In order to understand the mechanism of stone genesis, it is essential to determine the characteristics of macromolecules constituting the urinary stones. We characterized proteins from the inner core and outer matrix of calcium oxalate (CaOx) renal stones.
Inner core and outer matrix of CaOx renal stones were separated and proteins were extracted with a buffer containing SDS and beta-mercaptoethanol. Proteins were analyzed and purified by SDS-PAGE and RP-HPLC respectively. The protein bands from gel and protein fractions were sequenced by MALDI TOF mass spectrometry. ELISA, western and slot blot immunoassays were performed to confirm the identity of the proteins in stones and urine of the stone formers. The potential of the identified protein as an effective promoter or inhibitor was assessed by observing their effects on CaOx crystallization using aggregometer.
The inner core extract predominantly exhibited protein species in the molecular weight range of 12-14 kDa. However, a 66 kDa band, identified as osteopontin was also detected in the inner core along with outer matrix and in the urine of stone formers and non stone formers. Purification of low molecular weight proteins was carried out by reversed phase HPLC. Tandem mass spectrometry analysis identified them as myeloperoxidase chain A (MPO-A), alpha-defensin, and calgranulin. ELISA, western blot and slot-blot immuno-assays further confirmed their presence restricted to the inner core and not in the outer matrix. Turbidity assays showed that low molecular weight renal stone proteins promoted the aggregation of CaOx crystals.
Persistent hyperoxaluria leads to tubular epithelial injury, resulting in the release of these anti-inflammatory proteins. These proteins could have been first adsorbed on CaOx crystals thereby become a part of nucleation process leading to inner matrix formation.