NAD⁺Supplementation Activates Renal Metabolism in the Proximal Tubules and Protects from Chronic Kidney Disease in a Model of Alport Syndrome

Background: Chronic kidney disease (CKD) is associated with renal metabolic disturbances, including impaired fatty acid oxidation (FAO). Nicotinamide adenine dinucleotide (NAD + ) is a small molecule that participates in hundreds of metabolism-related reactions. NAD + levels are decreased in CKD, and NAD + supplementation is protective. However, both the mechanism of how NAD + supplementation protects from CKD, as well as the cell types involved, are poorly understood. Drawing inspiration from investigations on NAD + supplementation in models of acute kidney injury, we hypothesized that NAD + supplementation protects from CKD by restoring renal metabolism in the proximal tubule. Methods: Control (Col4a3 +/- ) and Alport (Col4a3 -/- ) mice on the C57BL/6J background were treated with or without nicotinamide riboside (NR), an NAD + precursor. Both sexes were investigated at two timepoints: moderate disease (25-wk) and end-stage (35-wk). Kidney NAD + levels, plasma creatinine, and 24-hr urinary albumin excretion were quantified with commercial kits. Picrosirius red staining was performed to visualize renal fibrosis. Podocytes and immune cells were identified by immunohistochemistry for p57 kip2 and CD45, respectively. Kidney PGC-1α, CPT1α, and MCAD were quantified by immunoblotting. Bulk RNA-sequencing (RNA-seq) was performed on isolated kidney cortex. Single nuclei RNA-seq (snRNA-seq) and spatial transcriptomics were performed on transverse kidney sections. Results: NR-treatment prevented kidney disease in both sexes and at both timepoints. Plasma creatinine and urinary albumin excretion were increased in Alport mice, and NR-treatment ameliorated this in both sexes. On histology, Alport mice had increased renal fibrosis, increased renal inflammatory infiltrate, mesangial matrix expansion, glomerular hypertrophy, podocyte hypertrophy, and reduced volumetric podocyte density. NR-treatment prevented these pathological changes in both sexes. Bulk RNA-seq identified renal metabolic pathways, including FAO, were reduced in vehicle-treated Alport mice (vs. vehicle-treated control mice) and restored in NR-treated Alport mice (vs. vehicle-treated Alport mice). Importantly, metabolic pathways were increased in NR-treated control mice (vs. vehicle-treated control mice), suggesting that these changes are the direct effect of NR treatment. Transcription factor analyses identified PPARα as responsible for these changes. NR treatment increased protein expression of the PPARα coactivator, PGC-1α, in both control and Alport mice. Kidney protein expression of CPT1α and MCAD, key players in mitochondrial FAO, were reduced in Alport mice and restored by NR treatment. On snRNA-seq, NR treatment restored several vital cellular functions in the proximal tubule cells: translation (ribosome), metabolism, endocrine function, fatty acid degradation, and PPAR signaling. In the podocytes, NR treatment also restored PPAR signaling and fatty acid degradation, although fewer genes were differentially expressed, possibly due to the smaller sample size. Cpt1a and Acadm (MCAD) expression by snRNA-seq were increased in the proximal tubule S1 and S2 cells of Alport mice after NR treatment. On spatial transcriptomics, Cpt1a and Acadm (MCAD) expression were reduced in the pseudobulk, cortex, and outer medullary stripe in Alport mice, and NR treatment restored their expression. Conclusions: NAD + supplementation protects from CKD via the NAD + PGC-1α–PPARα–FAO axis, and the proximal tubule cells substantially contribute to this benefit. This work was supported by NIH award numbers F30DK129003 (BJ), TL1TR001431 (BJ), R01DK116567 (ML), R01DK127830 (ML), P50DK133943 (SJ), U01DK114923 (ME), and P30CA051008. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.