006 - Increased Gentamicin Uptake and Renal Toxicity in Mice with Congenital Nephron Deficits

Publication Number: 6.5271

Jacob Silberman, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, United States; David A. Bateman, Columbia University Vagelos College of Physicians and Surgeons, New Rochelle, NY, United States; Fangming Lin, Columbia University, New York, NY, United States; Pamela I. Good, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, United States

Background: Humans born preterm have low nephron number and an increased risk of acute kidney injury (AKI). We used gentamicin to induce AKI in a newly-generated mouse model that simulates the spectrum of preterm human nephron deficits (named RetUB del, Good et al, JCI Insight, 2023). Gentamicin is nephrotoxic and commonly prescribed to preterm infants, making this a highly clinically relevant model. We previously showed that after gentamicin-induced AKI, RetUB del pups have more cell injury than controls with normal nephron number. We also found that in the absence of injury, adult RetUB del mice develop nephron hypertrophy and increased expression of proximal tubular (PT) lysosomal proteins, suggesting more endocytosis.

Objective: We hypothesized that increased PT endocytosis causes more gentamicin uptake in low nephron number pups, leading to cell death. We focused on ferroptosis, which is triggered by lipid oxidative injury. Since gentamicin is cationic and binds to negatively charged phospholipids, this interaction may cause lipid damage and activation of ferroptosis-mediated cell death.

Design/Methods: We injected 5 day old control and RetUB del pups with Texas-red conjugated gentamicin (TR-Gent, 2 ug/g) and measured fluorescent density per cell 1 h after injection. To induce AKI, 2 day old pups were injected with 100 mg/kg of gentamicin daily for 2 days, and kidneys were analyzed on day 4. Immunostaining of the established ferroptosis markers transferrin receptor 1 (TfR1) and malondialdehyde adduct (MDA) was used to assess for ferroptosis.

Results: We found increased proximal tubular TR-Gent uptake in pups with low nephron number, with mean fluorescent density per cell of 29.23 x10^5 in RetUB del (n=4) vs. 20.96 x10^5 in controls (n=3, p=0.007, Welch’s t - test). We next determined whether increased megalin expression contributes to increased gentamicin uptake. We found a 1.8-fold increase in megalin mRNA expression on P5 in RetUB del mice compared to controls (qRT-PCR, n=6, p < 0.001, Welch’s t - test). Qualitative assessments of TR-Gent showed that trafficking from endosomes to lysosomes was similar in RetUB del and controls. We discovered that damaged PT cells co-express MDA and TfR1 (n=9), suggesting activation of ferroptosis.

Conclusion(s): Neonatal mice with congenitally low nephron number have increased megalin-mediated gentamicin uptake, resulting in greater toxicity and activation of ferroptosis. Ferroptosis has not been implicated in neonatal AKI, making this a novel discovery and suggesting the utility of preclinical trials using ferroptosis inhibitors to reduce gentamicin toxicity.