Expression of bikunin mRNA in renal epithelial cells after oxalate exposure

Purpose: To determine the changes in expression of bikunin in renal epithel ial cells on exposure to oxalate and calcium oxalate crystals. Materials and Methods: This study used reverse transcription polymerase cha in reaction (RT-PCR) to examine bikunin mRNA expression levels in MDCK cell s exposed to oxalate or calcium oxalate crystals. Poly(A)(+) RNA was isolat ed directly from renal epithelial cells, then converted to cDNA with random primers and reverse transcriptase. To quantify the expression level of bik unin mRNA, we developed a competitive DNA template for competitive PCR anal ysis, The PCR products were resolved by electrophoresis on 1.3% agarose gel and visualized with ethidium bromide. In this system,we could quantify the exact number of bikunin mRNA transcripts. Bikunin mRNA from rat liver was expressed as a positive control. Results: Bikunin mRNAs and competitive templates from renal epithelial cell s were expressed in all samples as 434 bp and 312 bp bands, respectively. B ikunin expression was significantly increased in oxalate exposed cells. Cel ls exposed to calcium oxalate monohydrate crystals or latex beads showed no significant change in expression of bikunin. Western blotting analysis als o showed increased expression of bikunin and inter-cu-inhibitor-related pro teins in the culture medium of oxalate exposed cells. Conclusions: These findings suggest that renal epithelial cells express bik unin gene and have the capability to produce bikunin when stimulated by cer tain agents such as oxalate. This increased expression and production of bi kunin may represent a protective response of renal epithelial cells to neph rotoxic challenges of oxalate.