The expression of wild-type pendrin (SLC26A4) in human embryonic kidney (HEK 293 Phoenix) cells leads to the activation of cationic currents

Objective: The SLC26A4 protein (pendrin) seems to be involved in the exchange of chloride with other anions, therefore being responsible for iodide organification in the thyroid gland and the conditioning of the endolymphatic fluid in the inner ear. Malfunction of SLC26A4 leads to Pendred syndrome, characterized by mild thyroid dysfunction often associated with goiter and/or prelingual deafness. The precise function of the SLC26A4 protein, however, is still elusive. An open question is still whether the SLC26A4-induced ion exchange mechanism is electrogenic or electroneutral. Recently, it has been shown that human pendrin expressed in monkey cells leads to chloride currents. Methods: We overexpressed the human SLC26A4 isoform in HEK293 Phoenix cells and measured cationic and anionic currents by the patch-clamp technique in whole cell configuration. Results: Here we show that human pendrin expressed in human cells does not lead to the activation of chloride currents, but, in contrast, leads to an increase of cationic currents. Conclusion: Our experiments suggest that the SLC26A4-induced chloride transport is electroneutral when expressed in human cellular systems

Functional characterization of wild-type and a mutated form of SLC26A4 identified in a patient with pendred syndrome

BACKGROUND: Malfunction of the SLC26A4 protein leads to prelingual deafness often associated with mild thyroid dysfunction and goiter. It is assumed that SLC26A4 acts as a chloride/anion exchanger responsible for the iodide organification in the thyroid gland, and conditioning of the endolymphatic fluid in the inner ear. METHODS: Chloride uptake studies were made using HEK293-Phoenix cells expressing human wild type SLC26A4 (pendrin) and a mutant (SLC26A4(S28R)) we recently described in a patient with hypothyroidism, goiter and sensorineural hearing loss. RESULTS: Experiments are summarized showing the functional characterization of wild type SLC26A4 and a mutant (S28R), which we described recently. This mutant protein is transposed towards the cell membrane, however, its transport capability is markedly reduced if compared to wild-type SLC26A4. Furthermore, we show that the SLC26A4 induced chloride uptake in HEK293-Phoenix cells competes with iodide, and, in addition, that the chloride uptake can be blocked by NPPB and niflumic acid, whereas DIDS is ineffective. CONCLUSIONS: The functional characteristics of SLC26A4(S28R) we describe here, are consistent with the clinical phenotype observed in the patient from which the mutant was derived

Evaluating human basal metabolism: the erroneous and misleading use of so-called "prediction equations"

Prediction (regression) equations are widely used, but their reliability as predictive tools is questionable as they provide contradicting results. The key point is that values calculated by regression equations are not precisely defined numbers but lie within a range of possible values in the standard deviation interval, none of which can be considered as the most probable. Ignoring this point leads to illicit/improper calculations, generating wrong results, which may have adverse consequences for human health. To demonstrate this, we applied the equations of Harris and Benedict in a reverse method, i.e. calculating (predicting) the daily energy expenditure in the same subjects used to obtain the equations and comparing values with the original measured data. We used the Bland-Altman and frequency distribution analyses. We found large differences in both individual data and population characteristics, showing that prediction equations are not predictive tools

Effetto di una dieta carente di iodio sulla funzione tiroidea in topi knock out per il gene della pendrina

La sindrome di Pendred \ue8 un disordine genetico recessivo caratterizzato da sordit\ue0 neurosensoriale, gozzo e difetti nell\u2019organificazione dello iodio. Tale sindrome \ue8 causata da mutazioni del gene PDS che codifica per un trasportatore anionico (Pendrina) espresso nella tiroide, nell\u2019orecchio interno e nel rene. Nella tiroide la Pendrina si localizza sulla superficie apicale delle cellule follicolari, dove regola il trasporto di iodio dal citoplasma alla colloide. La Sindrome di Pendred presenta un\u2019 estrema variabilit\ue0 inter- ed intrafamiliare in termini di gravit\ue0 dei sintomi ed et\ue0 di insorgenza. In particolare le disfunzioni tiroidee sono presenti solo nel 50% circa dei pazienti e variano da un leggero aumento del volume della ghiandola alla presenza di gozzo di notevoli dimensioni. Tale variabilit\ue0 \ue8 probabilmente dovuta all\u2019interazione tra fattori genetici e ambientali, quali l\u2019apporto di iodio. In questo studio abbiamo valutato l\u2019effetto di una dieta carente di iodio sul fenotipo tiroideo di topi knock-out per la Pendrina (PDS -/-). I topi PDS -/- sviluppano nel corso della prime settimane di vita alterazioni a carico dell\u2019orecchio interno tipiche della sindrome di Pendred , ma la funzione tiroidea \ue8 riportata essere nella norma. Tuttavia \ue8 da considerare il fatto che tali studi sono stati condotti su animali alimentati con un mangime ad alto contenuto di iodio mentre non \ue8 stata finora valutata la funzionalit\ue0 tiroidea in presenza di insufficiente apporto di iodio. Per chiarire questo aspetto abbiamo mantenuto una colonia di topi con diverso genotipo (PDS +/+; PDS +/-; PDS -/-) suddivisa in due gruppi di trattamento: gruppo sperimentale (S, n=21) e gruppo controllo (C, n=22). Il gruppo S \ue8 stato sottoposto ad alimentazione controllata con mangime semisintetico a basso contenuto di iodio e l\u2019aggiunta di una dose minima di ioduro nell\u2019acqua (KI 2ng/ml). Il gruppo C ha ricevuto invece una dieta normale, tipicamente ad elevato contenuto di iodio. Tutti gli animali sono stati genotipizzati ed \ue8 stato eseguito un prelievo di sangue a 6 mesi per la valutazione della funzionalit\ue0 tiroidea tramite dosaggio di TSH e TT4 mediante RIA. I valori di TSH non rivelano differenze significative tra i vari gruppi. In particolare non sembrano influenzati n\ue9 dal genotipo, n\ue9 dall\u2019apporto di iodio (PDS -/- gruppo S: 203,7 +/- 14,09 ng/ml; PDS -/- gruppo C: 217,1 +/- 18,86 ng/ml). Non sono state osservate differenze significative anche per quanto riguarda i valori di TT4 (PDS -/- gruppo S: 55,57 +/- 12,75 nmol/L; PDS-/- gruppo C : 74,03 +/- 7,54 nmol/L). Sulla base di questi dati \ue8 possibile concludere che una dieta carente di iodio non sembra influenzare in maniera rilevante il fenotipo tiroideo di topi PDS -/-. \uc8 pertanto ipotizzabile che nel topo esistano altri sistemi di trasporto in grado di compensare la mancanza della Pendrina. Studi futuri sono necessari per identificare ulteriori meccanismi di trasporto apicale dello iodio e chiarire le eventuali differenze presenti tra la patologia umana e il modello murino

ICln channels reconstituted in heart-lipid bilayer are selective to chloride

ICln is an ion channel cloned from renal epithelial cells. The reconstitution of the protein in 1,2-diphytanoyl- sn-glycero-3-phosphocholine (Diph-PC) bilayer membranes reveals potassium-selective channels, which become more chloride selective in the presence of calcium. Here we show that the ion selectivity of ICln also depends on the lipid environment in which the channels are reconstituted. Diph-PC is a synthetic lipid commonly used for reconstituting ion channels. However, since this lipid is not found in native membranes, we reconstituted the ICln ion channels in a polar heart-lipid extract. Using this lipid mixture the reconstituted ICln ion channels are chloride selective in the presence of calcium and an acidic pH. The relative ion selectivity of ICln under these conditions is similar to the cation versus anion selectivity of native ion channels activated by cell swelling

S-CMC-Lys Protective Effects on Human Respiratory Cells During Oxidative Stress

The mucoactive drug S-carbocysteine lysine salt monohydrate (S-CMC-Lys) stimulates glutathione (GSH) efflux from respiratory cells. Since GSH is one of the most important redox regulatory mechanisms, the aim of this study was to evaluate the S-CMC-Lys effects on GSH efflux and intracellular concentration during an oxidative stress induced by the hydroxyl radical (center dot OH). Experiments were performed on cultured human respiratory WI-26VA4 cells by means of patch-clamp experiments in whole-cell configuration and of fluorimetric analyses at confocal microscope. center dot OH exposure induced an irreversible inhibition of the GSH and chloride currents that was prevented if the cells were incubated with S-CMC-Lys. In this instance, the currents were inhibited by the specific blocker CFTRinh-172. CFT1-C2 cells, which lack a functional CFTR channel, were not responsive to S-CMC-Lys, but the stimulatory effect of the drug was restored in LCFSN-infected CFT1 cells, functionally corrected to express CFTR. Fluorimetric measurements performed on the S-CMC-Lys-incubated cells revealed a significant increase of the GSH concentration that was completely hindered after oxidative stress and abolished by CFTRinh-172. The cellular content of reactive oxygen species was significantly lower in the S-CMC-Lystreated cells either before or after center dot OH exposure. As a conclusion, S-CMC-Lys could exert a protective function during oxidative stress, therefore preventing or reducing the ROS-mediated inflammatory response

Absence of primary hypothyroidism and goiter in Slc26a4(-/-) mice fed on a low iodine diet

Background: Mutations in the SLC26A4 gene, coding for the anion transporter pendrin, are responsible for Pendred syndrome, characterized by congenital sensorineural deafness and dyshormonogenic goiter. The physiological role of pendrin in the thyroid is still unclear and the lack of a thyroid phenotype in some patients with SLC26A4 mutations and in Slc26a4 (-/-) mice indicate the existence of environmental or individual modifiers able to compensate for pendrin inactivation in the thyroid. Since pendrin can transport iodide in vitro, variations in iodide supply have been claimed to account for the thyroid phenotype associated with pendrin defects. Aim: the Slc26a4 (-/-) mouse model was used to test the hypothesis that iodide supply may influence the penetrance and expressivity of SLC26A4 mutations. Materials and methods: Slc26a4 (-/-) and (+/+) mice were fed up to 6 months on a standard or low iodine diet and were evaluated for thyroid structural abnormalities or biochemical hypothyroidism. Results: a 27-fold iodide restriction induced similar modifications in thyroid histology, but no differences in thyroid size, T4 or TSH levels were observed between between Slc26a4 (-/-) and (+/+) mice, either in standard conditions and during iodine restriction. Conclusions: iodide restriction is not able to induce a thyroid phenotype in Slc26a4 (-/-) mice. These experimental data, together with those coming from a review of familial Pendred cases leaving in regions either with low or sufficient iodide supply, support the idea that the expression of thyroid phenotype in Pendred syndrome is more powerfully influenced by individual factors than by dietary iodide