Modulation of renal superoxide dismutase by telmisartan therapy in C57BL/6-Ins2(Akita) diabetic mice

Abstract

Renal superoxide excess, which is induced by an imbalance of the superoxide-producing enzyme NAD(P)H oxidase and the superoxide-scavenging enzyme superoxide dismutase (SOD) under hyperglycemia, increases oxidative stress and contributes to the development of diabetic nephropathy. In this study, we treated non-obese and hypoinsulinemic C57BL/6-Ins2(Akita) (C57BL/6-Akita) diabetic mice with telmisartan (5 mg kg(-1) per day), an angiotensin II type 1 receptor blocker, or amlodipine (5 mg kg(-1) per day), a calcium channel blocker, for 4 weeks and compared the effects of these two anti-hypertensive drugs on renal NAD(P)H oxidase, SOD and transcription factor Nrf2 (NF-E2-related factor 2), which is known to upregulate several antioxidant enzymes including SOD. Vehicle-treated C57BL/6-Akita mice exhibited higher renal NAD(P)H oxidase and lower renal SOD activity with increased levels of renal superoxide than the C57BL/6-wild-type non-diabetic mice. Interestingly, telmisartan treatment not only reduced NAD(P)H oxidase activity but also enhanced SOD activity in C57BL/6-Akita mouse kidneys, leading to a reduction of renal superoxide levels. Furthermore, telmisartan-treated C57BL/6-Akita mice increased the renal protein expression of SOD and Nrf2. In parallel with the reduction of renal superoxide levels, a reduction of urinary albumin levels and a normalization of elevated glomerular filtration rate were observed in telmisartan-treated C57BL/6-Akita mice. In contrast, treatment with amlodipine failed to modulate renal NAD(P)H oxidase, SOD and Nrf2. Finally, treatment of C57BL/6-Akita mice with apocynin, an NAD(P)H oxidase inhibitor, also increased the renal protein expression of SOD and Nrf2. Collectively, our data suggest that NAD(P)H oxidase negatively regulates renal SOD, possibly by downregulation of Nrf2, and that telmisartan could upregulate renal SOD by the suppression of NAD(P)H oxidase and subsequent upregulation of Nrf2, leading to the amelioration of renal oxidative stress and diabetic renal changes.

Figure 1

Effects of telmisartan treatment on renal superoxide production, NAD(P)H oxidase activity and SOD activity in C57BL/6-Akita diabetic mice. The C57BL/6-Akita diabetic mice were treated with the vehicle, amlodipine or telmisartan for 4 weeks. The treatment started at 10 weeks of age and ended at 14 weeks of age. The data from C57BL/6-Akita diabetic mice were compared with those from age-matched C57BL/6-wild-type non-diabetic mice. (A) Representative glomerular DHE staining after a 4-week treatment with telmisartan. (a) C57BL/6-wild-type; (b) vehicle-treated C57BL/6-Akita; (c) amlodipine-treated C57BL/6-Akita; (d) telmisartan-treated C57BL/6-Akita. (B) Renal superoxide production after a 4-week treatment with telmisartan. Data are presented as the mean±s.e.m. SOD+, kidney tissue pre-incubated with SOD-PEG protein; SOD−, kidney tissue without SOD-PEG protein. n=5 per group. ^*P<0.001 vs. wild-type. ⁺P<0.01 vs. vehicle. (C) Renal NAD(P)H oxidase activity after a 4-week treatment with telmisartan. Data are presented as the mean±s.e.m. n=5 per group. ^*P<0.001 vs. wild-type. ^‡P<0.001 vs. vehicle. (D) Renal SOD activity after a 4-week treatment with telmisartan. Data are presented as the mean±s.e.m. n=5 per group. ^*P<0.001 vs. wild-type. ^‡P<0.001 vs. vehicle.

Figure 2

Western blot analysis of renal cortical SOD isoforms and Nrf2 expression after a 4-week treatment with the vehicle (VE), amlodipine (AM) or telmisartan (TE) in C57BL/6-Akita diabetic mice. WT indicates non-diabetic C57BL/6-wild-type mice. The relative intensity of the SOD-to-actin or Nrf2-to-actin ratios to WT is also shown in the lower panels. Data are presented as the mean±s.e.m. n=4 per group. ^*P<0.001 vs. VE.

Figure 3

Immunofluorescence SOD isoform staining of kidney sections after a 4-week treatment with the vehicle, amlodipine or telmisartan in C57BL/6-Akita diabetic mice. (a–d) SOD1; (e–h) SOD2; (i–l) SOD3; (a, e, i) C57BL/6-wild-type; (b, f, j) vehicle-treated C57BL/6-Akita; (c, g, k) amlodipine-treated C57BL/6-Akita; (d, h, l) telmisartan-treated C57BL/6-Akita.

Figure 4

Western blot analysis of renal cortical SOD isoform and Nrf2 expression after an 8-week treatment with the vehicle (VE) or apocynin (AP) in C57BL/6-Akita diabetic mice. WT indicates non-diabetic C57BL/6-wild-type mice. The relative intensity of the SOD-to-actin or Nrf2-to-actin ratios to WT is also shown in the lower panels. Data are presented as the mean±s.e.m. n=4 per group. ^*P<0.001 vs. VE.

Figure 5

Renal cortical PGE2 levels after a 4-week treatment with the vehicle, amlodipine or telmisartan and after an 8-week treatment with apocynin in C57BL/6-Akita diabetic mice. Data are presented as the mean±s.e.m. n=5 per group. ^*P<0.001.

Figure 6

The proposed mechanism by which telmisartan upregulates renal SOD in diabetes.