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BACKGROUND: The endocytic reabsorption of proteins in the proximal tubule requires a complex machinery and defects can lead to tubular proteinuria. The precise mechanisms of endocytosis and processing of receptors and cargo are incompletely understood. EHD1 belongs to a family of proteins presumably involved in the scission of intracellular vesicles and in ciliogenesis. However, the relevance of EHD1 in human tissues, in particular in the kidney, was unknown.METHODS: Genetic techniques were used in patients with tubular proteinuria and deafness to identify the disease-causing gene. Diagnostic and functional studies were performed in patients and disease models to investigate the pathophysiology.RESULTS: We identified six individuals (5-33 years) with proteinuria and a high-frequency hearing deficit associated with the homozygous missense variant c.1192C>T (p.R398W) in EHD1. Proteinuria (0.7-2.1 g/d) consisted predominantly of low molecular weight proteins, reflecting impaired renal proximal tubular endocytosis of filtered proteins. Ehd1 knockout and Ehd1R398W/R398W knockin mice also showed a high-frequency hearing deficit and impaired receptor-mediated endocytosis in proximal tubules, and a zebrafish model showed impaired ability to reabsorb low molecular weight dextran. Interestingly, ciliogenesis appeared unaffected in patients and mouse models. In silico structural analysis predicted a destabilizing effect of the R398W variant and possible inference with nucleotide binding leading to impaired EHD1 oligomerization and membrane remodeling ability.CONCLUSIONS: A homozygous missense variant of EHD1 causes a previously unrecognized autosomal recessive disorder characterized by sensorineural deafness and tubular proteinuria. Recessive EHD1 variants should be considered in individuals with hearing impairment, especially if tubular proteinuria is noted.

Background For many patients with kidney failure, the cause and underlying defect remain unknown. Here, we describe a novel mechanism of a genetic order characterized by renal Fanconi syndrome and kidney failure. Methods We clinically and genetically characterized members of five families with autosomal dominant renal Fanconi syndrome and kidney failure. We performed genome-wide linkage analysis, sequencing, and expression studies in kidney biopsy specimens and renal cells along with knockout mouse studies and evaluations of mitochondrial morphology and function. Structural studies examined the effects of recognized mutations. Results The renal disease in these patients resulted from monoallelic mutations in the gene encoding glycine amidinotransferase (GATM), a renal proximal tubular enzyme in the creatine biosynthetic pathway that is otherwise associated with a recessive disorder of creatine deficiency. In silico analysis showed that the particular GATM mutations, identified in 28 members of the five families, create an additional interaction interface within the GATM protein and likely cause the linear aggregation of GATM observed in patient biopsy specimens and cultured proximal tubule cells. GATMaggregates-containing mitochondria were elongated and associated with increased ROS production, activation of the NLRP3 inflammasome, enhanced expression of the profibrotic cytokine IL-18, and increased cell death. Conclusions In this novel genetic disorder, fully penetrant heterozygous missense mutations in GATM trigger intramitochondrial fibrillary deposition of GATM and lead to elongated and abnormal mitochondria. We speculate that this renal proximal tubular mitochondrial pathology initiates a response from the inflammasome, with subsequent development of kidney fibrosis.

TASK-1 potassium channels have been implicated in central and peripheral chemoreception; however, the precise contribution of TASK-1 for the control of respiration is still under debate. Here, we investigated the respiration of unrestrained adult and neonatal TASK-1 knockout mice (TASK-1−/−) using a plethysmographic device. Respiration in adult female TASK-1−/− mice under control (21% O2), hypoxia and hypercapnia was unaffected. Under acute hypoxia male TASK-1−/− mice exhibited a reduced increase of the respiratory frequency (fR) compared to wildtypes. However, the tidal volume (VT) of male TASK-1−/− mice was strongly enhanced. The volatile anesthetic isoflurane induced in male TASK-1−/− and male wild type mice (TASK-1+/+) a similar respiratory depression. Neonatal TASK-1−/− mice demonstrated a 30–40% decrease of the minute volume, caused by a reduction of the fR under control condition (21% O2). Under hypoxia, neonatal TASK-1−/− mice more frequently stopped breathing (apnea > 3s) suggesting an increased hypoxia-sensitivity. As reported before, this increased hypoxia sensitivity had no influence on the survival rate of neonatal TASK-1−/− mice. In adult and neonatal mice, TASK-1 gene deletion induced a significant prolongation of the relaxation time (RT), which is a parameter for expiration kinetics. Additionally, screening for mutations in the human TASK-1 gene in 155 cases of sudden infant death syndrome (SIDS) was inconclusive. In conclusion, these data are suggestive for an increased hypoxia-sensitivity of neonatal TASK-1−/− mice, however, without causing an increase in neonatal lethality. In adult female TASK-1−/− mice respiration was unaffected, whereas adult male TASK-1−/− mice showed a modified breathing pattern. These results are suggestive for sex-specific mechanisms for compensating the inactivation of TASK-1 in mice.

Mutations of the KCNJ10 ( Kir4.1 ) K + channel underlie autosomal recessive epilepsy, ataxia, sensorineural deafness, and (a salt-wasting) renal tubulopathy (EAST) syndrome. We investigated the localization of KCNJ10 and the homologous KCNJ16 in kidney and the functional consequences of KCNJ10 mutations found in our patients with EAST syndrome. Kcnj10 and Kcnj16 were found in the basolateral membrane of mouse distal convoluted tubules, connecting tubules, and cortical collecting ducts. In the human kidney, KCNJ10 staining was additionally observed in the basolateral membrane of the cortical thick ascending limb of Henle's loop. EM of distal tubular cells of a patient with EAST syndrome showed reduced basal infoldings in this nephron segment, which likely reflects the morphological consequences of the impaired salt reabsorption capacity. When expressed in CHO and HEK293 cells, the KCNJ10 mutations R65P, G77R, and R175Q caused a marked impairment of channel function. R199X showed complete loss of function. Single-channel analysis revealed a strongly reduced mean open time. Qualitatively similar results were obtained with coexpression of KCNJ10/KCNJ16, suggesting a dominance of KCNJ10 function in native renal KCNJ10/KCNJ16 heteromers. The decrease in the current of R65P and R175Q was mainly caused by a remarkable shift of pH sensitivity to the alkaline range. In summary, EAST mutations of KCNJ10 lead to impaired channel function and structural changes in distal convoluted tubules. Intriguingly, the metabolic alkalosis present in patients carrying the R65P mutation possibly improves residual function of KCNJ10, which shows higher activity at alkaline pH.

Five children from two consanguineous families presented with epilepsy beginning in infancy and severe ataxia, moderate sensorineural deafness, and a renal salt-losing tubulopathy with normotensive hypokalemic metabolic alkalosis. We investigated the genetic basis of this autosomal recessive disease, which we call the EAST syndrome (the presence of epilepsy, ataxia, sensorineural deafness, and tubulopathy).Whole-genome linkage analysis was performed in the four affected children in one of the families. Newly identified mutations in a potassium-channel gene were evaluated with the use of a heterologous expression system. Protein expression and function were further investigated in genetically modified mice.Linkage analysis identified a single significant locus on chromosome 1q23.2 with a lod score of 4.98. This region contained the KCNJ10 gene, which encodes a potassium channel expressed in the brain, inner ear, and kidney. Sequencing of this candidate gene revealed homozygous missense mutations in affected persons in both families. These mutations, when expressed heterologously in xenopus oocytes, caused significant and specific decreases in potassium currents. Mice with Kcnj10 deletions became dehydrated, with definitive evidence of renal salt wasting.Mutations in KCNJ10 cause a specific disorder, consisting of epilepsy, ataxia, sensorineural deafness, and tubulopathy. Our findings indicate that KCNJ10 plays a major role in renal salt handling and, hence, possibly also in blood-pressure maintenance and its regulation.

Epilepsy, ataxia, sensorineural deafness, tubulopathy, and KCNJ10 mutations. N Engl J Med 360: 1960–1970, 2009 Bockenhauer D, Feather S, Stanescu HC, Bandulik S, Zdebik AA, Reichold M, Tobin J, Lieberer E, Sterner C, Landoure G, Arora R, Sirimanna T, Thompson D, Cross JH, van’t Hoff W, Al Masri O, Tullus K, Yeung S, Anikster Y, Klootwijk E, Hubank M, Dillon MJ, Heitzmann D, ArcosBurgos M, Knepper MA, Dobbie A, Gahl WA, Warth R, Sheridan E, Kleta R

Aldosterone-producing adenoma (APA) is a major cause of primary aldosteronism, leading to secondary hypertension. Somatic mutations in the gene for the α1 subunit of the Na(+)/K(+)-ATPase were found in about 6% of APAs. APA-related α1 subunit of the Na(+)/K(+)-ATPase mutations lead to a loss of the pump function of the Na(+)/K(+)-ATPase, which is believed to result in membrane depolarization and Ca(2+)-dependent stimulation of aldosterone synthesis in adrenal cells. In addition, H(+) and Na(+) leak currents via the mutant Na(+)/K(+)-ATPase were suggested to contribute to the phenotype. The aim of this study was to investigate the cellular pathophysiology of adenoma-associated Na(+)/K(+)-ATPase mutants (L104R, V332G, G99R) in adrenocortical NCI-H295R cells. The expression of these Na(+)/K(+)-ATPase mutants depolarized adrenal cells and stimulated aldosterone secretion. However, an increase of basal cytosolic Ca(2+) levels in Na(+)/K(+)-ATPase mutant cells was not detectable, and stimulation with high extracellular K(+) hardly increased Ca(2+) levels in cells expressing L104R and V332G mutant Na(+)/K(+)-ATPase. Cytosolic pH measurements revealed an acidification of L104R and V332G mutant cells, despite an increased activity of the Na(+)/H(+) exchanger. The possible contribution of cellular acidification to the hypersecretion of aldosterone was supported by the observation that aldosterone secretion of normal adrenocortical cells was stimulated by acetate-induced acidification. Taken together, mutations of the Na(+)/K(+)-ATPase depolarize adrenocortical cells, disturb the K(+) sensitivity, and lower intracellular pH but, surprisingly, do not induce an overt increase of intracellular Ca(2+). Probably, the autonomous aldosterone secretion is caused by the concerted action of several pathological signaling pathways and incomplete cellular compensation.

Elucidation of the molecular mechanisms leading to autonomous aldosterone secretion is a prerequisite to define potential targets and biomarkers in the context of primary aldosteronism. After a genome-wide association study with subjects from the population-based Cooperative Health Research in the Region of Augsburg F4 survey, we observed a highly significant association ( P =6.78×10 –11 ) between the aldosterone to renin ratio and a locus at 5q32. Hypothesizing that this locus may contain genes of relevance for the pathogenesis of primary aldosteronism, we investigated solute carrier family 26 member 2 (SLC26A2), a protein with known transport activity for sulfate and other cations. Within murine tissues, adrenal glands showed the highest expression levels for SLC26A2, which was significantly downregulated on in vivo stimulation with angiotensin II and potassium. SLC26A2 expression was found to be significantly lower in aldosterone-producing adenomas in comparison with normal adrenal glands. In adrenocortical NCI-H295R cells, specific knockdown of SLC26A2 resulted in a highly significant increase in aldosterone secretion. Concomitantly, expression of steroidogenic enzymes, as well as upstream effectors including transcription factors such as NR4A1, CAMK1, and intracellular Ca 2+ content, was upregulated in knockdown cells. To substantiate further these findings in an SLC26A2 mutant mouse model, aldosterone output proved to be increased in a sex-specific manner. In summary, these findings point toward a possible effect of SLC26A2 in the regulation of aldosterone secretion potentially involved in the pathogenesis of primary aldosteronism.

Somatic mutations of the potassium channel KCNJ5 are found in 40% of aldosterone producing adenomas (APAs). APA-related mutations of KCNJ5 lead to a pathological Na(+) permeability and a rise in cytosolic Ca(2+), the latter presumably by depolarizing the membrane and activating voltage-gated Ca(2+) channels. The aim of this study was to further investigate the effects of mutated KCNJ5 channels on intracellular Na(+) and Ca(2+) homeostasis in human adrenocortical NCI-H295R cells. Expression of mutant KCNJ5 led to a 2-fold increase in intracellular Na(+) and, in parallel, to a substantial rise in intracellular Ca(2+). The increase in Ca(2+) appeared to be caused by activation of voltage-gated Ca(2+) channels and by an impairment of Ca(2+) extrusion by Na(+)/Ca(2+) exchangers. The mutated KCNJ5 exhibited a pharmacological profile that differed from the one of wild-type channels. Mutated KCNJ5 was less Ba(2+) and tertiapin-Q sensitive but was inhibited by blockers of Na(+) and Ca(2+)-transporting proteins, such as verapamil and amiloride. The clinical use of these drugs might influence aldosterone levels in APA patients with KCNJ5 mutations. This might implicate diagnostic testing of APAs and could offer new therapeutic strategies.

In renal Fanconi's syndrome, dysfunction in proximal tubular cells leads to renal losses of water, electrolytes, and low-molecular-weight nutrients. For most types of isolated Fanconi's syndrome, the genetic cause and underlying defect remain unknown.We clinically and genetically characterized members of a five-generation black family with isolated autosomal dominant Fanconi's syndrome. We performed genomewide linkage analysis, gene sequencing, biochemical and cell-biologic investigations of renal proximal tubular cells, studies in knockout mice, and functional evaluations of mitochondria. Urine was studied with the use of proton nuclear magnetic resonance ((1)H-NMR) spectroscopy.We linked the phenotype of this family's Fanconi's syndrome to a single locus on chromosome 3q27, where a heterozygous missense mutation in EHHADH segregated with the disease. The p.E3K mutation created a new mitochondrial targeting motif in the N-terminal portion of EHHADH, an enzyme that is involved in peroxisomal oxidation of fatty acids and is expressed in the proximal tubule. Immunocytofluorescence studies showed mistargeting of the mutant EHHADH to mitochondria. Studies of proximal tubular cells revealed impaired mitochondrial oxidative phosphorylation and defects in the transport of fluids and a glucose analogue across the epithelium. (1)H-NMR spectroscopy showed elevated levels of mitochondrial metabolites in urine from affected family members. Ehhadh knockout mice showed no abnormalities in renal tubular cells, a finding that indicates a dominant negative nature of the mutation rather than haploinsufficiency.Mistargeting of peroxisomal EHHADH disrupts mitochondrial metabolism and leads to renal Fanconi's syndrome; this indicates a central role of mitochondria in proximal tubular function. The dominant negative effect of the mistargeted protein adds to the spectrum of monogenic mechanisms of Fanconi's syndrome. (Funded by the European Commission Seventh Framework Programme and others.).

Genetically modified mice represent important models for elucidating renal pathophysiology, but gene deletions frequently cause severe failure to thrive. In such cases, the analysis of the phenotype is often limited to the first weeks of life when renal excretory function undergoes dramatic physiological changes. Here, we investigated the postnatal dynamics of urinary ion excretion in mice. The profiles of urinary electrolyte excretion of mice were examined from birth until after weaning using an automated ion chromatography system. Postnatally, mice grew about 0.4 g/day, except during two phases with slower weight gain: (i) directly after birth during adaptation to extrauterine conditions (P0-P2) and (ii) during the weaning period (P15-P21), when nutrition changed from mother's milk to solid chow and water. During the first 3 days after birth, remarkable changes in urinary Na+, Ca2+, Mg2+, and phosphate concentrations occurred, whereas K+ and Cl- concentrations hardly changed. From days 4-14 after birth, Na+, Ca2+, Mg2+, K+, and Cl- concentrations remained relatively stable at low levels. Urinary concentrations of creatinine, NH4+, phosphate, and sulfate constantly increased from birth until after weaning. Profiles of salt excretion in KCNJ10-/- mice exemplified the relevance of age-dependent analysis of urinary excretion. In conclusion, the most critical phases for analysis of renal ion excretion during the first weeks of life are directly after birth and during the weaning period. The age dependence of urinary excretion varies for the different ions. This should be taken into consideration when the renal phenotype of mice is investigated during the first weeks of life., OA-Komponente aus Allianzlizenz

Primary aldosteronism is a heterogeneous group of disorders comprising both sporadic and familial forms. Mutations in the KCNJ5 gene, which encodes the inward rectifier K(+) channel 4 (G protein-activated inward rectifier K(+) channel 4, Kir3.4), cause familial hyperaldosteronism type III (FH-III) and are involved in the pathogenesis of sporadic aldosterone-producing adenomas.The objective of the study was to characterize the effects of a newly described KCNJ5 mutation in vitro.The index case is a 62-year-old woman affected by primary aldosteronism, who underwent left adrenalectomy after workup for adrenal adenoma. Exon 1 of KCNJ5 was PCR amplified from adrenal tissue and peripheral blood and sequenced. Electrophysiological and gene expression studies were performed to establish the functional effects of the new mutation on the membrane potential and adrenal cell CYP11B2 expression.KCNJ5 sequencing in the index case revealed a new p.Y152C germline mutation; interestingly, the phenotype of the patient was milder than most of the previously described FH-III families. The tyrosine-to-cysteine substitution resulted in pathological Na(+) permeability, cell membrane depolarization, and disturbed intracellular Ca(2+) homeostasis, effects similar, albeit smaller, to the ones demonstrated for other KCNJ5 mutations. Gene expression studies revealed an increased expression of CYP11B2 and its transcriptional regulator NR4A2 in HAC15 adrenal cells overexpressing KCNJ5(Y152C) compared to the wild-type channel. The effect was clearly Ca(2+)-dependent, because it was abolished by the calcium channel blocker nifedipine.Herein we describe a new germline mutation in KCNJ5 responsible for FH-III.

Task2 K + channel expression in the central nervous system is surprisingly restricted to a few brainstem nuclei, including the retrotrapezoid (RTN) region. All Task2-positive RTN neurons were lost in mice bearing a Phox2b mutation that causes the human congenital central hypoventilation syndrome. In plethysmography, Task2 −/− mice showed disturbed chemosensory function with hypersensitivity to low CO 2 concentrations, leading to hyperventilation. Task2 probably is needed to stabilize the membrane potential of chemoreceptive cells. In addition, Task2 −/− mice lost the long-term hypoxia-induced respiratory decrease whereas the acute carotid-body-mediated increase was maintained. The lack of anoxia-induced respiratory depression in the isolated brainstem–spinal cord preparation suggested a central origin of the phenotype. Task2 activation by reactive oxygen species generated during hypoxia could silence RTN neurons, thus contributing to respiratory depression. These data identify Task2 as a determinant of central O 2 chemoreception and demonstrate that this phenomenon is due to the activity of a small number of neurons located at the ventral medullary surface.

TASK1 (KCNK3) and TASK3 (KCNK9) are two-pore domain potassium channels highly expressed in adrenal glands. TASK1/TASK3 heterodimers are believed to contribute to the background conductance whose inhibition by angiotensin II stimulates aldosterone secretion. We used task1-/- mice to analyze the role of this channel in adrenal gland function. Task1-/- exhibited severe hyperaldosteronism independent of salt intake, hypokalemia, and arterial 'low-renin' hypertension. The hyperaldosteronism was fully remediable by glucocorticoids. The aldosterone phenotype was caused by an adrenocortical zonation defect. Aldosterone synthase was absent in the outer cortex normally corresponding to the zona glomerulosa, but abundant in the reticulo-fasciculata zone. The impaired mineralocorticoid homeostasis and zonation were independent of the sex in young mice, but were restricted to females in adults. Patch-clamp experiments on adrenal cells suggest that task3 and other K+ channels compensate for the task1 absence. Adrenal zonation appears as a dynamic process that even can take place in adulthood. The striking changes in the adrenocortical architecture in task1-/- mice are the first demonstration of the causative role of a potassium channel in development/differentiation. ©2008 European Molecular Biology Organization., The study was supported by the Deutsche Forschungsgemeinschaft (SFB699 to RW), the Centre National de la Recherche scientifique (JB), and by the European Section of Aldosterone Council (11AD5B to JB).

Heteromeric KCNEx/KCNQ1 (=KvLQT1, Kv7.1) K(+) channels are important for repolarization of cardiac myocytes, endolymph secretion in the inner ear, gastric acid secretion, and transport across epithelia. They are modulated by pH in a complex way: homomeric KCNQ1 is inhibited by external acidification (low pH(e)); KCNE2/KCNQ1 is activated; and for KCNE1/KCNQ1, variable effects have been reported.The role of KCNE subunits for the effect of pH(e) on KCNQ1 was analyzed in transfected COS cells and cardiac myocytes by the patch-clamp technique.In outside-out patches of transfected cells, hKCNE2/hKCNQ1 current was increased by acidification down to pH 4.5. Chimeras with the acid-insensitive hKCNE3 revealed that the extracellular N-terminus and at least part of the transmembrane domain of hKCNE2 are needed for activation by low pH(e). hKCNE1/hKCNQ1 heteromeric channels exhibited marked changes of biophysical properties at low pH(e): The slowly activating hKCNE1/hKCNQ1 channels were converted into constitutively open, non-deactivating channels. Experiments on guinea pig and mouse cardiac myocytes pointed to an important role of KCNQ1 during acidosis implicating a significant contribution to cardiac repolarization under acidic conditions.External pH can modify current amplitude and biophysical properties of KCNQ1. KCNE subunits work as molecular switches by modulating the pH sensitivity of human KCNQ1.

Tubulin, the building block of microtubules, consists of an alpha and beta subunit, each in itself a family of several highly homologous isotypes. Abundance, tissue specificity, developmental regulation, and possibly function vary between isotypes. Six isotypes of beta tubulin (class I to class VI) have been cloned from several vertebrate species. Class I beta tubulin is believed to be widely expressed, but has not been studied by in situ hybridization in any vertebrate species so far. We have cloned a beta tubulin from zebrafish that appears most similar to other vertebrate class I tubulins and name it zbeta1 tubulin, accordingly. We report a distinct expression pattern of zbeta1 tubulin in the zebrafish embryo in restricted regions of the peripheral and central nervous system that comprise early-differentiating neurons. The expression pattern changes during development and in the adult zebrafish expression mostly is confined to a subset of proliferative zones that include the subependymal zone around the telencephalic ventricle, zones in the preoptic and hypothalamic area and in the olfactory epithelium. Thus, zbeta1 tubulin is expressed with remarkable selectivity during neuronal differentiation and neurogenesis in the embryonic and adult nervous system, respectively.

RFamides constitute a large family of neuromodulatory peptides. We have cloned a zebrafish gene, which is presumably a homologue to the mammalian PQRF subfamily of RFamides, and named it zfPQRF for its species and subfamily allocation. We report that in contrast to its mammalian counterparts zfPQRF is expressed in the olfactory bulb and the nucleus olfactoretinalis in the telencephalon, but absent in more caudal regions, including hypothalamus, brain stem and spinal cord. zfPQRF-expressing neurons originate in the vicinity of the olfactory placode and populate the nuclei of the terminal nerve during later development, as demonstrated by co-expression of zebrafish salmon-type gonadotropin releasing hormone, which was found to exclusively label terminal nerve neurons.