| ObjectivesTo understand the mechanism that Cyprinus carpio decoction (CCD) improves edema in Adriamycin (ADR) nephropathy rats, by observing the effects of CCD on nutritional status, the level of interleukin-4(IL-4) and interferon-gamma (IFN-y) in serum and expression of CD2AP and nephrin in kidney of these rats.Methods70healthy8-week-old Wistar rats were randomly divided into5groups:Control group (group N), group M, Fosinopril group (group F), group YD and group YG, in which all rats except group N received ADR6.5mg/kg via injecting from vena caudlis, to introduce ADR induced nephropathy. Then begin the treatment one week later. Rats in group YD and group YG receive CCD by lavage; and group F receive Fosinopril by lavage. Rats in group N and group M are given the same amount of water. During the experiment, the rats in all groups are weighted and put into metabolic cage to gather urine in12h. These70rats were sacrificed7weeks after intervention, and blood were obtained. The serum biochemical values of the total protein (TP), albumin (ALB), globulin (G), glucose (Glu), cholesterol (CHO), low density lipoprotein (LDL), blood urea nitrogen (BUN), creatinine (Cr), electrolyte levels and the urinary protein (UP) content in12h urine were measured. Interleukin-4and Interferon were measured by enzyme-liked immunosorbent assay (ELISA). The pathomorphological analysis was observed using light and electron microscopy, and the expressions of CD2-associated protein (CD2AP) and nephrin in renal tissues were determined by immunohistochemical assay.Results(1) General ObservationAt week3, rats in YD, YG groups eat8,9g diet per day, respectively. It was more than M and F groups (5g)(P<0.05). The consumption gradually increased in every group from week1to week5. However, there was no difference in each rat (P>0.05) afterwards. They ate about20g chow one day. Rats grew with time. At the end of the third week, the weights of rats in the experimental groups were0.230±0.029, 0.225±0.027,0.252±0.045,0.249±0.017kg. They began lower than normal group (0.328±0.023kg)(P<0.05). At the next week, it occurred that significant differences existed among these four experimental groups (P<0.05). However, there was no statistically differences between YD and YG groups (P>0.05).(2) Biochemical indicatorAdministration of high dose of CCD significantly prevented the decrease in serum albumin and total protein (P<0.05, compared with group M), but the differences between group YD/YG and group F. Concentrations of CHO and LDL in serum of rats receiving injection of ADR are higher than group N (P<0.05), but no differences are found among these rats. The level of Glu in group F, group YD and group YG is higher than group M (P<0.05). There was no significant difference in serum Cr, BUN, Cr, Na+, K+, CI-concentration among each group (P>0.05).(3) Urinary protein excretion in12h urineThe value of Urinary protein excretion in12h urine is gradually increasing as time goes by. Before injection of ADR, there is no significant difference among all rats (P <0.05=. At the first week after induction, the rats excreted plenty of protein in urine,76.78±8.63,69.66±15.29,77.49±12.83,88.25±15.05mg. And rats receiving injection of ADR is higher than that of rats in group N. Although Urinary protein excretion in12h urine of the rats received Fosinopril and CCD was higher than that of the normal rats (P＜0.05), it was less than group M since the second week after injection of ADR. And since the sixth week, the difference is significant (P<0.05).(4) IL-4and IFN-yThe results show that CCD can increase the amount of IFN-y production, although the difference was not statistically significant. The level of IL-4in model group (32.13±2.84pg/ml) was greatly higher than that in normal group (12.55±1.21pg/ml, P<0.05). Fosinopril and CCD can dramatically decrease the level of IL-4, when compared to the model group. However, there was no significant difference among group F, YD and YG. It was shown that CCD was as effective as the Fosinopril.(5) Renal tissue morphological analysisLight-microscopy measurement:It showed that in the rat kidney of M group there were obvious pathological lesions characterized by glomerular swelling, stenosis of the renal glomerulus capsular space, blurry margin between each part, many inflammatory cells around glomerulus, abundant protein exudation in the renal tubular lumen. In the rats’kidney of F, YD and YG group, these pathological damages were lighter than those of M group, although glomerular swelling, inflammatory cells around glomerulus, protein cast in the renal tubular lumen were also sporadically observed. The severity of pathological damage was reduced. No pathological changes were found in the kidney of control group.Electron Microscopy measurement:The ultrastructural changes of rat kidney in the five groups were comparatively analyzed by electron microscopy. In control group, there were no apparent damages as judged by intactness of renal glomerulus of rats’ kidney tissue. Clear foot process was observed on the visceral surface of renal glomerular epithelial cells, which was no swelling, hypertrophy and inflammatory cells around them. In M group, the basal structure of renal glomerulus was damaged. Foot process diffusely changed, and became thin and flat, and fused, even disappeared. The line of glomerular basement membrane (GBM) was unclear and there were a lot of inflammatory cells in renal interstitial cells. In F, YD and YG group, the lesions were significantly relieved, although foot process effacement and inflammatory cells were observed in the renal glomerulus. There was no difference in electron microscopic morphology during these three groups.(6) CD2AP and nephrinBrown or yellow granular mass, in a continuous shape of thick line, was found in the glomerular capillary wall, the attachment of podocytes, in each group. The expressions of CD2AP and nephrin in renal tissues of ADNs were decreased with comparsion of that in normal group, especially the model group the lowest. Its positive expressive area was distributed in punctuation or irregular clumps. In the rats received Fosinopril and CCD, the area distribution was more than that in group M. Semi-quantitative analysis of staining intensity showed that the expressions of CD2AP and nephrin decreased in group M, F, YD and YG, compared with the normal group. However, it had some degree increase in the three treatment groups (P<0.05, compared with group M). ConclusionIn conclusion, the present study shows that CCD has a protective effect against ADR-induced nephropathy, and this effect may be attributed to the improvement of nutrition status and regulation of cytokines and immune balance. Moreover, the roles of CCD are related to the reduction of proteinuria through promoting CD2AP and expressions. The findings may shed light on the pharmacological basis for the clinical nephrin application of the traditional Chinese medicine in the treatment of chronic kidney disease. |
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