Diabetic Endothelial Nitric Oxide Synthase Knockout Mice Develop Advanced Diabetic Nephropathy

Abstract

The pathogenesis of diabetic nephropathy remains poorly defined, and animal models that represent the human disease have been lacking. It was demonstrated recently that the severe endothelial dysfunction that accompanies a diabetic state may cause an uncoupling of the vascular endothelial growth factor (VEGF)-endothelial nitric oxide (eNO) axis, resulting in increased levels of VEGF and excessive endothelial cell proliferation. It was hypothesized further that VEGF-NO uncoupling could be a major contributory mechanism that leads to diabetic vasculopathy. For testing of this hypothesis, diabetes was induced in eNO synthase knockout mice (eNOS KO) and C57BL6 controls. Diabetic eNOS KO mice developed hypertension, albuminuria, and renal insufficiency with arteriolar hyalinosis, mesangial matrix expansion, mesangiolysis with microaneurysms, and Kimmelstiel-Wilson nodules. Glomerular and peritubular capillaries were increased with endothelial proliferation and VEGF expression. Diabetic eNOS KO mice showed increased mortality at 5 mo. All of the functional and histologic changes were improved with insulin therapy. Inhibition of eNO predisposes mice to classic diabetic nephropathy. The mechanism likely is due to VEGF-NO uncoupling with excessive endothelial cell proliferation coupled with altered autoregulation consequent to the development of preglomerular arteriolar disease. Endothelial dysfunction in human diabetes is common, secondary to effects of glucose, advanced glycation end products, C-reactive protein, uric acid, and oxidants. It was postulated that endothelial dysfunction should predict nephropathy and that correction of the dysfunction may prevent these important complications.

Selective Stimulation of VEGFR2 Accelerates Progressive Renal Disease

“…53 This last possibility seems unlikely, however, given that thrombomodulin is associated with the endothelial marker CD34 in eNOSKO mice. 24 Last of all, IHC was used to assess the levels of soluble factors such as PDGF. This may not be the most reliable method, because of secretion of these molecules; other strategies, such as glomerular isolation, should be considered in the future.…”

“…Renal histology was examined at 4 months with blood and urine collected to measure blood urea nitrogen and urinary albumin/creatinine ratio, as described previously. 24 The scheme of the experimental design is presented in Figure 1.…”

“…Mesangiolysis was determined as presence of dissolution of the mesangial matrix. 24 Mesangial cell number was quantified using the Oxford classification, 27 in which each glomerulus is assessed by the most cellular mesangial area, omitting the mesangial area immediately adjacent to the vascular stalk. In addition, capillary lumen number per glomerulus was also counted, to evaluate angiogenic responses.…”

“…We therefore examined BrdU incorporation, as a marker of cell proliferation, 33 and thrombomodulin, as a specific endothelial cell marker 24,34 (Figure 4). Administration of rAAV1-Flk-sel tended to increase thrombomodulin-positive endothelial cells in glomeruli and tubulointerstitium in UNx-WT mice.…”

Differential contribution of diabetes and the Ren2 gene to glomerular pathology in diabetic (mREN-2)27 rats

“…1,2 Streptozotocin (STZ), which induces hyperglycemia by selectively damaging pancreatic b cells, has been administered to genetically modified rodents to create a variety of DN models. [3][4][5][6] The validity of one of these models, the diabetic (mREN-2)27 rat, has been questioned because of the apparent overwhelming impact of its genetically mediated hypertension on renal pathology. 7 One group of investigators reported that STZ-induced diabetes in female heterozygous (mREN-2)27 rats recapitulates the proteinuria, nodular glomerulosclerosis, and progressive loss of renal function that are cardinal features of DN in human beings.…”