Expression of bikunin mRNA in renal epithelial cells after oxalate exposure

Shizuka Iida, Ammon B. Peck, Karen J. Byer, Saeed R. Khan

Research output: Contribution to journalArticle

Abstract

Purpose: To determine the changes in expression of bikunin in renal epithelial cells on exposure to oxalate and calcium oxalate crystals. Materials and Methods: This study used reverse transcription polymerase chain reaction (RT-PCR) to examine bikunin mRNA expression levels in MDCK cells exposed to oxalate or calcium oxalate crystals. Poly(A)+ RNA was isolated directly from renal epithelial cells, then converted to cDNA with random primers and reverse transcriptase. To quantify the expression level of bikunin mRNA, we developed a competitive DNA template for competitive PCR analysis. 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 cells were expressed in all samples as 434 bp and 312 bp bands, respectively. Bikunin expression was significantly increased in oxalate exposed cells. Cells exposed to calcium oxalate monohydrate crystals or latex beads showed no significant change in expression of bikunin. Western blotting analysis also showed increased expression of bikunin and inter-α-inhibitor-related proteins in the culture medium of oxalate exposed cells. Conclusions: These findings suggest that renal epithelial cells express bikunin gene and have the capability to produce bikunin when stimulated by certain agents such as oxalate. This increased expression and production of bikunin may represent a protective response of renal epithelial cells to nephrotoxic challenges of oxalate.

Original languageEnglish (US)
Pages (from-to)1480-1486
Number of pages7
JournalJournal of Urology
Volume162
Issue number4
DOIs
StatePublished - Oct 1999
Externally publishedYes

Fingerprint

Oxalates
Epithelial Cells
Kidney
Calcium Oxalate
Messenger RNA
Polymerase Chain Reaction
Madin Darby Canine Kidney Cells
Ethidium
RNA-Directed DNA Polymerase
Microspheres
Sepharose
Reverse Transcription
Culture Media
Electrophoresis
Complementary DNA
Western Blotting
Gels
Liver
DNA
Genes

Keywords

  • Bikunin
  • Calcium oxalate
  • Inter-α-inhibitor
  • Kidney stones

ASJC Scopus subject areas

  • Urology

Cite this

Expression of bikunin mRNA in renal epithelial cells after oxalate exposure. / Iida, Shizuka; Peck, Ammon B.; Byer, Karen J.; Khan, Saeed R.

In: Journal of Urology, Vol. 162, No. 4, 10.1999, p. 1480-1486.

Research output: Contribution to journalArticle

Iida, Shizuka ; Peck, Ammon B. ; Byer, Karen J. ; Khan, Saeed R. / Expression of bikunin mRNA in renal epithelial cells after oxalate exposure. In: Journal of Urology. 1999 ; Vol. 162, No. 4. pp. 1480-1486.
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abstract = "Purpose: To determine the changes in expression of bikunin in renal epithelial cells on exposure to oxalate and calcium oxalate crystals. Materials and Methods: This study used reverse transcription polymerase chain reaction (RT-PCR) to examine bikunin mRNA expression levels in MDCK cells exposed to oxalate or calcium oxalate crystals. Poly(A)+ RNA was isolated directly from renal epithelial cells, then converted to cDNA with random primers and reverse transcriptase. To quantify the expression level of bikunin mRNA, we developed a competitive DNA template for competitive PCR analysis. 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 cells were expressed in all samples as 434 bp and 312 bp bands, respectively. Bikunin expression was significantly increased in oxalate exposed cells. Cells exposed to calcium oxalate monohydrate crystals or latex beads showed no significant change in expression of bikunin. Western blotting analysis also showed increased expression of bikunin and inter-α-inhibitor-related proteins in the culture medium of oxalate exposed cells. Conclusions: These findings suggest that renal epithelial cells express bikunin gene and have the capability to produce bikunin when stimulated by certain agents such as oxalate. This increased expression and production of bikunin may represent a protective response of renal epithelial cells to nephrotoxic challenges of oxalate.",
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N2 - Purpose: To determine the changes in expression of bikunin in renal epithelial cells on exposure to oxalate and calcium oxalate crystals. Materials and Methods: This study used reverse transcription polymerase chain reaction (RT-PCR) to examine bikunin mRNA expression levels in MDCK cells exposed to oxalate or calcium oxalate crystals. Poly(A)+ RNA was isolated directly from renal epithelial cells, then converted to cDNA with random primers and reverse transcriptase. To quantify the expression level of bikunin mRNA, we developed a competitive DNA template for competitive PCR analysis. 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 cells were expressed in all samples as 434 bp and 312 bp bands, respectively. Bikunin expression was significantly increased in oxalate exposed cells. Cells exposed to calcium oxalate monohydrate crystals or latex beads showed no significant change in expression of bikunin. Western blotting analysis also showed increased expression of bikunin and inter-α-inhibitor-related proteins in the culture medium of oxalate exposed cells. Conclusions: These findings suggest that renal epithelial cells express bikunin gene and have the capability to produce bikunin when stimulated by certain agents such as oxalate. This increased expression and production of bikunin may represent a protective response of renal epithelial cells to nephrotoxic challenges of oxalate.

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