Personalised Medicine in ANCA-Associated Vasculitis: The Role of Urine Soluble CD163

Abstract

In this thesis I have identified a highly sensitive and specific association between urinary sCD163 and active renal vasculitis in the clinical setting of renal vasculitis flare in both retrospective and prospective studies. To define clinical caveats in usCD163 interpretation, I have explored the influence of high-grade proteinuria on usCD163 interpretation. There is a strong biologic rationale for usCD163 in the monitoring of renal vasculitis activity as it is a marker of macrophages and is strongly expressed in glomerular crescents. In crescentic glomerulonephritis there is direct shedding of soluble CD163 protein from the glomerular crescent cell surface directly into the urinary space leads to elevated levels. usCD163 is easily measured by commercial ELISA with this thesis validating a diagnostic grade assay. usCD163 is elevated in subtle renal vasculitis flare and is superior to uMCP-1: In collaboration with the Vasculitis Clinical Research Consortium usCD163 and uMCP-1 were measured in a serially sampled longitudinal multicentre cohort with clinically mild renal vasculitis. Both biomarkers were elevated in the presence of active renal vasculitis, with usCD163 displaying superior area under the curve than uMCP1, 0.794 and 0.687, respectively. usCD163 and uMCP1 correlated poorly with r2 of 0.11, highlighted their differing roles in glomerular macrophage recruitment and activation. In subtle active renal vasculitis, the moderate clinical utility of each biomarker in isolation was enhanced by using usCD163 to exclude active vasculitis, and then grouping the ?usCD163 positive / uMCP1 positive? and ?usCD163 / new proteinuria? as the two ?Yes? nodes, giving a positive LR of 19. This decision tree approach increased diagnostic precision and incorporated proteinuria. usCD163 is diagnostic of renal vasculitis flare: Prospective enrolment of patients with known AAV presenting with potential renal vasculitis flare was performed in a multicentre cohort. usCD163 was measured by diagnostic and research grade ELISAs. usCD163 was elevated in renal vasculitis flare with concentrations remaining low in renal vasculitis flare mimics such as sepsis, isolated hematuria and non-vasculitic acute kidney injury. usCD163 displayed exceptional biomarker characteristics in this setting with AUC of 0.95 with superiority to RBC casts, BVAS criteria and changes in serum creatinine. usCD163 displayed similar biomarker characteristics to the current ?gold standard? of kidney biopsy. The use of a diagnostic grade sCD163 assay has enhanced the potential clinical translationof usCD163 as a diagnostic test for active renal vasculitis.usCD163 is elevated in high-grade proteinuria but correction for urinary protein attenuates usCD163 concentrations in those without active renal vasculitis: Loss of integrity of the glomerular filtration barrier may lead to detection of serum sCD163 in urine. To address this diagnostically relevant potential caveat in usCD163 interpretation we studied usCD163 in (1) primary nephrotic syndrome and (2) renal AAV with and without proteinuria. In primary nephrotic syndrome usCD163 concentrations were hypothesised to be undetectable as there is no local source of sCD163 production, however there is extensive foot process effacement with potential for passage of serum sCD163 across the glomerular filtration barrier leading to detection in urine. In primary nephrotic syndrome with proteinuria >3.5g/day, usCD163 concentrations were elevated. This signal was subsequently attenuated when sCD163 concentrations were corrected for urine protein and albumin concentrations. In renal AAV usCD163 concentration was increased in remission AAV with persistent proteinuria compared to remission AAV without proteinuria, but concentrations remained significantly less than those with active renal vasculitis. The sCD163 ratio of serum to urine protein and albumin values was calculated to provide an estimate of local glomerular sCD163 production. Normalising usCD163 to total urine protein, albumin and sCD163 ratio of serum to urine protein and albumin values yielded similar results, with no significant difference between remission proteinuric and remission non-proteinuric subjects, but concentrations remained elevated in active renal vasculitis. The simple normalisation of usCD163 to total urine protein performing marginally better than the more complex fractional excretion of protein: sCD163 ratio with AUC values of 0.93 and 0.91, respectively. Therefore, in patients with nephrotic range proteinuria, normalisation of the usCD163 value to total urine protein is the method of greatest clinical utility for non-invasive identification of active renal vasculitis. Validation of a Diagnostic Grade Test: A key step in the translation of usCD163 from research to diagnostic grade assay was collaboration with industry to develop a diagnostic usCD163 assay. We rigorously validated both commercially available research grade and in-house research grade assays and determined that the R&D Systems Duoset sCD163 assay had the most optimal operating characteristics for detection of active renal vasculitis. We then partnered with industry to develop a diagnostic grade usCD163 ELISA. The optimal research grade usCD163 assay was used to validate the diagnostic grade assay. This assay has received the CE marking, ISO 15189 and NEQAS accreditation allowing widespread adoption by clinical laboratories and is now commercially available.