Abstract: SA-PO105
In Vitro and Ex Vivo Exploration of the Expression of MicroRNAs to Assess the Progression of AKI
Session Information
- AKI: Mechanisms - Primary Injury and Repair - II
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Masih, Annie, University College Dublin, Dublin, Ireland
- Ng, Su wei, University College Dublin, Dublin, Ireland
- Murray, Patrick T., UCD School of Medicine and Medical Science, Dublin 7, Ireland
- Doran, Peter P., University College Dublin, Dublin, Ireland
Background
MicroRNAs are endogenous short, non-protein coding RNAs that post-transcriptionally/translationally control protein expression by binding to target mRNA. There is increasing interest in miRNAs in disease research because of their ability to coordinate the regulation of protein expression by influencing multiple signalling pathways. Some miRNAs have previously been shown to be responsive to AKI, suggesting they may be mediators of the damage and repair processes and potential markers. To build on this work, we have sought to determine miRNA expression in AKI using a combination of in vitro and ex vivo models.
Methods
We first sought to categorise the urinary expression of selected miRNAs to determine their biological importance in AKI. Recognising the challenges of recovering low abundance transcripts in urine, we sought to firstly validate our experimental approach. We found that RNA recovery was similar in urine before centrifugation and after centrifugation. We further compared the RNA recovery rates between normal sample and exosome enriched samples, demonstrating that whilst exosome enrichment did not increase the overall yield of miRNAs, it did result in improved amplification profiles. Having demonstrated experimental validity, miRNA was assessed in urine samples obtained from patients with KDIGO Stage 2 and 3 and samples from non-AKI donors. Expression of miR-30a-e, miR-192, miR-101-3p was reduced in KDIGO 3 and KDIGO 2 compared to healthy control samples.
Results
Having shown that these miRNAs are differentially regulated in AKI ex vivo, we next sought to categorise in vitro. For these investigations, HK2 tubule epithelial cells were cultured with 10 µg/ml, 1 mg/ml and 0.1 mg/ml of LPS for 2, 4, 6, 12 and 24 hours. Following stimulation, RNA was extracted, cDNA prepared, and RT-PCR conducted to determine the expression of miR-30c, miR-101-3p and miR-192.
Conclusion
These investigations demonstrated LPS dysregulation of these target miRNAs in a time and concentration dependent manner. These data suggest these mediators respond to tubule injury and thus may be useful as early markers of AKI, as well as contributing to the changes in transcription which underpin the initiation, progression and outcome of AKI. Further investigations will focus on delineating the functional importance of MiRNAs as molecular drivers of AKI.