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.This work was supported by the Wellcome Trust (grant 088489/Z/09/Z to FEKF and Strategic award 100140/Z/12/Z to the Cambridge Institute for Medical Research). The sequencing facility is supported by the Cambridge Biomedical Research Centre

Novel desmoplakin mutation: juvenile biventricular cardiomyopathy with left ventricular non-compaction and acantholytic palmoplantar keratoderma

Two sons of a consanguineous marriage developed biventricular cardiomyopathy. One boy died of severe heart failure at the age of 6 years, the other was transplanted because of severe heart failure at the age of 10 years. In addition, focal palmoplantar keratoderma and woolly hair were apparent in both boys. As similar phenotypes have been described in Naxos disease and Carvajal syndrome, respectively, the genes for plakoglobin (JUP) and desmoplakin (DSP) were screened for mutations using direct genomic sequencing. A novel homozygous 2 bp deletion was identified in an alternatively spliced region of DSP. The deletion 5208_5209delAG led to a frameshift downstream of amino acid 1,736 with a premature truncation of the predominant cardiac isoform DSP-1. This novel homozygous truncating mutation in the isoform-1 specific region of the DSP C-terminus caused Carvajal syndrome comprising severe early-onset heart failure with features of non-compaction cardiomyopathy, woolly hair and an acantholytic form of palmoplantar keratoderma in our patient. Congenital hair abnormality and manifestation of the cutaneous phenotype in toddler age can help to identify children at risk for cardiac death

Arrhythmogenic right ventricular cardiomyopathy/dysplasia

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a heart muscle disease clinically characterized by life-threatening ventricular arrhythmias. Its prevalence has been estimated to vary from 1:2,500 to 1:5,000. ARVC/D is a major cause of sudden death in the young and athletes. The pathology consists of a genetically determined dystrophy of the right ventricular myocardium with fibro-fatty replacement to such an extent that it leads to right ventricular aneurysms. The clinical picture may include: a subclinical phase without symptoms and with ventricular fibrillation being the first presentation; an electrical disorder with palpitations and syncope, due to tachyarrhythmias of right ventricular origin; right ventricular or biventricular pump failure, so severe as to require transplantation. The causative genes encode proteins of mechanical cell junctions (plakoglobin, plakophilin, desmoglein, desmocollin, desmoplakin) and account for intercalated disk remodeling. Familiar occurrence with an autosomal dominant pattern of inheritance and variable penetrance has been proven. Recessive variants associated with palmoplantar keratoderma and woolly hair have been also reported. Clinical diagnosis may be achieved by demonstrating functional and structural alterations of the right ventricle, depolarization and repolarization abnormalities, arrhythmias with the left bundle branch block morphology and fibro-fatty replacement through endomyocardial biopsy. Two dimensional echo, angiography and magnetic resonance are the imaging tools for visualizing structural-functional abnormalities. Electroanatomic mapping is able to detect areas of low voltage corresponding to myocardial atrophy with fibro-fatty replacement. The main differential diagnoses are idiopathic right ventricular outflow tract tachycardia, myocarditis, dialted cardiomyopathy and sarcoidosis. Only palliative therapy is available and consists of antiarrhythmic drugs, catheter ablation and implantable cardioverter defibrillator. Young age, family history of juvenile sudden death, QRS dispersion ≥ 40 ms, T-wave inversion, left ventricular involvement, ventricular tachycardia, syncope and previous cardiac arrest are the major risk factors for adverse prognosis. Preparticipation screening for sport eligibility has been proven to be effective in detecting asymptomatic patients and sport disqualification has been life-saving, substantially declining sudden death in young athletes

Mutations with pathogenic potential in proteins located in or at the composite junctions of the intercalated disk connecting mammalian cardiomyocytes: a reference thesaurus for arrhythmogenic cardiomyopathies and for Naxos and Carvajal diseases

In the past decade, an avalanche of findings and reports has correlated arrhythmogenic ventricular cardiomyopathies (ARVC) and Naxos and Carvajal diseases with certain mutations in protein constituents of the special junctions connecting the polar regions (intercalated disks) of mature mammalian cardiomyocytes. These molecules, apparently together with some specific cytoskeletal proteins, are components of (or interact with) composite junctions. Composite junctions contain the amalgamated fusion products of the molecules that, in other cell types and tissues, occur in distinct separate junctions, i.e. desmosomes and adherens junctions. As the pertinent literature is still in an expanding phase and is obviously becoming important for various groups of researchers in basic cell and molecular biology, developmental biology, histology, physiology, cardiology, pathology and genetics, the relevant references so far recognized have been collected and are presented here in the following order: desmocollin-2 (Dsc2, DSC2), desmoglein-2 (Dsg2, DSG2), desmoplakin (DP, DSP), plakoglobin (PG, JUP), plakophilin-2 (Pkp2, PKP2) and some non-desmosomal proteins such as transmembrane protein 43 (TMEM43), ryanodine receptor 2 (RYR2), desmin, lamins A and C, striatin, titin and transforming growth factor-β3 (TGFβ3), followed by a collection of animal models and of reviews, commentaries, collections and comparative studies

The desmosome and pemphigus

Desmosomes are patch-like intercellular adhering junctions (“maculae adherentes”), which, in concert with the related adherens junctions, provide the mechanical strength to intercellular adhesion. Therefore, it is not surprising that desmosomes are abundant in tissues subjected to significant mechanical stress such as stratified epithelia and myocardium. Desmosomal adhesion is based on the Ca2+-dependent, homo- and heterophilic transinteraction of cadherin-type adhesion molecules. Desmosomal cadherins are anchored to the intermediate filament cytoskeleton by adaptor proteins of the armadillo and plakin families. Desmosomes are dynamic structures subjected to regulation and are therefore targets of signalling pathways, which control their molecular composition and adhesive properties. Moreover, evidence is emerging that desmosomal components themselves take part in outside-in signalling under physiologic and pathologic conditions. Disturbed desmosomal adhesion contributes to the pathogenesis of a number of diseases such as pemphigus, which is caused by autoantibodies against desmosomal cadherins. Beside pemphigus, desmosome-associated diseases are caused by other mechanisms such as genetic defects or bacterial toxins. Because most of these diseases affect the skin, desmosomes are interesting not only for cell biologists who are inspired by their complex structure and molecular composition, but also for clinical physicians who are confronted with patients suffering from severe blistering skin diseases such as pemphigus. To develop disease-specific therapeutic approaches, more insights into the molecular composition and regulation of desmosomes are required

Spinster Homolog 2 (Spns2) Deficiency Causes Early Onset Progressive Hearing Loss

Spinster homolog 2 (Spns2) acts as a Sphingosine-1-phosphate (S1P) transporter in zebrafish and mice, regulating heart development and lymphocyte trafficking respectively. S1P is a biologically active lysophospholipid with multiple roles in signalling. The mechanism of action of Spns2 is still elusive in mammals. Here, we report that Spns2-deficient mice rapidly lost auditory sensitivity and endocochlear potential (EP) from 2 to 3 weeks old. We found progressive degeneration of sensory hair cells in the organ of Corti, but the earliest defect was a decline in the EP, suggesting that dysfunction of the lateral wall was the primary lesion. In the lateral wall of adult mutants, we observed structural changes of marginal cell boundaries and of strial capillaries, and reduced expression of several key proteins involved in the generation of the EP (Kcnj10, Kcnq1, Gjb2 and Gjb6), but these changes were likely to be secondary. Permeability of the boundaries of the stria vascularis and of the strial capillaries appeared normal. We also found focal retinal degeneration and anomalies of retinal capillaries together with anterior eye defects in Spns2 mutant mice. Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals. These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing

Loss of desmoplakin isoform I causes early onset cardiomyopathy and heart failure in a Naxos-like syndrome

Background: Desmosomes are cellular junctions important for intercellular adhesion and anchoring the intermediate filament (IF) cytoskeleton to the cell membrane. Desmoplakin (DSP) is the most abundant desmosomal protein with 2 isoforms produced by alternative splicing. Methods: We describe a patient with a recessively inherited arrhythmogenic dilated cardiomyopathy with left and right ventricular involvement, epidermolytic palmoplantar keratoderma, and woolly hair. The patient showed a severe heart phenotype with an early onset and rapid progression to heart failure at 4 years of age. Results: A homozygous nonsense mutation, R1267X, was found in exon 23 of the desmoplakin gene, which results in an isoform specific truncation of the larger DSPI isoform. The loss of most of the DSPI specific rod domain and C-terminal area was confirmed by Western blotting and immunofluorescence. We further showed that the truncated DSPI transcript is unstable, leading to a loss of DSPI. DSPI is reported to be an obligate constituent of desmosomes and the only isoform present in cardiac tissue. To address this, we reviewed the expression of DSP isoforms in the heart. Our data suggest that DSPI is the major cardiac isoform but we also show that specific compartments of the heart have detectable DSPII expression. Conclusions: This is the first description of a phenotype caused by a mutation affecting only one DSP isoform. Our findings emphasise the importance of desmoplakin and desmosomes in epidermal and cardiac function and additionally highlight the possibility that the different isoforms of desmoplakin may have distinct functional properties within the desmosome