A mutation creating an out-of-frame alternative translation initiation site in the GRHPR 5'UTR causing primary hyperoxaluria type II.

Primary hyperoxaluria type II is a recessive genetic disorder caused by mutations in the GRHPR gene. Although several dozen mutations have been described, all affect coding or transcript splicing. A man suspected of having primary hyperoxaluria type II was heterozygous for a novel single-nucleotide deletion (c.694delC) in GRHPR affecting Gln(232) , which introduced a pre-mature termination (p.Gln232Argfs*3). Two 5'untranslated region (UTR) variants of unknown significance were also noted. We show that these two variants occur in cis, on the opposite allele, and introduce - immediately upstream of the canonical translation initiation site - a novel out-of-frame translational start site. In vitro studies using the GRHPR 5'UTR fused to a luciferase reporter show that the variant start site pre-empted initiation at the canonical translational start site, and this was corroborated within the broader context of 1.3 kb of the GRHPR proximal promoter. This latter mechanism may be underappreciated in general; reports of clinically significant functional variation of this type are extremely rare.

Metabolism of fructose to oxalate and glycolate.

Much attention has been recently directed at fructose consumption because of its association with obesity and subsequent development of chronic diseases. It was recently reported that an increased fructose intake increases the risk of forming kidney stones. It was postulated that fructose consumption may increase urinary oxalate, a risk factor for calcium oxalate kidney stone disease. However, conflicting results have been obtained in human studies examining the relationship between fructose metabolism and oxalate synthesis. To test whether fructose intake influences urinary excretions impacting kidney stone risk, healthy subjects consumed diets controlled in their contents of fructose, oxalate, calcium, and other nutrients. Subjects consumed diets containing 4, 13, and 21% of calories as fructose in a randomized order. No changes in the excretions of oxalate, calcium, and uric acid were observed. In vitro investigations with cultured liver cells incubated with (13)C-labeled sugars indicated that neither fructose nor glucose was converted to oxalate by these cells. Fructose metabolism accounted for 12.4 ± 1.6% of the glycolate detected in the culture medium and glucose 6.4 ± 0.9%. Our results suggest that mechanisms for stone risk associated with fructose intake may lie in factors other than those affecting the major stone risk parameters in urine.

Hydroxyproline ingestion and urinary oxalate and glycolate excretion.

Endogenous synthesis of oxalate is an important contributor to calcium oxalate stone formation and renal impairment associated with primary hyperoxaluria. Although the principal precursor of oxalate is believed to be glyoxylate, pathways in humans resulting in glyoxylate synthesis are not well defined. Hydroxyproline, a component amino acid of collagen, is a potential glyoxylate precursor. We have investigated the contribution of dietary hydroxyproline derived from gelatin to urinary oxalate and glycolate excretion. Responses to the ingestion of 30 g of gelatin or whey protein were compared on controlled oxalate diets. The time course of metabolism of a 10 g gelatin load was determined as well as the response to varying gelatin loads. Urinary glycolate excretion was 5.3-fold higher on the gelatin diet compared to the whey diet and urinary oxalate excretion was 43% higher. Significant changes in plasma hydroxyproline and urinary oxalate and glycolate were observed with 5 and 10 g gelatin loads, but not 1 and 2 g loads. Extrapolation of these results to daily anticipated collagen turnover and hydroxyproline intake suggests that hydroxyproline metabolism contributes 20-50% of glycolate excreted in urine and 5-20% of urinary oxalate derived from endogenous synthesis. Our results also revealed that the kidney absorbs significant quantities of hydroxyproline and glycolate, and their metabolism to oxalate in this tissue warrants further consideration.

Contribution of dietary oxalate to urinary oxalate excretion.

BACKGROUND: The amount of oxalate excreted in urine has a significant impact on calcium oxalate supersaturation and stone formation. Dietary oxalate is believed to make only a minor (10 to 20%) contribution to the amount of oxalate excreted in urine, but the validity of the experimental observations that support this conclusion can be questioned. An understanding of the actual contribution of dietary oxalate to urinary oxalate excretion is important, as it is potentially modifiable. METHODS: We varied the amount of dietary oxalate consumed by a group of adult individuals using formula diets and controlled, solid-food diets with a known oxalate content, determined by a recently developed analytical procedure. Controlled solid-food diets were consumed containing 10, 50, and 250 mg of oxalate/2500 kcal, as well as formula diets containing 0 and 180 mg oxalate/2500 kcal. Changes in the content of oxalate and other ions were assessed in 24-hour urine collections. RESULTS: Urinary oxalate excretion increased as dietary oxalate intake increased. With oxalate-containing diets, the mean contribution of dietary oxalate to urinary oxalate excretion ranged from 24.4 +/- 15.5% on the 10 mg/2500 kcal/day diet to 41.5 +/- 9.1% on the 250 mg/2500 kcal/day diet, much higher than previously estimated. When the calcium content of a diet containing 250 mg of oxalate was reduced from 1002 mg to 391 mg, urinary oxalate excretion increased by a mean of 28.2 +/- 4.8%, and the mean dietary contribution increased to 52.6 +/- 8.6%. CONCLUSIONS: These results suggest that dietary oxalate makes a much greater contribution to urinary oxalate excretion than previously recognized, that dietary calcium influences the bioavailability of ingested oxalate, and that the absorption of dietary oxalate may be an important factor in calcium oxalate stone formation.

(L)-2-oxothiazolidine-4-carboxylate in the treatment of primary hyperoxaluria type 1.

OBJECTIVE: To evaluate the short-term efficacy of (l)-2-oxothiaolidine-4-carboxylate (OTZ, which reduces urinary oxalate excretion in normal subjects) in the treatment of primary hyperoxaluria type 1 (PH1) in a phase II study. PATIENTS AND METHODS: Two patients with PH1 received intravenous infusions of OTZ (100 mg/kg body weight for 2 h) given every 8 h for four doses. One patient also received a placebo treatment. Urine samples (24-h collections) were obtained before and during OTZ treatment and assayed for oxalate, citrate, creatinine, sulphate and pH. Daily blood samples were assayed for plasma oxalate and serum creatinine. RESULTS: Urinary oxalate excretion was unaffected by OTZ treatment. Plasma oxalate declined in both individuals with OTZ treatment, but the effect was small. Plasma cysteine was normal in one patient, rising from a mean (sd) of 36 (3.7) micromol/L before treatment to a peak of 141 micromol/L after OTZ, but was not detected in samples from the other patient. The ratio of oxalate to creatinine clearances was high in both patients, with mean values of 3.1 and 3.8. CONCLUSIONS: Treatment with OTZ did not lead to clinically significant changes in urinary oxalate excretion. The high clearance of oxalate in these patients suggests a substantial renal secretion of oxalate.

Severe oligohydramnios induced by cyclooxygenase-2 inhibitor nimesulide.

BACKGROUND: Cyclooxygenase-2 inhibitors might have fewer adverse fetal effects than conventional nonsteroidal anti-inflammatory drugs that inhibit both isoforms of the enzyme. Although cyclooxygenase-2 is expressed in fetal kidneys, there are no reports of adverse effects in human pregnancy. CASE: A 27-year-old woman, gravida 2, para 0, with a twin pregnancy at 24 weeks' gestation had placement of a cervical cerclage. Nimesulide was prescribed for postoperative preterm labor prophylaxis. Three weeks later, severe oligohydramnios was identified in both sacs, despite normal growth, renal anatomy, and umbilical artery and renal artery Doppler flow velocimetry. After stopping the drug, amniotic fluid volumes returned to normal over 2 weeks. There were no adverse neonatal renal effects. CONCLUSION: Selective cyclooxygenase-2 inhibition might cause severe oligohydramnios. If it is used, we advise close fetal surveillance.

Identification of missense, nonsense, and deletion mutations in the GRHPR gene in patients with primary hyperoxaluria type II (PH2).

Primary hyperoxaluria type II (PH2) is a rare disease characterized by the absence of an enzyme with glyoxylate reductase, hydroxypyruvate reductase, and D-glycerate dehydrogenase activities. The gene encoding this enzyme (GRHPR) has been characterized, and a single mutation has been detected in four PH2 patients. In this report, we have identified five novel mutations. One nonsense mutation (C295T) results in a premature stop codon at codon 99. A 4-bp deletion mutation has been found in the 5' consensus splice site of intron D, resulting in a predicted splicing error. Three missense mutations have been detected, including a missense transversion (T965G) in exon 9 (Met322Arg), a missense transition (G494A) in the putative co-factor binding site in exon 6 (Gly165Asp), and a substitution of an adenosine for a guanine in the 3' splice site of intron G. The functional consequences of the missense transversion and transition mutations have been investigated by transfection of cDNA encoding the mutated protein into COS cells. Cells transfected with either mutant construct have no enzymatic activity, a finding that is not significantly different from the control (empty) vector (P<0.05). These results further confirm that mutations in the GRHPR gene form the genetic basis of PH2. Ten of the 11 patients that we have genotyped are homozygous for one of the six mutations identified to date. Because of this high proportion of homozygotes, we have used microsatellite markers in close linkage with the GRHPR gene to investigate the possibility that the patients are the offspring of related individuals. Our data suggest that two thirds of our patients are the offspring of either closely or distantly related persons. Furthermore, genotyping has revealed the possible presence of a founder effect for the two most common mutations and the location of the gene near the marker D9S1874.

Role of diet in the therapy of urolithiasis.

The data reviewed in this paper indicate that there is compelling direct and indirect evidence that certain dietary modifications can limit the risk for stone formation. Fluid therapy should be a front-line approach for all stone formers, because it is safe, cheap, and effective. Restricting sodium and animal-protein consumption produces changes in the urinary environment that should benefit the majority of stone formers, including a decrease in calcium and increase in citrate excretion. Minimizing the intake of processed goods limits sodium gluttony. These dietary modifications also reduce cardiovascular risks. Indiscriminant calcium restriction should be avoided, because it could accelerate stone formation and violate skeletal integrity. Oxalate restriction should be considered for calcium oxalate stone formers, especially those with hyperoxaluria. Specific recommendations for modifying the consumption of other nutrients cannot be made at this time because of the limited available information about the resultant effects. The aforementioned goals can be achieved within the context of a nutritionally balanced diet providing adequate sources of fruits and vegetables. There is a definite need for better designed studies of the nutritional effects on stone disease. This would promote a better understanding of the interplay between the genetic and environmental components of this disorder.

Estimation of the oxalate content of foods and daily oxalate intake.

BACKGROUND: The amount of oxalate ingested may be an important risk factor in the development of idiopathic calcium oxalate nephrolithiasis. Reliable food tables listing the oxalate content of foods are currently not available. The aim of this research was to develop an accurate and reliable method to measure the food content of oxalate. METHODS: Capillary electrophoresis (CE) and ion chromatography (IC) were compared as direct techniques for the estimation of the oxalate content of foods. Foods were thoroughly homogenized in acid, heat extracted, and clarified by centrifugation and filtration before dilution in water for analysis. Five individuals consuming self-selected diets maintained food records for three days to determine their mean daily oxalate intakes. RESULTS: Both techniques were capable of adequately measuring the oxalate in foods with a significant oxalate content. With foods of very low oxalate content (<1.8 mg/100 g), IC was more reliable than CE. The mean daily intake of oxalate by the five individuals tested was 152 +/- 83 mg, ranging from 44 to 352 mg/day. CONCLUSIONS: CE appears to be the method of choice over IC for estimating the oxalate content of foods with a medium (>10 mg/100 g) to high oxalate content due to a faster analysis time and lower running costs, whereas IC may be better suited for the analysis of foods with a low oxalate content. Accurate estimates of the oxalate content of foods should permit the role of dietary oxalate in urinary oxalate excretion and stone formation to be clarified. Other factors, apart from the amount of oxalate ingested, appear to exert a major influence over the amount of oxalate excreted in the urine.

Maternal insulin-like growth factor binding protein-1, body mass index, and fetal growth.

AIM: To examine the hypothesis that the maternal insulin-like growth factor system may constrain fetal growth. METHODS: A prospective observational study of maternal serum insulin-like growth factor binding protein-1 (IGFBP-1) and fetal growth was undertaken in neonates with birthweights below the 5th centile. They had been classified either as having fetal growth restriction (FGR) due to placental dysfunction (increased umbilical artery Doppler pulsatility index (PI); n = 25) or as being small for gestational age (SGA; normal umbilical artery PI, growth velocity and amniotic fluid; n = 27). Eighty nine controls had normal birthweights (5th-95th centile), umbilical artery PI, growth velocity, and amniotic fluid. IGFBP-1 was measured by radioimmunoassay. RESULTS: Among the controls, there was no significant correlation between IGFBP-1 and birthweight after allowing for body mass index (BMI). Maternal BMI was high in FGR and after adjusting for this, IGFBP-1 was increased (109 ng/ml) compared with SGA babies (69 ng/ml) and controls (57 ng/ml) and correlated with the umbilical artery PI. CONCLUSIONS: Maternal IGFBP-1 is probably not part of normal placental function. Its increase in FGR could be the cause or consequence of impaired placental perfusion, but high IGFBP-1 concentrations might further reduce the availability of maternal IGF-I to the placenta. This could worsen placental function and so adversely affect fetal growth.

Oxalate synthesis in humans: assumptions, problems, and unresolved issues.

The reactions that culminate in the synthesis of oxalate in human cells have not yet been clarified. Glycolate and glyoxylate appear to be major precursors. Experimental problems and unresolved issues are highlighted in this review. Assumptions that have been made with out experimental support are identified. The recognition of these assumptions and the testing of their validity should advance our knowledge of the pathways involved, their regulation, and their physiologic significance.

Guaifenesin- and ephedrine-induced stones.

PURPOSE: We report a new type of drug-induced stone that is caused by overconsumption of preparations containing guaifenesin and ephedrine. MATERIALS AND METHODS: Clinical and stone analysis data from the Molecular Structure Laboratory at the Veterans Affairs Medical Center in Milwaukee, Wisconsin, were reviewed. Stone analysis was performed by Fourier transform infrared spectroscopy, high-resolution X-ray crystallographic powder diffraction, or both. The urine and stone material from one of the subjects were analyzed with high-performance liquid chromatography. RESULTS: Stone analysis from seven patients demonstrated metabolites of guaifenesin. High-performance liquid chromatography revealed that the stone and urine from one subject had a high content of guaifenesin metabolites and a small amount of ephedrine. Demographic data were available on five patients. Three had a history of alcohol or drug dependency. All were consuming over-the-counter preparations containing ephedrine and guaifenesin. Four admitted to taking excessive quantities of these agents, mainly as a stimulant. Hypocitraturia was identified in two individuals subjected to urinary metabolic testing. These stones are radiolucent on standard X-ray imaging but can be demonstrated on unenhanced CT. Shockwave lithotripsy was performed in two patients, and the calculi fragmented easily. CONCLUSIONS: Individuals consuming large quantities of preparations containing ephedrine and guaifenesin may be at risk to develop stones derived mainly from metabolites of guaifenesin and small quantities of ephedrine. These patients may be prone to drug or alcohol dependency.

Glycolate metabolism by Hep G2 cells.

The pathways of oxalate synthesis in humans are not well defined despite their clinical significance in primary hyperoxaluria and idiopathic calcium oxalate nephrolithiasis. Furthermore, the functional roles, if any, of this synthesis have not been elucidated. This study examines pathways of oxalate synthesis from glycolate in Hep G2 cells, a human hepatoma cell line. Incubation of these cells with glycolate has revealed that a pathway may function to synthesize oxalate from glycolate that does not depend on the oxidation of glycolate to glyoxylate by glycolate oxidase. Labeling cells with 14C-glycolate and chromatographic analyses indicated that detectable amounts of 14C-glyoxylate were not formed. A radioactive peak that coeluted with oxalate on ion exclusion chromatography was the only peak yet identified. A detailed examination of glycolate metabolism in these cells should help clarify the terminal steps associated with oxalate synthesis and aid in our understanding of two-carbon metabolism.

The gene encoding hydroxypyruvate reductase (GRHPR) is mutated in patients with primary hyperoxaluria type II.

Primary hyperoxaluria type II (PH2) is a rare monogenic disorder that is characterized by a lack of the enzyme that catalyzes the reduction of hydroxypyruvate to D-glycerate, the reduction of glyoxylate to glycolate and the oxidation of D-glycerate to hydroxypyruvate. The disease is characterized by an elevated urinary excretion of oxalate and L-glycerate. The increased oxalate excretion can cause nephrolithiasis and nephrocalci-nosis and can, in some cases, result in renal failure and systemic oxalate deposition. We identified a glyoxylate reductase/hydroxypyruvate reductase (GRHPR) cDNA clone from a human liver expressed sequence tag (EST) library. Nucleotide sequence analysis identified a 1198 nucleotide clone that encoded a 984 nucleotide open reading frame. The open reading frame encodes a predicted 328 amino acid protein with a mass of 35 563 Da. Transient transfection of the cDNA clone into COS cells verified that it encoded an enzyme with hydroxy-pyruvate reductase, glyoxylate reductase and D-glycerate dehydrogenase enzymatic activities. Database analysis of human ESTs reveals widespread tissue expression, indicating that the enzyme may have a previously unrecognized role in metabolism. The genomic structure of the human GRHPR gene was determined and contains nine exons and eight introns and spans approximately 9 kb pericentromeric on chromosome 9. Four PH2 patients representing two pairs of siblings from two unrelated families were analyzed for mutations in GRHPR by single strand conformation polymorphism analysis. All four patients were homozygous for a single nucleotide deletion at codon 35 in exon 2, resulting in a premature stop codon at codon 45. The cDNA that we have identified represents the first characterization of an animal GRHPR sequence. The data we present will facilitate future genetic testing to confirm the clinical diagnosis of PH2. These data will also facilitate heterozygote testing and prenatal testing in families affected with PH2 to aid in genetic counseling.

Maternal serum insulin-like growth factor binding protein-2 and -3 and fetal growth.

This was a prospective observational study of maternal insulin-like growth factor binding protein-2 and -3 and fetal growth in 141 pregnant women after 24 weeks gestation who were scanned and venesected fortnightly. Cases (birthweight <5th centile) were sub-divided into those with growth restriction due to placental dysfunction (n = 25) and normal small (n = 27) and there were 89 normally grown controls. Maternal binding protein-3 was measured by radioimmunoassay and the overall pattern of the binding proteins and their proteolytic modifications were assessed by Western ligand blotting and immunoblotting followed by densitometric analysis. In controls, there was no correlation between binding protein-3 and birthweight, and binding protein-3 was elevated in the normal small but not the placental dysfunction group. Complete proteolysis of the 40 kDa doublet of binding protein-3 was observed in all pregnancies. Maternal serum binding protein-2 concentrations were unchanged in normal pregnancy compared to non-pregnant controls but elevated in the growth-restricted group and in all pregnancies binding protein-2 was predominantly present as a 14 kDa proteolysed fragment. These results suggest that compensatory changes in binding protein-2 and -3 or their proteolysis do not increase bioavailability and so do not confound the low maternal insulin-like growth factor-I in growth restricted pregnancies.

Direct quantification of the enteric bacterium Oxalobacter formigenes in human fecal samples by quantitative competitive-template PCR.

Homeostasis of oxalic acid appears to be regulated, in part, by the gut-associated bacterium Oxalobacter formigenes. The loss of this bacterium from the gut flora is associated with an increased susceptibility to hyperoxaluria, a condition which can lead to the formation of calcium oxalate crystalluria and kidney stones. In order to identify and quantify the presence of O. formigenes in clinical specimens, a quantitative-PCR-based assay system utilizing a competitive DNA template as an internal standard was developed. This quantitative competitive-template PCR test allows for the rapid, highly specific, and reproducible quantification of O. formigenes in fecal samples and provides a prototype for development of DNA-based quantitative assays for enteric bacteria.

Urinary oxalate and citrate.

The amounts of oxalate and citrate excreted in urine, and their urinary concentrations are important risk factors for the development of calcium oxalate kidney stones (1). The most widely used procedures to estimate these analytes are enzyme-based procedures using commercially available reagent kits (2,3). Components in the urine matrix may interfere with these assays, and some sample cleanup is required to remove them for oxalate analysis. Ion chromatography, although well suited to these determinations (4,5), is less widely used presumably because of long assay times, the need for expensive equipment, and the maintenance costs associated with the procedure. Capillary electrophoresis enables the rapid determination of oxalate and citrate in the same run, as well as the simultaneous measurement of chloride and sulfate. Estimation of these anions is useful for the calculation of relative supersaturations of urine with calcium oxalate and calcium phosphate. The method developed here utilizes indirect absorption to detect anions (6), and relies on the change in absorption observed when oxalate and citrate migrate through the detection window, and displace chromate in the electrolyte. It is important that the chromophoric electrolyte chosen has a migration time similar to that of the anions of interest. Pyromellitic acid is another suitable chromophore that can be used as the electrolyte (7).

Glyoxylate synthesis, and its modulation and influence on oxalate synthesis.

PURPOSE: We define the major pathways of hepatic oxalate synthesis in humans, examine the association with other metabolic pathways and identify ways that oxalate synthesis may be modified. In addition, we suggest what is required for further progress in this area. MATERIALS AND METHODS: We consolidated relevant data primarily from recently published literature, considered new pharmacological approaches to decrease oxalate synthesis, and formulated an overview of the regulation and modification of oxalate synthesis pathways. RESULTS: Experiments with animals, including humans, animal cells and in vitro preparations of cellular components, support the existence of a major metabolic pathway linking the amino acids serine, glycine and alanine. Oxalate synthesis is a minor, secondary reaction of a cascade of reactions termed the glyoxylate pathway, which has a prominent role in gluconeogenesis and ureagenesis. The enzymatic steps and effectors which regulate glyoxylate and oxalate synthesis are not well characterized. Pharmacological approaches can reduce oxalate synthesis by diminishing the glyoxylate pool and possibly modifying enzymatic reactions leading to glyoxylate synthesis. CONCLUSIONS: The individual steps associated with glyoxylate and oxalate synthesis can be identified. The glyoxylate pathway has a significant functional role in intermediary liver metabolism but the way it is regulated is uncertain. Oxalate synthesis can be modified by drugs, indicating that primary and idiopathic hyperoxaluria may respond to pharmacological intervention.

Genetic and dietary influences on urinary oxalate excretion.

Several genes contribute to the development of calcium oxalate nephrolithiasis as it is a polygenic disease. To explore the influence of genetic factors on oxalate excretion we have examined the distribution of oxalate excretions in 101 normal individuals who consumed self-selected diets. The distribution was apparently trimodal, consistent with the existence of three classes of oxalate excretors reflecting two allelic genes determining high and low oxalate excretion occurring with frequencies of 0.32 and 0.68 respectively. The pattern of inheritance in eight families was compatible with the expression of a pair of codominant alleles. A comparison of the distribution of excretory classes among the 101 normal individuals with that of 101 calcium oxalate stone formers suggests that high oxalate excretion may be associated with a 4-fold increased risk of stone disease and intermediate excretion with a 1.6-fold increase. Control of dietary factors influencing oxalate excretion apparently improved the discrimination between excretory classes in 17 individuals but the intra-individual variability in oxalate excretion was not reduced in four of nine individuals in whom this parameter was evaluated. More stringent dietary control than that applied in this study may be required before more extensive genotyping of individuals is attempted.