MKP-2调控肾小管上皮细胞JNK信号通路在吡非尼酮抗间质纤维化肾脏保护中的作用及机制研究

Renal interstitial fibrosis (RIF) in obstructive nephropathy has a large contribution to the deterioration of renal function. Therefore, how to alleviate renal interstitial fibrosis is very important to inhibit the progressive renal injury, even to promote the recovery of renal function after relief of urinary tract obstruction. The mechanical stretching of tubules dilated by downstream obstruction to urine flow leads to the activation of c-Jun N-terminal kinase (JNK) in the tubular epithelial cells. Phosphorylated-JNK results in expression of transforming growth factor-β1 (TGF-β1) and its downstream signal, which leads to the formation of renal fibrosis. Mitogen-activated protein kinase phosphatase 2 (MKP-2) is able to deactivate JNK specifically by dephosphorylation of JNK. However, the role of MKP-2 in renal interstitial fibrosis has not been reported. Moreover, recent investigation on the renoprotective effects of pirfenidone suggests that it inhibits TGF-β1 induced fibrosis cascade and likely exerts antifibrotic effects in rat proximal tubular epithelial cells. However the concrete mechanism is not clear. In the previous experiment we have demonstrated MKP-2 express in renal tubular epithelial cells for the first time. Moreover, our results have shown that pirfenidone could induce the expression of MKP-2. So we put forward the following hypothesis: pirfenidone could alleviate renal fibrosis by up-regulated MKP-2 inhibiting JNK phosphorylation and secondary reduction of TGF-β1 in tubular epithelial cells, which may be a new mechanism to treat RIF. In this proposal we will perform studies using tubular epithelial cells treated by pirfenidone and siMKP-2 transfection, as well as unliateral ureteric obstruction (UUO) rat model treated by pirfenidone to verify our hypotheses. Our findings will reveal the new therapeutic mechanism of pirfenidone on renal interstitial fibrosis and provide new insights into the prevention and treatment of renal interstitial fibrosis and new pharmacological targets.

梗阻性肾病中肾间质纤维化（RIF）程度与肾功能减退密切相关，延缓其进程是抑制肾功能恶化及促进术后肾功能恢复的关键。梗阻所致肾小管扩张并激活肾小管上皮细胞JNK致TGF-β1上调是RIF发生的重要环节。双特异性磷酸酶MKP-2可特异性的将JNK去磷酸化而失活，但其在RIF中的作用，迄今国内外尚无报道。研究显示吡非尼酮具有抗RIF作用，但具体机制不详。前期预实验中，我们首次发现肾小管上皮细胞有MKP-2的表达，且吡非尼酮干预可使MKP-2的表达增加。我们推测吡非尼酮通过上调MKP-2表达来抑制肾小管上皮细胞JNK磷酸化从而抑制TGF-β1及其下游信号，最终减缓RIF，这可能是抗RIF的一个新机制。我们拟在肾小管上皮细胞和输尿管梗阻大鼠模型上通过吡非尼酮干预、siMKP-2转染等分子生物学方法，探索证明前述假说。本课题将从新的视角揭示吡非尼酮的作用机制，为RIF的防治提供新思路和药物作用新靶点。

梗阻性肾病中肾间质纤维化（RIF）程度与肾功能减退密切相关，延缓其进程是抑制肾功能恶化及促进术后肾功能恢复的关键。细胞外基质过度沉积和肾间质肌成纤维细胞增多是RIF形成的两个最重要的环节，MyoFb是一种介于平滑肌细胞和成纤维细胞之间的过渡型细胞，比普通的成纤维细胞具有更强的增殖和分泌胶原的能力，是RIF时细胞外基质的主要来源，而肾小管上皮细胞通过上皮-间质转分化（EMT）形成MyoFb是病理条件下肾间质中MyoFb十分重要的来源之一。研究显示吡非尼酮（PFD）有抗纤维化的作用，但具体机制不详。双特异性磷酸酶MKP-2参与TGF-beta诱导的EMT的过程，但目前大部分的研究都集中在MKP-2与肿瘤上皮组织发生EMT有关，结果均提示其可能在EMT过程中发挥着一定的作用。此外作为MAPK信号通路的关键负性调控酶MKP-2在不同细胞系中会选择性的表达抑制。目前关于MKP-2在病理条件下肾小管上皮细胞发生EMT中的作用，迄今国内外尚无报道。在本课题中，我们首先利用肾小管上皮细胞和输尿管梗阻大鼠模型观察不同浓度的PFD干预对肾小管上皮细胞间质转分化，继而对肾纤维化的影响及其机制进行探讨。该部分研究结果显示PFD通过拮抗MAPK通路进而抑制TGF-β1及其下游信号，最终在一定程度上抑制肾小管上皮细胞间质转分化过程，并减缓RIF，提示这可能是PFD抗肾纤维化的一个新机制。之后我们进一步利用上述体内、体外模型，结合siMKP-2和MKP-2过表达质粒载体来观察作为MAPK信号通路的关键负性调控酶MKP-2对肾小管上皮细胞转分化的影响及其机制。该部分结果显示MKP-2过表达可以选择性地通过抑制JNK信号通路抑制TGF-β1诱导的肾小管上皮细胞间质转分化，进而抑制肾纤维化进程。本课题研究从新的视角揭示吡非尼酮以及MKP-2在肾小管上皮细胞出现间质转分化过程中的作用及其机制，为RIF的防治提供新思路和药物作用新靶点。