Alanine-glyoxylate Aminotransferase Research Articles

CRISPR/Cas9–mediated metabolic pathway reprogramming in a novel humanized rat model ameliorates primary hyperoxaluria type 1

Primary hyperoxaluria type I is caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT), leading to accumulation of glyoxylate and subsequent production of oxalate and urolithiasis. Here, we generated a novel rat model of primary hyperoxaluria type I that carries a D205N mutation in the partially humanized Agxt gene through the CRISPR/Cas9 system. The AgxtD205N mutant rats showed undetectable alanine glyoxylate aminotransferase protein expression, developed hyperoxaluria at 1 month of age and exhibited severe renal calcium oxalate deposition after ethylene glycol challenge. This suggests our novel model is more relevant to the human disease than existing animal models. To test whether this model could be used for the development of innovative therapeutics, SaCas9 targeting hydroxyacid oxidase 1, responsible for metabolizing glycolate into glyoxylate, was delivered via adeno-associated viral vectors into newborn rats with primary hyperoxaluria type 1. This approach generated nearly 30% indels in the Hao1 gene in the liver, leading to 42% lower urine oxalate levels in the treated group than in the control group and preventing the rats with primary hyperoxaluria type 1 from undergoing severe nephrocalcinosis for at least 12 months. Thus, our results demonstrate that this partially humanized AgxtD205N rat strain is a high-performing model of primary hyperoxaluria type 1 for understanding pathology, and the development of novel therapeutics, such as reprogramming of the metabolic pathway through genome editing.

AGXT2L1 is downregulated in carcinomas of the digestive system

Alanine-glyoxylate aminotransferase 2-like 1 (AGXT2L1) is a modulator of phospholipid metabolism, and its role in tumor biology is obscure. Previously, significant downregulation of AGXT2L1 has been observed in hepatocellular carcinoma. The aim of the present study was to investigate AGXT2L1 expression and its association with the clinical characteristics of common carcinomas of the digestive system. In the present study, the expression levels of AGXT2L1 were detected by immunohistochemical staining in colorectal cancer (CRC), gastric cancer and pancreatic cancer tissues. The associations between AGXT2L1 expression and clinicopathological features were analyzed using public gene expression datasets. Small interfering RNA was transfected into SW480 and HCT116 cells to explore the role of AGXT2L1 in CRC cells. AGXT2L1 expression was significantly decreased in cancerous tissues compared with in normal tissues, and low AGXT2L1 expression was associated with an unfavorable prognosis in patients. Furthermore, it was revealed that AGXT2L1 may regulate phosphatidylinositol and phosphatidylserine metabolism in cancerous tissues, and that decreased AGXT2L1 expression could induce autophagy in CRC cells. Overall, the present study provides a basis for further understanding of the role of AGXT2L1 and its association with autophagy in cancer.

SUN-331 COMBINED LIVER KIDNEY TRANSPLANTATION FOR PRIMARY HYPEROXALURIA: SINGLE CENTRE EXPERIENCE IN SOUTH INDIA

Type 1 primary hyperoxaluria is a rare inherited autosomal recessive disorder, characterized by deficiency of the hepatic enzyme alanine glyoxylate aminotransferase, leading to increased oxalate production, nephrocalcinosis, urolithiasis, and systemic oxalosis. Early diagnosis and management is pivotal as medical management and definitive treatment in the form of combined or sequential liver-kidney transplantation, isolated liver or kidney transplantation have been studied to transform disease outcomes. Here, we review the experience from our institution of combined liver-kidney transplantation among patients with primary hyperoxaluria from February, 2014 to October, 2019. Among three children with primary hyperoxaluria, two underwent deceased donor liver-kidney transplantation, and the third live related donor sequential liver-kidney transplantation. Case 1 was that of a 17 year old boy, who had been on intensive daily dialysis for two years prior to deceased donor sequential liver-kidney transplantation, with a history of bladder calculus in childhood. He had a relatively uneventful post -transplant period and is doing well on follow up five years following transplantation. Case 2 was a 4 year old boy, diagnosed to have oxalosis two years prior to transplant, on dialysis five days a week through tunnelled catheter, prior to deceased donor liver-kidney transplantation, with a history of tonic-clonic seizures. Post-operatively, he had a stormy course with cytomegalovirus infection of the hepatic allograft, and hepatic artery thrombosis, and expired a month following transplantation. Case 3 was that of a 13 year old girl, diagnosed at 10 years of age with primary hyperoxaluria, who underwent live related donor sequential liver-kidney transplantation. She developed obstructive jaundice following liver transplantation, owing to biliary stricture, and underwent ERCP and biliary stenting. She also had an upper gastro -intestinal bleed, and profound anaemia, and later developed sepsis and cholangitis, which were conservatively managed. Subsequently, she underwent renal transplantation and later developed gram negative septicemia, and recurrent proximal bile duct stricture, which was subjected to biliary stenting. Presently, she has stable graft function, and is clinically stable. The experience from our institution re-emphasises the significance of early diagnosis, and pre-transplant intensive dialysis in reducing oxalate load if planning for sequential liver-kidney transplantation to improve outcomes in primary hyperoxaluria. Isolated liver or kidney transplantation have not been adopted in our centre. In all patients, Scheinman protocol was followed in the immediate post-transplant period. Renal biopsies in the recipient showed only mild oxalate deposits which did not affect graft survival.

Natural history of GM1 gangliosidosis mouse models generated by The Jackson Laboratory Rare and Orphan Disease Center

Primary Hyperoxaluria type I (PH1) is a rare disease caused by mutations in the AGXT gene encoding alanine:glyoxylate aminotransferase (AGT), a liver enzyme involved in the detoxification of glyoxylate, the failure of which results in accumulation of oxalate and kidney stones formation. The role of protein misfolding in the AGT deficit caused by most PH1-causing mutations is increasingly being recognized. In addition, the genetic background in which a mutation occurs is emerging as a critical risk factor for disease onset and/or severity. Based on these premises, in this study we have analyzed the clinical, biochemical and cellular effects of the p.Ile56Asn mutation, recently described in a PH1 patient, as a function of the residue at position 11, a hot-spot for both polymorphic (p.Pro11Leu) and pathogenic (p.Pro11Arg) mutations. We have found that the p.Ile56Asn mutation induces a structural defect mostly related to the apo-form of AGT. The effects are more pronounced when the substitution of Ile56 is combined with the p.Pro11Leu and, at higher degree, the p.Pro11Arg mutation. As compared with the non-pathogenic forms, AGT variants display reduced expression and activity in mammalian cells. Vitamin B6, a currently approved treatment for PH1, can overcome the effects of the p.Ile56Asn mutation only when it is associated with Pro at position 11. Our results provide a first proof that the genetic background influences the effects of PH1-causing mutations and the responsiveness to treatment and suggest that molecular and cellular studies can integrate clinical data to identify the best therapeutic strategy for PH1 patients.

Gene therapy PR006 increased progranulin levels and improved lysosomal related phenotypes in model systems

Primary Hyperoxaluria type I (PH1) is a rare disease caused by mutations in the AGXT gene encoding alanine:glyoxylate aminotransferase (AGT), a liver enzyme involved in the detoxification of glyoxylate, the failure of which results in accumulation of oxalate and kidney stones formation. The role of protein misfolding in the AGT deficit caused by most PH1-causing mutations is increasingly being recognized. In addition, the genetic background in which a mutation occurs is emerging as a critical risk factor for disease onset and/or severity. Based on these premises, in this study we have analyzed the clinical, biochemical and cellular effects of the p.Ile56Asn mutation, recently described in a PH1 patient, as a function of the residue at position 11, a hot-spot for both polymorphic (p.Pro11Leu) and pathogenic (p.Pro11Arg) mutations. We have found that the p.Ile56Asn mutation induces a structural defect mostly related to the apo-form of AGT. The effects are more pronounced when the substitution of Ile56 is combined with the p.Pro11Leu and, at higher degree, the p.Pro11Arg mutation. As compared with the non-pathogenic forms, AGT variants display reduced expression and activity in mammalian cells. Vitamin B6, a currently approved treatment for PH1, can overcome the effects of the p.Ile56Asn mutation only when it is associated with Pro at position 11. Our results provide a first proof that the genetic background influences the effects of PH1-causing mutations and the responsiveness to treatment and suggest that molecular and cellular studies can integrate clinical data to identify the best therapeutic strategy for PH1 patients.

Transcriptomic profile analysis of non-coding RNAs involved in Capsicum chinense Jacq. fruit ripening

Capsicum species are highly diverse plants, with over 200 species. Their fruits are important, both nutritionally and economically. Plant non-coding RNAs (ncRNAs) and microRNAs (miRNAs) significantly influence several plant biological processes, including regulating their gene expression. Specifically, in pepper plants, in excess of 300 conserved and novel miRNAs have previously been described. However, the functional role and influence of ncRNAs in pepper fruit ripening processes have not been sufficiently addressed. Herein, we used high-throughput sequencing to identify ncRNAs in Capsicum chinense Jacq. at three distinct fruit ripening stages (green ripening stage, the colour transition stage and the red ripening stage). 142 novel miRNAs, 9928 circRNAs and 20,563 lncRNAs were identified. DE analysis revealed that 4627 mRNA, 5918 lncRNA and 85circRNA were upregulated. Two miRNAs were associated with pigment biosynthetic process hence are linked to carotenoid synthesis in pepper fruits. Similarly, two known miRNAs; miR157d-3p and miR396h, that target beta-glucosidase – a vital enzyme in the fruit ripening process were identified. Further, circRNA that targets alanine-glyoxylate aminotransferase 2 (Capana00g005022); which contributes to pepper aroma formation was identified. In conclusion, CircRNAs, lncRNAs and mRNAs can perform as ceRNAs in gene regulation in plants. Overall, the findings from this study provide knowledge on ncRNAs functional and regulation dynamism during Capsicum chinense Jacq. pepper fruit ripening process.

Первичная гипероксалурия: диагностика, лечение, исходы

Primary hyperoxaluria (PH) is a rare autosomal recessive disease caused by defects in liver glyoxylate metabolism and leading to overproduction of oxalates. Of the three types of PH, type I is the most common and severe form of the disease, which is caused by deficiency or loss of the liver-specific, vitamin B6-dependent, peroxisomal enzyme alanine-glyoxylateaminotransferase (AGT). In all types of PH, urinary excretion of oxalate is strongly elevated (> 1 mmol /1,73 m2/24 h), which results in recurrent urolithiasis and/or progressive nephrocalcinosis and subsequently, with a decrease in glomerular filtration rate (GFR), to the deposition of oxalates in the tissues of the body and the development of systemic oxalosis. PH type I is diagnosed late, in > 30% of patients already at the terminal stage of renal disease (ESRD). Every fourth patient with PH type II achieves ESRD, but cases of ESRD in type III are extremely rare. The diagnosis of PH is based on clinical and imaging (ultrasound, X-ray, CT scan) findings, urine oxalate assessment, genetic analysis. Early initiation of conservative treatment (high fluid intake, sodium citrate, etc.) is aimed at preserving renal function. Pyridoxine treatment can be effective in about 30% of patients with PH type I. Time on dialysis in anticipation of transplantation should be short to avoid overt systemic oxalosis. Transplantation methods depend on the type of PH and on the degree of GFR reduction, but combined liver and kidney transplantation is the method of choice in patients with primary hyperoxaluria type I. High index of clinical suspicion of PH must be in patients with nephrocalcinosis and/or recurrent urolithiasis, especially if urinary stones are predominantly whewellite (calcium oxalate monohydrate) in order to start early conservative treatment and preserve kidney function.

Cycloserine enantiomers are reversible inhibitors of human alanine:glyoxylate aminotransferase: implications for Primary Hyperoxaluria type 1.

Peroxisomal alanine:glyoxylate aminotransferase (AGT) is responsible for glyoxylate detoxification in human liver and utilizes pyridoxal 5'-phosphate (PLP) as coenzyme. The deficit of AGT leads to Primary Hyperoxaluria Type I (PH1), a rare disease characterized by calcium oxalate stones deposition in the urinary tract as a consequence of glyoxylate accumulation. Most missense mutations cause AGT misfolding, as in the case of the G41R, which induces aggregation and proteolytic degradation. We have investigated the interaction of wild-type AGT and the pathogenic G41R variant with d-cycloserine (DCS, commercialized as Seromycin), a natural product used as a second-line treatment of multidrug-resistant tuberculosis, and its synthetic enantiomer l-cycloserine (LCS). In contrast with evidences previously reported on other PLP-enzymes, both ligands are AGT reversible inhibitors showing inhibition constants in the micromolar range. While LCS undergoes half-transamination generating a ketimine intermediate and behaves as a classical competitive inhibitor, DCS displays a time-dependent binding mainly generating an oxime intermediate. Using a mammalian cellular model, we found that DCS, but not LCS, is able to promote the correct folding of the G41R variant, as revealed by its increased specific activity and expression as a soluble protein. This effect also translates into an increased glyoxylate detoxification ability of cells expressing the variant upon treatment with DCS. Overall, our findings establish that DCS could play a role as pharmacological chaperone, thus suggesting a new line of intervention against PH1 based on a drug repositioning approach. To a widest extent, this strategy could be applied to other disease-causing mutations leading to AGT misfolding.

Loss of alanine-glyoxylate and serine-pyruvate aminotransferase expression accelerated the progression of hepatocellular carcinoma and predicted poor prognosis

BackgroundAccumulated studies reported abnormal gene expression profiles of hepatocellular carcinoma (HCC) by cDNA microarray. We tried to merge cDNA microarray data from different studies to search for stably changed genes, and to find out better diagnostic and prognostic markers for HCC.MethodsA systematic review was performed by searching publications indexed in Pubmed from March 1, 2001 to July 1, 2016. Studies that reporting cDNA microarray profiles in HCC, containing both tumor and nontumor data and published in English-language were retrieved. The differentially expressed genes from eligible studies were summarized and ranked according to the frequency. High frequency genes were subjected to survival analyses. The expression and prognostic value of alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT) was further evaluated in HCC datasets in Oncomine and an independent HCC tissue array cohort. The role of AGXT in HCC progression was evaluated by proliferation and migration assays in a human HCC cell line.ResultsA total of 43 eligible studies that containing 1917 HCC patients were included, a list of 2022 non redundant abnormally expressed genes in HCC were extracted. The frequencies of reported genes were ranked. We finally obtained a list of only five genes (AGXT; ALDOB; CYP2E1; IGFBP3; TOP2A) that were differentially expressed in tumor and nontumor tissues across studies and were significantly correlated to HCC prognosis. Only AGXT had not been reported in HCC. Reduced expression of AGXT reflected poor differentiation of HCC and predicts poor survival. Knocking down of AGXT enhanced cell proliferation and migration of HCC cell line.ConclusionsThe present study supported the feasibility and necessity of systematic review on discovering new and reliable biomarkers for HCC. We also identified a list of high frequency prognostic genes and emphasized a critical role of AGXT deletion during HCC progression.

Diet and Adaptive Evolution of Alanine-Glyoxylate Aminotransferase Mitochondrial Targeting in Birds.

Adaptations to different diets represent a hallmark of animal diversity. The diets of birds are highly variable, making them an excellent model system for studying adaptive evolution driven by dietary changes. To test whether molecular adaptations to diet have occurred during the evolution of birds, we examined a dietary enzyme alanine-glyoxylate aminotransferase (AGT), which tends to target mitochondria in carnivorous mammals, peroxisomes in herbivorous mammals, and both mitochondria and peroxisomes in omnivorous mammals. A total of 31 bird species were examined in this study, which included representatives of most major avian lineages. Of these, 29 have an intact mitochondrial targeting sequence (MTS) of AGT. This finding is in stark contrast to mammals, which showed a number of independent losses of the MTS. Our cell-based functional assays revealed that the efficiency of AGT mitochondrial targeting was greatly reduced in unrelated lineages of granivorous birds, yet it tended to be high in insectivorous and carnivorous lineages. Furthermore, we found that proportions of animal tissue in avian diets were positively correlated with mitochondrial targeting efficiencies that were experimentally determined, but not with those that were computationally predicted. Adaptive evolution of AGT mitochondrial targeting in birds was further supported by the detection of positive selection on MTS regions. Our study contributes to the understanding of how diet drives molecular adaptations in animals, and suggests that caution must be taken when computationally predicting protein subcellular targeting.

ESGCT 27th Annual Congress In collaboration with SETGyc Barcelona, Spain October 22–25, 2019 Abstracts

Xenotransplantation of transgenic porcine pancreatic islets offers a promising alternative source to circumvent current limitations posed by the scarcity of allogeneic donors. We have investigated the feasibility to generate tissue engineered SLA silenced islet cell clusters (ICC) to decrease xenogeneic immune responses Pancreatic islets single cell suspensions were generated by enzymatic digestion of porcine ICCs. Single cells were silenced for SLA expression by lentiviral vectors encoding for Nanoluciferase as reporter gene and for short hairpin RNAs targeting beta2-microglobulin or class II transactivator, respectively. SLA transcripts were evaluated by real-time PCR and protein levels by flow cytometry and fluorescence microscopy analyses. The effect of SLA silencing was evaluated in human T and NK cell cytotoxicity assays. SLA-silenced pancreatic beta-cells were then reassembled into ICCs in stirred bioreactors. SLA class I silencing reached a level of up to 84% and class II by up to 50% on pancreatic islet cell. Silencing SLA expression did not affect cell viability and the insulin-producing beta-cell phenotype. Xenogeneic T-cell immune responses (p < 0.05) as well as antibody-mediated cellular-dependent immune responses (p < 0.01) were significantly decreased. Silencing SLA class I expression did not increase susceptibility to NK-cell cytotoxicity. In stirred bioreactors, tissue engineered islets showing the typical 3D-structure and morphology of ICC were assembled from SLA-silenced pancreatic cell suspensions. These data shows the feasibility to generate low immunogenic porcine ICC from transgenic pigs after single cell engineering and post-transduction islet reassembling that might serve as a robust alternative to allogeneic pancreatic islet cell transplantation.

Generation of an induced pluripotent stem cell line (CIMAi001-A) from a compound heterozygous Primary Hyperoxaluria Type I (PH1) patient carrying p.G170R and p.R122* mutations in the AGXT gene.

Primary Hyperoxaluria Type I (PH1) is a rare autosomal recessive metabolic disorder characterized by defects in enzymes involved in glyoxylate metabolism. PH1 is a life-threatening disease caused by the absence, deficiency or mistargeting of the hepatic alanine-glyoxylate aminotransferase (AGT) enzyme. A human induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of a PH1 patient being compound heterozygous for the most common mutation c.508G>A (G170R), a mistargeting mutation, and c.364C>T (R122*), a previously reported nonsense mutation in AGTX. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for drug development.

Primary Hyperoxaluria-Imaging of Renal Oxalosis

Primary Hyperoxaluria is a rare autosomal recessive hereditary disorder due to deficient alanine-glyoxylate aminotransferase enzyme with defective glyoxylate metabolism leading to excessive oxalate production and deposition into the tissues (oxalosis). Deposition of excessive calcium oxalates in nephrons leads to crystallization (nephrocalcinosis) which increases risk for end-stage renal disease. We are presenting a case of primary hyperoxaluria type I confirmed with genetic studies.

Targeted gene therapy in human-induced pluripotent stem cells from a patient with primary hyperoxaluria type 1 using CRISPR/Cas9 technology

Primary hyperoxaluria type 1 (PH1) is an inherited metabolic disorder caused by a deficiency of the peroxisomal enzyme alanine-glyoxylate aminotransferase (AGT), which leads to overproduction of oxalate by the liver and results in urolithiasis, nephrocalcinosis and renal failure. The only curative treatment for PH1 is combined liver and kidney transplantation, which is limited by the lack of suitable organs, significant complications, and the life-long requirement for immunosuppressive agents to maintain organ tolerance. Hepatocyte-like cells (HLCs) generated from CRISPR/Cas9 genome-edited human-induced pluripotent stem cells would offer an attractive unlimited source of autologous gene-corrected liver cells as an alternative to orthotopic liver transplantation (OLT). Here we report the CRISPR/Cas9 nuclease–mediated gene targeting of a single-copy AGXT therapeutic minigene into the safe harbour AAVS1 locus in PH1-induced pluripotent stem cells (PH1-iPSCs) without off-target inserts. We obtained a robust expression of a codon-optimized AGT in HLCs derived from AAVS1 locus-edited PH1-iPSCs. Our study provides the proof of concept that CRISPR/Cas9-mediated integration of an AGXT minigene into the AAVS1 safe harbour locus in patient-specific iPSCs is an efficient strategy to generate functionally corrected hepatocytes, which in the future may serve as a source for an autologous cell-based gene therapy for the treatment of PH1.

Induction of enteric oxalate secretion by Oxalobacter formigenes in mice does not require the presence of either apical oxalate transport proteins Slc26A3 or Slc26A6.

Oxalobacter sp. promotion of enteric oxalate excretion, correlating with reductions in urinary oxalate excretion, was previously reported in rats and mice, but the mechanistic basis for this affect has not been described. The main objective of the present study was to determine whether the apical oxalate transport proteins, PAT1 (slc26a6) and DRA (slc26a3), are involved in mediating the Oxalobacter-induced net secretory flux across colonized mouse cecum and distal colon. We measured unidirectional and net fluxes of oxalate across tissues removed from colonized PAT1 and DRA knockout (KO) mice and also across two double knockout (dKO) mouse models with primary hyperoxaluria, type 1 (i.e., deficient in alanine-glyoxylate aminotransferase; AGT KO), including PAT1/AGT dKO and DRA/AGT dKO mice compared to non-colonized mice. In addition, urinary oxalate excretion was measured before and after the colonization procedure. The results demonstrate that Oxalobacter can induce enteric oxalate excretion in the absence of either apical oxalate transporter and urinary oxalate excretion was reduced in all colonized genotypes fed a 1.5% oxalate-supplemented diet. We conclude that there are other, as yet unidentified, oxalate transporters involved in mediating the directional changes in oxalate transport across the Oxalobacter-colonized mouse large intestine.

Generation of induced pluripotent stem cells-derived hepatocyte-like cells for ex vivo gene therapy of primary hyperoxaluria type 1

Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disorder of the liver metabolism due to functional deficiency of the peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). AGT deficiency results in overproduction of oxalate which complexes with calcium to form insoluble calcium-oxalate salts in urinary tracts, ultimately leading to end-stage renal disease. Currently, the only curative treatment for PH1 is combined liver-kidney transplantation, which is limited by donor organ shortage and lifelong requirement for immunosuppression. Transplantation of genetically modified autologous hepatocytes is an attractive therapeutic option for PH1. However, the use of fresh primary hepatocytes suffers from limitations such as organ availability, insufficient cell proliferation, loss of function, and the risk of immune rejection. We developed patient-specific induced pluripotent stem cells (PH1-iPSCs) free of reprogramming factors as a source of renewable and genetically defined autologous PH1-hepatocytes. We then investigated additive gene therapy using a lentiviral vector encoding wild-type AGT under the control of the liver-specific transthyretin promoter. Genetically modified PH1-iPSCs successfully provided hepatocyte-like cells (HLCs) that exhibited significant AGT expression at both RNA and protein levels after liver-specific differentiation process. These results pave the way for cell-based therapy of PH1 by transplantation of genetically modified autologous HLCs derived from patient-specific iPSCs.

Genetic regulation of dimethylarginines and endothelial dysfunction in rheumatoid arthritis.

Rheumatoid Arthritis (RA) confers an increased cardiovascular disease (CVD) risk which accounts for much of the premature morbidity and mortality observed in this population. Alterations in vascular function and morphology leading to increased atherosclerotic burden are considered the main drivers of CVD in RA individuals with systemic inflammation playing a key role in the dysregulation of endothelial homeostasis and initiation of vascular injury. Dimethylarginines are endogenous inhibitors of nitric oxide (NO) synthase and have emerged as novel, independent biomarkers of CVD in a wide range of conditions associated with vascular pathology. In RA several reports have demonstrated abnormal dimethylarginine metabolism attributable to various factors such as systemic inflammation, decreased degradation or upregulated synthesis. Although a causal relationship between dimethylarginines and vascular damage in RA has not been established, the tight interrelations between inflammation, dimethylarginines and endothelial dysfunction suggest that determination of dimethylarginine regulators may shed more light in the pathophysiology of the atherosclerotic process in RA and may also provide new therapeutic targets. The Alanine-Glyoxylate Aminotransferase 2 (AGTX2)-dependent pathway is a relatively recently discovered alternative pathway of dimethylarginine catabolism and its role on RA-related atherosclerotic disease is yet to be established. As factors affecting dimethylarginine concentrations linked to CVD risk and endothelial dysfunction are of prominent clinical relevance in RA, we present preliminary evidence that gene variants of AGTX-2 may influence dimethylarginine levels in RA patients and provide the rationale for larger studies in this field.

769 – Identifying Small Molecules to Correct Abnormal Protein Trafficking of Alanine-Glyoxylate Aminotransferase (AGT) in a Human Cell Model of Primary Hyperoxaluria Type I to Avoid Need for Liver Transplantation

769 – Identifying Small Molecules to Correct Abnormal Protein Trafficking of Alanine-Glyoxylate Aminotransferase (AGT) in a Human Cell Model of Primary Hyperoxaluria Type I to Avoid Need for Liver Transplantation

Metabolism of Oxalate in Humans: A Potential Role Kynurenine Aminotransferase/Glutamine Transaminase/Cysteine Conjugate Betalyase Plays in Hyperoxaluria.

Hyperoxaluria, excessive urinary oxalate excretion, is a significant health problem worldwide. Disrupted oxalate metabolism has been implicated in hyperoxaluria and accordingly, an enzymatic disturbance in oxalate biosynthesis can result in the primary hyperoxaluria. Alanine-glyoxylate aminotransferase-1 and glyoxylate reductase, the enzymes involving glyoxylate (precursor for oxalate) metabolism, have been related to primary hyperoxalurias. Some studies suggest that other enzymes such as glycolate oxidase and alanine-glyoxylate aminotransferase-2 might be associated with primary hyperoxaluria as well, but evidence of a definitive link is not strong between the clinical cases and gene mutations. There are still some idiopathic hyperoxalurias, which require a further study for the etiologies. Some aminotransferases, particularly kynurenine aminotransferases, can convert glyoxylate to glycine. Based on biochemical and structural characteristics, expression level, and subcellular localization of some aminotransferases, a number of them appear able to catalyze the transamination of glyoxylate to glycine more efficiently than alanine glyoxylate aminotransferase-1. The aim of this minireview is to explore other undermining causes of primary hyperoxaluria and stimulate research toward achieving a comprehensive understanding of underlying mechanisms leading to the disease. Herein, we reviewed all aminotransferases in the liver for their functions in glyoxylate metabolism. Particularly, kynurenine aminotransferase-I and III were carefully discussed regarding their biochemical and structural characteristics, cellular localization, and enzyme inhibition. Kynurenine aminotransferase-III is, so far, the most efficient putative mitochondrial enzyme to transaminate glyoxylate to glycine in mammalian livers, which might be an interesting enzyme to look for in hyperoxaluria etiology of primary hyperoxaluria and should be carefully investigated for its involvement in oxalate metabolism.

Human MiR-4660 regulates the expression of alanine-glyoxylate aminotransferase and may be a biomarker for idiopathic oxalosis.

Dysfunction of oxalate synthesis can cause calcium oxalate stone disease and inherited primary hyperoxaluria (PH) disorders. PH type I (PH1) is one of the most severe hyperoxaluria disorders, which results in urolithiasis, nephrocalcinosis, and end-stage renal disease. Here, we sought to determine the role of microRNAs in regulating AGXT to contribute to the pathogenesis of mutation-negative idiopathic oxalosis. We conducted bioinformatics to search for microRNAs binding to AGXT, and examined the expression of the highest hit (miR-4660) in serum samples of patients with oxalosis, liver tissue samples, and determined the correlation and regulation between the microRNA and AGXT in vitro. MiR-4660 expression was downregulated in patients with oxalosis compared with healthy controls (84.03 copies/µL vs 33.02 copies/µL, P < 0.0001). Moreover, miR-4660 epigenetically decreased the expression of AGT in human liver tissues (Rho = - 0543, P = 0.037). Overexpression of miR-4660 in HepG2 and L02 cell lines led to dysregulation of AGXT at both the mRNA (by 71% and 81%, respectively; P < 0.001) and protein (by 49% and 42%, respectively; P < 0.0001) levels. We confirmed the direct target site of miR-4660 binding to the 3'UTR of AGXT by a luciferase assay. MiR-4660 is probably a new biomarker for mutation-negative idiopathic oxalosis by regulating the post-transcription of AGXT, providing a potential treatment target of mutation-negative idiopathic oxalosis.