Despite the fact that hyperoxaluria is an established risk factor for calcium oxalate (CaOx) urolithiasis, except for extreme cases, oxalate's exact role in urolithiasis is not well understood. There has been little progress in determining the genetic defects involved in idiopathic hyperoxaluria or urolithiasis, in part because oxalate's effects on the nephron are not well understood. In vivo models allow study of oxalate's effects on the renal epithelium with the intercellular associations and regulatory cascades intact. We will use a graded series of hyperoxaluric rat models 1) vehicle control; 2) hyperoxaluria, 3) hyperoxaluria+crystalluria and 4) hyperoxaluria+crystalluria+crystals deposited in the kidneys) and microarray analysis to develop a solid data base to be used as a springboard for future studies.
In Specific Aim I we will develop a new model for the study of hyperoxaluric rats by using osmotic minipumps to deliver the oxalate subcutaneously.
Specific Aim 2 will validate the microarray analysis by determining the number of rats needed for each pool of tissue, the number of pools/treatment group and the number of chips on which a pool must be run to control for technical and biological variability. It will also determine the limits of sensitivity for detecting changes between control and treated rats, by comparing the results obtained by microarray analysis vs RT-PCR.
Specific Aim 3 will use microarray analysis and the models developed above to determine how the pattern of gene expression changes as hyperoxaluria progresses into urolithiasis, with particular emphasis on those genes regulated by oxalate in the absence of crystals. Data will be analyzed by SAM(statistical analysis) and the data mining programs SpotFire & GeneSpring. Kidney function will be monitored by creatinine clearance and fraction sodium excretion. Renal morphology will be evaluated by light and electron microscopy. Markers of tubular function will be evaluated in the urine and kidney to determine those segments of the nephron that are the most sensitive to damage by oxalate. Depending on the marker, expression will be measured at the level of the message (RT-PCR, Northern blot) and protein (Western blot, immunohistochemistry or enzymatic assays). The long term objective of this work is to establish the mechanism of oxalate induced damage in the kidney.
