MicroRNA expression in mesangial proliferative glomerulonephritis (MsGN)

Laura Denby, Hollie Robinson, Dianne Z Hillyard, Alan G Jardine, Andrew H Baker. BHF Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, United Kingdom.

It is estimated that 10% of the global population are affected by chronic kidney disease. Patients with renal dysfunction have a high cardiovascular risk, ∼10x that of the normal population. MsGN affects ∼2% of the population and transforming growth factor β (TGFβ) plays a pivotal role in disease pathogensis. MicroRNAs (miRNAs) are endogenous non-coding RNAs which are ∼22 nucleotides in length and regulate gene expression by binding to the 3’UTR of the mRNA which results in translational repression or target degradation. They have now been found to play a fundamental role in the pathophysiogy of many diverse diseases including cancer and cardiac hypertrophy. The aim of this work was to determine if microRNA expression is altered in the anti-Thy1.1 model of MsGN. The model provides a reproducible immune-mediated glomerulonephritis which is self-limiting.

MsGN was induced by a single or multiple (x3, 1 week apart) injections of anti-Thy1.1 antibody, ER4, with sacrifice at 7 or 14 days. As matrix expansion and mesangial proliferation are key features of MsGN we examined the gene expression of collagen and several members of the metzincin superfamily of metalloendopeptidases in the kidney by qRT-PCR. Animals who received multiple ER4 injections at 7 days exhibited a significant 3-fold upregulation of tissue inhibitor matrix metalloproteinase-1 while expression of matrix metalloproteinase (MMP)-9 was reduced by 88%. Furthermore, 14 day animals exhibited significant upregulation of multiple genes involved in matrix metabolism and turnover with MMP-9 expression reduced to 93% of control animals. We also examined the expression of TGFβ regulated disintegrin and metalloproteinases (ADAMs), ADAM-12, -17,-19 and -33. ADAM-12 and -19 had moderate but significant increases in gene expression at 7 and 14 days. To examine miRNA expression a screen was performed on animals with MsGN induced by single ER4 injection, multiple injections or age-matched controls and sacrificed at 7 days. In animals who received 3 injections miR-214 was significantly increased and miR-30 family members were significantly decreased. Validation of miR-214 demonstrated that expression was significantly increased by 2-fold at 7 days, rising to 5-fold at 14 days. MiR-21 expression was 3-fold that of control animals at day 7 and 14. We also used an in vitro model and quantified miR-214 and -21 expression in a rat tubular epithelial cell line (NRK52E) and mesangial cell line (CRL-2753) stimulated with TGFβ (10ng/ml). MiR-214 expression was increased 4-fold 24 hrs-post stimulation in CRL-2753 cells and remained upregulated over the time course. Similarly in NRK52E cells miR-214 was increased by a maximum of 3-fold during the 4 day time course. MiR21 expression was unaltered in CRL-2753 cells, however, was increased to a maximum 2.5-fold in NRK52E cells.

In a rat model of MsGN we show altered expression of the metzincin superfamily and collagen. MiRNA-214 and -21 expression is significantly increased and are regulated by TGFβ suggesting miRNAs and their targets are important in disease development.