Resistance to Aristolochic Acid-Induced AKI in Female Mice

Background: Females are less susceptible to acute kidney injury (AKI) compared to males, in both humans and mice. The resistance of female mice to developing AKI poses a significant barrier to pre-clinical therapeutic studies, and many studies only use male mice. Our aim was to establish a protocol to induce AKI in female mice using the proximal tubule (PT)-specific nephrotoxin, aristolochic acid I (AAI). Methods: Male and female C57Bl/6 mice aged 12 weeks were injected intraperitoneally with 2 doses of 3mg/kg AAI, and female mice were also given 2 doses of 4mg/kg – 6.5mg/kg AAI. All doses were given 72 hours apart, followed by euthanasia 24 hours after the last injection. Body and kidney weights were recorded and serum collected for creatinine and urea nitrogen measurements. Kidneys were analyzed using hematoxylin and eosin (H&E) and periodic acid Schiff staining, and immunofluorescence for cytokeratin-20 (KRT-20; injured PT) and Lotus lectin (LL; uninjured PT). Liver morphology was assessed using H&E. Results: Female mice given the standard male AKI dose of 3mg/kg AAI did not show any histological injury, whereas male mice had extensive PT death. Female mice given 4-6.5mg/kg AAI lost weight, but there was no difference between different doses, and kidney weights did not change. Serum creatinine and urea nitrogen concentrations were not elevated in response to any of the AAI doses. Histologically, kidneys showed minimal injury at 4mg/kg and 5mg/kg, and only small areas of PT cell death and mild loss of brush border at 6mg/kg and 6.5mg/kg AAI. LL and KRT-20 staining showed a linear trend towards reduced LL across increasing AAI doses (one-way ANOVA test for trend), and minimal, non-significant increases in KRT-20. AAI is activated in the liver and H&E showed significant liver vacuolization in mice treated with 6mg/kg AAI, despite only mild kidney injury. Conclusion: Female mice had mild kidney injury and no loss of kidney function at AAI doses double that needed to cause AKI in male mice. Furthermore, high doses caused liver injury, thus limiting their utility. Studies using high doses of AAI should be interpreted with caution due to the likelihood of liver injury. Further studies are needed to identify a protocol to induce robust AKI in female mice without liver injury, to facilitate future pre-clinical therapeutic studies. Funding: NIDDK Support