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Updated: Jul 6, 2025
Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration
Published on: June 7, 2014
[m6A methylase WTAP participates in renal ischemia-reperfusion injury by regulating FOXO1 expression]
1Department of Urology, First Affiliated Hospital of Xi'an Medical University, Xi'an 710000, China.
Objective:
To investigate the expression of WTAP, a m6A methylase, in a mouse model of renal ischemia-reperfusion (I/R) injury and the effect of WTAP knockdown on biological behavior of renal tubular epithelial cells exposed to I/R injury.
Methods:
Sixteen C57BL/6 mice with renal I/R injury or sham operation (n=8) were examined for blood urea nitrogen (BUN) and creatinine (Scr) levels to assess renal function, and renal pathologies were observed with HE staining. The expressions of WTAP and FOXO1 proteins in the kidneys of the mice were detected using immunohistochemistry. Human renal tubular epithelial cells (HK-2) were transfected with si-WTAP or si-NC followed by hypoxia-reoxygenation (H/R) exposure, Protein and mRNA expression were assessed by Western blot and qRT-PCR, and changes and changes in cell viability and apoptosis were assessed using CCK8 assay and TUNEL staining, respectively; LDH release level and caspase-3 activity of the cells were measured using commercial assay kits. FOXO1 m6A modification sites were predicted using SRAMP website (http://www.cuilab.cn/sramp/), and the interaction between WTAP and FOXO1 mRNA was analyzed with RIP experiment; the level of FOXO1 modified by m6A was detected by MeRIP-qPCR.
Results:
Compared with sham-operated mice, the mice with renal I/R injury showed significantly increased Scr and BUN levels (P < 0.001) and renal expressions of WTAP mRNA and protein (P < 0.001). In cultured HK-2 cells, H/R exposure significantly decreased the cell viability (P < 0.001) and increased cellular LDH release (P < 0.001) and expressions of WTAP mRNA and protein (P < 0.001). WTAP knockdown obviously reduced the cell damage induced by I/R injury and significantly decreased the mRNA and protein levels of FOXO1 in the cells (P < 0.001). RIP experiment confirmed WTAP binding to FOXO1 mRNA, and inhibition of WTAP expression significantly reduced FOXO1 m6A level in HK-2 cells (P < 0.001).
Conclusion:
WTAP expression is up-regulated in the kidneys of mice with renal I/R injury and in HK-2 cells with H/R exposure. Inhibition of WTAP alleviates H/R-induced apoptotic damage in HK-2 cells possibly by inhibiting FOXO1 expression.
Insights
WTAP expression increases in renal ischemia-reperfusion injury. Inhibiting WTAP reduces kidney cell damage and apoptosis by affecting FOXO1 expression, offering a potential therapeutic target for kidney injury.
Area of Science:
- Molecular Biology
- Renal Pathophysiology
- Epithelial Cell Biology
Context:
- Renal ischemia-reperfusion (I/R) injury is a significant cause of acute kidney injury.
- The role of RNA methylation, specifically N6-methyladenosine (m6A), in I/R injury is an emerging area of research.
- WTAP is a key component of the m6A methyltransferase complex.
Purpose:
- To investigate the expression of WTAP in a mouse model of renal I/R injury.
- To determine the effect of WTAP knockdown on renal tubular epithelial cells under hypoxia-reoxygenation (H/R) conditions.
- To explore the potential mechanism involving FOXO1 in WTAP-mediated cellular responses.
Summary:
- WTAP expression was significantly upregulated in the kidneys of mice subjected to I/R injury and in human renal tubular epithelial cells (HK-2) exposed to H/R.
- Knockdown of WTAP in HK-2 cells attenuated H/R-induced cell damage, reduced apoptosis, and decreased cell viability.
- WTAP was found to bind to FOXO1 mRNA, and its inhibition led to reduced m6A modification of FOXO1, suggesting a regulatory role in FOXO1 expression.
Impact:
- This study identifies WTAP as a key mediator in renal I/R injury.
- The findings suggest that targeting WTAP and its downstream effects on FOXO1 may offer a novel therapeutic strategy for mitigating kidney injury.
- Provides mechanistic insights into the role of RNA m6A modification in the cellular response to ischemic stress.
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