Temporal Changes in mRNA Expression for Bikunin in the Kidneys of Rats during Calcium Oxalate Nephrolithiasis

Inter-α-inhibitor and other bikunin-containing proteins are synthesized in relatively large quantities by the liver. These proteins function as Kunitz-type serine protease inhibitors and appear capable of inhibiting calcium oxalate (CaOx) crystallization in vitro. Preliminary studies have shown that renal tubular epithelial cells synthesize bikunin in response to CaOx challenge. To examine this response in vivo, a sensitive reverse transcription-quantitative competitive template-PCR was developed to detect and quantify poly(A)+ -tailed bikunin mRNA expression in kidney tissue from normal rats and rats developing CaOx nephrolithiasis after challenge with ethylene glycol. Bikunin mRNA expression in rat liver tissue was assessed as a positive control. The expression of bikunin mRNA in liver did not differ significantly between normal control rats and experimental rats with induced hyperoxaluria and renal CaOx crystallization. In contrast, there were significant temporal increases in the levels of bikunin mRNA expression in rat kidneys during CaOx nephrolithiasis after challenge with ethylene glycol. Urinary excretion of bikunin-containing proteins seemed to increase concomitantly. These findings indicate an association between the induction of hyperoxaluria/CaOx nephrolithiasis and the expression of the bikunin gene in rat kidneys.

Direct Quantification of the Enteric BacteriumOxalobacter formigenes in Human Fecal Samples by Quantitative Competitive-Template PCR

Homeostasis of oxalic acid appears to be regulated, in part, by the gut-associated bacterium Oxalobacter formigenes. The loss of this bacterium from the gut flora is associated with an increased susceptibility to hyperoxaluria, a condition which can lead to the formation of calcium oxalate crystalluria and kidney stones. In order to identify and quantify the presence of O. formigenes in clinical specimens, a quantitative-PCR-based assay system utilizing a competitive DNA template as an internal standard was developed. This quantitative competitive-template PCR test allows for the rapid, highly specific, and reproducible quantification of O. formigenesin fecal samples and provides a prototype for development of DNA-based quantitative assays for enteric bacteria.