基于PKC/PDLIM2/p65信号通路的Klotho抑制慢性环孢素A肾病的巨噬细胞侵润作用及机制

Invasion of macrophage mediated by activating NF-kBp65 is the main reason which reduces the life expectancy of a transplanted kidney in chronic CsA nephropathy. Recent studies have shown that the novel protein PDLIM2 promoted ubiquitination of p65, repressed its transcription, and inhibited the inflammation in a PKC dependent pathway. Anti-aging Protein klotho is mainly expressed in the kidney and secreted by renal cells。 It could suppress TNF-α-induced p65, but the roles of klotho in chronic CsA nephropathy are still unclear. We have reported that there was close relationship between klotho and renal interstitial fibrosis induced by oxidative stress. The preliminary study has indicated that klotho could inhibit macrophage accumulation, suppress renal fibrosis,ameliorate renal function. It suggests that klotho might regulate the invasion of macrophage through PKC/PDLIM2/p65 pathway, improve progression of CsA nephropathy. To investigate the roles of klotho, we will use chronic CsA nephropathy mouse model and macrophage (RAW264.7),clarify the protective effects of klotho on chronic CsA nephropathy,reveal the regulatory mechanism of klotho on PKC/PDLIM2/p65 pathway, which could mediate the infiltration of macrophage in chronic CsA nephropathy. We believe it will provide the theoretical basis for the prevention and treatment of CsA-induced renal toxicity.

慢性环孢素A肾病中NF-kBp65活化介导的巨噬细胞大量侵润是降低移植肾寿命的主要原因。已知PDLIM2依赖PKC活化泛素化p65，抑制其转录，降低炎症反应。抗衰老蛋白Klotho主要在肾脏中合成和分泌，通过降低p65活性抑制炎性介质的释放，降低炎症反应，但其在慢性CsA肾病中的作用机制尚未阐明。我们曾报道klotho与氧化应激诱导的肾纤维化密切相关，且预实验发现klotho显著抑制巨噬细胞聚集，减轻肾纤维化,改善肾功能，提示:klotho可能通过PKC/PDLIM2/p65信号通路调节巨噬细胞侵润，拮抗CsA肾病的发展。为此，本课题拟利用klotho过表达的腺病毒，在慢性CsA肾病小鼠模型和巨噬细胞RAW264.7中，阐明klotho改善慢性CsA肾病的作用，揭示klotho调节PKC/PDLIM2/p65信号通路，抑制巨噬细胞侵润的分子机制，为CsA肾毒性的防治提供理论依据和实验基础。

研究报道klotho具有改善氧化应激和降低细胞凋亡的以及维持Ca/P代谢等重要的生理调节作用。我们也曾报道慢性CsA肾病中，klotho的降低与氧化应激诱导的肾纤维化密切相关，且发现klotho显著抑制巨噬细胞的聚集，并减轻肾纤维化和改善肾功能。我们进一步利用转染klotho过表达的腺病毒，在慢性CsA肾病小鼠模型和巨噬细胞RAW264.7中，分析klotho改善慢性CsA肾病的分子机制，结果显示klotho通过调节PKC/PDLIM2/p65信号通路，降低肾脏炎性细胞因子的分泌和巨噬细胞侵润的分子机制。结果发现，过表达klotho具有降低巨噬细胞分泌iNOS,TNFa等促炎性细胞因子的作用,且巨噬细胞自身也表达klotho,其表达变化与NOD2蛋白负相关。但klotho如何调节巨噬细胞的极化，其调节机制是什么，还有待进一步研究。以上结果可能为慢性CsA肾毒性的防治及改善肾纤维化，提供一定的实验基础。