Analysis of Purine Metabolism to Elucidate the Pathogenesis of Acute Kidney Injury in Renal Hypouricemia.

Renal hypouricemia is a disease caused by the dysfunction of renal urate transporters. This disease is known to cause exercise-induced acute kidney injury, but its mechanism has not yet been established. To analyze the mechanism by which hypouricemia causes renal failure, we conducted a semi-ischemic forearm exercise stress test to mimic exercise conditions in five healthy subjects, six patients with renal hypouricemia, and one patient with xanthinuria and analyzed the changes in purine metabolites. The results showed that the subjects with renal hypouricemia had significantly lower blood hypoxanthine levels and increased urinary hypoxanthine excretion after exercise than healthy subjects. Oxidative stress markers did not differ between healthy subjects and hypouricemic subjects before and after exercise, and no effect of uric acid as a radical scavenger was observed. As hypoxanthine is a precursor for adenosine triphosphate (ATP) production via the salvage pathway, loss of hypoxanthine after exercise in patients with renal hypouricemia may cause ATP loss in the renal tubules and consequent tissue damage.

Modified forearm ischemic test in hypouricemic patients.

Renal hypouricemia sometimes leads to exercise-induced acute kidney injury (EIAKI) of unknown pathogenesis. In order to elucidate the various pathological conditions associated with hypouricemia, we analyzed the effects of low uric acid level on energy metabolism. We have modified semi-ischemic forearm exercise test and performed this test in one Japanese healthy volunteer, three patients with hereditary renal hypouricemia and one patient with hereditary xanthinuria of Czech origin. Forearm exercise was performed by squeezing a hand dynamometer with the sphygmomanometer cuff pressure kept at the mean arterial pressure. Venous blood was drawn five times (before exercise, 3, 10, 30, 45 minutes after the start of exercise) in each tests. The mean plasma lactate concentration increased from a baseline of 1.3 (range 0.7-1.8 mmol/L) to 4.0 (range 2.0-5.5 mmol/L) at 3 minutes after the start of exercise. The plasma hypoxanthine concentrations were quite low before exercise (0-2.9 µmol/L), but increased markedly to a range of 13.6-28.8 µmol/L after 10 minute forearm ischemia. Our protocol allowed us to conclude that the load was sufficient for observing metabolic changes in temporally hypoxia and in following recovery phase. The test was well tolerated and safe, we did not observe any adverse reactions including EIAKI.

Genetic background of uric acid metabolism in a patient with severe chronic tophaceous gout.

Hyperuricemia depends on the balance of endogenous production and renal excretion of uric acid. Transporters for urate are located in the proximal tubule where uric acid is secreted and extensively reabsorbed: secretion is principally ensured by the highly variable ABCG2 gene. Enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) plays a central role in purine metabolism and its deficiency is an X-linked inherited metabolic disorder associated with clinical manifestations of purine overproduction. Here we report the case of a middle-aged man with severe chronic tophaceous gout with a poor response to allopurinol and requiring repeated surgical intervention. We identified the causal mutations in the HPRT1 gene, variant c.481G>T (p.A161S), and in the crucial urate transporter ABCG2, a heterozygous variant c.421C>A (p.Q141K). This case shows the value of an analysis of the genetic background of serum uric acid.

Functional analysis of novel allelic variants in URAT1 and GLUT9 causing renal hypouricemia type 1 and 2.

BACKGROUND: Renal hypouricemia is a rare heterogeneous inherited disorder characterized by impaired tubular uric acid transport with severe complications, such as acute kidney injury and nephrolithiasis. Type 1 is caused by a loss-of-function mutation in the SLC22A12 gene (URAT1), while type 2 is caused by defects in the SLC2A9 gene (GLUT9). METHODS AND RESULTS: In this article we present clinical, biochemical and molecular genetics of two Czech patients. The serum uric acid in the probands was 57 and 98 µmol/l and expressed as an increase in the fractional excretion of uric acid (40 and 18 %). The sequencing analysis of SLC22A12 and SLC2A9 revealed novel variants p.R92C and p.R203C in URAT1 and p.G72D in GLUT9. Functional studies were performed for these novel variants and for previously reported variants p.I118HfsX27, p.G216R and p.N333S in GLUT9 responsible for renal hypouricemia in three probands from Czech Republic and United Kingdom. Functional studies showed significantly decreased urate uptake for all variants. However, urate uptake of GLUT9 variants prepared for both isoforms were not significantly different. CONCLUSIONS: This is the first complex function characterization of non-synonymous allelic variants in patients with renal hypouricemia regarding both GLUT9 isoforms. Our finding of defects in the SLC2A9 and SLC22A12 genes show the following: renal hypouricemia is not restricted to East Asia populations; urate uptake of GLUT9 variants prepared for both isoforms were not significantly different; renal hypouricemia type 2 has more wide clinical variability than type 1; the phenotypic severity of renal hypouricemia is not correlated with results of functional characterizations of URAT1 and GLUT9 variants.

Purine disorders with hypouricemia.

Hypouricemia is defined as a serum urate levels less than 2 mg/dL (119 µmol/L). Primary hypouricemia is caused by disorders of purine metabolism and transport. This laboratory finding is sometimes overlooked and, following two genetic defects, should be considered in differential diagnosis of unexplained hypouricemia. Hereditary xanthinuria is autosomal recessive and due to mutations in xanthine oxidase, leading to over-production of xanthine and minimal production of urate. Patients have very low serum urate levels and suffer from elevated levels of xanthine in the urine, leading to xanthine stones, haematuria, and sometimes occult chronic kidney failure. Hypouricemia is the key to diagnosis. Hereditary renal hypouricemia is a new genetic defect of renal transport of uric acid. Two types were distinguished: a) renal hypouricemia type 1, caused by the defects in the SLC22A12 gene coding the human urate transporter 1 (hURAT1) and b) renal hypouricemia type 2, caused by the defects in the SLC2A9 gene, which encodes GLUT9 transporter. This disorder predisposes patients to exercise-induced acute renal failure and/or nephrolithiasis. Diagnosis is based on two markers: hypouricemia (<119 µmol/L) and increased fractional excretion of uric acid (>10%). Over one hundred cases were identified in Japan and and this number is unique worldwide. Several patients were described in Macedonia. We were able to detect four Czech families with hereditary xanthinuria and eight cases of hereditary renal hypouricemia. In conclusion, hereditary xanthinuria and hereditary renal hypouricemia are still unrecognized conditions. Patients with unexplained hypouricemia need detailed purine metabolic investigations.

Novel allelic variants and evidence for a prevalent mutation in URAT1 causing renal hypouricemia: biochemical, genetics and functional analysis.

Renal hypouricemia (RHUC) is a heterogeneous inherited disorder characterized by impaired tubular uric acid (UA) transport with severe complications, such as acute kidney injury (AKI). Type 1 is caused by a loss-of-function mutation in the SLC22A12 gene (URAT1), type 2 in the SLC2A9 gene (GLUT9). This article describes three Czech families with RHUC type 1. The serum UA in the probands was 0.9, 1.1 and 0.5 mg/dl and expressed as an increase in the fractional excretion of UA (48, 43 and 39%). The sequencing analysis of SLC22A12 revealed three novel variants: p.G366R, p.T467M and a deletion p.L415_G417del. A detailed metabolic investigation in proband C for progressive visual failure supported suspicion of neuronal ceroid lipofuscinosis type 7 conditioned by the mutation in the MFSD8 gene. Functional studies showed significantly decreased urate uptake and a mis-localized URAT1 signal in p.G366R, p.L415_G417del and p.T467M. Furthermore, colocalization studies showed accumulation of URAT1 protein in the endoplasmic reticulum. The findings suggest that loss-of-function mutations cause RHUC via loss of UA absorption partly by protein misfolding. However, they do not necessarily lead to AKI and a possible genotype-phenotype correlation was not proposed. Furthermore, results confirm an uneven geographical and ethnic distribution of SLC22A12 variants; the p.L415_G417del mutation predominates in the Roma ethnic group in the Czech Republic.

Genetic disorders resulting in hyper- or hypouricemia.

Serum uric acid concentrations are governed by the balance of urate production and excretion. Besides well-known secondary causes of hyperuricemia, such as myeloproliferative diseases, decreased renal function, and excessive dietary purine intake, there are a number of genetic disorders that result in hyper- or hypouricemia. Renal impairment in these disorders may be associated with the development of chronic kidney disease, acute kidney injury, or urate nephrolithiasis. These conditions are frequently misdiagnosed, not because the diagnosis is complicated and difficult to ascertain, but rather because of a lack of awareness of the particular condition. The first important step in the diagnosis is obtaining a detailed family history, with evaluation of serum and urinary urate concentrations. This review will aid physicians in identifying these inherited kidney disorders associated with hyperuricemia and hypouricemia. Identification of these conditions will help to explain the pathogenesis of different types of gout, and may extend insights into the urate transport and chronic kidney disease.

Acute kidney injury in two children caused by renal hypouricaemia type 2.

BACKGROUND: Renal hypouricaemia is a heterogeneous inherited disorder characterized by impaired tubular uric acid transport with severe complications, such as acute kidney injury and nephrolithiasis. Type 1 is caused by a loss-of-function mutation in the SLC22A12 gene (OMIM #220150), while type 2 is caused by defects in the SLC2A9 gene (OMIM #612076). CASE-DIAGNOSIS/TREATMENT: The cases of two children, a 12- and a 14-year-old boy with acute kidney injury (proband 1: urea 9.4 mmol/l, creatinine 226 µmol/l; proband 2: urea 11.7 mmol/l, creatinine 202 µmol/l) are described. Both are offspring of nonconsanguineous couples in the UK. The concentrations of serum uric acid were consistently below the normal range (0.03 and 0.04 mmol/l) and expressed as an increase in the fractional excretion of uric acid (46 and 93 %). CONCLUSIONS: A sequencing analysis of the coding region of uric acid transporters SLC22A12 and SLC2A9 was performed. Analysis of genomic DNA revealed two unpublished missense transitions, p.G216R and p.N333S in the SLC2A9 gene. No sequence variants in SLC22A12 were found. Our findings suggest that homozygous and/or compound heterozygous loss-of-function mutations p.G216R and p.N333S cause renal hypouricaemia via loss of uric acid absorption and do lead to acute kidney injury.

Novel mutations in xanthine dehydrogenase/oxidase cause severe hypouricemia: biochemical and molecular genetic analysis in two Czech families with xanthinuria type I.

BACKGROUND: The article describes the clinical, biochemical, enzymological and molecular genetics findings in two patients from two families with xanthinuria type I. METHODS: Biochemical analysis using high performance liquid chromatography, allopurinol loading test and analysis of xanthine oxidase activity in plasma and of uromodulin excretion in urine were performed. Sequencing analysis of the xanthine dehydrogenase gene and the haplotype and statistical analyses of consanguinity were performed. RESULTS: Probands showed extremely low concentrations of uric acid, on seven occasions under the limit of detection. The concentration of uric acid in 38-year-old female was 15 µmol/L in serum and 0.04 mmol/L in urine. Excretion of xanthine in urine was 170 mmol/mol creatinine. The concentration of uric acid in 25-year-old male was 0.03 mmol/L in urine. Excretion of xanthine in urine was 141 mmol/mol creatinine. The allopurinol loading test confirmed xanthinuria type I. The xanthine oxidase activities in patients were 0 and 0.4 pmol/h/mL of plasma. We found three nonsense changes: p.P214QfsX4 and unpublished p.R825X and p.R881X. CONCLUSIONS: We found two nonconsanguineous compound heterozygotes with xanthinuria type I caused by three nonsense changes. The methods used did not confirm consanguinity in the probands, thus there might be an unconfirmed biological relationship or mutational hotspot.

Diagnostic tests for primary renal hypouricemia.

Primary renal hypouricemia is a genetic disorder characterized by defective renal uric acid (UA) reabsorption with complications such as nephrolithiasis and exercise-induced acute renal failure. The known causes are: defects in the SLC22A12 gene, encoding the human urate transporter 1 (hURAT1), and also impairment of voltage urate transporter (URATv1), encoded by SLC2A9 (GLUT9) gene. Diagnosis is based on hypouricemia (<119 µmol/L) and increased fractional excretion of UA (>10%). To date, the cases with mutations in hURAT1 gene have been reported in East Asia only. More than 100 Japanese patients have been described. Hypouricemia is sometimes overlooked; therefore, we have set up the flowchart for this disorder. The patients were selected for molecular analysis from 620 Czech hypouricemic patients. Secondary causes of hyperuricosuric hypouricemia were excluded. The estimations of (1) serum UA, (2) excretion fraction of UA, and (3) analysis of hURAT1 and URATv1 genes follow. Three transitions and one deletion (four times) in SLC22A12 gene and one nucleotide insertion in SLC2A9 gene in seven Czech patients were found. Three patients had acute renal failure and urate nephrolithiasis. In addition, five nonsynonymous sequence variants and three nonsynonymous sequence variants in SLC2A9 gene were found in two UK patients suffering from acute renal failure. Our finding of the defects in SLC22A12 and SLC2A9 genes gives further evidence of the causative genes of primary renal hypouricemia and supports their important role in regulation of serum urate levels in humans.

Novel homozygous insertion in SLC2A9 gene caused renal hypouricemia.

Renal hypouricemia is a heterogeneous inherited disorder characterized by impaired uric acid handling in the renal tubules. Patients are usually asymptomatic; however, some may experience urolithiasis and/or acute kidney injury. Most of the described patients (compound heterozygous and/or homozygous) are Japanese with mutations in the SLC22A12 gene (OMIM #220150). Four patients with renal hypouricemia caused by heterozygous defects and two families with homozygous mutations in the SLC2A9 gene have been recently described (OMIM #612076). We describe the clinical history, biochemical and molecular genetics findings of a Czech family with renal hypouricemia. The concentration of serum uric acid in the proband (16-year-old Czech girl with unrelated parents) was 0.17 ± 0.05 mg/dl and expressed as an increase in the fractional excretion of uric acid (194 ± 99%). The sequencing analysis of the coding region of uric acid transporters SLC22A12, SLC2A9, SLC17A3, ABCC4 and ABCG2, was performed. Analysis of genomic DNA revealed novel one nucleotide homozygote insertion in exon 3 in the SLC2A9 gene in proband and her brother resulting in a truncated protein (p.Ile118HisfsX27). No sequence variants in other candidate uric acid transporter were found. Homozygous loss-of-function mutations cause massive renal hypouricemia via total loss of uric acid absorption; however, they do not necessarily lead to nephrolithiasis and acute kidney injury. In contrast to previously reported heterozygous patients with renal hypouricemia type 2, we did not find even slight hypouricemia and found no decrease in the FE-UA of the heterozygous parents of the reported siblings.

Polymorphisms of the TPMT gene in the Czech healthy population and patients with inflammatory bowel disease.

Genetic variation in thiopurine S-methyltransferase (TPMT) is a major factors for wide variation in the metabolism and safety of thiopurine drugs. We investigated the frequency of functional gene polymorphisms in 396 patients with inflammatory bowel disease and 300 healthy subjects. Frequencies of functionally deficient alleles TPMT*2, TPMT*3A, TPMT*3B, and TPMT*3B in the patient group were 0.1%, 4.3%, 0.1%, and 0.4%, respectively, and were similar to those of healthy subjects in the Czech population. Our results provide necessary information for pharmacoeconomic studies in the Czech Republic.

Studies of biochemistry and clinical biochemistry. Studies at sample medical schools in 13 EU countries regarding biochemistry and clinical biochemistry teaching.

The study summarizes the results obtained during personal visits to 53 medical schools in the 13 original EU countries during 2004--2006. Data from the Czech Republic is shown for comparison. The possibilities of acquiring information from the websites of the medical schools in the local language and English are assessed. The admission process to medical schools and the organization of studies of medicine, dentistry, and non-medical healthcare fields are briefly characterized. Significant attention is paid to the forms of education in biochemistry and clinical (bio)chemistry in the medical study field. The position of these subjects in the studies of dentistry and non-medical healthcare fields is also noted. In addition, the course of subject exams is described. The methods of funding and postgraduate studies at the medical schools are also briefly addressed.

Familial juvenile hyperuricaemic nephropathy (FJHN): linkage analysis in 15 families, physical and transcriptional characterisation of the FJHN critical region on chromosome 16p11.2 and the analysis of seven candidate genes.

Familial juvenile hyperuricaemic nephropathy (FJHN) is an autosomal dominant renal disease characterised by juvenile onset of hyperuricaemia, gouty arthritis, and progressive renal failure at an early age. Recent studies in four kindreds showed linkage of a gene for FJHN to the same genomic interval on chromosome 16p11.2, where the gene for the phenotypically similar medullary cystic disease type 2 (MCKD2) has been localised. In this study we performed linkage analysis in additional 15 FJHN families. Linkage of FJHN to 16p11.2 was confirmed in six families, which suggests that, in a large proportion of FJHN kindreds, the disease is likely to be caused by a gene or genes located outside of 16p11.2. Haplotype analysis of the new and previously analysed families provided two non-overlapping critical regions on 16p11.2-FJHN1, delimited by markers D16S499-D16S3036 and FJHN2, delimited by markers D16S412-D16S3116. Considering MCKD2 to be a distinct molecular entity, the analysis suggests that as many as three kidney disease genes may be located in close proximity on 16p11.2. From genomic databases we compiled integrated physical and transcription maps of whole critical genomic region in which 45 known genes and 129 predicted loci have been localised. We selected, analysed and found no pathogenic mutations in seven candidate genes. The linkage and haplotype analysis reported here demonstrates the genetic heterogeneity of FJHN. The report of integrated physical and mostly in-silico predicted transcription maps of the FJHN critical region provides a basis for precise experimental annotation of the current transcript map, which is essential for final identification of the FJHN gene(s).