HSPA8 dampens SCAP/INSIG split and SREBP activation by reducing PKR-mediated INSIG phosphorylation. HSPA8 dampens SCAP/INSIG split and SREBP activation by reducing PKR-mediated INSIG phosphorylation

Lipid accumulation in the renal tubules is a major determinant of diabetic kidney disease (DKD), and activation of SREBPs plays a central role in this process. Our study aimed to explore whether HSPA8, a molecular chaperone, is the master regulator of INSIG/SREBPs function in DKD. Here, we showed that tubular epithelial cell (TEC)-specific knockout of HSPA8 upregulated the phosphorylation of INSIG1 and INSIG2, which disrupted the interaction between INSIG proteins and SCAP, leading to the translocation of the SCAP-SREBP complex to the Golgi apparatus, and the activation of SREBPs. TEC-specific overexpression of HSPA8 restrained these changes. INSIG1 and INSIG2 can be phosphorylated by protein kinase R (PKR), while HSPA8 can recognize PKR and recruit the E3 ubiquitin ligase (CHIP) to promote PKR ubiquitination and degradation. SREBP1 transcriptionally activated HSPA8 expression under temporary hyperglycemic stimulation. However, persistent hyperglycemia reduced HSPA8 levels by promoting nuclear factor (NF)-κB-mediated transcriptional inhibition and reducing USP43-mediated deubiquitination of HSPA8. Collectively, these findings indicate that the molecular chaperone HSPA8 serves as a negative feedback regulator of SREBPs, lipogenesis, and DKD development. Overall design: 1. First, establish diabetic kidney disease (DKD) models in rodents and cell lines. For in vivo, use streptozotocin (STZ) injection. For in vitro, expose tubular epithelial cells (TECs) to high glucose. 2. Then, in TECs, knockdown HSPA8 using siRNA and overexpress it using plasmids. 3. Measure the phosphorylation levels of INSIG1 and INSIG2, the interaction between INSIG and SCAP, and the activation of SREBPs. 4. Examine the ubiquitination of PKR and HSPA8. 5. Analyze the transcriptional regulation of HSPA8 by SREBP1 and the effect of NF - κB and USP43 on HSPA8 levels. 6. Finally, assess lipid accumulation in TECs. Through these steps, we can clarify the role of HSPA8 as a negative feedback regulator in DKD.

肾小管脂质蓄积是糖尿病肾病（Diabetic Kidney Disease, DKD）的核心致病特征之一，而固醇调节元件结合蛋白（Sterol Regulatory Element-Binding Proteins, SREBPs）的激活在该进程中发挥中枢调控作用。本研究旨在探究分子伴侣热休克蛋白71 kDa蛋白8（Heat Shock Protein 71 kDa Protein 8, HSPA8）是否为DKD中INSIG/SREBPs功能的核心调控因子。本研究结果显示，肾小管上皮细胞（Tubular Epithelial Cell, TEC）特异性敲除HSPA8可上调INSIG1与INSIG2的磷酸化水平，破坏INSIG蛋白与SCAP（SREBP裂解激活蛋白，SREBP Cleavage-Activating Protein）的相互作用，进而促使SCAP-SREBP复合物转位至高尔基体，最终激活SREBPs；而TEC特异性过表达HSPA8则可显著抑制上述病理变化。机制研究表明，INSIG1与INSIG2可被蛋白激酶R（Protein Kinase R, PKR）磷酸化，而HSPA8可识别PKR并招募E3泛素连接酶CHIP（羧基末端HSP70相互作用蛋白，Carboxyl-Terminal Hsp70-interacting Protein）以促进PKR的泛素化降解。此外，在暂时性高糖刺激下，SREBP1（固醇调节元件结合蛋白1，Sterol Regulatory Element Binding Protein 1）可转录激活HSPA8的表达；但持续性高糖则通过促进核因子κB（Nuclear Factor-κB, NF-κB）介导的转录抑制，以及降低USP43（泛素特异性蛋白酶43，Ubiquitin-Specific Protease 43）介导的HSPA8去泛素化水平，下调HSPA8的表达量。综上，本研究证实分子伴侣HSPA8可作为SREBPs、脂质生成及DKD进展的负反馈调控因子。整体实验设计如下：1. 首先在啮齿类动物与细胞系中构建糖尿病肾病（DKD）模型：体内实验采用链脲佐菌素（Streptozotocin, STZ）注射造模；体外实验则将肾小管上皮细胞（TECs）置于高糖环境中培养。2. 随后在TECs中，通过小干扰RNA（small interfering RNA, siRNA）敲低HSPA8的表达，并通过质粒载体实现HSPA8的过表达。3. 检测INSIG1与INSIG2的磷酸化水平、INSIG与SCAP的相互作用，以及SREBPs的激活状态。4. 分析PKR与HSPA8的泛素化修饰情况。5. 探究SREBP1对HSPA8的转录调控作用，以及NF-κB与USP43对HSPA8表达水平的影响。6. 最后评估TECs中的脂质蓄积情况。通过上述实验步骤，本研究明确了HSPA8作为负反馈调控因子在DKD中的作用机制。