The protective role of Nrf2 in streptozotocin-induced diabetic nephropathy

Abstract

Objective: Diabetic nephropathy is one of the major causes of renal failure, which is accompanied by the production of reactive oxygen species (ROS). Nrf2 is the primary transcription factor that controls the antioxidant response essential for maintaining cellular redox homeostasis. Here, we report our findings demonstrating a protective role of Nrf2 against diabetic nephropathy.

Research design and methods: We explore the protective role of Nrf2 against diabetic nephropathy using human kidney biopsy tissues from diabetic nephropathy patients, a streptozotocin-induced diabetic nephropathy model in Nrf2(-/-) mice, and cultured human mesangial cells.

Results: The glomeruli of human diabetic nephropathy patients were under oxidative stress and had elevated Nrf2 levels. In the animal study, Nrf2 was demonstrated to be crucial in ameliorating streptozotocin-induced renal damage. This is evident by Nrf2(-/-) mice having higher ROS production and suffering from greater oxidative DNA damage and renal injury compared with Nrf2(+/+) mice. Mechanistic studies in both in vivo and in vitro systems showed that the Nrf2-mediated protection against diabetic nephropathy is, at least, partially through inhibition of transforming growth factor-beta1 (TGF-beta1) and reduction of extracellular matrix production. In human renal mesangial cells, high glucose induced ROS production and activated expression of Nrf2 and its downstream genes. Furthermore, activation or overexpression of Nrf2 inhibited the promoter activity of TGF-beta1 in a dose-dependent manner, whereas knockdown of Nrf2 by siRNA enhanced TGF-beta1 transcription and fibronectin production.

Conclusions: This work clearly indicates a protective role of Nrf2 in diabetic nephropathy, suggesting that dietary or therapeutic activation of Nrf2 could be used as a strategy to prevent or slow down the progression of diabetic nephropathy.

FIG. 1.

Significant pathological changes and activation of Nrf2 pathway in the glomeruli of human diabetic nephropathy patients. Renal biopsy samples were fixed and cut into 2-μm sections; the sections were subjected to H&E staining (A and B) and immunohistochemical analysis with anti-Nrf2 (C and D), anti-NQO1 (E and F), and anti–8-Oxo-dG (G and H) antibodies. The images shown are representative of eight normal kidney tissue patients (A, C, E, and G) and eight kidney tissues from diabetic nephropathy patients (B, D, F, and H). Magnification is 400×. White and black arrows (B and D) indicate K-W nodules and the Nrf2-positive nuclei, respectively. (A high-quality color representation of this figure is available in the online issue.)

FIG. 2.

Nrf2^−/− mice suffered greater renal damage by STZ compared with Nrf2^+/+ mice. During the course of 16 weeks after STZ injection, the survival rate of mice was recorded and plotted (A). Tail-vein blood glucose levels were monitored at 3, 5, 8, 12, and 16 weeks post-injection (B). Mice were killed at 16 weeks post-injection. Whole-body and kidney weight were measured. The body weight in four different groups at 16 weeks post-injection is shown in C. The ratio of kidney to body weight was calculated (D). UACR was also measured at 0, 8, and 16 weeks post-injection (E). *P < 0.05 untreated vs. STZ-treated; #P < 0.05 Nrf2^+/+ vs. Nrf2^−/−. Data are expressed as means ± SD (n = 8).

FIG. 3.

Higher levels of oxidative stress and oxidative damage occurred in the glomeruli of Nrf2^−/− mice than in Nrf2^+/+ mice in response to STZ. At 16 weeks post-injection, Nrf2^+/+ and Nrf2^−/− mice were killed and kidneys were isolated. Kidney tissue sections from each mouse were used for immunohistochemical analysis with anti-Nrf2 (A, a–d), anti–8-Oxo-dG (A, e–h), and anti-NQO1 (A, i–L) antibodies. Nuclear staining of Nrf2 is shown in the insert (Fig. A, b). Each image is a representative of eight kidney tissue sections from eight mice in each group. Urinary 8-Oxo-dG was detected by liquid chromatography–mass spectrometry (B). *P < 0.05 vs. Nrf2^+/+ mice; #P < 0.05. (A high-quality color representation of this figure is available in the online issue.)

FIG. 4.

Nrf2^−/− mice had more severe glomerular injury than Nrf2^+/+ mice. Kidney tissue sections were subject to H&E (Fig. A, a–d), PAS (A, e–h), and trichrome staining (A, i–L), as well as immunohistochemical analysis with an anti-FN antibody (A, m–p). Each image is a representative of eight kidney tissue sections from eight mice in each group. PAS-stained tissues were used for semiquantitative scoring as described in the research design and methods. Glomerulosclerosis index for four different groups is shown in B. In total, 30 glomeruli were scored for each mouse. *P < 0.05 untreated vs. STZ-treated; #P < 0.05 Nrf2^+/+ vs. Nrf2^−/−. Data are expressed as means ± SD (n = 8). (A high-quality color representation of this figure is available in the online issue.)

FIG. 5.

Nrf2^−/− mice had higher TGF-β1 transcription and FN expression. The mRNA level of Nrf2, NQO1, and GST (A) and TGF-β1, FN, and collagen IV (B) was measured by qRT-PCR. The data presented are relative mRNA level normalized to β-actin mRNA level, and the value from the untreated Nrf2^+/+ group was set as 1. *P < 0.05 untreated vs. STZ-treated; #P < 0.05 Nrf2^+/+ vs. Nrf2^−/−. Data are expressed as means ± SD (n = 8). The protein level of FN, NQO1, and GAPDH was measured by immunoblot analysis (C, upper panel). Each lane contained total proteins from the kidney of different individual mice. The band intensity was calculated and normalized to GAPDH (C, lower panel). The value from the untreated Nrf2^+/+ group was set as 1. *P < 0.05 untreated vs. STZ-treated; #P < 0.05 Nrf2^+/+ vs. Nrf2^−/−. Data are expressed as means ± SD (n = 3).

FIG. 6.

Nrf2 was activated by high glucose–induced ROS production in HRMCs. Before exposure to low-glucose (LG) (1 g/l) or high-glucose (HG) (5.4 g/l) DMEM, cells were starved for 24 h with low-glucose DMEM containing 0.5% FBS. Cells were then incubated in low- and high-glucose DMEM for an additional 24 h. Nuclear and cytosolic fractions were extracted and subjected to immunoblot analysis using anti-Nrf2, anti-lamin A, and anti-tubulin antibodies (A, left panel). The intensity of the bands was calculated and quantified (A, right panel). Total mRNA was extracted and used for qRT-PCR for measurement of the mRNA level of Nrf2, HO-1, NQO1, or GST (B). ROS level was also measured in these cells growing in low- or high-glucose medium by dichlorofluorescein (DCF)/flow cytometry analysis described in research design and methods (C). HRMCs were incubated with NAC (50 μmol/l) for 24 h. Total cell lysates were subjected to immunoblot analysis using anti-Nrf2, anti-NQO1, and anti-GAPDH antibodies (D, left panel). The intensity of the bands was quantified (D, right panel).

FIG. 7.

Nrf2 negatively regulated TGF-β1 and FN in HRMCs. HRMCs growing were transfected with plasmids for TGF-β1–promoter-firefly luciferase and renilla luciferase (internal control), along with different amounts of an expression vector for Nrf2. At 48 h post-transfection, both firefly and renilla luciferase activities were measured (A, upper panel). An aliquot of cell lysates was used for immunoblot analysis (A, lower panel). HRMCs were transfected with plasmids for TGF-β1–promoter-firefly luciferase and renilla luciferase. Cells were then treated with tert-butylhydroquinone (tBHQ) for 16 h before the measurement of luciferase at 48 h post-transfection (B, upper panel). An aliquot of cell lysates was used for immunoblot analysis (B, lower panel). HRMCs were transfected with control- or Nrf2-siRNA. Then 24 h later, cells were starved before incubation with low- or high-glucose medium for an additional 24 h. Total mRNAs were extracted, and qRT-PCR was performed to measure the mRNA level of TGF-β1 (C). Another parallel set of cells was collected in lysis buffer for immunoblot analysis with antibodies against Nrf2, aldose reductase (AR), γGCS, NQO1, FN, and GAPDH (D, upper panel). The intensity of the bands was calculated and quantified (D, lower panel). All the experiments were repeated three times and data represent means ± SD.