Quantification of Proteins Extracted from Calcium Oxalate and Calcium Phosphate Crystals Induced in vitro in the Urine of Healthy Controls and Stone-Forming Patients

Abstract

We have previously identified proteins extracted from calcium oxalate (CaOx) and calcium phosphate (CaP) crystals generated experimentally in vitro in whole urine of healthy controls and stone formers. No significant differences were detected between protein components in matrices of crystals obtained from both groups. The aim of the present study was to estimate the amounts of six proteins identified earlier in order to investigate the differences, if any, between healthy controls and lithiasis patients. CaOx and CaP crystals were generated in the urine samples by adding an oxalate and phosphate load, respectively. Crystals were harvested, washed, dried, and analyzed. Crystal matrix protein was extracted by demineralizing crystals with EDTA solution, analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and identified immunochemically using Western blot analysis. The quantity of each protein was estimated by densitometric analysis. The predominant proteins found in organic matrices of CaOx crystals induced in the urine of healthy controls were prothrombin-related proteins followed by albumin and osteopontin. In matrices of CaP crystals, the principal proteins were Tamm-Horsfall protein followed by albumin, prothrombin-related proteins and osteopontin. However, when crystals were induced in the urine of stone formers, albumin was the major component of the organic matrix of both CaOx and CaP crystals. We concluded that a subset of urinary proteins is present in crystal matrix. Among them, albumin seems to play a crucial role in kidney stone formation.