Protein Analysis Core

University Of Chicago, Chicago IL

Investigators

Linked publications, trials & patents

Abstract

Summary Core D will conduct all protein-related analyses for Projects 1, 2, and 3. Human urine samples will be collected by Projects 1 and 2 from highly selected and well-characterized stone-former and normal patient pools. Human kidney interstitial tissue will be collected by biopsy (Project 2) and subsequent laser microdissection (LMD) (Project 3) from those same patient pools. We will quantify inflammation in the urine samples using high sensitivity multiplexed magnetic particle-based enzyme-linked immunosorbent assay (MP-ELISA) assays in 1) idiopathic hypercalciuria (IH) of both papillary types and in normal subjects with high salt diet; 2) conditions of high and low sodium diets with and without potassium citrate; 3) whether high papillary injury scores correlate with inflammation in either urine or LMD samples; 4) patients with and without non-obstructive stone pain; and 5) patients with non-obstructive stone pain before and after surgical removal of collecting duct plugs. Exploiting our ability to obtain highly homogeneous LMD samples, we will use MP-ELISA to measure key toll-like receptor pathway-related molecules in the interstitium and thin limb cells away from and near plaque. We will measure osteopontin, THP, CD59, bikunin, and calgranulin (A, B and C) expression in regions of plaque and crystal-free interstitium near plaque and compare them to non-stone forming controls. We will assess oxidative injury in areas near or distant from plaque compared to plugging by targeting oxidized proteins and quantifying them by LFQMS of biotin hydrazide-derivatized protein carbonyls. We will also exploit our unique ability to combine sensitive LFQMS with the specificity of LMD to investigate currently unknown pathways or cellular mechanisms of plugging or plaque that could serve as biomarker panels or drug targets. We will analyze regions of interstitium from papillary biopsies that do and do not contain apatite plaque comparing affected areas to non- affected sites within the same individuals in the papillae with plaque and/or plugging calcium stone disease and extend these investigations to plaque overgrowth sites to identify and quantify proteins unique to Randall's plaque to examine the protein content of the plaque-stone interface