Leslie Gewin, M.D.

ABSTRACT

The U.S. population is aging, and patients lose almost 50% of their renal function between their 20s and age 70. Nearly 15% of Americans suffer from chronic kidney disease (CKD) and age-related kidney dysfunction is an important contributor, along with hypertension and diabetes. Progressive CKD leading to end-stage kidney disease (ESKD) places a huge economic burden on the healthcare system and imparts significant mortality and morbidity on patients. Those with CKD who do not progress to ESKD still have an increased risk of cardiovascular disease and death from other causes (e.g., COVID-19). Metabolic dysfunction is a common feature of age-related dysfunction and aging-associated diseases (e.g., Alzheimer’s disease, Parkinson’s disease).

Most studies have focused on the role of mitochondria in metabolic dysfunction, but the overlooked peroxisome also plays an important role, particularly with fatty acid oxidation (FAO). The proximal tubule, highly metabolically active due to its reabsorptive capacity, prefers FAO to generate the required ATP to support its functions.

This proposal investigates the role of peroxisomal FAO in kidney aging and tests the hypothesis that impaired peroxisomal FAO contributes to the pathophysiology of kidney aging. The proposal has two aims:

• Aim 1: The first aim will determine how peroxisomal FAO affects the aging of primary proximal tubule-enriched (PT) cells by multiple passages in culture, an in vitro model that has similar molecular signatures to in vivo Primary PT cells generated from mice lacking tubular ACOX1, the rate-limiting enzyme for peroxisomal FAO, will be compared to PT cells from floxed control mice. Both male and female mice will be used to assess sex-specific differences, and some mice will be fed a high-fat diet prior to generation of primary PT cells.
• The second aim will investigate how drugs known to promote FAO, including SGLT2 inhibitors, alter the aging response of primary PT cells and whether the response is dependent upon intact peroxisomal FAO (i.e., ACOX1).

Concurrent with these studies, we will start the process of aging mice with and without tubular ACOX1. Based on data from this pilot project, we will treat the mice in the final three to four months with the drugs (identified in Aim 2) deemed most promising to slow the aging-related tubular injury. This pilot project will generate the necessary preliminary data for an R01 grant to the National Institute of Aging.

Progress Report — Final (Year 1 of 1)

We were pleased to receive LLF funding for our pilot project on Peroxisomal Fatty Acid Metabolism and Kidney Aging (October 1, 2023 – September 30, 2024). This one-year grant focused on a proximal tubule in vitro model of aging using serial passaging of primary cells to induce senescence, with two proposed aims: (1) determine how peroxisomal fatty acid oxidation affects aging tubule cells in vitro, and (2) define how metabolism-modulating drugs — including a PPARα agonist, SGLT2 inhibitors, and nicotinamide mononucleotide (NMN) — alter aging and senescence in primary proximal tubules.

Aim 1: Peroxisomal Fatty Acid Oxidation and Kidney Aging

We encountered delays during the funding year in generating mice with tubule-specific deletion of ACOX1, the rate-limiting enzyme in peroxisomal fatty acid oxidation (FAO). Subsequent to the funding period, we demonstrated that Acox1CKO murine kidneys accumulate both long-chain and very-long-chain fatty acids and show impaired mitochondrial respiration (assessed by high-resolution respirometry via Oroboros) and altered mitochondrial structure (by electron microscopy). Bulk RNA sequencing revealed significant changes in aging-associated genes, suggesting that loss of ACOX1 and impaired peroxisomal FAO may accelerate the aging process. Acox1CKO mice aged to one year are now established in our colony.

Aim 2: Metabolism-Modulating Drugs and Senescence

We established a robust in vitro aging model with progressive increases in senescence-associated β-galactosidase (SA-βgal) staining and upregulation of p16 and p21 expression with successive passages of primary proximal tubule cells. Initial treatment with the proposed agents showed high variability, prompting a switch to a mitochondrial pyruvate carrier (MPC) inhibitor developed in collaboration with Dr. Brian Finck. The MPC inhibitor significantly reduced SA-βgal staining, indicating a protective effect against cellular senescence.

Following the funding year, we obtained aged kidneys from mice treated with MPC inhibitor or control chow through the NIH-funded Interventions Testing Program (ITP). MPC inhibitor-treated mice showed a significantly increased median lifespan (9% improvement). Single-nuclear RNA sequencing (snRNA-seq) of these tissues is underway to identify mechanisms underlying this protective effect.

Path to External Funding

These findings served as preliminary data for an R01 submission on “Metabolic Reprogramming in Kidney Aging” in June 2025. The A0 submission received a 13% score, and a resubmission is in preparation for March 2026. LLF support was instrumental in launching this project and generating the critical preliminary data needed to compete for NIH funding.

To read the full Progress Report, please click here.