Study on the mechanism of cyclosporine A nephrotoxicity : Application of microarray and microdissection

本研究旨在阐明环孢素慢性肾毒性的机制。我们利用基因芯片(ATRAS大鼠1.2阵列，ATRAS 1.2 Array2，Clontech)检测了另一种钙调神经磷酸酶抑制剂他克莫司对慢性肾毒性大鼠肾脏2,352个基因的表达。我们发现慢性肾毒性大鼠肾脏中有数百个基因表达增强。它们包括E-选择素、VCAM-1、Fas抗原配体、Fas抗原、IL-6、G-CSF和u-PA，它们都受转录因子核因子-kB的调节。病变肾脏核蛋白的凝胶迁移率改变分析表明，无论是环孢素还是他克莫司处理的肾脏，其核转录因子-kB和AP-1的DNA结合活性均显著增强。当大鼠同时喂食高镁饲料或假定的NF-kB抑制剂PDTC时，这些变化几乎消失。慢性肾毒性所见的肾间质纤维化也可通过这些治疗而减轻。这些结果表明，核因子-kB在钙调神经磷酸酶抑制剂慢性肾毒性的发病机制中起重要作用。此外，补充镁可能通过抑制核因子-kB的激活来减轻慢性肾毒性。为了进一步证明核因子-kB在肾间质纤维化发病机制中的作用，我们采用了梗阻性肾病大鼠肾纤维化模型。PDTC或通过不同机制抑制NF-kB活化的蛋白酶体抑制剂均可显着减轻肾纤维化。此外，口服吸附剂AST-120显著减轻了残留肾组织中核因子-kB的激活和肾纤维化的发展。因此，我们的研究表明，核因子-kB的激活是治疗肾纤维化的可能靶点之一，而肾纤维化是进展性肾脏疾病的主要决定因素。

The present study was conducted to elucidate the mechanism of chronic cyclosporine nephrotoxicity. We utilized cDNA microarray (Atras Rat 1.2 array, Atras 1.2 Array2, Clontech) and examined expression of 2,352 genes from rat kidneys suffering chronic nephrotoxicity with another calcineurin inhibitor, tacrolimus. We found enhanced gene expression of hundreds of gene in the kidney of chronic nephrotoxicity. Those included E-selectin, VCAM-1, fas antigen ligand, fas antigen, IL-6,G-CSF and u-PA which were known to be regulated by a transcription factor, nuclear factor kappaB (NF-kB). Electrophoretic mobility shift assay of nuclear protein from diseased kidney showed that DNA binding activity of not only NF-kB but also AP-1 were markedly enhanced in the kidney treated with either cyclosporine or tacrolimus. These changes were almost abolished when rats were simultaneously maintained on high magnesium diet or putative NF-kB inhibitor, PDTC. Renal interstitial fibrosis seen in chronic nephrotoxicity was also attenuated by these treatments.These results suggested that NF-kB play an important role in the pathogenesis of chronic nephrotoxicity of calcineurin inhibitors. Furthermore, Mg supplementation attenuated chronic nephrotoxocity possibly via inhibition of NF-kB activation. To further demonstrate the role of NF-kB in the pathogenesis of renal ineterstitial fibrosis, we utilized rat model of renal fibrosis, obstructive nephropathy. Either PDTC or a proteasome inhibitor that inhibits NF-kB activation through different mode of mechanism significantly attenuated renal fibrosis. Further, oral adsorbent, AST-120 markedly attenuated NF-kB activation and development of renal fibrosis observed in remnant kidney. Thus, our study suggested that NF-kB activation is one of possible targets in treating renal fibrosis that is a main determinant of progressive renal disease.