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Adhesion of calcium oxalate (CaOx) crystals onto renal tubular epithelial cells is one of the critical steps in kidney stone formation. However, effects of crystal size on the crystal adhesive capability remained unclear. This study compared the adhesive capabilities of CaOx monohydrate (COM) crystals with various sizes (10 μm, 20-30 μm, 50-60 μm, and 80 μm). Crystal-cell adhesion assay showed size-dependent increase of COM crystal adhesion onto epithelial cell surface using the larger crystals. Identification of apical membrane proteins that could bind to COM crystals by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) demonstrated size-specific sets of the COM crystal-binding proteins. Among these, numbers of known oxalate-binding proteins and COM crystal receptors were greatest in the set of the largest size (80 μm). Atomic force microscopy (AFM) revealed that adhesive forces between carboxylic-immobilized AFM tip and COM crystal surface and between COM-mounted AFM tip and renal epithelial cell surface were size-dependent (greater for the larger crystals). In summary, the adhesive capability of COM crystals is size-dependent - the larger the greater adhesive capability. These data may help better understanding of the pathogenic mechanisms of kidney stone formation at an initial stage when renal tubular cells are exposed to various sizes of COM crystals.

Background/Aims: The interactions between calcium oxalate monohydrate (COM) crystals and renal tubular epithelial cells are important for renal stone formation but still unclear. This study aimed to investigate changes of epithelial cell phenotype after COM attachment and whether L-carnitine could protect cells against subsequent COM crystals adhesion. Methods: Cultured MDCK cells were employed and E-cadherin and Vimentin were used as markers to estimate the differentiate state. AlexaFluor-488-tagged COM crystals were used in crystals adhesion experiment to distinguish from the previous COM attachment, and adhesive crystals were counted under fluorescence microscope, which were also dissolved and the calcium concentration was assessed by flame atomic absorption spectrophotometry. Results: Dedifferentiated MDCK cells induced by transforming growth factor β1 (TGF-β1) shown higher affinity to COM crystals. After exposure to COM for 48 hours, cell dedifferentiation were observed and more subsequent COM crystals could bind onto, mediated by Akt/GSK-3β/Snail signaling. L-carnitine attenuated this signaling, resulted in inhibition of cell dedifferentiation and reduction of subsequent COM crystals adhesion. Conclusions: COM attachment promotes subsequent COM crystals adhesion, by inducing cell dedifferentiation via Akt/GSK-3β/Snail signaling. L-carnitine partially abolishes cell dedifferentiation and resists COM crystals adhesion. L-carnitine, may be used as a potential therapeutic strategy against recurrence of urolithiasis.

Studied is the influence of amino acids (L-Asp, L-Arg, L-Thr) and B-group vitamins (B1, B6, B12) on the processes of nucleation and the value of surface energy of calcium oxalate monohydrate (COM) crystals. It is established that L-Asp and L-Arg molecules inhibit nucleation of COM, that increases their induction time tenfold in comparison with the one of the pure crystals. L-Thr amino acid is shown to promote the processes of COM nucleation and to diminish the induction time by two times. At the introduction of L-Asp and L-Arg molecules into the solution the degree of COM crystal growth inhibition is 32.0–90.2% and 10.2–86.0% respectively. It is found that B1 and B6 vitamins inhibit the growth of COM crystals practically completely (the inhibition degree exceeds 95%) and lead to 70-fold increase in the induction time of pure COM. The value of surface energy of COM crystals diminishes to 19.8–20.8 mJ/m2 at the introduction of the crystallization inhibitor molecules (L-Asp, L-Arg, B1, B6) into COM solution.

Objective To evaluate the effect of an extract obtained from Dolichos biflorus (Fabaceae) on calcium oxalate crystallization in vitro. Materials and methods A hydro-methanolic extract of (30:70, v/v) of D. biflorus seeds at different concentrations (1–10 mg/ml) was subjected to in vitro anticrystallization activity using a synthetic urine system. The results were compared with a parallel study conducted with the herbal medicinal product cystone under identical dosage conditions. The nucleation and aggregation of calcium oxalate crystals were measured using spectrophotometric methods and crystals generated in the urine were also analyzed by light microscopy. Statistical differences and percent inhibitions were calculated using GraphPad prism 5 software. Results The seed extract was significantly more effective than cystone at inhibiting the nucleation, as well as the aggregation of calcium oxalate monohydrate crystals in a dose-dependent manner. These results were also confirmed by the microscopic analysis. Conclusion The results showed that the selected herb, D. biflorus has excellent anticrystallization activity and therefore, might be beneficial if used in formulating a strategy for the dissolution and thereby prevention of urinary stones.

Adsorption of EDTA onto calcium oxalate monohydrate crystals was investigated and the adsorption characteristics were analyzed. Experiments were carried out to study the effects of factors such as contact time, initial concentration of EDTA, temperature and solution pH on EDTA adsorption. The experimental results showed that EDTA adsorption on calcium oxalate monohydrate crystals increased with its initial concentration in the aqueous solutions. A high temperature was beneficial to the adsorption process. However, the adsorption capacity of EDTA on calcium oxalate monohydrate crystals decreased as the pH increased from 5 to 8. The kinetic data fitting analysis suggested a pseudo-second-order kinetic model best described the adsorption process. The adsorption equilibrium data analysis indicated that the Langmuir isotherm model fit better than the Freundlich isotherm model, based on the R2, RMSE and χ2 values. Thermodynamic parameters such as ΔG0, ΔH0 and ΔS0 for EDTA adsorption have also been estimated. The results demonstrated that nature of the adsorption was feasible, spontaneous and endothermic. This study would provide a foundation for the modification of an oxalate-degrading enzyme using EDTA or its derivatives, in order to endow the enzyme with the adsorption ability of calcium oxalate stones.

An 80-year-old man presented to the emergency department after a fall at home. Serum studies demonstrated an anion and osmolal gap. Examination of the urine sediment showed calcium oxalate monohydrate crystals, suggestive of ethylene glycol toxicity.

Investigated is the effect of L-threonine (L-thr) amino acid on the growth of pathogenic calcium oxalate monohydrate (СОМ) crystals. It is found that L-thr molecules inhibit the growth of COM crystals and prevent their aggregation. Additions of 2-4 mM L-threonine in the investigated system decreases the crystal size (5-6 μm) in comparison with that of СОМ crystals without additives (9 μm). Increase of L-thr concentration in the solution up to 8-20 mM does not lead to further decrease of the crystal size.

Crystallization process has a major role in urolithiasis. In the present study, effect of two indigenous plants extracts namely Boerhavia diffusa and Bryophyllum pinnatum extract was determined on the crystallization of calcium oxalate crystals. Effect on the number, size and type of calcium oxalate crystals was observed. Results showed significant activity of both extracts against calcium oxalate crystallization at different concentrations (P < 0.05). Size of the crystals gradually reduced with the increasing concentration of both extracts. The number of calcium oxalate monohydrate crystals which are injurious to epithelial cells gradually reduced and at the highest concentration of extracts (100 mg/ml) completely disappeared (P < 0.05). These results confirm that B. diffusa and B. pinnatum extracts have antiurolithic activity and have the ability to reduce crystal size as well as to promote the formation of calcium oxalate dihydrate (COD) crystals rather than monohydrate (COM) crystals. Control of crystal size and formation of COD rather than COM crystals, in combination with the diuretic action of extracts is an important way to control urolithiasis.

When seen, some habits of calcium oxalate monohydrate crystals (whewellite) are so typical of ethylene glycol intoxication that they may be helpful for its diagnosis when circumstances are not clearly established.

A large number of people in this world are suffering from urinary stone problem. Calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) containing stones (calculi) are commonly found. In the present study, COM crystals were grown by a double diffusion gel growth technique using U-tubes. The gel was prepared from hydrated sodium metasilicate solution. The gel framework acts like a three-dimensional crucible in which the crystal nuclei are delicately held in the position of their formation, and nutrients are supplied for the growth. This technique can be utilized as a simplified screening static model to study the growth, inhibition and dissolution of urinary stones in vitro. The action of putative litholytic medicinal plants, Tribulus terrestris Linn. (T.t) and Bergenia ligulata Linn. (B.l.), has been studied in the growth of COM crystals. Tribulus terrestris and Bergenia ligulata are commonly used as herbal medicines for urinary calculi in India. To verify the inhibitive effect, aqueous extracts of Tribulus terrestris and Bergenia ligulata were added along with the supernatant solutions. The growth was measured and compared, with and without the aqueous extracts. Inhibition of COM crystal growth was observed in the herbal extracts. Maximum inhibition was observed in Bergenia ligulata followed by Tribulus terrestris. The results are discussed.

Knoop microhardness studies were carried out on urinary calculi and synthesized calcium oxalate monohydrate (COM) crystals. COM calculi showed a higher value of microhardness than the synthesized crystals. Calcium oxalate dihydrate (COD) and uric acid (UA) stones showed microhardness values close to that of COM. In the case of mixed stones, the stone with COM showed higher hardness value than the stone with COD component. The presence of calcium phosphate (hydroxyapatite, HAP) in the mixed stones did not contribute in anyway to the overall hardness of the stones. The variation of microhardness with the orientation of COM calculi showed a cyclic pattern indicating the presence of alternate layers of crystalline and matrix materials.

Large size (9.0 × 6.0 × 1.0 mm) transparent single crystals of calcium oxalate monohydrate (CaC2O2·H2O), which were suitable for hardness studies have been grown by a double diffusion technique. The crystallization conditions to obtain bigger crystals have been optimized. The crystals grown were characterized by XRD, FTIR, TGA/DTG and microhardness increased with an increase in load and could withstand up to a load of 200 g, revealing the high hardness nature of the calcium oxalate monohydrate crystals.

A 59-year-old man with diabetes mellitus Type II, arterial hypertension and depressive disorder was hospitalized due to unexplained severe inebriation followed by sedation. Regular medications consisted of metformin 1500 mg, enalapril 20 mg and paroxetine 20 mg. Blood tests showed a creatinine of 0.9 mg/dL (79 μmol/L), glucose 110 mg/dL (6.1 mmol/L) and potassium 6.4 mmol/L. Blood gas test showed a pronounced metabolic acidosis, with pH 7.04, bicarbonate 3 mmol/L, PCO2 10 mmHg, lactic acid 4.8 mmol/L, Na 145 mmol/L, chloride 105.4 mmol/L and anion gap 43 mmol/L. Cerebrospinal fluid analysis and cerebral computerized tomography scan were normal. The patient’s condition, including cognitive status, was rapidly deteriorating, requiring orotracheal intubation. Anuria developed in the following few hours, requiring the referral to our hospital. Laboratory data confirmed the high anion gap metabolic acidosis and showed a serum creatinine of 3.8 mg/dL (336 μmol/L). Renal replacement therapy was soon initiated with continuous venovenous haemodiafiltration; after 21 h acid–base status was normalized, but anuria persisted requiring intermittent haemodialysis. Examination of urine sediment (Figure 1) showed both needle-shaped and dumbbell-shaped calcium oxalate monohydrate crystals; these crystals are considered a clue to ethylene glycol poisoning since oxalate derives from the metabolism of ethylene glycol [1, 2]. Furthermore, renal damage most likely results from a crystal-induced injury to proximal tubule cells [3]. Fig. 1. Urine sediment showing both needle-shaped and dumbbell-shaped (upper right side) calcium oxalate dihydrate crystals. The coma lasted 2 weeks; upon regaining consciousness the patient admitted to having ingested automobile anti-freeze (containing ethylene glycol) in a suicide attempt. Progressive recovery of renal function occurred 4 weeks after admission; renal replacement therapy was interrupted 2 weeks later. A prompt examination of urine sediment, often forgotten in the current ‘high-tech’ medicine, would have suggested the correct diagnosis and allowed administration of fomepizole or ethanol to prevent brain and renal damage.

Ureterocele calculi are developed in cavities with urinary retention but far from the upper renal cavities. The structural features of three ureterocele calcium oxalate stones were observed by scanning electron microscope coupled with X ray microanalysis. The urinary parameters of the three patients were also determined. The stone A consisted of loose structure of large calcium oxalate dihydrate crystals and small spheres of hydroxyapatite. The interior contains disorganized plate-like calcium oxalate monohydrate crystals. The stone B was formed by a compact outer layer of calcium oxalate monohydrate columnar crystals. The structure of stone interior was similar to the stone A. The stone C was formed by concentric layers composed of either calcium oxalate monohydrate columnar crystals or hydroxyapatite. The core consisted of agglomerated calcium oxalate monohydrate crystals, hydroxyapatite and organic matter. From the urinary biochemical data it was deduced that two ureterocele patients (who formed A and B stones) were hypercalciuric (calcium > 300 mg/24 h), being 6.5 the urinary pH value of the patient that formed the A stone, and 7.0 the urinary pH of the patient that formed the C stone. The rest of urinary parameters for the three patients were normal. Thus, one of the requisite conditions for unattached stone development is the existence of a place inside the urinary tract where the solid particles that act as calculus initiator of the stone can be retained enough time to exert this action.

Aggregation of crystals is the most important process during stone formation in the kidney and can be influenced by different proteins of renal origin. Tamm-Horsfall glycoprotein (THP) is produced in the cells of the thick ascending limb of Henle’s loop and is excreted into the tubular lumen. One of its major physiological roles is the inhibition of calcium oxalate monohydrate (COM) crystal aggregation. However, under specific conditions this protein may also act as a promotor of crystallization. The interaction between THP and the crystal surface during aggregation is not known and is the topic of this study.

By energy-filtering transmission electron microscopy (EFTEM) electrons can be separated by their energy losses. An electron-energy filter, added to the microscope column allows the measurement of the energy distribution of transmitted electrons that have lost energy (< 2,000 eV, with an energy resolution of ~ 1 eV). These filtered electrons, recorded either as a spectrum or as an image, are composed of two parts superimposed on top of each other: (a) the unspecific energy-loss population (= the continuum) and (b) the specific element-related energy-loss population (= the edges). At the edges, electron data in spectra and images are mathematically processed, to obtain the desired element-related net-intensity values or images. These data are related to the total transmitted electron intensity, from the zero-and low-loss spectral region giving the relative spectral-or image intensity ratios (SR*x, IR*x), which can be related to the element concentration. The acquisition of the zero-loss and low-loss data is hampered by the restricted dynamic range of the TV camera. By improvements through the introduction of calibrated attenuation filters in the optical path to the TV-camera, more reliable values for SR*x and IR*x can be acquired. By addition of Bio-standards adjacent to the tissue, a "known" and "unknown" concentration of the element present in the same ultrathin section and the "bias" in the concentration estimation, can be obtained. Some practical examples are given for the estimation of the iron concentration in siderosomes, boron in melanosomes and calcium in calcium oxalate monohydrate crystals.

Calcium oxalate (CaOx) is the main compound of human kidney stones, specifically calcium oxalate monohydrate crystals (COM). Normal human urine contains small crystals of calcium oxalate dihydrate (COD), which have reduced capacity to form stable aggregates and strong adhesion contacts to renal epithelial cell thus protecting kidney stone formation. Here, we report the preparation of a new monomethylitaconate grafted polymethylsiloxaner, which was able to promote and stabilize COD crystals. Although the mechanisms involved in control of CaOx polymorphism still remain unclear, we believe that research using synthetic polymers would provide interesting chemical clues to develop calculi preventing-compounds.

Human urine, and nephrocalcin (NC), a glycoprotein of probable kidney cell origin, greatly reduce consumption of calcium and oxalate from metastably supersaturated solutions seeded with calcium oxalate crystals, a phenomenon usually referred to as inhibition of crystal growth. We seeded metastably supersaturated calcium oxalate solutions with calcium oxalate monohydrate crystals under conditions of ion clamping to maintain constant composition and measured ion consumption from pump delivery rates. Consumption rates increased continuously with time as if the solutions were autocatalytic. After incubation, the seeds were covered with innumerable crystallites, which were also free and numerous in the solution, reflecting self-nucleation. The addition of 20% whole, dialyzed urine, or purified NC reduced ion consumption rates markedly, and the only crystals observed at the end of incubation were the large original seeds. Crystals precoated with concentrated dialyzed urine or NC also showed reduced ion consumption. Urine and NC from patients with nephrolithiasis inhibited nucleation less than normal controls. Self-nucleation seems to be the preferred response in sparsely seeded, ion-clamped, supersaturated solutions, such as exist in the nephron. Urine and NC suppress self-nucleation in vitro by adsorbing to the surface of calcium oxalate crystals.

This 73-year-old man presented with a self-inflicted stab wound, plasma ethanol level of 27 mg per deciliter, and a pH of 6.91. Analysis of the urine showed calcium oxalate monohydrate crystals. Free crystals were extensive.

Crystal-induced membranolysis of human red blood cells has been quantltated for calcium oxalate monohydrate and calcium oxalate dihydrate crystals. Calcium oxalate monohydrate crystals are significantly more membranolytic than calcium oxalate dihydrate crystals at constant surface area. If the crystal morphology of calcium oxalate monohydrate is altered by grinding, the lytic potential at constant surface area is markedly reduced. However, altered calcium oxalate dihydrate crystals are as lytic as natural calcium oxalate dihydrate crystals at constant surface area. Differences in the calcium oxalate monohydrate and dihydrate crystal structures, specifically the structural characteristics of the disordered water channel in calcium oxalate dihydrate, can explain these different membranolytic characteristics.

Studies of calcium oxalate crystals in urine suggest similarities to crystal growth in calcium oxalate renal calculi. Previous reports indicate that urinary crystals in patients in whom stones form are larger than those in normal subjects. We report herein a study on crystal size by structure and habit (shape) based on direct microscopic measurement of crystals in the urine of 27 normal subjects and in 6 of 22 patients in whom stones form. The mean size of all crystals in normal subjects is 12.0 plus or minus 7.8 µm. Calcium oxalate monohydrate crystals are significantly smaller than calcium oxalate dihydrate (p less than 0.01). In 22 patients with stones there was no correlation between crystalluria and severity or duration of disease. The mean crystal size in 6 patients did not support the conclusion that patients in whom stones form excrete larger crystals than normal subjects.

Previous studies have shown that magnesium deficiency accelerates renal tubular calcium oxalate monohydrate deposition in rats on chronic hyperoxaluric, lithogenic protocols. The present study was conducted to investigate the effect of magnesium deficiency on intratubular calcium oxalate formation in rats from the 1st day of administration of a hyperoxaluric agent. The objectives were to delineate early ultrastructural features of the formation, mechanisms of retention, and development of renal tubular crystal deposits and to characterize the crystalluria in rats on the hyperoxaluric/hypomagnesuric protocol. Intratubular calcium oxalate monohydrate deposits were found in magnesium deficient rats after only 24 hours of ad libitum administration of 1 per cent ethylene glycol drinking water. Animals on regular food diet did not display renal tubular deposition after 11 days of ethylene glycol administration. Strand- and sheet-like organic material emanating from the luminal wall of the tubules was adherent to the crystals, thereby serving to immobilize them within the tubule. Calcium oxalate monohydrate crystals predominated in the urines of hyperoxaluric/hypomagnesuric animals with intratubular deposits while dihydrate crystals were the primary constituent of urines from rats administered ethylene glycol alone (no intratubular deposition). The results support the supposition that under certain conditions magnesium deficiency is a significant risk factor for intrarenal calcium oxalate deposition and stone formation. Furthermore the identification of calcium oxalate monohydrate crystalluria may be an important indicator of the propensity toward intranephronic calcium oxalate formation and urolithiasis.

Urine sediments of dogs with experimentally induced ethylene glycol poisoning were examined by light microscopy and X-ray diffraction. Massive calcium oxalate crystalluria was observed in all poisoned dogs. By light microscopy, the frequency with which six-sided hippurate-like prisms and envelope forms of calcium oxalate dihydrate occurred was approximately equal. The hippurate-like crystals were shown to be calcium oxalate monohydrate by X-ray diffractometry.

Rat renal inner papillary collecting tubule cells (RPCT) have been isolated and maintained in primary culture. The cells have been found to be of only one type and they have maintained the characteristics of RPCT cells. The RPCT cells in culture appear as a monolayer with intermittent clumps of rounded cells. When small calcium oxalate monohydrate crystals (COM) or calcium oxalate dihydrate crystals (COD) are added to the monolayer of RPCT cells, the crystals bind on or about these clumps of rounded-up cells. The use of this system as a model for the study of crystal membrane interactions in crystalluria and urolithiasis is discussed. (J. Urol., 138: 640–643, 1987)

We report a case of primary oxalosis in a 4-year-old boy with intensive deposition of calcium oxalate monohydrate crystals in the kidney and the myocardium. In addition severe fibroplastic inflammatory reaction in form of intensive myocarditis was found. It is an extremely rare complication at this age and represents a particularly rare case of oxalosis. Disorders of oxalate metabolism are discussed in relation to extrarenal deposition of oxalate crystals.

Seed experiments on the growth of calcium oxalate monohydrate in vitro have given the following results. Chondroitin sulphate does not influence the growth but only the agglomeration. The main influence of heparin at all concentrations is to inhibit the agglomeration, but at concentrations greater than about 10−8 mol/l the growth is also inhibited. In the range 0.05–30 μmol/l there is a linear dependence between the percentage inhibition of the growth and the concentration of heparin.

Methods are described for growing calcium oxalate in silica and gelatin gels under different conditions. The results obtained indicate that, in silica gel, calcium oxalate grows into single individual crystals, twins, and rosettes. Bipyramidal calcium oxalate dihydrate crystals similar to those present in the urine of stone formers were prepared in the silica gel system. The gelatin gel offered a suitably structured substrate on which calcium oxalate monohydrate crystals grow into aggregates. The orientation pattern of calcium oxalate crystals suggests that the growth process is controlled by the stereospecificity of the gelatin medium supporting growth.

An attempt has been made to elucidate the role of alkalinization and chelation in the dissolution of both calcium oxalate monohydrate crystals and stones. Dissolution by these agents has been, measured at various concentrations over the pH range from 4 to 8. The alkalinizing agents used were sodium orthophosphate and sodium bicarbonate. The chelating agents were sodium ethylenediaminetetraacetate (EDTA) and sodium citrate.