The ß3-AR agonist BRL37344 ameliorates the main symptoms of X-linked nephrogenic diabetes insipidus in the mouse model of the disease.

X-linked nephrogenic diabetes insipidus (X-NDI) is a rare congenital disease caused by inactivating mutations of the vasopressin type-2 receptor (AVPR2), characterized by impaired renal concentrating ability, dramatic polyuria, polydipsia and risk of dehydration. The disease, which still lacks a cure, could benefit from the pharmacologic stimulation of other GPCRs, activating the cAMP-intracellular pathway in the kidney cells expressing the AVPR2. On the basis of our previous studies, we here hypothesized that the ß3-adrenergic receptor could be such an ideal candidate. We evaluated the effect of continuous 24 h stimulation of the ß3-AR with the agonist BRL37344 and assessed the effects on urine output, urine osmolarity, water intake and the abundance and activation of the key renal water and electrolyte transporters, in the mouse model of X-NDI. Here we demonstrate that the ß3-AR agonism exhibits a potent antidiuretic effect. The strong improvement in symptoms of X-NDI produced by a single i.p. injection of BRL37344 (1 mg/kg) was limited to 3 h but repeated administrations in the 24 h, mimicking the effect of a slow-release preparation, promoted a sustained antidiuretic effect, reducing the 24 h urine output by 27%, increasing urine osmolarity by 25% and reducing the water intake by 20%. At the molecular level, we show that BRL37344 acted by increasing the phosphorylation of NKCC2, NCC and AQP2 in the renal cell membrane, thereby increasing electrolytes and water reabsorption in the kidney tubule of X-NDI mice. Taken together, these data suggest that human ß3-AR agonists might represent an effective possible treatment strategy for X-NDI.

Targeting unfolded protein response reverts ER stress and ER Ca2+ homeostasis in cardiomyocytes expressing the pathogenic variant of Lamin A/C R321X.

BACKGROUND: We previously demonstrated that an Italian family affected by a severe dilated cardiomyopathy (DCM) with history of sudden deaths at young age, carried a mutation in the Lmna gene encoding for a truncated variant of the Lamin A/C protein (LMNA), R321X. When expressed in heterologous systems, such variant accumulates into the endoplasmic reticulum (ER), inducing the activation of the PERK-CHOP pathway of the unfolded protein response (UPR), ER dysfunction and increased rate of apoptosis. The aim of this work was to analyze whether targeting the UPR can be used to revert the ER dysfunction associated with LMNA R321X expression in HL-1 cardiac cells. METHODS: HL-1 cardiomyocytes stably expressing LMNA R321X were used to assess the ability of 3 different drugs targeting the UPR, salubrinal, guanabenz and empagliflozin to rescue ER stress and dysfunction. In these cells, the state of activation of both the UPR and the pro-apoptotic pathway were analyzed monitoring the expression levels of phospho-PERK, phospho-eIF2α, ATF4, CHOP and PARP-CL. In addition, we measured ER-dependent intracellular Ca2+ dynamics as indicator of proper ER functionality. RESULTS: We found that salubrinal and guanabenz increased the expression levels of phospho-eIF2α and downregulated the apoptosis markers CHOP and PARP-CL in LMNA R321X-cardiomyocytes, maintaining the so-called adaptive UPR. These drugs also restored ER ability to handle Ca2+ in these cardiomyocytes. Interestingly, we found that empagliflozin downregulated the apoptosis markers CHOP and PARP-CL shutting down the UPR itself through the inhibition of PERK phosphorylation in LMNA R321X-cardiomyocytes. Furthermore, upon empagliflozin treatment, ER homeostasis, in terms of ER ability to store and release intracellular Ca2+ was also restored in these cardiomyocytes. CONCLUSIONS: We provided evidence that the different drugs, although interfering with different steps of the UPR, were able to counteract pro-apoptotic processes and to preserve the ER homeostasis in R321X LMNA-cardiomyocytes. Of note, two of the tested drugs, guanabenz and empagliflozin, are already used in the clinical practice, thus providing preclinical evidence for ready-to-use therapies in patients affected by the LMNA R321X associated cardiomyocytes.

ß3 Adrenergic Receptor Agonist Mirabegron Increases AQP2 and NKCC2 Urinary Excretion in OAB Patients: A Pleiotropic Effect of Interest for Patients with X-Linked Nephrogenic Diabetes Insipidus.

We previously reported the novel finding that ß3-AR is functionally expressed in the renal tubule and shares its cellular localization with the vasopressin receptor AVPR2, whose physiological stimulation triggers antidiuresis by increasing the plasma membrane expression of the water channel AQP2 and the NKCC2 symporter in renal cells. We also showed that pharmacologic stimulation of ß3-AR is capable of triggering antidiuresis and correcting polyuria, in the knockout mice for the AVPR2 receptor, the animal model of human X-linked nephrogenic diabetes insipidus (XNDI), a rare genetic disease still missing a cure. Here, to demonstrate that the same response can be evoked in humans, we evaluated the effect of treatment with the ß3-AR agonist mirabegron on AQP2 and NKCC2 trafficking, by evaluating their urinary excretion in a cohort of patients with overactive bladder syndrome, for the treatment of which the drug is already approved. Compared to baseline, treatment with mirabegron significantly increased AQP2 and NKCC2 excretion for the 12 weeks of treatment. This data is a step forward in corroborating the hypothesis that in patients with XNDI, treatment with mirabegron could bypass the inactivation of AVPR2, trigger antidiuresis and correct the dramatic polyuria which is the main hallmark of this disease.

Neuroprotective Effect of Antiapoptotic URG7 Protein on Human Neuroblastoma Cell Line SH-SY5Y.

Up-regulated Gene clone 7 (URG7) is a protein localized in the endoplasmic reticulum (ER) and overexpressed in liver cells upon hepatitis B virus (HBV) infection. Its activity has been related to the attenuation of ER stress resulting from HBV infection, promoting protein folding and ubiquitination and reducing cell apoptosis overall. While the antiapoptotic activity of URG7 in HBV-infected cells may have negative implications, this effect could be exploited positively in the field of proteinopathies, such as neurodegenerative diseases. In this work, we aimed to verify the possible contribution of URG7 as a reliever of cellular proteostasis alterations in a neuronal in vitro system. Following tunicamycin-induced ER stress, URG7 was shown to modulate different markers of the unfolded protein response (UPR) in favor of cell survival, mitigating ER stress and activating autophagy. Furthermore, URG7 promoted ubiquitination, and determined a reduction in protein aggregation, calcium release from the ER and intracellular ROS content, confirming its pro-survival activity. Therefore, in light of the results reported in this work, we hypothesize that URG7 offers activity as an ER stress reliever in a neuronal in vitro model, and we paved the way for a new approach in the treatment of neurodegenerative diseases.

TRPML1-Induced Lysosomal Ca2+ Signals Activate AQP2 Translocation and Water Flux in Renal Collecting Duct Cells.

Lysosomes are acidic Ca2+ storage organelles that actively generate local Ca2+ signaling events to regulate a plethora of cell functions. Here, we characterized lysosomal Ca2+ signals in mouse renal collecting duct (CD) cells and we assessed their putative role in aquaporin 2 (AQP2)-dependent water reabsorption. Bafilomycin A1 and ML-SA1 triggered similar Ca2+ oscillations, in the absence of extracellular Ca2+, by alkalizing the acidic lysosomal pH or activating the lysosomal cation channel mucolipin 1 (TRPML1), respectively. TRPML1-dependent Ca2+ signals were blocked either pharmacologically or by lysosomes' osmotic permeabilization, thus indicating these organelles as primary sources of Ca2+ release. Lysosome-induced Ca2+ oscillations were sustained by endoplasmic reticulum (ER) Ca2+ content, while bafilomycin A1 and ML-SA1 did not directly interfere with ER Ca2+ homeostasis per se. TRPML1 activation strongly increased AQP2 apical expression and depolymerized the actin cytoskeleton, thereby boosting water flux in response to an hypoosmotic stimulus. These effects were strictly dependent on the activation of the Ca2+/calcineurin pathway. Conversely, bafilomycin A1 led to perinuclear accumulation of AQP2 vesicles without affecting water permeability. Overall, lysosomal Ca2+ signaling events can be differently decoded to modulate Ca2+-dependent cellular functions related to the dock/fusion of AQP2-transporting vesicles in principal cells of the CD.

Pro-inflammatory cytokines as emerging molecular determinants in cardiolaminopathies.

Mutations in Lamin A/C gene (lmna) cause a wide spectrum of cardiolaminopathies strictly associated with significant deterioration of the electrical and contractile function of the heart. Despite the continuous flow of biomedical evidence, linking cardiac inflammation to heart remodelling in patients harbouring lmna mutations is puzzling. Therefore, we profiled 30 serum cytokines/chemokines in patients belonging to four different families carrying pathogenic lmna mutations segregating with cardiac phenotypes at different stages of severity (n = 19) and in healthy subjects (n = 11). Regardless lmna mutation subtype, high levels of circulating granulocyte colony-stimulating factor (G-CSF) and interleukin 6 (IL-6) were found in all affected patients' sera. In addition, elevated levels of Interleukins (IL) IL-1Ra, IL-1ß IL-4, IL-5 and IL-8 and the granulocyte-macrophage colony-stimulating factor (GM-CSF) were measured in a large subset of patients associated with more aggressive clinical manifestations. Finally, the expression of the pro-inflammatory 70 kDa heat shock protein (Hsp70) was significantly increased in serum exosomes of patients harbouring the lmna mutation associated with the more severe phenotype. Overall, the identification of patient subsets with overactive or dysregulated myocardial inflammatory responses could represent an innovative diagnostic, prognostic and therapeutic tool against Lamin A/C cardiomyopathies.

Effect of Quercetin on ABCC6 Transporter: Implication in HepG2 Migration.

Quercetin is a member of the flavonoid group of compounds, which is abundantly present in various dietary sources. It has excellent antioxidant properties and anti-inflammatory activity and is very effective as an anti-cancer agent against various types of tumors, both in vivo and in vitro. Quercetin has been also reported to modulate the activity of some members of the multidrug-resistance transporters family, such as P-gp, ABCC1, ABCC2, and ABCG2, and the activity of ecto-5'-nucleotidase (NT5E/CD73), a key regulator in some tumor processes such as invasion, migration, and metastasis. In this study, we investigated the effect of Quercetin on ABCC6 expression in HepG2 cells. ABCC6 is a member of the superfamily of ATP-binding cassette (ABC) transporters, poorly involved in drug resistance, whose mutations cause pseudoxanthoma elasticum, an inherited disease characterized by ectopic calcification of soft connective tissues. Recently, it has been reported that ABCC6 contributes to cytoskeleton rearrangements and HepG2 cell motility through purinergic signaling. Gene and protein expression were evaluated by quantitative Reverse-Transcription PCR (RT-qPCR) and western blot, respectively. Actin cytoskeleton dynamics was evaluated by laser confocal microscopy using fluorophore-conjugated phalloidin. Cell motility was analyzed by an in vitro wound-healing migration assay. We propose that ABCC6 expression may be controlled by the AKT pathway as part of an adaptative response to oxidative stress, which can be mitigated by the use of Quercetin-like flavonoids.

Aquaporin-1 Facilitates Transmesothelial Water Permeability: In Vitro and Ex Vivo Evidence and Possible Implications in Peritoneal Dialysis.

We previously showed that mesothelial cells in human peritoneum express the water channel aquaporin 1 (AQP1) at the plasma membrane, suggesting that, although in a non-physiological context, it may facilitate osmotic water exchange during peritoneal dialysis (PD). According to the three-pore model that predicts the transport of water during PD, the endothelium of peritoneal capillaries is the major limiting barrier to water transport across peritoneum, assuming the functional role of the mesothelium, as a semipermeable barrier, to be negligible. We hypothesized that an intact mesothelial layer is poorly permeable to water unless AQP1 is expressed at the plasma membrane. To demonstrate that, we characterized an immortalized cell line of human mesothelium (HMC) and measured the osmotically-driven transmesothelial water flux in the absence or in the presence of AQP1. The presence of tight junctions between HMC was investigated by immunofluorescence. Bioelectrical parameters of HMC monolayers were studied by Ussing Chambers and transepithelial water transport was investigated by an electrophysiological approach based on measurements of TEA+ dilution in the apical bathing solution, through TEA+-sensitive microelectrodes. HMCs express Zo-1 and occludin at the tight junctions and a transepithelial vectorial Na+ transport. Real-time transmesothelial water flux, in response to an increase of osmolarity in the apical solution, indicated that, in the presence of AQP1, the rate of TEA+ dilution was up to four-fold higher than in its absence. Of note, we confirmed our data in isolated mouse mesentery patches, where we measured an AQP1-dependent transmesothelial osmotic water transport. These results suggest that the mesothelium may represent an additional selective barrier regulating water transport in PD through functional expression of the water channel AQP1.

Astaxanthin anticancer effects are mediated through multiple molecular mechanisms: A systematic review.

During the latest decades, the interest on the effectiveness of natural compounds and their impact on human health constantly increased, especially on those demonstrating to be effective on cancer. Molecules coming from nature are currently used in chemotherapy like Taxol, Vincristine or Vinblastine, and several other natural substances have been showed to be active in reducing cancer cell progression and migration. Among them, astaxanthin, a xanthophyll red colored carotenoid, displayed different biological activities including, antinflammatory, antioxidant, proapoptotic, and anticancer effects. It can induce apoptosis through downregulation of antiapoptotic protein (Bcl-2, p-Bad, and survivin) expression and upregulation of proapoptotic ones (Bax/Bad and PARP). Thanks to these mechanisms, it can exert anticancer effects towards colorectal cancer, melanoma, or gastric carcinoma cell lines. Moreover, it possesses antiproliferative activity in many experimental models and enhances the effectiveness of conventional chemotherapic drugs on tumor cells underling its potential future use. This review provides an overview of the current knowledge on the anticancer potential of astaxanthin by modulating several molecular targets. While it has been clearly demonstrated its multitarget activity in the prevention and regression of malignant cells in in vitro or in preclinical investigations, further clinical studies are needed to assess its real potential as anticancer in humans.

A Novel Formulation of Glucose-Sparing Peritoneal Dialysis Solutions with l-Carnitine Improves Biocompatibility on Human Mesothelial Cells.

The main reason why peritoneal dialysis (PD) still has limited use in the management of patients with end-stage renal disease (ESRD) lies in the fact that the currently used glucose-based PD solutions are not completely biocompatible and determine, over time, the degeneration of the peritoneal membrane (PM) and consequent loss of ultrafiltration (UF). Here we evaluated the biocompatibility of a novel formulation of dialytic solutions, in which a substantial amount of glucose is replaced by two osmometabolic agents, xylitol and l-carnitine. The effect of this novel formulation on cell viability, the integrity of the mesothelial barrier and secretion of pro-inflammatory cytokines was evaluated on human mesothelial cells grown on cell culture inserts and exposed to the PD solution only at the apical side, mimicking the condition of a PD dwell. The results were compared to those obtained after exposure to a panel of dialytic solutions commonly used in clinical practice. We report here compelling evidence that this novel formulation shows better performance in terms of higher cell viability, better preservation of the integrity of the mesothelial layer and reduced release of pro-inflammatory cytokines. This new formulation could represent a step forward towards obtaining PD solutions with high biocompatibility.

Functional study of a KCNH2 mutant: Novel insights on the pathogenesis of the LQT2 syndrome.

The K+ voltage-gated channel subfamily H member 2 (KCNH2) transports the rapid component of the cardiac delayed rectifying K+ current. The aim of this study was to characterize the biophysical properties of a C-terminus-truncated KCNH2 channel, G1006fs/49 causing long QT syndrome type II in heterozygous members of an Italian family. Mutant carriers underwent clinical workup, including 12-lead electrocardiogram, transthoracic echocardiography and 24-hour ECG recording. Electrophysiological experiments compared the biophysical properties of G1006fs/49 with those of KCNH2 both expressed either as homotetramers or as heterotetramers in HEK293 cells. Major findings of this work are as follows: (a) G1006fs/49 is functional at the plasma membrane even when co-expressed with KCNH2, (b) G1006fs/49 exerts a dominant-negative effect on KCNH2 conferring specific biophysical properties to the heterotetrameric channel such as a significant delay in the voltage-sensitive transition to the open state, faster kinetics of both inactivation and recovery from the inactivation and (c) the activation kinetics of the G1006fs/49 heterotetrameric channels is partially restored by a specific KCNH2 activator. The functional characterization of G1006fs/49 homo/heterotetramers provided crucial findings about the pathogenesis of LQTS type II in the mutant carriers, thus providing a new and potential pharmacological strategy.

Inhibiting the urokinase-type plasminogen activator receptor system recovers STZ-induced diabetic nephropathy.

The urokinase-type plasminogen activator (uPA) receptor (uPAR) participates to the mechanisms causing renal damage in response to hyperglycaemia. The main function of uPAR in podocytes (as well as soluble uPAR -(s)uPAR- from circulation) is to regulate podocyte function through αvß3 integrin/Rac-1. We addressed the question of whether blocking the uPAR pathway with the small peptide UPARANT, which inhibits uPAR binding to the formyl peptide receptors (FPRs) can improve kidney lesions in a rat model of streptozotocin (STZ)-induced diabetes. The concentration of systemically administered UPARANT was measured in the plasma, in kidney and liver extracts and UPARANT effects on dysregulated uPAR pathway, αvß3 integrin/Rac-1 activity, renal fibrosis and kidney morphology were determined. UPARANT was found to revert STZ-induced up-regulation of uPA levels and activity, while uPAR on podocytes and (s)uPAR were unaffected. In glomeruli, UPARANT inhibited FPR2 expression suggesting that the drug may act downstream uPAR, and recovered the increased activity of the αvß3 integrin/Rac-1 pathway indicating a major role of uPAR in regulating podocyte function. At the functional level, UPARANT was shown to ameliorate: (a) the standard renal parameters, (b) the vascular permeability, (c) the renal inflammation, (d) the renal fibrosis including dysregulated plasminogen-plasmin system, extracellular matrix accumulation and glomerular fibrotic areas and (e) morphological alterations of the glomerulus including diseased filtration barrier. These results provide the first demonstration that blocking the uPAR pathway can improve diabetic kidney lesion in the STZ model, thus suggesting the uPA/uPAR system as a promising target for the development of novel uPAR-targeting approaches.

New insights on the functional role of URG7 in the cellular response to ER stress.

BACKGROUND INFORMATION: Up-regulated Gene clone 7 (URG7) is an ER resident protein, whose expression is up-regulated in the presence of hepatitis B virus X antigen (HBxAg) during HBV infection. In virus-infected hepatocytes, URG7 shows an anti-apoptotic activity due to the PI3K/AKT signalling activation, does not seem to have tumorigenic properties, but it appears to promote the development and progression of fibrosis. However, the molecular mechanisms underlying URG7 activity remain largely unknown. RESULTS: To shed light on URG7 activity, we first analysed its interactome in HepG2 transfected cells: this analysis suggests that URG7 could have a role in affecting protein synthesis, folding and promoting proteins degradation. Moreover, keeping into account its subcellular localisation in the ER and that several viral infections give rise to ER stress, a panel of experiments was performed to evaluate a putative role of URG7 in ER stress. Our main results demonstrate that in ER-stressed cells URG7 is able to modulate the expression of Unfolded Protein Response (UPR) markers towards survival outcomes, up-regulating GRP78 protein and down-regulating the pro-apoptotic protein CHOP. Furthermore, URG7 reduces the ER stress by decreasing the amount of unfolded proteins, by increasing both the total protein ubiquitination and the AKT activation and reducing Caspase 3 activation. CONCLUSIONS: All together these data suggest that URG7 plays a pivotal role as a reliever of ER stress-induced apoptosis. SIGNIFICANCE: This is the first characterisation of URG7 activity under ER stress conditions. The results presented here will help to hypothesise new strategies to counteract the antiapoptotic activity of URG7 in the context of the viral infection.

Human ß3-Adrenoreceptor is Resistant to Agonist-Induced Desensitization in Renal Epithelial Cells.

BACKGROUND/AIMS: We recently showed that the ß3-adrenoreceptor (ß3AR) is expressed in mouse kidney collecting ducts (CD) cells along with the type-2 vasopressin receptor (AVPR2). Interestingly, a single injection of a ß3AR selective agonist promotes a potent antidiuretic effect in mice. Before considering the feasibility of chronic ß3AR agonism to induce antidiuresis in vivo, we aimed to evaluate in vitro the signaling and desensitization profiles of human ß3AR. METHODS: Human ß3AR desensitization was compared with that of human AVPR2 in cultured renal cells. Video imaging and FRET experiments were performed to dissect ß3AR signaling under acute and chronic stimulation. Plasma membrane localization of ß3AR, AVPR2 and AQP2 after agonist stimulation was studied by confocal microscopy. Receptors degradation was evaluated by Western blotting. RESULTS: In renal cells acute stimulation with the selective ß3AR agonist mirabegron, induced a dose-dependent increase in cAMP. Interestingly, chronic exposure to mirabegron promoted a significant increase of intracellular cAMP up to 12 hours. In addition, a slow and slight agonist-induced internalization and a delayed downregulation of ß3AR was observed under chronic stimulation. Furthermore, chronic exposure to mirabegron promoted apical expression of AQP2 also up to 12 hours. Conversely, long-term stimulation of AVPR2 with dDAVP showed short-lasting receptor signaling, rapid internalization and downregulation and apical AQP2 expression for no longer than 3 h. CONCLUSIONS: Overall, we conclude that ß3AR is less prone than AVPR2 to agonist-induced desensitization in renal collecting duct epithelial cells, showing sustained cAMP production, preserved membrane localization and delayed degradation after 12 hours agonist exposure. These results may be important for the potential use of chronic pharmacological stimulation of ß3AR to promote antidiuresis overcoming in vivo renal concentrating defects caused by inactivating mutations of the AVPR2.

Dandelion Root Extract Induces Intracellular Ca2+ Increases in HEK293 Cells.

Dandelion (Taraxacum officinale Weber ex F.H.Wigg.) has been used for centuries as an ethnomedical remedy. Nonetheless, the extensive use of different kinds of dandelion extracts and preparations is based on empirical findings. Some of the tissue-specific effects reported for diverse dandelion extracts may result from their action on intracellular signaling cascades. Therefore, the aim of this study was to evaluate the effects of an ethanolic dandelion root extract (DRE) on Ca2+ signaling in human embryonic kidney (HEK) 293 cells. The cytotoxicity of increasing doses of crude DRE was determined by the Calcein viability assay. Fura-2 and the fluorescence resonance energy transfer (FRET)-based probe ERD1 were used to measure cytoplasmic and intraluminal endoplasmic reticulum (ER) Ca2+ levels, respectively. Furthermore, a green fluorescent protein (GFP)-based probe was used to monitor phospholipase C (PLC) activation (pleckstrin homology [PH]-PLCδ-GFP). DRE (10-400 µg/mL) exposure, in the presence of external Ca2+, dose-dependently increased intracellular Ca2+ levels. The DRE-induced Ca2+ increase was significantly reduced in the absence of extracellular Ca2+. In addition, DRE caused a significant Ca2+ release from the ER of intact cells and a concomitant translocation of PH-PLCδ-GFP. In conclusion, DRE directly activates both the release of Ca2+ from internal stores and a significant Ca2+ influx at the plasma membrane. The resulting high Ca2+ levels within the cell seem to directly stimulate PLC activity.

A Comparative Study on Phytochemical Profiles and Biological Activities of Sclerocarya birrea (A.Rich.) Hochst Leaf and Bark Extracts.

Sclerocarya birrea (A.Rich.) Hochst (Anacardiaceae) is a savannah tree that has long been used in sub-Saharan Africa as a medicinal remedy for numerous ailments. The purpose of this study was to increase the scientific knowledge about this plant by evaluating the total content of polyphenols, flavonoids, and tannins in the methanol extracts of the leaves and bark (MLE and MBE, respectively), as well as the in vitro antioxidant activity and biological activities of these extracts. Reported results show that MLE is rich in flavonoids (132.7 ± 10.4 mg of quercetin equivalents/g), whereas MBE has the highest content of tannins (949.5 ± 29.7 mg of tannic acid equivalents/g). The antioxidant activity was measured using four different in vitro tests: ß-carotene bleaching (BCB), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), O2-•, and nitric oxide (NO•) assays. In all cases, MBE was the most active compared to MLE and the standards used (Trolox and ascorbic acid). Furthermore, MBE and MLE were tested to evaluate their activity in HepG2 and fibroblast cell lines. A higher cytotoxic activity of MBE was evidenced and confirmed by more pronounced alterations in cell morphology. MBE induced cell death, triggering the intrinsic apoptotic pathway by reactive oxygen species (ROS) generation, which led to a loss of mitochondrial membrane potential with subsequent cytochrome c release from the mitochondria into the cytosol. Moreover, MBE showed lower cytotoxicity in normal human dermal fibroblasts, suggesting its potential as a selective anticancer agent.

Functional Characterization of a Novel Truncating Mutation in Lamin A/C Gene in a Family with a Severe Cardiomyopathy with Conduction Defects.

BACKGROUND/AIMS: Truncating LMNA gene mutations occur in many inherited cardiomyopathy cases, but the molecular mechanisms involved in the disease they cause have not yet been systematically investigated. Here, we studied a novel frameshift LMNA variant (p.D243Gfs*4) identified in three members of an Italian family co-segregating with a severe form of cardiomyopathy with conduction defects. METHODS: HEK293 cells and HL-1 cardiomyocytes were transiently transfected with either Lamin A or D243Gfs*4 tagged with GFP (or mCherry). D243Gfs*4 expression, cellular localization and its effects on diverse cellular mechanisms were evaluated with western blotting, laser-scanning confocal microscopy and video-imaging analysis in single cells. RESULTS: When expressed in HEK293 cells, GFP- (or mCherry)-tagged LMNA D243Gfs*4 colocalized with calnexin within the ER. ER mislocalization of LMNA D243Gfs*4 did not significantly induce ER stress response, abnormal Ca2+ handling and apoptosis when compared with HEK293 cells expressing another truncated mutant of LMNA (R321X) which similarly accumulates within the ER. Of note, HEK293-LMNA D243Gfs*4 cells showed a significant reduction of connexin 43 (CX43) expression level, which was completely rescued by activation of the WNT/ß-catenin signaling pathway. When expressed in HL-1 cardiomyocytes, D243Gfs*4 significantly impaired the spontaneous Ca2+ oscillations recorded in these cells as result of propagation of the depolarizing waves through the gap junctions between non-transfected cells surrounding a cell harboring the mutation. Furthermore, mCh-D243Gfs*4 HL-1 cardiomyocytes showed reduced CX43-dependent Lucifer Yellow (LY) loading and propagation. Of note, activation of ß-catenin rescued both LY loading and LMNA D243Gfs*4 -HL-1 cells spontaneous activity propagation. CONCLUSION: Overall, the present results clearly indicate the involvement of the aberrant CX43 expression/activity as a pathogenic mechanism for the conduction defects associated to this LMNA truncating alteration.

ABCC6 knockdown in HepG2 cells induces a senescent-like cell phenotype.

BACKGROUND: Pseudoxanthoma elasticum (PXE) is characterized by progressive ectopic mineralization of elastic fibers in dermal, ocular and vascular tissues. No effective treatment exists. It is caused by inactivating mutations in the gene encoding for the ATP-binding cassette, sub-family C member 6 transporter (ABCC6), which is mainly expressed in the liver. The ABCC6 substrate (s) and the PXE pathomechanism remain unknown. Recent studies have shown that overexpression of ABCC6 in HEK293 cells results in efflux of ATP, which is rapidly converted into nucleoside monophosphates and pyrophosphate (PPi). Since the latter inhibits mineralization, it was proposed that the absence of circulating PPi in PXE patients results in the characteristic ectopic mineralization. These studies also demonstrated that the presence of ABCC6 modifies cell secretory activity and suggested that ABCC6 can change the cell phenotype. METHODS: Stable ABCC6 knockdown HepG2 clones were generated using small hairpin RNA (shRNA) technology. The intracellular glutathione and ROS levels were determined. Experiments using cell cycle analysis, real-time PCR and western blot were performed on genes involved in the senescence phenotype. RESULTS: To shed light on the physiological role of ABCC6, we focused on the phenotype of HepG2 cells that lack ABCC6 activity. Interestingly, we found that ABCC6 knockdown HepG2 cells show: 1) intracellular reductive stress; 2) cell cycle arrest in G1 phase; 3) upregulation of p21Cip p53 independent; and 4) downregulation of lamin A/C. CONCLUSIONS: These findings show that the absence of ABCC6 profoundly changes the HepG2 phenotype, suggesting that the PXE syndrome is a complex metabolic disease that is not exclusively related to the absence of pyrophosphate in the bloodstream.

Hereditary Nephrogenic Diabetes Insipidus: Pathophysiology and Possible Treatment. An Update.

Under physiological conditions, excessive loss of water through the urine is prevented by the release of the antidiuretic hormone arginine-vasopressin (AVP) from the posterior pituitary. In the kidney, AVP elicits a number of cellular responses, which converge on increasing the osmotic reabsorption of water in the collecting duct. One of the key events triggered by the binding of AVP to its type-2 receptor (AVPR2) is the exocytosis of the water channel aquaporin 2 (AQP2) at the apical membrane the principal cells of the collecting duct. Mutations of either AVPR2 or AQP2 result in a genetic disease known as nephrogenic diabetes insipidus, which is characterized by the lack of responsiveness of the collecting duct to the antidiuretic action of AVP. The affected subject, being incapable of concentrating the urine, presents marked polyuria and compensatory polydipsia and is constantly at risk of severe dehydration. The molecular bases of the disease are fully uncovered, as well as the genetic or clinical tests for a prompt diagnosis of the disease in newborns. A real cure for nephrogenic diabetes insipidus (NDI) is still missing, and the main symptoms of the disease are handled with s continuous supply of water, a restrictive diet, and nonspecific drugs. Unfortunately, the current therapeutic options are limited and only partially beneficial. Further investigation in vitro or using the available animal models of the disease, combined with clinical trials, will eventually lead to the identification of one or more targeted strategies that will improve or replace the current conventional therapy and grant NDI patients a better quality of life. Here we provide an updated overview of the genetic defects causing NDI, the most recent strategies under investigation for rescuing the activity of mutated AVPR2 or AQP2, or for bypassing defective AVPR2 signaling and restoring AQP2 plasma membrane expression.

Cytotoxic Activity of Origanum Vulgare L. on Hepatocellular Carcinoma cell Line HepG2 and Evaluation of its Biological Activity.

The potential of plant essential oils (EOs) in anticancer treatment has recently received many research efforts to overcome the development of multidrug resistance and their negative side effects. The aims of the current research are to study (i) the cytotoxic effect of the crude EO extracted from Origanum vulgare subsp hirtum and its main constituents (carvacrol, thymol, citral and limonene) on hepatocarcinoma HepG2 and healthy human renal cells HEK293; (ii) the antibacterial and phytotoxic activities of the above EO and its main constituents. Results showed that cell viability percentage of treated HepG2 by EO and its main constituents was significantly decreased when compared to untreated cells. The calculated inhibition concentration (IC50) values for HepG2 were lower than healthy renal cells, indicating the sort of selectivity of the studied substances. Citral is not potentially recommended as an anticancer therapeutic agent, since there are no significant differences between IC50 values against both tested cell lines. Results showed also that oregano EO and its main constituents have a significant antibacterial activity and a moderate phytotoxic effect. The current research verified that oregano EO and its main constituents could be potentially utilized as anticancer therapeutic agents.