Genome-wide Linkage Analysis And GeneChip～(?) Technology For Susceptibility Genes Contributing To Diabetic Nephropathy In KK/Ta Mice

Part One: Chromosomal Mapping of a Quantitative Trait Locus for theDevelopment of Albuminuria in Diabetic KK/Ta miceBackgroundThe KK/Ta mouse strain serves as a suitable polygenic model for human type 2 diabetes. We previously reported a genome-wide linkage analysis of KK/ Ta alleles contributing to type 2 diabetes and related phenotypes such as fasting hyperglycemia, glucose intolerance and hyperinsulinemia, obesity and dyslipi-demia. Since KK/Ta mice spontaneously develop renal lesions closely resembling those in human diabetic nephropathy, we investigated the susceptibility loci using the KK/Ta x ( BALB/c x KK/Ta) F1 backcross progeny in the present study. MethodsBALB/c female mice were mated with KK/Ta males to produce the Fl hybrid mice in the animal facility of Juntendo University. KK/Ta x (BALB/c x KK/Ta) Fl backcross mice were obtained by crossing female KK/Ta mice with male (BALB/c x KK/Ta) Fl mice. Genome screening of KK/Ta-derived loci contributing to the development of albuminuria entailed genotyping of 208 male KK/Ta x ( BALB/c x KK/Ta) Fl backcross mice with 101 microsatellite markers polymorphic between the two strains. Linkage analysis was performed using the Map Manager QT package program. ResultsA genome-wide analysis of susceptibility loci for albuminuria with microsat-ellite-based chromosomal maps showed a contributing KK/Ta locus, provisionally designated UA-1 , with a significant linkage with the interval on chromosome 2 at 83.0 cM close to the microsatellite marker D2Mit311 with a maximum LOD of3.5 (x2 = 13. 2, P = 0. 0003 ). UA-1 was different from the susceptibility loci contributing to type 2 diabetes, which we earlier identified. The mode of inheritance differed from that of hypertension. The progeny homozygous for UA-l showed significantly higher urinary albumin levels. Although there were no significant correlations between urinary albumin levels and other diabetic pheno-types, the group of progeny homozygous for both UA-l and alleles for fasting hy-perglycemia showed the highest urinary albumin levels. ConclusionWe identified a susceptibility locus, provisionally designated UA-l , contributing to the development of albuminuria in diabetic KK/Ta mice. UA-l appears to increase the risk of diabetic nephropathy, particularly in individuals susceptible to fasting hyperglycemia, in a gene dosage-dependent manner. There are potentially important candidate genes that may be relevant to diabetic nephropathy.Part Two Gene Expression Profile in Diabetic KK/Ta Mice Background.To identify susceptibility genes for diabetic nephropathy, GeneChip Ex-pression Analysis was employed to survey the gene expression profile of diabetic KK/Ta mouse kidneys. MethodsKidneys from three KK/Ta and two BALB/c mice at 20 weeks of age were dissected. Total UNA was extracted and labeled for hybridizing to the Affymetrix Murine Genome U74Av2 array. The gene expression profile was compared between KK/Ta and BALB/c mice using GeneChip expression analysis software. Competitive RT-PCR was used to confirm the results of GeneChip for a selected number of genes. ResultsOut of 12,490 probe pairs present on GeneChip , 98 known genes and 31 expressed sequence tags ( ESTs) were found to be differentially expressed between KK/Ta and BALB/c kidneys. Twenty-one known genes and 7 expressed sequence tags ( ESTs) that increased in expression and 77 known genes and 24ESTs that decreased in KK/Ta kidneys were identified. These genes are related to renal function, extracellular matrix expansion and degradation, signal trans-duction, transcription regulation, ion transport, glucose and lipid metabolism, and protein synthesis and degradation. In the vicinity of UA-1 (Quantitative trait locus for the development of albuminuria in KK/Ta mice) , candidate genes that showed differential expression were identified, including the Sdc4 gene for syn-decan-4, Ahcy gene for S-adenosylhomocysteine hydrolase, Sstr4 gene for soma-tostatin receptor 4 and MafB gene for Kreisler leucine zipper protein.