Nephrocalcin Research Articles

Defective urinary crystallization inhibition and urinary stone formation

Nephrocalcin (NC) is a glycoprotein produced in the kidney and inhibits calcium oxalate crystal formation. It has been separated into 4 isoforms (A, B, C, and D) and found that (A + B) are more abundant than (C + D) in urine of healthy subjects, but the reverse is seen in human urine of kidney stone patients. To further examine the role of this protein in inhibition of urinary crystallization, nephrocalcin isoforms were purified from 2 genetically pure dog species. We studied healthy Beagles, known to be non-stone forming dogs, and Mini-Schnauzers, known to be calcium oxalate stone formers. NC was isolated and purified from each group. Urinary biochemistry and calcium oxalate crystal growth inhibition were measured. Specific crystal growth inhibition activity was significantly higher in non-stone forming dogs (9.79 +/- 2.25 in Beagles vs. 2.75 +/- 1.34 of Mini-Schnauzers, p < 0.005). Dissociation constants toward calcium oxalate monohydrate were 10-fold different, with Beagles' isoforms being 10 times stronger inhibitors compare to those of Mini-Schnauzers'. Isoforms C + D of NC were the main isoforms isolated in stone-forming dogs. NC of these two species of dogs differently affects calcium oxalate crystallization and might have a role in determining ulterior urinary stone formation.

Urinary Nephrocalcin Excretion in Children with Urolithiasis

The aim of this study was to investigate the role of nephrocalcin in childhood urolithiasis. Forty-one patients with urinary stones and 25 age- and sex-matched healthy controls were admitted to the study. Blood and timed urine samples were taken from both patient and control groups for biochemical analysis. Serum and urine creatinine (Cr) and urinary nephrocalcin (NC) were measured. NC excretion was expressed as a NC/Cr (mg/g) ratio. NC-PreA/Cr and NC-D/Cr ratios were found to be significantly higher in patients than in the control group. No statistically significant differences were found in NC-A/Cr, NC-B/Cr, NC-C/Cr ratios between the patient and control groups. The high NC-PreA/Cr ratio (p = 0.012) observed in stone-forming patients indicates that this ratio may also be an important stimulatory factor for urinary stone disease.

Nephrocalcin isoforms coat crystal surfaces and differentially affect calcium oxalate monohydrate crystal morphology, growth, and aggregation

Calcium oxalate crystals were grown in the presence of each of the four isoforms of nephrocalcin (NC), a urinary protein proposed to inhibit kidney stone growth. Crystal size, morphology, and surface topography were assessed using optical microscopy, Coulter counter measurements, scanning electron microscopy (SEM), and atomic force microscopy (AFM). All crystals grown in the presence of NC isoforms were calcium oxalate monohydrates (COMs). Crystals formed in the presence of NC-A were smaller than control crystals, which were grown without NC, according to optical and SEM results, suggesting that NC-A restricts crystal growth. In contrast, samples grown with NC-C and NC-D exhibit more large crystals and several crystal aggregates, suggesting that NC-C and -D promote crystal growth and aggregation. Crystals grown with NC-B are not significantly larger or smaller than controls. AFM images of the crystals reveal significantly different surface textures on the control crystals relative to those grown with NC isoforms, indicating that NC acts by coating nascent calcium oxalate crystals. These are the first reported AFM images that show topography of NC-coated crystals. These findings suggest that NC isoforms have distinct interactions with different COM crystal faces, which may be responsible for their different effects on crystal growth and morphology.

The reappraisal of nephrocalcin--its role in the inhibition of calcium oxalate crystal growth and interaction with divalent metal ions.

Nephrocalcin (NC) is a potent crystal growth inhibitor of calcium oxalate monohydrate. However, the sequence is undefined owing to its multiple glycosylations. Although there have been many biochemical studies of the binding capacity of calcium, the study of the function of the domain is still deferred. By using S-200 gel filtration and Mono Q ion-exchange chromatographic procedures, NC can be purified without further treatment for the removal of urobilirubin. The kinetic study of crystal growth in calcium oxalate monohydrate is performed using a seed slurry system. NC was cut into two peptides through interaction with copper ion and ascorbic acid. The interaction site of the copper ion is presumed to be located between 8 and 6 kDa of molecular weight in NC. The data suggest that divalent metal ions may be involved in the calcium oxalate crystallization through interaction with NC. The role of ascorbic acid in the formation of urinary stones should be reappraised for its association in the redox reaction, with resultant protein digestion in the presence of copper ions.

Immunohistochemical localization of nephrocalcin, a kidney-specific glycoprotein, to renal cell carcinoma

Objectives. Nephrocalcin (NC), an acidic glycoprotein produced by renal proximal tubule cells and functioning as an inhibitor of calcium oxalate monohydrate crystalization, has been previously shown to have increased urinary excretion in patients with renal cell carcinoma (RCC). The current study uses immunohistochemical techniques to localize NC to cells of primary RCC. Methods. We studied 29 kidneys removed because of RCC. Slides were deparaffinized and stained after incubating with anti-NC antibody by using the avidin-biotin-peroxidase complex techniques. Uptake of stain by tumor cells and adjacent normal renal cells was compared. Results. Twenty-seven kidneys (93%) showed positive staining for RCC tumor cells; 2 kidneys staining positive for normal proximal tubule cells failed to stain adjacent RCC tumor cells (7%). Conclusions. The data suggest that enhanced production of urinary NC in patients with RCC derives from cells of the primary tumor.

Properties and function of nephrocalcin: mechanism of kidney stone inhibition or promotion.

Nephrocalcin (NC), an acidic glycoprotein with molecular weight 14,000, is present in urine and prevents kidney stone formation. Histoimmunochemical staining shows that NC is localized in the proximal tubles in kidneys. Isolated NC from mammalian urine, revealed at least 4 isoforms of NC (we call these isoforms NC-A, NC-B, NC-C, and NC-D in the order of elution) during DEAE cellulose column chromatography with a linear gradient of NaCl elution step. Non-stone forming people excrete more NC-A and NC-B isoforms in urine; however, more NC-C and NC-D isoforms were found in stone formers' urine. When the organic matrix was extracted from surgically removed calcium oxalate kidney stones, we found greater quantities of NC-C and NC-D isoforms than those of NC-A and NC-B isoforms. Amino acid compositions and carbohydrate contents of these 4 isoforms were similar with the exception of the gamma-carboxyglutamic acid (GLA) residues. Only the NC-A and NC-B isoforms contained residues of GLA. There were more phosphate residues present in NC-C and NC-D than in NC-A and NC-B. Upon removal of phosphate residues by alkaline phosphatase, NC-C eluted at the same salt concentrations as NC-A eluted. This indicates that the backbone protein could be similar, but the NC-C isoform is modified by excess phosphate residues. Surface tension measurements using a Lauda film balance indicated that NC-A and -B were strongly amphiphilic while NC-C and -D were less amphiphilic. NC-A has an elongated shape, and occupies a smaller area per molecule; whereas NC-C is a bulky molecule. Using NC-A as a model of a "good" inhibitor and NC-C as a model of a "poor" inhibitor, both bound with 4 atoms of Ca2+ per molecule as investigated by equilibrium dialysis method, 31P-NMR, and electron spin resonance spectrometry. Isoforms A and B changed their conformation upon Ca2+ binding, but C and D did not change their conformation. All these observations suggest that isoforms A and B are strong inhibitors of calcium oxalate monohydrate (COM) crystal growth and aggregation. However, isoforms C and D act as promotors for COM crystal growth-kidney stone formation. Measuring the amount of NC in urine from renal cell carcinoma patients and from NC isolated from a supernatant of a primary renal cell carcinoma cells demonstrated the amount of NC increased with disease progression.

Characterization of Ca(2+)-binding sites in the kidney stone inhibitor glycoprotein nephrocalcin using vanadyl ions: different metal binding properties in strong and weak inhibitor proteins revealed by EPR and ENDOR.

Nephrocalcin (NC), a calcium-binding glycoprotein of 14,000 molecular weight as a monomer, is known to inhibit the growth of calcium oxalate monohydrate (COM) crystals in renal tubules. We have isolated NC from bovine kidney tissue and purified into four isoforms, fractions A-D. NC-A and NC-B strongly inhibit the growth of COM crystals, and NC-C and NC-D inhibit crystal growth weakly. The strongly inhibitor proteins are abundant in normal subjects, whereas stone formers excrete less of NC-A and NC-B and more of NC-C and NC-D. NC-C was characterized with respect to its metal binding sites by using vanadyl ion (VO2+) as a paramagnetic probe in electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopic studies. We demonstrated that VO2+ binds to NC-C with a stoichiometry of metal:protein binding of 4:1 and that VO2+ competes with Ca2+ in binding to NC-C. In NC-C, the metal ion is exposed to solvent water molecules and two water molecules are detected in the inner coordination sphere of the metal ion by ENDOR. In the metal binding environment of NC-A, as reported previously (Mustafi, D., & Nakagawa, Y. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11323-11327), inner sphere coordinated water is completely excluded. Based on the results of the metal binding properties in both strong and weak inhibitor proteins, a probable mechanism of inhibition of COM crystal growth by NC has been outlined.

Is nephrocalcin related to the urinary derivative (bikunin) of inter-alpha-trypsin inhibitor?

To isolate, purify, sequence and characterize nephrocalcin (NC), a urinary protein that may be an important determinant of calcium oxalate (CaOx) kidney-stone disease. Proteins were isolated from human urine using cellulose and resin columns and were sequenced using Edman degradation and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition of CaOx crystal growth by the isolated proteins was assessed by measuring the deposition of 14C-labelled CaOx. A protein assumed to be NC on the basis of SDS-PAGE, inhibitory and gel filtration properties was isolated from healthy human urine. Its molecular weight and the amino acid sequences of two of its peptides suggested it was identical to fragment HI-14 of the light chain (bikunin) of inter-alpha-trypsin inhibitor (ITI). NC represents a portion of the light chain of ITI, although this conclusion must remain tentative until confirmed using authentic NC.

Characterization of calcium-binding sites in the kidney stone inhibitor glycoprotein nephrocalcin with vanadyl ions: electron paramagnetic resonance and electron nuclear double resonance spectroscopy.

Nephrocalcin (NC) is a calcium-binding glycoprotein of 14,000 molecular weight. It inhibits the growth of calcium oxalate monohydrate crystals in renal tubules. The NC used in this study was isolated from bovine kidney tissue and purified with the use of DEAE-cellulose chromatography into four isoforms, designated as fractions A-D. They differ primarily according to the content of phosphate and gamma-carboxy-glutamic acid. Fractions A and B are strong inhibitors of the growth of calcium oxalate monohydrate crystal, whereas fractions C and D inhibit crystal growth weakly. Fraction A, with the highest Ca(2+)-binding affinity, was characterized with respect to its metal-binding sites by using the vanadyl ion (VO2+) as a paramagnetic probe in electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopic studies. By EPR spectrometric titration, it was shown that fraction A of NC bound VO2+ with a stoichiometry of metal:protein binding of 4:1. Also, the binding of VO2+ to NC was shown to be competitive with Ca2+. Only protein residues were detected by proton ENDOR as ligands, and these ligands bound with complete exclusion of solvent from the inner coordination sphere of the metal ion. This type of metal-binding environment, as derived from VO(2+)-reconstituted NC, differs significantly from the binding sites in other Ca(2+)-binding proteins.

The effect of warfarin on urine calcium oxalate crystal growth inhibition and urinary excretion of calcium and nephrocalcin.

Urine contains inhibitors of calcium oxalate (CaOx) crystal growth. One such inhibitor is nephrocalcin (NC), a glycoprotein which is made in the kidney and contains several residues of gamma-carboxyglutamic acid (Gla) per molecule. The presence of Gla may be important to its ability to inhibit crystal growth. Several studies suggest that vitamin K-dependent proteins may also play a role in renal calcium (Ca) handling, and that vitamin D deficiency may lead to excess urinary Ca loss, but the effect of the vitamin K antagonist warfarin on urinary Ca excretion and CaOx growth inhibition in humans is not known. We studied 11 men while they were taking warfarin for a mean of 252 days, and again a mean of 64 days after its discontinuation. Urinary Ca excretion did not differ between those on or off warfarin, or between those on warfarin and normal controls. The ability of the subjects' urine to inhibit CaOx crystal growth did not differ on or off warfarin, or from that of control urine, and the excretion of immunoreactive NC also did not differ between these groups. NC was found to be responsible for approximately 16% of the CaOx growth inhibition seen. These results do not suggest that vitamin K-dependent proteins play a major role in renal Ca excretion in men, or that interference with vitamin K alters NC excretion or inhibitory activity of the urine.

Isolation and purification of a new glycoprotein from human urine inhibiting calcium oxalate crystallization.

A calcium oxalate crystal growth inhibitor was isolated from human urine using DEAE-Sephacel gel followed by Sephacryl S-300 chromatography and FPLC column. The isolated inhibitor was a uronic-acid-rich protein (UAP). It was found to be a glycoprotein with a molecular weight of 35,000 Da as determined by SDS-polyacrylamide gel electrophoresis. Inhibitory activity was demonstrated using a calcium oxalate crystallization system. In addition UAP, nephrocalcin (NC) or nephrocalcin-like (NC-like) activity was an effective inhibitor in this system. However, the inhibitory activity of UAP appeared to be higher than that of NC or NC-like activity. This finding suggests that NC or NC-like activity is not only urinary protein with strong inhibitory activity. UAP and probably other proteins also play a role in the control of urinary crystal growth.

Calcium oxalate monohydrate crystals are endocytosed by renal epithelial cells and induce proliferation.

Monkey kidney epithelial cells of the nontransformed BSC-1 line were used as a model system with which to search for biological responses to urinary crystals commonly found in renal stones. Calcium oxalate monohydrate (COM), the most common urinary crystal, was avidly internalized, initiated DNA synthesis, and stimulated cell multiplication. The increase in DNA synthesis observed after exposure to COM crystals was equivalent in magnitude to that of 10% calf serum, but occurred 8 h later. Maximal stimulation of DNA synthesis by COM was associated with crystal endocytosis by 50% of the cell monolayer. COM crystals also stimulated DNA synthesis and multiplication of canine kidney epithelial cells (MDCK line). As COM stimulated growth of both monkey and canine renal cells but not fibroblasts, the mitogenic effect of this crystal appeared cell-type specific. Hydroxyapatite also enhanced multiplication of BSC-1 cells, whereas brushite, another calcium-containing urinary crystal, did not. In the presence of nephrocalcin (NC), a glycoprotein in normal human urine that inhibits nucleation, aggregation, and growth of COM crystals, the capacity of these crystals to initiate DNA synthesis was blocked. This is the first demonstration that specific calcium-containing urinary crystals can induce proliferation of renal epithelial cells and that NC can inhibit this effect.

Evidence that nephrocalcin and urine inhibit nucleation of calcium oxalate monohydrate crystals

Human urine, and nephrocalcin (NC), a glycoprotein of probable kidney cell origin, greatly reduce consumption of calcium and oxalate from metastably supersaturated solutions seeded with calcium oxalate crystals, a phenomenon usually referred to as inhibition of crystal growth. We seeded metastably supersaturated calcium oxalate solutions with calcium oxalate monohydrate crystals under conditions of ion clamping to maintain constant composition and measured ion consumption from pump delivery rates. Consumption rates increased continuously with time as if the solutions were autocatalytic. After incubation, the seeds were covered with innumerable crystallites, which were also free and numerous in the solution, reflecting self-nucleation. The addition of 20% whole, dialyzed urine, or purified NC reduced ion consumption rates markedly, and the only crystals observed at the end of incubation were the large original seeds. Crystals precoated with concentrated dialyzed urine or NC also showed reduced ion consumption. Urine and NC from patients with nephrolithiasis inhibited nucleation less than normal controls. Self-nucleation seems to be the preferred response in sparsely seeded, ion-clamped, supersaturated solutions, such as exist in the nephron. Urine and NC suppress self-nucleation in vitro by adsorbing to the surface of calcium oxalate crystals.

Inhibitors Within the Nephron

Kidney-derived inhibitors of crystal growth and aggregation prevent supersaturations created by water conservation from expressing themselves in pathological soft tissue calcifications and intranephronal crystallizations. These inhibitors include nephrocalcin (NC), an acidic glycoprotein produced in proximal tubules and thick ascending limbs of Henle's loop, and the Tamm-Horsfall glycoprotein (THP), produced only in the thick ascending limb. NC inhibits growth and aggregation of calcium oxalate monohydrate (COM), the major crystalline component of human renal stones, THP inhibits only COM aggregation. Patients who form COM stones produce abnormal NC molecules that lack gamma-carboxyglutamic acid and fail to inhibit COM crystallizations normally.

Isolation of nephrocalcin from kidney tissue of nine vertebrate species

Nephrocalcin (NC), a urinary calcium oxalate monohydrate (COM) crystal growth inhibitor, was purified from kidneys of nine vertebrate species including humans. All isolates were glycoproteins with high contents of aspartic and glutamic acids and small amounts of basic and aromatic amino acids. Carbohydrate contents ranged from 4 to 16 wt% among the different species, but all contained fucose, mannose, galactose, glucose, galactosamine, glucosamine, and N-acetylneuraminic acid. Although amino acid and carbohydrate compositions were similar, dissociation constants derived from calcium oxalate monohydrate inhibition varied between 10(-7) and 10(-8) M, and highest affinity could be related to highest ability of the kidney of origin to concentrate urine. Using an antibody raised against NC from human kidney tissue culture medium, we found strong immunoreactivity with two species, pigs and sheep. Thus far all vertebrate kidneys possess COM growth-inhibiting material that seems to be of glycoprotein character, and all the glycoproteins isolated so far share similar features, suggesting NC-like proteins are a well-conserved trait.

Evidence that mouse renal proximal tubule cells produce nephrocalcin.

Nephrocalcin (NC) is a glycoprotein inhibitor of calcium oxalate monohydrate crystal growth present in urine and kidney tissue. To determine if kidney cells can produce NC, we have isolated proximal tubules from mouse kidney and cultured them in a serum-free medium containing supplements. The tubules accumulate p-aminohippurate and respond with increase in adenosine 3',5'-cyclic monophosphate to parathyroid hormone but not to arginine vasopressin. They produce 1,25 dihydroxyvitamin D3 when 25 hydroxyvitamin D3 is added as substrate. Medium conditioned for 3 days reacted by direct enzyme-linked immunosorbent assay (ELISA) with a rabbit antiserum to human urinary NC; the same antiserum was localized to the cells using immunoperoxidase staining. Using ion exchange and molecular sieve chromatography, we purified a glycoprotein calcium oxalate crystal-growth inhibitor the chromatographic behavior and amino acid and carbohydrate compositions of which closely resembled that of purified human urinary NC. Its dissociation constant toward the calcium oxalate crystal was 0.4-10 X 10(-7) M. Purified fractions of the protein cross-reacted with the antiserum tested by ELISA. Cultured proximal tubule cells produce a molecule that resembles NC from human urine, rat urine, and rat kidneys; proximal tubule is a probable site of NC production in vivo.

Inhibition of calcium oxalate monohydrate crystal aggregation by urine proteins.

Normal urine inhibits both the growth and the aggregation of calcium oxalate monohydrate (COM) crystals but the molecules that inhibit aggregation are not well defined. We have developed a spectrophotometric assay method to measure the aggregation of COM crystals in vitro under conditions that avoid simultaneous crystal growth. At pH 7.2 and 90 mM NaCl, Tamm-Horsfall glycoprotein (THP) and nephrocalcin (NC), a major urinary inhibitor of COM crystal growth, inhibit COM crystal aggregation at concentrations as low as 2 X 10(-9) and 1 X 10(-8) M, respectively. When increasing NaCl to 270 mM or lowering pH to 5.7, inhibition by both glycoproteins, but more markedly by THP, is decreased. Urinary NC from calcium oxalate renal stone formers (SF NC) and NC isolated from calcium oxalate renal stones (stone NC) both inhibit COM crystal aggregation 10-fold less than NC from normal urine. Citrate is ineffective even at millimolar concentrations. Thus THP and NC are two major inhibitors of COM crystal aggregation in normal urine; SF NC and stone NC are defective aggregation inhibitors.