Biochimica et Biophysica Acta (BBA) - General Subjects
The association of different urinary proteins with calcium oxalate hydromorphs. Evidence for non-specific interactions
Introduction
The associations between biominerals and proteins have been the subject of much investigation in recent years. Examples in controlled systems include the highly acidic proteins found within the structure of sea urchin spines [1] and collagen which modulates calcium phosphate crystallisation and influences bone formation [2]. As well as functional biominerals, proteins also associate with pathological minerals. Osteopontin, for example, interacts with the calcium phosphate that contributes to atherolsclerotic plaques and oral plaque [3]. Urolithiasis is another example of uncontrolled biomineralisation, and, as is true in controlled systems, organic matter is now believed to play a fundamental role in the structural control of the mineral phase. Whether such biological materials are part of a mechanism that usually prevents such mineralisation and has become overwhelmed by the excess crystal formation of the system or whether the organic matter associates with the mineral phase through chance interactions remains debated. It is argued that evidence of specific interactions would imply a degree of protein-mediated control in the prevention of such processes.
Kidney stones are common, affecting up to 12% of adult American males [4]. The majority of kidney stones have calcium oxalate (CaOx) as their principal component. It is recognised however, that as well as the mineral phase, kidney stones contain between 2 and 5 wt.% of an organic matrix, a majority of which is protein [5]. Many urinary proteins inhibit or promote crystal nucleation, growth and aggregation [6], [7], they could control crystal attachment to the lumen of kidney tubules [8] and they could form intra-crystalline inclusions through which crystal structures can be degraded via protease actions [9], [10]. The possibility that proteins have a role in the prevention, or indeed, promotion of urolithiasis, and the observations that only a few of the many urinary proteins are found associated with calcium salts grown in vitro [11], [12] imply that specific interactions occur. On the other hand, in vitro studies have demonstrated that similar urinary proteins associate with both calcium phosphate and CaOx crystals when grown in human urine [13]. The interpretation of protein–CaOx interactions are further complicated as three hydromorphs of CaOx can exist. Two of these–the monohydrate (COM) and dihydrate (COD)–are commonly found in stones, with the trihydrate occurring rarely. COM and COD stones have different physical properties, with COM stones often consisting of radial concretions [14] whereas COD stones are often made of aggregates of individual crystals [15]. It is possible that some of the differences are due to the influence of proteins and the organisation of the matrix. If specific proteins were to interact with particular hydromorphs this would imply that the formation of different types of stone is controlled by distinct mechanisms or growth modifications.
Growing crystals in urine has been adopted as the basis for a wide number of studies into the protein–crystal interactions that are deemed relevant to stone formation [11], [12], [13], [16], [17]. Few reports, however, consider which CaOx hydrates are present or try to examine more than one hydromorph using the same method. Where this approach has been taken it has been suggested that osteopontin (OP) primarily associates with COD and that urinary prothrombin fragment 1 (UPTF1) has a greater affinity for COM [18], [19]. The different growth conditions used to achieve the formation of specific CaOx hydromorphs in vitro may, however, affect the protein–crystal interactions and these observations could be artefacts of the experimental design. Another distinction that needs to be considered is the difference between crystal-matrix (internal) protein and crystal-binding (surface associated) protein [11], [16]. When both types of interaction are possible, as with crystals grown in urine, this may add to the difficulties in the interpretation of results. The present investigation was devised to control the hydromorphs used and to avoid crystal growth by using pure COM or COD. These crystals were then incubated in CaOx-saturated urine samples to allow the identification of protein–surface interactions.
Section snippets
Growth of pure calcium oxalate hydromorphs
COM and COD were grown as described previously [20]. In brief, COM was grown by adding 30 ml of 0.5 M (COOH)2 dropwise to 500 ml of a stirred solution of 0.2 M CaCl2.6H2O at pH 6. This mix was incubated at 30 °C for 30 min. COD was grown in a 1 l solution of 38.5 mM Na3 citrate, 46.2 mM MgSO4.7H2O and 254.8 mM KCl. 500 ml of 25.1 mM CaCl2 was added, mixed, adjusted to pH 6 and left to equilibrate to 22 °C. 500 ml of 6.4 mM Na2(COO)2 was added dropwise, with continuous stirring and the solution
Results
TGA and XRD analyses confirmed that the crystal populations consisted of pure COM and pure COD with no XRD peaks corresponding to other CaOx hydromorphs. The TGA decomposition curves of the appropriate samples matched theoretical decomposition data for COM and COD (Fig. 1).
The average surface area of the COM crystals was 0.76 m2/g (n = 3, range = 0.53–0.91 m2/g). The average surface area of the COD crystals was 0.45 m2/g (n = 3, range = 0.31–0.54 m2/g).
SEM of the CaOx crystals showed two distinct
Discussion
When exploring the interactions between urinary proteins and CaOx, previous publications have focused on either one specific hydromorph [26], [27], on mixed (or uncharacterised) populations grown in human urine [16], [28], or populations grown in urine under very different conditions to obtain the preferential growth of either COM or COD [18], [19]. The present study differs as it has used the same protocol with pure COM and COD, and, as no further crystal growth occurred in the experiments,
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