Renal Tubular-Specific NRF2 Activity Protects Against Aristolochic Acid Nephropathy

Background: Aristolochic Acid Nephropathy (AAN) is a kidney disease caused by nephrotoxin exposure leading to tubulointerstitial injury, inflammation, fibrosis, and end stage renal disease. AAN can be induced in mice as a model of kidney disease. The KEAP1/NRF2 pathway is a cytoprotective pathway responsible for upregulating antioxidant and detoxifying genes at times of cellular stress. KEAP1 is an inhibitor of NRF2 and prevents this transcription factor from upregulating target genes including NAD(P)H quinone oxidoreductase 1 (Nqo1). Previous studies have shown that NRF2 activity protects against tubular injuries, but the role of NRF2 in AAN is unknown. We enhanced tubular-specific NRF2 activity by generating conditional Keap1 knockouts and exposed these mice and wild-type littermates to AAN injury. We hypothesized that tubular NRF2 activity is protective against AAN in mice. Methods: To generate tubular-specific Keap1 knockout mice we crossed Keap1 floxed animals with tubular-specific Cre expressing mice using the Pax8-Lc1 system. Doxycycline was administered for two weeks (2g/L in drinking water) to induce knockout prior to injection of aristolochic acid type I (5mg/kg i.p. thrice weekly x 1-2 weeks) to induce AAN. Glomerular filtration rates (GFR) were measured using transcutaneous probes and FITC-sinistrin. Serum and kidney tissue were collected to measure blood urea nitrogen (BUN) and injury biomarkers. Results: Keap1 knockout mice exhibited increased Nqo1 protein levels in renal tubules at baseline, indicating higher NRF2 activity. As expected, AAN decreased GFR and increased BUN. Keap1 knockout mice were protected from these changes and exhibited improvements in biomarkers of kidney injury including α-SMA, fibronectin, TNF-α, MCP-1, and IL-1β. Conclusion: Tubular-specific conditional Keap1 knockout increases NRF2 activity. These mice are protected from AAN, suggesting that tubular NRF2 is protective against this injury. Targeting NRF2 may be an effective treatment for this disease. Funding: NIDDK Support, Veterans Affairs Support