MicroRNA-5010-5p ameliorates high-glucose induced inflammation in renal tubular epithelial cells by modulating the
Sunghee Choi1, Mithun Kumer Sarker1, Mi Ra Yu1
1Hyonam Kidney Laboratory, Seoul, Korea (the Republic of).
Introduction:
We previously reported the significant upregulation of eight circulating exosomal microRNAs (miRNAs) in patients with diabetic kidney disease (DKD). However, their specific roles and molecular mechanisms in the kidney remain unknown. Among the eight miRNAs, we evaluated the effects of miR-5010-5p on renal tubular epithelial cells under diabetic conditions in this study.
Research Design And Methods:
We transfected the renal tubular epithelial cell line, HK-2, with an miR-5010-5p mimic using recombinant plasmids. The target gene of hsa-miR-5010-5p was identified using a dual-luciferase assay. Cell viability was assessed via the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Moreover, mRNA and protein expression levels were determined via real-time PCR and western blotting, respectively.
Results:
High glucose levels did not significantly affect the intracellular expression of miR-5010-5p in HK-2 cells. Transfection of the miR-5010-5p mimic caused no change in cell viability. However, miR-5010-5p-transfected HK-2 cells exhibited significantly decreased expression levels of inflammatory cytokines, such as the monocyte chemoattractant protein-1, interleukin-1β, and tumor necrosis factor-ɑ, under high-glucose conditions. These changes were accompanied by the restored expression of phosphorylated AMP-activated protein kinase (AMPK) and decreased phosphorylation of nuclear factor-kappa B. Dual-luciferase assay revealed that miR-5010-5p targeted the gene, protein phosphatase 2 regulatory subunit B delta (PPP2R2D), a subunit of protein phosphatase 2A, which modulates AMPK phosphorylation.
Conclusions:
Our findings suggest that increased miR-5010-5p expression reduces high glucose-induced inflammatory responses in renal tubular epithelial cells via the regulation of the target gene, PPP2R2D, which modulates AMPK phosphorylation. Therefore, miR-5010-5p may be a promising therapeutic target for DKD.
Insights
This study shows that miR-5010-5p reduces inflammation in diabetic kidney disease (DKD) by targeting PPP2R2D and modulating AMPK phosphorylation. This microRNA may offer a new therapeutic strategy for DKD.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Diabetic kidney disease (DKD) is associated with upregulated circulating exosomal microRNAs (miRNAs).
- The specific roles and mechanisms of these miRNAs in kidney pathology remain largely unknown.
- This study focuses on miR-5010-5p, one of the eight upregulated miRNAs, to investigate its function in renal tubular epithelial cells under diabetic conditions.
Purpose of the Study:
- To investigate the functional role of miR-5010-5p in renal tubular epithelial cells under high glucose conditions.
- To identify the molecular targets and pathways regulated by miR-5010-5p in the context of diabetic kidney disease.
- To evaluate the potential of miR-5010-5p as a therapeutic target for DKD.
Main Methods:
- Transfection of HK-2 renal tubular epithelial cells with an miR-5010-5p mimic.
- Dual-luciferase assay to identify the target gene of miR-5010-5p.
- Cell viability assessment using MTT assay.
- Real-time PCR and western blotting to determine mRNA and protein expression levels.
Main Results:
- High glucose did not affect intracellular miR-5010-5p expression or cell viability.
- miR-5010-5p mimic transfection significantly decreased inflammatory cytokines (MCP-1, IL-1β, TNF-α) under high glucose.
- miR-5010-5p restored phosphorylated AMP-activated protein kinase (AMPK) and decreased phosphorylated nuclear factor-kappa B (NF-κB).
- Dual-luciferase assay identified PPP2R2D as a direct target of miR-5010-5p, which modulates AMPK phosphorylation.
Conclusions:
- Increased miR-5010-5p expression mitigates high glucose-induced inflammation in renal tubular epithelial cells.
- This effect is mediated through the regulation of PPP2R2D, which influences AMPK phosphorylation.
- miR-5010-5p represents a potential therapeutic target for managing diabetic kidney disease.
