Wang Dapeng, Fang Ming, Shen Nan, Li Longkai, Wang Weidong, Wang Lingyu, Lin Hongli
Department of Nephrology, The First Affiliated Hospital, Institute for Nephrology Research of Dalian Medical University, Center for Kidney Diseases Translational Medicine of Liaoning ProvinceDalian, China.
Postgraduate School of Dalian Medical UniversityDalian, China.
Am J Transl Res. 2017 Oct 15;9(10):4415-4427. eCollection 2017.
Albuminuria is an independent risk factor for renal interstitial fibrosis (RIF). Glomerular-filtered albumin in endocytic and non-endocytic pathways may injure proximal tubular epithelial cells (PTECs) via megalin and TGFβRII, respectively. Since megalin and TGFβRII are both modified by post-translational core fucosylation, which plays a critical role in RIF. Thus, we sought to identify whether core fucosylation is a potential target for reducing albumin-induced injury to PTECs. We constructed a human PTEC-derived cell line (HK-2 cells) and established an in vitro model of bovine serum albumin (BSA) injury. RNAi was used to inhibit the expression of megalin, TGFβRII, and Fut8. Western blotting, immunostaining, ELISA, lectin blotting, and fluorescence-activated cell sorting were used to identify BSA-induced endocytic and non-endocytic damage in HK-2 cells. Fut8 is a core fucosylation-related gene, which is significantly increased in HK-2 cells following an incubation with BSA. Fut8 siRNA significantly reduced the core fucosylation of megalin and TGFβRII and also inhibited the activation of the TGFβ/TGFβRII/Smad2/3 signaling pathway. Furthermore, Fut8 siRNA could reduce monocyte chemotactic protein-1, reactive oxygen species, and apoptosis, as well as significantly decrease the fibronectin and collagen I levels in BSA-overloaded HK-2 cells. Core fucosylation inhibition was more effective than inhibiting either megalin or TGFβRII for the prevention of albumin-induced injury to PTECs. Our findings indicate that post-translational core fucosylation is essential for the albumin-induced injury to PTECs. Thus, the inhibition of core fucosylation could effectively alleviate albumin-induced endocytic and non-endocytic injury to PTECs. Our study provides a potential therapeutic target for albuminuria-induced injury.
蛋白尿是肾间质纤维化（RIF）的独立危险因素。内吞和非内吞途径中肾小球滤过的白蛋白可能分别通过巨膜蛋白和转化生长因子β受体II（TGFβRII）损伤近端肾小管上皮细胞（PTECs）。由于巨膜蛋白和TGFβRII都通过翻译后核心岩藻糖基化修饰，而这种修饰在RIF中起关键作用。因此，我们试图确定核心岩藻糖基化是否是减少白蛋白诱导的PTECs损伤的潜在靶点。我们构建了一种人PTECs来源的细胞系（HK - 2细胞），并建立了牛血清白蛋白（BSA）损伤的体外模型。RNA干扰用于抑制巨膜蛋白、TGFβRII和岩藻糖基转移酶8（Fut8）的表达。采用蛋白质免疫印迹法、免疫染色、酶联免疫吸附测定、凝集素印迹法和荧光激活细胞分选法来鉴定HK - 2细胞中BSA诱导的内吞和非内吞损伤。Fut8是一个与核心岩藻糖基化相关的基因，在HK - 2细胞与BSA孵育后显著增加。Fut8小干扰RNA（siRNA）显著降低了巨膜蛋白和TGFβRII的核心岩藻糖基化，还抑制了TGFβ/TGFβRII/Smad2/3信号通路的激活。此外，Fut8 siRNA可以降低单核细胞趋化蛋白 - 1、活性氧和细胞凋亡，以及显著降低BSA过载的HK - 2细胞中纤连蛋白和I型胶原的水平。对于预防白蛋白诱导的PTECs损伤，抑制核心岩藻糖基化比抑制巨膜蛋白或TGFβRII更有效。我们的研究结果表明，翻译后核心岩藻糖基化对于白蛋白诱导的PTECs损伤至关重要。因此，抑制核心岩藻糖基化可以有效减轻白蛋白诱导的PTECs内吞和非内吞损伤。我们的研究为蛋白尿诱导的损伤提供了一个潜在的治疗靶点。
