Susan Sheehan

Date of Award

12-2007

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

Advisor

Beverly Paigen

Second Committee Member

Mary Ann Handel

Third Committee Member

Sharon L. Ashworth

Abstract

Chronic kidney disease (CKD) is a growing medical problem and a significant risk factor for the development of end-stage renal disease, cardiovascular disease, and cardiovascular mortality; thus it is also a substantial economic burden. The genetic basis of CKD is recognized, but knowledge of the specific genes that contribute to the onset and progression of kidney disease is limited, mainly due to the difficulty and expense of identifying genes underlying CKD in humans. Animal models, including mice, are a crucial component of kidney disease research; however, few animal models exist for CKD. In an effort to study CKD using mouse models, we validated autoanalyzer methods for measuring blood urea nitrogen (BUN) and urinary albumin concentrations, two common markers of kidney disease, in samples from mice. We observed a significant, linear correlation between BUN and albumin concentrations measured by autoanalyzer and high-performance liquid chromatography, although the autoanalyzer method underestimated the known amount of albumin by 3.5- to 4-fold. We used our validated method of assessing kidney function in the mouse in two different but complementary studies. First, we used quantitative trait locus (QTL) analysis to detect genomic regions affecting albuminuria in a cross between C57BL/6J and DBA/2J mice, strains resistant and susceptible to CKD, respectively. We identified several independent and interacting loci affecting albuminuria, including one QTL on mouse chromosome (Chr) 2 that is concordant with QTL influencing urinary albumin excretion on rat Chr 3 and diabetic nephropathy on human Chr 20p. Because this QTL was identified in multiple mouse crosses, as well as in rats and humans, we used comparative genomics, haplotype analysis, and expression profiling to narrow the initial QTL interval from 386 genes to 10 genes with known coding sequence polymorphisms or expression differences between the strains. This method allows us to identify naturally variant genetic components of CKD. Our complementary methodology involves developing novel mouse models to enhance the knowledge of CKD and facilitate the effort to uncover the underlying genetics. To find such new models, male C57BL/6J mice were treated with a mutagen, N-ethyl-N-nitrosourea, and bred to obtain third generation offspring. For blood urea nitrogen, we found 36 offspring (1.5%) with levels greater than 33 mg/dL; for urinary albumin/creatinine we found 22 animals (0.6%) with levels greater than 14 mg/g. So far, three proven heritable lines show recessive inheritance (RF5, RF11, RF14); four lines show dominant inheritance (RF10, RF12, RF13, RF15); and four additional lines are likely to be heritable because multiple offspring from the same Gl founder male showed the aberrant phenotype. These new models of kidney disease are available to the scientific community without restrictions. Mutagenesis programs allow us to use a phenotype to identify the regulatory genetic components; QTL studies allow us to use the natural genetic variation to ascertain information about a phenotype. By using these complementing methods, knowledge of the specific genes that contribute to the onset and progression of kidney disease can be discovered.

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