Functional analysis of the left-right determinant inv (inversion of embryonic turning) gene

Satsuki Shirota, Takumi Yoshida, Hidekazu Sugiura, Ken Tsuchiya, Hiroshi Nihei

Research output: Contribution to journalArticle

Abstract

We identified the inv gene that encodes left and right asymmetry and regulates kidney development based on the information of the inv mutant mouse. However, functional properties and the modulator of gene expression of inv have been unclear. We used the tissue injury model for assessing the functional roles of inv in ischemia reperfusion injury (IRI). The kidney tissue taken from rats with IRI showed reciprocal changes in mRNA expression of inv: a 0.25-fold decrease at 6 hours and then a gradual increase to a maximum 1.8-fold rise at 10 days of reperfusion. Next, oxidative stress was induced by exposing mouse inner medullary collecting duct (mIMCD-3) cells to hydrogen peroxide (H 2O2) in the medium. Real-time PCR showed that mRNA expression of inv decreased 0.52-fold at 3 hours with 0.2 mM H2O 2 in the medium, and then increased 3.1-fold at 24 hours with 0.1 mM H2O2 in the medium. RNA interference (RNAi) is a powerful tool to inhibit gene expression in experimental model systems. We knocked down inv gene expression in mIMCD-3 cells using RNAi to investigate the function of the inv gene. We designed a small interfering RNA (siRNA) to target the coding region of inv (inv-siRNA) and random-sequence scrambled siRNA (control siRNA). mIMCD-3 cells transfected with either the inv-siRNA or control siRNA were observed by microscopy. The cells transfected with inv-siRNA progressively lost cell-to-cell contact and the cell population significantly diminished approximately 48 hours post-transfection. The changes in gene expression profile were observed at time points (36 hours) using real-time PCR-based gene screening with categorized primer sets. Several genes related to structural protein of the matrix were downregulated. In contrast, repairing related genes were upregulated. In conclusion, gene expression of inv was modulated under oxidative stress and the inv gene may play a role in repairing and regenerating renal epithelial cells.

Original languageEnglish (US)
Pages (from-to)676-684
Number of pages9
JournalJapanese Journal of Nephrology
Volume46
Issue number7
StatePublished - 2004

Fingerprint

Small Interfering RNA
Genes
Gene Expression
RNA Interference
Reperfusion Injury
Kidney
Real-Time Polymerase Chain Reaction
Oxidative Stress
Messenger RNA
Transcriptome
Hydrogen Peroxide
Reperfusion
Transfection
Microscopy
Theoretical Models
Down-Regulation
Epithelial Cells
Wounds and Injuries
Population
Proteins

Keywords

  • Cystic kidney
  • Inv
  • Ischemia reperfusion injury
  • Real-time PCR
  • RNA interference

ASJC Scopus subject areas

  • Nephrology

Cite this

Shirota, S., Yoshida, T., Sugiura, H., Tsuchiya, K., & Nihei, H. (2004). Functional analysis of the left-right determinant inv (inversion of embryonic turning) gene. Japanese Journal of Nephrology, 46(7), 676-684.

Functional analysis of the left-right determinant inv (inversion of embryonic turning) gene. / Shirota, Satsuki; Yoshida, Takumi; Sugiura, Hidekazu; Tsuchiya, Ken; Nihei, Hiroshi.

In: Japanese Journal of Nephrology, Vol. 46, No. 7, 2004, p. 676-684.

Research output: Contribution to journalArticle

Shirota, S, Yoshida, T, Sugiura, H, Tsuchiya, K & Nihei, H 2004, 'Functional analysis of the left-right determinant inv (inversion of embryonic turning) gene', Japanese Journal of Nephrology, vol. 46, no. 7, pp. 676-684.
Shirota, Satsuki ; Yoshida, Takumi ; Sugiura, Hidekazu ; Tsuchiya, Ken ; Nihei, Hiroshi. / Functional analysis of the left-right determinant inv (inversion of embryonic turning) gene. In: Japanese Journal of Nephrology. 2004 ; Vol. 46, No. 7. pp. 676-684.
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AB - We identified the inv gene that encodes left and right asymmetry and regulates kidney development based on the information of the inv mutant mouse. However, functional properties and the modulator of gene expression of inv have been unclear. We used the tissue injury model for assessing the functional roles of inv in ischemia reperfusion injury (IRI). The kidney tissue taken from rats with IRI showed reciprocal changes in mRNA expression of inv: a 0.25-fold decrease at 6 hours and then a gradual increase to a maximum 1.8-fold rise at 10 days of reperfusion. Next, oxidative stress was induced by exposing mouse inner medullary collecting duct (mIMCD-3) cells to hydrogen peroxide (H 2O2) in the medium. Real-time PCR showed that mRNA expression of inv decreased 0.52-fold at 3 hours with 0.2 mM H2O 2 in the medium, and then increased 3.1-fold at 24 hours with 0.1 mM H2O2 in the medium. RNA interference (RNAi) is a powerful tool to inhibit gene expression in experimental model systems. We knocked down inv gene expression in mIMCD-3 cells using RNAi to investigate the function of the inv gene. We designed a small interfering RNA (siRNA) to target the coding region of inv (inv-siRNA) and random-sequence scrambled siRNA (control siRNA). mIMCD-3 cells transfected with either the inv-siRNA or control siRNA were observed by microscopy. The cells transfected with inv-siRNA progressively lost cell-to-cell contact and the cell population significantly diminished approximately 48 hours post-transfection. The changes in gene expression profile were observed at time points (36 hours) using real-time PCR-based gene screening with categorized primer sets. Several genes related to structural protein of the matrix were downregulated. In contrast, repairing related genes were upregulated. In conclusion, gene expression of inv was modulated under oxidative stress and the inv gene may play a role in repairing and regenerating renal epithelial cells.

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