Diet-induced renal changes in Zucker rats are ameliorated by the superoxide dismutase mimetic TEMPOL

Abstract

Diabetic nephropathy is the leading cause of renal failure in the United States. The obese Zucker rat (OZR; fa/fa) is a commonly used model of type 2 diabetes and metabolic syndrome (MetS), and of the nephropathy and renal oxidative stress commonly seen in these disorders. Heterozygous lean Zucker rats (LZRs; fa/+) are susceptible to high-fat diet (HFD)-induced obesity and MetS. The present study was designed to investigate whether 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL), a membrane-permeable radical scavenger, could alleviate the renal effects of MetS in OZR and LZR fed a HFD, which resembles the typical "Western" diet. OZR and LZR were fed a HFD (OZR-HFD and LZR-HFD) or regular diet (OZR-RD and LZR-RD) and allowed free access to drinking water or water containing 1 mmol/l TEMPOL for 10 weeks. When compared to OZR-RD animals, OZR-HFD animals exhibited significantly higher levels of total renal cortical reactive oxygen species (ROS) production, plasma lipids, insulin, C-reactive protein, blood urea nitrogen (BUN), creatinine (Cr), and urinary albumin excretion (P < 0.05); these changes were accompanied by a significant decrease in plasma high-density lipoprotein levels (P < 0.05). The mRNA expression levels of desmin, tumor necrosis factor-alpha (TNF-alpha), nuclear factor kappaB (NFkappaB), and NAD(P)H oxidase-1 (NOX-1) were significantly higher in the renal cortical tissues of OZR-HFD animals; NFkappaB p65 DNA binding activity as determined by electrophoretic mobility shift assay was also significantly higher in these animals. The same trends were noted in LZR-HFD animals. Our data demonstrate that TEMPOL may prove beneficial in treating the early stages of the nephropathy often associated with MetS.

Figure 1

(a) Total reactive oxygen species (ROS) production rates in renal cortical tissues from 15-week-old experimental animals as measured by electron paramagnetic resonance (EPR) spectroscopy. Values are expressed as means ± s.e.m. High-fat diet (HFD) significantly increased total ROS production in cortical tissues from both LZR and OZR; these increases were attenuated by TEMPOL treatment. (b) NAD(P) H-dependent superoxide production in homogenates of renal cortical tissues from 15-week-old experimental animals as measured by lucigenin assay. Values are expressed as means ± s.e.m., and are reported as relative light units/milligram protein. HFD significantly increased NAD(P) H-dependent superoxide production in both LZR and OZR; TEMPOL attenuated these increases. ^*P < 0.05 vs. LZR-RD, ^#P < 0.05 vs. LZR-HFD, ^@P < 0.05 vs. LZR-HFD-TEM, ^$P < 0.05 vs. OZR-RD, ^†P < 0.05 vs. OZR-HFD, ^‡P < 0.05 vs. OZR-HFD-TEM. a.u., arbitrary unit; LZR, lean Zucker rat; OZR, obese Zucker rat; RD, regular diet; TEMPOL, superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy.

Figure 2

Glomeruli of 15-week-old experimental animals as examined by transmission electron microscopy (×40,000 magnification; bar = 500nm). (a) Normal podocyte ultrastructure, with ordered and upright foot processes, in LZR-RD animals; (b) LZR-HF animals exhibited minimal podocyte damage, and (c) LZR-HFD-TEMPOL animals exhibited almost normal podocyte architecture. (d) Normal podocyte ultrastructure was observed in OZR-RD animals. (e) OZR-HFD animals exhibited fusion and shortening of foot processes, variation of the width of filtration slits, broadening of foot processes, fewer filtration slits per unit length, and multiple inclusion cysts or vacuoles within the cytoplasm of the podocytes. (f) OZR-HFD-TEMPOL animals exhibited almost normal podocyte ultrastructure. HFD, high-fat diet; LZR, lean Zucker rat; OZR, obese Zucker rat; RD, regular diet; TEMPOL, superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy.

Figure 3

(a) Immunofluorescent micrographs (×20 original magnification) of glomeruli of 15-week-old experimental animals stained for desmin. Negative controls are depicted in a and e. (b) LZR-RD glomeruli exhibited no detectable desmin expression; (f) few desmin positive cells were found in OZR-RD glomeruli; (c) LZR-HFD glomeruli exhibited increased expression of desmin; (g) OZR-HFD glomeruli exhibited a marked increase in desmin expression; (d) and (h) TEMPOL treatment attenuated desmin expression in both LZR-HFD-TEMPOL and OZR-HFD-TEMPOL animals. HFD, high-fat diet; LZR, lean Zucker rat; OZR, obese Zucker rat; RD, regular diet; TEMPOL, superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy.

Figure 4

(a) Gene expression levels of desmin, TNF-α, NFκB p65, and NOX-1 mRNA in renal cortical tissues of 15-week-old experimental animals. Values are expressed as means ± s.e.m. Expression levels of all four genes were increased in LZR-HFD animals when compared with LZR-RD animals, and were also increased in OZR-HFD animals when compared with OZR-RD animals. TEMPOL treatment attenuated these increases in both LZR and OZR animals. (b) NFκB p65 DNA binding activity in renal cortical nuclear extracts of experimental animals as determined by electrophoretic mobility shift assay. Densitometry values are expressed as means ± s.e.m. HFD increased NFκB p65 DNA binding activity in both LZR and OZR; TEMPOL treatment attenuated these increases. ^*P < 0.05 vs. LZR-RD, ^#P < 0.05 vs. LZR-HFD, ^@P < 0.05 vs. LZR-HFD-TEM, ^$P < 0.05 vs. OZR-RD, ^†P < 0.05 vs. OZR-HFD, ^‡P < 0.05 vs. OZR-HFD-TEM. HFD, high-fat diet; LZR, lean Zucker rat; NFκB, nuclear factor κB; NOX-1, NAD(P)H oxidase-1; OZR, obese Zucker rat; RD, regular diet; TEMPOL, superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy; TNF-α, tumor necrosis factor-α.