Defining and Systematic Analyses of Aggregation Indices to Evaluate Degree of Calcium Oxalate Crystal Aggregation

Calcium oxalate (CaOx) can be crystallized in several forms and morphologies. We evaluated factors that determine differential types and shapes of CaOx crystals generated in vitro. CaCl2 and Na2C2O4 solutions at various molar concentrations were mixed in different conditions (with or without Tris-HCl buffer and varying pH, temperature and speed of stirring) and incubated overnight. A total of 78 conditions were evaluated. The most frequently observed type of CaOx crystals was calcium oxalate monohydrate (COM). In 18.2 MOmega.cm water, typical monoclinic prismatic form of COM was found when 0.5-1 mmol/l CaCl2 and 0.5-1 mmol/l Na2C2O4 were mixed, whereas the COM dendrites were found when higher concentrations were used. Calcium oxalate dihydrate (COD) crystals were observed when 5 mmol/l CaCl2 and 0.5 mmol/l Na2C2O4 were employed. With the same molar concentrations of CaCl2 and Na2C2O4, the sequence of adding these 2 chemicals into the chamber had some effects on crystal types and morphologies. The presence of Tris-HCl buffer in the solution enhanced COM crystal growth and aggregation. The pH greater than 5.0 was associated with the presence of weddellite COD. Magnetic stirring of the supersaturated solution resulted to reduction in size of all crystal forms; the higher speed provided the smaller crystals. Finally, crystallization of CaOx at 4 degrees C was more efficient than performing the experiment at 25 and 37 degrees C. Molar concentrations, order of adding the substrates, buffering, pH, stirring and temperature have significant effects on CaOx crystal formation, types and morphologies. Cataloging these differential forms of crystals generated in different conditions will be useful for further study on modulations of CaOx crystals and kidney stone disease.

Supersaturation (SS) with respect to calcium oxalate monohydrate (COM), brushite (Br) and uric acid (UA), obtained in three 24-hour pretreatment urine samples from patients with stone disease were compared to the mineral composition of stones passed by the same patients to determine whether sparse urine SS measurements accurately reflect the long-term average SS values in the kidney and final urine. Among males and females elevation of SS above same sex normals corresponded to composition. As well, treatments that reduced stone rates also reduced these SS values. The degree of calcium phosphate (CaP) admixture was accurately matched by shifting magnitudes of COM and Br SS. As well, increasing CaP content was associated with falling urine citrate and rising urine pH, suggesting renal tubular acidosis. We conclude that sparse urine SS measurements accurately track stone admixtures, and are a reliable index of average renal and urine SS.

Our previous report showed that uropathogenic bacteria, e.g., Escherichia coli, are commonly found inside the nidus of calcium oxalate (CaOx) kidney stones and may play pivotal roles in stone genesis. The present study aimed to prove this new hypothesis by direct examining CaOx lithogenic activities of both Gram-negative and Gram-positive bacteria. CaOx was crystallized in the absence (blank control) or presence of 10(5) CFU/ml E. coli, Klebsiella pneumoniae, Staphylococcus aureus, or Streptococcus pneumoniae. Fragmented red blood cell membranes and intact red blood cells were used as positive and negative controls, respectively. The crystal area and the number of aggregates were measured to initially screen for effects of bacteria on CaOx crystal growth and aggregation. The data revealed that all the bacteria tested dramatically increased the crystal area and number of crystal aggregates. Validation assays (spectrophotometric oxalate-depletion assay and an aggregation-sedimentation study) confirmed their promoting effects on both growth (20.17 ± 3.42, 17.55 ± 2.27, 16.37 ± 1.38, and 21.87 ± 0.85 % increase, respectively) and aggregation (57.45 ± 2.08, 51.06 ± 5.51, 55.32 ± 2.08, and 46.81 ± 3.61 % increase, respectively) of CaOx crystals. Also, these bacteria significantly enlarged CaOx aggregates, with the diameter greater than the luminal size of distal tubules, implying that tubular occlusion might occur. Moreover, these bacterial effects were dose-dependent and specific to intact viable bacteria, not intact dead or fragmented bacteria. In summary, intact viable E. coli, K. pneumoniae, S. aureus, and S. pneumoniae had significant promoting effects on CaOx crystal growth and aggregation. This functional evidence supported the hypothesis that various types of bacteria can induce or aggravate metabolic stone disease, particularly the CaOx type.