Abstract: SA-PO718
Inhibiting STAT3-Mediated Glutathione Metabolism Attenuates Parietal Epithelial Cell Activation in Proliferative Glomerulopathy
Session Information
- Glomerular Diseases: Therapeutic Strategies
October 26, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Authors
- Gowthaman, Yogesh, Stony Brook University, Stony Brook, New York, United States
- Kim, Joseph, Stony Brook University, Stony Brook, New York, United States
- Guo, Yiqing, Stony Brook University, Stony Brook, New York, United States
- Salant, David J., Boston University, Boston, Massachusetts, United States
- Mallipattu, Sandeep K., Stony Brook University, Stony Brook, New York, United States
Background
The activation of STAT3 is seen in podocytes and parietal epithelial cells (PECs) in mouse models of proliferative glomerulopathy and human RPGN and subtypes of FSGS. We reported that the loss of STAT3 in PECs reduced PEC activation, and eventual FSGS in nephrotoxic serum (NTS) treated mice. To elucidate the mechanims of STAT3-mediated PEC activation, we performed bulk RNA-sequencing in Stat3 knockout PECs and identified glutathione (GSH) metabolism as a significantly downregulated pathway. GSH is the major antioxidant in the body which functions to maintain cellular redox homeostasis, however, an overabundance of GSH may fuel pathologic PEC activation.
Methods
CRISPR/Cas9 generated PECs with deletion of Stat3. ChIP-qPCR was performed for GSH metabolism genes on Stat3 KO and control PECs after incubation with total STAT3 and phospho-STAT3 antibodies. GSH metabolism was targeted using an inhibitor of glutamate–cysteine ligase, L-buthionine sulfoximine (BSO). Control PECs were treated with increasing doses of BSO (62.5 - 500 μM), and MTT and mitostress seahorse assays were performed. BSO was given to mice injected with NTS, a mouse model of proliferative glomerulopathy. Osmotic pump released BSO at a dose of 10 mg/kg daily until the mice were collected on day 7, and proteinuria and PEC activation were evaluated.
Results
Stat3 KO PECs showed a reduction in GSH synthesis and peroxidase activity levels compared to control PECs. STAT3 occupancy in the promoter region of 8 genes involved in GSH metabolism (Gpx4, Gss, Gstz1, Gsta4, Gstm4, Gstm5, and Mgst2) was significantly reduced in Stat3 KO PECs as compared to control PECs. Increasing doses of BSO resulted in significantly lower cell viability via MTT assay, and in significantly lower OCR via seahorse assay. Compared to vehicle, BSO treatment reduced albuminuria and crescent formation 7 days post-NTS. Immunostaining and quantification for markers of activated PECs and quiescent PECs, CD44 and AKAP12, respectively, reveals that BSO treatment reduces CD44 expression, but increases AKAP12 expression, suggesting a reduction in pathogenic PEC activation post-NTS treatment.
Conclusion
These data suggest that inhibition of GSH metabolism might be a key target in attenuating PEC activation, proliferation, and crescent formation in proliferative glomerulopathy.
Funding
- NIDDK Support