Acute hemodialysis therapy in neonates with inborn errors of metabolism.

BACKGROUND: Inborn errors of metabolism (IEM), including organic acidemias and urea cycle defects, are characterized by systemic accumulation of toxic metabolites with deleterious effect on the developing brain. While hemodialysis (HD) is most efficient in clearing IEM-induced metabolic toxins, data regarding its use during the neonatal period is scarce. METHODS: We retrospectively summarize our experience with HD in 20 neonates with IEM-induced metabolic intoxication (seven with maple syrup urine disease, 13 with primary hyperammonia), over a 16-year period, between 2004 and 2020. All patients presented with IEM-induced neurologic deterioration at 48 h to 14 days post-delivery, and were managed with HD in a pediatric intensive care setting. HD was performed through an internal jugular acute double-lumen catheter (6.5-7.0 French), using an AK-200S (Gambro, Sweden) dialysis machine and tubing, with F3 or FXpaed (Fresenius, Germany) dialyzers. RESULTS: Median (interquartile range) age and weight at presentation were 5 (3-8) days and 2830 (2725-3115) g, respectively. Two consecutive HD sessions decreased the mean leucine levels from 2281 ± 631 to 179 ± 91 µmol/L (92.1% reduction) in MSUD patients, and the mean ammonia levels from 955 ± 444 to 129 ± 55 µmol/L (86.5% reduction), in patients with hyperammonemia. HD was uneventful in all patients, and led to marked clinical improvement in 17 patients (85%). Three patients (15%) died during the neonatal period, and four died during long-term follow-up. CONCLUSIONS: Taken together, our results indicate that HD is safe, effective, and life-saving for most neonates with severe IEM-induced metabolic intoxication, when promptly performed by an experienced and multidisciplinary team. A higher resolution version of the Graphical abstract is available as Supplementary information.

Long-term hemodialysis therapy in neonates and infants with end-stage renal disease: a 16-year experience and outcome.

BACKGROUND: Peritoneal dialysis is the preferred mode of renal replacement therapy in infants with end-stage renal disease (ESRD). Hemodialysis (HD) is seldom used in neonates and infants due to the risk of major complications in the very young. METHODS: Demographic, clinical, laboratory, and imaging data on all infants younger than 12 months with ESRD who received HD in our Pediatric Dialysis Unit between January 1997 and June 2013 were analyzed. RESULTS: Eighteen infants (n = 6 male) with ESRD (median age 3 months; median weight 4.06 kg) received HD through a central venous catheter (CVC) for a total of 543 months (median duration per infant 16 months). Seven of the infants (39%) were neonates, and five (28%) had serious comorbidities. There were five episodes of CVC infection, which is a rate of 0.3/1000 CVC days. Median catheter survival time was 320 days. Most infants had good oral intake, and only four (22%) required a gastric tube; 14 (78%) infants displayed normal growth. Fourteen (78%) infants had hypertension, of whom four (22%) had severe cardiac complications; eight (44%) showed delayed psychomotor development. Eleven (61%) of the infants, including six (86%) of the neonates, survived. Five (28%) infants underwent renal transplantation; 10-year graft survival was 80%. CONCLUSIONS: Based on these results, long-term HD in neonates and infants with ESRD is technically feasible, can be implemented without major complications, carries a very low rate of CVC infection and malfunction, and results in adequate nutrition, good growth, as well as good kidney graft and patient survivals. Future efforts should aim to prevent hypertension and its cardiac sequelae, improve neurodevelopmental outcome, and lower mortality rate in these infants.

The claudin-16 channel gene is transcriptionally inhibited by 1,25-dihydroxyvitamin D.

NEW FINDINGS: What is the central question of this study? In the kidney, the bulk of the filtered Mg(2+) is reabsorbed in the thick ascending limb by paracellular conductance, mediated by the tight junction protein, claudin-16, which is encoded by the gene CLDN16. The role of 1,25-dihydroxyvitamin D [1,25(OH)2 VitD] in renal Mg(2+) handling is unclear. We aimed to explore the molecular mechanisms underlying the effect of 1,25(OH)2 VitD on claudin-16-mediated Mg(2+) transport. What is the main finding and its importance? Paracellular, claudin-16-mediated Mg(2+) transport is transcriptionally repressed by 1,25(OH)2 VitD, probably via a Ca(2+)-sensing receptor-dependent mechanism. This renal effect of 1,25(OH)2 VitD may serve as an adaptive mechanism to the 1,25(OH)2 VitD-induced enteric hyperabsorption of dietary Mg(2+). Magnesium is reabsorbed in the thick ascending limb by paracellular conductance, mediated by the CLDN16-encoded tight junction protein, claudin-16. However, the role of 1,25-dihydroxyvitamin D [1,25(OH)2 VitD] in renal Mg(2+) handling is unclear. We have shown that Mg(2+) depletion increases and 1,25(OH)2 VitD inhibits CLDN16 transcription. We have now explored further the molecular mechanisms underlying the effect of 1,25(OH)2 VitD on claudin-16-mediated Mg(2+) transport. Adult mice received parenteral 1,25(OH)2 VitD or 1,25(OH)2 VitD combined with either high-Mg(2+) or low-Mg(2+) diets. Administration of 1,25(OH)2 VitD enhanced urinary excretion of Mg(2+) and Ca(2+). The 1,25(OH)2 VitD also increased renal Ca(2+)-sensing receptor (CaSR) mRNA and decreased renal claudin-16 and claudin-19 mRNA and claudin-16 protein, but did not affect renal claudin-2 mRNA. The 1,25(OH)2 VitD reversed the expected increase in claudin-16 mRNA in Mg(2+)-depleted animals. Comparably treated HEK 293 cells showed similar changes in claudin-16 mRNA, but 1,25(OH)2 VitD did not alter mRNA of the TRPM6 Mg(2+) channel. A luciferase reporter vector containing 2.5 kb of 5'-flanking DNA sequence from human CLDN16 (hCLDN16) was transfected into HEK 293 and OK cells. The hCLDN16 promoter activity was modestly decreased by 1,25(OH)2 VitD, but markedly inhibited in HEK 293 cells coexpressing CaSR. Coexpression in OK cells of dominant-negative CaSR completely abolished inhibition of hCLDN16 promoter activity by 1,25(OH)2 VitD. The 1,25(OH)2 VitD-induced decrease in hCLDN16 promoter activity was attenuated in Mg(2+)-depleted HEK 293 cells. In conclusion, 1,25(OH)2 VitD transcriptionally inhibits claudin-16 expression by a mechanism sensitive to CaSR and Mg(2+). This renal effect of 1,25(OH)2 VitD may serve as an adaptive response to the 1,25(OH)2 VitD-induced increase in intestinal Mg(2+) absorption.

Molecular dynamics simulations of the STAS domains of rat prestin and human pendrin reveal conformational motions in conserved flexible regions.

BACKGROUND: Molecular dynamics (MD) simulations provide valuable information on the conformational changes that accompany time-dependent motions in proteins. The reported crystal structure of rat prestin (PDB 3LLO) is remarkable for an α1-α2 inter-helical angle that differs substantially from those observed in bacterial STAS domains of SulP anion transporters and anti-sigma factor antagonists. However, NMR data on the rat prestin STAS domain in solution suggests dynamic features at or near the α1-α2 helical region (Pasqualetto et al JMB, 2010). We therefore performed a 100 ns 300K MD simulation study comparing the STAS domains of rat prestin and (modeled) human pendrin, to explore possible conformational flexibility in the region of the α1 and α2 helices. METHODS: The conformation of the loop missing in the crystal structure of rat prestin STAS (11 amino acids between helix α1 and strand ß3) was built using Modeller. MD simulations were performed with GROMACSv4.6 using GROMOS96 53a6 all-atom force field. RESULTS: A subset of secondary structured elements of the STAS domains exhibits significant conformational changes during the simulation time course. The conformationally perturbed segments include the majority of loop regions, as well as the α1 and α2 helices. A significant decrease in the α1-α2 inter-helical angle observed across the simulation trajectory leads to closer helical packing at their C-termini. The end-simulation conformations of the prestin and pendrin STAS domains, including their decreased α1-α2 inter-helical angles, resemble more closely the packing of corresponding helices in the STAS structures of bacterial SulP transporters Rv1739c and ychM, as well as those of the anti-sigma factor antagonists. Several structural segments of the modeled human pendrin STAS domain exhibit larger atomic motions and greater conformational deviations than the corresponding regions of rat prestin, predicting that the human pendrin STAS domain in solution structure may be more dynamic than rat prestin STAS. Regions of prestin and pendrin identified by RMS fluctuation data as exhibiting larger atomic fluctuations corresponded to nominal GDP-binding regions of the aligned Rv1739c STAS domain of M. tuberculosis. CONCLUSIONS: MD simulations of mammalian STAS domains reveal substantial predicted conformational heterogeneity. These predicted conformational dynamics serve to supplement the reported crystal structure of the rat prestin STAS domain, and extend our understanding of the roles of STAS domains in SLC26 anion transporter function.

A loss-of-function mutation in NaPi-IIa and renal Fanconi's syndrome.

We describe two siblings from a consanguineous family with autosomal recessive Fanconi's syndrome and hypophosphatemic rickets. Genetic analysis revealed a homozygous in-frame duplication of 21 bp in SLC34A1, which encodes the renal sodium-inorganic phosphate cotransporter NaPi-IIa, as the causative mutation. Functional studies in Xenopus laevis oocytes and in opossum kidney cells indicated complete loss of function of the mutant NaPi-IIa, resulting from failure of the transporter to reach the plasma membrane. These findings show that disruption of the human NaPi-IIa profoundly impairs overall renal phosphate reabsorption and proximal-tubule function and provide evidence of the critical role of NaPi-IIa in human renal phosphate handling.

Pendrin, a novel transcriptional target of the uroguanylin system.

Guanylin (GN) and uroguanylin (UGN) are low-molecular-weight peptide hormones produced mainly in the intestinal mucosa in response to oral salt load. GN and UGN (guanylin peptides) induce secretion of electrolytes and water in both intestine and kidney. Thought to act as "intestinal natriuretic factors", GN and UGN modulate renal salt secretion by both endocrine mechanisms (linking the digestive system and kidney) and paracrine/autocrine (intrarenal) mechanisms. The cellular function of GN and UGN in intestine and proximal tubule is mediated by guanylyl cyclase C (GC-C)-, cGMP-, and G protein-dependent pathways, whereas, in principal cells of the cortical collecting duct (CCD), these peptide hormones act via GC-C-independent signaling through phospholipase A2 (PLA2). The Cl(-)/HCO(-)3 exchanger pendrin (SLC26A4), encoded by the PDS gene, is expressed in non-α intercalated cells of the CCD. Pendrin is essential for CCD bicarbonate secretion and is also involved in NaCl balance and blood pressure regulation. Our recent studies have provided evidence that pendrin-mediated anion exchange in the CCD is regulated at the transcriptional level by UGN. UGN exerts an inhibitory effect on the pendrin gene promoter likely via heat shock factor 1 (HSF1) action at a defined heat shock element (HSE) site. Recent studies have unraveled novel roles for guanylin peptides in several organ systems including involvement in appetite regulation, olfactory function, cell proliferation and differentiation, inflammation, and reproductive function. Both the guanylin system and pendrin have also been implicated in airway function. Future molecular research into the receptors and signal transduction pathways involved in the action of guanylin peptides and the pendrin anion exchanger in the kidney and other organs, and into the links between them, may facilitate discovery of new therapies for hypertension, heart failure, hepatic failure and other fluid retention syndromes, as well as for diverse diseases such as obesity, asthma, and cancer.

Mutations in GALNT3, encoding a protein involved in O-linked glycosylation, cause familial tumoral calcinosis.

Familial tumoral calcinosis (FTC; OMIM 211900) is a severe autosomal recessive metabolic disorder that manifests with hyperphosphatemia and massive calcium deposits in the skin and subcutaneous tissues. Using linkage analysis, we mapped the gene underlying FTC to 2q24-q31. This region includes the gene GALNT3, which encodes a glycosyltransferase responsible for initiating mucin-type O-glycosylation. Sequence analysis of GALNT3 identified biallelic deleterious mutations in all individuals with FTC, suggesting that defective post-translational modification underlies the disease.

The pendrin anion exchanger gene is transcriptionally regulated by uroguanylin: a novel enterorenal link.

The pendrin/SLC26A4 Cl(-)/HCO(3)(-) exchanger, encoded by the PDS gene, is expressed in cortical collecting duct (CCD) non-A intercalated cells. Pendrin is essential for CCD bicarbonate secretion and is also involved in NaCl balance and blood pressure regulation. The intestinal peptide uroguanylin (UGN) is produced in response to oral salt load and can function as an "intestinal natriuretic hormone." We aimed to investigate whether UGN modulates pendrin activity and to explore the molecular mechanisms responsible for this modulation. Injection of UGN into mice resulted in decreased pendrin mRNA and protein expression in the kidney. UGN decreased endogenous pendrin mRNA levels in HEK293 cells. A 4.2-kb human PDS (hPDS) promoter sequence and consecutive 5' deletion products were cloned into luciferase reporter vectors and transiently transfected into HEK293 cells. Exposure of transfected cells to UGN decreased hPDS promoter activity. This UGN-induced effect on the hPDS promoter occurred within a 52-bp region encompassing a single heat shock element (HSE). The effect of UGN on the promoter was abolished when the HSE located between nt -1119 and -1115 was absent or was mutated. Furthermore, treatment of HEK293 cells with heat shock factor 1 (HSF1) small interfering RNA (siRNA) reversed the UGN-induced decrease in endogenous PDS mRNA level. In conclusion, pendrin-mediated Cl(-)/HCO(3)(-) exchange in the renal tubule may be regulated transcriptionally by the peptide hormone UGN. UGN exerts its inhibitory activity on the hPDS promoter likely via HSF1 action at a defined HSE site. These data define a novel signaling pathway involved in the enterorenal axis controlling electrolyte and water homeostasis.

Transcriptional regulation of the pendrin gene.

Pendrin (SLC26A4), a Cl(-)/anion exchanger encoded by the gene PDS, is highly expressed in the kidney, thyroid and inner ear epithelia and is essential for bicarbonate secretion/chloride reabsorption, iodide accumulation and endolymph ion balance, respectively. The molecular mechanisms controlling pendrin activity in renal, thyroid and inner ear epithelia have been the subject of recent studies. The effects of ambient pH, the hormone aldosterone and the peptide uroguanylin (UGN; the "intestinal natriuretic hormone"), known modulators of electrolyte balance, on transcription of the pendrin gene, have been investigated. Luciferase reporter plasmids containing different length fragments of the human PDS (hPDS) promoter were transfected into renal HEK293, thyroid LA2, and inner ear VOT36 epithelial cells. Acidic pH decreased and alkaline pH increased hPDS promoter activity in transfected HEK293 and VOT36, but not in LA2 cells. Aldosterone reduced hPDS promoter activity in HEK293 but had no effect in LA2 and VOT36 cells. These pH and aldosterone-induced effects on the hPDS promoter occurred within 96-bp and 89-bp regions, respectively, which likely contain distinct response elements to these modulators. Injection of UGN into mice resulted in decreased pendrin mRNA and protein expression in the kidney. Exposure of transfected HEK293 to UGN decreased hPDS promoter activity. The findings provided evidence for the presence of a UGN response element within the 96-bp region overlapping with the pH response element on the hPDS promoter. Pendrin is also expressed in airway epithelium. The cytokins interleukin 4 (IL-4) and interleukin-13 (IL-13), known regulators of airway surface function, have been shown to increase hPDS promoter activity by a STAT6-dependent mechanism. In conclusion, systemic pH, the hormone aldosterone, and the peptide UGN influence renal tubular pendrin gene expression and, perhaps, pendrin-mediated Cl(-)/HCO(3)(-) exchange at the transcriptional level. Pendrin-driven anion transport in the endolymph and at the airway surface may be regulated transcriptionally by systemic pH and IL-3/IL-4, respectively. The distinct response elements and the corresponding transcription factors mediating the effect of these modulators on the PDS promoter remain to be identified and characterized.

Pendrin function and regulation in Xenopus oocytes.

SLC26A4/PDS mutations cause Pendred Syndrome and non-syndromic deafness. but some aspects of function and regulation of the SLC26A4 polypeptide gene product, pendrin, remain controversial or incompletely understood. We have therefore extended the functional analysis of wildtype and mutant pendrin in Xenopus oocytes, with studies of isotopic flux, electrophysiology, and protein localization. Pendrin mediated electroneutral, pH-insensitive, DIDS-insensitive anion exchange, with extracellular K((1/2)) (in mM) of 1.9 (Cl(-)), 1.8 (I(-)), and 0.9 (Br(-)). The unusual phenotype of Pendred Syndrome mutation E303Q (loss-of-function with normal surface expression) prompted systematic mutagenesis at position 303. Only mutant E303K exhibited loss-of-function unrescued by forced overexpression. Mutant E303C was insensitive to charge modification by methanethiosulfonates. The corresponding mutants SLC26A2 E336Q, SLC26A3 E293Q, and SLC26A6 E298Q exhibited similar loss-of-function phenotypes, with wildtype surface expression also documented for SLC26A2 E336Q. The strong inhibition of wildtype SLC26A2, SLC26A3, and SLC26A6 by phorbol ester contrasts with its modest inhibition of pendrin. Phorbol ester inhibition of SLC26A2, SLC26A3, and SLC26A6 was blocked by coexpressed kinase-dead PKCδ but was without effect on pendrin. Mutation of SLC26A2 serine residues conserved in PKCδ -sensitive SLC26 proteins but absent from pendrin failed to reduce PKCδ sensitivity of SLC26A2 (190).

Transcriptional regulation of the claudin-16 gene by Mg2+ availability.

BACKGROUND/AIMS: Renal tubular Mg(2+) reabsorption is mediated predominantly by the tight junction channel protein claudin-16 which is encoded by the gene CLDN16. Hypermagnesemia decreases, whereas hypomagnesemia increases Mg(2+) reabsorption. This study examines the role of claudin-16 in the adaptive response of the kidney to Mg(2+) availability. METHODS/RESULTS: Mice received a low-, normal- or high Mg(2+) diet for up to 3 days. Mg(2+)-loaded animals displayed hypermagnesemia with increasing urine Mg(2+)/Ca(2+) levels paralleled by a decrease in claudin-16 protein and mRNA in the kidney. Mg(2+)- deprived animals developed hypomagnesemia with decreasing urine Mg(2+)/Ca(2+) levels associated with an increase in claudin-16 protein and mRNA abundance. Mg(2+) depletion markedly increased and Mg(2+) load decreased endogenous claudin-16 mRNA levels in calcium-sensing receptor-transfected HEK293 cells compared with native HEK293 cells. The effect of Mg(2+) availability on the human CLDN16 (hCLDN16) gene promoter was examined. Using a 2.5kb hCLDN16 5'-flanking DNA sequence, we show that magnesium depletion increases and Mg(2+) load decreases hCLDN16 promoter activity in transfected HEK293 cells. CONCLUSIONS: Changes in Mg(2+) availability may influence claudin-16 mediated Mg(2+) transport at the transcriptional level. The possible involvement of the cell membrane bound Ca(2+)/Mg(2+) sensing receptor or the potential role of a hypothetical Mg(2+) response element on the CLDN16 promoter in the Mg(2+)-induced response remains to be explored.

Distinct and novel SLC26A4/Pendrin mutations in Chinese and U.S. patients with nonsyndromic hearing loss.

Mutations of the human SLC26A4/PDS gene constitute the most common cause of syndromic and nonsyndromic hearing loss. Definition of the SLC26A4 mutation spectrum among different populations with sensorineural hearing loss is important for development of optimal genetic screening services for congenital hearing impairment. We screened for SLC26A4 mutations among Chinese and U.S. subjects with hearing loss, using denaturing HPLC (DHPLC) and direct DNA sequencing. Fifty-two of 55 Chinese subjects with deafness accompanied by enlargement of the vestibular aqueduct (EVA) exhibited at least one mutant SLC26A4 allele, whereas SLC26A4 mutations were found in only 2 of 116 deaf Chinese patients without EVA. The spectrum of SLC26A4 mutations differed among Chinese and U.S. subjects and included 10 previously unreported SLC26A4 variants: 4 in the Chinese population (p.E303Q, p.X329, p.X467, p.X573) and 6 in the U.S. population (p.V250A, p.D266N, p.F354S, p.D697A, p.K715N, p.E737D). Among the seven novel in-frame missense mutations, five encoded SLC26A4 proteins with substantially reduced Cl(-)/anion exchange activity as expressed and measured in Xenopus oocytes, but four of these were sufficiently active to allow study of anion selectivity. The only mutant polypeptide exhibiting complete loss of anion exchange function, p.E303Q, was expressed at or near the oocyte surface at near-wild-type levels. Two variants, p.F354S and p.E737D, displayed selective reduction in relative rate of Cl(-)/HCO(3)(-) exchange compared with similarly measured rates of Cl(-)/Cl(-) and Cl(-)/I(-) exchange. Our data show that mutation analysis of the SLC26A4 gene is of high diagnostic yield among subjects with deafness and bilateral EVA in both China and the U.S. However, the pathogenicity of monoallelic SLC26A4 gene variants in patients with hearing loss remains unclear in many instances.

Early performance of voiding cystourethrogram after urinary tract infection in children.

BACKGROUND: Voiding cystourethrogram is performed 3-6 weeks after urinary tract infection. This prolongs the interval of prophylactics, reducing the likelihood of having to perform the procedure. OBJECTIVES: To investigate the yield and potential risks/benefits of early compared to late performance of VCUG after UTI. METHODS: We conducted a prospective study of 84 previously healthy children < 5 years old admitted from October 2001 to November 2002 with first documented UTI. We then divided the 78 patients who had VCUG into two groups and compared them to a control group: group A--49 children in whom VCUG was performed within 10 days, group B--29 children in whom VCUG was performed > 10 days after UTI, and a historical control group C--82 children in whom VCUG was performed > 4 weeks following UTI. RESULTS: VCUG was performed in 48/48 (100%), 6/35 patients (17.1%) and 34/116 patients (29.3%), and vesicoureteral reflux was demonstrated in 38.8%, 37.9% and 39% in groups A, B and C respectively. No significant difference was found between these groups in terms of incidence of VUR and severity and grading of reflux within each group. One case of UTI secondary to VCUG occurred in a patient in whom the procedure was performed 4 months after the diagnosis. CONCLUSIONS: Performing VCUG early does not influence the detection rate, severity of the VUR, or risk of secondary infection; it shortens the period of prophylactic use and increases performance rate of VCUG, thereby minimizing the risk of failure to detect VUR. The traditional recommendation of performing VCUG 3-6 weeks after the diagnosis of UTI should be reevaluated.

Human SLC26A4/Pendrin STAS domain is a nucleotide-binding protein: Refolding and characterization for structural studies.

Mutations in the human SLC26A4/Pendrin polypeptide (hPDS) cause Pendred Syndrome /DFNB4, syndromic deafness with enlargement of the vestibular aqueduct and low-penetrance goiter. Here we present data on cloning, protein overexpression and purification, refolding, and biophysical characterization of the recombinant hPDS STAS domain lacking its intrinsic variable sequence (STAS-ΔIVS). We report a reproducible protein refolding protocol enabling milligram scale expression and purification of uniformly 15N- and 13C/15N-enriched hPDS STAS-ΔIVS domain suitable for structural characterization by solution NMR. Circular dichroism, one-dimensional 1H, two-dimensional 1H-15N HSQC, and 1H-13C HSQC NMR spectra confirmed the well-folded state of purified hPDS STAS-ΔIVS in solution. Heteronuclear NMR chemical shift perturbation of select STAS-ΔIVS residues by GDP was observed at fast-to-intermediate NMR time scales. Intrinsic tryptophan fluorescence quench experiments demonstrated GDP binding to hPDS STAS-ΔIVS with Kd of 178 µM. These results are useful for structure/function characterization of hPDS STAS, the cytoplasmic subdomain of the congenital deafness protein, pendrin, as well as for studies of other mammalian STAS domains.

Autosomal recessive renal proximal tubulopathy and hypercalciuria: a new syndrome.

BACKGROUND: The best described primary inherited proximal tubulopathies include X-linked hypercalciuric nephrolithiasis (XLHN), caused by a mutation in the chloride channel gene CLCN5, and classic Fanconi's syndrome, the genetic basis of which is unknown. The aim of this study is to examine the clinical, biochemical, and genetic characteristics of a highly consanguineous Druze family with autosomal recessive proximal tubulopathy and hypercalciuria (ARPTH), a syndrome not reported previously. METHODS: Three children (2 girls, 1 boy) of the family referred for evaluation of renal glycosuria and hypercalciuria and 10 of their close relatives were evaluated clinically and biochemically. All study participants underwent genetic analysis to exclude involvement of the CLCN5 gene. RESULTS: Evaluation of the 3 affected children showed glycosuria, generalized aminoaciduria, hypouricemia, uricosuria, low molecular weight (LMW) proteinuria, and hypercalciuria in all 3 children and phosphaturia in 2 children. They had no metabolic acidosis or renal insufficiency. One affected girl had nephrocalcinosis. Two children had a history of growth retardation and radiological findings of metabolic bone disease. Parathyroid hormone and 1,25-dihydroxyvitamin D [1,25(OH)2Vit D] blood levels in affected children were normal. Unaffected family members examined had no renal tubular defects or LMW proteinuria. Genetic linkage analysis excluded cosegregation of the ARPTH phenotype with the CLCN5 locus. CONCLUSION: ARPTH is a new syndrome characterized by nonacidotic proximal tubulopathy, hypercalciuria, metabolic bone disease, and growth retardation. It can be distinguished from XLHN by its autosomal recessive mode of inheritance and normal serum levels of calciotropic hormones, as well as the absence of LMW proteinuria in obligate carriers. The gene mutated in ARPTH remains to be identified.

Fanconi-Bickel syndrome and autosomal recessive proximal tubulopathy with hypercalciuria (ARPTH) are allelic variants caused by GLUT2 mutations.

CONTEXT: Many inherited disorders of calcium and phosphate homeostasis are unexplained at the molecular level. OBJECTIVE: The objective of the study was to identify the molecular basis of phosphate and calcium abnormalities in two unrelated, consanguineous families. PATIENTS: The affected members in family 1 presented with rickets due to profound urinary phosphate-wasting and hypophosphatemic rickets. In the previously reported family 2, patients presented with proximal renal tubulopathy and hypercalciuria yet normal or only mildly increased urinary phosphate excretion. METHODS: Genome-wide linkage scans and direct nucleotide sequence analyses of candidate genes were performed. Transport of glucose and phosphate by glucose transporter 2 (GLUT2) was assessed using Xenopus oocytes. Renal sodium-phosphate cotransporter 2a and 2c (Npt2a and Npt2c) expressions were evaluated in transgenically rescued Glut2-null mice (tgGlut2-/-). RESULTS: In both families, genetic mapping and sequence analysis of candidate genes led to the identification of two novel homozygous mutations (IVS4-2A>G and R124S, respectively) in GLUT2, the gene mutated in Fanconi-Bickel syndrome, a rare disease usually characterized by renal tubulopathy, impaired glucose homeostasis, and hepatomegaly. Xenopus oocytes expressing the [R124S]GLUT2 mutant showed a significant reduction in glucose transport, but neither wild-type nor mutant GLUT2 facilitated phosphate import or export; tgGlut2-/- mice demonstrated a profound reduction of Npt2c expression in the proximal renal tubules. CONCLUSIONS: Homozygous mutations in the facilitative glucose transporter GLUT2, which cause Fanconi-Bickel syndrome, can lead to very different clinical and biochemical findings that are not limited to mild proximal renal tubulopathy but can include significant hypercalciuria and highly variable degrees of urinary phosphate-wasting and hypophosphatemia, possibly because of the impaired proximal tubular expression of Npt2c.

Low infection rates and prolonged survival times of hemodialysis catheters in infants and children.

BACKGROUND AND OBJECTIVES: Hemodialysis (HD) catheter-related complications are regarded as the main cause of HD failure in infants and children with ESRD. In this study, we determined HD catheter infection rates and survival times in children. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We analyzed demographic, clinical, laboratory, and microbiologic data on all infants and children with ESRD who received HD therapy through a tunneled central venous catheter (CVC) in our Pediatric Dialysis Unit between January 2001 and December 2009. Our strict care of HD-CVCs makes no use of any kind of prophylactic antibiotic therapy. RESULTS: Twenty-nine children with ESRD (median age, 10 years) received HD through a CVC, for a total of 22,892 days during the study period. Eleven (38%) children were infants (<1 year of age) who received HD for a cumulative 3779 days (16% of total). Fifty-nine CVCs were inserted, of which 13 (22%) were in infants. There were 12 episodes of CVC infection-a rate of 0.52/1000 CVC days. Four (33%) episodes occurred in infants-a rate of 1.06/1000 CVC days. Only three (5%) of the CVCs were removed because of infection. Median catheter survival time for all children was 310 days and for infants was 211 days. CONCLUSIONS: Very low CVC infection rates (one infection per 5 CVC years) and prolonged CVC survival times (around 1 year) are achievable in infants and children with ESRD receiving HD therapy by adhering to a strict catheter management protocol and without using prophylactic antibiotic therapy.

Molecular mechanisms of epithelial cell-specific expression and regulation of the human anion exchanger (pendrin) gene.

Pendrin, a Cl(-)/anion exchanger encoded by the gene PDS, is highly expressed in the kidney, thyroid, and inner ear epithelia and is essential for bicarbonate secretion, iodide accumulation, and endolymph ion balance, respectively. This study aimed to define promoter regulatory elements essential for renal, thyroid, and inner ear epithelial cell-specific expression of human PDS (hPDS) and to explore the effect of ambient pH and aldosterone on hPDS promoter activity. Endogenous pendrin mRNA and protein were detected in renal HEK293, thyroid LA2, and inner ear VOT36 epithelial cell lines, but not in the fibroblast cell line, NIH3T3. A 4.2-kb hPDS 5'-flanking DNA sequence and consecutive 5'-deletion products were cloned into luciferase reporter vectors and transiently transfected into the above cell lines. Distinct differences in expression/activity of deduced positive/negative regulatory elements within the hPDS promoter between HEK293, LA2, and VOT36 cells were demonstrated, with only basal activity in NIH3T3 cells. Acidic pH (7.0-7.1) decreased and alkaline pH (7.6-7.7) increased hPDS promoter activity in transfected HEK293 and VOT36, but not in LA2 cells. Aldosterone (10(-8) M) reduced hPDS promoter activity in HEK293 but had no effect in LA2 and VOT36 cells. These pH and aldosterone-induced effects on the hPDS promoter occurred within 96-bp and 89-bp regions, respectively, which likely contain distinct response elements to these modulators. Acidic pH and aldosterone decreased, and alkaline pH increased, endogenous pendrin mRNA level in HEK293 cells. In conclusion, pendrin-mediated HCO3(-) secretion in the renal tubule and anion transport in the endolymph may be regulated transcriptionally by systemic pH and aldosterone.

The human paracellin-1 gene (hPCLN-1): renal epithelial cell-specific expression and regulation.

Tubular reabsorption of Mg2+ is mediated by the tight junction protein paracellin-1, which is encoded by the gene PCLN-1 (CLDN16) and exclusively expressed in the kidney. Tubular Mg2+ reclamation is modulated by many hormones and factors. The aim of this study was to define regulatory elements essential for renal tubular cell-specific expression of human PCLN-1 (hPCLN-1) and to explore the effect of Mg2+ transport modulators on the paracellin-1 gene promoter. Endogenous paracellin-1 mRNA and protein were detected in renal cell lines opossom kidney (OK), HEK293, and MDCT, but not in the fibroblast cell line NIH3T3. A 7.5-kb hPCLN-1 5'-flanking DNA sequence along with seven 5'-deletion products were cloned into luciferase reporter vectors and transiently transfected into the renal and nonrenal cells. The highest levels of luciferase activity resulted from transfection of a 5'-flanking 2.5-kb fragment (pJ2M). This activity was maximal in OK cells, was orientation dependent, and was absent in NIH3T3 cells. Mg2+ deprivation significantly increased pJ2M-driven activity in transfected OK cells, whereas Mg2+ load decreased it compared with conditions of normal Mg2+. Deletion analysis along with electrophoretic mobility-shift assay demonstrated that OK cells contain nuclear proteins, which bind a 70-bp region between -1633 and -1703 of major functional significance. Deleting this 70-bp segment, which contains a single peroxisome proliferator-response element (PPRE), or mutating the PPRE, caused a 60% reduction in luciferase activity. Stimulating the 70-bp sequence with 1,25(OH)2 vitamin D decreased luciferase activity by 52%. This effect of 1,25(OH)2 vitamin D was abolished in the absence of PPRE or in the presence of mutated PPRE. We conclude that the PPRE within this 70-bp DNA region may play a key role in the cell-specific and regulatory activity of the hPCLN-1 promoter. Ambient Mg2+ concentration and 1,25(OH)2 vitamin D may modulate paracellular, paracellin-1-mediated, Mg2+ transport at the transcriptional level. 1,25(OH)2 vitamin D exerts its activity on the hPCLN-1 promoter likely via the PPRE site.