Adenine Phosphoribosyltransferase Research Articles

590-P: Modulation of Adenine Phosphoribosyltransferase-Mediated Purine Salvage to Accelerate Diabetic Wound Healing

Aim: Adenine phosphoribosyltransferase (APRT) is the key enzyme in purine salvage by the incorporation of adenine and phosphoribosyl pyrophosphate to provide adenylate nucleotide. Our aim was to evaluate the role of APRT in diabetic wound healing. Methods: The molecular mechanism of APRT-mediated cell survival was validated by the haploid HAP1 cell. The adenylate nucleotides in NIH-3T3 fibroblast response to adenine was monitoring by HPLC-MS/MS method. The effects of cell behaviors were evaluated by mice NIH-3T3 fibroblast and human epithelial keratinocytes. Genetically diabetic mice received surgery for full-thickness of the cutaneous wound and the adenine addition as the substrate for APRT on the wound. Results: In the wild type and APRT knockout HAP1 cell, our data revealed that APRT utilized adenine to rescue cellular ATP levels and proliferation against hydrogen peroxide-induced oxidative damage. The time-course of monitoring the total adenylate nucleotides in NIH-3T3 fibroblast showed that adenine addition enlarged the cellular adenylate pool, reduced the adenylate energy charge, and provided more AMP for the generation of ATP in further. In mice NIH-3T3 fibroblasts and human NHEK keratinocytes, adenine elevated cellular ATP levels in a dose-dependent manner and facilitated cell migration. The up-regulated APRT found in wound skin was correlated with the demands of wound repair in diabetic mice. Administration of adenine on the wound of diabetic mice exhibited elevated ATP levels in organismic skin and accelerated wound healing. Conclusions: Our data indicated the role of APRT during diabetic wound healing by regulating the nucleotide pool after injury and demonstrated the improvement by topical adenine, which highlights its value as a promising agent in therapeutic intervention. This study provided an explanation for the up-regulation of APRT in tissue repair and adenine supplement resulted in an enlargement of the adenylate pool for ATP generation. Disclosure G. Young: Employee; Self; Energenesis-Biomedical. J. Lin: Employee; Self; Energenesis-Biomedical. Y. Cheng: Employee; Self; Energenesis-Biomedical. H. Chen: Board Member; Self; Energenesis-Biomedical.

Are conventional stone analysis techniques reliable for the identification of 2,8-dihydroxyadenine kidney stones? A case series.

We have recently encountered patients incorrectly diagnosed with adenine phosphoribosyltransferase (APRT) deficiency due to misidentification of kidney stones as 2,8-dihydroxyadenine (DHA) stones. The objective of this study was to examine the accuracy of stone analysis for identification of DHA. Medical records of patients referred to the APRT Deficiency Research Program of the Rare Kidney Stone Consortium in 2010-2018 with a diagnosis of APRT deficiency based on kidney stone analysis were reviewed. The diagnosis was verified by measurement of APRT enzyme activity or genetic testing. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectra of pure crystalline DHA and a kidney stone obtained from one of the confirmed APRT deficiency cases were generated. The ATR-FTIR spectrum of the kidney stone matched the crystalline DHA spectrumand was used for comparison with available infrared spectra of stone samples from the patients. Of 17 patients referred, 14 had sufficient data available to be included in the study. In all 14 cases, the stone analysis had been performed by FTIR spectroscopy. The diagnosis of APRT deficiency was confirmed in seven cases and rejected in the remaining seven cases. Comparison of the ATR-FTIR spectrum of the DHA stone with the FTIR spectra from three patients who did not have APRT deficiency showed no indication of DHA as a stone component. Misidentification of DHA as a kidney stone component by clinical laboratories appears common among patients referred to our program. Since current clinical protocols used to interpret infrared spectra for stone analysis cannot be considered reliable for the identification of DHA stones, the diagnosis of APRT deficiency must be confirmed by other methods.

Cellular and Molecular Mechanisms of Kidney Injury in 2,8-Dihydroxyadenine Nephropathy.

Hereditary deficiency of adenine phosphoribosyltransferase causes 2,8-dihydroxyadenine (2,8-DHA) nephropathy, a rare condition characterized by formation of 2,8-DHA crystals within renal tubules. Clinical relevance of rodent models of 2,8-DHA crystal nephropathy induced by excessive adenine intake is unknown. Using animal models and patient kidney biopsies, we assessed the pathogenic sequelae of 2,8-DHA crystal-induced kidney damage. We also used knockout mice to investigate the role of TNF receptors 1 and 2 (TNFR1 and TNFR2), CD44, or alpha2-HS glycoprotein (AHSG), all of which are involved in the pathogenesis of other types of crystal-induced nephropathies. Adenine-enriched diet in mice induced 2,8-DHA nephropathy, leading to progressive kidney disease, characterized by crystal deposits, tubular injury, inflammation, and fibrosis. Kidney injury depended on crystal size. The smallest crystals were endocytosed by tubular epithelial cells. Crystals of variable size were excreted in urine. Large crystals obstructed whole tubules. Medium-sized crystals induced a particular reparative process that we term extratubulation. In this process, tubular cells, in coordination with macrophages, overgrew and translocated crystals into the interstitium, restoring the tubular luminal patency; this was followed by degradation of interstitial crystals by granulomatous inflammation. Patients with adenine phosphoribosyltransferase deficiency showed similar histopathological findings regarding crystal morphology, crystal clearance, and renal injury. In mice, deletion of Tnfr1 significantly reduced tubular CD44 and annexin two expression, as well as inflammation, thereby ameliorating the disease course. In contrast, genetic deletion of Tnfr2, Cd44, or Ahsg had no effect on the manifestations of 2,8-DHA nephropathy. Rodent models of the cellular and molecular mechanisms of 2,8-DHA nephropathy and crystal clearance have clinical relevance and offer insight into potential future targets for therapeutic interventions.

Crystalline nephropathy due to adenine phosphoribosyl transferase deficiency as a cause of renal allograft dysfunction

APRT deficiency is a rare but under recognized genetic disease. Recurrent urolithiasis and DHA nephropathy are the two clinical manifestations of APRT deficiency and diagnosis can be made at any age and recurs after renal transplant. Allopurinol is the cornerstone in preventing recurrence. APRT activity assay and genetic testing are useful for confirmation of diagnosis, for family screening and in difficult cases of urolithias or crystalline nephropathy.

Kidney Transplant Outcomes in Patients With Adenine Phosphoribosyltransferase Deficiency.

Adenine phosphoribosyltransferase (APRT) deficiency is a rare, hereditary cause of kidney stones and chronic kidney disease (CKD) which is characterized by 2,8-dihydroxyadenine renal parenchymal crystal deposition. The aim of this study was to examine outcomes of kidney transplantation in APRT deficiency patients. Included were 13 patients in the APRT Deficiency Registry of the Rare Kidney Stone Consortium, 2 from Westmead Hospital in Sydney, Australia, and 2 from Necker Hospital in Paris, France. The CKD-EPI and CKiD equations were used to calculate glomerular filtration rate estimates. Allograft survival was analyzed employing the Kaplan-Meier method. The Wilcoxon-Mann-Whitney test was used to compare alllograft outcomes according to xanthine oxidoreductase (XOR) inhibitor treatment status at transplantation. Seventeen patients (9 females) received 22 kidney transplants. Age at first transplantation was 47.2 (14.9-67.0) years. Ten patients received XOR inhibitor therapy pretransplant (11 allografts), while 8 patients did not receive such treatment before transplantation (11 allografts). Two-year allograft survival was 91% and 55% in the 2 groups, respectively (P = 0.16). The median (range) estimated glomerular filtration rate at 2 years posttransplant was 61.3 (24.0-90.0) mL/min/1.73 m when XOR inhibitor therapy was initiated before transplantation, and 16.2 (10.0-39.0) mL/min/1.73 m (P = 0.009) when such treatment was not administered pretransplant. Kidney allograft outcomes are good in APRT deficiency patients beginning XOR inhibitor therapy pretransplant. Delay in such treatment is a major cause of premature graft loss in these patients. Increased awareness among clinicians is imperative, promoting early diagnosis of APRT deficiency and pharmacotherapy initiation before kidney transplantation.

Transcriptome analysis of two inflorescence branching mutants reveals cytokinin is an important regulator in controlling inflorescence architecture in the woody plant Jatropha curcas

BackgroundIn higher plants, inflorescence architecture is an important agronomic trait directly determining seed yield. However, little information is available on the regulatory mechanism of inflorescence development in perennial woody plants. Based on two inflorescence branching mutants, we investigated the transcriptome differences in inflorescence buds between two mutants and wild-type (WT) plants by RNA-Seq to identify the genes and regulatory networks controlling inflorescence architecture in Jatropha curcas L., a perennial woody plant belonging to Euphorbiaceae.ResultsTwo inflorescence branching mutants were identified in germplasm collection of Jatropha. The duo xiao hua (dxh) mutant has a seven-order branch inflorescence, and the gynoecy (g) mutant has a three-order branch inflorescence, while WT Jatropha has predominantly four-order branch inflorescence, occasionally the three- or five-order branch inflorescences in fields. Using weighted gene correlation network analysis (WGCNA), we identified several hub genes involved in the cytokinin metabolic pathway from modules highly associated with inflorescence phenotypes. Among them, Jatropha ADENOSINE KINASE 2 (JcADK2), ADENINE PHOSPHORIBOSYL TRANSFERASE 1 (JcAPT1), CYTOKININ OXIDASE 3 (JcCKX3), ISOPENTENYLTRANSFERASE 5 (JcIPT5), LONELY GUY 3 (JcLOG3) and JcLOG5 may participate in cytokinin metabolic pathway in Jatropha. Consistently, exogenous application of cytokinin (6-benzyladenine, 6-BA) on inflorescence buds induced high-branch inflorescence phenotype in both low-branch inflorescence mutant (g) and WT plants. These results suggested that cytokinin is an important regulator in controlling inflorescence branching in Jatropha. In addition, comparative transcriptome analysis showed that Arabidopsis homologous genes Jatropha AGAMOUS-LIKE 6 (JcAGL6), JcAGL24, FRUITFUL (JcFUL), LEAFY (JcLFY), SEPALLATAs (JcSEPs), TERMINAL FLOWER 1 (JcTFL1), and WUSCHEL-RELATED HOMEOBOX 3 (JcWOX3), were differentially expressed in inflorescence buds between dxh and g mutants and WT plants, indicating that they may participate in inflorescence development in Jatropha. The expression of JcTFL1 was downregulated, while the expression of JcLFY and JcAP1 were upregulated in inflorescences in low-branch g mutant.ConclusionsCytokinin is an important regulator in controlling inflorescence branching in Jatropha. The regulation of inflorescence architecture by the genes involved in floral development, including TFL1, LFY and AP1, may be conservative in Jatropha and Arabidopsis. Our results provide helpful information for elucidating the regulatory mechanism of inflorescence architecture in Jatropha.

How a purine salvage enzyme singles out the right base

Two phosphoribosyltransferases in the purine salvage pathway exhibit exquisite substrate specificity despite the chemical similarity of their distinct substrates, but the basis for this discrimination was not fully understood. Ozeir et al. now employ a complementary biochemical, structural, and computational approach to deduce the chemical constraints governing binding and propose a distinct mechanism for catalysis in one of these enzymes, adenine phosphoribosyltransferase. These insights, built on data from an unexpected finding, finally provide direct answers to key questions regarding these enzymes and substrate recognition more generally.

Structural basis for substrate selectivity and nucleophilic substitution mechanisms in human adenine phosphoribosyltransferase catalyzed reaction

The reversible adenine phosphoribosyltransferase enzyme (APRT) is essential for purine homeostasis in prokaryotes and eukaryotes. In humans, APRT (hAPRT) is the only enzyme known to produce AMP in cells from dietary adenine. APRT can also process adenine analogs, which are involved in plant development or neuronal homeostasis. However, the molecular mechanism underlying substrate specificity of APRT and catalysis in both directions of the reaction remains poorly understood. Here we present the crystal structures of hAPRT complexed to three cellular nucleotide analogs (hypoxanthine, IMP, and GMP) that we compare with the phosphate-bound enzyme. We established that binding to hAPRT is substrate shape-specific in the forward reaction, whereas it is base-specific in the reverse reaction. Furthermore, a quantum mechanics/molecular mechanics (QM/MM) analysis suggests that the forward reaction is mainly a nucleophilic substitution of type 2 (SN2) with a mix of SN1-type molecular mechanism. Based on our structural analysis, a magnesium-assisted SN2-type mechanism would be involved in the reverse reaction. These results provide a framework for understanding the molecular mechanism and substrate discrimination in both directions by APRTs. This knowledge can play an instrumental role in the design of inhibitors, such as antiparasitic agents, or adenine-based substrates.

Inverted Regulation of Multidrug Efflux Pumps, Acid Resistance, and Porins in Benzoate-Evolved Escherichia coli K-12.

Benzoic acid, a partial uncoupler of the proton motive force (PMF), selects for sensitivity to chloramphenicol and tetracycline during the experimental evolution of Escherichia coli K-12. Transcriptomes of E. coli isolates evolved with benzoate showed the reversal of benzoate-dependent regulation, including the downregulation of multidrug efflux pump genes, the gene for the Gad acid resistance regulon, the nitrate reductase genes narHJ, and the gene for the acid-consuming hydrogenase Hyd-3. However, the benzoate-evolved strains had increased expression of OmpF and other large-hole porins that admit fermentable substrates and antibiotics. Candidate genes identified from benzoate-evolved strains were tested for their roles in benzoate tolerance and in chloramphenicol sensitivity. Benzoate or salicylate tolerance was increased by deletion of the Gad activator ariR or of the acid fitness island from slp to the end of the gadX gene encoding Gad regulators and the multidrug pump genes mdtEF Benzoate tolerance was also increased by deletion of multidrug component gene emrA, RpoS posttranscriptional regulator gene cspC, adenosine deaminase gene add, hydrogenase gene hyc (Hyd-3), and the RNA chaperone/DNA-binding regulator gene hfq Chloramphenicol resistance was decreased by mutations in genes for global regulators, such as RNA polymerase alpha subunit gene rpoA, the Mar activator gene rob, and hfq Deletion of lipopolysaccharide biosynthetic kinase gene rfaY decreased the rate of growth in chloramphenicol. Isolates from experimental evolution with benzoate had many mutations affecting aromatic biosynthesis and catabolism, such as aroF (encoding tyrosine biosynthesis) and apt (encoding adenine phosphoribosyltransferase). Overall, benzoate or salicylate exposure selects for the loss of multidrug efflux pumps and of hydrogenases that generate a futile cycle of PMF and upregulates porins that admit fermentable nutrients and antibiotics.IMPORTANCE Benzoic acid is a common food preservative, and salicylic acid (2-hydroxybenzoic acid) is the active form of aspirin. At high concentrations, benzoic acid conducts a proton across the membrane, depleting the proton motive force. In the absence of antibiotics, benzoate exposure selects against proton-driven multidrug efflux pumps and upregulates porins that admit fermentable substrates but that also allow the entry of antibiotics. Thus, evolution with benzoate and related molecules, such as salicylates, requires a trade-off for antibiotic sensitivity, a trade-off that could help define a stable gut microbiome. Benzoate and salicylate are naturally occurring plant signal molecules that may modulate the microbiomes of plants and animal digestive tracts so as to favor fermenters and exclude drug-resistant pathogens.

SUN-328 ADENINE PHOSPHORIBOSYLTRANSFERASE DEFICIENCY: A POTENTIALLY REVERSIBLE CAUSE OF CHRONIC KIDNEY DISEASE IN THE PRE AND POST TRANSPLANT SETTING

Adenine phosphoribosyltransferase (APRT) deficiency is a rare, autosomal recessive disorder which results in increased production of 2,8-dihydroxyadenine (DHA) viathe purine salvage pathway. Excess DHA is excreted in the urine, where it forms highly insoluble crystals and manifests as either urolithiasis or crystalline nephropathy. The onset and presentation of disease is variable and may present as chronic kidney disease of unknown aetiology; renal calculi; misdiagnosed oxalate nephropathy; graft dysfunction following renal transplantation; or through family screening. DHA crystals have distinct appearance on renal biopsy and urine microscopy, but is easily misdiagnosed or unrecognized due to low clinical suspicion. Diagnosis is of paramount importance; as chronic renal injury can be potentially can be minimized with xanthine oxidase inhibitors (allopurinol or feboxustat). We present cases of 3 cases of APRT deficiency (2 patients were post cadevaric renal transplantation). Patient characteristics pre and post transplantation will be presented in a table, including information on renal function, oxipurinol levels and urine microscopy. Indication and protocol transplant renal biopsies were processed as standard protocol and evaluated for rejection (according to Banff criteria) and crystal nephropathy (percentage of biopsy affected and distribution in luminal, epithelial and interstitial compartments). Both transplant patients had early recurrence of crystalline nephropathy (figure 1) and necessitated rapid escalation of xanthine oxidase inhibitors to levels exceeding recommendations for renal impairment. Both patients were also treated for borderline rejection and this highlights the difficulties in delineating rejection from crystal-associated inflammation. One patient developed Banff 2A rejection but both patients suffered infective complications of excess immunosuppression. Review of all available transplant biopsies revealed interesting kinetics of crystal formation and deposition in the kidney. Early in the disease, with maximal excretion of DHA metabolites in urine leads to crystal precipitation, luminal aggregate with tubular obstruction and injury. These crystals then are seen in the epithelial layer (either by encapsulation or endocytosis). Later in the disease, there is associated tubular rupture and interstitial deposition with associated interstitial fibrosis and tubular atrophy (figure 2). The amount of luminal crystals reduce over time with increasing dose of allopurinol and/or feboxustat. Lack of awareness and clinical suspicion appear to be a major barrier to timely diagnosis, although multiple management challenges post diagnosis remains. The current target levels and biomarkers of adequacy of therapy with allopurinol and/or feboxustat remains to be determined.

Glutamate Dehydrogenase from Thermus thermophilus Is Activated by AMP and Leucine as a Complex with Catalytically Inactive Adenine Phosphoribosyltransferase Homolog.

Glutamate dehydrogenase (GDH) from a thermophilic bacterium, Thermus thermophilus, is composed of two heterologous subunits, GdhA and GdhB. In the heterocomplex, GdhB acts as the catalytic subunit, whereas GdhA lacks enzymatic activity and acts as the regulatory subunit for activation by leucine. In the present study, we performed a pulldown assay using recombinant T. thermophilus, producing GdhA fused with a His tag at the N terminus, and found that TTC1249 (APRTh), which is annotated as adenine phosphoribosyltransferase but lacks the enzymatic activity, was copurified with GdhA. When GdhA, GdhB, and APRTh were coproduced in Escherichia coli cells, they were purified as a ternary complex. The ternary complex exhibited GDH activity that was activated by leucine, as observed for the GdhA-GdhB binary complex. Furthermore, AMP activated GDH activity of the ternary complex, whereas such activation was not observed for the GdhA-GdhB binary complex. This suggests that APRTh mediates the allosteric activation of GDH by AMP. The present study demonstrates the presence of complicated regulatory mechanisms of GDH mediated by multiple compounds to control the carbon-nitrogen balance in bacterial cells.IMPORTANCE GDH, which catalyzes the synthesis and degradation of glutamate using NAD(P)(H), is a widely distributed enzyme among all domains of life. Mammalian GDH is regulated allosterically by multiple metabolites, in which the antenna helix plays a key role to transmit the allosteric signals. In contrast, bacterial GDH was believed not to be regulated allosterically because it lacks the antenna helix. We previously reported that GDH from Thermus thermophilus (TtGDH), which is composed of two heterologous subunits, is activated by leucine. In the present study, we found that AMP activates TtGDH using a catalytically inactive APRTh as the sensory subunit. This suggests that T. thermophilus possesses a complicated regulatory mechanism of GDH to control carbon and nitrogen metabolism.

Urinary 2,8-dihydroxyadenine excretion in patients with adenine phosphoribosyltransferase deficiency, carriers and healthy control subjects

BackgroundAdenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder of adenine metabolism that results in excessive urinary excretion of the poorly soluble 2,8-dihydroxyadenine (DHA), leading to kidney stones and chronic kidney disease. The purpose of this study was to assess urinary DHA excretion in patients with APRT deficiency, heterozygotes and healthy controls, using a recently developed ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) assay. MethodsPatients enrolled in the APRT Deficiency Registry and Biobank of the Rare Kidney Stone Consortium (http://www.rarekidneystones.org/) who had provided 24-h and first-morning void urine samples for DHA measurement were eligible for the study. Heterozygotes and healthy individuals served as controls. Wilcoxon-Mann-Whitney test was used to compare 24-h urinary DHA excretion between groups. Associations were examined using Spearman's correlation coefficient (rs). ResultsThe median (range) 24-h urinary DHA excretion was 138 (64–292) mg/24 h and the DHA-to-creatinine (DHA/Cr) ratio in the first-morning void samples was 13 (4–37) mg/mmol in APRT deficiency patients who were not receiving xanthine oxidoreductase inhibitor therapy. The 24-h DHA excretion was highly correlated with the DHA/Cr ratio in first-morning void urine samples (rs = 0.84, p < .001). DHA was detected in all urine samples from untreated patients but not in any specimens from heterozygotes and healthy controls. ConclusionsHigh urinary DHA excretion was observed in patients with APRT deficiency, while urine DHA was undetectable in heterozygotes and healthy controls. Our results suggest that the UPLC-MS/MS assay can be used for diagnosis of APRT deficiency.

Adenine Phosphoribosyltransferase Deficiency: A Potentially Reversible Cause of CKD

Adenine Phosphoribosyltransferase Deficiency: A Potentially Reversible Cause of CKD

Efficient Editing of the Nuclear APT Reporter Gene in Chlamydomonas reinhardtii via Expression of a CRISPR-Cas9 Module.

The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is a versatile and useful tool to perform genome editing in different organisms ranging from bacteria and yeast to plants and mammalian cells. For a couple of years, it was believed that the system was inefficient and toxic in the alga Chlamydomonas reinhardtii. However, recently the system has been successfully implemented in this model organism, albeit relying mostly on the electroporation of ribonucleoproteins (RNPs) into cell wall deficient strains. This requires a constant source of RNPs and limits the application of the technology to strains that are not necessarily the most relevant from a biotechnological point of view. Here, we show that transient expression of the Streptococcus pyogenes Cas9 gene and sgRNAs, targeted to the single-copy nuclear apt9 gene, encoding an adenine phosphoribosyl transferase (APT), results in efficient disruption at the expected locus. Introduction of indels to the apt9 locus results in cell insensitivity to the otherwise toxic compound 2-fluoroadenine (2-FA). We have used agitation with glass beads and particle bombardment to introduce the plasmids carrying the coding sequences for Cas9 and the sgRNAs in a cell-walled strain of C. reinhardtii (CC-125). Using sgRNAs targeting exons 1 and 3 of apt9, we obtained disruption efficiencies of 3 and 30% on preselected 2-FA resistant colonies, respectively. Our results show that transient expression of Cas9 and a sgRNA can be used for editing of the nuclear genome inexpensively and at high efficiency. Targeting of the APT gene could potentially be used as a pre-selection marker for multiplexed editing or disruption of genes of interest.

Structural and functional characterization of thermostable biocatalysts for the synthesis of 6-aminopurine nucleoside-5′-monophospate analogues

The present work describes the functional and structural characterization of adenine phosphoribosyltransferase 2 from Thermus thermophilus HB8 (TtAPRT2). The combination of structural and substrate specificity data provided valuable information for immobilization studies. Dimeric TtAPRT2 was immobilized onto glutaraldehyde-activated MagReSyn®Amine magnetic iron oxide porous microparticles by two different strategies: a) an enzyme immobilization at pH 8.5 to encourage the immobilization process by N-termini (MTtAPRT2A, MTtAPRT2B, MTtAPRT2C) or b) an enzyme immobilization at pH 10.0 to encourage the immobilization process through surface exposed lysine residues (MTtAPRT2D, MTtAPRT2E, MTtAPRT2F). According to catalyst load experiments, MTtAPRT2B (activity: 480 IU g−1biocatalyst, activity recovery: 52%) and MTtAPRT2F (activity: 507 IU g−1biocatalyst, activity recovery: 44%) were chosen as optimal derivatives. The biochemical characterization studies demonstrated that immobilization process improved the thermostability of TtAPRT2. Moreover, the potential reusability of MTtAPRT2B and MTtAPRT2F was also tested. Finally, MTtAPRT2F was employed in the synthesis of nucleoside-5′-monophosphate analogues.

Genetic and Biochemical Features of the Monogenic Hereditary Kidney Stone Disease

Kidney stone disease (KSD) is a common urological problem. In most cases, this multifactorial pathology develops due to the combination of inherited low-penetrance gene variants and environment factors such as urinary tract infections, unbalanced diet, and gastrointestinal tract diseases with malabsorption. However, some cases of KSD represent monogenic diseases in which mutations are inherited from one or both parents. These hereditary forms of KSD, manifested in childhood, are characterized by multiple, bilateral or recurrent kidney stones and the earlier development of chronic kidney disease. Introduction of methods for exome and gene panels (NGS—Next Generation Sequencing) sequencing, significantly increased the proportion of detectable genetically identifiable hereditary forms of KSD in the group of patients under 18 year old, which exceeded 20%. In this review we have analyzed genetic and biochemical mechanisms of the main hereditary forms of KSD, associated with enzymopathies (primary hyperoxaluria, adenine phosphoribosyltransferase deficiency, phosphoribosylpyrophosphate synthetase superactivity, xanthinuria, Lesch-Nyhan syndrome) and impaired membrane transport (Dent’s disease, familial hypomagnesemia with hypercalciuria and nephrocalcinosis, hypophosphatemic urolithiasis, distal tubular acidosis, cystinuria, Bartter’s syndrome). We suggest a comprehensive gene panel for NGS diagnostics of the hereditary KSD. Accurate and timely diagnosis of hereditary forms of urolithiasis makes it possible to identify a pathological germinal mutation and make an accurate diagnosis, to analyze the heterozygous mutation carriage in affected families and evaluate the prognosis of KSD development in family members, as well as to perform personalized management of the patients with KSD.

Thermophilic phosphoribosyltransferases Thermus thermophilus HB27 in nucleotide synthesis.

Phosphoribosyltransferases are the tools that allow the synthesis of nucleotide analogues using multi-enzymatic cascades. The recombinant adenine phosphoribosyltransferase (TthAPRT) and hypoxanthine phosphoribosyltransferase (TthHPRT) from Thermus thermophilus HB27 were expressed in E.coli strains and purified by chromatographic methods with yields of 10–13 mg per liter of culture. The activity dependence of TthAPRT and TthHPRT on different factors was investigated along with the substrate specificity towards different heterocyclic bases. The kinetic parameters for TthHPRT with natural substrates were determined. Two nucleotides were synthesized: 9-(β-D-ribofuranosyl)-2-chloroadenine 5'-monophosphate (2-Сl-AMP) using TthAPRT and 1-(β-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine-4-one 5'-monophosphate (Allop-MP) using TthНPRT.

Dependence of Leishmania parasite on host derived ATP: an overview of extracellular nucleotide metabolism in parasite.

The widespread role of ATP in humans has been characterized as a substrate for metabolism as well as other key processes occurring in the human system. Hence, the ATP pool may also be accessible to the invaders, including Leishmania donovani (a protozoan parasite), considering the fact that ATP is not scarce at the cellular location where the pathogen resides. The protozoan parasite survives in the human host by utilizing purines from its extracellular environment, due to its inability to synthesize purines de novo. The purines are accessible in the form of nucleoside triphosphates (NTPs), for example, adenosine (ADO) in the form of ATP molecules. These NTPs are processed by the ecto-nucleotidases in the transmembrane region of the parasite, which are then transported inside via nucleoside/nucleobase transporters belonging to the ENT family of transporters. Besides, the breakdown of NTPs by ecto-nucleotidases also yields inorganic phosphate (Pi) as by-product which is utilized by the parasite to maintain Pi homeostasis. These transporters have been characterized in protozoan parasites exhibiting homology in various species of Leishmania. Once inside the parasite, these purines (or their derivatives) are fluxed into purine metabolic pathways with the help of several cytosolic enzymes, prominently, adenosine deaminase, adenine amino hydrolase, phosphoribosyl transferases (APRT, HGPRT and XPRT) and adenosine kinase. This review outlines the predominant role of extracellular nucleotide metabolism and intracellular metabolic machinery in the containment of leishmanial infection. It also highlights the importance of inorganic phosphate transporter in relation to the purine transport.

Long-term renal outcomes of APRT deficiency presenting in childhood.

Adenine phosphoribosyltransferase (APRT) deficiency is a hereditary purine metabolism disorder that causes kidney stones and chronic kidney disease (CKD). The purpose of this study was to examine the course of APRT deficiency in patients who presented in childhood. The disease course of 21 (35%) patients in the APRT Deficiency Registry of the Rare Kidney Stone Consortium, who presented with manifestations of APRT deficiency and/or were diagnosed with the disorder before the age of 18years, was studied. The effect of pharmacotherapy on renal manifestations and outcomes was thoroughly assessed. Fourteen children were placed on allopurinol, 100 (25-200) mg/day, at the age of 2.6 (0.6-16.5) years. Six of these patients had experienced kidney stone events and three had developed acute kidney injury (AKI) prior to allopurinol treatment. During 18.9 (1.7-31.5) years of pharmacotherapy, stones occurred in two patients and AKI in three. Six adult patients started allopurinol treatment, 200 (100-300) mg/day, at age 29.8 (20.5-42.4) years. Five of these patients had experienced 28 stone episodes and AKI had occurred in two. Stone recurrence occurred in four patients and AKI in two during 11.2 (4.2-19.6) years of allopurinol therapy. Lack of adherence and insufficient dosing contributed to stone recurrence and AKI during pharmacotherapy. At latest follow-up, estimated glomerular filtration rate (eGFR) was 114 (70-163) and 62 (10-103) mL/min/1.73m2 in those who initiated treatment as children and adults, respectively. All three patients with CKD stages 3-5 at the last follow-up were adults when pharmacotherapy was initiated. Timely diagnosis and treatment of APRT deficiency decreases renal complications and preserves kidney function.

Dihydroxyadenine crystal-induced nephropathy presenting with rapidly progressive renal failure.

Adenine phosphoribosyltransferase enzyme deficiency is a rare, autosomal recessive disorder. It is a disease limited to the renal system and usually presents with urolithiasis. Herein, we report a young female with dihydroxyadenine (DHA) crystal-induced nephropathy presenting with rapidly progressive renal failure. DHA crystals can be easily diagnosed by their pathognomic color and shape in urine and biopsy specimens. A high index of clinical suspicion helps in the early diagnosis of this potentially treatable renal disease.