Abstract:

Objective：To learn the effect of NCTD on Calcineurin (CaN) /NFAT pathway and to explore whether the anti-fibrogenic effect of NCTD on renal interstitium in diabetic nephropathy is dependent of NCDT’s inhibition to CaN pathway. Methodology: HK-2 cells were cultured and transfected with CaN small interference RNA, then divided into 5 groups: (1) Normal glucose group (5.5mmol/L D-Glucose), (2) High glucose group (30 mmol/L D-Glucose), (3) High glucose +CaN siRNA groups: (4) High glucose +CaN siRNA+ NCTD (5ug/ml) group， (5) High glucose + NCTD(5ug/ml) group. The effect of NCTD on CaN /NFAT pathway in HK-2 cells and CaN siRNA transfection efficiency was examined by Western-blot, immunofluorescence and real-time PCR. The effects of NCTD on the protein expression of FN, ColⅣ and TGF-β1 in HK-2 cells transfected by CaNsiRNA  were observed by Western blot. Results: The expressions of CaN mRNA and protein in HK-2 cells were elevated when exposed to 30mmol/L glucose, and significant inhibition of the up-regulated expression of CaN was observed after treatment with NCTD（P<0.05）. NFATc, a downstream molecular of CaN, was predominantely visualized in cytoplasm of HK-2 cell in normal control group, whereas it was markedly decreased in the cytoplasm of high glucose group and was found mainly in nucleus, which peaked at 30min, suggesting the translocation of NFATc to the nucleus. NCTD treatment could inhibit the nuclear translocation of NFATc and reduce its protein level in the nucleus. Following CaN siRNA transfection, the mRNA and protein expression of CaN was significantly decreased, however, the protein levels of FN, ColIV and TGF-β were obviously increased（P<0.05）. NCTD treatment could down-regulated the increase of FN, ColIV and TGF-β protein expression in HK-2 cells stimulated by high glucose after transfecting CaN-SiRNA . Conclusions: NCTD could down-regulate CaN expression in HK-2 cells, and block CaN/NFAT signaling pathway. However, the effect of NCTD inhibiting the expression of extracellular matrixs in HK-2 cells induced by high glucose was not associated with its inhibition to CaN/NFAT pathway