A method for studying inhibitory activity in whole urine

Summary A method has been developed for inducing and quantifying calcium oxalate crystallisation in whole human urine. The propensity of a given urine to induce crystal formation was described in two ways: 1) its ability to resist spontaneous nucleation of calcium oxalate crystals was assessed by titrating 20 mls of the urine with increasing quantities of sodium oxalate (0–150 mol) to determine its practical metastable limit. This limit was inversely related to the endogenous calcium concentration. 2) its capacity to inhibit crystal growth was quantified by determining the rate of growth of calcium oxalate crystals precipitated in response to a fixed oxalate load (30 mol) above its metastable limit. The crystals produced were predominantly calcium oxalate dihydrate and were morphologically identical to those occurring naturally in urine. Citrate had no effect on the metastable limits of 3 urines examined, but markedly inhibited crystal growth. Pyrophosphate had a similar effect on crystal growth, and in addition, raised the metastable limit of one of the urine samples.     

Crystalluria in marathon runners

Summary Epidemiological evidence suggests that marathon runners have a higher incidence of renal stone formation than occurs in the general population. Since crystalluria and stone disease are thought to be related, we subjected urine samples from a group of marathon runners to particle counting and sizing in a Coulter Counter equipped with a population accessory unit. The volume-size distribution curves so obtained were bimodal with one peak occurring in the 2–5 m diameter range and a second in the 15–32 m diameter range—a pattern that is remarkably similar to the distributions reported for recurrent idiopathic stone formers and distinctly different to those recorded for control subjects. Analyses by scanning electron microscopy and X-ray powder diffraction revealed other features which are regarded as typical of stone formers' crystalluria. These physicochemical data indicate that marathon runners may be at increased risk of urinary stone formation.     

Vitamin C—Sources,Physiological Role,Kinetics, Deficiency,Use, Toxicity,and Determination

Vitamin C (L-ascorbic acid) has been known as an antioxidant for most people. However, its physiological role is much larger and encompasses very different processes ranging from facilitation of iron absorption through involvement in hormones and carnitine synthesis for important roles in epigenetic processes. Contrarily, high doses act as a pro-oxidant than an anti-oxidant. This may also be the reason why plasma levels are meticulously regulated on the level of absorption and excretion in the kidney. Interestingly, most cells contain vitamin C in millimolar concentrations, which is much higher than its plasma concentrations, and compared to other vitamins. The role of vitamin C is well demonstrated by miscellaneous symptoms of its absence—scurvy. The only clinically well-documented indication for vitamin C is scurvy. The effects of vitamin C administration on cancer, cardiovascular diseases, and infections are rather minor or even debatable in the general population. Vitamin C is relatively safe, but caution should be given to the administration of high doses, which can cause overt side effects in some susceptible patients (e.g., oxalate renal stones). Lastly, analytical methods for its determination with advantages and pitfalls are also discussed in this review.     

草酸钙的形成机理及影响因素

目的 :探讨草酸钙结石的形成机理和影响因素。方法 :根据微溶电解质动力学原理 ,利用草酸与氯化钙作用形成草酸钙的性质 ,加入其它尿液中常见组分 ,测定草酸钙诱导期与过饱和溶液浓度的关系 ,并分析结石的显微结构。结果 :草酸钙的诱导期与溶液过饱和浓度呈反比关系 ,且不同干扰因素对草酸钙的过饱和比影响也不同。结论 :钙和草酸是草酸钙形成的必要条件 ;溶液的过饱和是草酸钙成核的基础 ;基质对草酸钙起胶结作用 ;氯化钠、氯化铵、氯化镁能促进草酸钙的形成 ,而氯化铁、尿素、甘氨酸能抑制草酸钙的沉淀。     

An unidentified macromolecular inhibitory constituent of calcium oxalate crystal growth in human urine

Summary We have detected and isolated a macromolecular constituent in normal human urine possessing calcium crystal growth inhibitory activity. The purification procedure consisted of two anion exchange chromatographies and one affinity chromatography. The crystal growth inhibitor was found to be heterogeneous in net charge as well as in size. It has not been identified. It is not an uronic acid-containing glycosaminoglycan, hitherto presumed to be responsible for the inhibitory activity. Whether an urinary fragment of inter--trypsin inhibitor is responsible has yet to be resolved.     

OXGR1 is a candidate disease gene for human calcium oxalate nephrolithiasis

PurposeNephrolithiasis (NL) affects 1 in 11 individuals worldwide, leading to significant patient morbidity. NL is associated with nephrocalcinosis (NC), a risk factor for chronic kidney disease. Causative genetic variants are detected in 11% to 28% of NL and/or NC, suggesting that additional NL/NC-associated genetic loci await discovery. Therefore, we employed genomic approaches to discover novel genetic forms of NL/NC.MethodsExome sequencing and directed sequencing of the OXGR1 locus were performed in a worldwide NL/NC cohort. Putatively deleterious, rare OXGR1 variants were functionally characterized.ResultsExome sequencing revealed a heterozygous OXGR1 missense variant (c.371T>G, p.L124R) cosegregating with calcium oxalate NL and/or NC disease in an autosomal dominant inheritance pattern within a multigenerational family with 5 affected individuals. OXGR1 encodes 2-oxoglutarate (α-ketoglutarate [AKG]) receptor 1 in the distal nephron. In response to its ligand AKG, OXGR1 stimulates the chloride-bicarbonate exchanger, pendrin, which also regulates transepithelial calcium transport in cortical connecting tubules. Strong amino acid conservation in orthologs and paralogs, severe in silico prediction scores, and extreme rarity in exome population databases suggested that the variant was deleterious. Interrogation of the OXGR1 locus in 1107 additional NL/NC families identified 5 additional deleterious dominant variants in 5 families with calcium oxalate NL/NC. Rare, potentially deleterious OXGR1 variants were enriched in patients with NL/NC compared with Exome Aggregation Consortium controls (χ² = 7.117, P = .0076). Wild-type OXGR1-expressing Xenopus oocytes exhibited AKG-responsive Ca²⁺ uptake. Of 5 NL/NC-associated missense variants, 5 revealed impaired AKG-dependent Ca²⁺ uptake, demonstrating loss of function.ConclusionRare, dominant loss-of-function OXGR1 variants are associated with recurrent calcium oxalate NL/NC disease.     

Characterization of uronic-acid-rich inhibitor of calcium oxalate crystallization isolated from rat urine

Human urine contains several macromolecules which inhibit calcium oxalate crystallization. Uronic-acid-rich protein (UAP), a glycoprotein with a molecular weight of approximately 35 kDa, is one such inhibitor. Here we report the characterization of UAP extracted from rat urine using three chromatographic steps including diethylaminoethanol (DEAE)-Sephacel, Sephacryl S-300 and Mono Q column and compare it with human UAP. The molecular weight of rat UAP (UAP_r) is similar to that of human UAP (UAP_h), being approximately 35 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Their amino acid compositions are identical, they contain a high percentage of aspartic and glutamic acids and they react positively in the carbazole reaction, suggesting that they contain uronic acid. The inhibitory activities of UAP_h and UAP_r were assayed on a calcium oxalate crystallization system in vitro using [⁴⁵Ca]calcium chloride. Both exert a strong inhibition, suggesting that UAP_r, like UAP_h, plays an important role in preventing and reducing calcium oxalate crystallization in the urine. On Western blot analysis, both UAP_h and UAP_r immunoreact with inter--trypsin inhibitor (ITI) antibody. Nevertheless, using the Ouchterlony immunodiffusion technique, there was no precipitation line between ITI antibody and UAP. Therefore, we hypothesize that UAP is related to ITI and that they may have the same epitope but are not completely identical. We conclude that UAP belongs to the ITI superfamily of macromolecules which contribute to the regulation of the calcium oxalate crystallization process.     

Crystallization in the nephron

Recent experimental studies on the crystallization of calcium salts at different nephron levels support the theory that the initial formation of calcium concrements starts with an intratubular crystallization of calcium phosphate (CaP) and calcium oxalate (CaOx). CaP seems to be the initial crystallization product in pure CaP and mixed calcium phosphate–calcium oxalate (CaPCaOx) concrements, with the formation of CaP crystals at a nephron level above the collecting duct. Urinary macromolecules and cellular degradation products most probably promote this process. During the passage through the collecting duct, CaP might partly or completely dissolve at the lower pH encountered there. This might result in an increased concentration of calcium and hence an increased supersaturation with CaOx, which in turn can bring about a heterogeneous nucleation of CaOx on or around preformed CaP crystals or crystal aggregates. The final result will be mixed CaOxCaP or pure CaOx concrements. Pure CaOx concrements might also be the result of an initial CaOx crystallization at nephron levels above or in the collecting duct under conditions with a high urinary excretion of oxalate. Whether intratubular crystallization of calcium salts results in the formation of small harmless crystals excreted with urine or calcium stones appears to be determined by a complex process, involving kinetic factors that influence crystal growth and crystal aggregation and crystal retention. Received: 24 December 1998 / Accepted: 11 March 1999     

Inhibitory effect of bikunin on calcium oxalate crystallization in vitro and urinary bikunin decrease in renal stone formers

Two proteins of 17 and 24 kDa, respectively, which were immunologically related to bikunin, were purified from urine of healthy men, using in the last step a trypsin CNBr-sepharose affinity column. These proteins strongly inhibited calcium oxalate (CaOx) crystallization in two in vitro models. In the first model, the presence of 8 μg/ml protein in a medium containing 0.76 mM CaCl₂ (with ⁴⁵Ca) and 0.76 mM ammonium oxalate inhibited the crystallization process by 80%, as estimated by supernatant radioactivity after 60 min of incubation. A similar inhibition was observed in the second turbidimetric model, where the CaOx crystallization kinetics were followed for 10 min at 620 nm in a medium containing 4 mM CaCl₂ and 0.5 mM Na₂Ox. These proteins were used as standard protein for the development of an enzyme-linked immunosorbent assay (ELISA) in urine. Mean (± SEM) urinary bikunin concentration in 18 healthy subjects was 5.01 ± 0.91 μg/ml. This was a concentration range of strong inhibitory activity in vitro. Bikunin values were nearly 50% lower (2.54 ± 0.42 μg/ml, P=0.007) in 31 CaOx renal stone formers (having weddelite crystals in their first morning urine) than in the healthy volunteers. A correlation was found between urinary bikunin and alpha-1 microglobulin concentrations in the control group (y=0.73x + 1.09, r ²=0.8) while no such correlation existed in the lithiasis group. In conclusion, bikunin exerts a strong inhibitory action of CaOx crystallization in vitro. Its involvement in urinary CaOx crystallization of stone formers is highly probable, based on the significant decrease in its urinary concentration in the majority of stone formers studied. Received: 16 December 1997 / Accepted: 23 June 1998     

Role of inter-α-inhibitor and its related proteins in urolithiasis. Purification of an inter-α-inhibitor related protein from the bovine kidney

Urine contains several macromolecules that inhibit calcium oxalate (CaOx) crystallization. Among them is bikunin, the light chain of most of the inter-α-inhibitor (IαI) family of glycoproteins. This study aimed to verify whether bikunin and other members of the IαI family are synthesized in the kidneys or derived exclusively from the plasma. Proteins extracted from homogenized bovine kidney were applied successively to three chromatographic steps on DEAE-Sephacel, Sephacryl S-300, and Mono Q column. The inhibitory activity was assayed using a CaOx crystallization system. The presence of IαI-related proteins was determined by␣electrophoresis and Western blotting. The results showed that kidney extract contained a 125-kDa protein that cross-reacted with anti-IαI antibodies. This protein inhibited CaOx crystallization efficiently. According to its molecular weight and immunoreaction with anti-IαI antibody, the 125-kDa protein could be pre-α-inhibitor. The latter is known to encompass a heavy chain and bikunin, which may explain its inhibitory activity against CaOx crystallization. Consequently, we hypothesize that kidneys may produce some IαI-related proteins that are involved in the inhibition of stone formation. Received: 18 February 1998 / Accepted: 9 July 1998