The University of Georgia College of Veterinary Medicine

Georgia Veterinary Scholar Program

2003 Scholars

Georgia Veterinary Scholar	Faculty Mentor

Lauren Reid University of Georgia Class of 2005	Dr. Scott Brown

Development of a Novel Pathophysiological and Diagnostic Tool for Feline Renal Disease

End-stage renal disease is a leading cause of death in cats and has a poor prognosis due to the progressive nature of the disease. Gene expression profiling is a recently developed technique that has been applied to the study of human renal biopsies. The aim of this study was to develop a protocol for studying changes in levels of gene expression associated with feline renal diseases. Since expression of certain genes may be differentially regulated during a disease process, detection of molecular changes may provide early diagnostic and prognostic information and allow for earlier therapeutic intervention.

Reverse transcription (RT)-coupled real-time PCR was used to quantify mRNA of select genes extracted from a normal feline kidney. Genes for erythropoietin and renin were selected for their important roles in normal and diseased kidney function. GAPDH, a housekeeping gene, expressed constitutively in all metabolically active cells, was selected as a marker for overall mRNA quantities present among samples. Feline specific cDNA sequences were available for GAPDH and erythropoietin, which were used to design PCR primers. Renin primers were designed from conserved regions of the human, rat, mouse, sheep, and dog renin genes. Standard RT-PCR was used to sequence feline renin cDNA, from which new feline-specific primers were designed for real-time RT-PCR. Real-time RT-PCR will be used to quantify the mRNA present between normal and diseased kidneys.

RNA is extremely labile and begins degrading within minutes after the death of tissue. Therefore, for RNA quantification to be of any utility as a diagnostic tool, it must be shipped to a diagnostic facility in a manner that prevents degradation of RNA. To preserve RNA, kidney tissue must be either immediately snap frozen in liquid nitrogen and maintained at -80 C or stored in an RNase inhibitor. The latter is the most feasible means of shipping diagnostic specimens in veterinary medicine. A commercially available RNAse inhibition system (RNAlaterTM, RNAlater-ICETM) was used to preserve freshly harvested renal tissue at both room temperature and at 4 C. These results were compared to freezing in liquid nitrogen and storage at -80 C to determine if there were any significant differences in quantities of mRNA recovered. Real-time RT-PCR was used to compare the amount of GAPDH and erythropoietin mRNA recovered from each preservation method.