Calcium Oxalate Crystal Growth Research Articles

Exploring the Anti-Urolithiatic Potential of White Seeds from Abrus Precatorius Via In Vitro Investigations

Medicinal plants are a valuable aspect of local heritage with global importance, often used as remedies for various health conditions. Their therapeutic properties are linked to a wide variety of complex chemical compounds, particularly secondary metabolites, found in different plant parts. Identifying the specific components responsible for medicinal effects is essential. Phytochemicals, which are abundant in medicinal plants, are generally considered safer and less toxic compared to synthetic alternatives. In line with this understanding, the present research investigates the in vitro anti-urolithiatic potential of white seeds of Abrusprecatorius. The seeds were chosen for their possible role in treating urolithiasis. Phytochemical screening of the plant extract revealed the presence of compounds such as flavonoids, coumarins, saponins, proteins, glycosides, quinones, and tannins.Two In Vitro assays, Crystal Nucleation and Aggregation, were performed, both with and without inhibitors. The study tested various concentrations of plant extracts (50mg/ml, 100mg/ml, 150mg/ml, 200mg/ml, and 250mg/ml) using four solvents: water, ethanol, chloroform, and petroleum ether, to assess their effect on calcium oxalate (CaOX) crystal formation, which is a primary component of kidney stones.Among the extracts tested, the water extract showed the highest inhibition of nucleation (85%) at a concentration of 250mg/ml. Aqueous, ethanol, and chloroform extracts exhibited notable anti-urolithiatic activity across different concentrations. Crystal aggregation was assessed using a spectrophotometer, and the water extract demonstrated the highest inhibition (78%) at the same concentration. Additionally, the chloroform extract showed significant anti-urolithiatic activity.When comparing extracts, the chloroform extract at 500mg/ml was the most effective in inhibiting the growth of calcium oxalate crystals. Overall, the study highlights the potential of Abrusprecatorius white seed extracts in inhibiting calcium oxalate crystal nucleation, aggregation, and growth, suggesting their potential as a treatment for urolithiasis. These results underscore the value of these extracts as promising agents in managing kidney stones

The Effect of Lidan Huashi Pills on the Glutathione Peroxidase Activity and Growth of Calcium Oxalate Crystals in the Kidneys of Rats

Objective: To explore the effects of Lidan Huashi Pills on the glutathione peroxidase (GSH-Px) activity and growth of calcium oxalate crystals in the kidneys of rats. Methods: 30 rats were randomly divided into a blank group, a model group, and an experimental group, with 10 rats in each group. The blank group was free to eat and drink water for 8 weeks; the ethylene glycol method was used for the standard calcium oxalate crystal modeling in the control group and experimental group for 4 weeks. The model group was given free feeding and physiological saline (2 ml/d) by continuous gavage for 4 weeks; the experimental group was given free feeding and Lidan Huashi Pills (450 mg/kg, 2 ml/d) by continuous gavage for 4 weeks. After 4 weeks, all rats were euthanized, and the left kidney was taken for GSH-Px level detection. The right kidney was stained with hematoxylin and eosin (H&E) to observe the formation of calcium oxalate crystals. Results: After 4 weeks of modeling, the urinary calcium levels in the model group and experimental group significantly increased compared to the blank group (868.00 ± 39.29 vs 929.40 ± 33.61, P < 0.05), indicating successful modeling. The urine calcium ion concentration in the experimental group after modeling was significantly lower than that in the model group (929.40 ± 33.61 vs 888.60 ± 25.92, P < 0.05). The grading score of calcium oxalate crystals in the model group was significantly higher than that in the blank group (P < 0.05); the grading score of calcium oxalate crystals in the experimental group was lower than that in the model group, and the difference was not statistically significant (P > 0.05). The GSH-Px activity in the left kidney of the model group was significantly lower than that of the blank group [203.49 (208.21, 144.22) vs 494.91 (431.32, 538.18), P < 0.05); the GSH-Px activity in the left kidney of the experimental group was significantly higher than that of the model group [433.60 (383.86, 504.49) vs 203.49 (208.21, 144.22), P < 0.05). Morphological observation and H&E staining suggest that the formation of right kidney crystals and inflammation in the experimental group are between the blank group and the model group. Conclusion: Lidan Huashi Pills can enhance the serum GSH-Px activity in rats and inhibit the growth of calcium oxalate crystals and inflammatory response in the kidneys, thus playing a role in preventing and treating urinary tract stones.

Targeting urinary calcium oxalate crystallization with inulin-type AOFOS from Aspidopterys obcordata Hemsl. for the management of rat urolithiasis

Calcium oxalate crystals play a key role in the development and recurrence of kidney stones (also known as urolithiasis); thus, inhibiting the formation of these crystals is a central focus of urolithiasis prevention and treatment. Previously, we reported the noteworthy in vitro inhibitory effects of Aspidopterys obcordata fructo oligosaccharide (AOFOS), an active polysaccharide of the traditional Dai medicine Aspidopterys obcordata Hemsl. (commonly known as Hei Gai Guan), on the growth of calcium oxalate crystals. To investigated the effectiveness and mechanism of AOFOS in treating kidney stones. A kidney stones rats model was developed, followed by examining AOFOS transport dynamics and effectiveness in live rats. Additionally, a correlation between the polysaccharide and calcium oxalate crystals was studied by combining crystallization experiments with density functional theory calculations. The results showed that the polysaccharide was transported to the urinary system. Furthermore, their accumulation was inhibited by controlling their crystallization and modulating calcium ion and oxalate properties in the urine. Consequently, this approach helped effectively prevent kidney stone formation in the rats. The present study emphasized the role of the polysaccharide AOFOS in modulating crystal properties and controlling crystal growth, providing valuable insights into their potential therapeutic use in managing kidney stone formation.

Effect of croton Tonkinensis Gagnep extract on calcium oxalate crystal formation causing kidney stones under in vitro conditions

The study investigated the use of Croton tonkinensis Gagnep extract to evaluate its ability to inhibit Calcium oxalate crystal formation, including three main stages: nucleation, growth, and aggregation. Croton tonkinensis Gagnep samples were extracted using a soaking method with 96% ethanol to obtain the concentrated extract. The percentage of inhibition of Calcium oxalate crystal nucleation by the Croton tonkinensis Gagnep extract was determined using spectrophotometry at a wavelength of 620 nm. Meanwhile, the inhibition efficacy on the growth of Calcium oxalate crystals was evaluated by measuring the optical density of the test sample at a wavelength of 214 nm over a period of 780 seconds. The inhibition efficacy on the aggregation of Calcium oxalate crystals by the extract was determined by measuring the optical density at a wavelength of 620 nm at different time intervals of 30, 60, 90, 180, 360, and 720 minutes. The research results showed that the extract had a high efficiency of 1.36%. The Croton tonkinensis Gagnep extract contained various compounds such as flavonoids, alkaloids, saponins, terpenoids, tannins, and phenols. The extract exhibited the ability to inhibit nucleation with an IC50 value of 7.78 mg/mL, as well as inhibit the growth and aggregation of Calcium oxalate crystals with times of 13 minutes and 90 minutes, respectively.

Crystallization ripening and erosion of calcium oxalate under the effect of bacteria and a polymer materials surface.

As typical examples of pathological biomineralization, urinary stones and stent encrustation have been associated with bacteria, yet the underlying mechanisms remain unclear. In this study, the effect of Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus on the nucleation and growth of calcium oxalate crystals both in solution and on material surfaces in vitro was investigated. Both bacteria can promote calcium oxalate crystallization, and E. coli shows a prominent ability to boost the nucleation and growth rate. Interestingly, we discovered an Ostwald ripening phenomenon after the initial nucleation on the material surfaces, where larger particles emerge upon the disappearance of small nuclei particles, evident in the case of S. aureas. Over an extended period of time, erosion and disintegration of the crystals was observed when bacteria were involved. Based on these understandings, we developed a new functional surface by synthesizing an antibacterial polypeptoid in-house and utilizing polyurethane as the substrate material. This surface exhibits a synergistic effect that inhibits the formation of calcium oxalate crystals. This study helps to elucidate the role of bacteria in calcium oxalate biomineralization and supports further development of treatment approaches such as anti-encrustation polymer materials.

Nickel as a modifier of calcium oxalate: an in situ liquid cell TEM investigation of nucleation and growth.

Despite extensive research on the nucleation and growth of calcium oxalate (CaOx) crystals, there are still several challenges and unknowns that remain. In particular, the role of trace metal elements in the promotion or inhibition of CaOx crystals is not well understood. In the present study, in situ graphene liquid cell transmission electron microscopy (in situ GLC TEM) was used to observe real-time, nanoscale transformations of CaOx crystals in the presence of nickel ions (Ni2+). The results showed that Ni2+ form Ni-water complexes, acting as a shape-directing species, generating a unique morphology and altering growth kinetics. Transient adsorption of Ni-water complexes resulted in a metastable phase formation of calcium oxalate trihydrate. Atomistic molecular dynamics simulations confirmed that Ni2+ acts as a weak inhibitor which slows down the CaOx crystallization, elucidating that Ni2+ impacts small-sized CaOx clusters by bringing more water into the clusters. This work highlighted the intricacies behind the effect of Ni2+ on CaOx biomineralization that were made possible to discern using in situ GLC TEM.

Resveratrol inhibits calcium oxalate crystal growth, reduces adhesion to renal cells and induces crystal internalization into the cells, but promotes crystal aggregation

Resveratrol is a natural phenolic compound that belongs to stilbenoid group found in diverse plants. Health benefits and therapeutic potentials of resveratrol have been widely recognized in various diseases. In kidney stone disease, it can alleviate oxalate-induced hyperproduction of free radicals in renal epithelial cells. Nevertheless, its direct effects on calcium oxalate (CaOx) crystal, which is the major stone component, remained unclear. This study therefore addressed the direct effects of resveratrol (at 1, 10 or 100μM) on each step of CaOx kidney stone formation. The results revealed that resveratrol had no significant effects on CaOx crystallization. However, resveratrol significantly decreased CaOx crystal growth and adhesion to renal epithelial cells at all concentrations, and induced crystal internalization into the cells (a process related to crystal degradation by endolysosomes) in a concentration-dependent manner. On the other hand, resveratrol promoted crystal aggregation. These data indicate that resveratrol serves as a dual modulator on CaOx stone formation. While it inhibits CaOx stone development by reducing crystal growth and adhesion to renal cells and by inducing crystal internalization into the cells, resveratrol promotes crystal aggregation, which is one of the mechanisms leading to kidney stone formation.

Antioxidant Activity of Auricularia auricula Polysaccharides with Different Molecular Weights and Cytotoxicity Difference of Polysaccharides Regulated CaOx to HK-2 Cells.

This study aimed to investigate the growth of calcium oxalate (CaOx) crystals regulated by Auricularia auricular polysaccharides (AAPs) with different viscosity-average molecular weights (Mv), the toxicity of AAP-regulated CaOx crystals toward HK-2 cells, and the prevention and treatment capabilities of AAPs for CaOx stones. The scavenging capability and reducing capacity of four kinds of AAPs (Mv of 31.52, 11.82, 5.86, and 3.34 kDa) on hydroxyl, ABTS, and DPPH free radicals and their capability to chelate divalent iron ions were detected. AAP-regulated CaOx crystals were evaluated by using zeta potential, thermogravimetric analysis, X-ray diffraction, and scanning electron microscopy. The cytotoxicity of AAP-regulated crystals was evaluated through examination of cell viability, cell death, malondialdehyde (MDA) content, and cell surface hyaluronic acid (HA) expression. The in vitro antioxidant activities of the four AAPs were observed in the following order: AAP0 < AAP1 < AAP2 < AAP3. Thus, AAP3, which had the smallest Mv, had the strongest antioxidant activity. AAPs can inhibit the growth of CaOx monohydrate (COM), induce the formation of CaOx dihydrate (COD), and reduce the degree of crystal aggregation, with AAP3 exhibiting the strongest capability. Cell experiments showed the lowest cytotoxicity in AAP3-regulated CaOx crystals, along with the lowest MDA content, HA expression, and cell mortality. In addition, COD presented less cytotoxicity than COM. Meanwhile, the cytotoxicity of blunt crystals was less than that of sharp crystals. AAPs, particularly AAP3, showed an excellent antioxidative capability in vitro, and AAP3-regulated CaOx crystals presented minimal cytotoxicity.

In-Vitro Analysis of Ethanol Extract of Saponaria Officinalis for Antiurolithiatic Activity

The goal of the current study was to assess Saponaria officinalis ethanol extracts' in-vitro anti-urolithiatic effectiveness as a potential natural therapy to combat adverse effects brought on by the use of contemporary synthetic medications. S. officinalis extracted with ethanol was put to the test for its ability to treat urolithiasis utilizing in vitro studies (nucleation, aggregation assays). This extract was evaluated and contrasted with the reference medication cystone at increasing concentrations of 200, 400, 600, 800, and 1000μg/ml. The extract's ability to suppress the growth of calcium oxalate crystals in preformed forms was evaluated. The results of the S. officinalis extract on the nucleation assay were significant and showed considerable inhibition 64.2%±0.5 compared to standard cystone drug 67.7%±0.8 at 1000μg/ml concentration. Additionally, the ethanol extract of S. officinalis showed significant inhibition in the aggregation assay with a difference of 74.2%±0.08 compared to cystone 76.7%±0.09 at 1000μg/ml concentration. At a dose level of 1000μg/ml, the ethanol extract of S. officinalis possesses anti-urolithiatic potentials in vitro. It is possible to further exploit the ethanol extract of S. officinalis as a potential anti-urolithiasis medication.

Himalayan Citrus jambhiri juice reduced renal crystallization in nephrolithiasis by possible inhibition of glycolate oxidase and matrix metalloproteinases.

Citrus fruits are a very rich source of electrolytes and citric acid. They have been used traditionally for treating urinary ailments and renal stones. Citrus jambhiri is indigenously used as a diuretic. Present study aimed at establishing the antiurolithiatic potential of the juice of Citrus jambhiri fruits along with the elucidation of the mechanism involved in the urolithiasis disease defying activity. The antiurolithiatic activity was established by means of nucleation, growth and aggregation assay in the in vitro settings and by means of ethylene glycol mediated calcium oxalate urolithiasis in the male Wistar rats. Docking studies were performed in an attempt to determine the mechanism of the antiurolithiatic action. Present study revealed the role of C. jambhiri fruit juice in reducing nucleation, growth and aggregation of calcium oxalate crystals by possible reduction in the urinary supersaturation relative to calcium oxalate and raising the zeta potential of the calcium oxalate crystals. C. jambhiri fruit juice treatment in experimental rats produced significant amelioration of hypercalciuria, hyperoxaluria, hyperphosphaturia, hyperproteinuria, hyperuricosuria, hypocitraturia and hypomagnesiuria and ion activity product of calcium oxalate. It exhibited nephroprotection against calcium oxalate crystals induced renal tubular dilation and renal tissue deterioration. Docking studies further revealed high binding potential of the phytoconstituents of C. jambhiri viz. narirutin, neohesperidin, hesperidin, rutin and citric acid with glycolate oxidase and matrix metalloproteinase-9. C. jambhiri fruit juice possesses excellent antiurolithiatic activity. The study reveals antiurolithiatic mechanism that involves restoration of equilibrium between the promoters and inhibitors of stone formation; and inhibition of matrix metalloproteinases and glycolate oxidase.

Antioxidant Activities and Cytotoxicity of the Regulated Calcium Oxalate Crystals on HK-2 Cells of Polysaccharides from Gracilaria lemaneiformis with Different Molecular Weights.

The antioxidant activities of seven degraded products (GLPs) with different molecular weights (Mw) of polysaccharides from Gracilaria lemaneiformis were compared. The Mw of GLP1-GLP7 were 106, 49.6, 10.5, 6.14, 5.06, 3.71 and 2.42 kDa, respectively. The results show that GLP2 with Mw = 49.6 kDa had the strongest scavenging capacity for hydroxyl radical, DPPH radical, ABTS radical and reducing power. When Mw < 49.6 kDa, the antioxidant activity of GLPs increased with the increase in Mw, but when Mw increased to 106 kDa, their antioxidant activity decreased. However, the ability of GLPs to chelate Fe2+ ions increased with the decrease in polysaccharide Mw, which was attributed to the fact that the polysaccharide active groups (-OSO3- and -COOH) were easier to expose, and the steric hindrance was smaller when GLPs chelated with Fe2+. The effects of GLP1, GLP3, GLP5 and GLP7 on the crystal growth of calcium oxalate (CaOx) were studied using XRD, FT-IR, Zeta potential and thermogravimetric analysis. Four kinds of GLPs could inhibit the growth of calcium oxalate monohydrate (COM) and induce the formation of calcium oxalate dihydrate (COD) in varying degrees. With the decrease in Mw of GLPs, the percentage of COD increased. GLPs increased the absolute value of the Zeta potential on the crystal surface and reduced the aggregation between crystals. Cell experiments showed that the toxicity of CaOx crystal regulated by GLPs to HK-2 cells was reduced, and the cytotoxicity of CaOx crystal regulated by GLP7 with the smallest Mw was the smallest, which was consistent with the highest SOD activity, the lowest ROS and MDA levels, the lowest OPN expression level and the lowest cell necrosis rate. These results suggest that GLPs, especially GLP7, may be a potential drug for the prevention and treatment of kidney stones.

Inhibitory activity of Enhydra fluctuans Lour. on calcium oxalate crystallisation through in silico and in vitro studies.

The decoction of the whole plant of Enhydra fluctuans is used ethno medicinally by various tribes for the treatment of kidney stones and urinary problems. However, no scientific studies were carried out to delineate its influence on urinary stone formation and crystallisation. Hence, the present study is proposed to investigate the effect of the aqueous extract of Enhydra fluctuans extract on in vitro crystallisation of calcium oxalate. The present study also evaluated. in silico studies of the metabolites with the target proteins present in the renal calcium oxalate stone matrix. The plant material was subjected to decoction to obtain an aqueous extract. The effect of the extract on calcium oxalate crystallization was evaluated by in vitro nucleation and aggregation assays. Further, the metabolites present in E. fluctuans were mined from the existing literature and their number was found to be 35. The selected 35 metabolites of E. fluctuans were subjected to molecular docking with the 5 proteins which are known to be responsible for calcium oxalate crystal growth. Results of in vitro studies indicated that the extract (50, 100, and 200μg/mL) and standard drug cystone (1,000μg/mL) exhibited an inhibitory role in the nucleation process where the percentage inhibitions were 52.69, 43.47, 21.98, and 31.67μg/mL respectively. The results of molecular docking studies revealed that 2 out of 35 metabolites i.e. Baicalein-7-O-diglucoside and 4',5,6,7-Tetrahydroxy-8-methoxy isoflavone-7-O-beta-D- galactopyranosyl-(1→3)-O-beta-D-xylopyranosyl-(1→4)- O-alpha-L-rhamnopyranoside showed modulatory effects on the four renal stone matrix-associated protein (Human CTP: Phosphoethanolamine Cytidylyltransferase (Protein Data Bank ID: 3ELB), UDP glucose: glycoprotein glucosyltransferase 2 (Gene: UGGT2) (AlphaFold) and RIMS-binding protein 3A (Gene: RIMBP3) (AlphaFold), and Ras GTPase activating-like protein (PDB: 3FAY) based on their docking scores which indicates that they may inhibit the crystallization process. Findings from this study show that Enhydra fluctuans may be effective in the prevention of the crystallization of calcium oxalate. However, further, in vivo studies as well as molecular studies are needed to be conducted to confirm and strengthen its anti-urolithiatic activity and to elucidate the possible mechanism of action involved therein.

Antiurolithiatic Potential of Three Sri Lankan Medicinal Plants by the Inhibition of Nucleation, Growth, and Aggregation of Calcium Oxalate Crystals In Vitro

Background Deposition and formation of stones in any part of the urinary system is called urolithiasis. CaOx is the predominant component of most stones, and the formation of these stones is a multistep process that includes supersaturation, nucleation, aggregation, growth, and retention. In ayurvedic medicine, medicinal plants are used for the management of kidney stones. The objective of this study was to determine the effect of aqueous, ethanol, and hexane extracts of Drymoglossum piloselloides leaves, Kalanchoe laciniata leaves, and Aegle marmelos flowers against CaOx urolithiasis in vitro. Methods The crystallization of CaOx monohydrate (COM) and dihydrate (COD) was induced in a synthetic urine system. The nucleation, growth, and aggregation of crystals were measured using spectrophotometric methods. The results were compared against the polyherbal drug, Cystone, under identical concentrations. Crystals generated in the urine were also observed under light microscopy. Statistical differences and percentage inhibitions were calculated using standard formulae and compared. A preliminary phytochemical screening was also performed to detect active phytoconstituents present in the three plants used in the study. Results The results obtained clearly demonstrated that Kalanchoe laciniata, Aegle marmelos, and Drymoglossum piloselloides have the capacity to inhibit the nucleation, growth, and aggregation of CaOx crystals. Microscopic examination of crystals revealed the presence of more COM than COD crystals but a dose-dependent reduction in crystals was observed in the presence of plant extracts. Hexane, ethanol, and aqueous extracts of all three plants had different capabilities to inhibit nucleation, growth, and aggregation of CaOx crystals but their activities were different at different concentrations. Preliminary phytochemical screening revealed the presence of reducing sugars, proteins, flavonoids, tannins, and polyphenol compound in Kalanchoe laciniata and Drymoglossum piloselloides and reducing sugars, proteins, anthracene glycosides, and saponins in Aegle marmelos. Conclusions This study provided evidence that Kalanchoe laciniata, Aegle marmelos, and Drymoglossum piloselloides have the potential to be developed as inhibitors of nucleation, growth, and aggregation of CaOx crystals in the treatment of urolithiasis.

Dual Mechanism of Natural Polyphenols on crystal whiskers formation on calcium oxalate monohydrate crystal surface

A new class of molecular modifiers, i.e. natural polyphenols, inspired from Chinese tea has been discovered for CaOx crystallization inhibition. These polyphenol modifiers display a unique dual action of growth promoter and inhibitor dependent upon their concentrations. The unexpected, remarkable growth promotion reaches a new record up to 75%, leading to the formation of macroscopic crystal whiskers by change in growth mode from spiral-mediated to generation of 2D islands and layer spreading mechanism and concurrent growth of step bunches (not seen commonly). • Calcium oxalate crystal whiskers were induced by natural polyphenols on COM (0 1 0) surfaces. • Step growth mode was changed from spiral dislocation to 2D layer growth. • Adsorbed polyphenols on surface inhibited step advancement and induced the formation of step bunches. • The governing regime of whisker formation is determined by inhibitors concentration and supersaturation. Designing (macro)molecules controlling crystallization is an essential approach to regulate structure and properties of crystalline materials and circumvent detrimental mineralization in nature, biological, and synthetic systems. Here we discovered four novel, natural polyphenols, including gallic acid, ellagic acid, punicalagin, and tannic acid, as a new class of inhibitors for nucleation and crystal growth of calcium oxalate (CaOx), a primary constitution of kidney stones. Study of time-resolved growth reveals that polyphenols regulate the kinetics of crystal-liquid interface acting as a dual role of growth promoter and inhibitor which displays a new record of growth promotion up to 75% or complete suppression, dependent upon the modifier concentration. Time-elapsed in situ atomic force microscopy measurements unveil these modifiers regulate the growth mode from spiral-mediated to generation of two-dimensional (2D) islands and layer spreading mechanism, leading to formation of step bunches growing out of (0 1 0) surface and forming macroscopically crystal whiskers. The governing regime between growth promotion and inhibition is determined by inhibitors concentration but can be influenced by crystallization driving force. Such unique growth mechanism of crystal whiskers may also provide potential implications for understanding and synthesizing branched and hierarchical structures of biomimetic materials.

Sulfated Undaria pinnatifida polysaccharide inhibits the formation of kidney stones by inhibiting HK-2 cell damage and reducing the adhesion of nano‑calcium oxalate crystals

The formation of kidney stone is closely related to cell injury and crystal adhesion. The sulfur trioxide-pyridine method was used to sulfate raw Undaria pinnatifida polysaccharide (UPP) with a molecular weight (Mw) of 8.33 kDa. Four polysaccharides with the sulfate group (-OSO3-) contents of 1.59% (UPP0), 6.03% (UPP1), 20.83% (UPP2), and 36.39% (UPP3) were obtained. The antioxidant activity of the four UPPs, the difference in oxidative damage inflicted by nano-CaOx monohydrate (nano-COM) on human proximal tubular epithelial (HK-2) cells before and after protection by UPPs, and the inhibitory effect on nano-COM adhesion were explored. Structural characterization showed that sulfation was successful. As the -OSO3- content in the UPPs was increased, the antioxidant activity and capability of the UPPs to regulate the growth of calcium oxalate (CaOx) crystals gradually increased. The damage caused by nano-COM crystals to HK-2 cells under protection by UPPs was weakened. This effect enhanced cell viability, enabled the maintenance of good cell morphology, reduced reactive oxygen species (ROS) levels, and inhibited the decrease in mitochondrial membrane potential, as well as decreased the eversion of phosphatidylserine (PS) and the expression of the adhesion proteins osteopontin (OPN), heat shock protein (HSP 90), and Annexin A1 (ANXA1). The adhesion of nano-COM to HK-2 cells was inhibited under the protection by UPPs. UPP3 with the highest content of -OSO3- presented the best antioxidant activity and crystal regulation ability, while UPP2 with the second highest -OSO3- content showed optimal cell protection ability and crystal adhesion inhibition ability. The biological activity of UPPs was regulated by Mw and -OSO3- content. UPP2 with moderate -OSO3- content may become a potential drug for preventing CaOx stones.

Hyaluronic acid promotes calcium oxalate crystal growth, crystal-cell adhesion, and crystal invasion through extracellular matrix

Hyaluronic acid (HA), a macromolecule in glycosaminoglycans family, is normally excreted into the urine with a small amount, but with much greater level in the urine from stone patients (formers). However, its precise roles in kidney stone pathogenesis remained unclear. This study examined its roles in calcium oxalate (CaOx) kidney stone formation processes, including neocrystallization, crystal growth, adhesion, internalization, aggregation and invasion. The results showed that HA (1, 10, 100, 1000, or 10,000ng/ml) did not affect CaOx neocrystallization, aggregation, and internalization into MDCK distal renal tubular cells. However, HA significantly promoted CaOx crystal growth (at 10-10,000ng/ml), adhesion onto renal tubular cells (at 1000-10,000ng/ml), and invasion through extracellular matrix (ECM) (at 1-10,000ng/ml). Analysis of the plasminogen sequence revealed six sites with the HA-binding motif "B(X7)B" that explained the HA ability to trigger the plasminogen-plasmin system required for crystal invasion. In summary, our systematic analysis highlights the promoting effects of HA on CaOx crystal growth, crystal-cell adhesion and invasion through the ECM. These effects of HA may explain its high level in the stone patients' urine, thereby promoting the stone formation.

Antioxidant activity of sulfated Porphyra yezoensis polysaccharides and their regulating effect on calcium oxalate crystal growth

The nucleation, growth and aggregation of calcium oxalate (CaOx) crystals and the oxidative damage of renal tubular epithelial cells are the key factors to induce kidney stones. In this study, degraded Porphyra yezoensis polysaccharide (PYP0) with 14.14% sulfate group (-OSO3-) content was modified via the sulfur trioxide-pyridine method to obtain three kinds of sulfated P. yezoensis polysaccharides (PYPs), namely, PYPS1, PYPS2, and PYPS3, with -OSO3- group contents of 17.11%, 20.28%, and 27.14% respectively. Fourier transform infrared spectroscopy, 1H NMR, and 13C NMR analyses showed that the -OSO3- groups replaced the hydroxyl groups at the C2, C4, and C6 positions on (1→3)-linked β-D-galactose, the basic structural skeleton unit of PYP0. The antioxidant activity of the PYPSs increased after sulfation, and their scavenging capacity for OH and DPPH free radicals was enhanced with the increase in their -OSO3- group content. Calcium oxalate (CaOx) crystal growth experiments showed that sulfated PYPs promoted the conversion of the thermodynamically stable and sharp CaOx monohydrate (COM) crystals into the thermodynamically unstable and round CaOx dihydrate crystals. With the increase in the -OSO3- group content of the polysaccharides, the concentration of soluble Ca2+ ions in the supernatant increased and the amount of CaOx precipitate decreased. PYPs were nontoxic to human kidney proximal tubular epithelial cells (HK-2) and could protect HK-2 from oxidative damage caused by nano-COM and reduce the level of reactive oxygen species in cells. PYPS3, which had the highest degree of sulfation, had the best protective capability. The results of this work showed that sulfation improved the biological activity of PYPs. This study could provide inspiration for the development of new drugs for the prevention and treatment of kidney stones.

Genetic Background but Not Intestinal Microbiota After Co-Housing Determines Hyperoxaluria-Related Nephrocalcinosis in Common Inbred Mouse Strains.

Calcium oxalate (CaOx) crystal formation, aggregation and growth is a common cause of kidney stone disease and nephrocalcinosis-related chronic kidney disease (CKD). Genetically modified mouse strains are frequently used as an experimental tool in this context but observed phenotypes may also relate to the genetic background or intestinal microbiota. We hypothesized that the genetic background or intestinal microbiota of mice determine CaOx crystal deposition and thus the outcome of nephrocalcinosis. Indeed, Casp1 -/-, Cybb -/- or Casp1 -/-/Cybb -/- knockout mice on a 129/C57BL/6J (B6J) background that were fed an oxalate-rich diet for 14 days did neither encounter intrarenal CaOx crystal deposits nor nephrocalcinosis-related CKD. To test our assumption, we fed C57BL/6N (B6N), 129, B6J and Balb/c mice an oxalate-rich diet for 14 days. Only B6N mice displayed CaOx crystal deposits and developed CKD associated with tubular injury, inflammation and interstitial fibrosis. Intrarenal mRNA expression profiling of 64 known nephrocalcinosis-related genes revealed that healthy B6N mice had lower mRNA levels of uromodulin (Umod) compared to the other three strains. Feeding an oxalate-rich diet caused an increase in uromodulin protein expression and CaOx crystal deposition in the kidney as well as in urinary uromodulin excretion in B6N mice but not 129, B6J and Balb/c mice. However, backcrossing 129 mice on a B6N background resulted in a gradual increase in CaOx crystal deposits from F2 to F7, of which all B6N/129 mice from the 7th generation developed CaOx-related nephropathy similar to B6N mice. Co-housing experiments tested for a putative role of the intestinal microbiota but B6N co-housed with 129 mice or B6N/129 (3rd and 6th generation) mice did not affect nephrocalcinosis. In summary, genetic background but not the intestinal microbiome account for strain-specific crystal formation and, the levels of uromodulin secretion may contribute to this phenomenon. Our results imply that only littermate controls of the identical genetic background strain are appropriate when performing knockout mouse studies in this context, while co-housing is optional.

POS-398 Oxidative modifications of urinary proteins promote calcium oxalate crystallization, crystal growth and aggregation

The incidence/prevalence of kidney stone disease has been increasing around the globe but its pathogenic mechanisms remain unclear. Herein, we examined effects of oxidative modifications of urinary proteins on calcium oxalate (CaOx) crystallization, crystal growth and aggregation. Urinary proteins derived from 20 healthy individuals were modified by performic oxidation and the presence of oxidatively modified urinary proteins was verified, quantified and characterized by Oxyblot assay and tandem mass spectrometry (nanoLC-ESI-LTQ-Orbitrap-MS/MS). Subsequently, activities of oxidatively modified urinary proteins on CaOx crystallization, crystal growth and aggregation were evaluated. Both Oxyblot assay and nanoLC-ESI-LTQ-Orbitrap-MS/MS confirmed that urinary proteins were successfully oxidized by performic oxidation. The results showed that the oxidatively modified urinary proteins significantly promoted CaOx crystallization, crystal growth and aggregation. In contrast, the non-modified urinary proteins had inhibitory activities against CaOx crystallization, crystal growth and aggregation. These data indicate that oxidative modifications of urinary proteins increase the risk of kidney stone disease by promoting CaOx crystallization, crystal growth and aggregation.

Antilithiatic effect of C. dactylon, E. officinalis, K. pinnata, and B. nutans ethyl acetate fraction on glyoxylate-induced nephrolithiasis

BackgroundThe plants Cynodon dactylon (C. dactylon), Emblica officinalis (E. officinalis), Kalanchoe pinnata (K. pinnata), and Bambusa nutans (B. nutans) have been reported to possess diuretic and antiurolithiatic potential against ethylene glycol and ammonium chloride along with in vitro calcium oxalate (CaOx) crystal growth inhibition property. Our previous research publications reported a rich presence of antioxidative phytocompounds like polyphenols and flavonoids in ethyl acetate fractions of these plants. This present study aims to explore antiurolithiatic potential of C. dactylon, E. officinalis, K. pinnata, and B. nutans ethyl acetate fraction following 7 days of sodium glyoxalate treatment on mice.ResultsSodium glyoxylate treatment caused significant (P < 0.01–0.001) reduction in the urine magnesium and creatinine and elevation in oxalate, citrate, calcium, and phosphate levels. Ethyl acetate fraction of K. pinnata and B. nutans showed a highly significant antilithiatic effect by increasing urine volume, normalizing disrupted urine parameters, increasing LDH level, and decreasing kidney tissue oxalate content. E. officinalis and K. pinnata ethyl acetate fraction treatment showed a pronounced reversal of tubular dilation and damage of epithelial cell in kidney tissue with very less inflammatory cell infiltration.ConclusionThe results signify the protective effect of K. pinnata and B. nutans ethyl acetate fraction rich with polyphenol and flavonoid on glyoxylate induced oxidative cell damage and morphological changes in mouse kidneys.