Reduction in podocyte density as a pathologic feature in early diabetic nephropathy in rodents: prevention by lipoic acid treatment

Abstract

Background: A reduction in the number of podocytes and podocyte density has been documented in the kidneys of patients with diabetes mellitus. Additional studies have shown that podocyte injury and loss occurs in both diabetic animals and humans. However, most studies in animals have examined relatively long-term changes in podocyte number and density and have not examined effects early after initiation of diabetes. We hypothesized that streptozotocin diabetes in rats and mice would result in an early reduction in podocyte density and that this reduction would be prevented by antioxidants.

Methods: The number of podocytes per glomerular section and the podocyte density in glomeruli from rats and mice with streptozotocin (STZ)-diabetes mellitus was determined at several time points based on detection of the glomerular podocyte specific antigens, WT-1 and GLEPP1. The effect of insulin administration or treatment with the antioxidant, alpha-lipoic acid, on podocyte number was assessed.

Results: Experimental diabetes resulted in a rapid decline in apparent podocyte number and podocyte density. A significant reduction in podocytes/glomerular cross-section was found in STZ diabetes in rats at 2 weeks (14%), 6 weeks (18%) and 8 weeks (34%) following STZ injection. Similar declines in apparent podocyte number were found in STZ diabetes in C57BL/6 mice at 2 weeks, but not at 3 days after injection. Treatment with alpha-lipoic acid substantially prevented podocyte loss in diabetic rats but treatment with insulin had only a modest effect.

Conclusion: STZ diabetes results in reduction in apparent podocyte number and in podocyte density within 2 weeks after onset of hyperglycemia. Prevention of these effects with antioxidant therapy suggests that this early reduction in podocyte density is due in part to increased levels of reactive oxygen species as well as hyperglycemia.

Figure 1

Immunohistological detection of WT1 in control and STZ-diabetic rat glomeruli. Podocytes were detected with antibodies to WT1. WT1 staining is indicated by intense immunoperoxidase activity in podocyte nuclei (arrows).

Figure 2

Podocyte number at 2, 6 and 8 weeks after STZ injection in rats. Data are presented as numbers of podocytes per glomerular section and of glomerular volume per podocyte (GV/P [μm³]). * = p < 0.01, and ** = p < 0.001 vs. control animals. N = 6 animals per group.

Figure 3

Podocyte number at 3 days and 2 weeks after end of the 5 day STZ injection protocol in mice. Data are presented as numbers of podocytes per glomerular section and of glomerular volume pre podocyte (GV/P [μm³]). * = p < 0.05 vs. control animals. N = 5 animals per group.

Figure 4

Podocyte number at 2 weeks after STZ injection in rats. Diab. + ins. = data for diabetic rats that received twice daily insulin injections beginning 3 days after STZ injection. Data are presented as numbers of podocytes per glomerular section and of glomerular volume pre podocyte (GV/P [μm³]). ** = p < 0.001 vs. control animals. N = 6 animals per group.

Figure 5

Podocyte number at 6 weeks after STZ injection in rats. Diab. + ins. = data for diabetic rats that received twice daily insulin injections beginning 3 days after STZ injection. Data are presented as numbers of podocytes per glomerular section and of glomerular volume pre podocyte (GV/P [μm³]). Six week trial data were combined for this analysis. N = 11 (Control), 10 (Diabetes) or 6 (Diab. + ins). Staining for both WT1 and GLEPP1 is displayed. * = p < 0.05, ** = p < 0.0001

Figure 6

Podocyte number at 6 weeks after STZ injection in rats. Diab. + LA = data for diabetic rats that received daily α-lipoic acid injections beginning 2 days after STZ injection. Data are presented as numbers of podocytes per glomerular section and of glomerular volume pre podocyte (GV/P [μm³]). * = p < 0.05; ** = p < 0.001. N = 5 animals per group except in Diabetes group which was 4 animals.