| Proteinuria is common characteristic of renal disease, but also as an independent factor, which would directly cause renal pathological lesion and loss of renal function, is an important clinic index of curative effect and prognosis. It focuses that how to decrease proteinuria and retard progressive renal disease in nephrology field. Glucocorticoid and immunodepressant are the main medicines to treat proteinuria, but many side-effects appear with long time use of them without full-contented.Cantharidin, active component extracted from blister bug, has distinct effect on cancer in clinic experience. Norcantharidin (NCTD), an important derivant of Cantharidin, has clear pharmacologyical actions with low deleteriousness. It has confirmed that NCTD counld inhibit cell proliferation and induce apoptosis of tumor cells. The signal transduction pathway which NCTD induce cell apoptosis by has close relation to Caspase, Bcl-2, ERK, JNK and survivin protein. Moreover, NCTD counld down-regulate expressions of nuclear factorκB (NF-κB) and Smad3, also balance the ratio of MMPs/TIMPs, thus NCTD would inhibit tumor cells infiltration and local blood vessel formation, keeping balance of extracellular matrix (ECM) synthesis and degration. In addition, NCTD has anti-inflammation effect and may regulate immune system, so it dose good to some immune diseases such as rheumatic disease and psoriasis. Present studies showed that NCTD inhibited fibroblast cell line (NIH/3T3) proliferation by dose-dependence, indicating that it has some positive effects to organ fibrosis prevention.Primary glomerulopathy is a kind of immue diseases. There is close relationship among proteinuria, inflammation and fibrosis. In theory, NCTD may treat renal diseases according to its pharmacologic characteristic mentioned above. So in the study below, NCTD was chosen to treat nephropathy rats with overload proteinuria, in order to observe the changes of proteinuria and renal pathology, also elucidate the mechanisms by vivo and vitro experiments.Objective: To study effect of NCTD on nephropathy rats with overload proteinuria. To detect expression of connective tissue growth factor (CTGF) and nuclear factor-κB (NF-κBp65) in renal tissues. To observe effect of NCTD on proliferation and fibronectin (FN) expression in human renal proximal tubular epithelial cell line (HK-2) induced by albumin. To provide experimental evidences on NCTD used to treat renal diseases.Methods: 1,uninephrectomized SD rats were received intraperitoneally injections of bovine serum albumin (BSA). The nephropathy rats were randomly divided into model group (BSA group, intraperitoneally injections of BSA 5g/(kg'd) ), NCTD group (intraperitoneally injections of BSA 5g/(kg·d)and NCTD 0.1mg/(kg·d) ), and MP group (BSA 5g/(kg·d) and MP lmg/(kg·d) ). An additional set of uninephrectomized rats were only given saline as control group. Twenty-four-hour urine protein was measured at every time point. At week 9, the rats were sacrificed after anticoagulant blood and serum were collected. The remnant kidney of each rat was harvested and weighed which was observed by light microscopy, immunofluorescence staining and electron microscopy.2,CTGF protein expression was detected by immunohistochemistry and western blot. CTGF mRNA expression in renal tissue was semi-quantitative analyzed by RT-PCR.3,NF-κBp65 protein expression in renal tissue was detected by immunohistochemistry. NF-κBp65 mRNA expression in renal tissue was semi-quantitative analyzed by RT-PCR.4,After cytotoxicity test, low concentration of NCTD which do no harm to HK-2 cell was to do later experiments. HK-2 cells were divided into 5 groups: control group (1640 group), albumin group (1640+albumin 5g/L), NCTD groups with different concentrations (1640+albumin 5g/L+NCTD0.5, 1, 2.5mg/L). Different concentration of NCTD and HK-2 cell induced by albumin were incubated together for 24 hours. Proliferation of HK-2 cell was detected by MTT. FN protein level in the culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA). FN mRNA expression in HK-2 cell was analyzed by RT-PCR.Results: 1,(1) There were no significant differences of blood routine and alanine aminotransferase (ALT) in all groups(P＞0.05). Compared with other groups, in BSA group globulin (Glo) increased, but creatinine clearance rate (Ccr) and ratio of albumin/globulin (A/G) decreased (P＜0.01 or 0.05). In NCTD group and MP group, biochemical indicators above all improved (P＜0.01 or 0.05). Compared with BSA group, CRP level significantly decresed in NCTD group and MP group (P＜0.01). Correlation analysis indicated that there was positive correlation between CRP level and proteinuria (r=0.65, P＜0.01). (2)At week 5, there were no significant differences of proteinuria between MP group and BSA group, while proteinuria in NCTD group was greatly lower than BSA group (P＜0.01). At week 9, compared with BSA group, proteinuria in NCTD group and MP group decreased greatly (P＜0.05). (3)By light microscope, in BSA group it was showed that mesangium region enlarged, and lots of inflammatory cell infiltration in renal interstitium with segmental interstitial fibrosis. Compared with Saline group, glomerular sclerosis(GS) index and tubulointerstitial lesion(TIL) score were significantly increased (P＜0.01). Immunofluorescence of IgG with (3+～4+) stained in mesangium region in BSA group. By electron microscope, obvious electron dense deposits existed and foot process fusion was seen extensively in BSA group. After intervention of NCTD, GS index and TIL score significantly decreaded (P＜0.05). While pathologic scores above in MP group were not significantly lower than BSA group (P＞0.05).2,(1) Compared with BSA group, protein expressions of CTGF both in NCTD group and MP group were down-regulated (P＜0.01 or 0.05) by immunohistochemistry test and western blot. (2) Compared with BSA group, CTGF mRNA expression in other two experimental groups were down-regulated (P＜0.01).3,(1)Compared with BSA group, deposited area and positive cell population of NF-κBp65 in NCTD and MP group all decreased (P＜0.01or 0.05). (2)NF-κBp65 mRNA expressions in NCTD group and MP group were down-regulated (P＜0.01). Moreover, NF-κBp65 mRNA expression in MP group was lower than in NCTD group (P＜0.01).4,(1)Cytotoxicity test showed that there were no significant differences of LDH among NCTD(0.5, 1.0, 2.5 mg/L)groups and control group (P＞0.05). (2)MTT score in albumin group (0.438±0.073) was higher than in 1640 group(0.330±0.060) (P＜0.05), while NCTD group (2.5mg/L) MTT score(0.327±0.080) decreased compared with albumin group (P＜0.05). (3)Compared with albumin group, FN protein concentration in NCTD group (2.Smg/L) was down-regulated (P＜0.01) and FN mRNA expressions in NCTD groups(lmg/L and 2.Smg/L) significantly decreased (P＜0.01). Conclusion: 1,NCTD with a certain concentration could treat nephropathy rats with overload proteinuria and ameliorate renal tubulointerstitial lesion without severe side-effects of liver, kidney and bone marrow.2,Compared with MP group, NCTD could effectively prevent renal tubulointerstitial fibrosis by significantly down-regulating expressions of CTGF and NF-κBp65.3,NCTD could improve the effect of cell proliferation and FN overexpression on HK-2 cell induced by albumin.
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