PROJECT 4

Objectives: 
ESR4 explores the hypothesis that uremic microcalcification is not only caused by phosphate retention, but also by persistent inflammation, vitamin K deficiency, inactive MGP, and increased vascular expression of apoptotic genes and bone proteins. We also plan to study 1) progerin expression and markers of DNA repair deficiency and premature senescence, 2) chemical anatomy of vascular lesions associated with CKD, 3) relate uremic calcification to markers of established and novel markers of bioageing, 4) test relation between uremic calcification and ex vivo endothelial function and 5) relate the uremic microbiota to premature vascular ageing.

Methodologies: 
1. About 120 ESRD patients are tested at time of LD-RTx. Vascular biopsies are scored for fibrosis and calcification and stained for inflammatory markers, bone proteins, elastin, collagen and progerin. We analyse inhibitors and promoters of calcification, TMAO, protein-bound uremic toxins, calcification propensity, miRNAs, apoptotic genes in calcified vs. non-calcified vessels, genotypes of established as well as novel candidate genes, and gene specific DNA methylation, allowing exploration of signalling pathways. In addition, results from ESR3 on novel biomarkers predicting calcification and miRNA data from ESR1 and data on fetuin-A and CPPs from ESR12 will be validated and tested.
2. Uremic and control epigastric arteries obtained from patients and controls will be quantified for progerin expression, and markers for DNA repair deficiency and premature senescence, using IF microscopy. Droplet digital PCR will be used to screen DNA for the presence of low-degree mosaicism for nucleotide variants that result in increased progerin production.
3. Small arteries will be isolated from sc uremic fat. Functional properties of vessels will be studied using isometric ‘wire’ and perfusion myography, which is a reliable method for acutely assessing endothelial function response to pharmacological agonists before and after application with MV, while allowing studies on long-term effects and evaluates shear stress, pressure-mediated pharmacological activation, with different agonists on endothelium-derived factors and uremic microvesicles.