2011
Isolation of MicroRNA from the Proximal Tubules of Archival Renal Biopsies utilising Laser Capture Microdissection
1, 2Geraint J.R. Dingley, 1Juan C. Mason, 1, 2Sara K. Campbell, Paul S. Bass3 and 2Jane E. Collins
1 WessexRenal and Transplant Service, Queen Alexandra Hospital, Portsmouth UK 2Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, UK.3Depatrment of Cellular Pathology, Royal Free Hospital, London, UK.
Introduction:MicroRNAs (miRs) are short noncoding RNAs that play important roles in regulating mammalian gene expression by inducing posttranscriptional gene repression by blocking protein translation or by inducing mRNA degradation. Altered regulation ofmiRs are implicated in mechanisms of tubulointerstitial fibrosis in renal disease. The samemiRs are however differentially expressed in different cell populations within the kidney and so to understand the role of miRs in the kidney it is necessary to quantify microRNA expression from individual cell types.
Methods: Three serial sections were cut through archival renal biopsy specimens from patients with the single diagnosis of Diabetic Nephropathy with evidence of moderate to severe tubulointerstitial fibrosis on biopsy. The top and bottom sections were stained with lectins to identify the proximal and distal tubulesrespectively and the middle section was stained with Cresyl Violet for Laser Capture Microdissection. Proximal tubules were identified on the lectin stained slides by light microscopy and the corresponding tubules on the Cresyl violet slide were excised using laser capture. Proximal tubules from morphologically normal kidney tissue, which had been fixed in formalin and embedded in paraffin for a similar period as the biopsies were also dissected using the same technique to provide a control group. RNA was extracted from the tissue using modifications to a commercially available kit and the RNA underwent reverse transcription prior to quantification withqPCR.
Results:In seven patients with diabetic nephropathy with evidence of moderate to severe tubulointerstitial fibrosis on biopsy, miRs192 (p=<0.0001), 194 (p=<0.0001)and 30c (p=<0.005)weredownregulated over two fold and miRs 23a (p=<0.003) and 146a (p=0.0008) were up regulated over two fold relative to expression in normal tissue from seven normal specimens. The findings with miR-192 and 146a are in keeping with work from other groups using alternative methods.
Conclusion: MicroRNAs can be isolated from the proximal tubules of archival renal biopsies specimens,quantified in a reproducible manner and show differential expression in disease. Understanding the role of microRNAs in the development of tubulointerstitial fibrosis may identify targets for therapy in the future.