FADD phosphorylation contributes to development of renal fibrosis by accelerating epithelial-mesenchymal transition

FADD, a classical apoptotic signaling adaptor, has recently been reported to exhibit a series of non-apoptotic functions. Here, we report that FADD may play a critical role in the development of renal fibrosis. Neutrophil infiltration in the renal interstitial part, glomerular mesangial cell proliferation, and base-membrane thickening were observed in FADD-D mice by H&amp;E, PAS, and PASM staining. Immunofluorescence analysis revealed that macrophage infiltration was significantly enhanced in FADD-D mice. Renal fibrosis might be induced by IgA nephritis in FADD-D mice as evidenced by increased Ki67 and type IV collagen. Additionally, the levels of α-SMA, Fibronectin, and Vimentin were also found to be elevated. Mechanism study indicated that the TLR4/myD88/NF-κB signaling pathway was activated in FADD-D mice. Moreover, FADD phosphorylation activated the mTOR and TGF-β/Smad pathway and accelerated the process of epithelial mesenchymal transition. Further studies indicated that the TGF-β1 pathway was also activated and the process of EMT was accelerated in both FADD-disrupted HEK293 cells and FADD-deficient MES cells. Thus, we concluded that FADD phosphorylation could lead to IgA nephritis and eventually result in renal fibrosis. Taken together, our study provides evidence, for the first time, that FADD, especially in its phosphorylated form, has an effect on the development of renal fibrosis. <b>Abbreviations:</b> FADD: FAS-associated protein with death domain; DED: death effector domain; DD: death domain; CKD: chronic kidney disease; ECM: extracellular matrix; ESRD: end-stage renal disease; RRT: renal replacement therapy; H&amp;E: hematoxylin and eosin; PASM: periodic acid silver methenamine

FAS死亡结构域相关蛋白（FADD）作为经典的凋亡信号衔接蛋白，近期被报道具备一系列非凋亡相关功能。本研究发现，FADD可能在肾纤维化的发生发展中发挥关键作用。通过苏木精-伊红（H&E）、过碘酸-雪夫（PAS）及过碘酸六胺银（PASM）染色检测，可见FADD敲除（FADD-D）小鼠肾间质存在中性粒细胞浸润、肾小球系膜细胞增殖及基底膜增厚。免疫荧光分析结果显示，FADD-D小鼠的巨噬细胞浸润程度显著升高。FADD-D小鼠体内Ki67抗原与Ⅳ型胶原表达上调，提示IgA肾病可能诱发其肾纤维化。此外，α平滑肌肌动蛋白（α-SMA）、纤连蛋白（Fibronectin）及波形蛋白（Vimentin）的表达水平同样显著升高。机制探究显示，FADD-D小鼠体内Toll样受体4（TLR4）/髓系分化因子88（MyD88）/核因子κB（NF-κB）信号通路被激活。此外，FADD磷酸化可激活哺乳动物雷帕霉素靶蛋白（mTOR）与转化生长因子β（TGF-β）/Smad信号通路，并加快上皮间质转化（EMT）进程。进一步研究表明，在FADD敲低的HEK293细胞与FADD缺陷的MES细胞中，转化生长因子β1（TGF-β1）通路同样被激活，且EMT进程得以加快。综上，本研究得出结论：FADD磷酸化可诱发IgA肾病，最终引发肾纤维化。综上所述，本研究首次提供证据证实，FADD——尤其是其磷酸化形式——可参与调控肾纤维化的发生发展。**缩写说明：** FADD：FAS死亡结构域相关蛋白；DED：死亡效应结构域；DD：死亡结构域；CKD：慢性肾脏病；ECM：细胞外基质；ESRD：终末期肾病；RRT：肾脏替代治疗；H&E：苏木精-伊红染色；PASM：过碘酸六胺银染色