Sequential activation of the reactive oxygen species/angiotensinogen/renin-angiotensin system axis in renal injury of type 2 diabetic rats

Abstract

1. The present study was performed to test the hypothesis that the reactive oxygen species (ROS)-angiotensinogen (AGT)-renin angiotensin system (RAS) axis is sequentially activated in the development of diabetic nephropathy in Zucker diabetic fatty (ZDF) obese rats. 2. Genetic pairs of male ZDF obese and control ZDF lean rats (n = 12 of each species) were killed every 3 weeks from 12 to 21 weeks of age (n = 6 at each time point). 3. The ZDF obese rats developed diabetes mellitus at 12 weeks. At that time, urinary excretion rates of 8-isoprostane were similar between the groups; however, urinary 8-isoprostane levels were significantly increased at 15 weeks in ZDF obese rats compared with controls (36 +/- 6 vs 15 +/- 2 ng/day, respectively). At 15 weeks, protein levels of cortical angiotensinogen were similar between groups; however, cortical angiotensinogen levels were significantly increased at 18 weeks in ZDF obese rats compared with controls (relative ratio of 2.32 +/- 0.21 vs 1.00 +/- 0.20, respectively). At 12 weeks, angiotensin (Ang) II-like immunoreactivity was similar between groups in both the glomeruli and tubules; however, AngII-like immunoreactivity was increased significantly at 21 weeks in ZDF obese rats compared with controls (relative ratios of 1.98 +/- 0.55 vs 1.00 +/- 0.03, respectively, for glomeruli and 1.58 +/- 0.16 vs 1.00 +/- 0.13, respectively, for tubules). Moreover, at 21 weeks, the desmin-positive area in the glomeruli (0.63 +/- 0.08 vs 0.22 +/- 0.05%) and Masson's trichrome stain-positive area in the interstitium (4.97 +/- 0.05 vs 3.18 +/- 0.41%) were significantly increased in ZDF obese rats compared with controls, even though these differences had not been observed earlier. 4. These data suggest that the sequential activation of the ROS-AGT-RAS axis plays an important role in the development of diabetic nephropathy in ZDF obese rats.

Fig. 1

Urinary 8-isoprostane excretion from Zucker diabetic fatty (ZDF) obese (■) and control lean (□) rats. Urinary excretion of 8-isoprostane, a marker of intrarenal oxidative stress, was similar between the two groups at 12 weeks of age; however, urinary excretion of 8-isoprostane was significantly increased at 15 weeks in ZDF obese rats compared with controls. *P < 0.05 compared with control lean rats at the same age.

Fig. 2

Intrarenal angiotensinogen protein levels in Zucker diabetic fatty (ZDF) obese (■) and control lean (□) rats. Intrarenal angiotensinogen protein levels were similar between the two groups at 15 weeks of age (a,e); however, intrarenal angiotensinogen protein levels were significantly increased at 18 weeks in ZDF obese rats compared with control (c,e). No differences in the amount of β-actin were observed among samples (b,d). *P < 0.05 compared with control lean rats at the same age.

Fig. 3

(a–e) Intrarenal angiotensin (Ang) II-like immunoreactivity in the glomeruli of Zucker diabetic fatty (ZDF) obese (b,d) and control lean (a,c) rats at 12 and 21 weeks of age. Intrarenal AngII-like immunoreactivity in the glomeruli was similar between the groups at 12 weeks of age (a,b,e); however, intrarenal AngII-like immunoreactivity in the glomeruli increased significantly at 21 weeks in ZDF obese rats compared with controls (c–e). (□), ZDF lean; (■), ZDF obese. (f–j) Intrarenal AngII-like immunoreactivity in tubules of ZDF obese (g,i) and control lean (f,h) rats at 12 and 21 weeks of age. Intrarenal AngII-like immunoreactivity in tubules was similar between the groups at 12 weeks of age (f,g,j) but, increased significantly at 21 weeks in ZDF obese rats compared with controls (h–j). *P < 0.05 compared with the control lean rats at the same age.

Fig. 4

(a–e) Desmin-positive area in the glomeruli of Zucker diabetic fatty (ZDF) obese (b,d) and control lean (a,c) rats at 12 and 21 weeks of age. The desmin-positive area was similar between the groups at 12 weeks of age (a,b,e); however, the desmin-positive area was increased significantly at 21 weeks in ZDF obese rats compared with controls (c,d,e). (□), ZDF lean; (■), ZDF obese. (f–j) Masson's trichrome (MT)-positive area in the interstitium of ZDF obese (g,i) and control lean (f,h) rats. The MT-positive area was similar between the groups at 12 weeks of age (f,g,j), but increased significantly increased at 21 weeks in ZDF obese rats compared with controls (h–j). *P < 0.05 compared with control lean rats at the same age.