414-P: A Novel Mutation in HNF1B Promotes Ferroptosis-Mediated Renal Mesangial Cells Fibrosis

Objective: In this study, we identified a novel HNF1B gene mutation (c.445C>A) in a young male MODY5 patient with and elevated serum creatinine levels and albuminuria. Method: We constructed the HNF1B wild type (HNF1B-WT) and the novel missense mutation (c.445C>A) Mutant (HNF1B-Mut) plasmid and no-loaded control plasmid (pCDH) were transfected into mouse mesangial cells (MMCs) and identified successfully by real-time fluorescent quantitative PCR (RT-qPCR). The changes of cell proliferation activity were detected by CCK-8, the changes of intracellular reactive oxygen species (ROS) were detected by DCFH-DA probe, and the expressions of fibronectin (FN) and iron death index 4-HNE were detected by immunofluorescence staining. The expression levels of long chain fatty acyl-CoA synthetase 4 (ACSL4) and GPX4, key regulatory proteins of iron death, were detected by RT-qPCR. Results: RT-qPCR confirmed that compared with the control group, the expression levels of CycD1, FN, COLI and other indicators of proliferation and fibrosis decreased in the HNF1B-WT group, but increased in the HNF1B-Mut group. Immunofluorescence co-staining indicated that FN and 4-HNE decreased in HNF-WT cells and increased in HNF1B-Mut cells. CCK-8 assay confirmed that HNF1B-Mut plasmid restored the proliferation activity of MMCs inhibited by HNF-1B. After loading with DCFH-DA probe, it was found that HNF1B-Mut group had higher ROS level than HNF1B-WT group, suggesting that HNF1B-Mut promoted cellular oxidative stress. RT-qPCR showed that the expression of ACSL4 was increased and GPX4 was decreased in HNF1B-Mut group compared with wild control group. Conclusion: HNF1B c.445C>A mutation can promote MMCs proliferation and fibrosis by improving intracellular oxidative stress and iron death sensitivity. These abnormal changes may be related to the abnormal renal function impairment of the clinical progenitor.