O81 Isolation of MicroRNA from the Proximal Tubules of Archival

Isolation of MicroRNA from the Proximal Tubules of Archival Renal Biopsies
utilising Laser Capture Microdissection
1, 2 Geraint
J.R. Dingley, 1Juan C. Mason, 1, 2Sara K. Campbell, Paul S. Bass3 and
E. Collins
Wessex Renal and Transplant Service, Queen Alexandra Hospital, Portsmouth UK
Clinical 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 of miRs are implicated in mechanisms of tubulointerstitial fibrosis in renal
disease. The same miRs 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 tubules respectively 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 with qPCR.
Results: In seven patients with diabetic nephropathy with evidence of moderate to
severe tubulointerstitial fibrosis on biopsy, miRs 192 (p=<0.0001), 194 (p=<0.0001)
and 30c (p=<0.005) were downregulated 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.