitoneal injection of7ml/kg, one time a day. Group C was given danhong injection (7ml. kg-1. day-1) through abdominal cavity, while group D was given losartan potassium (20mg. kg-1. day-1) by gavage,equally to8ml/kg losartan potassium suspension.12weeks later, the rats were sacrificed. Urine was collected to analyse for quantitative test of urinary protein. Blood serum was tested by biochemical assay for serum creatinine,urea nitrogen,uric acid,cystic C, alumin, triglycerid,cholesterol,high density lipoprotein cholestral, low density lipoprotein cholesterol. Renal cortex plasm was analysed for protein concentration (coomassie light blue technique), malondialdeyde (thiobarbituric acid method), superoxide dismutase activity (hydroxylamine method), nitric oxide synthase activity(colorimetric method), endothelin-1, angiotensinⅡ, transforming growth factor-(3(ELISA method). Renal tissue was observed to find glomerular sclerosis, renal interstitial fibrosis, and lesions in glomerular basement membrane by HE stain, PAS stain, PASM stain, and Masson stain. Immunohistochemical technique was used to determine the position and quantity of transforming growth factor beta1in kidney tissue.Results1、Each group had lost one rat.2、Compared with Group A,the kidney weight (1.478±0.273g vs2.356±0.246g, P<0.01), kidney coefficient (0.325±0.053%vs0.662±0.088%, P<0.01), Cystatin C levels (0.256±0.053mg/1vs0.389±0.093mg/1, P<0.01) and24-hour urinary protein quantitaty (5.422±2.903mg/d vs160.158±34.907mg/d, P<0.01) of Group B were increased significantly, while serum creatinine, creatinine clearance rate, and serum uric acid level showed no significant difference (P>0.05). Compared with Group B, the kidney weight (2.356±0.246g vs1.967±0.415g, P<0.01), kidney coefficient (0.662±0.088%vs0.457±0.109%, P<0.01), and24-hour urinary protein quantitaty(160.158±34.907mg/d vs34.578±11.839mg/d, P<0.01) of Group C were decreased significantly. There is no significant difference between Group C and Group D (P>0.05).3、 There was no obvious pathological changes in the kidney of Group A. Lesions of glomerular sclerosis, glomerular basement membrane thickening, interstitial fibrosis were observed in Group B,and compared with Group A, the half quantitative analysis of renal interstitial fibrosis, glomerular basement membrane thickness, glomerular sclerosis,and the total integral is higher (P<0.01). Lesions of interstitial fibrosis, glomerular basement membrane thickening, glomerular sclerosis were alleviated in Group C,and compared with Group B, the half quantitative analysis of renal interstitial fibrosis, glomerular basement membrane thickness, glomerular sclerosis,and the total integral were reduced (P<0.01). The half quantitative analysis of renal interstitial fibrosis, glomerular basement membrane thickness, glomerular sclerosis,and the total integral in Group D were reduced (P<0.01). There was no significant difference between Group C and Group D(P>0.05).4、 Compared with Group A,SOD activity of Group B was down significantly (122.558±18.733U/mgprot vs72.834±8.855U/mgprot, P<0.01),while MDA in renal plasm of Group B was increased significantly (logarithm:0.763±0.219vs1.095±0.236, P<0.01). Compared with Group B, SOD activityof Group B was up significantly (72.834±8.855U/mgprot vs135.070±63.721U/mgprot, P<0.01),while MDA in renal plasm of Group C was decreased significantly (logarithm:1.095±0.236vs-0.031±0.598, P<0.01).SOD activity of Group D was up significantly(72.834±8.855U/mgprot vs125.233±30.284U/mgprot, P<0.01),while MDA in renal plasm of Group D was decreased significantly (logarithm:1.095±0.236vs-0.194±0.515, P<0.01).There is no significant difference between Group C and Group D (P>0.05)5、Compared with Group A, ET-1level in renal cortex of Group B (21.657±6.421ng/L vs66.716±6.704ng/L, P<0.01),TNOS activity (-0.123±0.147U/mgprot vs1.370±0.403U/mgprot, P<0.01) and iNOS activity (logarithm：-1.210±0.523vs0.607±0.394, P<0.01) were increased significantly. Compared with Group B, ET-1level (66.716±6.704ng/L vs26.542±7.193ng/L, P<0.01), TNOS activity (1.370±0.403U/mgprot vs0.312±0.519U/mgprot, P<0.05) and iNOS activity (logarithm:0.607±0.394vs-1.094±0.555, P<0.01) in renal cortex of Group C was decreased significantly. ET-1level (66.716±6.704vs29.883±9.283, P<0.01), TNOS activity (1.370±0.403U/mgprot vs0.264±0.626U/mgprot, P<0.01) and iNOS activity (0.607±0.394vs-8.695±0.654, P<0.01) in renal cortex of Group D was decreased significantly. There is no significant difference between Group C and Group D (P>0.05)6、Compared with Group A, Angll level in renal cortex of Group B was increased significantly (102.706±11.448ng/L vs263.255±10.730ng/L, P<0.01). Compared with Group B, Angll level in renal cortex of Group C was decreased significantly (263.255±10.730ng/L vs108.604±7.231ng/L, P<0.01). Angll levelin renal cortex of Group D was decreased significantly (263.255±10.730ng/L vs103.615±10.608ng/L, P<.01).There is no significant difference between Group C and Group D (P>0.05)7、Compared with Group A, TGF-β level in renal cortex of Group B was increased significantly (185.701±4.609ng/L vs272.364±34.858ng/L, P<0.01). Compared with Group B, TGF-β levelin renal cortex of Group C was decreased significantly (272.364±34.858ng/L vs196.862±35.519ng/L, P<0.01). TGF-β levelin renal cortex of Group D was decreased significantly (272.364±34.858ng/L vs202.881±42.743ng/L, P<0.01).There is no significant difference between Group C and Group D (P>0.05) 8、The results of immunohistochemistry of TGF-β1showed that:TGF-β1brown granules were observed in renal tubular epithelial cells, renal interstitial cells, mesangial cells of four groups of rats, especially Group B.Compared with Group A, the percentage of TGF-β1positive areain Group B was increased significantly (4.574±1.074%vs14.632±1.291%, P<0.01).Compared with Group B, the percentage of TGF-β1positive area in Group C was decreased obviously (14.632±1.291%vs11.239Q0.784%, P<0.01),the percentage of TGF-β1positive area in Group D was increased significantly(14.632±1.291%vs7.071±1.113%, P<0.01). There is no significant difference between Group C and Group D (P>0.05)9、Compared with Group A, serum concentration of cholesterol (1.500±0.332mmol/l vs4.544±2.587mmol/l, P<0.01),HDL-C(0.859±0.344mmol/l vs2.318±1.779mmol/l, P<0.05), LDL-C (0.252±0.078mmol/l vs0.931±0.551mmol/l, P<0.01) in Group B was increased significantly (P<0.01).Compared with Group B, serum concentration of cholesterol(4.544±2.587mmol/l vs1.667±0.781mmol/l, P<0.01),HDL-C (2.318±1.779mmol/l vs0.827±0.352mmol/l, P<0.01), LDL-C (0.931±0.551mmol/l vs0.331±0.129mmol/l, P<0.01) in Group C was decreased significantly (P<0.01), serum concentration of cholesterol (4.544±2.587mmol/l vs2.213±1.621mmol/l, P<0.05, LDL-C (0.931±0.551mmol/l vs0.420±0.407mmol/l, P<0.05) in Group D was decreased significantly (P<0.01). There is no significant difference between Group C and Group D (P>0.05)ConclusionBy right-side nephrectomy for12weeks, we successfully established the animal model of early CKD. We find that Danhong has renalprotective effect of ameliorating glomerular sclerosis, renal interstitial fibrosis in rats with CKD, by inhibiting oxidative stress, improving endothelial function, inhibiting the renin-angiotensin system activaty, reducing the secretion and expression of TGF-β and regulating lipid metabolism.