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Urat transportosome: recent knowledge of physiology and pathophysiology
Eisenreichová, Bára ; Stibůrková, Blanka (advisor) ; Janečková, Hana (referee)
The main topic and aim of this bachelor thesis is the description of the urate transportosome, its physiology and pathophysiology. The term "urate transportosome" collectively refers to the urate transporters that are predominantly located on the apical or basolateral membrane of the proximal tubule of the kidney. Here their main role is the transport of uric acid in the body. Uric acid is the end product of purine metabolism, if its physiological serum level decreases or increases, this condition is referred to as dysuricemia. Primary dysuricemia can be divided into hypouricemia and hyperuricemia. Both of these conditions result from a disorder of uric acid metabolism or transport. Key words: Uric acid, urate transport, dysuricemia, gout
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Identification of basolateral localization motifs of the urate transporter GLUT9 using functional studies.
Pácalová, Eliška ; Krylov, Vladimír (advisor) ; Pavelcová, Kateřina (referee)
The GLUT9 transporter, coded by the SLC2A9 gene, is one of the key proteins enabling the trasnfer of uric acid across the membrane in epithelial cells of the proximal tubule. In humans, this protein is naturally expressed in two variants: long (GLUT9L) and short (GLUT9S), which differ from one another by their N-terminus sequence. Each of these isoforms is localized on a different pole of the epithelial cell. The signal sequence/motif responsible for this difference is presumed to be located in the aforementioned N-terminus domain. Numerous allelic variants influencing the transport properties of the protein have also been described. The first aim of this thesis is to verify the influence of a newly discovered variant, characterized by substituting of valine for leucine in the 114th position (V114L, in the short form its corresponding variant V85L) on the ability to transport uric acid. Second aim is to verify the influence of mutations in selected motifs, which could be responsible for the localization of the protein, thus also changing its transport properties. Two dileucine motifs 12LGL14 and 33LL34 and one tyrosine motif 84YIKA87 were tested. Functional studies using 14 C radiolabeled urate demonstrated significant decrease of transport ability for the V114L/V85L allelic variant in both...
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Epithelial uric acid transport proteins and their allelic variants
Vrzáčková, Aneta ; Tlapáková, Tereza (advisor) ; Pavelcová, Kateřina (referee)
In humans, uric acid is the end product of purine metabolism and it is excreted from the body on the one hand by the epithelium lining the tubules of the nephrons, and on the other hand by the intestinal epithelium. This work aims to verify the activity of three candidate transporters from the SLC family (SLC2215, SLC22A18 and SLC47A1) that could transport uric acid in the intestinal epithelium. Preliminary results did not demonstrate that these transporters are involved in uric acid transport in the intestinal epithelium. Furthermore, the work deals with the influence of the W75C allelic variant of the SLC17A1 transporter and the mechanism by which this variant affects the transport of urate in cells. Key words: SLC22A15, SLC22A18, SLC47A1, SLC17A1, transport, epithel, uric acid
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Metabolism of uric acid during mammalian evolution
Mančíková, Andrea ; Krylov, Vladimír (advisor) ; Stibůrková, Blanka (referee)
Uric acid is the end product of purine metabolism of humans and some higher primates. In other mammals enzyme urate oxidase is capable to further convert uric acid to allantoin. During evolution of primates several mutations in urate oxidase gene had occured, led the enzyme to decrease its activity or to complete loss of function. What was the initial impetus for the utilization of uric acid and what biochemical traits of uric acid led to a tendency to retain a major of this metabolite production in the bloodstream? Uric acid has antioxidant capabilities and it is scavanger of free radicals. Increased levels of uric acid in blood has an effect on acute and chronic elevations of blood pressure. It is possible that hyperuricemia helped to maintain blood pressure under low-salt dietary conditions of primates during the Miocene. This mechanism probably have allowed to stabilize bipedalism our ancestors. Uric acid has an important role as a neuroprotector. As inhibitor the permeability blood-brain barrier, uric acid limits the infiltration of undesirable substances to the neurons and prevents central nervous system against the formation of inflammatory diseases. Such as neurodegenerative diseases may be caused by reduced serum uric acid levels. Uric acid protects against peroxinitrite damage tissues in the...
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Molecular Pathophysiology of Primary Hyperuricemia and Gout
Bohatá, Jana ; Stibůrková, Blanka (advisor) ; Hrnčíř, Zbyněk (referee) ; Friedecký, David (referee)
Primary hyperuricemia, as a condition of elevated serum uric acid levels, is caused by various factors and necessarily precedes a form of inflammatory arthritis referred to as gout. Uric acid is the end product of purine catabolism and requires specialized proteins for its transport. Pathogenic variants in the genes for these transport proteins can have a major negative impact on their function, thereby affecting the resulting serum uric acid levels. However, chronically elevated uric acid levels are not the only predisposition to the development of gout. Other factors, such as epigenetic mechanisms or genetic predispositions to inflammatory conditions caused by immune dysregulation, are likely to play a role in disease progression. The aim of the study was to analyse damaging variants in genes for important urate transporters ABCG2, SLC22A12 and SLC2A9, which may cause impaired excretion or reabsorption of uric acid and thus contribute to the development of primary hyperuricemia and gout, or rare hereditary renal hypouricemia. We also focused on circulating miRNAs in the plasma of patients with primary hyperuricemia, gout and gout attack. We identified and functionally characterized over ten rare nonsynonymous variants in the ABCG2 gene. Most of these variants had a negative impact on protein...
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The effect of urate transporter polymorphisms on uric acid excretion
Mančíková, Andrea ; Krylov, Vladimír (advisor) ; Novotný, Jiří (referee) ; Ježek, Petr (referee)
Uric acid excretion disorders are the most common cause of primary dysuricemia. The kidneys eliminate two-thirds of uric acid production and the other third is eliminated in the gastrointestinal tract. Renal reabsorption and secretion occur through the polarised epithelial cells in the proximal tubules. Uric acid transporters are expressed on these cell membranes. Reabsorption deficiency leads to hypouricemia and elevated fraction excretion associated with urolithiasis, nephrolithiasis or acute renal injury. Decreased uric acid secretion in the kidneys and small intestine leads to hyperuricemia, which develops into gout in 10% of individuals. Genome wide association studies detected a strong effect of SLC22A12 (URAT1), SLC2A9 (GLUT9) reabsorbing transporters and ABCG2 (ABCG2) secreting transporter on uric acid serum concentration variability. This thesis aimed to map out urate transporter allelic variants in a cohort of primary dysuricemia patients and identification of the variants causing defective uric acid excretion. Six non-synonymous variants were described in SLC22A12 (URAT1) and SLC2A9 (GLUT9) genes in hypouricemic individuals, which had not been identified previously in any population studies. Significant decreases in uric acid transport have been demonstrated experimentally in vitro,...
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Pathophysiology of urate transporters in primary gout
Pavelcová, Kateřina ; Stibůrková, Blanka (advisor) ; Doležel, Zdeněk (referee) ; Hrnčíř, Zbyněk (referee)
There are localised proteins (so-called urate transporters) in the renal proximal tubules and in the intestine, which excrete and reabsorb uric acid. Polymorphisms in the genes coding these proteins can result in the disruption of the transport function and development of hyperuricemia and gout. However the serum level of uric acid is also determined by other factors which include the intake of exogenous purines in food, synthesis of endogenous purines and degradation of nucleic acids, but also certain conditions. In 250 patients with primary hyperuricemia and gout we used Sanger sequencing to analyse the exons and adjacent intron regions in ten genes coding urate transporters: ABCG2, ABCC4, SLC2A9, SLC22A12, SLC22A11, SLC22A13, SLC17A1, SLC17A3, SLC22A6 and SLC22A8. We examined a possible connection between the identified genetic variants and primary hyperuricemia and gout based on a comparison of allele frequencies with the European population, according to topological models, according to programs predicting the functional impacts of variants and searches in specialised literature. We also took into account the conclusions of functional studies analysing the impact of nonsynonymous variants in the ABCG2 and SLC2A9 genes. We also focused on the effect of the concomitant occurrence of several...
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The effect of urate transporter polymorphisms on uric acid excretion
Mančíková, Andrea
Uric acid excretion disorders are the most common cause of primary dysuricemia. The kidneys eliminate two-thirds of uric acid production and the other third is eliminated in the gastrointestinal tract. Renal reabsorption and secretion occur through the polarised epithelial cells in the proximal tubules. Uric acid transporters are expressed on these cell membranes. Reabsorption deficiency leads to hypouricemia and elevated fraction excretion associated with urolithiasis, nephrolithiasis or acute renal injury. Decreased uric acid secretion in the kidneys and small intestine leads to hyperuricemia, which develops into gout in 10% of individuals. Genome wide association studies detected a strong effect of SLC22A12 (URAT1), SLC2A9 (GLUT9) reabsorbing transporters and ABCG2 (ABCG2) secreting transporter on uric acid serum concentration variability. This thesis aimed to map out urate transporter allelic variants in a cohort of primary dysuricemia patients and identification of the variants causing defective uric acid excretion. Six non-synonymous variants were described in SLC22A12 (URAT1) and SLC2A9 (GLUT9) genes in hypouricemic individuals, which had not been identified previously in any population studies. Significant decreases in uric acid transport have been demonstrated experimentally in vitro,...
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Determination of selected components in human urine with electrophoresis in short capillary.
Makrlíková, Anna
Capillary zone electrophoresis is frequently used in various analyses. In this diploma thesis a hydrodynamic sample introduction method controlled by pressure pulse has been proposed for short-capillary electrophoresis. The base electrolyte flushes sample from the loop of a six-way sampling valve and is carried to the injection end of the capillary. At the time when the sample zone reached the capillary, a short pressure impulse is generated in the electrolyte stream, which provides injection of the sample into the capillary. Then the electrolyte flow is stopped and the separation voltage is turned on. The amount of sample introduced to the capillary is controlled by the duration of the pressure pulse. This new sample introduction method was tested in the determination of ammonia, histidine, creatinine, uric acid and hippuric acid in human urine and for rapid screening of the contents of the inorganic ions in cerebrospinal fluid and blood plasma. The determination was performed in a capillary with an overall length of 10,5 cm and two base electrolytes was tested - 50 mM MES + 5 mM NaOH (pH 5,10) and 1 M acetic acid + 1,5 mM crown ether 18-crown-6 (pH 2,40). Using dual detection techniques contactless conductivity and UV spectrometric detection, anorganic and organic substances in the sample could...
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Research of vitreous and vitreoretinal interface in microvascular retinal disorders focussed on eye complications of diabetes mellitus
Křížová, Libuše ; Zima, Tomáš (advisor) ; Průša, Richard (referee) ; Sosna, Tomáš (referee)
In this work I present conclusions of clinical-laboratory research focused on the patients with diabetic macular edema (DME). We performed biochemical and immunochemical analyses of vitreous samples that were collected during the pars plana vitrectomy. Moreover, at patients with non-proliferative diabetic retinopathy (NPDR) we assessed morphological characteristics of DME using optical coherence tomography (OCT). According to our findings, the vitreous and serum concentrations of uric acid and glucose were significantly higher in patients with diabetic retinopathy and DME compared to controls. Also total ratio (serum/ vitreous concentration) of uric acid and glucose was in diabetics significantly higher than in controls. The most important determinant of increasing concentration of both uric acid and glucose in the vitreous was the grade of diabetic retinopathy. Moreover, we demonstrated significant correlation between vitreous concentration of uric acid and concentration of the vascular endothelial growth factor (VEGF) in patients with DME and NPDR. We found further, that the volume of the macula (cube volume - CV) computed with the software of Cirrus HD-OCT correlates in diabetics significantly with the vitreous VEGF concentration, but not with uric acid. This OCT parameter could be used to...
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