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)-?B-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 - ?B 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.