Identification of a macromolecular crystal growth inhibitor in human urine as osteopontin

Macromolecules occurring in human urine inhibit the growth and/or aggregation of calcium oxalate crystals and may prevent the formation of kidney stones. Attention has focused particularly on proteins, as these seem to be most responsible for the inhibitory activity; three proteins, nephrocalcin, an unidentified protein rich in uronic acid, and uropontin have all been described as possessing such activity. We have recently isolated an unknown inhibitor of calcium oxalate crystal growth that co-eluted with trypsin inhibitor in several separation steps, which suggested its identity. The aim of the present study was to outline a simple procedure for isolating and identifying this inhibitor. Purification was done as follows: precipitation of the major proteins (albumin and uromucoid) with trichloroacetic acid, followed by anion exchange chromatography, hydroxyapatite chromatography, anion exchange chromatography, negative affinity chromatography, and twice reversed phase chromatographies of the supernatant. By this procedure, the inhibitor was identified as being a fragment of osteopontin; urinary trypsin inhibitor and nucleic acids were excluded as being responsible for inhibitory action.     

The influence of multidentate organic phosphonates on the crystal growth of hydroxyapatite

The rate of crystal growth of hydroxyapatite seed crystals in stable supersaturated solutions of calcium phosphate has been studied under reproducible conditions at 25° and at a constant pH of 7.40 in the presence of the organic phosphonates HEDP, 1-hydroxyethylidine 1,1-diphosphonic acid, NTMP, nitrilotri (methylene phosphonic acid); ENTMP, N,N,NN ethylenediamine-tetra (methylene phosphonic acid); TENTMP, triethylenediamine tetra (methylene phosphonic acid). It is suggested that the marked inhibitory influence of the additives upon the rate of crystal growth is due to the formation of strong, substitution inert chelate bonds with the calcium ions present at kinks and dislocations on the crystal surface of HAP. The results of this study show that the potentially hexadentate ligand ENTMP is more effective as a crystal growth inhibitor than the tetradentate NTMP or the tridentate HEDP. The general ineffectiveness of the monophosphonates as crystal growth inhibitors supports the conclusion that the calcium ions are chelated at the surface thereby preventing further deposition of calcium phosphate at that growth site. The relatively low concentration of added phosphonate as compared with the calcium ion concentration rules out calcium chelation in the bulk of the solution as a significant factor in the observed crystal growth inhibition.This investigation was supported in part by grant DE 3223-01 from the National Institute of Health.     

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

Summary 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.     

An electron microscope study of the formation of the nacreous layer in the shell of certain bivalve molluscs

An electron-microscopic study was made of nacreous shell growth in several species of marine molluscs. Studies of sections of mantle-shell preparations show that the first step in crystal formation is the polymerization of part of the pallial fluid to form lamellae parallel to the surface of the epithelium. These lamellae form compartments enclosing a modified apallial fluid. Initiation of crystals occurs in these compartments in contact with a crystal in an adjacent layer. During crystal growth the organic matrix present in the compartment is displaced by the growing surface of the crystal. When growth is complete the crystal is entirely enveloped by a delicate organic sheath. These studies show that the pallial fluid with its organic constituents is responsible for supplying a matrix or substrate for crystal initiation and growth. It serves as a regulatory device for guiding the orderly growth and arrangement of crystals and, further, it may participate in the induction of new crystals. The formation of compartments during shell growth accounts for the uniform thickness, preferred exhibited orientation and mineralogy of the crystals as well as other features exhibited by the mature nacre.This investigation was aided (in part) by Grant DE-01825, N.I.D.R., U.S.P.H Service.     

ISOLATION AND PURIFICATION OF A HIGH MOLECULAR WEIGHT SUBSTANCE FROM HUMAN URINE WHICH INHIBITS CALCIUM OXALATE CRYSTAL GROWTH

A high molecular weight inhibitor of calcium oxalate crystal growth in human urine was investigated. Three inhibitors were isolated by DEAE-Sephacel ion-exchange chromatography and, of these, the substance we named Peak 3 protein seemed to be the main inhibitor in human urine. Peak 3 protein was purified by fast protein liquid chromatography and polyacrylamide gel electrophoresis. This substance, with a molecular weight of 30 kDa, did not contain uronic acid and its inhibitory activity decreased after digestion with proteinase. The difference between Peak 3 protein and several inhibitors previously reported was investigated but no clear difference could be found. The fact that it was the protein structure which was responsible for the inhibitory activity and the fact that Peak 3 protein probably possessed many side-chains which did not contribute to the inhibitory activity influenced the outcome of the investigation.     

The inhibition of calcium oxalate crystal growth by multidentate organic phosphonates

Summary The effect of a number of structurally related multidentate organic phosphonates on the rate of crystal growth of calcium oxalate was studied as a function of pH. Rate constants were obtained at various concentrations for the phosphonates ethane-1-hydroxy-1,1-diphosphonate (EHDP), nitrilotri(methylenephosphonic acid) (NTMP), N,N,N,N-ethylenediaminetetra(methylenephosphonic acid) (ENTNP), and N,N,N,N-hexamethylenediaminetetra(methylenephosphonic acid (HMTMP), at pH 5.00, 6.00, and 7.00. The effect of pH on the inhibitory activity of each of the phosphonates was considerable with effective concentrations of inhibitor decreasing two orders of magnitude, in some cases, as the pH was increased. At a given pH the potentially hexadentate ligands, ENTMP and HMTMP, were generally the most effective inhibitors. The results suggested that EHDP, at currently administered doses, provides only a moderate increase in the capacity of human urine to inhibit calcium oxalate crystal growth.This investigation was supported in part by Research Grant AM-17717 from the National Institutes of Health, Public Health Service.     

Possible role of Tamm-Horsfall glycoprotein in calcium oxalate crystallisation

The role of Tamm-Horsfall glycoprotein (THGP) in the crystallisation of calcium oxalate was investigated. The results showed THGP to have a weak inhibitory effect on crystal growth. In contrast, urinary macromolecules showed a strong inhibitory effect. THGP should, therefore, not be considered the main component of urinary macromolecules which have a strong inhibitory effect on crystal growth. However, THGP did enhance crystal production in the early phase of crystallisation. It appears that THGP should be viewed as a promoter rather than an inhibitor of calcium oxalate crystallisation at physiological concentrations.     

Pentosan polysulfate as an inhibitor of calcium oxalate crystal growth

Pentosan polysulfate was studied as a calcium oxalate crystal growth inhibitor. The inhibition from pentosan polysulfate at concentrations ranging from 3.5 to 110 mg./l. was measured in simple inorganic calcium oxalate solutions at pH 5, 6 and 7. Pentosan polysulfate also was mixed with urine at concentrations from 10 to 350 mg./l. and inhibition determined. Measurement of calcium oxalate crystal growth inhibition was performed in a seeded crystal growth system. One inhibitory unit (concentration of pentosan polysulfate necessary to give a 50 per cent reduction in the rate of crystal growth) was 5.7 +/- 2 mg./l. at pH 5, 7.2 +/- 1.1 mg./l. at pH 6 and 6.0 +/- 2.1 mg./l. at pH 7. Urine-pentosan polysulfate mixtures showed more inhibitory activity than the predicted inhibition present. Addition of pentosan polysulfate to urine at a concentration of 10 mg./l. increased the inhibitory activity from 45 +/- 3 to 59 +/- 3 IU/l. Pentosan polysulfate is a potent calcium oxalate crystal growth inhibitor in urine at physiologic pH levels. If the urinary concentration of pentosan polysulfate after oral administration reaches 10 mg./l., the increase in inhibitory activity in urine that may occur might be important in the treatment of patients who form calcium oxalate calculi within the urinary tract.     

Measurement of the risk of calcium oxalate crystallization in urine

Summary The risk of calcium crystallization (CaOx-CR) in urine was analyzed by means of crystal counting following standardized addition of oxalate. CaOx-CR was determined in 24h urine samples from 21 stone formers and 26 normal subjects following dilution of urine to a creatinine concentration of 5 mol per ml. The mean (±SD) CaOx-CR was in stone formers 1.42±0.57 and in normal subjects 1.29±0.40. CaOx-CR was also analyzed in 16 fresh urine samples diluted to 80 per cent of the original concentration whereby values between 0.36 and 3.6 were recorded. There was a good correlation between CaOx-CR and estimates of the ion-activity product of CaOx, both in urine diluted to 5 mol of creatinine per ml and in 80 per cent diluted urine. It ist suggested that the method described is of value for evalution and follow up of patients with CaOx urolithiasis.     

The effect of uraemic “middle-sized molecules” on T lymphocyte functions

The activity of the leucocyte inhibitory factor (LIF) stimulated with Concanavalin A was examined in chronic uraemic, haemodialysed patients. It was found that the LIF activity decreased in chronic uraemic patients as compared with the normal controls.The absolute T lymphocyte count and active E rosette formation also decreased. Inin vitro experiments the authors have also examined the inhibitory effect of the socalled middle-sized molecule (MSM) on uraemic materials separated from the serum and the haemofiltrate. They observed that the materials with a molecular weight of 1000–1500 isolated both from the serum and the haemofiltrate significantly inhibited the Con A stimulated LIF production of normal lymphocytes and decreased the ratio of the active E rosette-forming T lymphocytes.On the basis of their exeeriments the MSM uraemic materials are considered responsible for the decreased immune reactivity of uraemic patients. These uraemic toxins can be dialysed.     

Nitric oxide synthase and nitric oxide-mediated effects in lower urinary tract smooth muscles

Summary In the lower urinary tract smooth muscles, both excitatory and inhibitory non-adrenergic, non-cholinergic (NANC) nerves and neurotransmission can be demonstrated. An inhibitory, relaxation-mediating system may serve not only the detrusor, the trigone, and the bladder neck/urethra, but may also be of importance for their integrated function. Available data suggest that nitric oxide synthase (NOS) is localized in nerve fibres of the lower urinary tract, preferably in the outflow region, and evidence has accumulated that l-arginine-derived nitric oxide (NO) is responsible for the main part of the inhibitory NANC response. Coinciding localization of NOS positive nerves with nerves expressing acetylcholine esterase, vasoactive intestinal peptide, and neuropeptide Y, suggests that NO may have a role both as a directly acting transmitter and as a modulator of efferent neurotransmission. In addition, NO may be involved in afferent neurotransmission. Theoretically, NO released from nerves in the detrusor, could be one factor keeping the bladder relaxed during filling. However, the detrusor has a low sensitivity to NO and agents acting via cyclic GMP, which makes it less likely that NO has a role as a relaxant neurotransmitter. This does not exclude that NO may modulate the effects of other transmitters, or that it has an afferent function. NO effectively relaxes isolated smooth muscle preparations from the outflow region, suggesting that it may be involved in the decrease in intraurethral pressure associated with normal micturition, and with the excessive urethral pressure variations (unstable urethra), which may be associated with certain voiding disturbances in women. The l-arginine/NO system may also control afferent activity in the outlet region, where lack of NO may lower the threshold for afferent firing leading to bladder instability. Another possible site where the l-arginine/NO pathway can have a functional role is in the urethral lamina propria. Here, NO-mediated relaxation may influence the inner urethral softness, and thereby the sealing function of the urethral mucosa. However, it should be stressed that the functional importance of the l-arginine/NO system in the central and peripheral pathways controlling micturition remains to be established.     

Effect of curcumin (<Emphasis Type="Italic">Curcuma longa</Emphasis> extract) on LPS-induced acute lung injury is mediated by the activation of AMPK

Purpose

Curcumin, a biphenolic compound extracted from turmeric (Curcuma longa), possesses potent anti-inflammatory activity. The present study investigated whether curcumin could increase 5′ adenosine monophosphate-activated protein kinase (AMPK) activity in macrophages and modulate the severity of lipopolysaccharide (LPS)-induced acute lung injury.

Methods

Macrophages were treated with curcumin and then exposed (or not) to LPS. Acute lung injury was induced by intratracheal administration of LPS in BALB/c mice.

Results

Curcumin increased phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), a downstream target of AMPK, in a time- and concentration-dependent manner. Curcumin did not increase phosphorylation of liver kinase B1, a primary kinase upstream of AMPK. STO-609, an inhibitor of calcium²⁺/calmodulin-dependent protein kinase kinase, diminished curcumin-induced AMPK phosphorylation, but transforming growth factor-beta-activated kinase 1 inhibitor did not. Curcumin also diminished the LPS-induced increase in phosphorylation of inhibitory κB-alpha and the production of tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein (MIP)-2, and interleukin (IL)-6 by macrophages. Systemic administration of curcumin significantly decreased the production of TNF-α, MIP-2, and IL-6 as well as neutrophil accumulation in bronchoalveolar lavage fluid, and also decreased pulmonary myeloperoxidase levels and the wet/dry weight ratio in mice subjected to LPS treatment.

Conclusion

These results suggest that the protective effect of curcumin on LPS-induced acute lung injury is associated with AMPK activation.

     

Assessment of urine specific gravity by reagent strip test in newborn infants

Specific gravity was measured with a strip test (N-Labstix SG, Ames Division, Miles, Puteaux, France) in 98 urine specimens obtained from 57 newborn infants; osmolality was measured with an osmometer. The strip test did not accurately predict urine osmolalities; a very weak correlation was found between the specific gravity and the osmolality (r ²=0.598,P<0.01). Specific gravity values up to 1.015 always indicated hypotonic urine with osmolality less than 211 mosmol/kg H₂O, whereas higher values could be associated with either hypotonic or hypertonic urine. Therefore, the strip test cannot be recommended in the neonatal period because its clinical usefulness is strictly restricted to urine samples with low specific gravities (1.015) and without confounding variables (urine pH6.5, glucosuria, proteinuria, haematuria).     

High urinary excretion level of citrate and magnesium in children: potential etiology for the reduced incidence of pediatric urolithiasis

It is well known that the incidence of calcium oxalate (CaOX) urolithiasis is much lower in children than in adults [2, 21]. One purpose of this study was to compare the inhibitory activity on CaOX crystal growth and nucleation of urine from children (ufC) with that of urine from adults (ufA). Another was to measure low molecular weight urinary substances related to CaOX lithiasis, including citrate and magnesium, which have been identified as stone inhibitors. The excretion volume per body weight of uric acid, phosphorus, magnesium and citrate was all significantly higher in 24-h ufC than in 24-h ufA, but that of calcium and oxalate was not. The growth inhibitory activities against CaOX crystals of ufC and ufA were measured in a whole urine system. The diameter of the crystals produced in this system was smaller for ufC (3.68 μm) than for ufA (4.66 μm). We also examined the metastable limit for CaOX with fresh spot urine, which was 3.15 mmol/l in ufC and 0.41 mmol/l in ufA. These results indicate that ufC has a more powerful inhibitory effect on CaOX crystal growth and nucleation than ufA. We also found that the excretion rate of citrate and magnesium in ufC was much higher than in ufA. We suggest that these two stone inhibitors are very likely to elevate the inhibitory activity of ufC against CaOX crystal growth and nucleation. The lower incidence of CaOX lithiasis in children might thus be partly attributed to citrate and magnesium. Received: 30 July 1997 / Accepted: 27 November 1997     

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

Summary 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.     

The anti-proliferative effect of proliferating cell nuclear antigen-specific antisense oligonucleotides on human gastric cancer cell lines

The proliferating cell nuclear antigen (PCNA) is a nuclear protein that leads DNA synthesis by the DNA polymerase delta. As the PCNA gene is strongly expressed in invasive gastric cancer cells with high proliferative activity, PCNA is suspected of playing an important role in the proliferation and advancement of gastric cancer. Thus, the effects of antisense oligonucleotides specific for PCNA mRNA were examined in seven gastric cancer cell lines. It was found that treatment with antisense oligonucleotides at concentrations of 10–40 M dose-dependently inhibited the growth of all cell lines; however, random sequence oligonucleotides did not modify the proliferation of any type of cells. These results indicate that PCNA is essential for cell proliferation in gastric cancer cells, and that the growth inhibitory effect results from the inhibition of PCNA gene expression. Therefore, PCNA-specific antisense oligonucleotides may be effective in the treatment of gastric cancer.     

Glycosylation of prothrombin fragment 1 governs calcium oxalate crystal nucleation and aggregation,but not crystal growth

Urinary glycoproteins play an important role in the modulation of calcium oxalate crystallisation. In several cases, this has been attributed to glycosylation of the proteins as evidenced by urinary prothrombin fragment 1 where there is a correlation between sialylation and calcium oxalate kidney stone disease. In the present study, plasma-derived prothrombin fragment 1 (PTF1) was enzymatically modified in order to generate its asialo and aglyco forms. The parent glycoprotein and its two glycoforms were used in calcium oxalate crystallisation studies to assess the role of the carbohydrate moeity in PTF1’s potent inhibitory activity. The glycans inhibited crystal aggregation and promoted crystal nucleation, but had no effect on crystal growth. The terminal sialic acid residues had a small effect on inhibition of crystal aggregation whereas they contributed significantly to promotion of nucleation. These results indicate that glycosylation of PTF1 governs calcium oxalate crystal nucleation and aggregation but it does not affect the protein’s role in inhibiting crystal growth. Since promotion of nucleation and inhibition of aggregation are both regarded as protective mechanisms against calcium oxalate urinary stone formation, the kringle domain on which the glycans are located is implicated in PTF1’s inhibitory activity. It is speculated that modifications in the glycosylation of urinary PTF1 in stone-formers may regulate its capacity to protect against calcium urolithiasis.     

Influence of chondroitin sulfate, heparin sulfate, and citrate on Proteus mirabilis-induced struvite crystallization in vitro

Struvite crystals were produced by Proteus mirabilis growth in artificial urine, in the presence of a number of naturally occurring crystallization inhibitors. The use of phase contrast light microscopy enabled the effects of added chondroitin sulfate A, chondroitin sulfate C, heparin sulfate, or sodium citrate, on struvite crystal growth rates to be rapidly monitored as changes in crystal habit. Struvite crystals formed as a consequence of the urease activity of P. mirabilis under all chemical conditions. In the absence of inhibitor, early crystal development was marked by large quantities of amorphous precipitate, followed immediately by the appearance of rapidly growing X-shaped or planar crystals. Addition of the glycosaminoglycans, chondroitin sulfate A, chondroitin sulfate C, or heparin sulfate to the artificial urine mixture had no effect on the rate of crystal growth or appearance. When sodium citrate was present in elevated concentrations, crystal appearance was generally slowed, and the crystals assumed an octahedral, slow growing appearance. None of the added compounds had any influence on bacterial viability, pH, or urease activity. It is therefore likely that the inhibitory activity displayed by sodium citrate might be related to its ability to complex magnesium or to interfere with the crystal structure during struvite formation. From these experiments it would appear that citrate may be a factor in the natural resistance of whole urine to struvite crystallization.     

The direct measurement of inhibitory capacity to crystal growth of calcium oxalate in undiluted urine and in other inhibitor containing solutions

Summary This paper describes a simple method to measure the capacity of undiluted urine and of other inhibitor containing solutions (PPi and EHDP) to protect a given mass of calcium oxalate crystals from growth. The method has also been used to determine relative urinary saturation with respect to calcium oxalate. It is based on titration with oxalate and measures the critical changes of concentration necessary to reach saturation or to induce crystal growth. From these changes inhibitory capacity as well as the level of urinary saturation can be calculated in terms of differences of concentration products. The use of the methods at present available to measure urinary oxalate are thereby avoided. In order to compare the results from different urines without the need for cumbersome calculations of activity products we have introduced a saturation-inhibition ratio.     

Proteoglycan core protein in human urine and its possible role on calcium oxalate urolithiasis

BACKGROUND: There are only a few papers reporting on the role of proteoglycan core protein in calcium oxalate stone formation. The present study was carried out to investigate the role of core protein of proteoglycan in human urine on calcium oxalate (CaOx) crystallization. METHODS: Proteoglycans were collected from whole human urine. The covalently bound glycosaminoglycans (GAG) of proteoglycans were then digested by GAG lyase. The inhibitory activity on CaOx crystal growth in vitro was measured before and after enzyme digestion of proteoglycans. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the core protein of proteoglycans and the analysis of amino acid sequence were performed. RESULTS: The core protein showed significant inhibitory activity on CaOx crystal growth, which scarcely changed when compared with that of proteoglycans before enzyme digestion. The SDS-PAGE revealed that the core protein was a single unit with a molecular weight of 26 kDa and amino acid sequencing demonstrated high homology to interalpha-trypsin inhibitor (ITI) light chain (bikunin) with Kunitz inhibitor domain as a core protein. CONCLUSIONS: The results suggested that human urine contains proteoglycans and a major part of them is ITI light chain (bikunin). The Kunitz inhibitor domain, a core protein of bikunin, has significant inhibitory activity on CaOx crystallization without GAG bound covalently to the core protein.