Metabolomic analyses of Drosophila models for human renal disease

Inborn errors of metabolism (IEMs) constitute a major class of genetic disorder. Most of IEMs are transmitted recessively, so consanguinity has a huge impact on disease prevalence, particularly in societies like Saudi Arabia, where consanguineous marriage is common. Understanding and treatment are very important in genetic diseases, and simple models would be helpful. Thus, the feasibility of applying the fruit fly, Drosophila melanogaster, as a model for a human renal genetic disease - xanthinuria - was investigated. Xanthinuria is a rare human genetic disease, caused by mutations in xanthine oxidase or molybdenum cofactor sulphurase; in Drosophila, the homologous genes are rosy (ry) and maroon-like (mal), respectively. The new Orbitrap technology of mass spectrometry has the potential to determine levels of many metabolites simultaneously by exact mass, and a major part of this thesis was to investigate the utility of Orbitrap technology in metabolomics of both wild-type and Drosophila mutant. Repeatable significant differences were identified between ry and wild-type flies, which recapitulated painstaking analytical biochemical determinations of the 1950s, but with greater precision. Additionally, completely novel impacts of the ry mutation (on pyrimidine metabolism, the urea cycle and osmolyte biosynthesis) were identified. As expected mal mutants showed more similar changes as ry, but with widespread metabolic perturbations.. The online resource, FlyAtlas.org, provides detailed microarray-based expression data for multiple tissues and life-stages of Drosophila. Downstream genes, such as urate oxidase, are utterly tubule-specific. Accordingly, the utility of Orbitrap technology in elucidating tissue-specific metabolomes was also investigated. Additionally, genetic interventions using designed RNAi constructs were also made and validated by QPCR and metabolomics. As urate is a potent antioxidant, survival of urate oxidase knockdowns was tested in vivo, and a significant impact on survival identified. An Affymetrix microarray was performed, comparing ry506 mutant flies against wild-type and differences were identified in a second experiment, the anti-gout drug allopurinol was used to phenocopy the effects of ry. Overall, the thesis showed that Orbitrap technology was highly suitable for metabolomic analysis of both wild-type and mutant Drosophila, and had potential in the analysis of metabolomes of single tissues. The possibility of using Orbitrap-based metabolomicsin human diagnosis is discussed