Recent studies show that podocyte nuclear density (N V ) and numbers of renal podocytes per glomerulus (N) are altered in experimental and spontaneous diabetes mellitus. N V and N are generally reduced, and it has been hypothesized that these morphological changes may relate to the loss of glomerular permselectivity in diabetic nephropathy (DN). In the current study, OVE26 transgenic diabetic mice and age-matched (FVB) controls (60, 150, or 450 days) were fixed by vascular perfusion and renal cortical tissues were prepared for morphometric analyses. ImageJ software and point counting analyses were carried out on light and transmission electron micrographs to determine glomerular volume (V G ), N V , and N. As expected, mean V G in OVE26 mice increased substantially ( 134%) over the course of the study and was significantly increased over FVB mice at all ages. At 60 days, N V and N were not statistically distinguishable in OVE26 and control mice, while at 150 days, N V was significantly reduced in diabetics but not N. In 450-day-old OVE26 animals, however, N V and N were both significantly decreased ( 231% and 99%, respectively) relative to age-matched FVB mice. These data suggest that in the OVE26 model of diabetes, significant podocyte loss occurs relatively late in the course of the disease. Moreover, it seems possible that these podocytic changes could play a role in sustaining the increased permeability of the blood-urine barrier in the later stages of diabetic renal decompensation. Anat Rec, 291:114-121, 2007. 2007 Wiley-Liss, Inc.

Background/Aims: Glomerular lesions in diabetic nephropathy (DN) have been studied in numerous murine diabetic models, but the critical feature of aging is often absent. Since histopathology indicates that in mice, DN glomerular lesions may just begin to develop at about 5 months of age, we utilized the long-lived OVE26 transgenic diabetic model for stereometric analyses of DN glomerulopathic aging. Methods: Albuminuria was determined by ELISA, and transmission electron microscopy stereometry was utilized exclusively to demonstrate changes in glomerular cell density and number, and alterations in the glomerular filtration barrier in OVE26 mice at 60, 150, and 450 days of age. Results: Compared to age-matched controls, albuminuria in diabetic mice is significant at 60 days. At 150 days, glomerular volume and mesangial, endothelial and total cell numbers, and podocyte effacement are significantly increased, while podocyte, endothelial, and total cell density are significantly decreased. Endothelial fenestrations are decreased, and glomerular basement membrane thickness is increased. At 450 days, stereometric alterations are exacerbated. Conclusion: Our data indicate that in OVE26 mice, albuminuria precedes morphological glomerular lesions and could be due to early-onset hyperglycemia. Moreover, in this model, most DN glomerulopathic lesions occur relatively late in life, and it is possible that they may result from prolonged hyperglycemia-induced oxidative stress.

The diabetic-prone BioBreeding Wistar (BB/DP) rat is an autoimmune model of insulin-dependent diabetes mellitus. Approximately 90% of the animals (BB/DPh) are hyperglycemic by 90-120 days of age, while the remaining ~10% (BB/DPn) and diabetes-resistant rats (BB/DR) are normoglycemic for life. The transmission electron microscope data from this study demonstrate expected significant age- and diabetes-related increases in retinal capillary basement membrane (RCBM) widths in (BB/DPh) rats relative to BB/DR animals. However, the data show, for the first time, an unexpected significant RCBM thickening in (BB/DPn) rats compared to BB/DR animals at 6 months and 1 year post-onset of hyperglycemia.

Glomerular lesions in diabetic nephropathy (DN) have been studied in numerous murine diabetic models, but the critical feature of aging is often absent. Since DN lesions may just begin to develop at about five months of age, we utilized the long‐lived OVE26 transgenic diabetic model for stereometric analyses of DN glomerulopathic aging. Albuminuria was determined by ELISA and TEM stereometry was utilized exclusively to demonstrate morphometric alterations in the glomerular filtration barrier (GFB) in OVE26 mice at 60, 150 and 450 days of age. Relative to age‐matched controls, albuminuria in diabetic mice is significant at 60 days and profound at 120 days. At 150 days, glomerular volume, mesangial, endothelial, and total cell number are significantly increased, while podocyte, endothelial, and total cell density are significantly decreased. Dystrophic changes in the GFB include podocyte effacement, glomerular basement membrane thickening, and increased non‐fenestrated areas of glomerular capillary endothelium. At 450 days most stereometric alterations are exacerbated. Our data indicate that in OVE26 mice, intense albuminuria precedes morphological lesions and could be due to early‐onset hyperglycemia. Most morphological changes occur relatively late in life and appear unrelated to early renal decompensation. It is possible that they may result from prolonged hyperglycemia‐induced oxidative stress.Grant Funding Source: Supported in part by grants R01DK072032, COBRE P20 RR024489, Juvenile Diabetes Research Foundation Grant 1‐200‐88 and the North Dakota Lions Foundation

Diabetes mellitus increases the risk for cardiovascular disease due, in part, to hyperglycemia‐induced oxidative stress. To investigate the potential for antioxidant protection of the vasculature, a transgenic mouse (Jtmt) that overexpresses the antioxidant metallothionein (MT) specifically in endothelial cells (EC) was produced. A 16kb transgene consisting of the murine Tie2 promoter and enhancer ligated to the human MTII gene was microinjected into FVB mouse embryos. Five Jtmt(1–5) founder mice were identified by PCR using human MTII primers. Two of these founder mice, Jtmt1 and Jtmt4, have generated numerous transgenic progeny. Further characterization of the Jtmt1 line showed that at 60 days of age heart, kidney and body weights as well as blood glucose, HbA1c, and urine albumin excretion levels were normal. Importantly, MT overexpression in EC in the micro‐ and macrovasculature of kidney, heart, and tail tissues was confirmed by immunohistochemical studies. Ongoing studies are assessing the amount of MT overexpression in renal, hepatic and myocardial tissues by competitive ELISA and Western blot analyses as well as the effect of MT overexpression on EC cytological features by TEM. Future studies include crossing Jtmt1 and OVE26 transgenic diabetic mice in an effort to determine the potential benefits of endothelial‐specific antioxidant protection of the diabetic vasculature.Grant Funding Source: North Dakota Lions Foundation