Pathogenesis Of Kidney Stones Research Articles

Modulatory effects of fibronectin on calcium oxalate crystallization, growth, aggregation, adhesion on renal tubular cells, and invasion through extracellular matrix.

Fibronectin, an extracellular matrix (ECM) protein, has been thought to be involved in pathogenic mechanisms of kidney stone disease, especially calcium oxalate (CaOx) type. Nevertheless, its precise roles in modulation of CaOx crystal remained unclear. We thus performed a systematic evaluation of effects of fibronectin on CaOx monohydrate (COM) crystal (the major causative chemical crystal in kidney stone formation) in various stages of kidney stone pathogenesis, including crystallization, crystal growth, aggregation, adhesion onto renal tubular cells, and invasion through ECM in renal interstitium. The data showed that fibronectin significantly decreased crystallization, growth and adhesive capability of COM crystals in a dose-dependent manner. In contrast, COM crystal aggregation and invasion through ECM migration chamber were significantly enhanced by fibronectin in a dose-dependent fashion. Sequence analysis revealed three calcium-binding and six oxalate-binding domains in fibronectin. Immunofluorescence study confirmed binding of fibronectin to COM crystals. Additionally, calcium- and oxalate-affinity assays confirmed depletion of both calcium and oxalate ions after incubation with fibronectin. Moreover, calcium-saturated and oxalate-saturated forms of fibronectin markedly reduced the modulatory activities of fibronectin on COM crystallization, crystal growth, aggregation, and adhesion onto the cells. These data strongly indicate the dual functions of fibronectin, which serves as an inhibitor for COM crystallization, crystal growth and adhesion onto renal tubular cells, but on the other hand, acts as a promoter for COM crystal aggregation and invasion through ECM. Finally, its COM crystal modulatory activities are most likely mediated through binding with calcium and oxalate ions on the crystals and in their environment.

Correction to: Testing the dogma that total phospholipid fatty acid composition of blood plays a role in kidney stone pathogenesis, using a high-low risk human model: results from a pilot study.

The authors would like to add the following paragraph in the acknowledgement section of the original version of the paper.

Changing expression profiles of long non-coding RNAs, mRNAs and circular RNAs in ethylene glycol-induced kidney calculi rats

BackgroundTo explore long non-coding RNA (lncRNA), mRNA and circular RNA (circRNA) expression profiles and their biological functions in the pathogenesis of kidney stones in ethylene glycol-induced urolithiasis rats.ResultsThe expression of 1440 lncRNAs, 2455 mRNAs and 145 circRNAs was altered in the kidneys of urolithiasis rats. GO and KEGG biological pathway analysis were performed to predict the functions of differentially expressed lncRNAs, circRNAs and co-expressed potential targeting genes. Co-expression networks of lncRNA-mRNA and circRNA-miRNA were constructed based on correlation analysis between differentially expressed RNAs. mRNAs coexpressed with lncRNAs were involved in many kidney diseases, e.g., Ephb6 was associated with the reabsorption ability of the kidney. Arl5b was associated with the dynamic changes in the podocyte foot process in podocyte injury. miRNAs co-expressed with circRNAs, such as rno-miR-138-5p and rno-miR-672-5p, have been proven to be functional in hypercalciuria urolithiasis.ConclusionThe expression profile provided a systematic perspective on the potential functions of lncRNAs and circRNAs in the pathogenesis of kidney stones. Differentially expressed lncRNAs and circRNAs might serve as treatment targets for kidney stones.

Testing the dogma that total phospholipid fatty acid composition of blood plays a role in kidney stone pathogenesis, using a high-low risk human model: results from a pilot study.

Previous studies have suggested that ω-3 and ω-6 polyunsaturated fatty acid (PUFA) composition in plasma and red blood cell (RBC) total phospholipids plays a role in urolithiasis. Our aim was to test the robustness of this dogma by retrospectively comparing baseline profiles of these parameters in subjects from high- and low-stone-risk groups. The documented difference in stone occurrence in white (relatively common) (W) and black (rare) (B) subjects prompted us to select these groups as the high-low risk model for the study. Blood and urine samples were obtained from ten subjects in each group and were analysed for PUFAs and stone risk factors, respectively. Concentrations of linoleic acid (LA), eicosadienoic acid (EDA) and arachidonic acid (AA) in plasma and or/RBC total phospholipids were significantly higher in B. Differences in other PUFA profiles were also observed. There was no inter-group difference in AA/LA ratios. Urinary oxalate was significantly higher while urinary phosphate was significantly lower in B. We speculate that elevated AA in B might arise because of a possibly enhanced elongation of LA to EDA, as well as an enhanced ∆-8-desaturation of EDA to dihomo-gamma-linolenic acid (DGLA), which is the immediate precursor of AA. Alternatively, we speculate that the ∆-5-desaturation step of DGLA to AA might be more highly activated in this group. Irrespective of the mechanism, our observed inter-group differences in phospholipid PUFA composition are in conflict with previously published dogma which relates PUFA characteristics to high- and low-stone risk.

Protein Network Analysis and Functional Studies of Calcium Oxalate Crystal-Induced Cytotoxicity in Renal Tubular Epithelial Cells.

Our previous expression study has reported a set of proteins with altered levels in renal tubular cells after exposure to calcium oxalate monohydrate (COM) crystals, which are the main composition of kidney stones. However, their functional significance remained largely unknown. In this study, protein network analysis revealed that the significantly altered proteins induced by COM crystals were involved mainly in three main functional networks, including i) cell proliferation and wound healing; ii) oxidative stress and mitochondrial function; and iii) cellular junction complex and integrity. Cell proliferation and wound healing assays showed that the COM-treated cells had defective proliferation and tissue healing capability, respectively. Oxyblot analysis demonstrated accumulation of the oxidized proteins, whereas intracellular ATP level was significantly increased in the COM-treated cells. Additionally, level of zonula occludens-1 (ZO-1), a tight junction protein, was significantly decreased, consistent with the significant declines in transepithelial resistance (TER) and level of RhoA signaling molecule in the COM-treated cells. These findings indicate significant perturbations in mitochondrial and oxidative stress axis that cause defective cell proliferation, tissue healing capability, junctional protein complex, and cellular integrity of renal tubular epithelial cells exposed to COM crystals that may play important roles in kidney stone pathogenesis.

Roles of Macrophage Exosomes in Immune Response to Calcium Oxalate Monohydrate Crystals.

In kidney stone disease, macrophages secrete various mediators via classical secretory pathway and cause renal interstitial inflammation. However, whether their extracellular vesicles, particularly exosomes, are involved in kidney stone pathogenesis remained unknown. This study investigated alterations in exosomal proteome of U937-derived macrophages (by phorbol-12-myristate-13-acetate activation) after exposure to calcium oxalate monohydrate (COM) crystals for 16-h using 2-DE-based proteomics approach. Six significantly altered proteins in COM-treated exosomes were successfully identified by nanoscale liquid chromatography–electrospray ionization–electron transfer dissociation tandem mass spectrometry as proteins involved mainly in immune processes, including T-cell activation and homeostasis, Fcγ receptor-mediated phagocytosis, interferon-γ (IFN-γ) regulation, and cell migration/movement. The decreased heat shock protein 90-beta (HSP90β) and increased vimentin were confirmed by Western blotting. ELISA showed that the COM-treated macrophages produced greater level of interleukin-1β (IL-1β), one of the markers for inflammasome activation. Functional studies demonstrated that COM-treated exosomes enhanced monocyte and T-cell migration, monocyte activation and macrophage phagocytic activity, but on the other hand, reduced T-cell activation. In addition, COM-treated exosomes enhanced production of proinflammatory cytokine IL-8 by monocytes that could be restored to its basal level by small-interfering RNA targeting on vimentin (si-Vimentin). Moreover, si-Vimentin could also abolish effects of COM-treated exosomes on monocyte and T-cell migration as well as macrophage phagocytic activity. These findings provided some implications to the immune response during kidney stone pathogenesis via exosomal pathway of macrophages after exposure to COM crystals.

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

Crystal aggregation is one of the most crucial steps in kidney stone pathogenesis. However, previous studies of crystal aggregation were rarely done and quantitative analysis of aggregation degree was handicapped by a lack of the standard measurement. We thus performed an in vitro assay to generate aggregation of calcium oxalate monohydrate (COM) crystals with various concentrations (25–800 μg/ml) in saturated aggregation buffer. The crystal aggregates were analyzed by microscopic examination, UV-visible spectrophotometry, and GraphPad Prism6 software to define a total of 12 aggregation indices (including number of aggregates, aggregated mass index, optical density, aggregation coefficient, span, number of aggregates at plateau time-point, aggregated area index, aggregated diameter index, aggregated symmetry index, time constant, half-life, and rate constant). The data showed linear correlation between crystal concentration and almost all of these indices, except only for rate constant. Among these, number of aggregates provided the greatest regression coefficient (r = 0.997; p < 0.001), whereas the equally second rank included aggregated mass index and optical density (r = 0.993; p < 0.001 and r = −0.993; p < 0.001, respectively) and the equally forth were aggregation coefficient and span (r = 0.991; p < 0.001 for both). These five indices are thus recommended as the most appropriate indices for quantitative analysis of COM crystal aggregation in vitro.

Characterizations of PMCA2-interacting complex and its role as a calcium oxalate crystal-binding protein.

Three isoforms of plasma membrane Ca2+-ATPase (PMCA) are expressed in the kidney. While PMCA1 and PMCA4 play major role in regulating Ca2+ reabsorption, the role for PMCA2 remains vaguely defined. To define PMCA2 function, PMCA2-interacting complex was characterized by immunoprecipitation followed by nanoLC-ESI-Qq-TripleTOF MS/MS (IP-MS). After subtracting non-specific binders using isotype-controlled IP-MS, 474 proteins were identified as PMCA2-interacting partners. Among these, eight were known and 20 were potential PMCA2-interacting partners based on bioinformatic prediction, whereas other 446 were novel and had not been previously reported/predicted. Quantitative immuno-co-localization assay confirmed the association of PMCA2 with these partners. Gene ontology analysis revealed binding activity as the major molecular function of PMCA2-interacting complex. Functional validation using calcium oxalate monohydrate (COM) crystal-protein binding, crystal-cell adhesion, and crystal internalization assays together with neutralization by anti-PMCA2 antibody compared to isotype-controlled IgG and blank control, revealed a novel role of PMCA2 as a COM crystal-binding protein that was crucial for crystal retention and uptake. In summary, a large number of novel PMCA2-interacting proteins have been defined and a novel function of PMCA2 as a COM crystal-binding protein sheds light onto its involvement, at least in part, in kidney stone pathogenesis.

49 - A classic paradigm shift: Seeking explanations for kidney stone pathogenesis in subjects from a relatively stone-free race group

49 - A classic paradigm shift: Seeking explanations for kidney stone pathogenesis in subjects from a relatively stone-free race group

6 - Seeking explanations for the pathogenesis of kidney stones in studies of a relatively stone-free race group

6 - Seeking explanations for the pathogenesis of kidney stones in studies of a relatively stone-free race group

The role of macromolecules in the formation of kidney stones.

The formation of crystal aggregates, one of the critical processes in kidney stone pathogenesis, involves interactions between crystals (predominantly calcium oxalate monohydrate, COM) and urinary constituents (e.g., proteins), which serve as an adhesive "glue" between crystals in stones. To develop a better understanding of the protein-crystal interactions that lead to crystal aggregation, we have measured the effect of model proteins on bulk COM crystal properties as well as their adsorption on crystal surfaces using three synthetic polyanions: poly(aspartic acid) (polyD), poly(glutamic acid) (polyE), and poly(acrylic acid) (polyAA). These anionic macromolecules reduced the amount of COM crystal aggregation in bulk solution to an extent similar to that observed for mixture of proteins from normal urine, with little difference between the polymers. In contrast, the polymers exhibited differences in measures of COM crystal growth. Polycations such as poly(arginine) (polyR) and poly(lysine) (polyK) reduced aggregation weakly and exerted negligible effects on crystal growth. All polyions were found to associate with COM crystal surfaces, as evidenced by changes in the zeta potential of COM crystals in electrophoretic mobility measurements. On the other hand, COM aggregation and possibly growth can be promoted by many binary mixtures of polycations and polyanions, which appeared to be mediated by polymer aggregate formation rather than loss of crystal charge stabilization. Similarly, crystal aggregation promotion behavior can be driven by forming aggregates of weakly charged polyanions, like Tamm-Horsfall protein, suggesting that polymer (protein) aggregation may play a critical role in stone formation. Sensitivity of polyanion-COM crystal surface interactions to the chemical composition of polymer side groups were demonstrated by large differences in crystal aggregation behavior between polyD and polyE, which correlated with atomic force microscopy (AFM) measurements of growth inhibition on various COM surfaces and chemical force microscopy (CFM) measurements of unbinding forces between COM crystal surfaces and AFM tips decorated with either carboxylate or amidinium moieties (mimicking polyanion and polyR side chains, respectively). The lack of strong interaction for polyE at the COM (100) surface compared to polyD appeared to be the critical difference. Finally, the simultaneous presence of polyanions and polycations appeared to alter the ability of polycations to mediate unbinding forces in CFM and promote crystal growth. In summary, polyanions strongly associated with COM surfaces and influenced crystallization, while polycations did not, though important differences were observed based on the physicochemical properties of polyanions. Observations suggest that COM aggregation with both polyanion-polycation mixtures and weakly charged polyanions is promoted by polymer aggregate formation, which plays a critical role in bridging crystal surfaces.

Does Milk Intake Play a Specific Role in the Pathogenesis of Kidney Stones?

Background: Diet plays a key role in the pathogenesis of kidney stones. In particular, recent findings have advanced knowledge on the protective role of a high calcium diet. However, not much is known about the specific role played by milk intake in the pathogenesis of kidney stones compared with other dairy products, especially cheese.

Effect of Urinary Macromolecules on l-Cystine Crystal Growth and Crystal Surface Adhesion

l-Cystine is the primary crystalline component of l-cystine kidney stones, which are a consequence of cystinuria, a genetic disorder that results from mutation in the SLC3A1 or the SLC7A9 gene. Urinary macromolecules present in the cellular matrix are thought to play a role in the pathogenesis of kidney stones affecting crystal nucleation, growth, crystal–crystal aggregation, and adhesion to epithelial cells. The effect of six prevalent urinary constituents—osteopontin, Tamm–Horsfall protein, albumin, apotransferrin, chondroitin sulfate, and lysozyme—on crystallization kinetics and adhesion events on the l-cystine (0001) surface was investigated with real-time in situ atomic force microscopy (AFM). These additives did not significantly change crystal morphology and crystallization yield, although slight reductions in step velocities were observed at nanogram per milliliter and microgram per milliliter concentrations. Chemical force microscopy performed with AFM tips decorated with terminal carboxylate, am...

Heritability of dietary traits that contribute to nephrolithiasis in a cohort of adult sibships.

Kidney stones and their risk factors aggregate in families, yet few studies have estimated the heritability of known risk factors. Estimate the heritability of dietary risk factors for kidney stones. Dietary intakes were assessed using the Viocare Food Frequency Questionnaire in sibships enrolled in the Rochester, MN cohort of the Genetic Epidemiology Network of Arteriopathy. Measures of urinary supersaturation were determined using 24h urine samples. Heritabilities and genetic correlations were estimated using variance components methods. Samples were available from 620 individuals (262 men, 358 women, mean (SD) age 65 (9)years). Dietary intakes of protein, sucrose, and calcium had strong evidence for heritability (p<0.01) after adjustment for age, sex, height and weight. Among the significantly heritable dietary intakes (p<0.05), genetic factors explained 22-50% of the inter-individual variation. Significant genetic correlations were observed among dietary protein, dietary sucrose, and dietary calcium intakes (p<0.001). Evidence from this relatively large cohort suggests a strong heritable component to dietary intakes of protein, sucrose and calcium that contributes to nephrolithiasis risk. Further efforts to understand the interplay of genetic and environmental risk factors in kidney stone pathogenesis are warranted.

Extracellular vesicles in urine of women with but not without kidney stones manifest patterns similar to men: a case control study

BackgroundThe lifetime incidence of kidney stones is about two times greater in men compared to women. Extracellular vesicles (EVs) shed from activated cells are present in the urine and may reflect or even mediate renal physiology and/or pathology. This study was designed to standardize methodology to characterize urinary EVs by digital flow cytometry and to identify possible sex differences in EVs in persons with and without their first symptomatic kidney stones.MethodsTwenty-four-hour urine collections were obtained from persons presenting with their first kidney stone episode (n = 50 women, 60 men; age 19–76 years) and sex- and age-matched controls from the general population (n = 24 women, 36 men).ResultsStandardization: Size of EV was variable within all groups. EV positivity was verified with two fluorophores for surface phosphatidylserine and/or using two different protein markers specific for renal-specific cells. The number of phosphatidylserine- and exosome marker-positive EVs did not correlate with urine osmolality and were similar in fresh vs. frozen and between two sequential urine collections from the same individual. Sex differences: Urine from women controls contained greater (P < 0.05) numbers of EVs positive for phosphatidylserine, exosomes, inflammatory factors and adhesion molecules, and cell-specific markers from different segments of the nephron, renal pelvis, and bladder compared to control men. In contrast, urine from women with kidney stones contained significantly (P < 0.05) lower numbers of EVs derived from podocytes, parietal cells, proximal convoluted tubule, thin and thick loop of Henle, distal tubule, collecting duct, renal pelvis, and bladder compared to control women and contained similar quantities of these types of EVs in men with and without kidney stones. There were also no sex differences in EVs positive for cell adhesion (E-cadherin and inter-cellular adhesion molecule-1 [ICAM-1]) molecules.ConclusionsUnlike women who do not have kidney stones, EVs in urine from women with nephrolithiasis are similar to men with and without kidney stones. Thus, EVs may mediate or reflect aspects of kidney stone pathogenesis and perhaps provide clues regarding sex differences in kidney stone incidence rates.

Kidney stone analysis techniques and the role of major and trace elements on their pathogenesis: a review.

Kidney stone disease is a polygenic and multifactorial disorder with a worldwide distribution, and its incidence and prevalence are increasing. Although significant progress has been made in recent years towards identifying the specific factors that contribute to the formation of kidney stone, many questions on the pathogenesis of kidney stones remain partially or completely unanswered. However, none of the proposed mechanisms specifically consider the role(s) of the trace elements and, consequently, the contribution of trace constituents to the pathogenesis of kidney stones remains unclear and under debate. The findings of some studies seem to support a role for some major and trace elements in the initiation of stone crystallization, including as a nucleus or nidus for the formation of the stone or simply as a contaminant of the stone structure. Thus, the analysis of kidney stones is an important component of investigations on nephrolithiasis in order to understand the role of trace constituents in the formation of kidney stones and to formulate future strategies for the treatment and prevention of stone formation and its recurrence. The aim of this review is to compare and evaluate the methods/procedures commonly used in the analysis of urinary calculi. We also highlight the role of major and trace elements in the pathogenesis of kidney stones.

Heritability of Urinary Traits That Contribute to Nephrolithiasis

Kidney stones and their risk factors aggregate in families, yet few studies have systematically estimated heritabilities and genetic correlations of the numerous urinary traits associated with risk of kidney stones. Twenty-four-hour urine samples were collected from the Genetic Epidemiology Network of Arteriopathy cohort of families in Rochester, Minnesota, to measure urinary determinants of supersaturation. Diet was assessed using the Viocare food frequency questionnaire. Heritabilities and genetic correlations among the urinary traits were estimated using variance components methods. Samples were available from 811 individuals (344 men, 467 women; mean age 66 ± 9 years). Age, sex, and weight were significantly correlated with the majority of urinary traits. Many urine excretions (calcium, magnesium, citrate excretion) had strong evidence for heritability (P<0.01) both before and after adjusting for the identified covariates. Among significantly heritable urinary traits, genetic factors explained 20%-36% of interindividual variation after adjustment for covariates. Urinary calcium excretion was significantly genetically correlated with urinary magnesium and with urinary citrate excretion (P<0.05). Although eGFR influenced many urinary traits, controlling for eGFR did not greatly affect estimated heritabilities. Evidence from this cohort suggests a strong heritable component to many urinary nephrolithiasis risk factors. Further study of genetic influences on urinary traits relevant for kidney stone pathogenesis is warranted.

Correlation of serum and ionized calcium in patients with calcium nephrolithiasis.

Introduction:Pathogenesis of kidney stones includes many factors, whereas uroliths, as a generic term for kidney stones, are of a different composition. In pathogenesis of calcium urolithiasis hypercalcemia/hypercalciuria takes a significant place. Hypercalcemia exists when the serum calcium is of increased values, along with measurement and calculation of physiologically active calcium, when there are differences in the Ph of the blood or albumin.Goal:the goal of this research is to determine the correlation of values of the serum (CaS) and ionized calcium (Ca++) in patients with the calcium nephrolithiasis, whom have been established not to have hyperparathyroidism and malign diseases.Material and methods:the research was prospective and implemented at the Clinical Center in Banja Luka, at the Urology Clinic, in the period between 1st April 2012 – 1st January 2013 and it included 120 patients with the calcium lithiasis of the upper part of the urinary tract, divided into three age categories. Diagnosis of the calcium lithiasis of the upper part of the urinary tract was established on the basis of the ultrasonography of the urinary tract as well as native urinary tract/intravenous urography and chemical analysis of the stone in patients with spontaneous stone emission or after some of the methods for active removal of the stone. Chemical laboratory analysis of the serum and ionized calcium was done for all the patients, with 3ml of blood being taken for establishing the aforementioned parameters (1-2 ml of the serum) in vacuumed test tubes or glass tubes of capillary blood. Increased parathormone values (PHT) and history of malignity were excluding factors.Results:out of the 120 patients observed, Cs(S) had the value in the reference interval with most of them, that is, in 110 patients (91.7%). Those, whose value was out of the interval, are of an older age (all above 40). Average value of this parameter amounted to 2.3017, with an average difference (the standard deviation) of 0.11391. Observing the value of Ca++, the value within the reference interval was found in 106 patients (88.3%). Out of the remaining 14 patients, only two simultaneously had the value of Ca(S) out of the permitted interval. The majority of this group consisted of older patients (a half of those whose values were outside the interval was over 60). Average value of Ca++ amounted to 1.22 mmol/L with an average difference of 0.06454. In the 2 aforementioned patients, who simultaneously had increased values of CaS and Ca++, the blood Ph was within the referential value limits, which suggests that, in line with the hyporcalcemia definition, only 2 patients with nephrolithiasis, in the total sample of 120 patients of our research sample, could have had a true hypercalcemia.Conclusion:the biggest number of patients with the calcium urolithiasis, who do not have hyperparathyroidism and history of malign diseases, do not have a real hypercalcemia.

1,2,3,4,6-Penta-O-galloyl-beta-D-glucose reduces renal crystallization and oxidative stress in a hyperoxaluric rat model

Adhesion of calcium oxalate (CaOx) crystals to kidney cells may be a key event in the pathogenesis of kidney stones associated with marked hyperoxaluria. Previously, we found that 1,2,3,4,6-penta-O-galloyl-β-D-glucose (PGG), isolated from a traditional medicinal herb, reduced CaOx crystal adhesion to renal epithelial cells by acting on the cells as well as on the crystal surface. Here we used the ethylene glycol (EG)-mediated hyperoxaluric rat model and found evidence of oxidant stress as indicated by decreases in the activities of the renal antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase, with increased kidney cell apoptosis and serum malondialdehyde levels, all evident by 21 days of EG treatment. These effects of hyperoxaluria were reversed by concurrent PGG treatment along with decreased urinary oxalate levels and CaOx supersaturation. Renal epithelial cell expression of the crystal binding molecule hyaluronan increased diffusely within 7 days of EG initiation, suggesting it is not a result of but precedes crystal deposition. Renal cell osteopontin (OPN) was also upregulated in EG-treated animals, and PGG significantly attenuated overexpression of both OPN and hyaluronan. Thus, our findings demonstrate that PGG reduces renal crystallization and oxidative renal cell injury, and may be a candidate chemopreventive agent for nephrolithiasis.

Effects of calcium oxalate monohydrate crystals on expression and function of tight junction of renal tubular epithelial cells

Tight junction has a crucial role in regulating paracellular transports (as a barrier) and in separating apical from basolateral compartments to maintain cell polarity (as a fence). Tight junction can be disrupted by various stimuli, including oxidative stress, pathogens and proinflammatory cytokines. However, association of defective tight junction with kidney stone pathogenesis remains unknown. We therefore examined whether calcium oxalate monohydrate (COM) crystals, which are the major crystalline composition in kidney stones, have any effects on expression and function of tight junction of polarized renal tubular epithelial cells. Western blot analysis revealed marked decrease in levels of occludin and zonula occludens-1 (ZO-1) in COM-treated polarized Madin-Darby canine kidney (MDCK) cells. Immunofluorescence staining revealed not only the decline of these tight junction proteins but also their redistribution and dissociation in COM-treated cells. Additionally, transepithelial resistance was significantly decreased, indicating impaired tight junction barrier and increased paracellular permeability in COM-treated cells. Subcellular fractionation followed by western blot analysis of Na+/K+-ATPase-α1 revealed that this basolateral membrane marker was also detectable in apical membrane fraction of COM-treated cells, but not in apical membrane fraction of control cells. Immunofluorescence study confirmed the translocation of Na+/K+-ATPase-α1 (from basolateral to apical membranes) in COM-treated cells, indicating impaired fence function of the tight junction. Moreover, dihydrorhodamine assay using flow cytometry revealed the significantly higher level of hydrogen peroxide in the COM-treated cells. These data provide the first evidence to demonstrate decreased expression and defective barrier and fence functions of the tight junction of renal tubular epithelial cells exposed to COM crystals that may be fundamental for subsequent renal tubulointerstitial injury, which in turn enhances the stone pathogenesis.