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Aggregation (AGN) of freshly precipitated calcium oxalate crystals was photometrically studied in urine of 30 calcium stone patients and 30 controls, in solutions containing urinary macromolecules (UMS) and in an inhibitor free control solution (CS). Crystals were produced by oxalate titration and crystallization was monitored measuring optical density (OD). Tests were repeated adding hydroxyapatite (HAP) to urine and UMS and adding citrate and pyrophosphate (PPi) to UMS of the controls. AGN was recognized as a rapid OD decrease being at least three times faster than sedimentation of single crystals ( p < 0.001) and used to calculate an extent of AGN (EA%). The time between the end of titration and the beginning of AGN was determined as suspension stability (SS). The main effect of urinary inhibitors was retardation of AGN without changing EA, SS being higher in urine than UMS ( p < 0.001) and in UMS than CS ( p < 0.001). In urine of 63% of controls but only in 33% of patients, no AGN was recorded ( p < 0.05). The high inhibitory activity of urine could not be reproduced in UMS even in combination with 3.5 mM citrate or 0.05 mM PPi. 0.05 mg/mL HAP reduced SS in all urine samples to low values and increased the rate of rapid OD decrease, being a measure for the size of aggregates. Retarding AGN of crystals during their passage through the kidney seems to be an important mechanism to prevent stone formation during crystalluria. The promotion of AGN by HAP reveals a new role of Randall's plaques in nephrolithiasis. [ABSTRACT FROM AUTHOR] more...

Osteopontin (OPN) is one of a group of proteins found in urine that are believed to limit the formation of kidney stones. In the present study, we investigate the roles of phosphate and carboxylate groups in the OPN-mediated modulation of calcium oxalate (CaOx), the principal mineral phase found in kidney stones. To this end, crystallization was induced by addition of CaOx solution to ultrafiltered human urine containing either human kidney OPN (kOPN; 7 consecutive carboxylates, 8 phosphates) or synthesized peptides corresponding to residues 65-80 (pSHDHMDDDDDDDDDGD; pOPAR) or 220-235 (pSHEpSTEQSDAIDpSAEK; P3) of rat bone OPN. Sequence 65-80 was also synthesized without the phosphate group (OPAR). Effects on calcium oxalate monohydrate (COM) and dihydrate (COD) formation were studied by scanning electron microscopy. We found that controls form large, partly intergrown COM platelets; COD was never observed. Adding any of the polyelectrolytes was sufficient to prevent intergrowth of COM platelets entirely, inhibiting formation of these platelets strongly, and inducing formation of the COD phase. Strongest effects on COM formation were found for pOPAR and OPAR followed by kOPN and then P3, showing that acidity and hydrophilicity are crucial in polyelectrolyte-affected COM crystallization. At higher concentrations, OPAR also inhibited COD formation, while P3, kOPN and, in particular, pOPAR promoted COD, a difference explainable by the variations of carboxylate and phosphate groups present in the molecules. Thus, we conclude that carboxylate groups play a primary role in inhibiting COM formation, but phosphate and carboxylate groups are both important in initiating and promoting COD formation. [ABSTRACT FROM AUTHOR] more...

Experimental animal model studies suggest that calcium oxalate (CaOx) crystal deposition in the kidneys is associated with the development of oxidative stress, epithelial injury and inflammation. There is increased production of inflammatory molecules including osteopontin (OPN), monocyte chemoattractant protein-1 (MCP-1) and various subunits of inter-alpha-inhibitor such as bikunin. What does the increased production of such molecules suggest? Is it a cause or consequence of crystal deposition? We hypothesized that over-expression and increased production of MCP-1 is a result of the interaction between renal epithelial cells and CaOx crystals after their deposition in the renal tubules. We induced hyperoxaluria in MCP-1 null as well as wild type mice and examined pathological changes in their kidneys and urine. Both wild type and MCP-1 null male mice became hyperoxaluric and demonstrated CaOx crystalluria. Neither of them developed crystal deposits in their kidneys. Both showed some morphological changes in their renal proximal tubules. Significant pathological changes such as cell death and increased urinary excretion of LDH were not seen. Results suggest that at least in mice (1) Increase in oxalate and decrease in citrate excretion can lead to CaOx crystalluria but not CaOx nephrolithiasis; (2) MCP-1 does not play a role in crystal retention within the kidneys; (3) Expression of OPN and MCP-1 is not increased in the kidneys in the absence of crystal deposition; (4) Crystal deposition is necessary for significant pathological changes and movement of monocytes and macrophages into the interstitium. [ABSTRACT FROM AUTHOR] more...

Tamm-Horsfall protein (THP) is thought to protect against calcium oxalate monohydrate (COM) stone formation by inhibiting COM aggregation. Several studies reported that stone formers produce THP with reduced levels of glycosylation, particularly sialic acid levels, which leads to reduced negative charge. In this study, normal THP was treated with neuraminidase to remove sialic acid residues, confirmed by an isoelectric point shift to higher pH. COM aggregation assays revealed that desialylated THP (ds-THP) promoted COM aggregation, while normal THP inhibited aggregation. The appearance of protein aggregates in solutions at ds-THP concentrations ≥1 μg/mL in 150 mM NaCl correlated with COM aggregation promotion, implying that ds-THP aggregation induced COM aggregation. The aggregation-promoting effect of the ds-THP was independent of pH above its isoelectric point, but was substantially reduced at low ionic strength, where protein aggregation was much reduced. COM aggregation promotion was maximized at a ds-THP to COM mass ratio of ~0.025, which can be explained by a model wherein partial COM surface coverage by ds-THP aggregates promotes crystal aggregation by bridging opposing COM surfaces, whereas higher surface coverage leads to repulsion between adsorbed ds-THP aggregates. Thus, desialylation of THP apparently abrogates a normal defensive action of THP by inducing protein aggregation, and subsequently COM aggregation, a condition that favors kidney stone formation. [ABSTRACT FROM AUTHOR] more...

Nephrocalcinosis is the deposition of calcium salts in renal parenchyma and can be intratubular or interstitial. Animal model studies indicate that intratubular nephrocalcinosis is a result of increased urinary supersaturation. Urinary supersaturation with respect to calcium oxalate (CaOx) and calcium phosphate (CaP) are generally achieved at different locations in the renal tubules. As a result experimental induction of hyperoxaluria in animals with CaP deposits does not lead to growth of CaOx over CaP. Interstitial nephrocalcinosis has been seen in mice with lack of crystallization modulators Tamm-Horsfall protein and osteopontin. Sodium phosphate co-transporter or sodiumhydrogen exchanger regulator factor-1 null mice also produced interstitial nephrocalcinosis. Crystals plug the tubules by aggregating and attaching to the luminal cell surface. Structural features of the renal tubules also play a role in crystal retention. The crystals plugging the terminal collecting ducts when exposed to the metastable pelvic urine may promote the formation of stone. [ABSTRACT FROM AUTHOR] more...

Our aim was to examine the attachment to, and incorporation of intact, highly phosphorylated osteopontin (OPN) into inorganic (i) and urinary (u) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals. uCOM and uCOD crystals were precipitated from ultrafiltered (UF) urine containing human milk OPN (mOPN) labelled with Alexa Fluor 647 fluorescent dye at concentrations of 0.1-5.0 mg/L. iCOM and iCOD crystals were generated in aqueous solutions at concentrations of 0.01-0.5 mg/L. Crystals were demineralised with EDTA and the resulting extracts analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and western blotting, or examined by fluorescent confocal microscopy and field emission scanning electron microscopy before and after washing to remove proteins bound reversibly to the crystal surfaces. Binding of mOPN to pre-formed iCOM crystals was also studied in phosphate-buffered saline (PBS) and ultrafiltered (UF) urine. mOPN attached to the {100} faces and to the {010} sides of the {100}/{010} edges of iCOM crystals was removed by washing, indicating that it was not incorporated into the mineral bulk. In both PBS and urine, mOPN was attached to the {021} faces of pre-formed iCOM crystals as well as to the {100}/{010} edges, but was concentrated at the intersection points of the {100} and {121} faces at the crystal tips. Attachment in UF urine appeared to be greater than in PBS and stronger at higher calcium concentrations than lower calcium concentrations. In uCOM crystals, the distribution of fluorescence and patterns of erosion after washing suggested attachment of mOPN to the four end faces, followed by interment within the mineral phase. Fluorescence distributions of mOPN associated with both iCOD and uCOD crystals were consistent with uniform binding of the protein to all equivalent {101} faces and concentration along the intersections between them. Persistence of fluorescence after washing indicated that most mOPN was incarcerated within the mineral phase. We concluded that attachment of mOPN to calcium oxalate crystals is face-specific and depends upon the anatomical and genetic source of the protein and whether the crystals are (1) COM or COD; (2) pre-formed or precipitated from solution, and (3) precipitated from urine or aqueous solutions. Our findings emphasise the need for caution when drawing conclusions about possible roles of OPN or other proteins in urolithiasis from experimental data obtained under inorganic conditions. [ABSTRACT FROM AUTHOR] more...

Intratubular nephrocalcinosis is defined as the histological observation of calcium oxalate and/or calcium phosphate deposits retained within the lumen of the renal tubules. As the tubular epithelium is the primary interaction partner of crystals formed in the tubular fluid, the role of the epithelial cells in nephrocalcinosis has been investigated intensively. This review summarizes our current understanding on how the tubular epithelium mechanistically appears to be involved both in the initiation and in the course of nephrocalcinosis, with emphasis on in vivo observations. [ABSTRACT FROM AUTHOR] more...

The mechanisms involved in biomineralization are modulated through interactions with organic matrix. In the case of stone formation, the role of the organic macromolecules in the complex urinary environment is not clear, but the presence of mineralogical ‘signatures’ suggests that some aspects of stone formation may result from a non-classical crystallization process that is induced by acidic proteins. An amorphous precursor has been detected in many biologically controlled mineralization reactions, which is thought to be regulated by non-specific interactions between soluble acidic proteins and mineral ions. Using in vitro model systems, we find that a liquid-phase amorphous mineral precursor induced by acidic polypeptides can lead to crystal textures that resemble those found in Randall’s plaque and kidney stones. This polymer-induced liquid-precursor process leads to agglomerates of coalesced mineral spherules, dense-packed spherulites with concentric laminations, mineral coatings and ‘cements’, and collagen-associated mineralization. Through the use of in vitro model systems, the mechanisms involved in the formation of these crystallographic features may be resolved, enhancing our understanding of the potential role(s) that proteins play in stone formation. [ABSTRACT FROM AUTHOR] more...

Attachment of stone crystals to tubular epithelium may initiate kidney stone formation. We previously reported that apical nucleolin related protein (NRP) expression during mitosis enhance attachment of Ca oxalate monohydrate crystals (COM). Some forms of injury may also increase affinity for crystals. We examined changes in subcellular localization of NRP during the course of cisplatin-induced apoptosis in cultured inner medullary collecting duct cells. Caspase-3 activation and chromatin condensation followed by nuclear fragmentation occurred after 20 h exposure to cisplatin, indicating the development of apoptosis. Cells were fixed without permeabilization and stained for surface NRP. Cells with condensed chromatin showed little or no cytoplasmic or apical NRP. Those at an early stage of nuclear fragmentation had cytoplasmic but not apical NRP and cells with advanced nuclear fragmentation were positively stained for apical NRP. Membrane proteins isolated by apical biotinylation and precipitated with avidin were analyzed by Western blot. Apical NRP was markedly increased after cisplatin compared to control, while expression of the apical marker, GP-135, and other putative attachment protein were unchanged. Hyaluronic acid was decreased. Cultures with apoptotic cells demonstrated increased adherence of COM that was inhibited by the polyanion (poly)aspartic acid. We conclude that pre-existing apoptotic injury may promote calcium oxalate crystals attachment to renal tubular epithelium via apical NRP expression. [ABSTRACT FROM AUTHOR] more...

Exposure to high levels of oxalate and calcium oxalate monohydrate (COM) crystals is injurious to renal epithelial cells and triggers serial responses related to stone formation. Multiple molecules and proteins are involved in this process, but previous studies have generally been limited, without an overall understanding of protein expression alteration after oxalate and/or crystal exposure as well as its role in stone formation. We used proteomic analysis to reveal the changes in the proteome of HK-2 cells induced by oxalate and COM crystals, so as to provide candidate proteins involved in the molecular mechanisms concerning HK-2 cell injury and kidney stone formation. HK-2 cells were exposed to oxalate plus COM crystals at different concentrations in various samples. Cell viability was determined using a Cell Counting Kit-8 assay kit. For proteomic analysis, cells were exposed to oxalate (2 mM) and COM crystals (200 ug/ml) for 12 h. The proteins were separated by two-dimensional electrophoresis and the differentially expressed proteins were identified by liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS). Validation of protein expression was further performed by Western blot analysis. Oxalate and COM crystals showed concentration-dependent toxicity on HK-2 cells. A total of 12 differentially expressed proteins in HK-2 cells induced by oxalate and COM crystals were identified, which were involved in various aspects of cellular processes. Our study provides a platform for further studying the molecular mechanism of renal epithelial cell injury and kidney stone formation. [ABSTRACT FROM AUTHOR] more...

Nephrolithiasis is a result of formation and retention of crystals within the kidneys. The driving force behind crystal formation is urinary supersaturation with respect to the stone-forming salts, which means that crystals form when the concentrations of participating ions are higher than the thermodynamic solubility for that salt. Levels of supersaturation are kept low and under control by proper functioning of a variety of cells including those that line the renal tubules. It is our hypothesis that crystal deposition, i.e., formation and retention in the kidneys, is a result of impaired cellular function, which may be intrinsic and inherent or triggered by external stimuli and challenges. Cellular impairment or dysfunction affects the supersaturation, by influencing the excretion of participating ions such as calcium, oxalate and citrate and causing hypercalciuria, hyperoxaluria or hypocitraturia. The production and excretion of macromolecular promoters and inhibitors of crystallization is also dependent upon proper functioning of the renal epithelial cells. Insufficient or ineffective crystallization modulators such as osteopontin, Tamm-Horsfall protein, bikunin, etc. are most likely produced by the impaired cells. [ABSTRACT FROM AUTHOR] more...

Cynodon dactylon (Poaceae family) decoction was used in the treatment of kidney stones. However, no scientific study was undertaken so far to demonstrate the beneficial effect of the plant. Thus, the aim of the current study is to evaluate the effect of Cynodon aqueous extract as a preventive and curative agent in experimentally induced nephrolithiasis in a rat model. Ethylene glycol (EG) was used in the experiment to induce calcium oxalate (CaOx) deposition into kidneys. In preventive protocol, Cynodon decoction was administered in the same day with EG to evaluate the ability of the extract to prevent crystal deposition. However, in curative protocol, rats were first rendered nephrolithiasic and then the extract was administered to assess the ability of the plant to eliminate the pre-existing crystal deposition. In both protocols, urinary biochemical and other variables were measured during the course of the study. Crystalluria and renal histology were examined as well. The results showed that, in both protocols, all measured variables were similar for both the rat groups. Nevertheless, urinary biochemical analysis was apparently unaffected by the extract except oxalate in preventive protocol, and calcium, sodium, and potassium in curative protocol which were significantly highly excreted in treated rats compared to untreated animals. Crystalluria was characterized mostly by the presence of large quantities of CaOx monohydrate and CaOx dihydrate particles in untreated rats. However, crystalluria was mainly dominated by the presence of CaOx dihydrate particles with reduced size. The most apparent beneficial effect of Cynodon extract was seen in kidney tissues where reduced levels of CaOx deposition have been noticed especially in medullary and papillary sections from treated rats. We concluded that C. dactylon extract has beneficial effect in preventing and eliminating CaOx deposition into kidneys. Such findings provide a scientific explanation for its use in the treatment of kidneys stones. [ABSTRACT FROM AUTHOR] more...

Crystal formation in mice could not be induced either by the administration of ethylene glycol or by glycolate. To clarify the reasons for the difference among these oxalate precursors in mice, we studied renal tubular epithelial injury by immunohistochemical staining of oxidative stress and observing microstructures. Daily intra-abdominal injection of saline solution [10 ml/(kg day)], ethylene glycol[(48.3 mmol/(kg day)], glycolate [1.31 mmol/(kg day)], and glyoxylate [1.35 mmol/(kg day)] into C57BL/6 male mice (8 weeks) was performed for 7 days. Immunohistochemical staining of superoxide dismutase (SOD) and malondialdehyde (MDA), and transmission electron microscopy (TEM) of renal tubular epithelial cells were performed to observe oxidative stress and morphological changes, respectively. Decreased SOD and increased MDA were shown only in glyoxylate-treated mouse kidneys. The TEM study with glyoxylate-treated mouse kidneys demonstrated that the internal structure of mitochondria in renal tubular cells underwent destruction and vacuolization, and microvilli density decreased. These changes in renal tubular cells were located in the crystal-forming area. However, such changes were not detected in the other groups. Each precursor of oxalate induces different changes in renal epithelial cells regarding oxidative stress and the microstructural changes. It is suggested that calcium oxalate crystal formation requires cell injury and morphological changes of renal epithelial tubular cells induced by glyoxylate administration in the mouse kidney. [ABSTRACT FROM AUTHOR] more...

Strong evidence that osteopontin (OPN) is a determinant of urolithiasis has prompted studies comparing the protein’s urinary excretion in healthy subjects and stone formers. However, reported mean urinary values have varied widely, from <1 μg/mL to more than 20 times that value. Since OPN binds to CaOx crystals, the presence of crystals in urine may cause underestimation of its urinary levels. Using a commercial ELISA, we measured urinary OPN levels in the presence of endogenous or exogenous CaOx monohydrate (COM) and dihydrate (COD) crystals. OPN concentrations decreased in the presence of endogenous and exogenous CaOx crystals, but never below 2 μg/mL. Increasing the urinary calcium concentration decreased detectable OPN levels, possibly as a result of changes in the three-dimensional conformation of the protein. Because calcium concentration and the formation of CaOx crystals cannot be controlled in urine, the use of urinary OPN levels as a biomarker for any human pathology must be seriously questioned, but particularly for the investigation of stone formers in whom hypercalciuria and crystalluria are more common than in healthy subjects. [ABSTRACT FROM AUTHOR] more...

Both scanning electron microscopy and atomic force microscopy (AFM) have shown that calcium oxalate monohydrate kidney stones are made up from arrangements of sub micron crystals. The purpose of this investigation was to determine the morphology of these crystals which was obscured by the presence of organic matrix in our earlier study. Sections of stones were treated to remove the protein component of the matrix and then imaged using AFM. Images obtained after proteolysis show that the crystals are in the form of plates stacked on (100) surfaces. These results were confirmed by scanning electron microscopy observations from selected regions of calcium oxalate kidney stone surfaces. The observed crystal sizes are consistent with both the known matrix mass fraction and crystallite growth in the passage through the collecting duct. [ABSTRACT FROM AUTHOR] more...

It is widely believed that proteins rich in Asp, Glu or Gla (γ carboxyglutamic acid) interact strongly with calcium oxalate surfaces and inhibit calcium oxalate crystal growth. An alternative hypothesis would be that the interaction of Asp, Glu and Gla residues with surfaces could facilitate nucleation and crystal aggregation. Prothrombin fragment 1 and bikunin have been studied extensively as inhibitors, β-microglobulin, transferrin and antitrypsin have been found in stone matrix and tubulin has been observed in the attachment of crystals to cell surfaces. The aim of this study is to examine how well carboxylate groups in proteins found either in stone matrix, or proposed as inhibitors, could fit with the calcium ion sub-lattice of both calcium oxalate monohydrate and dihydrate surfaces. The carboxylate groups in the acidic Asp, Glu and Gla residues were marked in the Protein Data Bank structures and matched to calcium oxalate surfaces using the Cerius 3D molecular modeling program. A contact was defined if a carboxylate oxygen atom approached a surface calcium atom in such a way that the separation was less than 6 Å but greater than 2.4 Å, the sum of the ionic radii. If the proteins maintain their 3D structure, the number of contacts was no more than 3 or 4 for all the proteins studied, irrespective of the calcium oxalate surface. [ABSTRACT FROM AUTHOR] more...

This study was undertaken to determine whether the use of different washing procedures could explain dissident findings in published studies examining the role of urinary macromolecules in urolithiasis. Calcium oxalate monohydrate (COM) crystals were deposited from or added to the same sieved urine, washed with copious or limited amounts of distilled water, or with methanol, and examined by field emission scanning electron microscopy (FESEM). Demineralized extracts were analysed by SDS-PAGE and Western blotting for Tamm–Horsfall glycoprotein (THG), human serum albumin (HSA), osteopontin (OPN) and prothrombin fragment 1 (PTF1). Synchrotron X-ray diffraction (SXRD) with Rietveld whole-pattern peak fitting and profile analysis was used to determine non-uniform crystal strain and crystallite size in crystals generated from inorganic solutions in the presence of increasing concentrations of THG and prothrombin (PT). HSA and PTF1 were present in all demineralized crystal extracts, confirming their inclusion within COM. OPN was present in all extracts except those derived from pure inorganic COM crystals, because of its occlusion within small numbers of calcium oxalate dihydrate (COD) crystals contaminating the COM population. THG was absent from the demineralized extracts of all crystals washed copiously with water, but present in those washed with methanol or limited amounts of water. FESEM showed extraneous organic material associated only with crystals whose extracts contained THG, confirming that the protein does not bind permanently to the COM crystal surface and is not occluded within the mineral bulk. This was confirmed by SXRD, which showed that non-uniform strain and crystallite size remained unaltered in crystals grown in the presence of increasing THG concentrations. However, non-uniform strain increased and crystallite size decreased with increasing PT concentrations, demonstrating unambiguously that PT is included in COM crystals. It was concluded that scrupulous care must be taken to ensure the complete removal of extraneous THG adventitiously associated with CaOx crystals in order to avoid inaccurate analysis of crystal matrix protein content and possible misinterpretation of experimental data. [ABSTRACT FROM AUTHOR] more...

Crystal adherence to the renal epithelium is widely regarded as a probable mechanism of stone formation. Intracrystalline proteins may provide access to the core of urinary crystals and thereby facilitate the dismantling of these crystals after their attachment to and phagocytosis by renal epithelial cells. The present study investigated the role of proteolysis and limited dissolution of urinary calcium oxalate (CaOx) crystals in South Africa’s white and black populations with a view to understanding the remarkably low stone incidence in the black population compared with the whites. CaOx crystals were precipitated from filtered urine or ultrafiltered urine dosed with an intracrystalline protein, urinary prothrombin fragment 1 (UPTF1), isolated from white and black subjects. The crystals were fractured and subjected to proteolysis and/or limited dissolution before examination using field emission scanning electron microscopy (FESEM). FESEM data showed that CaOx crystals from white and black subjects were eroded by treatment with proteases. Cathepsin D caused the most significant crystal erosion, and more noticeable degradation of CaOx monohydrate (COM) crystals compared to CaOx dihydrate (COD). Limited dissolution studies showed the unique ultrastructures and fragmentation processes of COM and COD crystals. COM crystals appeared to be more susceptible to degradation and dissolution than CODs. Since COMs are predominant in blacks, compared with COD crystals from whites, it is speculated that the lower stone rate amongst South African blacks might be attributed partly to their more efficient destruction of retained COM crystals. [ABSTRACT FROM AUTHOR] more...

Nitrosative stress plays a role in calcium oxalate stone formation, as nitrosated proteins have been identified in stone formers. Nitric oxide (NO•), the common precursor for reactive nitrogen species, is synthesized in the juxtaglomerular apparatus of the kidneys. The present study is aimed to determine the role of nitric oxide synthase (NOS) in an experimental hyperoxaluric condition by histological and biochemical techniques. Hyperoxaluria was induced by 0.75% ethylene glycol in drinking water. L-arginine ( L-arg) was supplemented at a dose of 1.25 g/kg body weight orally for 28 days. Nitric oxide metabolites (NOx), protein content in the urine and lipid peroxidation in the kidney were determined at the end of the experimental period. Histopathological examination of the rat kidneys was then carried out. NADPH-diaphorase and eNOS expression studies were carried out in control and hyperoxaluric rat kidneys using histochemical and immunohistochemical techniques. Significant amounts of NOx were present in the urine of hyperoxaluric animals when compared to control rats. Histopathological examinations revealed membrane injury, tubular dilatation and edema in the hyperoxaluric rats, whereas co-supplementation of L-arg to the hyperoxaluric rats significantly reduced these changes. The results of histochemical analysis for NADPH-diaphorase staining demonstrate the role of NOS in hyperoxaluric rats. Hyperoxaluric rats showed intense staining for NADPH-diaphorase when compared to control and L-arg co-supplemented hyperoxaluric rats. Immunohistochemical demonstration confirmed that eNOS expression was markedly increased in L-arg supplemented rats, when compared to EG treated rat kidney sections. Thus, from the present study, we conclude that supplementation of L-arg to the hyperoxaluric animals minimizes the cellular injury mediated by ethylene glycol, prevents oxidative/nitrosative damage to the membranes and reduces the incidence of calcium oxalate stone formation. [ABSTRACT FROM AUTHOR] more...

Based on the structure of kidney stones, it is likely that they form as aggregations of preformed crystals, mostly calcium oxalate monohydrate (COM). In this study, we examined the ability of a macromolecular mixture isolated from the urine of normal individuals and stone formers to inhibit aggregation of preformed COM seed crystals in a simple ionic solution using measurements of changes in the particle size distribution (PSD) of preformed COM crystal aggregates. We also examined the effect in this assay of a number of synthetic homopolymers, naturally occurring urine macromolecules, and binary mixtures thereof. The macromolecular mixtures from urine of normals and most stone formers reduced the degree of aggregation of the seed crystals, whereas 22% of stone former urine macromolecules either did not disaggregate or actually promoted further aggregation. Stone formers within one family shared this property, but a non-stone forming sibling did not. Polyanions, either synthetic or naturally occurring, induced disaggregation to an extent similar to that exhibited by normal urine macromolecules, while polycations had no effect on the PSD. However, mixing a polyanion, either poly-aspartate or osteopontin, with the polycation poly-arginine, changed their behavior from disaggregation to aggregation promotion. The disaggregating behavior of normal urinary macromolecules provides a defense against aggregation, but a minority of stone forming individuals lacks this defense, which may contribute to stone formation. [ABSTRACT FROM AUTHOR] more...

Urinary oxalate is a biomarker for calcium oxalate kidney stone disease; however, its assay is insensitive and nonspecific. Calcium oxalate monohydrate (COM) binding protein (45 kDa) is a promoter of calcium oxalate kidney disease, which is markedly upregulated by oxalate induced oxidative stress. The current study was carried out to evaluate whether COM binding protein can serve as a diagnostic marker for calcium oxalate kidney stone formers. COM binding protein was isolated, purified and antibody was raised against it in rabbits. Urine samples (24 h) were collected from patients suffering from various kidney diseases such as acute nephritis, chronic nephritis, nephrotic syndrome, calcium oxalate (CaOx) stone formers, uric acid stone formers, struvite stone formers and calcium phosphate stone formers. This COM binding protein was quantified by an in house ELISA method and the excretion was found to lie between 2 and 3 mg in control samples, while in CaOx stone formers it was detected between 11 and 19 mg. Urinary risk factors were assayed. We conclude that COM binding protein can serve as a diagnostic marker for CaOx stone formers. [ABSTRACT FROM AUTHOR] more...

Phyllanthus niruri is a plant used in Brazilian folk medicine for the treatment of urolithiasis. It was previously observed that P. niruri shows no toxicity, potentially increases calculus voiding by stone forming patients and inhibits the endocytosis of calcium oxalate (CaOx) crystals by MDCK cells. In addition, in a rat model of urolithiasis it reduced calculus growth. In the present study, we evaluated the effect of an aqueous extract of P. niruri on CaOx crystallization in vitro. CaOx precipitation was induced by the addition of 0.1 M sodium oxalate to unfiltered urine samples from Wistar rats (n=14) and normal humans (n=18) in the presence or absence of P. niruri extract (0.25 mg/ml of urine). The presence of CaOx crystals was evaluated immediately and 24 h later. In vitro crystallization of human urine produced typical mono- and dihydrated CaOx crystals, but only a few typical CaOx crystals were found in rat urine. The presence of P. niruri extract did not inhibit CaOx precipitation and even more crystals were obtained, although they were significantly smaller than those in the control urine. Crystal aggregation observed 24 h after crystallization was also inhibited by P. niruri extract. The results showed an inhibitory effect of P. niruri extract on CaOx crystal growth and aggregation in human urine, suggesting that it may interfere with the early stages of stone formation and may represent an alternative form of treatment and/or prevention of urolithiasis [ABSTRACT FROM AUTHOR] more...