In vivo oxalate degradation by liposome encapsulated oxalate oxidase in rat model of hyperoxaluria.

BACKGROUND & OBJECTIVES: High level of urinary oxalate substantially increases the risk of hyperoxaluria, a significant risk factor for urolithiasis. The primary goal of this study was to reduce urinary oxalate excretion employing liposome encapsulated oxalate oxidase in animal model. METHODS: A membrane bound oxalate oxidase was purified from Bougainvillea leaves. The enzyme in its native form was less effective at the physiological pH of the recipient animal. To increase its functional viability, the enzyme was immobilized on to ethylene maleic anhydride (EMA). Rats were injected with liposome encapsulated EMA- oxalate oxidase and the effect was observed on degradation of oxalic acid. RESULTS: The enzyme was purified to apparent homogeneity with 60-fold purification and 31 per cent yield. The optimum pH of EMA-derivative enzyme was 6.0 and it showed 70 per cent of its optimal activity at pH 7.0. The EMA-bound enzyme encapsulated into liposome showed greater oxalate degradation in 15 per cent casein vitamin B 6 deficient fed rats as compared with 30 per cent casein vitamin B 6 deficient fed rats and control rats. INTERPRETATION & CONCLUSIONS: EMA-oxalate oxidase encapsulated liposome caused oxalate degradation in experimental hyperoxaluria indicating that the enzyme could be used as a therapeutic agent in hyperoxaluria leading to urinary stones.

Molecular Insight into the Synergism between the Minor Allele of Human Liver Peroxisomal Alanine:Glyoxylate Aminotransferase and the F152I Mutation.

Human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that converts glyoxylate into glycine. AGT deficiency causes primary hyperoxaluria type 1 (PH1), a rare autosomal recessive disorder, due to a marked increase in hepatic oxalate production. Normal human AGT exists as two polymorphic variants: the major (AGT-Ma) and the minor (AGT-Mi) allele. AGT-Mi causes the PH1 disease only when combined with some mutations. In this study, the molecular basis of the synergism between AGT-Mi and F152I mutation has been investigated through a detailed biochemical characterization of AGT-Mi and the Phe(152) variants combined either with the major (F152I-Ma, F152A-Ma) or the minor allele (F152I-Mi). Although these species show spectral features, kinetic parameters, and PLP binding affinity similar to those of AGT-Ma, the Phe(152) variants exhibit the following differences with respect to AGT-Ma and AGT-Mi: (i) pyridoxamine 5'-phosphate (PMP) is released during the overall transamination leading to the conversion into apoenzymes, and (ii) the PMP binding affinity is at least 200-1400-fold lower. Thus, Phe(152) is not an essential residue for transaminase activity, but plays a role in selectively stabilizing the AGT-PMP complex, by a proper orientation of Trp(108), as suggested by bioinformatic analysis. These data, together with the finding that apoF152I-Mi is the only species that at physiological temperature undergoes a time-dependent inactivation and concomitant aggregation, shed light on the molecular defects resulting from the association of the F152I mutation with AGT-Mi, and allow to speculate on the responsiveness to pyridoxine therapy of PH1 patients carrying this mutation.

Detection of endothelial nitric oxide synthase and NADPH-diaphorase in experimentally induced hyperoxaluric animals.

Nitrosative stress plays a role in calcium oxalate stone formation, as nitrosated proteins have been identified in stone formers. Nitric oxide (NO(*)), the common precursor for reactive nitrogen species, is synthesized in the juxtaglomerular apparatus of the kidneys. The present study is aimed to determine the role of nitric oxide synthase (NOS) in an experimental hyperoxaluric condition by histological and biochemical techniques. Hyperoxaluria was induced by 0.75% ethylene glycol in drinking water. L-arginine (L-arg) was supplemented at a dose of 1.25 g/kg body weight orally for 28 days. Nitric oxide metabolites (NOx), protein content in the urine and lipid peroxidation in the kidney were determined at the end of the experimental period. Histopathological examination of the rat kidneys was then carried out. NADPH-diaphorase and eNOS expression studies were carried out in control and hyperoxaluric rat kidneys using histochemical and immunohistochemical techniques. Significant amounts of NOx were present in the urine of hyperoxaluric animals when compared to control rats. Histopathological examinations revealed membrane injury, tubular dilatation and edema in the hyperoxaluric rats, whereas co-supplementation of L-arg to the hyperoxaluric rats significantly reduced these changes. The results of histochemical analysis for NADPH-diaphorase staining demonstrate the role of NOS in hyperoxaluric rats. Hyperoxaluric rats showed intense staining for NADPH-diaphorase when compared to control and L-arg co-supplemented hyperoxaluric rats. Immunohistochemical demonstration confirmed that eNOS expression was markedly increased in L-arg supplemented rats, when compared to EG treated rat kidney sections. Thus, from the present study, we conclude that supplementation of L-arg to the hyperoxaluric animals minimizes the cellular injury mediated by ethylene glycol, prevents oxidative/nitrosative damage to the membranes and reduces the incidence of calcium oxalate stone formation.

Crystal structure of alanine:glyoxylate aminotransferase and the relationship between genotype and enzymatic phenotype in primary hyperoxaluria type 1.

A deficiency of the liver-specific enzyme alanine:glyoxylate aminotransferase (AGT) is responsible for the potentially lethal hereditary kidney stone disease primary hyperoxaluria type 1 (PH1). Many of the mutations in the gene encoding AGT are associated with specific enzymatic phenotypes such as accelerated proteolysis (Ser205Pro), intra-peroxisomal aggregation (Gly41Arg), inhibition of pyridoxal phosphate binding and loss of catalytic activity (Gly82Glu), and peroxisome-to-mitochondrion mistargeting (Gly170Arg). Several mutations, including that responsible for AGT mistargeting, co-segregate and interact synergistically with a Pro11Leu polymorphism found at high frequency in the normal population. In order to gain further insights into the mechanistic link between genotype and enzymatic phenotype in PH1, we have determined the crystal structure of normal human AGT complexed to the competitive inhibitor amino-oxyacetic acid to 2.5A. Analysis of this structure allows the effects of these mutations and polymorphism to be rationalised in terms of AGT tertiary and quaternary conformation, and in particular it provides a possible explanation for the Pro11Leu-Gly170Arg synergism that leads to AGT mistargeting.

Control of urinary risk factors of stones by betulin and lupeol in experimental hyperoxaluria.

Urolithiasis, the process of formation of stones in the kidney and the urinary tract, is the major clinical manifestation of hyperoxaluria. Crystal deposition, as indicated by increased stone-forming constituents in urine, such as calcium, oxalate and uric acid, and decreased concentration of inhibitors, such as magnesium and glycosaminoglycans, was observed in pyridoxine-deficient hyperoxaluric rats. Renal tubular damage was indicated by increased excretion of enzymes such as alkaline phosphatase, lactate dehydrogenase, gamma-glutamyl transferase, beta-glucuronidase and N-acetyl glucosaminidase. Fibrinolytic activity was found to be reduced. Administration of pentacyclic triterpenes such as lupeol and its structural analogue betulin to hyperoxaluric rats minimised the tubular damage and reduced the markers of crystal deposition in the kidneys. In this connection, lupeol was found to be more effective than betulin.

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.

N-acetyl-beta-D-glucosaminidase excretion in healthy children and in pediatric patients with urolithiasis.

Hyperoxaluria was reported to induce renal damage, probably due to toxic effects on renal tubules. Such tubular damage might be expressed by an increase in urinary excretion of marker enzymes such as N-acetyl-beta-D-glucosaminidase (NAG). We set out to examine a possible relationship between the excretion of NAG and that of urinary lithogenic and stone-inhibitory substances by analyzing 24-h urine specimens from 56 children with urolithiasis and 25 healthy children with normal renal function and without a history of urolithiasis. The NAG excretion was higher in patients with urolithiasis (3.5 +/- 0.51 U/g creatinine) as compared with healthy subjects (1.33 +/- 0.14 U/g creatinine, P < 0.05). A positive correlation between NAG and oxalate excretion was observed in female patients (r = 0.56: P < 0.01). In conclusion, the increase in urinary NAG in children with urolithiasis might express renal tubular damage. It seemed, however, not to be specifically related to the excretion of a single lithogenic substance.

Variable peroxisomal and mitochondrial targeting of alanine: glyoxylate aminotransferase in mammalian evolution and disease.

Under the putative influence of dietary selection pressure, the subcellular distribution of alanine:glyoxylate aminotransferase 1 (AGT) has changed on many occasions during the evolution of mammals. Depending on the particular species, AGT can be found either in peroxisomes or mitochondria, or in both peroxisomes and mitochondria. This variable localization depends on the differential expression of N-terminal mitochondrial and C-terminal peroxisomal targeting sequences by the use of alternative transcription and translation initiation sites. AGT is peroxisomal in most humans, but it is mistargeted to the mitochondria in a subset of patients suffering from the rare hereditary disease primary hyperoxaluria type 1. Mistargeting is due to the unlikely combination of a normally occurring polymorphism that generates a functionally weak mitochondrial targeting sequence and a disease-specific mutation which, in combination with the polymorphism, inhibits AGT dimerization. The mechanisms by which AGT can be targeted differentially to peroxisomes and/or mitochondria highlight the different molecular requirements for protein import into these two organelles.

Effect of cyclosporin A on tissue lipid peroxidation and membrane bound phosphatases in hyperoxaluric rat and the protection by vitamin E pretreatment.

The effect of cyclosporin A, a highly effective immunosuppressant, was investigated on hyperoxaluric rats with and without vitamin E pretreatment. Hyperoxaluria was induced by oral feeding of 3% ammonium oxalate in water for 3 days. Cyclosporin A (50 mg/kg body wt.) was administered for 3 days. Pretreatment with vitamin E (50 mg/100 g body wt., once a week for 3 weeks) was carried out before the administration of cyclosporin A and ammonium oxalate. Nonenzymatic ascorbate-induced lipid peroxidation was increased to 1.55-fold in either cyclosporin A-administered or hyperoxaluric rat kidney and liver when compared to control. The lipid peroxidation was further elevated to 1.9-fold when both cyclosporin A and ammonium oxalate were coadministered. The activities of renal and hepatic ATPase, glucose-6-phosphatase as well as the concentrations of thiols were decreased significantly (p < 0.001) when cyclosporin A was administered under hyperoxaluric condition. On pretreatment with vitamin E the cyclosporin A-induced biochemical changes observed in the presence of hyperoxaluria were abolished.

A unique molecular basis for enzyme mistargeting in primary hyperoxaluria type 1.

The intermediary metabolic enzyme alanine:glyoxylate aminotransferase (AGT) is normally targeted to the peroxisomes in human liver cells. However, in a third of patients suffering from the autosomal recessive disease primary hyperoxaluria type 1 (PH1), AGT is mistargeted to the mitochondria. Such organelle-to-organelle mistargeting is without parallel in human genetic disease. AGT mistargeting results from the combination of a common Pro11-->Leu polymorphism and a rare Gly170-->Arg mutation. The former generates a functionally weak mitochondrial targeting sequence (MTS) while the latter, in combination with the former, increases the efficiency of this MTS by slowing the rate at which AGT dimerises. The fact that the intracellular compartmentation of AGT can be determined, at least in part, by its oligomeric status highlights the fundamental differences in the molecular requirements for protein import into two intracellular organelles--the peroxisomes and mitochondria.

Mammalian alanine/glyoxylate aminotransferase 1 is imported into peroxisomes via the PTS1 translocation pathway. Increased degeneracy and context specificity of the mammalian PTS1 motif and implications for the peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1.

Alanine/glyoxylate aminotransferase 1 (AGT) is peroxisomal in most normal humans, but in some patients with the hereditary disease primary hyperoxaluria type 1 (PH1), AGT is mislocalized to the mitochondria. In an attempt to identify the sequences in AGT that mediate its targeting to peroxisomes, and to determine the mechanism by which AGT is mistargeted in PH1, we have studied the intracellular compartmentalization of various normal and mutant AGT polypeptides in normal human fibroblasts and cell lines with selective deficiencies of peroxisomal protein import, using immunofluorescence microscopy after intranuclear microinjection of AGT expression plasmids. The results show that AGT is imported into peroxisomes via the peroxisomal targeting sequence type 1 (PTS1) translocation pathway. Although the COOH-terminal KKL of human AGT was shown to be necessary for its peroxisomal import, this tripeptide was unable to direct the peroxisomal import of the bona fide peroxisomal protein firefly luciferase or the reporter protein bacterial chloramphenicol acetyltransferase. An ill-defined region immediately upstream of the COOH-terminal KKL was also found to be necessary for the peroxisomal import of AGT, but again this region was found to be insufficient to direct the peroxisomal import of chloramphenicol acetyltransferase. Substitution of the COOH-terminal KKL of human AGT by the COOH-terminal tripeptides found in the AGTs of other mammalian species (SQL, NKL), the prototypical PTS1 (SKL), or the glycosomal PTS1 (SSL) also allowed peroxisomal targeting, showing that the allowable PTS1 motif in AGT is considerably more degenerate than, or at least very different from, that acceptable in luciferase. AGT possessing the two amino acid substitutions responsible for its mistargeting in PH1 (i.e., Pro11-->Leu and Gly170-->Arg) was targeted mainly to the mitochondria. However, AGTs possessing each amino acid substitution on its own were targeted normally to the peroxisomes. This suggests that Gly170-->Arg-mediated increased functional efficiency of the otherwise weak mitochondrial targeting sequence (generated by the Pro11-->Leu polymorphism) is not due to interference with the peroxisomal targeting or import of AGT.

Effect of lupeol, a pentacyclic triterpene, on urinary enzymes in hyperoxaluric rats.

Investigations were undertaken to study the role of lupeol, a pentacyclic triterpene from Crataeva nurvala stem bark, in calcium oxalate experimental rat urolithiasis. A 2% solution of ammonium oxalate was administered by gastric intubation for inducing hyperoxaluric condition in adult male rats of Wistar strain. The duration of treatment was for 15 days. This resulted in increased urinary excretion of oxalate associated with reduction in citrate and glycosaminoglycans. The urinary marker enzymes which indicate renal tissue damage namely--lactate dehydrogenase, inorganic pyrophosphatase, alkaline phosphatase, gamma glutamyl transferase, beta-glucuronidase and N-acetyl beta-D glucosaminidase were found to be elevated. Lupeol administration (25 mg/kg body weight/day) reduced significantly the renal excretion of oxalate. It also reduced the extent of renal tubular damage as evidenced from the decreased levels of the above enzymes in urine. Such a reduction is likely to be beneficial in minimizing the deposition of stone-forming constituents in the kidney which provides antilithic effect.

Advances in the enzymology and molecular genetics of primary hyperoxaluria type 1. Prospects for gene therapy.

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive inborn error of glyoxylate metabolism caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). At the enzymic level, PH1 is usually heterogeneous. Several novel enzymic phenotypes have been identified, including the mistargeting of AGT from the peroxisomes to mitochondria, and the aggregation of AGT in the peroxisomal matrix. Seven PH1-specific point mutations, as well as a number of clinically useful normal polymorphisms, have been found so far in the AGT gene. The molecular elucidation of PH1 has led to changes in almost all aspects of its clinical management, most notably treatment. Liver transplantation as a form of enzyme replacement therapy has been used successfully in the treatment of PH1 over the last 10 years, but the long-term solution lies in gene therapy. Although PH1 is, in many respects, an ideal candidate for gene therapy, the strategies eventually adopted will need to take into account its unique metabolic and enzymic characteristics.

Cytosolic compartmentalization of hepatic alanine:glyoxylate aminotransferase in patients with aberrant peroxisomal biogenesis and its effect on oxalate metabolism.

Two patients with atypical manifestations of aberrant peroxisomal biogenesis are described. Contrary to previous studies, which had shown that Zellweger syndrome patients usually have normal levels of urinary oxalate excretion, the patients in the present study had evidence of abnormal oxalate metabolism in the form of hyperoxaluria and, in one of the patients, calcium oxalate urolithiasis. Activity of the liver-specific peroxisomal enzyme alanine:-glyoxylate aminotransferase (AGT), which is a major determinant of the level of endogenous oxalate synthesis in humans, was normal in one patient and markedly supranormal in the other. Using the technique of post-embedding protein A-colloidal gold immunoelectron microscopy, AGT was found to be mainly cytosolic in the livers of both patients, with significant amounts also localized in the nuclei. In a small minority of the hepatocytes of one patient, who was homozygous for the more common (major) AGT allele, large numbers of unidentified fibrillar arrays were found in the cytosol, which labelled heavily for immunoreactive AGT. The background cytosolic AGT labelling was markedly reduced in such cells when compared to the majority of cells that did not contain fibrils. In the other patient, who was heterozygous for the major and minor AGT alleles, there appeared to be low levels of mitochondrial AGT labelling. In the light of these data, the possible metabolic function of cytosolic AGT in the livers of panperoxisomal disease patients is discussed.

[Molecular pathology of type 1 primary hyperoxaluria]. / Pathologie moléculaire de l'hyperoxalurie primitive de type 1.

Type 1 is the most common form of primary hyperoxaluria, also called oxalosis when systemic involvement has occurred. This recessive autosomal inherited inborn error of metabolism is characterized by a defect of alanine: glyoxylate aminotransferase (AGT), which is a specific liver enzyme. This protein is responsible for glyoxylate detoxification only when it is located in the peroxisome. The clinical and biochemical phenotypes are neither correlated with the residual catalytic activity of AGT nor with its immunoreactivity. Most patients display less than 2% catalytic activity (enz-) or no immunoreactive protein (crm-); peroxisome-to-mitochondrion mistargeting is the main feature of patients crm+/enz+ or crm+/enz-. The cDNA and genomic DNA have been cloned and sequenced and the gene has been located on the long arm of chromosome 2 in the q36-37 region. Three polymorphisms have been identified which are preferentially associated, leading to two alleles; six point mutations have been currently reported.

High-performance liquid chromatographic microassay for L-alanine:glyoxylate aminotransferase activity in human liver.

We examine the suitability of a rapid and sensitive liquid chromatographic technique to determine L-alanine:glyoxylate aminotransferase (AGT) activity in human liver. Homogenised tissue was incubated for 30 min in the presence of substrates and the generated pyruvate was converted into the corresponding phenylhydrazone which was determined using reversed-phase high-performance liquid chromatography (HPLC). The procedure allowed the detection of the enzyme activity expressed by 10 micrograms of liver protein and was rapid enough resulting more sensitive and less time-consuming than the previous colorimetric one. We found that AGT activity in two hyperoxaluria type 1 patients was reduced as compared with controls. Also, cirrhotic patients had very low enzyme activities, even in the absence of detectable disorders of oxalate metabolism and this was ascribed to abnormal liver morphology. This may represent a misleading drawback if diagnosis of type 1 primary hyperoxaluria (PH1) uniquely relies on AGT assay.

Mistargeting of peroxisomal L-alanine:glyoxylate aminotransferase to mitochondria in primary hyperoxaluria patients depends upon activation of a cryptic mitochondrial targeting sequence by a point mutation.

In approximately one-third of primary hyperoxaluria type 1 patients, disease is associated with a unique protein sorting defect in which hepatic L-alanine:glyoxylate aminotransferase (AGT; EC 2.6.1.44), which is normally peroxisomal, is mistargeted to mitochondria. In all such patients analyzed to date, the gene encoding the aberrantly targeted AGT carries three point mutations, each of which specifies an amino acid substitution. In this paper we show that one of these substitutions, a proline-to-leucine at residue 11, is necessary and sufficient for the generation of a mitochondrial targeting sequence in the AGT protein. AGT with this substitution appears to interact specifically with the mitochondrial protein import machinery, via a discrete N-terminal domain of the AGT protein. The N-terminal 19 amino acids of AGT with this substitution are sufficient to direct mouse cytosolic dihydrofolate reductase to mitochondria, and a synthetic peptide corresponding to this same 19-amino acid region reversibly inhibits mitochondrial protein import, not only of AGT but also of ornithine transcarbamoylase, a genuine cytoplasmically synthesized mitochondrial protein. We have extended these studies to analyze a region of normal human AGT cDNA directly upstream of the coding region. This sequence appears to correspond to an ancestral mitochondrial targeting sequence deleted from the human coding region by point mutation at the initiation codon. We show that reestablishment of this initiation codon produces an active mitochondrial targeting sequence that is different to that found in the hyperoxaluria patients. These results are discussed with reference to the AGT targeting defect in primary hyperoxaluria and also in relation to the highly unusual species specificity of subcellular distribution of AGT among mammals.