Background In lupus, disease phenotype results from gene expression changes not only in the immune effector cells but also in the target organs. We have previously used miRNA analysis in the peripheral blood of patients with SLE in order to identify novel molecular pathways contributing to immune response deregulation.
Objectives We sought to identify novel genes within the kidney in lupus nephritis by miRNA analysis.
Methods We isolated RNA from renal biopsy samples of 12 subjects with proliferative or membranous lupus nephritis and 5 healthy controls. MiRNA expression was analysed by TaqMan Low Density Arrays (TLDA human miRNA v1.0). We used two different bioinformatic algorithms (TargetScan, PicTar) for the prediction of the gene targets of selected differentially expressed microRNAs. Validation of the miRNA-gene target interaction was performed by luciferase assay and real-time PCR analysis. MiRNA expression levels were assessed in kidney extracts from 2 months and 6 months lupus-prone (NZB/W F1) mice by real-time PCR.
Results Compared to normal tissue a 24-miRNA signature defines human lupus nephritis with 9 miRNAs up-regulated and 15 miRNAs down-regulated, with miR-422a exhibiting the highest up-regulation (17.2-fold). Bioinformatic analysis predicted that miR-422a has a binding site in the 3’UTR of kallikrein 4 (KLK4) gene. This was validated by overexpression of miR-422a, which suppressed by 65% KLK4 luciferase activity and by 82% KLK4 mRNA levels in 293HEK cells. To monitor miR-422a/KLK4 expression during lupus nephritis progression, we used NZB/W F1 lupus mice. At early stages (2 months old NZB/W F1 mice) miR-422a was 4.1-fold up-regulated, while KLK4 mRNA levels were 3.4-fold down-regulated while at later stages (6 months) miR-422a was 9.4-fold up-regulated and KLK4 mRNA levels were 7.6-fold down-regulated. Ongoing experiments using antagomirs for miR-422 will further define its contribution to nephritis together with the construction of gene networks -by combining peripheral blood and renal microRNA data, are in progress.
Conclusions The kallikrein family of genes has an important role in regulating inflammation, apoptosis, coagulation and fibrosis in the kidneys. Our data implicate KLK4, a secreted serine esterase with angiogenic and extracellular matrix remodeling properties in the pathogenesis of immune-mediated nephritis.
Disclosure of Interest None Declared