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Nuclear factor erythroid 2-related factor 2 (NRF2) functions as a master regulator of redox balance, conferring cellular cytoprotective responses. The effects of NRF2 activation are, however, controversial in animals and humans with diabetes. We reported previously that overexpression of NRF2 in renal proximal tubular cells (RPTCs) increases expression of sodium-glucose co-transporter 2 (SGLT2) and angiotensinogen (AGT, the sole precursor of angiotensins), exacerbates dysglycemia and progression of diabetic kidney disease (DKD) in type 1 diabetic (T1D) Akita Nrf2-/-/Nrf2-RPTC transgenic mice (Diabetes 2021). However, the pathophysiological role of renal NRF2 in the progression of DKD is not completely understood. We studied the impact of Nrf2 deletion specifically in renal tubule (RT) of Akita mice on progression of DKD.
Akita RT-specific Nrf2 knock-out (Akita Nrf2-RT KO) mice were generated by cross-breeding Akita with Nrf2-RT KO mice using Pax8-Cre (male Nrf2 floxed mice were crossbred with female RT-specific Cre deleter (Pax8-Cre) mice). Renal functional and morphological changes were assessed in male Akita Nrf2-RT KO mice vs. Akita Nrf2-lox/lox, non-diabetic Nrf2-lox/lox and Nrf2-RT KO mice at 10 to 20 weeks of age. Immunostaining on kidney sections, Western blot (WB) and real-time qPCR (RT-qPCR) were used to assess protein and gene expression in isolated renal proximal tubules (RPTs).
Glomerular filtration rate (GFR) (estimated with fluorescein isothiocyanate inulin) was increased in Akita mice but was normalized (indicating reversal of glomerular hyperfiltration) in Akita Nrf2-RT KO mice. Fasting blood glucose (FBG), systolic blood pressure (SBP, monitored with a BP-2000 tail-cuff pressure monitor), kidney hypertrophy, glomerular tuft volume, RPTC volume, tubular luminal dilatation, tubular injury score, podocyte loss (assessed by p57 and WT-1 immunofluorescence staining) and urinary albumin-creatinine ratio were significantly increased in Akita mice vs. nondiabetic Nrf2-lox/lox and Nrf2-RT KO mice. These abnormalities were greatly attenuated in Akita Nrf2-RT KO mice, except for FBG and SBP. Fractional excretion of glucose was increased in Akita mice vs. non-diabetic Nrf2-lox/lox and Nrf2-RT KO mice and increased further in Akita Nrf2-RT KO mice. Treatment with a selective A1 adenosine receptor inhibitor (A1aRi; blockade of tubuloglomerular feedback (TGF)) of Akita and Akita Nrf2-RT KO mice resulted in increases in GFR vs. untreated Akita and Akita Nrf2-RT KO mice, respectively. However, the increases in GFR were significantly greater in Akita Nrf2-RT KO mice than in Akita mice. SGLT2 and AGT expression in RPTs were significantly lower in Akita Nrf2-RT KO mice vs. Akita mice.
Our data demonstrated that selective RT-Nrf2 deletion ameliorates glomerular hyperfiltration and kidney injury in Akita mice, indicating that renal NRF2 signaling plays an important role in modulating GFR and DKD progression, at least in part, via the regulation of SGLT2 and AGT expression in RPTs and TGF in diabetic mice. These data were presented, in part, as a poster communication at the Annual American Society of Nephrology congress, Philadelphia, PA, USA, Nov. 1-5, 2023.