The Effect And Mechanism Of Norcantharidin On Tubulointerstitial Fibrosis Of Diabetic Nephropathy

Objective Diabetic nephropathy (DN) is one of the common causes of chronic renal disease. Much more researches have focused on glomerular lesions, little emphasized the crucial role of tubulointerstitial injury in diabetic kidney in the past years. It has recently been demonstrated that the development of tubulointerstitial fibrosis is more closely correlated with a progressive decline in renal function compared to glomerularsclerosis and tubulointerstitial injury serves as an important mediator of chronic renal failure and predictor of outcome in patients with diabetic nephropathy. However, there is no effective drugs to reverse the tubulointerstitial injury and fibrosis.Norcantharidin (NCTD),a demethylated analog of cantharidin, possesses potential anticancer activity. NCTD not only decreased the targeted gene expression involved in tumor metastasis such as VEGF,FAK and TGF-β1 but also maintained the balance of matrix metalloproteinase 2 and tissue inhibitor metalloproteinase. Our studies previously found that NCTD inhibited the tubulointerstitial lesions which attenuated the development of renal interstitial fibrosis by inhibiting the expressions of NF-κB and CTGF in protein overload nephropathy. NCTD inhibited the expression of fibronectin in tubular epithelial cells induced by albumin. NCTD could attenuate the tubulointerstitial fibrosis and inhibit tubular epithelial-mesenchymal transition in rat model of obstructive nephropathy as well as inhibiting EMT of tubular cells by down-regulating TGF-β1 in vitro. Since it inhibited tubulointerstitial fibrosis in several models, we wondered if NCTD could have a similar effect in DN tubulointerstitial fibrosis.Calcineurin expression is observed at glomeruli as well as renal tubular epithelial cells and it is up-regulated in DN. It is reported calcineurin iinhibitor can attenuate the glomerular fibrosis, but there is no report about if calcineurin plays a role in tubulointerstitial injury and fibrosis. NCTD is a calcineurin inhibitor and we wonder if NCTD could attenuate the tubulointerstitial fibrosis by down-regulating calcineurin pathway. To investigate the effect of norcantharidin in tubulointersitial firosis in DN and the possible and mechanism, the expression of FN(fibronectin),collagenⅣand TGF-β1 were analysed in STZ-induced diabetic model and in human renal proximal tubular epithelial cell line treated with high glucose and norcantharidin. The expression of calcineurin is also observed in the model. Furthermore, the role of calcineurin was investigated as a potent target of NCTD by small interfering RNA. It is aimed at providing experimentals evidence to develop NCTD as a potential treatment in DN fibrosis.Methods:1Sprague-Dawley rats were randomly divided into control(n=8), DN(n=9), low dose NCTD (0.05mg/kg/d) groups(n=12), and high dose NCTD(0.1mg/kg/d) groups(n=11).The DN model was induced by injection intra-peritoneally with 30 mg/kg body weight STZ in 0.1mol/L sodium citrate solution(pH 4.5),after high calorie given for two months. The rats in the control group were injected with 0.1mol/L sodium citrate solution. Diabetic model was considered to be successful when the blood glucose was≥16.7mmol/L and the urine glucose was＋＋＋～＋＋＋＋after 72 hours of the injection. NCTD was administered daily after the model was built. Rats were sacrificed at day 21and day 56 after and the kidneys were harvested and subjected to the studies. Renal lesions and the expression of FN,collagenⅣ, TGF-β1 and CaN were detected by HE, Masson and immunohistochemistry respectively. 2 Human kidney proximal tubular epithelial cell line(HK-2)cells were incubated with serum-free DMEM for 24 hours to synchronize the cell growth, then the cells were divided into 4 groups:NG group(media containing 5.5mM glucose);mannitol control group(media containing 5.5mM glucose and 24.5mM mannitol);HG group(media containing 30mM glucose);NCTD group(treated with NCTD at varied dose and 30mM glucose). Trypan blue dye exclusive assay and MTT assay were carried out to determin the effective and safe dose of NCTD in high glucose. The cells were collected to extracte total RNA and protein at 6,24 and 48 hours after incubation.The expression of FN,collagenⅣ, TGF-β1 and CaN were examined by realtime-PCR and Western-blot. NFATc(an important downstream molecular of CaN) nuclear translocation was examined by immunofluorescence. 3 Small interfering RNA(siRNA)targeting human calcineurin gene sequences were synthesized to silence calcineurin gene expression. Transient transfection was carried out using Lipofectamine 2000 according to the manufacturer's instruction. Fluorescent microscopy was used to examine GFP expression.. HK-2 cells were divided into 5 groups:NG group, HG group, HG+ CaN-siRNA group, HG+CaN-siRNA+NCTD group and HG+NCTD group. The CaN interference efficiency is deteced by realtime-PCR and Western-blot. FN, collagenIV and TGF-β1 expression was analyzed by Western-blot after the cells were treated for 24h.Results:1 Extracellular matrix deposition and tubulointerstitial fibrosis were observed in DN rats, while the pathological changes were reduced in that of NCTD-treated group(P<0.05). The expressions of FN, collagenⅣand TGF-β1 were increased in the tubulointerstitial field of DN rats compared with the control. NCTD treatment can reverse the fibrosis to a certein degree(P<0.05).2 Expression of CaN was detected in normal kidney tubular fields and the expression increased in the kidney tubulointerstitial field in DN rats. NCTD treatment down-reguated its expression in a dose dependent manner(P<0.05).3 The mRNA and protein expression of FN, collagenⅣincreased in HK-2 cells treated with HG in a time-depedent manner, while that in cells treated by 5μg/mlNCTD was dramatically inhibited(P<0.05). The expression of TGF-β1 in HK-2 treated by HG was also inhibit by NCTD in a time-dependent manner.4 The mRNA and protein expression of CaN increased in HK-2 cells treated with HG and NCTD inhibited its expression in a dose-dependent manner.5μg/ml NCTD had an impressive inhibiting effect(P<0.01). NCTD can also inhibit NFATc nuclear translocation induced by 30mM glucose in HK-2 cells5 After CaN-siRNA transfection, green fluorescent protein(GFP)was observed in about 70% HK-2 cells, showing that the transfection is successful. Compared with control group, the CaN mRNA expression was reduced to 48±12% in CaN-siRNA groups (P<0.05).Correspondingly, protein expression of CaN was decreased to 62±15%(P<0.05).Western blot showed that CaN-siRNA increase the expression of FN,collagenⅣand TGF-β1 induced by HG,whereas NCTD combined with CaN siRNA can lower the level of these proteins(p<0.05).Conclusions:1 NCTD can down-regulate FN, collagen IV and TGF-β1 expression in tubulointerstitial fields and may have an positive effect in attenuating tubulointerstitial fibrogenesis of in the early stage of DN rats. NCTD could significantly decreased the mRNA and protein expression of FN, collagenⅣand TGF-β1 in HK-2 stimulated by 30mM glucose.2 NCTD can down-regulate CaN/NFATc pathway activated by high glucose in the proximal tubular epithelial cells.3. The effect of NCTD on tubulointerstitial fibrosis of DN is not by inhibiting CaN.