Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities.

Hypertension affects one billion people and is a principal reversible risk factor for cardiovascular disease. Pseudohypoaldosteronism type II (PHAII), a rare Mendelian syndrome featuring hypertension, hyperkalaemia and metabolic acidosis, has revealed previously unrecognized physiology orchestrating the balance between renal salt reabsorption and K(+) and H(+) excretion. Here we used exome sequencing to identify mutations in kelch-like 3 (KLHL3) or cullin 3 (CUL3) in PHAII patients from 41 unrelated families. KLHL3 mutations are either recessive or dominant, whereas CUL3 mutations are dominant and predominantly de novo. CUL3 and BTB-domain-containing kelch proteins such as KLHL3 are components of cullin-RING E3 ligase complexes that ubiquitinate substrates bound to kelch propeller domains. Dominant KLHL3 mutations are clustered in short segments within the kelch propeller and BTB domains implicated in substrate and cullin binding, respectively. Diverse CUL3 mutations all result in skipping of exon 9, producing an in-frame deletion. Because dominant KLHL3 and CUL3 mutations both phenocopy recessive loss-of-function KLHL3 mutations, they may abrogate ubiquitination of KLHL3 substrates. Disease features are reversed by thiazide diuretics, which inhibit the Na-Cl cotransporter in the distal nephron of the kidney; KLHL3 and CUL3 are expressed in this location, suggesting a mechanistic link between KLHL3 and CUL3 mutations, increased Na-Cl reabsorption, and disease pathogenesis. These findings demonstrate the utility of exome sequencing in disease gene identification despite the combined complexities of locus heterogeneity, mixed models of transmission and frequent de novo mutation, and establish a fundamental role for KLHL3 and CUL3 in blood pressure, K(+) and pH homeostasis.

Uromodulin exclusion list improves urinary exosomal protein identification.

Advances in mass spectrometry (MS) have encouraged interest in its deployment in urine biomarker studies, but success has been limited. Urine exosomes have been proposed as an ideal source of biomarkers for renal disease. However, the abundant urinary protein, uromodulin, cofractionates with exosomes during isolation and represents a practical contaminant that limits MS sensitivity. Uromodulin depletion has been attempted but is labor- and time-intensive and may remove important protein biomarkers. We describe the application of an exclusion list (ExL) of uromodulin-related peptide ions, coupled with high-sensitivity mass spectrometric analysis, to increase the depth of coverage of the urinary exosomal proteome. Urine exosomal protein samples from healthy volunteers were subjected to tandem MS and abundant uromodulin peptides identified. Samples were run for a second time, while excluding these uromodulin peptides from fragmentation to allow identification of peptides from lower-abundance proteins. Uromodulin exclusion was performed in addition to dynamic exclusion. Results from these two procedures revealed 222 distinct proteins from conventional analysis, compared with 254 proteins after uromodulin exclusion, of which 188 were common to both methods. By unmasking a previously unidentified protein set, adding the ExL increased overall protein identifications by 29.7% to a total of 288 proteins. A fixed ExL, used in combination with conventional methods, effectively increases the depth of urinary exosomal proteins identified by MS, reducing the need for uromodulin depletion.

Life-threatening metabolic alkalosis in Pendred syndrome.

INTRODUCTION: Pendred syndrome, a combination of sensorineural deafness, impaired organification of iodide in the thyroid and goitre, results from biallelic defects in pendrin (encoded by SLC26A4), which transports chloride and iodide in the inner ear and thyroid respectively. Recently, pendrin has also been identified in the kidneys, where it is found in the apical plasma membrane of non-α-type intercalated cells of the cortical collecting duct. Here, it functions as a chloride-bicarbonate exchanger, capable of secreting bicarbonate into the urine. Despite this function, patients with Pendred syndrome have not been reported to develop any significant acid-base disturbances, except a single previous reported case of metabolic alkalosis in the context of Pendred syndrome in a child started on a diuretic. CASE REPORT: We describe a 46-year-old female with sensorineural deafness and hypothyroidism, who presented with severe hypokalaemic metabolic alkalosis during inter-current illnesses on two occasions, and who was found to be homozygous for a loss-of-function mutation (V138F) in SLC26A4. Her acid-base status and electrolytes were unremarkable when she was well. CONCLUSION: This case illustrates that, although pendrin is not usually required to maintain acid-base homeostasis under ambient condition, loss of renal bicarbonate excretion by pendrin during a metabolic alkalotic challenge may contribute to life-threatening acid-base disturbances in patients with Pendred syndrome.

Glyceraldehyde 3-phosphate dehydrogenase is required for band 3 (anion exchanger 1) membrane residency in the mammalian kidney.

The mammalian kidney isoform of the essential chloride-bicarbonate exchanger AE1 differs from its erythrocyte counterpart, being shorter at its N terminus. It has previously been reported that the glycolytic enzyme GAPDH interacts only with erythrocyte AE1, by binding to the portion not found in the kidney isoform. (Chu H, Low PS. Biochem J 400:143-151, 2006). We have identified GAPDH as a candidate binding partner for the C terminus of both AE1 and AE2. We show that full-length AE1 and GAPDH coimmunoprecipitated from both human and rat kidney as well as from Madin-Darby canine kidney (MDCK) cells stably expressing kidney AE1, while in human liver, AE2 coprecipitated with GAPDH. ELISA and glutathione S-transferase (GST) pull-down assays using GST-tagged C-terminal AE1 fusion protein confirmed that the interaction is direct; fluorescence titration revealed saturable binding kinetics with Kd 2.3±0.2 µM. Further GST precipitation assays demonstrated that the D902EY residues in the D902EYDE motif located within the C terminus of AE1 are important for GAPDH binding. In vitro GAPDH activity was unaffected by C-terminal AE1 binding, unlike in erythrocytes. Also, differently from red cell N-terminal binding, GAPDH-AE1 C-terminal binding was not disrupted by phosphorylation of AE1 in kidney AE1-expressing MDCK cells. Importantly, small interfering RNA knockdown of GAPDH in these cells resulted in significant intracellular retention of AE1, with a concomitant reduction in AE1 at the cell membrane. These results indicate differences between kidney and erythrocyte AE1/GAPDH behavior and show that in the kidney, GAPDH is required for kidney AE1 to achieve stable basolateral residency.

Importance of early audiologic assessment in distal renal tubular acidosis.

Autosomal recessive distal renal tubular acidosis is usually a severe disease of childhood, often presenting as failure to thrive in infancy. It is often, but not always, accompanied by sensorineural hearing loss, the clinical severity and age of onset of which may be different from the other clinical features. Mutations in either ATP6V1B1 or ATP6V0A4 are the chief causes of primary distal renal tubular acidosis with or without hearing loss, although the loss is often milder in the latter. We describe a kindred with compound heterozygous alterations in ATP6V0A4, where hearing loss was formally diagnosed late in both siblings such that they missed early opportunities for hearing support. This kindred highlights the importance of routine audiologic assessments of all children with distal renal tubular acidosis, irrespective either of age at diagnosis or of which gene is mutated. In addition, when diagnostic genetic testing is undertaken, both genes should be screened irrespective of current hearing status. A strategy for this is outlined.

Disorders of water and acid-base homeostasis.

Disorders of water balance lead either to dehydration or overhydration. Because there is an intimate relationship between water and sodium concentration (water generally following salt), one can distinguish hypotonic, isotonic and hypertonic dehydration and the same for overhydration. The vast majority of water balance disorders are acquired. In this article, the focus is on the inherited disorders both of water (nephrogenic diabetes insipidus) and acid-base balance. Both acidosis and alkalosis can arise from primary tubular ion transport abnormalities. The alkaloses are usually secondary to salt handling problems, whereas the renal tubular acidoses are often a consequence of primary abnormalities of acid-base transporters.

An extended nomenclature for mammalian V-ATPase subunit genes and splice variants.

The vacuolar-type H(+)-ATPase (V-ATPase) is a multisubunit proton pump that is involved in both intra- and extracellular acidification processes throughout the body. Multiple homologs and splice variants of V-ATPase subunits are thought to explain its varied spatial and temporal expression pattern in different cell types. Recently subunit nomenclature was standardized with a total of 22 subunit variants identified. However this standardization did not accommodate the existence of splice variants and is therefore incomplete. Thus, we propose here an extension of subunit nomenclature along with a literature and sequence database scan for additional V-ATPase subunits. An additional 17 variants were pulled from a literature search while 4 uncharacterized potential subunit variants were found in sequence databases. These findings have been integrated with the current V-ATPase knowledge base to create a new V-ATPase subunit catalogue. It is envisioned this catalogue will form a new platform on which future studies into tissue- and organelle-specific V-ATPase expression, localization and function can be based.

Cellular physiology of the renal H+ATPase.

PURPOSE OF REVIEW: Vacuolar-type H+ATPases are multisubunit macromolecules that play an essential role in renal acid-base homeostasis. Other cellular processes also rely on the proton pumping ability of H+ATPases to acidify organellar or lumenal spaces. Several diseases, including distal renal tubular acidosis, osteoporosis and wrinkly skin syndrome, are due to mutations in genes encoding alternate subunits that make up the H+ATPase. This review highlights recent key articles in this research area. RECENT FINDINGS: Further insights into the structure, expression and regulation of H+ATPases have been elucidated, within the kidney and elsewhere. This knowledge may enhance the potential for future drug targeting. SUMMARY: Novel findings concerning tissue-specific subunits of the H+ATPase that are important in the kidney and more general lessons of H+ATPase function and regulation are slowly emerging, though the paucity of cellular tools available has to date limited progress.

Uromodulin mutations causing familial juvenile hyperuricaemic nephropathy lead to protein maturation defects and retention in the endoplasmic reticulum.

Familial juvenile hyperuricaemic nephropathy (FJHN), an autosomal dominant disorder, is caused by mutations in the UMOD gene, which encodes Uromodulin, a glycosylphosphatidylinositol-anchored protein that is expressed in the thick ascending limb of the loop of Henle and excreted in the urine. Uromodulin contains three epidermal growth factor (EGF)-like domains, a cysteine-rich region which includes a domain of eight cysteines and a zona pellucida (ZP) domain. Over 90% of UMOD mutations are missense, and 62% alter a cysteine residue, implicating a role for protein misfolding in the disease. We investigated 20 northern European FJHN probands for UMOD mutations. Wild-type and mutant Uromodulins were functionally studied by expression in HeLa cells and by the use of western blot analysis and confocal microscopy. Six different UMOD missense mutations (Cys32Trp, Arg185Gly, Asp196Asn, Cys217Trp, Cys223Arg and Gly488Arg) were identified. Patients with UMOD mutations were phenotypically similar to those without UMOD mutations. The mutant Uromodulins had significantly delayed maturation, retention in the endoplasmic reticulum (ER) and reduced expression at the plasma membrane. However, Gly488Arg, which is the only mutation we identified in the ZP domain, was found to be associated with milder in vitro abnormalities and to be the only mutant Uromodulin detected in conditioned medium from transfected cells, indicating that the severity of the mutant phenotypes may depend on their location within the protein. Thus, FJHN-causing Uromodulin mutants are retained in the ER, with impaired intracellular maturation and trafficking, thereby indicating mechanisms whereby Uromodulin mutants may cause the phenotype of FJHN.

RhCG is the major putative ammonia transporter expressed in the human kidney, and RhBG is not expressed at detectable levels.

Rhesus glycoprotein homologs RhAG, RhBG, and RhCG comprise a recently identified branch of the Mep/Amt ammonia transporter family. Animal studies have shown that RhBG and RhCG are present in the kidney distal tubules. Studies in mouse and rat tissue suggest a basolateral localization for RhBG in cells of the distal tubules including the alpha-intercalated cells (alpha-IC), but no localization of RhBG has been reported in human tissue. To date RhCG localization has been described as exclusively apical plasma membrane in mouse and rat kidney, or apical and basolateral in humans, and some mouse and rat tissue studies. We raised novel antibodies to RhBG and RhCG to examine their localization in the human kidney. Madin-Darby canine kidney (MDCKI) cell lines stably expressing human green fluorescent protein-tagged RhBG or RhCG and human tissue lysates were used to demonstrate the specificity of these antibodies for detecting RhBG and RhCG. Using immunoperoxidase staining and antigen liberation techniques, both apical and basolateral RhCG localization was observed in the majority of the cells of the distal convoluted tubule and IC of the connecting tubule and collecting duct. Confocal microscopic imaging of normal human kidney cryosections showed that RhCG staining was predominantly localized to the apical membrane in these cells with some basolateral and intracellular staining evident. A proportion of RhCG staining labeled kAE1-positive cells, confirming that RhCG is localized to the alpha-IC cells. Surprisingly, no RhBG protein was detectable in the human kidney by Western blot analysis of tissue lysates, or by immunohistochemistry or confocal microscopy of tissue sections. The same antibodies, however, could detect RhBG in rat tissue. We conclude that under normal conditions, RhCG is the major putative ammonia transporter expressed in the human kidney and RhBG is not expressed at detectable levels.

Mechanisms in hyperkalemic renal tubular acidosis.

The form of renal tubular acidosis associated with hyperkalemia is usually attributable to real or apparent hypoaldosteronism. It is therefore a common feature in diabetes and a number of other conditions associated with underproduction of renin or aldosterone. In addition, the close relationship between potassium levels and ammonia production dictates that hyperkalemia per se can lead to acidosis. Here I describe the modern relationship between molecular function of the distal portion of the nephron, pathways of ammoniagenesis, and hyperkalemia.

The d subunit plays a central role in human vacuolar H(+)-ATPases.

The multi-subunit vacuolar-type H(+)-ATPase consists of a V(1) domain (A-H subunits) catalyzing ATP hydrolysis and a V(0) domain (a, c, c', c", d, e) responsible for H(+) translocation. The mammalian V(0) d subunit is one of the least-well characterized, and its function and position within the pump are still unclear. It has two different forms encoded by separate genes, d1 being ubiquitous while d2 is predominantly expressed at the cell surface in kidney and osteoclast. To determine whether it forms part of the pump's central stalk as suggested by bacterial A-ATPase studies, or is peripheral as hypothesized from a yeast model, we investigated both human d subunit isoforms. In silico structural modelling demonstrated that human d1 and d2 are structural orthologues of bacterial subunit C, despite poor sequence identity. Expression studies of d1 and d2 showed that each can pull down the central stalk's D and F subunits from human kidney membrane, and in vitro studies using D and F further showed that the interactions between these proteins and the d subunit is direct. These data indicate that the d subunit in man is centrally located within the pump and is thus important in its rotary mechanism.

Human H+ATPase a4 subunit mutations causing renal tubular acidosis reveal a role for interaction with phosphofructokinase-1.

The vacuolar-type ATPase (H+ATPase) is a ubiquitously expressed multisubunit pump whose regulation is poorly understood. Its membrane-integral a-subunit is involved in proton translocation and in humans has four forms, a1-a4. This study investigated two naturally occurring point mutations in a4's COOH terminus that cause recessive distal renal tubular acidosis (dRTA), R807Q and G820R. Both lie within a domain that binds the glycolytic enzyme phosphofructokinase-1 (PFK-1). We recreated these disease mutations in yeast to investigate effects on protein expression, H+ATPase assembly, targeting and activity, and performed in vitro PFK-1 binding and activity studies of mammalian proteins. Mammalian studies revealed complete loss of binding between the COOH terminus of a4 containing the G-to-R mutant and PFK-1, without affecting PFK-1's catalytic activity. In yeast expression studies, protein levels, H+ATPase assembly, and targeting of this mutant were all preserved. However, severe (78%) loss of proton transport but less decrease in ATPase activity (36%) were observed in mutant vacuoles, suggesting a requirement for the a-subunit/PFK-1 binding to couple these two functions. This role for PFK in H+ATPase function was supported by similar functional losses and uncoupling ratio between the two proton pump domains observed in vacuoles from a PFK-null strain, which was also unable to grow at alkaline pH. In contrast, the R-to-Q mutation dramatically reduced a-subunit production, abolishing H+ATPase function completely. Thus in the context of dRTA, stability and function of the metabolon composed of H+ATPase and glycolytic components can be compromised by either loss of required PFK-1 binding (G820R) or loss of pump protein (R807Q).

Regulation of the basolateral chloride/base exchangers AE1 and SLC26A7 in the kidney collecting duct in potassium depletion.

In the present study, the effect of potassium depletion on the expression of acid-base transporters in the collecting duct was examined. Toward this end rats were fed a potassium-free diet for 3 weeks. Thereafter, the expression of the basolateral chloride/bicarbonate exchangers AE1 and SLC26A7 and the apical H(+)-ATPase was examined by northern hybridization, immunoblot analysis and immunofluorescence labelling. The mRNA expression of AE1 increased by a robust approximately 500% in the cortex and approximately 70% in the outer medulla, which translated into a huge increase in AE1 protein abundance in the cortex and a moderate increase in the outer medulla in K-depletion. The mRNA expression of SLC26A7 did not change significantly but its protein abundance showed a robust increase in the outer medulla. The expression of SLC26A7 remained undetected in the cortex in K-depleted rats. The post translational increase in SLC26A7 membrane abundance in potassium depletion was recapitulated in vitro using epitope-tagged SLC26A7. H(+)-ATPase displayed enhanced apical plasma membrane immunoreactivity in the OMCD in K-depletion. We suggest that the up-regulation of SLC26A7 and AE1 on the basolateral membrane of A-intercalated cells in the OMCD and CCD, respectively, along with H(+)-ATPase on the apical membrane, contributes to enhanced bicarbonate absorption in the collecting duct in K-depletion.

Inherited renal acidoses.

Inherited acidosis may result from a primary renal defect in acid-base handling, emphasizing the central role of the kidney in control of body pH; as a secondary phenomenon resulting from abnormal renal electrolyte handling; or from excess production of acid elsewhere in the body. Here, we review our current understanding of the inherited renal acidoses at a genetic and molecular level.

Molecular cloning and characterization of a novel form of the human vacuolar H+-ATPase e-subunit: an essential proton pump component.

Several of the 13 subunits comprising mammalian H(+)-ATPases have multiple alternative forms, encoded by separate genes and with differing tissue expression patterns. These may play an important role in the intracellular localization and activity of H(+)-ATPases. Here we report the cloning of a previously uncharacterized human gene, ATP6V0E2, encoding a novel H(+)-ATPase e-subunit designated e2. We demonstrate that in contrast to the ubiquitously expressed gene encoding the e1 subunit (previously called e), this novel gene is expressed in a more restricted tissue distribution, particularly kidney and brain. We show by complementation studies in a yeast strain deficient for the ortholog of this subunit, that either form of the e-subunit is essential for proper proton pump function. The identification of this novel form of the e-subunit lends further support to the hypothesis that subunit differences may play a key role in the structure, site and function of H(+)-ATPases within the cell.

V1 and V0 domains of the human H+-ATPase are linked by an interaction between the G and a subunits.

The specialized H(+)-ATPases found in the inner ear and acid-handling cells in the renal collecting duct differ from those at other sites, as they contain tissue-specific subunits, such as a4 and B1, and in the kidney, C2, d2, and G3 as well. These subunits replace the ubiquitously expressed forms. Previously, we have shown that, in major organs of both mouse and man, G3 subunit expression is limited to the kidney. Here we have shown wide-spread transcription of murine G3 in specific segments of microdissected nephron, and demonstrated additional G3 expression in epithelial fragments from human inner ear. We raised a polyclonal G3-specific antibody, which specifically detects G3 from human, mouse, and rat kidney lysates, and displays no cross-reactivity with G1 or G2. However, immunolocalization using this antibody on human and mouse kidney sections was unachievable, suggesting epitope masking. Phage display analysis and subsequent enzyme-linked immunosorbent assay, using the G3 antibody epitope peptide as bait, identified a possible interaction between the G3 subunit and the a4 subunit of the H(+)-ATPase. This interaction was verified by successfully using purified, immobilized full-length G3 to pull down the a4 subunit from human kidney membrane preparations. This confirms that a4 and G3 are component subunits of the same proton pump and explains the observed epitope masking. This interaction was also found to be a more general feature of human H(+)-ATPases, as similar G1/a1, G3/a1, and G1/a4 interactions were also demonstrated. These interactions represent a novel link between the V(1) and V(0) domains in man, which is known to be required for H(+)-ATPase assembly and regulation.

A serum potassium level above 10 mmol/l in a patient predisposed to hypokalemia.

BACKGROUND: A 58-year-old man, previously diagnosed with Bartter's syndrome, presented with a short history of vomiting, diarrhea and weakness. He had severe hyperkalemia (serum potassium levels >10 mmol/l), which was successfully managed. Post hoc investigation suggested that the patient had Gitelman's rather than Bartter's syndrome. INVESTIGATIONS: Physical examination, urine and blood analyses, chest radiography, electrocardiogram, renal ultrasound, and genetic analysis focusing on the SLC12A3 gene, which encodes the thiazide-sensitive Na/Cl cotransporter. DIAGNOSIS: Gitelman's syndrome and hyperkalemia secondary to acute renal failure plus exogenous potassium supplementation. MANAGEMENT: Intravenous calcium gluconate, insulin and dextrose administration. Temporary continuous venovenous hemodiafiltration. Genetic confirmation of the underlying molecular defect. Long-term treatment for Gitelman's syndrome with oral potassium and magnesium supplements and epithelial sodium channel-blocking drugs. Review of patient education regarding renal salt-wasting syndromes.

Salt handling and hypertension.

The kidney plays a central role in our ability to maintain an appropriate sodium balance, which is critical for the determination of blood pressure. The kidney's capacity for salt conservation may not be widely appreciated, and in general we consume vastly more salt than we need. Here we consider the socioeconomics of salt consumption, outline current knowledge of renal salt handling at the molecular level, describe some of the disease entities associated with abnormal sodium handling, give an overview of some of the animal models and their relevance to human disease, and examine the evidence that lowering our salt intake can help combat hypertension and cardiovascular disease.

Polarisation, key to good localisation.

Polarisation of cells is crucial for vectorial transport of ions and solutes. In literature, however, proteins specifically targeted to the apical or basolateral membrane are often studied in non-polarised cells. To investigate whether these data can be extrapolated to expression in polarised cells, we studied several membrane-specific proteins. In polarised MDCK cells, the Aquaporin-2 water channel resides in intracellular vesicles and apical membrane, while the vasopressin-type 2 receptor, anion-exchanger 1 (AE1) protein and E-Cadherin mainly localise to the basolateral membrane. In non-polarised MDCK cells, however, Aquaporin-2 localises, besides plasma membrane, mainly in the Golgi complex, while the others show a dispersed staining throughout the cell. Moreover, while AQP2 mutants in dominant nephrogenic diabetes insipidus are missorted to different organelles in polarised cells, they all predominantly localise to the Golgi complex in non-polarised MDCK cells. Additionally, the maturation of V2R, and likely its missorting, is affected in transiently-transfected compared to stably-transfected cells. In conclusion, we show that the use of stably-transfected polarised cells is crucial in interpreting the processing and the localisation of membrane targeted proteins.