AbstractCurrent biomarkers in Lupus nephritis (LN) are not sensitive or specific enough to predict renal outcome. Recent data support the use of urinary exosomes as a source of biomarkers of renal origin, with special relevance in their miRNA content. Some studies have identified several miRNAs associated with disease activity and fibrosis formation, but no studies on prognostic biomarkers have been conducted. Methods To identify prognostic biomarkers, we performed a miRNA microarray on urinary exosomes from patients with active LN, analyzing their results according to the response to standard therapy (7 responders and 7 non-responders). Validation studies were performed by RT-qPCR technique in a new LN cohort (responders=21 and non-responders=22). Among the miRNA of interest, a comparative study was conducted with serum levels in the same cohort as well as with a healthy control group and non-lupus nephropathy cohort. Subsequently, in situ renal tissue hybridization and in vitro studies were performed to identify the origin and cell target of these exosomes at renal level and to understand their mechanism of action. For each differentially expressed miRNA, potential target genes were predicted through miRNA-target datasets. Results Responder patients expressed significantly increased levels of miR-31-5p, miR-107 and miR- 135b-5p in urine and renal tissue compared to non-responders. MiR-135b-5p exhibited the best predictive value to discriminate responder patients (AUC= 0.783 [95% confidence interval, 0.640 - 0.926], cut-off > 0.0884 with 77.8% sensitivity and 71.4% specificity). MiRNAs expression was mainly located in the tubular structure, demonstrating, in addition, greater formation on exosomes enriched with miR-31-5p, miR-107 and miR-135b-5p present in this lineage compared to endothelial glomerular and mesangial cells (p < 0.0001). Regarding the target of these exosomes, a faster rate of internalization of responders' urinary-derived exosomes was observed in both endothelial glomerular and mesangial cells (p = 0,001 y 0,0002), and there was greater capacity of internalization on mesangial cells (90% vs. 50%, p < 0.0001). The miR-135b-5p demonstrated ability to inhibit the proliferation of mesangial cells (p < 0.01). In addition, the analysis of the different biological targets demonstrate globally a role of these miRNAs in the modulation of the inflammatory response, with reduction of cytokines, chemokines and adhesion molecules levels, a modulatory effect on angiogenesis, as well as a marked antifibrosing capacity. The pathway analysis identified nine common targets relevant to renal recovery, one of them, HIF-1α, common for the 3 miRNA of interest. The 3 miRNAs demonstrated a modulating effect on the HIF-1α pathway through its inhibition. Conclusions Levels of miR-135b-5p, miR-31-5p and miR-107 are able to predict the response to treatment at the time of the renal flare and during follow-up in patients with NL, specifically. The origin of the exosomes enriched with these miRNAs appears to be at the level of tubular cells while the mesangial and glomerular endothelial cells appear as the main effectors of their biological actions. HIF-1α was identified as the common target of these 3 miRNAs, causing the presence of these miRNAs suppression of the expression levels of this pathway, considered key in the pathogenesis of NL.
|Date of Award||10 Oct 2019|
|Supervisor||Josefina Cortés Hernández (Director) & Albert Selva O Callaghan (Tutor)|