Redox dysregulation and oxidative stress in schizophrenia: genetic and functional anomalies of glutathione synthesis in patients and experimental models involving GABA interneurons and NMDA receptors

Objective: Converging evidence speak in favor of an abnormal susceptibility to oxidative stress in schizophrenia. A decreased level of glutathione (GSH), the principal non-protein antioxidant and redox regulator, was observed both in cerebrospinal-fluid and prefrontal cortex of schizophrenia patients (Do et al., 2000). Results: Schizophrenia patients have an abnormal GSH synthesis most likely of genetic origin: Two independent case-control studies showed a significant association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the GSH key synthesizing enzyme glutamate-cysteine-ligase (GCL) catalytic subunit (GCLC) gene. The most common TNR genotype 7/7 was more frequent in controls, whereas the rarest TNR genotype 8/8 was three times more frequent in patients. The disease-associated genotypes correlated with a decrease in GCLC protein expression, GCL activity and GSH content. Such a redox dysregulation during development could underlie the structural and functional anomalies in connectivity: In experimental models, GSH deficit induced anomalies similar to those observed in patients. (a) morphology: In animal models with GSH deficit during the development we observed in prefrontal cortex a decreased dendritic spines density in pyramidal cells and an abnormal development of parvalbumine (but not of calretinine) immunoreactive GABA interneurones in anterior cingulate cortex. (b) physiology: GSH depletion in hippocampal slices induces NMDA receptors hypofunction and an impairment of long term potentiation. In addition, GSH deficit affected the modulation of dopamine on NMDA-induced Ca 2+ response in cultured cortical neurons. While dopamine enhanced NMDA responses in control neurons, it depressed NMDA responses in GSH-depleted neurons. Antagonist of D2-, but not D1-receptors, prevented this depression, a mechanism contributing to the efficacy of antipsychotics. The redox sensitive ryanodine receptors and L-type calcium channels underlie these observations. (c) cognition: Developing rats with low [GSH] and high dopamine lead deficit in olfactory integration and in object recognition which appears earlier in males that females, in analogy to the delay of the psychosis onset between man and woman. Conclusion: These clinical and experimental evidence, combined with the favorable outcome of a clinical trial with N-Acetyl Cysteine, a GSH precursor, on both the negative symptoms (Berk et al., submitted) and the mismatch negativity in an auditory oddball paradigm supported the proposal that a GSH synthesis impairment of genetic origin represent, among other factors, one major risk factor in schizophrenia.

Time of injection determines the effect of alpha-MSH antiserum on DA neurons in psychological stress

Male rats were subjected to "psychological stress" which consisted in 10 sec footshock on the first day followed 24 hr later by a 10 sec stay in the experimental chamber without shock. Intravenous antiserum against alpha-MSH markedly changed the functional state of mesencephalic and hypothalamic DA neurons (assessed by histochemical microfluorimetry) when administered before the second session but not when given before the first session. These observations reveal an interesting parallelism in the temporal characteristics of the effects of alpha-MSH on avoidance behavior and central DA systems.

Immunocytochemical and pharmacological characterization of metabotropic glutamate receptors of the vestibular end organs in the frog.

Using immunocytochemistry and multiunit recording of afferent activity of the whole vestibular nerve, we investigated the role of metabotropic glutamate receptors (mGluR) in the afferent neurotransmission in the frog semicircular canals (SCC). Group I (mGluR1alpha) and group II (mGluR2/3) mGluR immunoreactivities were distributed to the vestibular ganglion neurons, and this can be attributed to a postsynaptic locus of metabotropic regulation of rapid excitatory transmission. The effects of group I/II mGluR agonist (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) and antagonist (R,S)-alpha-methyl-4-carboxyphenylglycine (MCPG) on resting and chemically induced afferent activity were studied. ACPD (10-100 microM) enhanced the resting discharge frequency. MCPG (5-100 microM) led to a concentration-dependent decrease of both resting activity and ACPD-induced responses. If the discharge frequency had previously been restored by L-glutamate (L-Glu) in high-Mg2+ solution, ACPD elicited a transient increase in the firing rate in the afferent nerve suggesting that ACPD acts on postsynaptic receptors. The L-Glu agonists, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA), were tested during application of ACPD. AMPA- and NMDA-induced responses were higher in the presence than absence of ACPD, implicating mGluR in the modulation of ionotropic glutamate receptors. These results indicate that activation of mGluR potentiates AMPA and NMDA responses through a postsynaptic interaction. We conclude that ACPD may exert modulating postsynaptic effects on vestibular afferents and that this process is activity-dependent.

Novel immunotherapeutic strategies against human papillomavirus type 16 (HPV 16) induced lesions and cervical cancer

RESUME Le cancer du col de l'utérus, deuxième cause de mort par cancer chez la femme, a pu être associé à une infection par plusieurs types de virus du Papillome Humain (HPV), et en particulier HPV 16. Les vaccins prophylactiques sont efficaces à prévenir le cancer du col utérin alors que les lésions de haut grade sont généralement traitées par ablation chirurgicale et par d'éventuels traitements additionnels. Les risques de récurrence liés aux ablations et le taux de mortalité (50%) lié au cancer, démontrent le besoin de développer des stratégies alternatives afin de cibler les lésions précancéreuses. A ce jour, les vaccins thérapeutiques ont démontré peu de résultats cliniques, contrastant avec les régressions de tumeurs ectopiques observées après vaccination dans des modèles murins avec tumeurs associées à HPV. L'induction de réponses immunitaires protectrices dans la muqueuse génitale semble être cruciale pour l'efficacité des vaccins thérapeutiques HPV et évaluer leur efficacité dans un modèle murin avec tumeurs-HPV génitales représente un pré-requis important avant de procéder à des études cliniques. Par conséquent, nous avons établi un modèle murin orthotopique où des tumeurs se développent dans (a muqueuse génitale après une instillation intra-vaginale (i.vag) de cellules tumorales exprimant les oncogènes E6/E7 d'HPV 16 et transduites par un vecteur lentiviral codant la luciferase afin de suivre le développement de ces tumeurs in vivo par imagerie. La caractérisation histologique a démontré que les tumeurs grandissaient dans l'épithélium vaginal et en accord avec leur localisation, des cellules Τ CD8 spécifiques à E7 induites par la tumeur n'étaient détectées que dans la muqueuse génitale et les ganglions drainants. Une infiltration de cellules Τ régulatrices a aussi été mise en évidence, empêchant la régression spontanée de ces tumeurs. Par conséquent, ce modèle devrait être plus adéquat pour tester des stratégies thérapeutiques, étant donné qu'il partage certaines similarités immunologiques avec les lésions génitales naturelles causées par HPV. Etant donné que les oncogènes E6 et E7 d'HPV sont nécessaires à la maintenance du phénotype cancéreux des cellules cervicales, elles représentent des antigènes cibles pour la vaccination thérapeutique. Nous avons démontré que des souris immunisées par voie sous-cutanée (s.c.) avec une dose d'un vaccin à base de polypeptide E7 d'HPV 16 et d'adjuvants, présentaient de nombreuses cellules Τ CD8 sécrétant de l'IFN-γ spécifiquement à E7 dans leurs organes lymphatiques mais également dans la muqueuse génitale. De plus, le manque de corrélation entre les réponses spécifiques mesurées dans la périphérie et dans la muqueuse génitale souligne la nécessité et l'importance de déterminer les réponses immunitaires localement là où les lésions dues à HPV se développent. Si une vaccination par voie muqueuse est plus propice à traiter/régresser des infections génitales/tumeurs que le voie parentérale est un sujet débattu. Nos données montrent que seule la voie s.c. était capable de régresser la quasi totalité des tumeurs génitales chez la souris bien que des réponses CD8 spécifiques à E7 similaires étaient mesurées dans la muqueuse génitale après des vaccinations intra-nasale et i.vag. Afin d'augmenter la réponse spécifique au vaccin dans la muqueuse génitale, des immunostimulants ont été administrés par voie i.vag après vaccination. Nous avons démontré qu'une application i.vag d'agonistes des Toll like receptors après une vaccination s.c. induisait de manière significative une augmentation des cellules Τ CD8 sécrétant de l'IFN-γ spécifiquement à E7 dans la muqueuse génitale. Plus précisément et concernant les CpG et Poly l:C, l'effet était probablement associé à une attraction locale des cellules Τ CD8 et deuxièmement dépendait respectivement des voies de signalisation TLR9 et TLR3/Mda5. Finalement, cette stratégie combinatoire a permis de régresser des grosses tumeurs génitales chez la souris, suggérant qu'une telle immunothérapie pourrait adéquatement traiter des lésions dues à HPV chez les femmes. SUMMARY Cervical cancer is the second leading cause of cancer mortality in women worldwide and results from an infection with a subset of Human Papillomavirus (HPV), HPV 16 representing the most prevalent type. The available prophylactic vaccines are an effective strategy to prevent cervical cancer while already established high grade lesions usually require surgical ablation of lesion with possible additional treatments. Recurrence risks linked to conventional ablations and the high mortality (50%) related to cervical cancer demonstrate the need for alternative strategies like immunotherapies to target pre¬cancerous lesions. Until now, therapeutic vaccines only showed limited clinical results, which strongly contrast with the regression of ectopic tumors observed in the available murine HPV tumor models after vaccination. Induction of protective immune responses in the genital mucosa (GM) may be crucial for efficacy of HPV therapeutic vaccines and evaluating their efficacy in a murine model with genital HPV- tumors represents an important prerequisite for clinical trials. Thus, we have here established an orthotopic mouse model where tumors in the GM develop after an intravaginal (i.vag) instillation of HPV 16 E6/E7 oncogenes-expressing tumor cells transduced with a luciferase encoding lentivirus vector for in vivo imaging of tumor growth. Histological characterization showed that tumor grew within the vaginal epithelium and according to their mucosal location tumor- induced E7-specific CD8 Τ cells were restricted to the GM and genital draining lymph nodes together with high Τ regulatory cells infiltrates preventing spontaneous regression. Consequently, sharing several immunological similarities with natural genital HPV lesions, this novel genital tumor model may be more adequate to test therapeutic strategies. As E6 and/or E7 HPV oncogenes expression is required for the maintenance of the cancerous phenotype of cervical cells, they represent target antigens for therapeutic vaccination. We reported that mice subcutaneously (s.c.) immunized once with an adjuvanted HPV 16 E7 polypeptide vaccine harbored high E7-specific IFN-γ secreting CD8 Τ cells in their lymphoid organs and more importantly in the GM. In addition, the lack of correlation between specific responses measured in the periphery with those measured in the GM highlighted the necessity and relevance to determine the immune responses locally where HPV 16-induced lesions develop. Whether a mucosal route of immunization is better to treat/regress genital infections/tumors than parenteral immunization is still debated. Our data shows that although similar E7-specific IFN-γ secreting CD8 Τ cells responses were measured in the GM upon mucosal routes of E7 vaccine delivery (nasal and vaginal immunizations), only the s.c immunization was able to regress at least all genital tumors in mice. To further increase the vaccine-specific responses in the GM, immunostimulatory agents were i.vag administrated after vaccination. We demonstrated that a single i.vag application of toll like receptor (TLR) agonists after a s.c. E7 vaccination induced a significant increase of E7-specific IFN-γ secreting CD8 Τ cells in the GM. More precisely, regarding CpG and Poly l:C, the effect is most probably associated with a local attraction of total CD8 Τ cells and secondly depends on TLR9 and TLR3/Mda5 signaling pathways, respectively. Finally, this combinatorial strategy induced tumor regression in mice harboring large genital tumors, suggesting that such an immunotherapy could be adequate to treat HPV-induced lesions in women.

Intravaginal live attenuated Salmonella increase local antitumor vaccine-specific CD8(+) T cells.

We have recently reported that the intravaginal instillation of synthetic Toll-like receptor 3 (TLR3) or TLR9 agonists after a subcutaneous vaccination against human papillomavirus E7 highly increases (~5-fold) the number of vaccine-specific CD8(+) T cells in the genital mucosa of mice, without affecting E7-specific systemic responses. Here, we show that the instillation of live attenuated Salmonella enterica serovar Typhimurium similarly, though more efficiently (~15- fold), increases both E7-specific and total CD8(+) T cells in the genital mucosa. Cancer immunotherapeutic strategies combining vaccination with local immunostimulation with live bacteria deserve further investigations.

Use of new biochemical plasmatic markers, plasma free and total metanephrines, for the diagnosis and follow-up of neuroblastoma in children with clinical correlation

Background: Neuroblastoma is a paediatrictumour derived from the neural crest. Biochemical diagnosis and follow up rely on quantitation of urinary catecholamines (dopamine and noradrenaline) and their metabolites vanillylmandelic acid (VMA) and homovanillic acid (HVA) (gold-standard). When combined, these analyses have a sensitivity of 95%. However, they are clearly limited by inaccuracy of urine collection in young children and normalisation of catecholamine concentrations by creatininuria. Recent development in biochemical diagnosis of pheochromocytoma, another neural crest tumour found in adults, shows that plasmatic measurement of methoxylated catecholamines called metanephrines are more sensitive and specific than other biomarkers. Moreover, a study to determine the reference intervals for metanephrines in a pediatric population has recently been completed. The aim of this work is to describe the role of metanephrines monitoring in the follow up of neuroblastoma. Method: This retrospective study included patients with neuroblastoma in whom the following parameters were determined: plasma free and total metanephrines, plasma catecholamines, 24h urinary catecholamines and metanephrines in absolute value and corrected by creatinine, VMA and HVA at the diagnosis and during treatment at the University Hospital of Lausanne (Switzerland). Eleven patients aged between the first day of life and 7 years old were followed between 2005 and 2012. Clinical outcome and biochemical concentrations of the analytes were correlated. Results: At diagnosis, plasma free and total normetanephrines and methoxytyramine have a sensitivity of 100% compared to 85% for the actual gold standard. Metanephrine remain below the upper reference limit as expected since these tumours do not produce adrenaline. The relationship between biochemical markers and clinical outcome is illustrated graphically. Plasma or urinary normetanephrine and methoxytyramine correlate better with the history of the patient than VMA and HVA, as evaluated by ordinal logistic regression. Concentrations of analytes in urine show a better correlation with clinical events when the results are corrected by creatininuria. Conclusion: Normetanephrine and methoxytyramine reflect disease history in neuroblastoma patients and could play a significant role in the follow up of this type of tumour. Formal studies in a sufficient number of patients are needed to confirm this preliminary observation.

Redox dysregulation in the pathophysiology of schizophrenia and bipolar disorder: insights from animal models.

Abstract Significance: Schizophrenia (SZ) and bipolar disorder (BD) are classified as two distinct diseases. However, accumulating evidence shows that both disorders share genetic, pathological, and epidemiological characteristics. Based on genetic and functional findings, redox dysregulation due to an imbalance between pro-oxidants and antioxidant defense mechanisms has been proposed as a risk factor contributing to their pathophysiology. Recent Advances: Altered antioxidant systems and signs of increased oxidative stress are observed in peripheral tissues and brains of SZ and BD patients, including abnormal prefrontal levels of glutathione (GSH), the major cellular redox regulator and antioxidant. Here we review experimental data from rodent models demonstrating that permanent as well as transient GSH deficit results in behavioral, morphological, electrophysiological, and neurochemical alterations analogous to pathologies observed in patients. Mice with GSH deficit display increased stress reactivity, altered social behavior, impaired prepulse inhibition, and exaggerated locomotor responses to psychostimulant injection. These behavioral changes are accompanied by N-methyl-D-aspartate receptor hypofunction, elevated glutamate levels, impairment of parvalbumin GABA interneurons, abnormal neuronal synchronization, altered dopamine neurotransmission, and deficient myelination. Critical Issues: Treatment with the GSH precursor and antioxidant N-acetylcysteine normalizes some of those deficits in mice, but also improves SZ and BD symptoms when given as adjunct to antipsychotic medication. Future Directions: These data demonstrate the usefulness of GSH-deficient rodent models to identify the mechanisms by which a redox imbalance could contribute to the development of SZ and BD pathophysiologies, and to develop novel therapeutic approaches based on antioxidant and redox regulator compounds. Antioxid. Redox Signal. 18, 1428-1443.

Stimulation of ENaC activity by rosiglitazone is PPARγ-dependent and correlates with SGK1 expression increase.

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists used to treat type 2 diabetes. TZD treatment induces side effects such as peripheral fluid retention, often leading to discontinuation of therapy. Previous studies have shown that PPARγ activation by TZD enhances the expression or function of the epithelial sodium channel (ENaC) through different mechanisms. However, the effect of TZDs on ENaC activity is not clearly understood. Here, we show that treating Xenopus laevis oocytes expressing ENaC and PPARγ with the TZD rosiglitazone (RGZ) produced a twofold increase of amiloride-sensitive sodium current (Iam), as measured by two-electrode voltage clamp. RGZ-induced ENaC activation was PPARγ-dependent since the PPARγ antagonist GW9662 blocked the activation. The RGZ-induced Iam increase was not mediated through direct serum- and glucocorticoid-regulated kinase (SGK1)-dependent phosphorylation of serine residue 594 on the human ENaC α-subunit but by the diminution of ENaC ubiquitination through the SGK1/Nedd4-2 pathway. In accordance, RGZ increased the activity of ENaC by enhancing its cell surface expression, most probably indirectly mediated through the increase of SGK1 expression.

Crosstalk between peroxisome proliferator-activated receptor delta and VEGF stimulates cancer progression.

Peroxisome proliferator-activated receptor (PPAR) delta is a member of the nuclear hormone receptor superfamily. PPARdelta may ameliorate metabolic diseases such as obesity and diabetes. However, PPARdelta's role in colorectal carcinogenesis remains controversial. Here, we present genetic and pharmacologic evidence demonstrating that deletion of PPARdelta decreases intestinal adenoma growth in Apc(Min/+) mice and inhibits tumor-promoting effects of a PPARdelta agonist GW501516. More importantly, we found that activation of PPARdelta up-regulated VEGF in colon carcinoma cells. VEGF directly promotes colon tumor epithelial cell survival through activation of PI3K-Akt signaling. These results not only highlight concerns about the use of PPARdelta agonists for treatment of metabolic disorders in patients who are at high risk for colorectal cancer, but also support the rationale for developing PPARdelta antagonists for prevention and/or treatment of cancer.

Pharmacological stimulation of edar signaling in the adult enhances sebaceous gland size and function.

Impaired ectodysplasin A (EDA) receptor (EDAR) signaling affects ectodermally derived structures including teeth, hair follicles, and cutaneous glands. The X-linked hypohidrotic ectodermal dysplasia (XLHED), resulting from EDA deficiency, can be rescued with lifelong benefits in animal models by stimulation of ectodermal appendage development with EDAR agonists. Treatments initiated later in the developmental period restore progressively fewer of the affected structures. It is unknown whether EDAR stimulation in adults with XLHED might have beneficial effects. In adult Eda mutant mice treated for several weeks with agonist anti-EDAR antibodies, we find that sebaceous gland size and function can be restored to wild-type levels. This effect is maintained upon chronic treatment but reverses slowly upon cessation of treatment. Sebaceous glands in all skin regions respond to treatment, although to varying degrees, and this is accompanied in both Eda mutant and wild-type mice by sebum secretion to levels higher than those observed in untreated controls. Edar is expressed at the periphery of the glands, suggesting a direct homeostatic effect of Edar stimulation on the sebaceous gland. Sebaceous gland size and sebum production may serve as biomarkers for EDAR stimulation, and EDAR agonists may improve skin dryness and eczema frequently observed in XLHED.

Fatty acid synthesis and PPARalpha hand in hand.

How can an ex-orphan be adopted? Is it possible to do so by attributing to it a key endogenous ligand that regulates its central functions? In the recent issue of Cell, Chakravarthy et al. attempted to answer this question by characterizing a new physiologically relevant ligand for the ex-orphan receptor peroxisome proliferator activated receptor alpha (PPARalpha).

Oxytocin selectively gates fear responses through distinct outputs from the central amygdala.

Central amygdala (CeA) projections to hypothalamic and brain stem nuclei regulate the behavioral and physiological expression of fear, but it is unknown whether these different aspects of the fear response can be separately regulated by the CeA. We combined fluorescent retrograde tracing of CeA projections to nuclei that modulate fear-related freezing or cardiovascular responses with in vitro electrophysiological recordings and with in vivo monitoring of related behavioral and physiological parameters. CeA projections emerged from separate neuronal populations with different electrophysiological characteristics and different response properties to oxytocin. In vivo, oxytocin decreased freezing responses in fear-conditioned rats without affecting the cardiovascular response. Thus, neuropeptidergic signaling can modulate the CeA outputs through separate neuronal circuits and thereby individually steer the various aspects of the fear response.

Diagnostic performance of urinary metanephrines for the postmortem diagnosis of hypothermia.

The purpose of this study was to assess the diagnostic potential of urinary metanephrines and 3-methoxytyramine compared to urinary catecholamine determination in diagnosing antemortem cold exposure and fatal hypothermia. 83 cases of fatal hypothermia and 144 control cases were included in this study. Catecholamines (adrenaline, noradrenaline and dopamine), metanephrines (metanephrine, normetanephrine) and 3-methoxytyramine were measured in urine collected during autopsy. All tested analytes were significantly higher in hypothermia cases compared to control subjects and displayed a generally satisfying discriminative value, thus indicating urinary catecholamines and their metabolites as reliable markers of cold-related stress and hypothermia related-deaths. Metanephrine and adrenaline had the best discriminative value between hypothermia and control cases compared to other tested analytes, though with different sensitivity and specificity. These can therefore be considered the most suitable markers of cold-related stress.

Beta(1)-selective agonist (-)-1-(3,4-dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [(-)-RO363] differentially interacts with key amino acids responsible for beta(1)-selective binding in resting and active states.

(-)-1-(3,4-Dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [(-)-RO363] is a highly selective beta(1)-adrenergic receptor (beta(1)AR) agonist. To study the binding site of beta(1)-selective agonist, chimeric beta(1)/beta(2)ARs and Ala-substituted beta(1)ARs were constructed. Several key residues of beta(1)AR [Leu(110) and Thr(117) in transmembrane domain (TMD) 2], and Phe(359) in TMD 7] were found to be responsible for beta(1)-selective binding of (-)-RO363, as determined by competitive binding. Based on these results, we built a three-dimensional model of the binding domain for (-)-RO363. The model indicated that TMD 2 and TMD 7 of beta(1)AR form a binding pocket; the methoxyphenyl group of N-substituent of (-)-RO363 seems to locate within the cavity surrounded by Leu(110), Thr(117), and Phe(359). The amino acids Leu(110) and Phe(359) interact with the phenyl ring of (-)-RO363, whereas Thr(117) forms hydrogen bond with the methoxy group of (-)-RO363. To examine the interaction of these residues with beta(1)AR in an active state, each of the amino acids was changed to Ala in a constitutively active (CA)-beta(1)AR mutant. The degree of decrease in the affinity of CA-beta(1)AR for (-)-RO363 was essentially the same as that of wild-type beta(1)AR when mutated at Leu(110) and Thr(117). However, the affinity was decreased in Ala-substituted mutant of Phe(359) compared with that of wild-type beta(1)AR. These results indicated that Leu(110) and Thr(117) are necessary for the initial binding of (-)-RO363 with beta(1)-selectivity, and interaction of Phe(359) with the N-substituent of (-)-RO363 in an active state is stronger than in the resting state.

Thiazolidinedione-induced fluid retention is independent of collecting duct alphaENaC activity.

Thiazolidinediones are agonists of peroxisome proliferator-activated receptor gamma (PPARgamma) that can induce fluid retention and weight gain through unclear mechanisms. To test a proposed role for the epithelial sodium channel ENaC in thiazolidinedione-induced fluid retention, we used mice with conditionally inactivated alphaENaC in the collecting duct (Scnn1a(loxloxCre) mice). In control mice, rosiglitazone did not alter plasma aldosterone levels or protein expression of ENaC subunits in the kidney, but did increase body weight, plasma volume, and the fluid content of abdominal fat pads, and decreased hematocrit. Scnn1a(loxloxCre) mice provided functional evidence for blunted Na+ uptake in the collecting duct, but still exhibited rosiglitazone-induced fluid retention. Moreover, treatment with rosiglitazone or pioglitazone did not significantly alter the open probability or number of ENaC channels per patch in isolated, split-open cortical collecting ducts of wild-type mice. Finally, patch-clamp studies in primary mouse inner medullary collecting duct cells did not detect ENaC activity but did detect a nonselective cation channel upregulated by pioglitazone. These data argue against a primary and critical role of ENaC in thiazolidinedione-induced fluid retention.