Calcium Oxalate Monohydrate Crystals Research Articles

Preliminary Evaluation of Nanobacteria on Crystal Retention, CaSR, and Claudin‐14 Expression in HK‐2 Cells

To evaluate cell morphology, crystal adhesion, cell damage, Calcium sensitive receptor (CaSR), and Claudin protein‐14 (Claudin‐14) expression at different time intervals and explore the role of nanobacteria in the formation of urinary calculi. In this experiment, HK‐2 cells were cocultured with nanobacteria (NB) in the absence or presence of tetracycline (Tet). Cells treated with calcium oxalate monohydrate (COM) crystals were used as a positive control of urinary stone‐induced cell damage. After which, cell morphology was evaluated by hematoxylin‐eosin staining in comparison to untreated HK‐2 cells (negative control). Use different methods to assess cell damage, crystal adhesion, and protein expression. (The degree of cell damage, crystal adhesion, and protein expression were evaluated by various methods). It was found that the degree of cell damage observed in Tet + NB‐treated cells was significantly lower than that in NB‐treated cells. Lactate dehydrogenase (LDH) leakage was higher in COM‐exposed than in control cells (P < 0.05). However, LDH release from both NB‐ and Tet + NB‐treated cells was significantly lower than from COM‐treated cells (P < 0.05). The relative expression of CaSR and Claudin‐14 proteins was higher in NB, COM, and TET + NB cells than in control cells (P < 0.001) and was lower in Tet + NB than in NB cells (P < 0.01). And P < 0.05 means that the difference was statistically significant, and P < 0.001 means that there was a significant difference between the both things. From the cell morphology, the cell damage in the COM group was greater than that in the NB group, and the cell damage markers in the COM group and the NB group were elevated. NB caused damage to HK‐2 cells by inducing lipid peroxidation, and the degree of damage was increased in processing time. The adhesion of HK‐2 cells to COM crystals increased after injury and was proportional to the duration of NB coculture. NB upregulated the expression of CaSR and Claudin‐14 in HK‐2 cells.

Ursolic acid treats renal tubular epithelial cell damage induced by calcium oxalate monohydrate via inhibiting oxidative stress and inflammation

ABSTRACT Ursolic acid (UA) has been proved to have antioxidant and anti-inflammatory effects. However, it is not clear whether it has a protective impact on kidney damage induced by crystals of calcium oxalate monohydrate (COM). This work aimed to make clear the potential mechanism of UA protecting COM-induced kidney damage. The results manifested that high- and low-dose UA reduced COM crystals in COM rats’ kidney, down-regulated urea, creatinine, and neutrophil gelatinase-associated lipocalin (NGAL) levels in rat plasma, declined kidney tissue and HK-2 cell apoptosis, inhibited Bax expression but elevated Bcl-2 expression. Additionally, UA alleviated renal fibrosis in COM rats, repressed α-SMA and collagen I protein expressions in the kidney and COM rats’ HK-2 cells, depressed COM-induced oxidative damage in vivo and in vitro via up-regulating Nrf2/HO-1 pathway, up-regulated SOD levels and reduced MDA levels, down-regulated TNF-α, IL-1β, and IL-6 levels in vivo and in vitro via suppressing activation of TLR4/NF-κB pathway. In summary, the results of this study suggest that COM-induced renal injury can be effectively improved via UA, providing powerful data support for the development of effective clinical drugs for renal injury in the future.

Theaflavin protects against oxalate calcium-induced kidney oxidative stress injury via upregulation of SIRT1.

Renal tubular cell injury induced by calcium oxalate (CaOx) is a critical initial stage of kidney stone formation. Theaflavin (TF) has been known for its strong antioxidative capacity; however, the effect and molecular mechanism of TF against oxidative stress and injury caused by CaOx crystal exposure in kidneys remains unknown. To explore the potential function of TF on renal crystal deposition and its underlying mechanisms, experiments were conducted using a CaOx nephrocalcinosis mouse model established by glyoxylate intraperitoneal injection, and HK-2 cells were subjected to calcium oxalate monohydrate (COM) crystals, with or without the treatment of TF. We discovered that TF treatment remarkably protected against CaOx-induced kidney oxidative stress injury and reduced crystal deposition. Additionally, miR-128-3p expression was decreased and negatively correlated with SIRT1 level in mouse CaOx nephrocalcinosis model following TF treatment. Moreover, TF suppressed miR-128-3p expression and further abolished its inhibition on SIRT1 to attenuate oxidative stress in vitro. Mechanistically, TF interacted with miR-128-3p and suppressed its expression. In addition, miR-128-3p inhibited SIRT1 expression by directly binding its 3'-untranslated region (UTR). Furthermore, miR-128-3p activation partially reversed the acceerative effect of TF on SIRT1 expression. Taken together, TF exhibits a strong nephroprotective ability to suppress CaOx-induced kidney damage through the recovery of the antioxidant defense system regulated by miR-128-3p/SIRT1 axis. These findings provide novel insights for the prevention and treatment of renal calculus.

Inhibition of Calcium Oxalate Formation and Antioxidant Activity of Carboxymethylated Poria cocos Polysaccharides.

Three carboxymethylated Poria cocos polysaccharides (PCP-C1, PCP-C2, and PCP-C3) with -COOH contents of 6.13%, 10.24%, and 16.22%, respectively, were obtained by carboxymethylation of the original polysaccharide (PCP-C0), which has a molecular weight of 4 kDa and a carboxyl (-COOH) content of 2.54%. The structure of the PCP-Cs was characterized by FT-IR, 1H NMR, and 13C NMR spectra. The four PCP-Cs exhibited antioxidant activity, and their ability to scavenge radicals (hydroxyl and DPPH) and chelate ferrous ions was positively correlated with the degree of carboxymethylation. As the content of -COOH groups in the PCP-Cs increases, their ability to regulate the growth of calcium oxalate (CaOx) crystals was enhanced, thus inhibiting the growth of calcium oxalate monohydrate (COM) crystals and inducing the formation of more calcium oxalate dihydrate (COD) crystals. The formed CaOx crystal was more round and blunt, the absolute value of the Zeta potential on the crystal surface increased, and the aggregation between crystals was inhibited. Thermogravimetric analysis curves showed that the proportions of PCP-C0, PCP-C1, PCP-C2, and PCP-C3 incorporated into the crystal were 20.52%, 15.60%, 10.65%, and 9.78%, respectively, in the presence of 0.4 g/L PCP-Cs. PCP-C protection resisted oxidative damages of human kidney proximal tubular epithelial cells (HK-2) caused by oxalate, resulting in increased cell viability and superoxide dismutase activity and decreased reactive oxygen species levels, malondialdehyde content, and 8-hydroxy-deoxyguanosine expression. Hence, PCP-Cs, especially PCP-C3, can inhibit the formation of CaOx crystals and may have the potential to be an alternative antistone drug.

Evaluation of the effect ofMoringa peregrinabark on the crystal habit and size of calcium oxalate monohydrate crystals in different stages of crystallization using experimental and theoretical methods

Moringa peregrinabark extract is found to successfully retard the nucleation and aggregation of calcium oxalate monohydrate crystals and distort their shape, a mechanism for which is proposed using molecular modeling.

Protective Effect of Degraded Porphyra yezoensis Polysaccharides on the Oxidative Damage of Renal Epithelial Cells and on the Adhesion and Endocytosis of Nanocalcium Oxalate Crystals.

The protective effects of Porphyra yezoensis polysaccharides (PYPs) with molecular weights of 576.2 (PYP1), 105.4 (PYP2), 22.47 (PYP3), and 3.89 kDa (PYP4) on the oxidative damage of human kidney proximal tubular epithelial (HK-2) cells and the differences in adherence and endocytosis of HK-2 cells to calcium oxalate monohydrate crystals before and after protection were investigated. Results showed that PYPs can effectively reduce the oxidative damage of oxalic acid to HK-2 cells. Under the preprotection of PYPs, cell viability increased, cell morphology improved, reactive oxygen species levels decreased, mitochondrial membrane potential increased, S phase cell arrest was inhibited, the cell apoptosis rate decreased, phosphatidylserine exposure reduced, the number of crystals adhered to the cell surface reduced, but the ability of cells to endocytose crystals enhanced. The lower the molecular weight, the better the protective effect of PYP. The results in this article indicated that PYPs can reduce the risk of kidney stone formation by protecting renal epithelial cells from oxidative damage and reducing calcium oxalate crystal adhesion, and PYP4 with the lowest molecular weight may be a potential drug for preventing kidney stone formation.

Preparation of novel chemically bonded ceramics with steel slag and potassium hydrogen oxalate

A novel chemically bonded ceramic (novel-CBC) is prepared based on the acid–base reaction of alkali metals in steel slag (SS) and oxalate anion (C2O42−) in potassium hydrogen oxalate (PO). The effects of SS/PO ratio and water–solid (W/S) ratio on the setting and compressive strength of novel-CBC were studied in this paper. Reaction products and microstructure of novel-CBC were characterized by X-ray diffractometer (XRD), field emission scanning electron microscope-energy dispersive spectroscopy (FESEM-EDS) and thermogravimetric analysis/differential scanning calorimetry (TG/DSC). An optimal formula is obtained at a SS/PO ratio of 3.0 and a W/S ratio of 0.20, which starts setting at 10 min and gives the strengths of 18.0, 25.0, 39.8 and 49.0 MPa at 1, 3, 7 and 28 days, respectively. The reactants from SS are mainly Ca-bearing phases, while only a small amount of RO phase is involved in reaction. The main reaction products of novel-CBC are calcium oxalate monohydrate (CaC2O4·H2O; whewellite) crystals and agglomerates consisting of K, Mg, Al, Si and O elements. The unreacted Ca-bearing phase particle and RO phase residue are embedded in a mixture of abundant CaC2O4·H2O crystals with smooth surfaces and a size of 0.5–1.0 µm with large amounts of the nanoscale agglomerates.

Differential bound proteins and adhesive capabilities of calcium oxalate monohydrate crystals with various sizes

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.

Comprehensive study of altered proteomic landscape in proximal renal tubular epithelial cells in response to calcium oxalate monohydrate crystals

BackgroundCalcium oxalate monohydrate (COM), the major crystalline composition of most kidney stones, induces inflammatory infiltration and injures in renal tubular cells. However, the mechanism of COM-induced toxic effects in renal tubular cells remain ambiguous. The present study aimed to investigate the potential changes in proteomic landscape of proximal renal tubular cells in response to the stimulation of COM crystals.MethodsClinical kidney stone samples were collected and characterized by a stone component analyzer. Three COM-enriched samples were applied to treat human proximal tubular epithelial cells HK-2. The proteomic landscape of COM-crystal treated HK-2 cells was screened by TMT-labeled quantitative proteomics analysis. The differentially expressed proteins (DEPs) were identified by pair-wise analysis. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEPs were performed. Protein interaction networks were identified by STRING database.ResultsThe data of TMT-labeled quantitative proteomic analysis showed that a total of 1141 proteins were differentially expressed in HK-2 cells, of which 699 were up-regulated and 442 were down-regulated. Functional characterization by KEGG, along with GO enrichments, suggests that the DEPs are mainly involved in cellular components and cellular processes, including regulation of actin cytoskeleton, tight junction and focal adhesion. 3 high-degree hub nodes, CFL1, ACTN and MYH9 were identified by STRING analysis.ConclusionThese results suggested that calcium oxalate crystal has a significant effect on protein expression profile in human proximal renal tubular epithelial cells.

Repair of Tea Polysaccharide Promotes the Endocytosis of Nanocalcium Oxalate Monohydrate by Damaged HK-2 Cells.

Endocytosis is a protective mechanism of renal epithelial cells to eliminate retained crystals. This research investigated the endocytosis of 100 nm calcium oxalate monohydrate crystals in human kidney proximal tubular epithelial (HK-2) cells before and after repair by four kinds of tea polysaccharides with molecular weights (MWs) of 10.88 (TPS0), 8.16 (TPS1), 4.82 (TPS2), and 2.31 kDa (TPS3), respectively. When HK-2 cells were repaired by TPSs after oxalic acid injury, the cell viability, wound healing ability, mitochondrial membrane potential, percentage of cells with endocytosed crystals, and dissolution rate of the endocytosed crystals increased; the cell morphology recovered; and the reactive oxygen level and lactate dehydrogenase release decreased. Most of the endocytosed crystals were found in the lysosomes. The repair effects of the four TPSs were ranked in the following order: TPS2>TPS1>TPS3>TPS0. TPS2 with moderate MW presented the optimal repair ability and strongest ability to promote endocytosis.

Effects of Mandelic and Propane-1,2,3-Tricarboxylic Acids on Calcium Oxalate Monohydrate Crystallization

The effects of mandelic and propane-1,2,3-tricarboxylic acids as additives on calcium oxalate monohydrate (COM) crystals were investigated in this study. The physicochemical properties of the COM crystals prepared with and without these additives were characterized using X-ray powder diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and zeta potential analysis. The XRD and SEM results showed that the crystals prepared in pure medium were monohydrates and consisted primarily of hexagonal crystals, respectively. The additives mandelic acid and propane-1,2,3-tricarboxylic acid in the crystallization media significantly changed the size and morphology of the COM crystals, the effects of which were more pronounced with higher concentrations of the additives. The results of FTIR suggested that both carboxylic acids were adsorbed onto the surface of the COM crystals. The zeta potential analysis showed a negative charge on the surface of the COM crystals in the mandelic acid medium, while the surface became more positive in the medium containing increasing concentrations of propane-1,2,3-tricarboxylic acid. In addition, an analysis was conducted to evaluate the thermal characteristics of the COM crystals prepared with and without the additives. The data obtained were used to calculate the kinetic parameters, such as the activation energy and pre-exponential factor, using the Coats-Redfern method. The calculated activation energies for stages I, II, and III in pure medium were 98.76, 270.69, 258.55 kJ/mol, respectively, which were lower than that of COM crystals prepared in the two media containing the additives.

Preparation and characterization of selenized Astragalus polysaccharide and its inhibitory effect on kidney stones.

Astragalus polysaccharide (APS) was modified using the Na2SeO3/HNO3 method to obtain selenized APS (Se-APS) with a selenium content of 1.75mg/g. The structure and physicochemical properties of APS and Se-APS were investigated through transmission electron microscopy-energy dispersive spectroscopy mapping, fourier transform infrared spectroscopy, nuclear magnetic resonance, nano-zetasizer analysis, atomic force microscopy, and scanning electron microscopy. APS and Se-APS did not exhibit toxic effects on human kidney proximal tubular epithelial (HK-2) cells and were able to remove hydroxyl and DPPH radicals, alleviate the damage caused by calcium oxalate (CaOx) monohydrate (COM) crystals to HK-2 cells, reduce intracellular reactive oxygen species levels, and restore cell viability and morphology. Both APS and Se-APS could inhibit COM growth, induce calcium oxalate dihydrate formation, and increase the absolute zeta potential of the crystals to inhibit crystal aggregation. However, the ability of Se-APS to regulate CaOx crystals and protect the cells from COM-induced damage was better than that of APS. These results suggested that Se-APS might be a candidate drug for the treatment and prevention of kidney stones.

Modulation of Calcium Oxalate Crystal Growth and Protection from Oxidatively Damaged Renal Epithelial Cells of Corn Silk Polysaccharides with Different Molecular Weights.

Corn silk polysaccharide (CSP0; molecular weight = 124 kDa) was degraded by ultrasonication to obtain five degraded polysaccharides, namely, CSP1, CSP2, CSP3, CSP4, and CSP5, with molecular weights of 26.1, 12.2, 6.0, 3.5, and 2.0 kDa, respectively. The structures of these polysaccharides were characterized by FT-IR, 1H NMR, and 13C NMR analyses. The antioxidant activities, including scavenging ability for hydroxyl radicals and DPPH free radicals, chelation ability for Fe2+ ions, and reducing ability of CSP increased with decreased molecular weight of CSPs within 6.0 to 124 kDa. However, antioxidant activity weakened when the molecular weight of CSPs reached 3.5 and 2 kDa. CSP3 with a molecular weight of 6.0 kDa exhibited the strongest antioxidant activity. After protection with 60 μg/mL CSPs, the viability of human renal proximal tubular epithelial cells (HK-2) damaged by nano-COM crystals increased, the level of reactive oxygen species decreased, and the amount of COM crystal adhered onto the cell surface decreased. The ability of CSPs to protect cells from CaOx crystal damage was consistent with their antioxidant activity. CSPs can specifically combine with CaOx crystal to inhibit the conversion of calcium oxalate dihydrate crystal to calcium oxalate monohydrate crystal. All these results showed that the activity of CSPs was closely correlated with molecular weight. A very high or low molecular weight of CSPs was not conducive to their activity. CSPs, especially CSP3 with a molecular weight of 6.0 kDa, can be used as a potential antistone drug.

Effect of bisphosphonates on the crystallization of stone-forming salts in synthetic urine.

PurposeWe investigated the inhibitory effect of bisphosphonates (BPs) on the crystallization of calcium oxalate monohydrate (COM), calcium phosphate (CaP), and magnesium ammonium phosphate (MAP) in synthetic urine, aiming to see 1) which specific BPs work best on a particular type of crystal and 2) what is the lowest concentration of BPs that inhibits crystal formation.Materials and MethodsCrystals from synthetic urine were exposed to different concentrations of BPs. Urinary turbidity was used as a marker of crystallization and was measured by spectrophotometry by use of a validated method in our laboratory. The percent inhibitory activity (IA) was calculated by using the formula: (a−b )/a×100, where a is baseline maximal turbidity and b is maximal turbidity with various concentrations of medication. Potassium citrate and magnesium citrate were used as positive controls.ResultsAt the lowest dose of 0.001 mg/mL, risedronate induced the highest IA of 37% on CaP, whereas ibandronate had the strongest IA on COM (24%). To initiate the inhibition of MAP crystallization, risedronate required a two-fold higher concentration (0.002 mg/mL) to reach 30% IA, whereas etidronate required a four-fold higher concentration (0.004 mg/mL) to reach 42% IA.ConclusionsBPs are good inhibitors of crystallization in synthetic urine, with risedronate and ibandronate being the most potent. At a low clinically acceptable dose, their highest inhibitory action was on CaP and COM crystals. Higher doses were needed to prevent MAP crystallization. Further investigation of the use of BPs in kidney stone prevention is warranted.

Influence of Caffeine on Crystallization and Amelioration of Oxidative Stress on in vitro Model of Urolithiasis

Urolithiasis is a disease characterized by formation of solid crystals within the urinary tract. Kidney stone formation is still not clear but it is mostly composed of calcium oxalate which can produce free radicals that are toxic to renal tubular cells. Oxidative stress is an important contributory mechanism in cell damage and is associated with a number of disorders. Several studies have shown antioxidative effects of caffeine, proposing its possible role in stopping the formation of calcium oxalate stones in urinary tract. Hence, the aim of this study was to evaluate the toxic effects of calcium oxalate monohydrate crystals (COM) on renal epithelial cell line; Madin-Darby canine kidney cells subtype I (MDCK I) and Epithelial-like pig kidney cell line (LLC-PK1), and to determine possible inhibition of COM that caused oxidative stress by antioxidant treatment with caffeine in different concentrations in a cell culture model of urolithiasis.

Effect of amino acids and B-group vitamins on nucleation of calcium oxalate monohydrate crystals

Abstract 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.

A Proteomic Network Approach across the Kidney Stone Disease Reveals Endoplasmic Reticulum Stress and Crystal-Cell Interaction in the Kidney.

Crystal-cell interactions are a vital step toward kidney stone formation. However, its mechanisms remained unclear. Here, a protein-protein interaction (PPI) network analysis of a kidney stone revealed that the proteins were enriched in a posttranslational protein modification process in the endoplasmic reticulum (ER). The in vitro study showed that the markers of ER stress, including Bip and CHOP, were upregulated, PERK and ATF6 were activated, and XBP-1 mRNA was spliced. An ER stress-specific protein, caspase-12, was activated in the apoptotic cells induced by calcium oxalate monohydrate (COM) crystals. The treatment with tunicamycin, an ER stress inducer, promoted the crystal-cell adhesion assayed by atomic absorption, reduced cell viability assayed by MTT, and downregulated the expression of proteins involved in the crystal formations. The treatment with salubrinal, an ER stress inhibitor, reversed the above effects for both tunicamycin and COM crystals. The aforementioned main observations were supported by in vivo study. These data demonstrated that ER stress was an essentially biological process of crystal-cell interactions. Our findings suggest that blocking ER stress may become a potential approach to preventing a kidney stone.

Idiopathic calcium nephrolithiasis with pure calcium oxalate composition: clinical correlates of the calcium oxalate dihydrate/monohydrate (COD/COM) stone ratio.

Pure calcium oxalate is the most frequent type of idiopathic kidney stone composition. Fourier transform infrared spectroscopy (FT-IR) allows to detect the ratio of calcium oxalate dihydrate (COD) and monohydrate (COM) crystals in stones, but the clinical significance of this parameter remains uncertain. The objective of this observational study was to verify the association of clinical and laboratory parameters of kidney stone disease with COD/COM ratio in a group of 465 (322M, age 46 ± 14) patients suffering from idiopathic calcium nephrolithiasis with pure calcium oxalate stones (≥ 97%). Each participant underwent a complete clinical examination, serum chemistry, 24-h urine collection for the determination of the profile of lithogenic risk, and had stones analyzed by FT-IR. Most (62%) of the stones had a COD/COM ratio ≤ 0.25, and the urine chemistry of the corresponding patients showed a low prevalence of urinary metabolic abnormalities. With increasing COD/COM ratio intervals (0-0.25, 0.26-0.50, 0.51-0.75, 0.76-1), a significant association was observed for the number of urological procedures, serum calcium, 24-h urinary calcium excretion, prevalence of hypercalciuria and relative calcium oxalate supersaturation, and a negative trend was detected for the age of the first stone episode (all p values < 0.05). A linear regression model showed that the only parameters significantly associated with COD/COM ratio were 24-h urinary calcium excretion (standardized β = 0.464, p < 0.001) and urine pH (standardized β = 0.103, p = 0.013). In pure calcium oxalate idiopathic stones, COD/COM ratio may reflect the presence of urinary metabolic risk factors, and represent a guide for the prescription of urinary analyses.

MiRNA-34a inhibits cell adhesion by targeting CD44 in human renal epithelial cells: implications for renal stone disease.

Nephrolithiasis is a very common disease in which cell-crystal adhesion is an essential mechanism for kidney stone formation. This study has explored the anti-adhesion function of the microRNA, miR-34a, by targeting CD44, a cell surface receptor, in human renal epithelial (HK-2) cells. The expression of CD44 was monitored by qPCR and western blot. A luciferase assay validated the target of miR-34a in CD44 3' UTR. Immunofluorescence staining under confocal microscopy was used to detect the cell-crystal adhesion effects in vitro. Pizzolato staining was performed to examine the adhesion role of miR-34a in vivo. In HK-2 cells, miR-34a was down-regulated and CD44 was up-regulated when exposed to calcium oxalate monohydrate crystals. Moreover, miR-34a negatively regulated the expression of CD44. According to the luciferase report assay, miR-34a direct targeted a binding site in the CD44 3'UTR. In vitro experiments, miR-34a overexpression inhibited CD44 expression and cell-crystals adhesion; whereas CD44 overexpression showed reversed results. Furthermore, miR-34a suppressed cell-crystals adhesion and stone formation in vivo. These findings indicate that miR-34a targets CD44 in HK-2 cells and inhibits cell-crystal adhesion both in vitro and in vivo. Based on these results, miR-34a may be a potential therapeutic target for renal stone disease.

Prevalence and chemical composition of uroliths in fattening pigs in Belgium

The present study investigated the prevalence of uroliths in fattening pigs and assessed the composition of these urinary tract concrements. In total, 2,432 urinary bladders were sampled in the slaughterhouse and checked for abnormal content. Urinary samples were analysed microscopically, and samples of the urinary bladder wall were tested for histological signs of inflammation. The composition of the concrements was examined by infrared spectrophotometry. Macroscopic and microscopic abnormalities were detected in 8.4% and 52.8% of the samples respectively. Magnesium ammonium phosphate (struvite), calcium oxalate dihydrate (COD), calcium carbonate (calcite), calcium oxalate monohydrate (COM) and amorphous crystals were detected. Analysis of stones showed COD in all samples in different proportions. The calcium content of examined stones was always considerable (up to 34%), in contrast to the magnesium content which represented max 1.9%. Struvite was found in one third of the samples, but was never part of stones and grit. COD crystals were the second most common microscopic crystal. These COD crystals and some COD stones had a rectangular shape, and therefore, they can be harmful to the bladder mucosa. In conclusion, uroliths are present in a large proportion of male fattening pigs, and consequently, urinary concrements pose a life-threatening risk for urethra obstruction in male pigs. Further research is warranted to identify potential risk factors for urolithiasis and microscopic crystals.