An outbred mouse strain to study diabetic nephropathy
To date, the AMDCC has nearly exclusively characterized inbred laboratory mice strains to study the development diabetes nephropathy. These studies support the influence of genetic risk factors as contributing to diabetic nephropathy in these inbred lines. In this regard some strains, like DBA2 develop more robust nephropathy than C57BL/6. Nevertheless, the complexity the genomes of inbred mouse strain are drastically reduced, as compared to the genetic complexity of outbred human populations. Inbred mice are homozygous at all loci; whereas in outbred populations, loci allowed to exist in both heterozygous and homozygous states . Furthermore during the process of brother-sister mating used to derive inbred mouse strains, any polymorphisms that either decrease fecundity or are lethal when homozygous, are lost. If similar mutations contribute, in a dominant or semi-dominant fashion, to human diabetic nephropathy, this variability would never be seen in inbred mouse models. In this regard, one of the specific suggestions of the AMDCC External Scientific Committee was to consider utilizing outbred mouse strains to investigate genetic predisposition for development of diabetic complications. We propose to characterize the progression of nephropathy in diabetic outbred CD-1 mice. A recent study has reported that CD-1 mice develop significant nephropathy in a streptozotocin-induced model of diabetes. This study reported that mice made diabetic by a high dose (single injection) streptozotocin protocol develop ESRD associated with prominent tubulointerstitial nephritis and fibrosis within 3 months and die because of diabetic complications by 6-7 months. In this regard, our own preliminary data in CD-1 mice indicate significant diversity in the levels of albuminuria (following STZ induced diabetes) among individual mice (see below). Such heterogeneity may indicate that outbred mice might more closely resemble the heterogeneity of human populations and might also provide the opportunity to capture dominant genes that contribute to increased albuminuria and diabetic nephropathy. For this reason, we propose to characterize the extent and heterogeneity of diabetic nephropathy in CD-1 mice and to begin to develop crosses for detecting dominant heritable traits predisposing to development and/or progression of diabetic nephropathy. Our first specific aim will be to characterize the variability of the development of nephropathy in the CD-1 strain. Our second specific aim is to to initiate a backcross of CD-1 mice with B6Akita mice, a strain resistant to development of diabetic nephropathy, in order to begin to identify potential dominant alleles that contribute to diabetic nephropathy.

Imaging Mass Spectroscopic Analysis of Human Diabetic Glomerulopathy
The spectrum of lesions in patients with diabetes who do not have classic or overt diabetic nephropathy is not established. Recently, there has been recognition that many patients with diabetes experience chronic kidney disease without significant albuminuria or evidence of other primary glomerulonephritis. Whether this injury is also attributable to diabetes or other underlying unrecognized injury processes is not established. The typical diabetic patient with nephropathy does not undergo renal biopsy. Current renal biopsies done in diabetic patients for clinical indications thus show a high incidence of disease superimposed on diabetes, such as IgA nephropathy, crescentic glomerulonephritis, proliferative glomerulonephritis, post-infectious glomerulonephritis, etc. Many show no other morphologic findings other than diabetic nephropathy, but diseases more advanced than predicted from the clinical data. We aim to use novel proteomic techniques to mine and phenotype diabetic nephropathy from existing archival renal biopsies, and prospectively to examine in tissues obtained from nephrectomies in diabetic and non-diabetic patients with or without nephropathy, and in autopsies. These approaches will allow us to map the spectrum of lesions that may be attributable to diabetes or to other unrecognized abnormalities in diabetic patients. These pilot studies will importantly interact with a companion pilot grant proposing to establish a pre-consenting cohort for tissue collection that will be linked to a synthetic derivative electronic medical record (BioVU). Validation of a detailed phenotype of existing diabetic nephropathy will allow detailed prospective mining of such samples that we plan to collect building on our expertise with BioVU. Our aims thus are as follows: 1) We aim to test the feasibility of examining the presence of proteins, metabolites, lipid derivatives in tissue, linked to the microenvironmental phenotype, by use of mass spectroscopy imaging and image fusion techniques. 2) We aim to link novel compounds identified linked to diabetes to detailed phenotypic analysis by light microscopy with morphometry and electron microscopy. Lay: We aim to use new techniques of mass spectroscopy to analyze the exact abnormalities present in kidney samples in patients with diabetes and kidney disease.

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