Proximal Tubule ETA Receptor Deletion Reduces Renal Iron Overload in Acute Iron Overload Murine Model

Background: Sickle cell disease, an inherited chronic hemolytic anemia, is associated with iron toxicity and increased mortality from prolonged iron overload. Activation of endothelin system (endothelin-1/endothelin A receptor (ETA)) has been reported in both sickle cell disease patients and humanized murine model. Moreover, pharmacological ETA receptor blockade ameliorated renal iron accumulation in humanized sickle cell mice. This study aims to investigate the impact of genetic, proximal tubule specific deletion of ETA receptor on kidney-specific iron handling and renal injury using acute iron overload mouse model. Methods: Experiments used 16 weeks old male and female proximal tubule ETA knockout (PT ETA KO) and proximal tubule ETA floxed control (PT ETA flox) mice. To induce acute hemolysis-associated iron overload, mice from both genotypes received intraperitoneal injections of phenylhydrazine (PhZ, 40 mg/kg) for two consecutive days, while control groups received saline. Three days following the last PhZ/saline injection, whole blood and tissues (spleen and kidney) from all 4 experimental groups were collected. Heparinized whole blood was used to assess hematological parameters using Heska analyzer. Right kidney was fixed, sectioned, and processed using standard histological protocols, including Prussian Blue and Periodic Acid-Schiff Hematoxilin (PASH) staining (n = 5 per each group). Ten and five images per kidney were acquired at 40x magnification for Prussian Blue and PASH staining, respectively. Renal iron accumulation was quantified as % of positive area using ImageJ, and tubular injury was assessed through histopathological evaluation of brush border loss. PASH staining was graded on a 0–4 scale, where 0 represents complete loss of brush border, and 4 indicates an intact tubular brush border. Statistical analysis was performed using GraphPad Prism 10 software. Data were compared using 2-way ANOVA with Tukey’s multiple comparisons test and presented as mean±standard deviation, with p< 0.05 being considered significant. Results: PT ETA KO and flox mice injected with PhZ had significantly reduced hemoglobin (7.8±0.5 and 6.7±0.3 g/dL, respectively) when compared to saline injected PT ETA KO and flox controls (11.6±0.3 and 11.4±0.2 g/dL, respectively; PPhZ< 0.0001, Pgenotype=0.10, Pinteraction=0.24). Also, PT ETA KO and flox mice injected with PhZ had significantly increased spleen/body weight ratio (23.6±2.0 and 20.4±1.1 mg/g, respectively) when compared to saline injected PT ETA KO and flox controls (3.4±0.3 and 3.3±0.2 mg/g, respectively; pPhZ< 0.001, Pgenotype=0.13, Pinteraction=0.18). Moreover, two-way ANOVA analysis revealed significant effects of PhZ and genotype on renal iron accumulation between the groups (pPhZ< 0.0001, Pgenotype=0.0018, Pinteraction=0.0013), with PT ETA KO mice treated with PhZ exhibiting significantly lower cortical iron accumulation when compared to PhZ given PT ETA flox mice (8.66±0.78 vs 11.44±1.42 % of area; p=0.0003). Assessment of brush border loss in cortical tubules showed evidence of tubular injury in PhZ treated mice (pPhZ< 0.0001, Pgenotype=0.0023, Pinteraction=0.045), whereas prevalence of these tubular defect was similar in saline injected mice. Importantly, PT ETA KO mice treated with PhZ had significantly lower brush border loss when compared to PT ETA flox mice treated with PhZ (2.84±0.22 vs 2.03±0.43, p=0.0033). Conclusion: These results demonstrate that the ETA receptor contributes substantially to renal iron overload and tubular structure impairment in acute iron overload murine model. Funding: NHLBI R01HL173038 and ASN Norman Siegel Research Grant to MK. This abstract was presented at the American Physiology Summit 2026 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.