Calcium Oxalate Monohydrate Crystals Research Articles

Tartronic Acid as a Potential Inhibitor of Pathological Calcium Oxalate Crystallization.

Kidney stones are a pervasive disease with notoriously high recurrence rates that require more effective treatment strategies. Herein, tartronic acid is introduced as an efficient inhibitor of calcium oxalate monohydrate (COM) crystallization, which is the most prevalent constituent of human kidney stones. A combination of in situ experimental techniques and simulations are employed to compare the inhibitory effects of tartronic acid with those of its molecular analogs. Tartronic acid exhibits an affinity for binding to rapidly growing apical surfaces of COM crystals, thus setting it apart from other inhibitors such as citric acid, the current preventative treatment for kidney stones. Bulk crystallization and in situ atomic force microscopy (AFM) measurements confirm the mechanism by which tartronic acid interacts with COM crystal surfaces and inhibits growth. These findings are consistent with in vivo studies that reveal the efficacy of tartronic acid is similar to that of citric acid in mouse models of hyperoxaluria regarding their inhibitory effect on stone formation and alleviating stone-related physical harm. In summary, these findings highlight the potential of tartronic acid as a promising alternative to citric acid for the management of calcium oxalate nephropathies, offering a new option for clinical intervention in cases of kidney stones.

The impact of crystal phase transition on the hardness and structure of kidney stones

Calcium oxalate kidney stones, the most prevalent type of kidney stones, undergo a multi-step process of crystal nucleation, growth, aggregation, and secondary transition. The secondary transition has been rather overlooked, and thus, the effects on the disease and the underlying mechanism remain unclear. Here, we show, by periodic micro-CT images of human kidney stones in an ex vivo incubation experiment, that the growth of porous aggregates of calcium oxalate dihydrate (COD) crystals triggers the hardening of the kidney stones that causes difficulty in lithotripsy of kidney stone disease in the secondary transition. This hardening was caused by the internal nucleation and growth of precise calcium oxalate monohydrate (COM) crystals from isolated urine in which the calcium oxalate concentrations decreased by the growth of COD in closed grain boundaries of COD aggregate kidney stones. Reducing the calcium oxalate concentrations in urine is regarded as a typical approach for avoiding the recurrence. However, our results revealed that the decrease of the concentrations in closed microenvironments conversely promotes the transition of the COD aggregates into hard COM aggregates. We anticipate that the suppression of the secondary transition has the potential to manage the deterioration of kidney stone disease.

Herbo-Mineral Medicine, Lithom Exhibits Anti-Nephrolithiasis Activity in Rat Model of Hyperoxaluria by Attenuating Calcium Oxalate Crystal Formation and Oxidative Stress.

Calcium oxalate monohydrate (COM) forms the most common type of kidney stones observed in clinics, elevated levels of urinary oxalate being the principal risk factor for such an etiology. The objective of the present study was to evaluate the anti-nephrolithiatic effect of herbo-mineral formulation, Lithom. The in vitro biochemical synthesis of COM crystals in the presence of Lithom was performed and observations were made by microscopy and Scanning Electron Microscope (SEM) based analysis for the detection of crystal size and morphology. The phytochemical composition of Lithom was evaluated by Ultra-High-Performance Liquid Chromatography (UHPLC). The in vivo model of Ethylene glycol-induced hyperoxaluria in Sprague-Dawley rats was used for the evaluation of Lithom. The animals were randomly allocated to 5 different groups namely Normal control, Disease control (ethylene glycol (EG), 0.75%, 28 days), Allopurinol (50 mg/kg, q.d.), Lithom (43 mg/kg, b.i.d.), and Lithom (129 mg/kg, b.i.d.). Analysis of crystalluria, oxalate, and citrate levels, oxidative stress parameters (malondialdehyde (MDA), catalase, myeloperoxidase (MPO)), and histopathology by hematoxylin and eosin (H&E) and Von Kossa staining was performed for evaluation of Lithom. The presence of Lithom during COM crystals synthesis significantly reduced the average crystal area, feret's diameter, and area-perimeter ratio, in a dose-dependent manner. SEM analysis revealed that COM crystals synthesized in the presence of 100 and 300 μg/mL of Lithom exhibited a veritable morphological transition from irregular polygons with sharp edges to smoothened smaller cuboid polygons. UHPLC analysis of Lithom revealed the presence of Trigonelline, Bergenin, Xanthosine, Adenosine, Bohoervinone B, Vanillic acid, and Ellagic acid as key phytoconstituents. In EG-induced SD rats, the Lithom-treated group showed a decrease in elevated urinary oxalate levels, oxidative stress, and renal inflammation. Von Kossa staining of kidney tissue also exhibited a marked reduction in crystal depositions in Lithom-treated groups. Taken together, Lithom could be a potential clinical-therapeutic alternative for management of nephrolithiasis.

Different Degrees of Sulfated Laminaria Polysaccharides Recovered Damaged HK-2 Cells and Inhibited Adhesion of Nano-COM and Nano-COD Crystals.

The crystal adhesion caused by the damage of renal tubular epithelial cells (HK-2) is the key to the formation of kidney stones. However, no effective preventive drug has been found. This study aims to explore the recovery effects of four Laminaria polysaccharides (SLPs) with different sulfate (-OSO3-) contents on damaged HK-2 cells and the difference in the adhesion of damaged cells to nanometer calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) before and after recovery. Sodium oxalate (2.6 mmol/L) was used to damage HK-2 cells to establish a damaged model. SLPs (LP0, SLP1, SLP2, and SLP3) with -OSO3- contents of 0.73%, 15.1%, 22.8%, and 31.3%, respectively, were used to restore the damaged cells, and the effects of SLPs on the adhesion of COM and COD, with a size of about 100 nm before and after recovery, were measured. The following results were observed after SLPs recovered the damaged HK-2 cells: increased cell viability, restored cell morphology, decreased reactive oxygen levels, increased mitochondrial membrane potential, decreased phosphatidylserine eversion ratio, increased cell migration ability, reduced expression of annexin A1, transmembrane protein, and heat shock protein 90 on the cell surface, and reduced adhesion amount of cells to COM and COD. Under the same conditions, the adhesion ability of cells to COD crystals was weaker than that to COM crystals. As the sulfate content in SLPs increases, the ability of SLPs to recover damaged HK-2 cells and inhibit crystal adhesion increases. SLP3 with high -OSO3- content may be a potential drug to prevent kidney stones.

The inhibitory effects of epigallocatechin-3-gallate on calcium oxalate monohydrate crystal growth, aggregation and crystal-cell adhesion

Epigallocatechin-3-gallate (EGCG), a predominant phytochemical in tea plant, has been reported to prevent kidney stone formation but with vague mechanism. We investigated modulatory effects of EGCG (at 0.1–100 µM) on calcium oxalate monohydrate (COM) crystals at various stages of kidney stone development. EGCG significantly increased crystal size (at 1–100 µM), but decreased crystal number (at 10–100 µM), resulting in unchanged crystal mass and volume. Interestingly, EGCG at 10–100 µM caused morphological change of the crystals from typical monoclinic prismatic to coffee-bean-like shape, which represented atypical/aberrant form of COM as confirmed by attenuated total reflection - Fourier transform infrared (ATR-FTIR) spectroscopy. EGCG at all concentrations significantly inhibited crystal growth in a concentration-dependent manner. However, only 100 µM and 10–100 µM of EGCG significantly inhibited crystal aggregation and crystal-cell adhesion, respectively. Immunofluorescence staining (without permeabilization) revealed that surface expression of heat shock protein 90 (HSP90) (a COM crystal receptor) on MDCK renal cells was significantly decreased by 10 µM EGCG, whereas other surface COM receptors (annexin A1, annexin A2, enolase 1 and ezrin) remained unchanged. Immunoblotting showed that 10 µM EGCG did not alter total level of HSP90 in MDCK cells, implicating that its decreased surface expression was due to translocation. Our data provide a piece of evidence explaining mechanism underlying the anti-lithiatic property of EGCG by inhibition of COM crystal growth, aggregation and crystal-cell adhesion via reduced surface expression of HSP90, which is an important COM crystal receptor.

Effect of Blumea Balsamifera Extract on the Kinetics of Calcium Oxalate Monohydrate (COM) Dissolution

Blumea balsamifera is a commercially available herbal drug that has anti-urolithic and diuretic properties. This study quantifies the effect of B. balsamifera extract on the dissolution kinetics of Calcium Oxalate Monohydrate (COM) crystals by modeling the dissolution of Ca2+. COM dissolution follows a first-order diffusion-controlled process. The extract did not change the dissolution phenomena. At 10 ppm, the extract had no significant effect on the rate constant (P = 0.166) and surface concentration (P = 0.372). Increasing the extract to 20 ppm did not change the dissolution phenomena and model parameters. The extract significantly decreases the equilibrium Ca2+ concentration with P = 0.0010 and P = 0.0179 at 10 and 20 ppm of extract, respectively. B. balsamifera extract binds free Ca2+ in the synthetic urine. This increases the amount of Ca2+ that dissolves but does not significantly increase the rate of dissolution suggesting that urine volume is more important for COM stone dissolution.

Boosting inhibition performance of natural polyphenols for the prevention of calcium oxalate kidney stones through synergistic cooperativity

Binary drug combination usually targets different pathways to achieve cooperative therapy, but the exploitation of synergistic cooperativity between crystal growth modifiers that bind to the same site for preventing pathological biomineralization has yet to be realized. Here, we report that the binary inhibitor combinations of citrate with natural polyphenols can boost the inhibitory efficacy of calcium oxalate monohydrate crystallization, a primary component of kidney stones, up to four-fold greater than citrate alone. A combination of experimental and simulation techniques shows a strong synergy of four citrate-polyphenol inhibitor pairs on suppressing calcium oxalate monohydrate growth with minimal amounts of inhibitor, resulting from the reduction of growth kinetic constant paralleled with suppressing the crystallization driving force. Further, the inhibitor pairs demonstrated both in vitro and in vivo synergistic reductions of crystal-cell interactions, renal calcium oxalate deposition, and kidney injury, collectively presenting an effective therapeutic strategy for preventing calcium oxalate stones by boosting the inhibition efficacy of potent inhibitor pairs.

Corn Silk Polysaccharides with Different Carboxyl Contents Reduce the Oxidative Damage of Renal Epithelial Cells by Inhibiting Endocytosis of Nano-calcium Oxalate Crystals.

Renal epithelial cell injury and cell-crystal interaction are closely related to kidney stone formation. This study aims to explore the inhibition of endocytosis of nano-sized calcium oxalate monohydrate (nano-COM) crystals and the cell protection of corn silk polysaccharides (CCSPs) with different carboxyl contents (3.92, 7.75, 12.90, and 16.38%). The nano-COM crystals protected or unprotected by CCSPs were co-cultured with human renal proximal tubular epithelial cells (HK-2), and then the changes in the endocytosis of nano-COM and cell biochemical indicators were detected. CCSPs could inhibit the endocytosis of nano-COM by HK-2 cells and reduce the accumulation of nano-COM in the cells. Under the protection of CCSPs, cell morphology is restored, intracellular superoxide dismutase levels are increased, lipid peroxidation product malondialdehyde release is decreased, and mitochondrial membrane potential and lysosomal integrity are increased. The release of Ca2+ ions in the cell, the level of cell autophagy, and the rate of cell apoptosis and necrosis are also reduced. CCSPs with higher carboxyl content have better cell protection abilities. CCSPs could inhibit the endocytosis of nano-COM crystals and reduce cell oxidative damage. CCSP3, with the highest carboxyl content, shows the best biological activity.

Improving the technique of protecting concrete floors in poultry houses

The object of the study was concrete floors in a poultry house with different types of litter. The paper addressed the problem of reducing microorganism contamination of concrete floors in poultry houses to prevent biological corrosion. Corrosion of the surface of the concrete floor in the form of the formation of calcium oxalate monohydrate crystals was established by scanning electron microscopy; microscopic fungi: A. pullulans, F. sporotrichioides, and A. niger were detected. The TPD MS method established that concrete samples obtained in a room with straw lose moisture by 51.52 % more, with granules – by 342.42 % (р≤0.05), with shavings by 6.06 %, compared to control. CO from concrete samples is released less with sawdust litter by 86.40 %, with straw – by 83.49 %, with shavings – by 76.69 %, with granules – by 69.90 % (р≤0.05). The CO2 content in concrete samples from the room with sawdust was lower by 86.88 % (р≤0.05), with straw – by 55.73 %, with shavings – by 38.52 %, with granules – by 23.77 %, compared to control without litter. Microbiological studies have established that 48 hours after disinfection, the total number of colonies of microorganisms on a concrete floor with a sawdust litter likely decreased by an average of 90.19 %, straw – by 91.62 %, shavings – by 79.76 %, granules – by 82.88 % (р≤0.05), in the control – by 83.73 %. It can be argued that the disinfectant destroys microorganisms on the concrete surface regardless of the type of substrate. The peculiarity of the experiment was the use of scanning electron microscopy and TPD MS methods to study structural changes in concrete. The research is distinguished by the use of a powdered disinfectant to reduce microbial damage to a concrete floor with different types of litter. The results of the experiment could be used in the aggressive environment of poultry houses to reduce the impact on concrete structures

In Situ Liquid-Phase AFM Observation of the Molecular Step Spiral Generation on the (1−01) Surface of Calcium Oxalate Monohydrate Crystal

Calcium oxalate monohydrate (COM) crystal is the major crystalline component of human kidney stones. Its growth event at the nanometer and micrometer scales, i.e., the growth of the COM molecular steps generated from the dislocation outcrop on the crystal surface and its inhibition by associated acidic organic molecules such as citrate, is now well understood by studies conducted using in situ liquid-phase atomic force microscopy (AFM). Yet, the detailed assessment of the evolution of the COM molecular steps at the dislocation outcrop has been poorly conducted. Herein, in situ liquid-phase AFM was used to primarily investigate how those COM molecular steps are generated on a COM broadest crystal surface (1−01) and influenced by a model acidic peptide, L-aspartic acid 6mer (L-Asp6) adsorbed onto the emerging steps and terraces on the surface. Like many other mineral crystals, a segment of the pseudo-triangle-shaped step spiral, in the process of its birth from the dislocation outcrop, starts to move after reaching the critical step length. When the budding step spiral got adsorption of L-Asp6 to it, it appeared rather with ellipse-like hexagonal morphology—which is reflected in the bulk crystal morphology—implying changes in orientation-dependent step edge energy and much-delayed spiral generation time.

Calcium oxalate crystal-induced secretome derived from proximal tubular cells, not that from distal tubular cells, induces renal fibroblast activation

BackgroundKidney stone disease (KSD) is commonly accompanied with renal fibrosis, characterized by accumulation and reorganization of extracellular matrix (ECM). During fibrogenesis, resident renal fibroblasts are activated to become myofibroblasts that actively produce ECM. However, such fibroblast–myofibroblast differentiation in KSD remained unclear. Our present study thus examined effects of secreted products (secretome) derived from proximal (HK-2) vs. distal (MDCK) renal tubular cells exposed to calcium oxalate monohydrate (COM) crystals on activation of renal fibroblasts (BHK-21).MethodsHK-2 and MDCK cells were treated with 100 µg/ml COM crystals under serum-free condition for 16 h. In parallel, the cells maintained in serum-free medium without COM treatment served as the control. Secretome derived from culture supernatant of each sample was mixed (1:1) with fresh serum-free medium and then used for BHK-21 culture for another 24 h.ResultsAnalyses revealed that COM-treated-HK-2 secretome significantly induced proliferation, caused morphological changes, increased spindle index, and upregulated fibroblast-activation markers (F-actin, α-SMA and fibronectin) in BHK-21 cells. However, COM-treated-MDCK secretome had no significant effects on these BHK-21 parameters. Moreover, level of transforming growth factor-β1 (TGF-β1), a profibrotic factor, significantly increased in the COM-treated-HK-2 secretome but not in the COM-treated-MDCK secretome.ConclusionsThese data indicate, for the first time, that proximal and distal tubular epithelial cells exposed to COM crystals send different messages to resident renal fibroblasts. Only the secretome derived from proximal tubular cells, not that from the distal cells, induces renal fibroblast activation after their exposure to COM crystals. Such differential effects are partly due to TGF-β1 secretion, which is induced by COM crystals only in proximal tubular cells.

English

The influence of aqueous extract of millet on the crystallization and the dissolution of calcium oxalate (CaOx) crystals were investigated in detail under supersaturated and artificial urinary circumstances in order to understand the effect of the water-soluble components of millet on CaOx kidney stones in humans. To investigate the inhibition effect, different volumes of millet extract were added separately to the combined supersaturated and artificial urinary solutions while the crystal deposition process was monitored by UV-Vis and conductivity measurements. The harvested crystals were characterized by FT-IR, SEM, TGA, XRD and redox titrations so as to determine the structure and the morphology of CaOx crystals formed. Our findings indicate that the crystals obtained from inhibition and dissolution experiments were mainly calcium oxalate monohydrate (COM), the thermodynamically more stable phase. Furthermore, the results show that the millet extract has a promising inhibition effect in the supersaturated solution and in artificial urinary circumstances showing ~34 and ~26% decrease in crystal deposition, respectively, with 50 mL of millet extract compared to control experiments. The dissolution effect of millet extract is far superior to that of the inhibition effect. One gram of COM crystals completely disappeared when the crystals were washed ten times with 300 mL millet extracts. Under the same conditions, the control experiment showed only ~44% dissolution of COM crystals. Further investigations conducted with actual kidney stones under artificial urinary circumstances showed approximately 20% dissolution when washings were carried out using 300-mL portions of millet over one week. This novel finding may shed the light on the possible use of millet as a potential natural antiurolithic remedy to treat CaOx kidney stones in humans. Key words: Calcium oxalate, kidney stones, millet, inhibition, dissolution effect.

Cell Protection and Crystal Endocytosis Inhibition by Sulfated Laminaria Polysaccharides Against Nano-COM-Induced Oxidative Damage in Renal Epithelial Cells.

Background: The damage to renal tubular epithelial cells is closely related to the formation of kidney stones. At present, research on drugs that can protect cells from damage remains limited. Methods: This study aims to explore the protective effects of four different sulfate groups (-OSO3 -) of Laminaria polysaccharides (SLPs) on human kidney proximal tubular epithelial (HK-2) cells and determine the difference in the endocytosis of nano-sized calcium oxalate monohydrate (COM) crystals before and after protection. COM with a size of 230 ± 80 nm was used to damage HK-2 cells to establish a damage model. The protection capability of SLPs (LP0, SLP1, SLP2, and SLP3) with -OSO3 - contents of 0.73, 15, 23, and 31%, respectively, against COM crystal damage and the effect of SLPs on the endocytosis of COM crystals were studied. Results: Compared with that of the SLP-unprotected COM-injured group, the cell viability of the SLP-protected group was improved, healing capability was enhanced, cell morphology was restored, production of reactive oxygen species was reduced, mitochondrial membrane potential and lysosome integrity were increased, intracellular Ca2+ level and autophagy were decreased, cell mortality was reduced, and internalized COM crystals were lessened. The capability of SLPs to protect cells from damage and inhibit the endocytosis of crystals in cells enhanced with an increase in the -OSO3 - content of SLPs. Conclusions: SLPs with a high -OSO3 - content may become a potential green drug for preventing the formation of kidney stones.

Calcineurin B inhibits calcium oxalate crystallization, growth and aggregation via its high calcium-affinity property

Calcineurin inhibitors (CNIs) are widely used in organ transplantation to suppress immunity and prevent allograft rejection. However, some transplant patients receiving CNIs have hypocitraturia, hyperoxaluria and kidney stone with unclear mechanism. We hypothesized that CNIs suppress activities of urinary calcineurin, which may serve as the stone inhibitor. This study aimed to investigate effects of calcineurin B (CNB) on calcium oxalate monohydrate (COM) stone formation. Sequence and structural analyses revealed that CNB contained four EF-hand (Ca2+-binding) domains, which are known to regulate Ca2+ homeostasis and likely to affect COM crystals. Various crystal assays revealed that CNB dramatically inhibited COM crystallization, crystal growth and crystal aggregation. At an equal amount, degrees of its inhibition against crystallization and crystal growth were slightly inferior to total urinary proteins (TUPs) from healthy subjects that are known to strongly inhibit COM stone formation. Surprisingly, its inhibitory effect against crystal aggregation was slightly superior to TUPs. While TUPs dramatically inhibited crystal-cell adhesion, CNB had no effect on this process. Ca2+-affinity assay revealed that CNB strongly bound Ca2+ at a comparable degree as of TUPs. These findings indicate that CNB serves as a novel inhibitor of COM crystallization, growth and aggregation via its high Ca2+-affinity property.

Ethylene glycol intoxication in a pregnant cat and a tomcat

Clinical cases of two cats, poisoned with ethylene glycol: one tomcat and a pregnant female in the last quarter of gestation, are presented. Cats were reared outdoor. The history included a sudden weakness, inappetence, lethargy and repeated vomiting in both animals. Physical examination showed reduced skin elasticity, hypothermia (37.3 С° and 37.8 С°), hyporeflexia, pale mucous coats. Blood biochemical changes comprised hyperglycaemia (7.74 and 10.1 mmol/L), hyperphosphataemia (6.3 and 5.67 mmol/L), increased urea (66.6 and 68.2 mmol/L) and creatinine concentrations (1408 and 918 µmol/L). Ultrasound examination showed severely increased corticomedullary echogenicity of kidneys, including foetal kidneys. Blood sample obtained post mortem from foetuses was characterised with dramatically increased phosphate (11.3 mmol/L), urea (66.2 mmol/L) and creatinine concentrations (642 µmol/L). Foetal urine had abundant calcium oxalate monohydrate crystals. Such crystals were present also in histological preparations from kidneys of both adult cats and foetuses.

The role of amino acids in the processes of nucleation of pathogenic crystals of calcium oxalate monohydrate under human body imitating conditions

The influence of amino acids such as leucine, methionine, and phenylalanine on the kinetics of the nucleation of pathogenic crystals of calcium oxalate monohydrate (COM) was studied. Leucine, methionine, and phenylalanine molecules were found to promote the processes of nucleation and reduce the induction time of pure COM by 2–3 times (the induction time of pure COM – 100 s). The boundary concentration of the amino acids leucine and phenylalanine, above which the amino acids can have a promoting effect on the kinetic parameters of nucleation of COM crystals, was estimated as 1 mM. It was found that Leu and Met amino acid in the concentration range of 1–20 mM do not affect the morphology of COM crystals.

Inhibition of crystallization of calcium oxalate monohydrate using leaves from different species of Moringa – Experimental and theoretical studies

The effect of Moringa leaves on the inhibition of crystallization of calcium oxalate, a key component of kidney stone, has been assessed by experimental and theoretical methods. Specifically, methanol-water extracts of Moringa oleifera and Moringa peregrina leaves have been considered as inhibitors for the crystallization of calcium oxalate monohydrate. Studies based on turbidity measurements, microscopy of crystals, as well as calcium content in mixtures with and without the leaf extracts have revealed greater inhibitory potential in the case of M. oleifera as compared to M. peregrina. Modelling of the interactions between a representative fragment of calcium oxalate monohydrate with selected phytochemicals present in both leaves enables us to propose a possible mechanism of inhibition.

Roles of heat-shock protein 90 and its four domains (N, LR, M and C) in calcium oxalate stone-forming processes.

Human heat-shock protein 90 (HSP90) has four functional domains, including NH2-terminal (N), charged linker region (LR), middle (M) and COOH-terminal (C) domains. In kidney stone disease (or nephrolithiasis/urolithiasis), HSP90 serves as a receptor for calcium oxalate monohydrate (COM), which is the most common crystal to form kidney stones. Nevertheless, roles of HSP90 and its four domains in kidney stone formation remained unclear and under-investigated. We thus examined and compared their effects on COM crystals during physical (crystallization, growth and aggregation) and biological (crystal–cell adhesion and crystal invasion through extracellular matrix (ECM)) pathogenic processes of kidney stone formation. The analyses revealed that full-length (FL) HSP90 obviously increased COM crystal size and abundance during crystallization and markedly promoted crystal growth, aggregation, adhesion onto renal cells and ECM invasion. Comparing among four individual domains, N and C domains exhibited the strongest promoting effects, whereas LR domain had the weakest promoting effects on COM crystals. In summary, our findings indicate that FL-HSP90 and its four domains (N, LR, M and C) promote COM crystallization, crystal growth, aggregation, adhesion onto renal cells and invasion through the ECM, all of which are the important physical and biological pathogenic processes of kidney stone formation.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-022-04483-z.

Trigonelline prevents kidney stone formation processes by inhibiting calcium oxalate crystallization, growth and crystal-cell adhesion, and downregulating crystal receptors

Trigonelline is the second most abundant bioactive alkaloid found in coffee. It is classified as a phytoestrogen with similar structure as of estradiol and exhibits an estrogenic effect. A previous study has reported that fenugreek seed extract rich with trigonelline can reduce renal crystal deposition in ethylene glycol-induced nephrolithiatic rats. However, direct evidence of such anti-lithogenic effects of trigonelline and underlying mechanisms have not previously been reported. Our study therefore addressed the protective effects and mechanisms of trigonelline against kidney stone-forming processes using crystallization, crystal growth, aggregation and crystal-cell adhesion assays. Also, proteomics was applied to identify changes in receptors for calcium oxalate monohydrate (COM), the most common stone-forming crystal, on apical membranes of trigonelline-treated renal tubular cells. The analyses revealed that trigonelline significantly reduced COM crystal size, number and mass during crystallization. Additionally, trigonelline dose-dependently inhibited crystal growth and crystal-cell adhesion, but did not affect crystal aggregation. Mass spectrometric protein identification showed the smaller number of COM crystal receptors on apical membranes of the trigonelline-treated cells. Western blotting confirmed the decreased levels of some of these crystal receptors by trigonelline. These data highlight the protective mechanisms of trigonelline against kidney stone development by inhibiting COM crystallization, crystal growth and crystal-cell adhesion via downregulation of the crystal receptors on apical membranes of renal tubular cells.

Systematic analysis of modulating activities of native human urinary Tamm-Horsfall protein on calcium oxalate crystallization, growth, aggregation, crystal-cell adhesion and invasion through extracellular matrix

Functions of Tamm-Horsfall protein (THP), the most abundant human urinary protein, have been studied for decades. However, its precise roles in kidney stone formation remain controversial. In this study, we aimed to clarify the roles of native human urinary THP in calcium oxalate monohydrate (COM) kidney stone formation. THP was purified from the human urine by adsorption method using diatomaceous earth (DE). Its effects on stone formation processes, including COM crystallization, crystal growth, aggregation, crystal-cell adhesion and invasion through extracellular matrix (ECM), were examined. SDS-PAGE and Western blotting confirmed that DE adsorption yielded 84.9% purity of the native THP isolated from the human urine. Systematic analyses revealed that THP (at 0.4–40 μg/ml) concentration-dependently reduced COM crystal size but did not affect the crystal mass during initial crystallization. At later steps, THP concentration-dependently inhibited COM crystal growth and aggregation, and prevented crystal-cell adhesion only at 40 μg/ml. However, THP did not affect crystal invasion through the ECM. Sequence analysis revealed two large calcium-binding domains (residues 65–107 and 108–149) and three small oxalate-binding domains (residues 199–207, 361–368 and 601–609) in human THP. Immunofluorescence study confirmed the binding of THP to COM crystals. Analyses for calcium-affinity and/or oxalate-affinity demonstrated that THP exerted a high affinity with only calcium, not oxalate. Functional validation revealed that saturation of THP with calcium, not with oxalate, could abolish the inhibitory effects of THP on COM crystal growth, aggregation and crystal-cell adhesion. These data highlight the inhibitory roles of the native human urinary THP in COM crystal growth, aggregation and crystal-cell adhesion, which are the important processes for kidney stone formation. Such inhibitory effects of THP are most likely mediated via its high affinity with calcium ions.