Research ArticleNephrology Open Access | 10.1172/jci.insight.181443

Bryce A. Jones,1 Debora L. Gisch,2 Komuraiah Myakala,3 Amber Sadiq,3 Ying-Hua Cheng,2 Elizaveta Taranenko,4,5 Julia Panov,5 Kyle Korolowicz,6 Ricardo Melo Ferreira,2 Xiaoping Yang,7 Briana A. Santo,7 Katherine C. Allen,3 Teruhiko Yoshida,8 Xiaoxin X. Wang,3 Avi Z. Rosenberg,7 Sanjay Jain,9 Michael T. Eadon,2 and Moshe Levi3,10

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Jones, B. in: JCI | PubMed | Google Scholar |

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Gisch, D. in: JCI | PubMed | Google Scholar

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Myakala, K. in: JCI | PubMed | Google Scholar |

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Sadiq, A. in: JCI | PubMed | Google Scholar

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Cheng, Y. in: JCI | PubMed | Google Scholar

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Taranenko, E. in: JCI | PubMed | Google Scholar

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Panov, J. in: JCI | PubMed | Google Scholar

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Korolowicz, K. in: JCI | PubMed | Google Scholar

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Melo Ferreira, R. in: JCI | PubMed | Google Scholar

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Yang, X. in: JCI | PubMed | Google Scholar

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Santo, B. in: JCI | PubMed | Google Scholar

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Allen, K. in: JCI | PubMed | Google Scholar

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Yoshida, T. in: JCI | PubMed | Google Scholar |

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Wang, X. in: JCI | PubMed | Google Scholar

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Rosenberg, A. in: JCI | PubMed | Google Scholar

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Jain, S. in: JCI | PubMed | Google Scholar

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Eadon, M. in: JCI | PubMed | Google Scholar |

1Department of Pharmacology and Physiology, Georgetown University, Washington, DC, USA.

2Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

3Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC, USA.

4Department of Biology, University of La Verne, La Verne, California, USA.

5Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel.

6Department of Microbiology and Immunology, Georgetown University, Washington, DC, USA.

7Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

8National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.

9Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

10Center for Biological and Biomedical Engineering, Georgetown University, Washington, DC, USA.

Address correspondence to: Moshe Levi, Basic Science Building, Room 353, 3900 Reservoir Rd. NW, Washington, DC, 20007, USA. Phone: 202.687.9296; Email: Moshe.Levi@georgetown.edu.

Find articles by Levi, M. in: JCI | PubMed | Google Scholar

Published March 10, 2025 - More info

Published in Volume 10, Issue 5 on March 10, 2025
JCI Insight. 2025;10(5):e181443. https://doi.org/10.1172/jci.insight.181443.
© 2025 Jones et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published March 10, 2025 - Version history
Received: March 25, 2024; Accepted: January 22, 2025 View PDF Abstract

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. Using a mouse model of Alport syndrome, we show that nicotinamide riboside (NR), an NAD+ precursor, stimulated renal PPARα signaling and restored FAO in the proximal tubules, thereby protecting from CKD in both sexes. Bulk RNA-sequencing showed that renal metabolic pathways were impaired in Alport mice and activated by NR in both sexes. These transcriptional changes were confirmed by orthogonal imaging techniques and biochemical assays. Single-nuclei RNA sequencing and spatial transcriptomics, both the first of their kind to our knowledge from Alport mice, showed that NAD+ supplementation restored FAO in proximal tubule cells. Finally, we also report, for the first time to our knowledge, sex differences at the transcriptional level in this Alport model. In summary, the data herein identify a nephroprotective mechanism of NAD+ supplementation in CKD, and they demonstrate that this benefit localizes to the proximal tubule cells.

Introduction

Chronic kidney disease (CKD) is a clinical diagnosis characterized by the gradual loss of renal function. The pathophysiology of CKD is complex, and kidney diseases of distinct etiologies can all converge on CKD (1). Nevertheless, several unifying mechanisms emerge when comparing healthy kidneys with their diseased counterparts. These include the progressive worsening of renal fibrosis, inflammation, and metabolic disturbances. This suggests that treatments that prevent these changes might also prevent the associated loss of renal function. Herein, we focus on preventing the progression of kidney disease by activating renal metabolism with the nicotinamide adenine dinucleotide (NAD+) precursor, nicotinamide riboside (NR).

Decreased NAD+ levels contribute to acute kidney injury (AKI), and NAD+ supplementation is protective in models of AKI (2–4). Using both gain- and loss-of-function transgenic mice, Tran et al. (5) comprehensively showed that the PPARγ coactivator 1-α (PGC-1α) protects from AKI by upregulating genes in the de novo NAD+ synthesis pathway in the renal tubules, and these effects are replicated by NAD+ supplementation. PGC-1α is a coactivator that controls metabolism-related gene regulatory networks, including by direct interaction with PPARα (6). Their data clearly indicate that improvements in renal tubular mitochondrial function, including fatty acid oxidation (FAO), substantially contribute to the protective effects of NAD+ supplementation in AKI (5).

Recent studies suggest that reduced NAD+ may also play a role in CKD. Endogenous NAD+ biosynthesis is impaired in CKD, and there is a corresponding decrease in levels of NAD+ and its related metabolites (7, 8). Consistent with this, both promoting NAD+ salvage and preventing NAD+ breakdown protect from CKD (9, 10). Furthermore, pharmacological NAD+ supplementation has generally been shown to protect against CKD (11–16). However, unlike in AKI, the mechanism of how NAD+ supplementation protects the kidney in CKD is still poorly understood. A key limitation is that no study thus far has investigated the cell type–specific effects of exogenous NAD+ supplementation in a model of CKD, which may affect the podocytes or the renal tubules to varying extents.

In a recent study, we demonstrated that NR treatment of diabetic mice restored mitochondrial function, including sirtuin 3 activation, thereby preventing mitochondrial damage. This reduced expression of the cyclic GMP-AMP synthase/stimulator of interferon genes pathway and thus protected the kidney. Importantly, we also identified changes in metabolic genes, including NR-mediated increases in mRNA transcripts for PGC-1α (Ppargc1a), nuclear respiratory factor 1 (Nrf1), mitochondrial transcription factor A (Tfam), carnitine palmitoyltransferase 1-α (Cpt1a), medium-chain acyl-coenzyme A dehydrogenase (MCAD; Acadm), and long-chain acyl-coenzyme A dehydrogenase (Acadl) (15). These genes are encoded in the nuclear genome, and thus, it is unlikely that the changes were a direct result of mitochondrial sirtuin 3. Instead, it implies a mechanism of NAD+ supplementation that acts at the transcriptional level to modulate expression of key metabolic genes. We therefore sought to identify the molecular mechanism underlying this change.

To begin to dissect this mechanism, as well as cell types responsible, we returned to the NAD+ supplementation data reported from models of AKI. We found the data reported by Tran et al. (5) very compelling evidence for the role of PGC-1α in the renal tubules and consistent with the transcriptional activation of metabolic genes that we observed in our recent study (15). Furthermore, mitochondrial dysfunction and associated FAO defects are key metabolic disturbances that drive CKD (17, 18), and restoring renal metabolism has been shown to be protective (19). Given this rationale, we hypothesized that NAD+ supplementation with NR protects from CKD by activating renal metabolism in the proximal tubules.

We tested this hypothesis with a 3-step sequential approach. We first tested NR in a mouse model of Alport syndrome to show that NAD+ supplementation protects against CKD at multiple time points. We then employed biochemical techniques, including bulk RNA sequencing (RNA-Seq) and immunoblotting, to assess metabolic dysregulation. Finally, we performed single-nuclei RNA-Seq (snRNA-Seq) and spatial transcriptomics (ST) to show that NAD+ supplementation enhances renal metabolism in the proximal tubules. The results presented herein provide strong evidence that NR activates the NAD+/PGC-1α/PPARα/FAO axis in the proximal tubules, thereby stimulating metabolism and protecting the kidney.

Results

Alport mice have reduced NAD+ levels and impaired renal metabolism. To verify that pathways related to both NAD+ and renal metabolism are dysregulated in Alport mice, we reanalyzed previously published RNA-Seq data from 2 independent experiments (20, 21). Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed on the 500 most downregulated genes in Alport mice, and both NAD+ biosynthetic pathways (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.181443DS1) and fatty acid metabolic pathways (Supplemental Figure 2) were significantly enriched. This is in stark contrast with the pathways identified from analyses of upregulated genes, most of which were related to inflammation and fibrosis (Supplemental Figure 1, C and D) (22). Finally, we verified that kidney NAD+ levels were lower in male and female Alport mice compared with control mice (Figure 1).

Figure 1

Kidney NAD+ is reduced in Alport mice. (A) Experimental design: Control and Alport mice of both sexes were sacrificed at 25 weeks of age. (B) Alport mice had lower levels of kidney NAD+ than control mice. Significance was determined by 1-way ANOVA with the Holm-Šídák correction for multiple comparisons. *P < 0.05, **P < 0.01. Data are expressed as the means ± SEM, and each datum represents 1 mouse.

NAD+ supplementation protects Alport mice from kidney disease. Given that kidney NAD+ levels were decreased in Alport mice compared with control mice, we hypothesized that NAD+ supplementation with NR would reduce the severity of kidney disease. Our colony of Alport mice on the C57BL/6J background slowly develop kidney disease until death at 35–40 weeks of age, and we investigated 2 time points.

In our initial experiment, mice were treated with or without NR between 10 and 25 weeks of age (Figure 2A). We did not observe any changes in echocardiography or blood pressure measurements between the groups, excluding these as potential confounding variables (Supplemental Tables 1 and 2). Twenty-four–hour urinary albumin excretion, a marker of kidney damage, was up to 1,000-fold increased in Alport mice, and NR treatment prevented this increase in both sexes (Figure 2B). Plasma creatinine was unchanged between control and Alport mice at the 25-week time point, consistent with the slowly progressing phenotype of Alport mice on the B6 background (Supplemental Table 3). As shown by polarized microscopy of kidney sections stained with Picrosirius red (PSR), a technique that is highly specific for collagen (23), NR treatment prevented the progression of overall renal fibrosis in both sexes (Figure 2, C and D). In addition, renal cortical tubulointerstitial fibrosis — quantified by excluding medullary, vascular bundle, and glomerular contributions — was increased in Alport mice and reduced by NR treatment in both sexes (Supplemental Figure 3, A and B). Renal inflammatory infiltrate was also increased in Alport mice and reduced by NR treatment (Supplemental Figure 3, C and D). Finally, the prevention of renal fibrosis was secondarily verified by immunoblotting for fibronectin, which was reduced in NR-treated male (significant, P < 0.001) and female (trend, P < 0.10) Alport mice (Supplemental Figure 4).

Figure 2

NAD+ supplementation protects the kidney in Alport mice. (A) Experimental design: Control and Alport mice of both sexes were treated with or without NR between 10 weeks and 25 weeks of age. (B) NR treatment reduced 24-hour urinary albumin excretion in Alport mice of both sexes. (C) Representative images of PSR-stained kidneys acquired with polarized light. Yellow-green-orange birefringence is highly specific for fibrosis. (D) Quantification of PSR-stained kidneys shows that NR treatment reduced renal fibrosis in both sexes. Significance was determined by 1-way ANOVA with the Holm-Šídák correction for multiple comparisons. Data are expressed as the means ± SEM, and each datum represents 1 mouse. *P < 0.05, **P < 0.01, ****P < 0.0001. NAD+, nicotinamide adenine dinucleotide; NR, nicotinamide riboside; PSR, Picrosirius red; Veh, vehicle.

We then repeated the experiment in both male and female mice, though we aged the mice longer to 35 weeks of age (Supplemental Figure 5A). Twenty-four–hour urinary albumin excretion was increased in Alport mice, and NR treatment ameliorated this increase in both sexes (Supplemental Figure 5B). At this later time point, plasma creatinine was greatly increased in Alport mice compared with control mice, and NR treatment prevented this increase in both sexes (Supplemental Figure 5C). Both our initial and replication experiments had substantial numbers of littermate-matched mice in each group, and they were temporally separated by greater than 1 year.

NAD+ supplementation protects from glomerular and tubular injury in Alport mice. Glomerular damage was further assessed by immunostaining for the podocyte marker p57kip2. Volumetric podocyte density is a podometric that controls for the thickness of the histological section, the size of the podocyte nucleus, and the size of the glomerulus (24–26). Compared with control mice, Alport mice had reduced volumetric podocyte density, both podocyte and glomerular hypertrophy, and an increased mesangial index. NR treatment prevented these pathologic changes in both sexes (Figure 3, A–D, and Supplemental Figure 6A). In females, but not males, the corrected podocyte number per glomerulus was reduced in Alport mice and restored with NR treatment (Supplemental Figure 6B). However, unlike the volumetric podocyte density, the corrected podocyte number per glomerulus does not control for glomerular hypertrophy and should be interpreted with caution. These results, in combination with the reduction in urinary albumin excretion, demonstrate that NR treatment protects from glomerular damage in the Alport model of kidney disease.

Figure 3

NAD+ supplementation prevents both glomerular and tubular injury in Alport mice. (A) Representative images of immunohistochemistry for p57kip2, followed by periodic acid–Schiff poststaining without hematoxylin counterstaining. Podocyte nuclei are stained brown. (B–D) Quantification of p57kip2 immunostaining with PodoCount, a validated algorithm to analyze p57kip2-stained whole-slide images. Podocyte volumetric density (B) was reduced in Alport mice and restored by NR treatment in both sexes. Alport mice had podocyte nuclear hypertrophy (C) and glomerular hypertrophy (D) that was reduced by NR treatment in both sexes. (E and F) Immunoblots for KIM-1, a tubular injury marker, in male (E) and female (F) kidney homogenate. Ponceau S, a nonspecific protein stain, was used as a loading control. (G and H) Quantification of immunoblots (E and F) shows that KIM-1 is increased in Alport mice and reduced by NR treatment in male (G) and female (H) mice. Scale bars represent 100 μm. Significance was determined by 1-way ANOVA with the Holm-Šídák correction for multiple comparisons. Data are expressed as the means ± SEM. Each datum represents 1 glomerulus (B–D) or 1 mouse (G and H). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. KIM-1, kidney injury molecule-1; NAD+, nicotinamide adenine dinucleotide; No., number; NR, nicotinamide riboside; Podo. Vol. Den., podocyte volumetric density; PSR