Reverse engineering the kidney: modelling calcium oxalate monohydrate crystallization in the nephron.

Crystallization of calcium oxalate monohydrate in a section of a single kidney nephron (distal convoluted tubule) is simulated using a model adapted from industrial crystallization. The nephron fluid dynamics is represented as a crystallizer/separator series with changing volume to allow for water removal along the tubule. The model integrates crystallization kinetics and crystal size distribution and allows the prediction of the calcium oxalate concentration profile and the nucleation and growth rates. The critical supersaturation ratio for the nucleation of calcium oxalate crystals has been estimated as 2 and the mean crystal size as 1 mum. The crystal growth order, determined as 2.2, indicates a surface integration mechanism of crystal growth and crystal growth dispersion. The model allows the exploration of the effect of varying the input calcium oxalate concentration and the rate of water extraction, simulating real life stressors for stone formation such as dietary loading and dehydration.

A nidus, crystalluria and aggregation: key ingredients for stone enlargement.

The in vitro study of calcium oxalate (CaOx) stone formation is usually based on crystallisation models but it is recognised that both healthy individuals and stone formers have crystalluria. We have established a robust in vitro stone growth model based on the principle of mixed suspension, mixed product removal system (MSMPR). Utilising this technique we studied the influence of CaOx crystallisation kinetics and the variation of calcium and oxalate concentrations on CaOx stone growth in vitro. Six stones received standard concentration of Ca (6 mM) and Ox (1.2 mM) in the medium while another six received variable concentrations of both Ca and Ox at various intervals. Stone mass was plotted against the experiment duration (typically 5-7 weeks). The stone growth was dependent on sufficient input calcium and oxalate concentrations and once triggered, stone growth could not be maintained at reduced calcium and oxalate inputs. The stone growth rate was positively correlated to the number of crystals in suspension around the stone and to the crystal nucleation rate and negatively correlated to the crystal growth rates. This leads to the conclusion that aggregation of crystals from the surrounding suspension was the dominant mechanism for stone enlargement.

Effects of inositol hexaphosphate (phytate) on calcium binding, calcium oxalate crystallization and in vitro stone growth.

PURPOSE: We measured and compared 3 activities of inositol hexaphosphate, also known as phytate, to explore their importance in relation to antilithogenic potential. MATERIALS AND METHODS: Calcium binding activity by inositol hexaphosphate was measured with a calcium electrode in artificial and whole urine. Calcium oxalate crystallization inhibition was measured by a 96-well plate turbidimetric method with artificial and whole urine. Effects on stone growth were measured in an in vitro system of 12 stones grown simultaneously (a stone farm) using artificial urine alone or supplemented with urinary macromolecules. RESULTS: Phytate decreased ionized calcium, increased the metastable limit, decreased the crystallization turbidity rate index and decreased the in vitro stone growth rate. The effective concentration for calcium binding reduction was about 2 orders of magnitude higher than that required for crystallization inhibition, which in turn was about 2 orders of magnitude higher than that required for stone growth inhibition. When human urine or artificial urine supplemented with urinary macromolecules was used, the effective concentration of phytate for inhibiting crystallization and stone growth was increased by about 1 order of magnitude. CONCLUSIONS: Inhibition of crystallization by phytate does not depend on decreasing the effective ionized calcium concentration and inhibition of in vitro stone growth does not depend on inhibiting crystallization of the suspended crystals. To our knowledge this is the first demonstration of a quantitative distinction between the inhibition of crystallization and stone growth. Inhibition of in vitro stone growth in the presence of macromolecules occurred at concentrations consistent with urinary phytate excretion.

The association of different urinary proteins with calcium oxalate hydromorphs. Evidence for non-specific interactions.

It has been proposed that various urinary proteins interact specifically with different calcium oxalate hydromorphs and these interactions have important implications regarding the understanding of the onset and progress of kidney stone disease. Calcium oxalate monohydrate and dihydrate crystals were grown and characterised thoroughly to establish sample purity. These crystals were then incubated in artificial urine samples containing isolated urinary macromolecules. Crystal growth was prevented by saturating the incubation mix with calcium oxalate, and this was confirmed through electron microscopy and calcium measurements of the incubation mix. The surface interactions between the different calcium oxalate hydrates and urinary proteins were investigated by the use of Western blots and immunoassays. The same proteins, notably albumin, Tamm-Horsfall protein, osteopontin and prothrombin fragment 1, associated with both hydrates. There was a trend for more protein to associate with calcium oxalate dihydrate, and greater quantities of different proteins associated with both hydrates when Tamm-Horsfall protein was removed from the incubation mix. There is no evidence from this study to indicate that particular proteins interact with specific calcium oxalate hydrates, which in turn suggests that these protein-mineral interactions are likely to be mediated through non-specific charge interactions.

A stone farm: development of a method for simultaneous production of multiple calcium oxalate stones in vitro.

We have previously shown how individual calcium oxalate stones of about 1 cm can be grown in vitro. While this proved a design concept, it was severely limited as an experimental tool because of the time required to undertake comparative studies. Here we describe a development of this system in which six parallel pairs of stone generators are supplied with feed solutions generating a medium that is supersaturated with calcium oxalate. Twelve stones were grown simultaneously in aseptically prepared artificial urine over a period of 32 days from 100 mg to about 250 mg. Flow rates, pH and [Ca(2+)] were stable and reproducible over the course of the experiment. Sodium azide (0.02%) was included in the growth medium of six stones and caused a modest decrease in growth rate from 5.5 to 3.4 mg/day. The experimental design is such that this was readily detectable both visually and statistically ( p<0.001). This multiple stone growing system ("a stone farm") shows improved consistency and illustrates the statistical power of the technique. Azide has only a minor effect on the growth kinetics and can be used as an antibacterial agent in studies involving urinary macromolecules. The technique is suitable for practical and meaningful investigation of calcium oxalate stone formation in vitro.

The density and protein content of calcium oxalate crystals precipitated from human urine: a tool to investigate ultrastructure and the fractional volume occupied by organic matrix.

One of the key debates in biomineralisation studies is the extent to which components of the organic matrix become occluded into the crystal lattice during growth. Here, the relationship between protein content and density of calcium oxalate crystals grown in human urine has been investigated in order to determine which fraction of crystal volume is non-mineral. The density of crystals varied from 1.84 to 2.08 g/cm3 while the protein content ranged from 0.1 to 2.1% (w/w). There was an inverse relationship between measured density and protein content which was qualitatively and quantitatively consistent with predictions based on reasonable densities for the mineral and non-mineral components. The coefficients of the fitted equation suggest that, at 2% protein (w/w), the volume of non-mineral would be 5.0% (v/v). The density values we observed are incompatible with fractional volumes of 20%. The results confirm that the occlusion of a small but possibly significant amount of protein into a crystal lattice is possible, but cast doubt on the hypothesis that protein acts as a major intracrystalline ultrastructural element. Moreover, the methodology developed for this study offers a simple and robust method for interrogating organic/inorganic associations in a range of biological and medical systems.

Enlargement of calcium oxalate stones to clinically significant size in an in-vitro stone generator.

OBJECTIVE: To develop and validate an in vitro method suitable for the quantitative investigation of the growth of calcium oxalate stones through to a clinically significant size. MATERIALS AND METHODS: Small fragments of calcium oxalate calculi were suspended in a mixed suspension/mixed product removal crystalliser supplied with artificial urine supersaturated with calcium oxalate. The fragments were weighed at regular intervals until they reached approximately equal 500 mg. The results were plotted as weight against time and fitted to equations corresponding to constant increase in diameter, surface area-controlled and constant-deposition growth patterns. The choice of the most appropriate model was based on the squared regression coefficient (r2). RESULTS: Eight fragments (2-6 mm in diameter) were grown to approximately 10 mm in diameter over periods from 137 to 369 h. Seven of the growth curves were best-fitted (r2 > or = 0.988) by the equation w = kt(3/2) + c, where w is the weight, k is a growth constant, t is the time and c is a constant approximating to the initial weight. This corresponds to a surface area-dependent mechanism. CONCLUSIONS: The growth of these small fragments to a clinically significant size accelerated throughout the experimental period in a way which was consistent with a surface area-dependent mechanism. We have developed a resilient model suitable for studying the kinetics of calcium oxalate stone growth in vitro.

Methods for measuring crystallization in urolithiasis research: why, how and when?

Whereas crystalluria does not distinguish between kidney stone formers and healthy people and thus can be considered a physiologic event, kidney stone formation is a pathologic incident and reflects a specific form of biomineralization. Both single urinary crystals as well as whole kidney stones form under exquisite control of organic macromolecules. Simple crystal formation in the urinary tract is distinguished from stone formation in the kidney by the process of particle retention. The latter occurs either because nucleated crystals strongly aggregate to particles too large to pass freely through the tubules ('free particle' theory), or because crystals become abnormally adherent to tubular cell surfaces ('fixed particle' theory). Since it is impossible to mimic all the processes involved in stone formation in vitro, it is highly important to carefully chose a specific crystallization process for in vitro studies, and to select the most appropriate experimental conditions for measuring the chosen process as reliably as possible. This overview aims at critically reviewing the principles of currently available assay systems for studying crystallization processes involved in stone formation. Consensus is reached by the experts that no in vitro system really mimics what happens in renal stone formation, but that carefully designed in vitro studies will always play an important part in urolithiasis research. For such studies, it is highly important to exactly control the appropriate experimental conditions that are relevant to a specific crystallization process under investigation. Practical guidelines for researchers working with crystallization systems are provided, and it is concluded that international efforts should be made to standardize the terminology, to agree on a set of basic experimental parameters (temperature, pH, artificial urine composition), and to adopt simple tests or conditions are reference points for quality and comparative control.

Calcium oxalate crystallization kinetics studied by oxalate-induced turbidity in fresh human urine and artificial urine.

We have studied the kinetics of oxalate-induced turbidity in fresh human urine and artificial urine. Assays are performed in 96-well plates, which allows many oxalate concentrations to be studied, repeatedly, in a short time. The metastable limit is defined in terms of the lowest oxalate concentration that gives a rate of change of attenuance significantly greater than the control. Interpretation of rates above this limit is based on ln/ln plots of initial rates against added oxalate concentration. This approach has a good theoretical basis, is well supported by our results and gives a turbidity rate index that is related to the product of the growth rate constant and a factor relating to the number and characteristics of the heteronuclei responsible for initiation of crystallization. This interpretation is posited upon the assumptions that second-order crystallization kinetics occur in unseeded urine when supersaturation exceeds the metastable limit and that aggregation during the initial phase of crystallization does not significantly contribute to changes in turbidity. Metastable limits of urine from healthy volunteers corresponded to a calcium oxalate supersaturation ratio of approx. 10. The turbidity rate index was higher in human urine than in artificial urine. The metastable limit, based on either oxalate concentration or supersaturation, for induction of calcium oxalate crystallization in normal human urine is higher than is likely to be found in normal subjects in vivo. The shape of the relationship between the metastable limit (based on oxalate concentration) and calcium concentration emphasizes the benefit of achieving a low urine calcium concentration. Comparison of the turbidity rate indices for human and artificial urine suggests that the role of nucleation promoters is more dominant than that of growth inhibitors.

Enlargement of a lower pole calcium oxalate stone: a theoretical examination of the role of crystal nucleation, growth, and aggregation.

BACKGROUND AND PURPOSE: Nucleation, growth, and aggregation are considered to be the principal crystallization mechanisms in stone development. It is important to understand their relative significance so that appropriate experimental models can be used, and to identify the best therapeutic targets. The aim of this study was to explore a simplistic model of precipitation and aggregation and to determine the impact of these processes on crystal size density (CSD) and stone growth. METHODS: The computer model represents a lower pole calcium oxalate stone and takes into account the limitations imposed by the amount of available oxalate. RESULTS: Stone enlargement is a result of direct precipitation onto the stone or aggregation of suspended crystals. About one third of the available oxalate needs to be precipitated to form a clinically significant stone within a realistic time frame. If all crystallization is directly at the stone surface, then crystal growth rates are limiting for stones less than about 1 mm, and aggregation of suspended particles would be required for significant enlargement. For crystals in suspension, the CSD is taken to result from two mixed-suspension, mixed-product removal crystallizers operating in series. Using realistic values for nucleation and growth rate, the CSD can be consistent with precipitation of a significant proportion of the available oxalate. This CSD is modified by aggregation, which is assumed to be proportional to the number and volume of crystals in each size range, and further modified by considering the aggregation to be shared by stone enlargement and polymerization in suspension. CONCLUSION: The variables in the model are the probability of aggregation, its rate, and the distribution between aggregation in suspension and onto the stone. Under some circumstances, the bimodal CSD of crystalluria seen in stone forming patients can be reproduced.

Calcium oxalate crystallization kinetics at different concentrations of human and artificial urine, with a constant calcium to oxalate ratio.

The effect of in vitro dilution of artificial urine or human urine on the crystallization of calcium oxalate was examined in a mixed suspension, mixed product removal crystallization system. Direct growth inhibition by components of artificial urine was not significant and supersaturation was the dominant factor in determining crystal nucleation and growth rates. Dilution of human urine caused a decrease in crystal growth rate that was independent of the input calcium and oxalate concentrations, suggesting that dilution of growth inhibitors could be physiologically more important than any reduction in supersaturation. This loss of growth inhibition was counteracted by a reduction in nucleation promotion, with the net effect that the mass of crystals declined. Correlation of crystallization measurements with urinary concentration (osmotic pressure) confirmed these observations, with a negative relationship for growth rate and a positive relationship for nucleation rate and suspension density. Increasing the concentration of urine shifts the crystallization balance from low nucleation/high growth to high nucleation/low growth. Calcium oxalate crystalluria in healthy urine is therefore less likely at early stages of urine development in the nephron and the likelihood can be further reduced by increased fluid output. Our results suggest that lowering the heterogeneous nucleation activity by dilution is more than sufficient to override the loss of growth inhibition.

Growth inhibition of prostate cell lines in vitro by phyto-oestrogens.

OBJECTIVE: To assess a range of phyto-oestrogens as moderators of growth and metabolism in several prostate cell lines. MATERIALS AND METHODS: Four prostate cell lines (PNT-1/A, PNT-2, PC-3 and DU145) were challenged with different doses of five phyto-oestrogens (biochanin A, daidzein, genistein, genistin and nordihydroguaiaretic acid) over 3 days in culture. Cell proliferation was assessed by incorporation of 5-bromo-2'-deoxyuridine (BrdU) and metabolic activity by cleavage of a tetrazolium salt (XTT). RESULTS: Growth and metabolism were inhibited with all compounds and cell lines (e.g. the dose for 50% inhibition of proliferation of PC-3 cells by genistein was 38 micromol/L); differences in the patterns of results suggested that different mechanisms operated, but there was no evidence for any synergistic activity on the inhibition of cell proliferation. CONCLUSION: These results offer further support for the hypothesized role of phyto-oestrogens as dietary protectors against prostatic cancer.

Kinetics of circulating TNF-alpha and TNF soluble receptors following surgery in a clinical model of sepsis.

The interrelationship between cytokines and their natural antagonists in patients with systemic sepsis are incompletely understood. We have followed the changes in serum levels of TNF-alpha and the two soluble receptors (TNF-sr) in a clinical model of post-operative sepsis. Serial blood samples were taken in patients undergoing percutaneous nephrolithotomy (PCNL) starting pre-operatively and continuing for 24 h thereafter. The levels of TNF-alpha and TNF-sr were raised in patients who became clinically septic and correlated well with the severity of sepsis (using the APACHE III score). In septic patients there was no difference in the pattern of changes in the two types of receptor (TNF-sr55 and TNF-sr75). However, in non-septic patients TNF-sr75 was higher in those with endotoxaemia than those without. This difference was not observed with TNF-sr55 which suggests a different mechanism of release or degree of sensitivity for the two soluble receptors. Regardless of severity of illness, the levels of all three molecules (TNF-alpha and the two receptors) appeared to start rising at about the same time point. The peak TNF-alpha level was reached earlier (2-4 h) than that of the two TNF-sr (4-8 h). The relative rise in TNF-alpha was greater than that of the soluble receptors and this difference was even more marked in those with more severe sepsis. The relationship between peak TNF-alpha and peak TNF-sr was non-linear and the concentration of each TNF-sr appeared to plateau at the higher levels of TNF-alpha. This suggests the exhaustion of a limited pool or saturation of the rate of release. Taken together, these results suggest sepsis develops because of delayed and insufficient secretion of TNF-sr compared with TNF-alpha.

Isocitric and citric acid in human prostatic and seminal fluid: implications for prostatic metabolism and secretion.

Human prostatic secretion is remarkably rich in citric acid but the mechanisms to account for this accumulation are not well understood. One factor may be the extent of citrate oxidation to isocitrate, catalyzed by aconitase. The citrate-to-isocitrate ratio will help characterize the relative significance of this reaction in prostatic production and secretion of citrate. Isocitric acid and citric acid were measured in samples of seminal fluid and expressed prostatic secretion (EPS). A constant ratio between citrate and isocitrate of about 33:1 was found (r = 0.93, P < 0.0001) despite the wide variation in concentrations. Citrate ranged from 1 to 180 mM in EPS and from 13 to 50 mM in seminal fluid while isocitrate varied between 0 to 4.8 mM in EPS and from 0.4 to 1.5 mM in seminal fluid. Isocitrate is present in EPS and semen at much higher levels than found in most other animal or plant tissues or fluids and may be actively secreted by the same mechanism as citrate. The high citrate to isocitrate ratio of about 33:1, compared to the expected value of about 10:1, supports suggestions that citrate to isocitrate oxidation by aconitase is a rate limiting step in prostatic citrate metabolism. A low aconitase activity will therefore play a significant role in enabling accumulation of high citrate levels in prostatic epithelia and acini.

Does fish oil benefit stone formers?

The possibility that dietary fish oil supplementation may benefit patients with hypercalciuric urolithiasis by decreasing calcium excretion and enhancing protective mechanisms has been studied in rats and humans. In experiments on rats in metabolic cages, fish oil inhibited experimental nephrocalcinosis induced by intraperitoneal calcium gluconate. There were no significant changes in urinary biochemistry. In a clinical study on 18 hypercalciuric recurrent stone patients fish oil significantly decreased urinary calcium excretion. This effect was accompanied by decreases in the excretion of magnesium and citrate. Oxalate excretion and urinary fibrinolytic activity were unchanged. Overall, fish oil had a limited impact on the risk profile for recurrent urolithiasis.

Crystallization kinetics of calcium oxalate in fresh, minimally diluted urine: comparison of recurrent stone formers and healthy controls in a continuous mixed suspension mixed product removal crystallizer.

A reproducible method has been developed for studying calcium oxalate crystallization from fresh, minimally diluted (92%) urine with the mixed suspension mixed product removal continuous crystallization technique. All samples were adjusted to give the same starting calcium and oxalate concentrations. Twenty-one recurrent male stone formers were compared with twenty-two healthy controls. There was no difference in crystal growth rates but crystal nucleation rates were much higher in the control group (p = 0.003). Using growth rate and nucleation rate results, the amount of crystalline material in suspension was shown to be lower in the urine from stone formers, and therefore the equilibrium supersaturation in the crystallizer was lower in the control group (p = 0.001). We propose that the ability of a healthy person's urine to maintain a lower supersaturation is a crucial protective factor distinguishing non-stone formers from stone formers.

Pyrophosphate in synovial fluid and urine and its relationship to urinary risk factors for stone disease.

Inorganic pyrophosphate (PPi) measurement in urine and synovial fluid has been established using the PPi-dependent phosphorylation of fructose-6-phosphate and subsequent reduction of dihydroxyacetone phosphate by NADH. The assay is linear up to 200 mumol/L, easy to perform and gives results comparable to more complex methods. Daily urinary output of PPi was independently related to both age (P = 0.0014) and sex (P = 0.0002). Men had higher values than women and older individuals excreted greater amounts. Male stone formers, younger than 45 years, had lower values than age matched male controls (P = 0.012). Younger female stone formers also tended to have lower values. In stone formers' urine significant and independent correlations were found of PPi excretion with urine volume (P = 0.004) and with phosphate excretion (P = 0.008). Oxalate excretion and that of other urine constituents and the degree of supersaturation with common stone-forming salts were not correlated with PPi. PPi excretion was markedly elevated in the urine of two patients with hypophosphatasia. The PPi concentration in synovial fluid from painful, swollen knee joints was elevated, but unrelated to the presence or absence of PPi or urate crystals.