Investigation of microRNAs as markers of human vascular dysfunction in kidney disease
Scullion, Kathleen Maria
Vascular dysfunction commonly co-exists with kidney disease, which results in a substantially increased risk of cardiovascular disease (CVD). A severe, acute form of this vascular-renal phenotype is seen in those presenting with systemic vasculitis associated with autoantibodies to neutrophil cytoplasmic antigens (ANCA) - a rare inflammatory disorder. The most frequent severe manifestation of ANCA vasculitis involves endothelial injury giving rise to rapidly progressive glomerulonephritis in the kidney. Despite current treatments, overall survival remains poor with many patients suffering chronic inflammation, a major contributor to the development and progression of both CVD and chronic kidney disease (CKD). Indeed, those with CKD have a substantially higher chance of dying from CVD than of progressing to end-stage renal disease (ESRD). Furthermore, those who respond to treatment remain at risk of further disease relapses. microRNAs (miRs) are selectively expressed in certain tissues and cell states, which makes them potential biomarker candidates. In the circulation, the miRs are bound to Argonaute 2 proteins or encapsulated in extracellular vesicles (ECVs). This makes them resistant to RNase degradation providing further support for their potential use as novel biomarkers. miR-126 is enriched in endothelial cells and is a regulator of vascular integrity and angiogenesis. miR-126 was measured in patients with active vasculitis and once in treatment-induced remission. At disease presentation, miR-126 was at a low concentration and increased with successful treatment. Patients with vasculitis - both active disease and in remission states - had lower circulating miR-126 than in healthy volunteers. miR-126 was also measured in patients with ESRD to establish whether it was also a marker of chronic vascular dysfunction. Circulating levels were measured in patients receiving haemodialysis before and after the treatment. Before haemodialysis, miR-126 was at a low level in circulating blood and increased after the treatment. The levels of miR-126 in patients with CKD were also measured and were at a higher concentration than patients with ESRD. These data suggest that miR-126 has a potential role as a biomarker of vascular health and could track the progression of vascular disease. Using small RNA sequencing, the miR profiles of patients with active vasculitis and once in remission were established. These results were analysed in order to determine appropriate miRs for biomarker discovery. Using the data obtained, the miR hits were measured in larger patient cohorts for validation. This did not result in the discovery of a specific miR biomarker for the identification of acute vascular dysfunction in humans. ECVs were isolated from healthy volunteers and patients with active vasculitis. Uptake of ECVs was established in human and mouse macrophages, as well as renal proximal tubules in vitro. This did not result in increased cytokine production in the cultured macrophages or renal cells. This suggests that uptake is possible, however, the ECV cargo does not activate immune and renal cells in vitro. These findings, therefore, show that circulating miRs are potential markers of acute and chronic human vascular dysfunction. miR-126 was a successful marker for differentiating between patients with active disease and treatment-induced remission. The miR profile of these patients did not differ as determined by RNA sequencing. In addition, ECVs can be isolated from patient blood and taken up by recipient macrophage and renal proximal tubule cells, however, this does not result in a phenotypic change. These data suggest that miR-126 has a potential role as a biomarker of vascular health and could track the progression of vascular disease and response to treatment in acute and chronic vascular dysfunction.