This portion of the proposed work will be performed at the Johns Hopkins School of Medicine in Dr. Charles Heilig's laboratory. The consortium proposes to create and study new transgenic mouse models of diabetes. One measure of the extent to which these mouse models reproduce and/or exacerbate a diabetic renal lesion, is the degree of glomerulosclerosis which develops. Dr. Heilig's laboratory will be responsible for morphometric measurements of pathologic changes in kidneys from transgenic and diabetic mice to be created in this proposed work. Dr. Heilig's laboratory will employ PAS and trichrome staining of formalin-fixed kidney sections from the mice, to measure percent mesangial sclerosis using computer-assisted microscopy with the Osteomeasure(R) software from Osteometrics, Inc. Glomerular and mesangial cross-sectional areas are traced and calculated using this program. Freshly harvested mouse kidneys are sectioned sagittally and fixed in 10% buffered formalin prior to preparation of microscopic slides. Measurements of mesangial sclerosis will be performed on both PAS-stained sections (extracellular matrix) and Gomori-trichrome-stained sections (collagen). A minimum of 50 sequential glomeruli from each kidney will be used for quantitation of cross-sectional mesangial and glomerular areas, and for calculation of the percent mesangial sclerosis as the ratio of the two measurements x 100. The mean percent mesangial sclerosis in glomeruli will be determined for each kidney. Subsequently, the mean +/- SEM percent mesangial sclerosis in glomeruli will be determined for kidney samples in each experimental group, prior to statistical analysis for detection of significant intergroup differences.
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