Mouse models of diabetic nephropathy

Abstract

Diabetic nephropathy is a major cause of ESRD worldwide. Despite its prevalence, a lack of reliable animal models that mimic human disease has delayed the identification of specific factors that cause or predict diabetic nephropathy. The Animal Models of Diabetic Complications Consortium (AMDCC) was created in 2001 by the National Institutes of Health to develop and characterize models of diabetic nephropathy and other complications. This interim report and our online supplement detail the progress made toward that goal, specifically in the development and testing of murine models. Updates are provided on validation criteria for early and advanced diabetic nephropathy, phenotyping methods, the effect of background strain on nephropathy, current best models of diabetic nephropathy, negative models, and views of future directions. AMDCC investigators and other investigators in the field have yet to validate a complete murine model of human diabetic kidney disease. Nonetheless, the critical analysis of existing murine models substantially enhances our understanding of this disease process.

Figure 1

Comparable appearance of four histologic and/or immunohistochemical stains utilized to characterize the morphologic changes in a 24 week old BKS db/db mouse. A. Jones’ silver methenamine/H&E stain shows expansion of mesangial matrix (black stain) and clear depiction of cellularity (cell nuclei). This stain also clearly delineates basement membranes, and allows good definition of capillary loops versus mesangial structures. B. PAS stain. Like the Jones’ stain depicted in A, this stain shows good delineation of basement membranes and capillary loops as distinct from mesangial regions. Expanded mesangial regions are well detected by this stain. Compared with the silver methenamine stain some cellular detail is lost with PAS as the distinction between matrix and cell cytoplasm can be obscured due to staining of carbohydrate structures in both compartments (see figure 2). C. H&E stain. This stain is valuable for identifying overall cellularity, but is unsatisfactory for quantification of glomerular matrix changes, particularly if they involve modest expansion of mesangial regions. This stain does not highlight basement membranes well, and does not clearly distinguish cellular versus matrix components in expanded mesangial regions. D. Immunohistochemical staining for collagen IV, with methyl green counter stain. This stain clearly defines increased matrix components within the glomerular tuft, and also is a good marker of basement membranes in the interstitium. Overall, Jones’ silver methenamine, PAS, and collagen IV immunohistochemical staining can be reliably used for morphometric analysis of glomerular changes that involve matrix expansion. When these changes are pronounced, and there is greater distortion of glomerular tuft architecture than depicted in this example, the PAS stain may be less reliable than the silver methenamine stain in separating cellular versus matrix contributions to expanded glomerular compartments due to its greater recognition of some cellular components.

Figure 2

Morphologically advanced murine diabetic nephropathy demonstrating prominant mesangiolysis. A. There are broad areas of lucency (arrows) within expanded mesangial regions. Mesangiolysis is most easily recognized with a Jones’ silver methenamine stain, in which the normally homogeneous and compact silver staining (black) matrix (shown in Figure 1A) is disrupted as indicated by areas of lucency and/or spongiform appearance. B. In contrast, a PAS stain shows equivalent mesangial expansion, but the demarcation of cellular and matrix components and lytic regions is less distinct with this stain as compared to the Jones’ stain.

Figure 3

Phenotypic comparison of the Ins2^Akita mutation in males of 3 stocks shows comparable hyperglycemia in Akita/+ males on all 3 backgrounds (A), but development of early signs of diabetic nephropathy [increased albumin/creatinine ratio (ACR)] was limited to the DBA/2 strain background (B) when necropsy was done at 6 months of age.

Figure 4

Representative glomerular histology at 24 weeks of age from eNOS^+/+m/m (control) mice, eNOS^−/− m/m mice (eNOS^−/−), eNOS^+/+db/db (db/db) and eNOS^−/−db/db (db/db eNOS^−/−) mice. All mice are on the BKS background. Note the markedly increased mesangial expansion and glomerulosclerosis in the BKS eNOS^−/−db/db mice.