Fiona E. Karet Frankl

Pregnancy, Primary Aldosteronism, and Adrenal CTNNB1 Mutations.

Recent discoveries of somatic mutations permit the recognition of subtypes of aldosterone-producing adenomas with distinct clinical presentations and pathological features. Here we describe three women with hyperaldosteronism, two who presented in pregnancy and one who presented after menopause. Their aldosterone-producing adenomas harbored activating mutations of CTNNB1, encoding β-catenin in the Wnt cell-differentiation pathway, and expressed LHCGR and GNRHR, encoding gonadal receptors, at levels that were more than 100 times as high as the levels in other aldosterone-producing adenomas. The mutations stimulate Wnt activation and cause adrenocortical cells to de-differentiate toward their common adrenal-gonadal precursor cell type. (Funded by grants from the National Institute for Health Research Cambridge Biomedical Research Centre and others.).

Human Urinary Exosomes as Innate Immune Effectors

Exosomes are small extracellular vesicles, approximately 50 nm in diameter, derived from the endocytic pathway and released by a variety of cell types. Recent data indicate a spectrum of exosomal functions, including RNA transfer, antigen presentation, modulation of apoptosis, and shedding of obsolete protein. Exosomes derived from all nephron segments are also present in human urine, where their function is unknown. Although one report suggested in vitro uptake of exosomes by renal cortical collecting duct cells, most studies of human urinary exosomes have focused on biomarker discovery rather than exosome function. Here, we report results from in-depth proteomic analyses and EM showing that normal human urinary exosomes are significantly enriched for innate immune proteins that include antimicrobial proteins and peptides and bacterial and viral receptors. Urinary exosomes, but not the prevalent soluble urinary protein uromodulin (Tamm-Horsfall protein), potently inhibited growth of pathogenic and commensal Escherichia coli and induced bacterial lysis. Bacterial killing depended on exosome structural integrity and occurred optimally at the acidic pH typical of urine from omnivorous humans. Thus, exosomes are innate immune effectors that contribute to host defense within the urinary tract.

Atp6v0a4 knockout mouse is a model of distal renal tubular acidosis with hearing loss, with additional extrarenal phenotype

Autosomal recessive distal renal tubular acidosis (dRTA) is a severe disorder of acid–base homeostasis, often accompanied by sensorineural deafness. We and others have previously shown that mutations in the tissue-restricted a4 and B1 subunits of the H+-ATPase underlie this syndrome. Here, we describe an Atp6v0a4 knockout mouse, which lacks the a4 subunit. Using β-galactosidase as a reporter for the null gene, developmental a4 expression was detected in developing bone, nose, eye, and skin, in addition to that expected in kidney and inner ear. By the time of weaning, Atp6v0a4−/− mice demonstrated severe metabolic acidosis, hypokalemia, and early nephrocalcinosis. Null mice were hypocitraturic, but hypercalciuria was absent. They were severely hearing-impaired, as shown by elevated auditory brainstem response thresholds and absent endocochlear potential. They died rapidly unless alkalinized. If they survived weaning with alkali supplementation, treatment could later be withdrawn, but −/− animals remained acidotic with alkaline urine. They also had an impaired sense of smell. Heterozygous animals were biochemically normal until acid-challenged, when they became more acidotic than +/+ animals. This mouse model recapitulates the loss of H+-ATPase function seen in human disease and can provide additional insights into dRTA and the physiology of the a4 subunit.

Mutation Conferring Apical-Targeting Motif on AE1 Exchanger Causes Autosomal Dominant Distal RTA

Mutations in SLC4A1 that mislocalize its product, the chloride/bicarbonate exchanger AE1, away from its normal position on the basolateral membrane of the α-intercalated cell cause autosomal dominant distal renal tubular acidosis (dRTA). We studied a family exhibiting dominant inheritance and defined a mutation (AE1-M909T) that affects the C terminus of AE1, a region rich in potential targeting motifs that are incompletely characterized. Expression of AE1-M909T in Xenopus oocytes confirmed preservation of its anion exchange function. Wild-type GFP-tagged AE1 localized to the basolateral membrane of polarized MDCK cells, but AE1-M909T localized to both the apical and basolateral membranes. Wild-type AE1 trafficked directly to the basolateral membrane without apical passage, whereas AE1-M909T trafficked to both cell surfaces, implying the gain of an apical-targeting signal. We found that AE1-M909T acquired class 1 PDZ ligand activity that the wild type did not possess. In summary, the AE1-M909T mutation illustrates the role of abnormal targeting in dRTA and provides insight into C-terminal motifs that govern normal trafficking of AE1.

Unexpected clinical sequelae of Gitelman syndrome: hypertension in adulthood is common and females have higher potassium requirements

Background Gitelman syndrome (GS) is a rare inherited disorder caused by mutations in SLC12A3, encoding the thiazide-sensitive transporter NCCT (sodium chloride co-transporter) in the distal tubule. It is characterized by renal potassium (K) and magnesium (Mg) wasting, relative hypotension and hypocalciuria. However, there is phenotypic variability and long-term studies are scarce. Methods We retrospectively assessed clinical and genetic characteristics, and electrolyte requirements, in a cohort of 36 patients with genetically proven GS. Results The 21 males and 15 females were of median age 39.5 years, range 17–66 years. Six were diagnosed in childhood. Among the 72 mutant alleles, 41 different sequence alterations were identified, of which 13 were previously unreported. Surprisingly, 44% (n = 16) of the cohort has developed hypertension (13 males, 3 females, P = 0.019; median age 53 versus 57 years, P = 0.95). One was already hypertensive by age 23 years. Currently normotensive patients were significantly younger: median 37 versus 55 years (P = 0.005). Hypertensive patients were more likely to harbour mutations in the C-terminal half of the NCCT protein (P = 0.016). Females required more K (median 128 versus 72 mmol/day; P = 0.01) but not Mg. Those with exon 26 and/or at least one destructive mutation had higher K requirements than those with neither: 108 versus 72 mmol (P = 0.016) and a tendency towards higher Mg needs: 30 versus 7.4 mmol (P = 0.07). Conclusions Our findings suggest that the development of secondary hypertension may be an expected feature of the ageing GS population despite the obligate salt wasting that characterizes the disorder. We hypothesize that this may be related to chronic secondary hyperaldosteronism. The apparently more severe phenotype in women may be related to the effects of female sex hormones on expression or function of NCCT.

Mice deficient in H+-ATPase a4 subunit have severe hearing impairment associated with enlarged endolymphatic compartments within the inner ear.

SUMMARY Mutations in the ATP6V0A4 gene lead to autosomal recessive distal renal tubular acidosis in patients, who often show sensorineural hearing impairment. A first Atp6v0a4 knockout mouse model that recapitulates the loss of H+-ATPase function seen in humans has been generated and recently reported (Norgett et al., 2012). Here, we present the first detailed analysis of the structure and function of the auditory system in Atp6v0a4−/− knockout mice. Measurements of the auditory brainstem response (ABR) showed significantly elevated thresholds in homozygous mutant mice, which indicate severe hearing impairment. Heterozygote thresholds were normal. Analysis of paint-filled inner ears and sections from E16.5 embryos revealed a marked expansion of cochlear and endolymphatic ducts in Atp6v0a4−/− mice. A regulatory link between Atp6v0a4, Foxi1 and Pds has been reported and we found that the endolymphatic sac of Atp6v0a4−/− mice expresses both Foxi1 and Pds, which suggests a downstream position of Atp6v0a4. These mutants also showed a lack of endocochlear potential, suggesting a functional defect of the stria vascularis on the lateral wall of the cochlear duct. However, the main K+ channels involved in the generation of endocochlear potential, Kcnj10 and Kcnq1, are strongly expressed in Atp6v0a4−/− mice. Our results lead to a better understanding of the role of this proton pump in hearing function.

Clinical utility of PKD2 mutation testing in a polycystic kidney disease cohort attending a specialist nephrology out-patient clinic

BackgroundADPKD affects approximately 1:1000 of the worldwide population. It is caused by mutations in two genes, PKD1 and PKD2. Although allelic variation has some influence on disease severity, genic effects are strong, with PKD2 mutations predicting later onset of ESRF by up to 20 years. We therefore screened a cohort of ADPKD patients attending a nephrology out-patient clinic for PKD2 mutations, to identify factors that can be used to offer targeted gene testing and to provide patients with improved prognostic information.Methods142 consecutive individuals presenting to a hospital nephrology out-patient service with a diagnosis of ADPKD and CKD stage 4 or less were screened for mutations in PKD2, following clinical evaluation and provision of a detailed family history (FH).ResultsPKD2 mutations were identified in one fifth of cases. 12% of non-PKD2 patients progressed to ESRF during this study whilst none with a PKD2 mutation did (median 38.5 months of follow-up, range 16–88 months, p < 0.03). A significant difference was found in age at ESRF of affected family members (non-PKD2 vs. PKD2, 54 yrs vs. 65 yrs; p < 0.0001). No PKD2 mutations were identified in patients with a FH of ESRF occurring before age 50 yrs, whereas a PKD2 mutation was predicted by a positive FH without ESRF.ConclusionsPKD2 testing has a clinically significant detection rate in the pre-ESRF population. It did not accurately distinguish those individuals with milder renal disease defined by stage of CKD but did identify a group less likely to progress to ESRF. When used with detailed FH, it offers useful prognostic information for individuals and their families. It can therefore be offered to all but those whose relatives have developed ESRF before age 50.

Urinary Exosomes Contain MicroRNAs Capable of Paracrine Modulation of Tubular Transporters in Kidney

Exosomes derived from all nephron segments are present in human urine, where their functionality is incompletely understood. Most studies have focused on biomarker discovery rather than exosome function. Through sequencing we identified the miRNA repertoire of urinary exosomes from healthy volunteers; 276 mature miRNAs and 345 pre-miRNAs were identified (43%/7% of reads). Among the most abundant were members of the miR-10, miR-30 and let-7 families. Targets for the identified miRNAs were predicted using five different databases; genes encoding membrane transporters and their regulators were enriched, highlighting the possibility that these miRNAs could modulate key renal tubular functions in a paracrine manner. As proof of concept, cultured renal epithelial cells were exposed to urinary exosomes and cellular exosomal uptake was confirmed; thereafter, reduced levels of the potassium channel ROMK and kinases SGK1 and WNK1 were observed in a human collecting duct cell line, while SPAK was unaltered. In proximal tubular cells, mRNA levels of the amino acid transporter gene SLC38A2 were diminished and reflected in a significant decrement of its encoded protein SNAT2. Protein levels of the kinase SGK1 did not change. Thus we demonstrated a novel potential function for miRNA in urinary exosomes.

Physical and Functional Links between Anion Exchanger-1 and Sodium Pump

Anion exchanger-1 (AE1) mediates chloride-bicarbonate exchange across the plasma membranes of erythrocytes and, via a slightly shorter transcript, kidney epithelial cells. On an omnivorous human diet, kidney AE1 is mainly active basolaterally in α-intercalated cells of the collecting duct, where it is functionally coupled with apical proton pumps to maintain normal acid-base homeostasis. The C-terminal tail of AE1 has an important role in its polarized membrane residency. We have identified the β1 subunit of Na(+),K(+)-ATPase (sodium pump) as a binding partner for AE1 in the human kidney. Kidney AE1 and β1 colocalized in renal α-intercalated cells and coimmunoprecipitated (together with the catalytic α1 subunit of the sodium pump) from human kidney membrane fractions. ELISA and fluorescence titration assays confirmed that AE1 and β1 interact directly, with a Kd value of 0.81 μM. GST-AE1 pull-down assays using human kidney membrane proteins showed that the last 11 residues of AE1 are important for β1 binding. siRNA-induced knockdown of β1 in cell culture resulted in a significant reduction in kidney AE1 levels at the cell membrane, whereas overexpression of kidney AE1 increased cell surface sodium pump levels. Notably, membrane staining of β1 was reduced throughout collecting ducts of AE1-null mouse kidney, where increased fractional excretion of sodium has been reported. These data suggest a requirement of β1 for proper kidney AE1 membrane residency, and that activities of AE1 and the sodium pump are coregulated in kidney.

A role for VAX2 in correct retinal function revealed by a novel genomic deletion at 2p13.3 causing distal Renal Tubular Acidosis: case report

BackgroundDistal Renal Tubular Acidosis is a disorder of acid-base regulation caused by functional failure of α-intercalated cells in the distal nephron. The recessive form of the disease (which is usually associated with sensorineural deafness) is attributable to mutations in ATP6V1B1 or ATP6V0A4, which encode the tissue-restricted B1 and a4 subunits of the renal apical H+-ATPase. ATP6V1B1 lies adjacent to the gene encoding the homeobox domain protein VAX2, at 2p13.3. To date, no human phenotype has been associated with VAX2 mutations.Case presentationThe male Caucasian proband, born of a first cousin marriage, presented at 2 months with failure to thrive, vomiting and poor urine output. No anatomical problems were identified, but investigation revealed hyperchloremic metabolic acidosis with inappropriately alkaline urine and bilateral nephrocalcinosis. Distal Renal Tubular Acidosis was diagnosed and audiometry confirmed hearing loss at 2 years. ATP6V0A4 was excluded from genetic causation by intragenic SNP linkage analysis, but ATP6V1B1 completely failed to PCR-amplify in the patient, suggesting a genomic deletion. Successful amplification of DNA flanking ATP6V1B1 facilitated systematic chromosome walking to ascertain that the proband harbored a homozygous deletion at 2p13.3 encompassing all of ATP6V1B1 and part of VAX2; gene dosage was halved in the parents. This results in the complete deletion of ATP6V1B1 and disruption of the VAX2 open reading frame. Later ocular examinations revealed bilateral rod / cone photoreceptor dystrophy and mild optic atrophy. Similar changes were not detected in an adult harbouring a disruptive mutation in ATP6V1B1.ConclusionsThe genomic deletion reported here is firstly, the only reported example of a whole gene deletion to underlie Distal Renal Tubular Acidosis, where the clinical phenotype is indistinguishable from that of other patients with ATP6V1B1 mutations; secondly, this is the first reported example of a human VAX2 mutation and associated ocular phenotype, supporting speculation in the literature that VAX2 is important for correct retinal functioning.