Human Calcium Oxalate Research Articles

2D map of proteins from human renal stone matrix and evaluation of their effect on oxalate induced renal tubular epithelial cell injury

Proteins constitute a major portion of the organic matrix of human calcium oxalate (CaOx) renal stones and the matrix is considered to be important in stone formation and growth. The present study evaluates the effect of these proteins on oxalate injured renal epithelial cells accompanied by a 2D map of these proteins. Proteins were isolated from the matrix of kidney stones containing CaOx as the major constituent using EGTA as a demineralizing agent. The effect of more than 3kDa proteins from matrix of human renal (calcium oxalate) CaOx stones was investigated on oxalate induced cell injury of MDCK renal tubular epithelial cells. A 2D map of >3kDa proteins was also generated followed by protein identification using MALDI-TOF MS. The >3kDa proteins enhanced the injury caused by oxalate on MDCK cells. Also, the 2D map of proteins having MW more than 3kDa suggested the abundance of proteins in the matrix of renal stone. Studies indicate that the mixture of >3kDa proteins in the matrix of human renal stones acts as promoter of calcium oxalate crystal nucleation and growth as it augments the renal epithelial cell injury induced by oxalate. The effect of promoters masks the inhibitors in the protein mixture thereby leading to enhanced renal cell injury. 2D map throws light on the nature of proteins present in the kidney stones.

Novel antilithiatic cationic proteins from human calcium oxalate renal stone matrix identified by MALDI-TOF-MS endowed with cytoprotective potential: An insight into the molecular mechanism of urolithiasis

BackgroundNo substantial work has been conducted to date in context to cationic proteins with antilithiatic activity. We explored the antilithiatic cationic proteins present in human calcium oxalate (CaOx) stones and also examined their molecular interactions with calcium oxalate crystals in silico. MethodsProteins were isolated from the matrix of human CaOx containing kidney stones. Proteins having MW>3kDa were subjected to cation exchange chromatography followed by molecular-sieve chromatography. The effect of these purified cationic proteins was tested against CaOx nucleation and growth and on oxalate injured MDCK cells for their activity. Proteins were identified by MALDI-TOF MS. Molecular interaction studies with COM crystals in silico were also investigated. ResultsThree antilithiatic cationic proteins were identified as histone-lysine N-methyltransferase, inward rectifier K channel and protein Wnt-2 (MW~53, ~44, and ~42kDa respectively) by MALDI-TOF MS based on database search with MASCOT server. Further molecular modeling calculations revealed the mode of interaction of these proteins with CaOx at the molecular level. ConclusionWe identified histone-lysine N-methyltransferase, inward rectifier K channel and protein Wnt-2 as novel antilithiatic proteins which play a vital role in the kidney function and have been associated with various kidney diseases.

In Vitro Comparison of Prototype Magnetic Tool with Conventional Nitinol Basket for Ureteroscopic Retrieval of Stone Fragments Rendered Paramagnetic with Iron Oxide Microparticles

We developed a prototype magnetic tool for ureteroscopic extraction of magnetized stone particles. We compared its efficiency for retrieving magnetized calcium oxalate monohydrate stone particles with that of a conventional nitinol basket from the pelvi-collecting system of a bench top ureteroscopic simulator. Iron oxide microparticles were successfully bound to 1 to 1.5, 1.5 to 2 and 2 to 2.5 mm human calcium oxalate monohydrate stones. Several coated fragments of each size were implanted in the collecting system of a bench top ureteroscopic simulator. Five-minute timed stone extraction trials were performed for each fragment size using a back loaded 8Fr magnetic tool mounted on a 0.038-inch guidewire or a conventional basket. The median number of fragments retrieved per timed trial was compared for the magnetic tool vs the basket using the Mann-Whitney U test. For 1 to 1.5 mm fragments the median number retrieved within 5 minutes was significantly higher for the prototype magnetic tool than for the nitinol basket (9.5 vs 3.5, p = 0.03). For 1.5 to 2 mm fragments the magnetic tool was more efficient but the difference in the number of fragments retrieved was not statistically significant (9.5 vs 4.5, p = 0.19). For 2 to 2.5 mm fragments there was no difference between the instruments in the number retrieved (6 per group, p = 1.0). The prototype magnetic tool improved the efficiency of retrieving stone particles rendered paramagnetic that were less than 2 mm but showed no advantage for larger fragments. This system has the potential to decrease the number of small retained fragments after ureteroscopic lithotripsy.

855 Proteomic analysis of human calcium oxalate renal stone by 2-D PAGE and in vitro analysis of bioactivity of the proteins

855 Proteomic analysis of human calcium oxalate renal stone by 2-D PAGE and in vitro analysis of bioactivity of the proteins

850 Identification of novel antilithiatic cationic proteins from human calcium oxalate renal stone matrix by MALDI TOF MS

850 Identification of novel antilithiatic cationic proteins from human calcium oxalate renal stone matrix by MALDI TOF MS

Novel Stone-Magnetizing Microparticles: In Vitro Toxicity and Biologic Functionality Analysis

We have developed novel iron-based microparticles (Fe-MP) that bind to calcium oxalate stone fragments, rendering them paramagnetic. Previously, we demonstrated enhanced efficiency of stone extraction in an inanimate model using magnetic instrumentation. Before in vivo stone extraction studies, we sought to further characterize Fe-MP with regard to cellular toxicity and to assess the influence of biologic fluids on binding performance. Monolayers of murine fibroblasts, human urothelium, and human transitional-cell carcinoma cells were exposed to 1 mg/mL of Fe-MP or saline via an agarose overlay. Cellular viability was assessed using neutral red staining and densitometry. Biologic functionality: Human calcium oxalate stone fragments were incubated with a solution of 1 mg/mL of Fe-MP containing varying concentrations of urine (10%-50%) or blood (0.5%-2%) for 10 minutes. Fragments were then extracted using an 8F magnetic tool. Assays of 10 stone fragments categorized as small (3-3.9 mg) or large (6-6.9 mg) were run in quadruplicate at each concentration. No toxicity was seen in any of the three cell lines after 48 hours of particle exposure, except in urothelial cells at the lowest cell concentration. Stone extraction success was 100% for all stones, regardless of concentration of urine or blood, and extractions were completed in less than 10 minutes. Preliminary toxicity testing revealed minimal to no cellular toxicity that was attributable to Fe-MP. The microparticles function well in the presence of clinically relevant concentrations of urine and blood that may be present during endoscopic stone surgery. Further toxicity and stone extraction testing in animal models is necessary.

Comparison of holmium:YAG and thulium fiber laser lithotripsy: ablation thresholds, ablation rates, and retropulsion effects

The holmium:YAG (Ho:YAG) laser lithotriptor is capable of operating at high pulse energies, but efficient operation is limited to low pulse rates (∼10 Hz) during lithotripsy. On the contrary, the thulium fiber laser (TFL) is limited to low pulse energies, but can operate efficiently at high pulse rates (up to 1000 Hz). This study compares stone ablation threshold, ablation rate, and retropulsion for the two different Ho:YAG and TFL operation modes. The TFL (λ = 1908 nm) was operated with pulse energies of 5 to 35 mJ, 500-μs pulse duration, and pulse rates of 10 to 400 Hz. The Ho:YAG laser (λ = 2120 nm) was operated with pulse energies of 30 to 550 mJ, 350-μs pulse duration, and a pulse rate of 10 Hz. Laser energy was delivered through 200- and 270-μm-core optical fibers in contact mode with human calcium oxalate monohydrate (COM) stones for ablation studies and plaster-of-Paris stone phantoms for retropulsion studies. The COM stone ablation threshold for Ho:YAG and TFL measured 82.6 and 20.8 J∕cm(2), respectively. Stone retropulsion with the Ho:YAG laser linearly increased with pulse energy. Retropulsion with TFL was minimal at pulse rates less than 150 Hz, then rapidly increased at higher pulse rates. For minimal stone retropulsion, Ho:YAG operation at pulse energies less than 175 mJ at 10 Hz and TFL operation at 35 mJ at 100 Hz is recommended, with both lasers producing comparable ablation rates. Further development of a TFL operating with both high pulse energies of 100 to 200 mJ and high pulse rates of 100 to 150 Hz may also provide an alternative to the Ho:YAG laser for higher ablation rates, when retropulsion is not a primary concern.

Effect of biomolecules from human renal matrix of calcium oxalate monohydrate (CaOx) stones on in vitro calcium phosphate crystallization

Investigate the activity of high and low molecular weight biomolecules present in the matrix of human calcium oxalate (CaOx) stones not only on the initial mineral phase formation of calcium and phosphate (CaP) but also on its growth and demineralization of the preformed mineral phase. Surgically removed renal stones were analyzed by Fourier Transform Infra Red (FTIR) spectroscopy and only CaOx stones were extracted with 0.05M EGTA, 1 mM PMSF and 1% Β-mercaptoethanol. Renal CaOx stone extract was separated into > 10 kDa and < 10 kDa fractions by dialysis. Activity of both the fractions along with whole extract was studied on the three mineral phases of CaP assay system. It was interesting to observe that both high and low molecular weight biomolecules extracted from human renal matrix of calcium oxalate (CaOx) stones exhibited different roles in the three mineral phases of CaP. Whole extract exhibited inhibitory activity in all the three assay systems; however, mixed (stimulatory and inhibitory) activity was exhibited by the > 10 kDa and < 10 kDa fractions. SDS-PAGE analysis showed bands of 66 kDa, 80 kDa, 42 kDa in whole EGTA extract lane and > 10 kDa fraction lane. Both high and low molecular weight biomolecules extracted from human renal matrix of calcium oxalate (CaOx) stones have a significant influence on calcium and phosphate (CaP) crystallization.

2108 NOVEL STONE –MAGNETIZING MICROPARTICLES (STONEMAG): TOXICITY AND BIOLOGIC FUNCTIONALITY ANALYSIS

You have accessJournal of UrologyStone Disease: New Technology1 Apr 20102108 NOVEL STONE –MAGNETIZING MICROPARTICLES (STONEMAG): TOXICITY AND BIOLOGIC FUNCTIONALITY ANALYSIS Saad Mir, Sara Best, Stacey McLeroy, Chester Donnally, Bruce Gnade, Margaret Pearle, and Jeffrey Cadeddu Saad MirSaad Mir Dallas, TX More articles by this author , Sara BestSara Best Dallas, TX More articles by this author , Stacey McLeroyStacey McLeroy Richardson, TX More articles by this author , Chester DonnallyChester Donnally Dallas, TX More articles by this author , Bruce GnadeBruce Gnade Richardson, TX More articles by this author , Margaret PearleMargaret Pearle Dallas, TX More articles by this author , and Jeffrey CadedduJeffrey Cadeddu Dallas, TX More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.2195AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES We have developed iron-based microparticles (StoneMag) that bind to calcium oxalate stone fragments, rendering them magnetic. The stones can then be manipulated and extracted with a magnetic instrument. This has previously been shown to dramatically improve the efficiency of stone extraction in an inanimate model. Our objective is to use a solution of the particles as the irrigation fluid during human stone surgeries to capitalize on this improvement in efficiency. To that end, we have further characterized our particles in terms of cellular toxicity and the influence of biological fluids (urine) on the efficacy of binding. METHODS Toxicity: Monolayers of murine fibroblast, human transitional cell carcinoma, and human urothelium were plated at 3 densities- 2x105, 5x105, and 1x106- on p60 petri dishes in T medium (5% P/C and 5% FBS). After 1 day of incubation, the medium was aspirated and 5 mL of T medium containing 1% agarose was plated. After cooling, 1 mg/mL of FeOH microparticles or equivalent volumes of PBS was placed on the agarose surface. Agarose was removed after 48 hours of incubation and each plate was stained with 1 mL of .01% neutral red for 3 hours and fluorescence was quantified. Biologic functionality: Human calcium oxalate stone fragments were incubated with a 1mg/ml solution of microparticles containing varying concentrations of urine (10-50%). After a 10 min incubation, we attempted to extract the stones from the solution using a 9 fr magnetic tool. Assays of 10 stone fragments categorized as small (3-3.9mg) or large (6-8mg) were run in triplicate at each concentration as well as in a saline control. RESULTS There was no evidence of toxicity in any of the 3 cell lines after 48 hours of particle exposure. The magnetization of the small stones was unaffected by urine, no matter the concentration. All of the large stones were able to be magnetically extracted at all urine concentrations. However, the extraction took longer as the concentration of urine increased (for example, 5.33 min in the 50% group, compared to 1.42 min in the control). CONCLUSIONS Preliminary toxicity testing results are promising. StoneMag appears to function well in the presence of clinically relevant concentrations of urine such as would likely be present if the microparticles are used in an irrigation solution during endourologic stone surgery. Further toxicity testing in animal models is warranted. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e819-e820 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Saad Mir Dallas, TX More articles by this author Sara Best Dallas, TX More articles by this author Stacey McLeroy Richardson, TX More articles by this author Chester Donnally Dallas, TX More articles by this author Bruce Gnade Richardson, TX More articles by this author Margaret Pearle Dallas, TX More articles by this author Jeffrey Cadeddu Dallas, TX More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Mechanism of Formation of Human Calcium Oxalate Renal Stones on Randall's Plaque

Although calcium oxalate (CaOx) renal stones are known to grow attached to renal papillae, and specifically to regions of papillae that contain Randall's plaque (interstitial apatite deposits), the mechanisms of stone overgrowth on plaque are not known. To investigate the problem, we have obtained biopsy specimens from two stone patients that included an attached stone along with its tissue base and have studied the ultrastructural features of the attachment point using light and transmission electron microscopy, Fourier transform infrared spectroscopy (mu-FTIR), and immunohistochemical analysis. The epithelium is disrupted at the attachment site. The denuded plaque that borders on the urinary space attracts an envelope of ribbon-like laminates of crystal and organic matrix arising from urine ions and molecules. Into the matrix of this ribbon grow amorphous apatite crystals that merge with and give way to the usual small apatite crystals imbedded in stone matrix; eventually CaOx crystals admix with apatite and become the predominant solid phase. Over time, urine calcium and oxalate ions gradually overgrow on the large crystals forming the attached stone.

Scanning Electron Microscopy and Atomic Force Microscopy of the Ring Structures in Human Calcium Oxalate Urinary Stones

Scanning Electron Microscopy and Atomic Force Microscopy of the Ring Structures in Human Calcium Oxalate Urinary Stones

Increased dietary oxalate does not increase urinary calcium oxalate saturation in hypercalciuric rats

Human calcium oxalate (CaOx) nephrolithiasis may occur if urine is supersaturated with respect to the solid-phase CaOx. In these patients, dietary oxalate is often restricted to reduce its absorption and subsequent excretion in an effort to lower supersaturation and to decrease stone formation. However, dietary oxalate also binds intestinal calcium which lowers calcium absorption and excretion. The effect of increasing dietary oxalate on urinary CaOx supersaturation is difficult to predict. To determine the effect of dietary oxalate intake on urinary supersaturation with respect to CaOx and brushite (CaHPO4), we fed 36th and 37th generation genetic hypercalciuric rats a normal Ca diet (1.2% Ca) alone or with sodium oxalate added at 0.5%, 1.0%, or 2.0% for a total of 18 weeks. We measured urinary ion excretion and calculated supersaturation with respect to the CaOx and CaHPO4 solid phases and determined the type of stones formed. Increasing dietary oxalate from 0% to 2.0% significantly increased urinary oxalate and decreased urinary calcium excretion, the latter presumably due to increased dietary oxalate-binding intestinal calcium. Increasing dietary oxalate from 0% to 2.0% decreased CaOx supersaturation due to the decrease in urinary calcium offsetting the increase in urinary oxalate and the decreased CaHPO4 supersaturation. Each rat in each group formed stones. Scanning electron microscopy revealed discrete stones and not nephrocalcinosis. X-ray and electron diffraction and x-ray microanalysis revealed that the stones were composed of calcium and phosphate; there were no CaOx stones. Thus, increasing dietary oxalate led to a decrease in CaOx and CaHPO4 supersaturation and did not alter the universal stone formation found in these rats, nor the type of stones formed. These results suggest the necessity for human studies aimed at determining the role, if any, of limiting oxalate intake to prevent recurrence of CaOx nephrolithiasis.

Ultrastructural immunodetection of osteopontin and osteocalcin as major matrix components of renal calculi.

The organic matrix of renal calculi has long been considered to influence the crystal growth that occurs in these pathological mineral deposits. Recent advances in characterizing individual organic moieties from mineralized tissues in general and the combined use of antibodies raised against these molecules with different immunocytochemical approaches have allowed their precise distribution to be visualized in a variety of normal and pathological mineralized tissues. The present ultrastructural study reports on the epithelial expression and extracellular localization of several noncollagenous proteins in rat and human kidney stones using high-resolution colloidal-gold immunocytochemistry. To this end, we have examined in an ethylene glycol-induced calcium oxalate model of urolithiasis in the rat, and in human kidney stones, the distribution of certain noncollagenous and plasma proteins known to accumulate in bone and other mineralized tissues that include osteopontin, osteocalcin, bone sialoprotein, albumin, and alpha 2HS-glycoprotein. Of these proteins, osteopontin (uropontin) and osteocalcin (or osteocalcin-related gene/protein) were prominent constituents of the calcium oxalate-associated crystal "ghosts" found in the nuclei, lamellae, and striations of the organic matrix of lumenal renal calculi in the rat and of small crystal ghosts found within epithelial cells. Immunocytochemical labeling for both proteins of the content of secretory granules in tubular epithelial cells from treated rats, together with labeling of a similarly textured organic material in the tubular lumen, provides evidence for cosecretion of osteopontin and osteocalcin by epithelial cells, their transit through the urinary filtrate, and ultimately their incorporation into growing renal calculi. In normal rat kidney, osteopontin was localized to the Golgi apparatus of thin loop of Henle cells. In human calcium oxalate monohydrate stones, osteopontin was similarly detected in the lamellae and striations of the organic matrix. Based on these data, it is proposed that during urolithiasis, secretion of osteopontin (uropontin) and osteocalcin (or osteocalcin-related gene/protein), and the subsequent incorporation of these proteins into kidney stone matrix, may influence the nucleation, growth processes, aggregation, and/or tubular adhesion of renal calculi in mammalian kidneys.

Mechanical impactor employing Nitinol probes to fragment human calculi: fragmentation efficiency with flexible endoscope deflection.

The Browne Pneumatic Impactor (BPI; Browne Medical Systems, Minneapolis, MN) is a new mechanical lithotripter that relies on a pneumatically driven metal probe. It can be employed through both rigid and actively deflectable flexible endoscopes. Prior in vivo studies have shown excellent fragmentation of hard human calculi while employing the BPI with active endoscope deflection of as much as 45 degrees. In this study, we set out to define the efficiency of stone fragmentation with active endoscope deflection and to quantify the extent of retrograde stone migration that is often noted when mechanical lithotripters are employed. Pressure transducer testing of the BPI revealed an inverse relation between increased deflection of the endoscope and transduced voltage. Calculus fragmentation tests showed that as the endoscope was actively deflected to 90 degrees, the BPI still was able to fragment human calcium oxalate monohydrate calculi into extractable fragments with as few as six pulses. The maximum active endoscope deflection was 95 degrees with the 0.020-inch Nitinol probe passed through the working channel. At this deflection, the BPI was able to fragment an 8 mm pure calcium oxalate monohydrate calculus into two fragments after 14 pulses. Retrograde migration was evaluated by employing the BPI in a simulated ureter in a waterbath. The travel distance of a 5-mm 5-g calculus with one pulse averaged 12.2 mm (range 1-44 mm). Smaller, less dense calculi travelled farther. A rebound effect was occasionally noted, and this was associated with an off-center delivery. In conclusion, the BPI is an efficient mechanical lithotrite able to fragment hard human calculi when employed with active endoscope deflection of as much as 95 degrees. Delivered energy decreased with active endoscope deflection, but this change did not prevent fragmentation. Retrograde migration was an important variable more notable with smaller calculi.

Role of Organic Matrix in Urinary Stone Formation: An Ltrastructural Study of Crystal Matrix Interface of Calcium Oxalate Monohydrate Stones

Human calcium oxalate monohydrate (COM) urinary stones were decalcified by treatment with a mixture of ethylenediaminetetraacetic acid (EDTA) solution and Karnovsky’s fixative after embedding in bactoagar. Decalcified stones were examined by light microscopy, and also by scanning and transmission electron microscopy. Stones had distinct nuclei that were occupied by amorphous or apatitic calcium phosphate or aggregates of spherulitic COM crystals. EDTA insoluble matrix was ubiquitous in stones and consisted largely of finely matted fibrous material. It was organized in concentric laminations in the peripheral area of the stone but appeared highly disorganized in the stone center. Crystals were replaced by crystal ghosts. Organic matrix was present both inside the crystals and in the intercrystalline spaces. The study indicates a very close association between crystals and organic matrix. The relationship appears to begin early in crystal formation and persists throughout the formative and growth phases of the urinary stones.

A New Model of Nephrolithiasis Involving Tubular Dysfunction/Injury

To better understand the pathogenesis of nephrolithiasis, we developed a new animal model that closely mimics human calcium oxalate stone disease. Rats were treated with a regimen that combines moderate hyperoxaluria (produced by 10 days of feeding with 3% ammonium oxalate) with mild proximal tubular injury/dysfunction (produced by 8 daily injections of gentamicin sulfate −40mg./kg.). This combined treatment caused a marked increase in the incidence of calcium oxalate crystals and stones over that seen in animals treated with oxalate or gentamicin alone. Using a semiquantitative scoring system for estimating the abundance of crystals in coronal sections of kidneys, we found that 63% of animals receiving gentamicin plus oxalate showed “moderate” numbers of crystal, as compared to 8% of animals receiving oxalate alone; and the majority of the crystals occurred in the papilla, a pattern similar to that seen in human stone disease. Untreated rats and rats treated with gentamicin alone did not exhibit calcium oxalate crystals or stones. Despite the abundance of crystals and stones, animals receiving gentamicin plus oxalate retained relatively normal renal function as judged by creatinine clearance. Thus, the model has several advantages over preexisting models of nephrolithiasis. Crystal and stone deposition develop rapidly (within 14 days). The pattern of deposition resembles that seen in human stone disease and renal function remains relatively normal. These findings indicate that this model of nephrolithiasis may prove useful for studies of the pathogenesis of stone disease. Moreover, they suggest that renal tubular injury and/or dysfunction may produce conditions conducive to the formation and growth of calcium oxalate stones.

Presence of lipids in urinary stones: results of preliminary studies.

The presence of lipids in urinary stones was determined by histochemical and biochemical methods. When crystals of calcium oxalate, made by mixing calcium chloride and potassium oxalate solutions and sections of human calcium oxalate urinary stones, were exposed to osmium vapors, there was no staining of the pure crystals whereas the stone sections were stained. De-paraffinized sections of demineralized calcium oxalate stones showed positive sudanophilia on staining with Sudan black B. Both these experiments indicate the presence of lipids in calcium oxalate stones. Lipids were extracted from uric acid, struvite, and calcium oxalate stones using standard techniques. Phospholipids were separated by one-dimensional thin layer chromatography. All the stones studied contained lipids. In calcium oxalate stones they accounted for 10.15% of the matrix. Calcium oxalate and struvite stones contained more phospholipids than uric acid stones. Cardiolipin, sphingomyelin, phosphatidyl choline, phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidyl serine, and phosphatidyl glycerol were identified in lipid extracts. Demineralization by ethylenediaminetetra-acetate (EDTA) treatment increased lipid output from calcium oxalate stones by 15.5%.

In Vitro Precipitation of Calcium Oxalate in the Presence of Whole Matrix or Lipid Components of the Urinary Stones

Organic matrix of human calcium oxalate urinary stones was obtained by demineralizing with EDTA. Lipids were extracted from the EDTA-insoluble matrix by chloroform methanol treatment. The whole matrix and its total lipid extract were then incubated in a metastable solution of calcium oxalate and depletion of calcium and oxalate ions from the calcifying solution was determined. Results of our studies described here show that urinary calcium oxalate stone matrix and its total lipid contents were capable of binding calcium and oxalate ions and of catalysing calcium oxalate crystal formation from a metastable calcium oxalate solution.

Isolation From Human Calcium Oxalate Renal Stones of Nephrocalcin, a Glycoprotein Inhibitor of Calcium Oxalate Crystal Growth. Evidence That Nephrocalcin From Patients With Calcium Oxalate Nephrolithiasis is Deficient in γ-Carboxyglutamic Acid

We have determined that the organic matrix of calcium oxalate kidney stones contains a glycoprotein inhibitor ofcalcium oxalate crystal growth (nephrocalcin) that resembles nephrocalcin present in the urine of patients with calcium oxalate stones and differs from nephrocalcin from the urine of normal people. Pulverized calcium oxalate renal stones were extracted with 0.05M EDTA, pH 8.0; nephrocalcin eluted in five peaks using DEAE-cellulose column chromatography, and each peak was further resolved by Sephacryl S-200 column chromatography. Four ofthe fiveDEAE peaks corresponded to those usually found in nephrocalcin from urine; the fifth eluted at a lower ionic strength than any found in urine. Amino acid compositions and surface properties of nephrocalcins isolated from kidney stones closely resembled those of nephrocalcins isolated from urine of stone-forming patients: they differed from normal in lacking y-carboxyglutamic acid residues, and in forming air-water interfacial films that were less stable than those formed by nephrocalcin from normal urine.

Isolation from human calcium oxalate renal stones of nephrocalcin, a glycoprotein inhibitor of calcium oxalate crystal growth. Evidence that nephrocalcin from patients with calcium oxalate nephrolithiasis is deficient in gamma-carboxyglutamic acid.

We have determined that the organic matrix of calcium oxalate kidney stones contains a glycoprotein inhibitor of calcium oxalate crystal growth (nephrocalcin) that resembles nephrocalcin present in the urine of patients with calcium oxalate stones and differs from nephrocalcin from the urine of normal people. Pulverized calcium oxalate renal stones were extracted with 0.05 M EDTA, pH 8.0; nephrocalcin eluted in five peaks using DEAE-cellulose column chromatography, and each peak was further resolved by Sephacryl S-200 column chromatography. Four of the five DEAE peaks corresponded to those usually found in nephrocalcin from urine; the fifth eluted at a lower ionic strength than any found in urine. Amino acid compositions and surface properties of nephrocalcins isolated from kidney stones closely resembled those of nephrocalcins isolated from urine of stone-forming patients: they differed from normal in lacking gamma-carboxyglutamic acid residues, and in forming air-water interfacial films that were less stable than those formed by nephrocalcin from normal urine.