The fact that organic material is always present and distributed throughout each renal calculus suggests that it may play a role in stone formation. The organic matrix of calcium oxalate (CaOx) crystals freshly generated in urine in vitro contains urinary prothrombin fragment 1 (UPTF1) as the principal protein. In this initial study, matrix was extracted from 12 renal calculi and evaluated for the presence of UPTF1 using Western blotting. UPTF1 was present in all eight stones whose principal component was CaOx, and in one of two stones which consisted mainly of calcium phosphate (CaP). UPTF1 was absent from the two struvite calculi examined. The relationship between CaP and UPTF1 was explored further. Matrix harvested from CaP crystals freshly generated in urine in vitro was also shown to contain UPTF1 as its principal component. Our inability to detect UPTF1 in one mixed CaOx/CaP stone may be related to our methods of matrix retrieval, while its absence from two struvite stones argues against it being present in the other stones merely as a consequence of passive inclusion. This absence may be related to the alkaline environment typical of struvite stone growth. The finding that UPTF1 is present in some renal stones provides the first direct evidence that links blood coagulation proteins with urolithiasis.
Demineralization of calcium oxalate (CaOx) crystals precipitated from human urine in vitro yields an organic crystal matrix extract (CME) consisting predominantly of a single protein which we originally named crystal matrix protein but have subsequently shown to be a urinary form of prothrombin activation peptide fragment 1 (F1). The aim of this study was to determine whether CME is a promoter or inhibitor of CaOx crystallization. The effect of CME on CaOx crystal growth and aggregation was tested using a standard seeded crystallization system, and its effect quantified by use of particle size analysis and a computer model. In addition, the effect of CME on the crystallization of CaOx was tested in undiluted, ultrafiltered human urine using Coulter Counter analysis and scanning electron microscopy. It was shown that CME is a potent inhibitor of CaOx crystal growth and aggregation in a seeded metastable solution. However, of greater significance is that at a concentration of 10 mg/l it completely reversed the formation of large crystalline aggregates that form upon the removal of urinary macromolecules from undiluted urine. It was concluded that CME is the most potent macromolecular urinary inhibitor yet to be tested in urine in vitro. By preventing the aggregation of newly formed crystals, the components of CME may significantly reduce the probability of particle retention in vivo and therefore the occurrence of urolithiasis.
1. A broad spectrum of proteins has been detected within calcium stones. A newcomer to the field of urolithiasis is the blood protein inter-alpha-inhibitor. Inter-alpha-inhibitor comprises three protein chains linked by chondroitin sulphate: two heavy chains, H1 (65 kDa) and H2 (70 kDa) and a light chain (approx. 30 kDa) most commonly known as bikunin. The physiological function of the two heavy chains is unknown; nor has their presence been reported in urine. However, bikunin has been implicated in various renal diseases, including urolithiasis. 2. This study was undertaken to determine which chains of inter-alpha-inhibitor are actually present in calcium kidney stones. Organic extracts were obtained from 10 calcium stones and analysed by SDS/PAGE and Western blotting. The H1 and H2 chains of inter-alpha-inhibitor were detected in 9 of the 10 stones, but only one stone contained a protein with a molecular mass close to that of bikunin (30-35 kDa). 3. These results demonstrate for the first time that H1 and H2 are present in stones and show that the bikunin chain of inter-alpha-inhibitor may not be the only part of the molecule implicated in stone formation.
Urinary prothrombin fragment 1 (UPTF1) is the principal protein in calcium oxalate (CaOx) crystals precipitated from human urine and is a potent inhibitor of CaOx crystallization, a property that should depend, at least in part, upon the extent of ␥-carboxylation of the 10 glutamic residues in its N-terminal region. Warfarin therapy limits full ␥-carboxylation of vitamin K-dependent proteins, including UPTF1. The aims of this study were to determine the effect of warfarin therapy on UPTF1, its occlusion into CaOx urinary crystals, and its influence on the crystallization of CaOx in undiluted human urine. In the first part of the study, urines were collected from six men prior to cardiac surgery and after stabilization on long-term warfarin treatment. Proteins in the urines and in the matrix of CaOx crystals precipitated from them were analyzed by two-dimensional SDS-PAGE and Western blotting. In urine, at least two charge variants of UPTF1 with low isoelectric point (pI) values were detected before and during warfarin therapy, but additional higher pI forms of the protein were also seen during anticoagulation. Nonetheless, the majority of UPTF1 was present in the more fully ␥-carboxylated state. CaOx crystals precipitated from the same urine samples contained only low pI forms of UPTF1. The effect of warfarin treatment on CaOx crystallization in urine was tested by collecting two consecutive 24-h urine samples from 16 men prior to cardiac surgery and during subsequent warfarin treatment. CaOx crystallization was induced in each sample by the addition of sodium oxalate. The size and volume of the particles deposited were determined using a Coulter counter, and the crystals were examined by scanning electron microscopy (SEM). There were no significant differences between the urinary metastable limits before or during warfarin treatment or in the total volume of crystals precipitated. A slight increase in the mean diameter of the crystalline particles precipitated from the urines during anticoagulant therapy was not significant. SEM showed little evidence of changes in overall particle size, although individual crystals of CaOx tended to be larger during warfarin treatment. It was concluded from these studies that the binding of UPTF1 to CaOx crystal surfaces is related to the degree of ␥-carboxylation of its Gla domain, which would also influence the protein's inhibitory effects on CaOx crystallization. However, during warfarin therapy the majority of UPTF1 exists in a highly charged state, indicating that it is completely, or almost completely, ␥-carboxylated, which would explain the lack of any difference between CaOx crystallization parameters in the urine of subjects before and during warfarin administration. We conclude that physiologically significant reductions in the inhibitory potency of UPTF1 would be likely to occur only as a result of proscription of ␥-carboxylation more extensive than that induced by
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