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cDNA微阵列（Atras Rat 1.2 array，Atras 1.2 Array 2，Clontech），检测了来自另一种钙调磷酸酶抑制剂他克莫司慢性肾毒性大鼠肾脏的2，352个基因的表达。我们发现慢性肾毒性肾脏中有数百个基因表达增强。这些细胞因子包括E-选择素、VCAM-1、Fas抗原配体、Fas抗原、IL-6、G-CSF和u-PA，已知它们受转录因子NF-κ B（nuclear factor kappaB，NF-κ B）调节。用电泳迁移率变动分析法检测了病肾组织核蛋白的DNA结合活性，结果表明，无论是用环孢素还是他克莫司处理的病肾组织，NF-κ B和AP-1的DNA结合活性均明显增强。当大鼠同时维持高镁饮食或假定的NF-κ B抑制剂PDTC时，这些变化几乎被消除。这些结果表明NF-κ B在钙调磷酸酶抑制剂慢性肾毒性的发病机制中起重要作用。此外，镁补充可能通过抑制NF-κ B活化来减轻慢性肾毒性。为了进一步证明NF-κ B在肾间质纤维化发病机制中的作用，我们利用大鼠肾纤维化模型，梗阻性肾病。PDTC或蛋白酶体抑制剂通过不同的机制抑制NF-κ B的活化，显著减轻肾纤维化。此外，口服吸附剂AST-120显著减弱了在残余肾中观察到的NF-kB活化和肾纤维化的发展。因此，我们的研究表明，NF-κ B活化是治疗肾纤维化的可能靶点之一，肾纤维化是进行性肾病的主要决定因素。

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.