Abstract: SA-PO0216
Epitranscriptomic Signature of Short-Term High Glucose in Renal Tubular Epithelial Cells
Session Information
- CKD: Mechanisms of Injury and Fibrosis - 3
October 24, 2026 | Location: Exhibit Hall A, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 2203 CKD (Non-Dialysis): Mechanisms
Authors
- Adams, Lauren T., University of Louisville School of Medicine, Louisville, Kentucky, United States
- Hart, Brian R., University of Louisville School of Medicine, Louisville, Kentucky, United States
- Li, Ming, University of Louisville School of Medicine, Louisville, Kentucky, United States
- Barati, Michelle T., University of Louisville School of Medicine, Louisville, Kentucky, United States
- Merchant, Michael, University of Louisville School of Medicine, Louisville, Kentucky, United States
Background
Diabetic nephropathy (DN) is a diabetic microvascular complication associated with hyperglycemia and glucose (glc)induced damage within renal tubular epithelial cells. N6-methyl-adenosine (m6A) is an epitranscriptomic modification catalyzed by METTL3 and METTL14 to methylate position six of adenine. m6A modified transcripts regulate oxidative stress, inflammation, and fibrosis. We tested the hypothesis that the spatial localization and relative abundance of METTL3, METTL14 and m6A varies in human renal tubular epithelial (HK11) cells exposed to hyperglycemic vs. normoglycemic conditions.
Methods
HK11 cells were plated onto coverslips in normal media + 5% FBS and then serum starved. The cells were exposed to: normal glc (NG, 5 mM glc), or high glc (HG, 25 mM glc), or normal glc + mannitol (Mann, 5 mM glc+20 mM mannitol). After 6h, 1° antibodies for METTL3 (n=12), METTL14 (n=9-12) and m6A (n=12) were added. DAPI added to stain nuclei. Confocal microscopy imaging of cells was used to quantify the total, nuclear, and cytoplasmic abundance using ImageJ (https://imagej.net/ij/). Differences in abundance of means between treatments and spatial localization was determined using one-way ANOVA and post-hoc t-test (p<0.05 considered significant).
Results
Total IF for METTL3, METTL14, or m6A did not differ significantly at 6h. All targets were significantly enriched in the nuclei compared to cytoplasm. Nuclear to cytoplasmic IF integrated densities ratios showed nuclear METTL3 fraction significantly increased with HG; nuclear METTL14 significantly increased with Mann, and nuclear m6A trended a significant reduction with Mann.
Conclusion
Early reports indicated chronic HG increases m6A or METTL3, we observed short term (6h) HG, bulk changes in m6A have not peaked. We do observe an association of HG with increased nuclear METTL3 and increased osmolality with nuclear METTL14. METTL14 has been shown to bind H3K27me3 independent of METTL3 to recruit lysine demethylase, KDM6B, promoting gene expression. Our collective data suggest in the tubular epithelium osmotic stress exposure may contribute to Mettl14-dependent regulation of gene expression outside of m6A generation. Ongoing studies will use m6A erasers (FTO) IB and m6A RNAseq to functionally dissect the osmotic versus metabolic impact of acute hyperglycemia on HK11 cells.
Acknowledgment
We aknowledge support from the University of Louisville Division of Nephrology & Hypertension and grant support through: P20GM113226, P30ES030283, P50AA024337, and DK130294.
Funding
- NIDDK Support