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. 2018 Feb 1;159(2):836-852.
doi: 10.1210/en.2017-00752.

Nrf2 Deficiency Upregulates Intrarenal Angiotensin-Converting Enzyme-2 and Angiotensin 1-7 Receptor Expression and Attenuates Hypertension and Nephropathy in Diabetic Mice

Shuiling Zhao  1 , Anindya Ghosh  1 , Chao-Sheng Lo  1 , Isabelle Chenier  1 , James W Scholey  2 , Janos G Filep  3 , Julie R Ingelfinger  4 , Shao-Ling Zhang  1 , John S D Chan  1
Affiliations

Nrf2 Deficiency Upregulates Intrarenal Angiotensin-Converting Enzyme-2 and Angiotensin 1-7 Receptor Expression and Attenuates Hypertension and Nephropathy in Diabetic Mice

Shuiling Zhao et al. Endocrinology. .

Abstract

We investigated the role of nuclear factor erythroid 2-related factor 2 (Nrf2) in renin-angiotensin system (RAS) gene expression in renal proximal tubule cells (RPTCs) and in the development of systemic hypertension and kidney injury in diabetic Akita mice. We used adult male Akita Nrf2 knockout mice and Akita mice treated with trigonelline (an Nrf2 inhibitor) or oltipraz (an Nrf2 activator). We also examined rat immortalized RPTCs (IRPTCs) stably transfected with control plasmids or plasmids containing rat angiotensinogen (Agt), angiotensin-converting enzyme (ACE), angiotensin-converting enzyme-2 (Ace2), or angiotensin 1-7 (Ang 1-7) receptor (MasR) gene promoters. Genetic deletion of Nrf2 or pharmacological inhibition of Nrf2 in Akita mice attenuated hypertension, renal injury, tubulointerstitial fibrosis, and the urinary albumin/creatinine ratio. Furthermore, loss of Nrf2 upregulated RPTC Ace2 and MasR expression, increased urinary Ang 1-7 levels, and downregulated expression of Agt, ACE, and profibrotic genes in Akita mice. In cultured IRPTCs, Nrf2 small interfering RNA transfection or trigonelline treatment prevented high glucose stimulation of Nrf2 nuclear translocation, Agt, and ACE transcription with augmentation of Ace2 and MasR transcription, which was reversed by oltipraz. These data identify a mechanism, Nrf2-mediated stimulation of intrarenal RAS gene expression, by which chronic hyperglycemia induces hypertension and renal injury in diabetes.

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Figures

Figure 1.
Figure 1.
Generation of Akita Nrf2 KO mice. (A) Genotyping of (panel i) mutated and normal Ins2 gene and (panel ii) Nrf2-LacZ (Nrf2 KO) and normal Nrf2 gene in WT, Nrf2 KO, Akita, and Akita Nrf2 KO mice by specific PCR analysis. (B) Longitudinal changes in mean SBP in male WT (●), Nrf2 KO (■), Akita (▲), and Akita Nrf2 KO (▼) mice. Values are means ± SEM; n = 10 per group. **P < 0.01 and ***P < 0.005 Akita mice compared with WT mice; +P < 0.05 and ++P < 0.01 Akita mice compared with Akita Nrf2 KO mice. (C–K) Immunohistochemical staining for (C) Nrf2, (D) NQO-1, and (E) Keap1 expression in kidney sections (magnification ×600); WB analysis of (F) Nrf2, (G) NQO-1, and (H) Keap1 protein expression and RT-qPCR analysis of (I) Nrf2, (J) NQO-1, and (K) Keap1 mRNA levels in RPTs of WT, Nrf2 KO, Akita, and Akita Nrf2 KO mice. Values are mean ± SEM; n = 6 per group. *P < 0.05; ***P < 0.005. WT (empty bars), Nrf2 KO mice (light gray bars), Akita (solid black bars), and Akita Nrf2 KO mice (dark gray bars). ns, not significant; PCR, polymerase chain reaction; SEM, standard error of the mean.
Figure 2.
Figure 2.
Characterization of Akita Nrf2 KO mice. (A) Periodic acid-Schiff (PAS) staining (magnification ×600), (B) DHE (red) and DAPI staining (blue) (magnification ×200), (C) Cat and (D) Nox4 immunostaining in kidney sections (magnification ×600) from male WT, Nrf2 KO, Akita, and Akita Nrf2 KO mouse kidneys at age 20 weeks. Semiquantification of (E) DHE fluorescence, (F) ROS production, (G) Cat activity, (H) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, (I) Cat mRNA, and (J) Nox4 mRNA expression in RPTs of WT controls, Nrf2 KO, Akita, and Akita Nrf2 KO mice. Values are expressed as mean ± SEM; n = 8 per group. *P < 0.05; **P < 0.01; ***P < 0.005. WT (empty bars), Nrf2 KO mice (light gray bars), Akita (solid black bars), and Akita Nrf2 KO mice (dark gray bars). DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant; SEM, standard error of the mean.
Figure 3.
Figure 3.
Agt, ACE, Ace2, and MasR expression in mouse kidneys at week 20. Immunohistochemical staining of (A) Agt, (B) ACE, (C) Ace2, and (D) MasR in mouse kidneys. Magnification ×600. WB of (E) Agt, (F) ACE, (G) Ace2, and (H) MasR expression and RT-qPCR analysis of (I) Agt, (J) ACE, (K) Ace2, and (L) MasR mRNA levels in RPTs of WT, Nrf2 KO, Akita, and Akita Nrf2 KO mice. Values are expressed as mean ± SEM; n = 8 per group. *P < 0.05; **P < 0.01; ***P < 0.005. WT (empty bars), Nrf2 KO mice (light gray bars), Akita (solid black bars), and Akita Nrf2 KO mice (dark gray bars). ns, not significant; SEM, standard error of the mean.
Figure 4.
Figure 4.
Effect of Nrf2 siRNA on Nrf2, NQO-1, Agt, ACE, Ace2, and MasR gene expression in IRPTCs in NG (normal glucose) and HG (high glucose) media. (A) Effect of Nrf2 siRNA or Scr siRNA on Nrf2 protein expression in cytoplasmic fraction and (B) nuclear fraction of IRPTCs incubated in NG and HG media and quantified by WB. NE-PER Nuclear and Cytoplasmic Extraction Reagents were used for the isolation of cytoplasmic and nuclear fractions from IRPTCs according to the manufacturer’s protocol (Catalog No. 78833; Thermo Scientific; Pierce Biotechnology, Rockford, IL). Effect of Nrf2 siRNA or Scr siRNA on (C) Nrf2, (D) NQO-1, (E) Agt, (F) ACE, (G) Ace2, and (H) MasR mRNA expression in IRPTCs incubated in NG and HG media and quantified by RT-qPCR. Cells were harvested after 24 hours of incubation. mRNA levels in cells incubated in NG medium with Scr RNA are expressed as arbitrary unit 1. The results are reported as percentages of control values (mean ± SEM; n = 3 for three separate experiments done in triplicates. *P < 0.05; **P < 0.01; ***P = 0.005. NG + Scr siRNA (empty bars), NG + Nrf2 siRNA (light gray bars), HG + Scr siRNA (solid black bars), and HG + Nrf2 siRNA (dark gray bars). ns, not significant; SEM, standard error of the mean.
Figure 5.
Figure 5.
Effect of Nrf2 siRNA and trigonelline on Agt, ACE, Ace2, and MasR promoter activity and mRNA in IRPTCs in HG medium. Dose-dependent effect of Nrf2 siRNA and Scr siRNA on (A) Agt, (B) ACE, (C) Ace2, and (D) MasR promoter activity in stably transfected IRPTCs incubated in HG medium and quantified by luciferase activity assay. Effect of trigonelline (Trig) ± oltipraz (Olz) on (E) Agt, (F) ACE, (G) Ace2, and (H) MasR mRNA expression in IRPTCs incubated in HG medium and quantified by RT-qPCR. Cells were harvested after 24 hours of incubation. Promoter activity and mRNA levels in cells incubated in NG medium are expressed as 100% control and arbitrary unit 1, respectively. The results are reported as percentages or fold of change of control values (mean ± SEM; n = 3 for three separate experiments done in triplicates). *P < 0.05; **P < 0.01; ***P < 0.005. ns, not significant; SEM, standard error of the mean.
Figure 6.
Figure 6.
Effect of trigonelline on Nrf2, Agt, ACE, Ace2, and MasR expression in Akita mice in vivo. Longitudinal changes in mean (A) BG and (B) SBP in male WT (●), Akita (■), Akita + trigonelline (Trig) (▲), and Akita + Trig + Oltipraz (Olz) (▼) mice. Values are mean ± SEM; n = 9 per group. ***P < 0.005 Akita mice compared with WT mice; +P < 0.05 Akita mice compared with Akita + Trig mice; #P < 0.05 Akita + Trig mice compared with Akita + Trig + Olz mice. (C) DHE (red) and DAPI staining (blue) (magnification ×200) and (D) PAS staining and immunohistochemical staining for (E) Nrf2, (F) Agt, (G) ACE, (H) Ace2, (I) MasR, and (J) Keap1 in the kidneys of WT and Akita ± Trig and Olz mice. Magnification ×600. SEM, standard error of the mean.
Figure 7.
Figure 7.
Effect of trigonelline (Trig) on oxidative stress and RPT Nrf2, Agt, ACE, Ace2, and MasR expression in mice in vivo. (A) Semiquantification of DHE fluorescence, (B) ROS production, (C) WB of (panel i) Nrf2, Agt, and Keap1 expression and (panel ii) ACE, Ace2, and MasR expression in RPTs of WT and Akita mice ± Trig and oltipraz (Olz). WB quantification of (D) Nrf2, (E) Agt, (F) Keap1, (G) ACE, (H) Ace2, and (I) MasR expression in RPTs of WT and Akita mice ± trigonelline and oltipraz. (J) WB of Nrf2 protein expression and quantitation in (K) nuclear fraction and (L) cytosolic fraction in RPTs of WT and Akita mice ± Trig and oltipraz, respectively. (M) WB of Nrf2 protein expression and quantitation in (N) nuclear fraction and (O) cytosolic fraction of IRPTCs incubated in NG, HG, and HG ± Trig and Olz, respectively. Values are expressed as mean ± SEM (n = 6 per group). *P < 0.05; **P < 0.01; ***P < 0.005. WT (empty bars), Akita mice (light gray bars), Akita mice treated with Trig (solid black bars), and Akita mice treated with Trig and Olz (dark gray bars). ns, not significant; SEM, standard error of the mean.
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