Olive Leaf Extract (OLE) as a Novel Antioxidant That Ameliorates the Inflammatory Response in Cystic Fibrosis.

The deletion of phenylalanine at position 508 (F508del) produces a misfolded CFTR protein that is retained in the ER and degraded. The lack of normal CFTR channel activity is associated with chronic infection and inflammation which are the primary causes of declining lung function in Cystic Fibrosis (CF) patients. Moreover, LPS-dependent oxidative stress downregulates CFTR function in airway epithelial cells. Olive leaf extract (OLE) is used in traditional medicine for its effects, including anti-oxidant and anti-inflammatory ones. We found that OLE decreased the intracellular ROS levels in a dose-response manner in CFBE cells. Moreover, OLE attenuates the inflammatory response to LPS or IL-1ß/TNFα stimulation, mimicking the infection and inflammatory status of CF patients, in CFBE and primary nasal epithelial (HNE) cells. Furthermore, we demonstrated that OLE restored the LPS-mediated decrease of TrikfaftaTM-dependent F508del-CFTR function in CFBE and HNE cultures. These findings provide strong evidence of OLE to prevent redox imbalance and inflammation that can cause chronic lung damage by enhancing the antioxidant activity and attenuating inflammation in CF airway epithelial cells. Additionally, OLE might be used in combination with CFTR modulators therapy to improve their efficacy in CF patients.

Uptake-Dependent and -Independent Effects of Fibroblasts-Derived Extracellular Vesicles on Bone Marrow Endothelial Cells from Patients with Multiple Myeloma: Therapeutic and Clinical Implications.

Extracellular vesicles (EVs) have emerged as important players in cell-to-cell communication within the bone marrow (BM) of multiple myeloma (MM) patients, where they mediate several tumor-associated processes. Here, we investigate the contribution of fibroblasts-derived EVs (FBEVs) in supporting BM angiogenesis. We demonstrate that FBEVs' cargo contains several angiogenic cytokines (i.e., VEGF, HGF, and ANG-1) that promote an early over-angiogenic effect independent from EVs uptake. Interestingly, co-culture of endothelial cells from MM patients (MMECs) with FBEVs for 1 or 6 h activates the VEGF/VEGFR2, HGF/HGFR, and ANG-1/Tie2 axis, as well as the mTORC2 and Wnt/ß-catenin pathways, suggesting that the early over-angiogenic effect is a cytokine-mediated process. FBEVs internalization occurs after longer exposure of MMECs to FBEVs (24 h) and induces a late over-angiogenic effect by increasing MMECs migration, chemotaxis, metalloproteases release, and capillarogenesis. FBEVs uptake activates mTORC1, MAPK, SRC, and STAT pathways that promote the release of pro-angiogenic cytokines, further supporting the pro-angiogenic milieu. Overall, our results demonstrate that FBEVs foster MM angiogenesis through dual time-related uptake-independent and uptake-dependent mechanisms that activate different intracellular pathways and transcriptional programs, providing the rationale for designing novel anti-angiogenic strategies.

Tamoxifen Affects Aquaporin-3 Expression and Subcellular Localization in Rat and Human Renal Collecting Ducts.

Sex hormones play an important role in the regulation of water homeostasis, and we have previously shown that tamoxifen (TAM), a selective estrogen receptor modulator (SERM), affects the regulation of aquaporin (AQP)-2. In this study, we investigated the effect of TAM on the expression and localization of AQP3 in collecting ducts using various animal, tissue, and cell models. The impact of TAM on AQP3 regulation was studied in rats subjected to 7 days of unilateral ureteral obstruction (UUO), with the rats fed a lithium-containing diet to induce nephrogenic diabetes insipidus (NDI), as well as in human precision-cut kidney slices (PCKS). Moreover, intracellular trafficking of AQP3 after TAM treatment was investigated in Madin-Darby Canine Kidney (MDCK) cells stably expressing AQP3. In all models, the expression of AQP3 was evaluated by Western blotting, immunohistochemistry and qPCR. TAM administration attenuated UUO-induced downregulation of AQP3 and affected the localization of AQP3 in both the UUO model and the lithium-induced NDI model. In parallel, TAM also affected the expression profile of other basolateral proteins, including AQP4 and Na/K-ATPase. In addition, TGF-ß and TGF-ß+TAM treatment affected the localization of AQP3 in stably transfected MDCK cells, and TAM partly attenuated the reduced AQP3 expression in TGF-ß exposed human tissue slices. These findings suggest that TAM attenuates the downregulation of AQP3 in a UUO model and a lithium-induced NDI model and affects the intracellular localization in the collecting ducts.

circPVT1 and PVT1/AKT3 show a role in cell proliferation, apoptosis, and tumor subtype-definition in small cell lung cancer.

Small cell lung cancer (SCLC) is treated as a homogeneous disease, although the expression of NEUROD1, ASCL1, POU2F3, and YAP1 identifies distinct molecular subtypes. The MYC oncogene, amplified in SCLC, was recently shown to act as a lineage-specific factor to associate subtypes with histological classes. Indeed, MYC-driven SCLCs show a distinct metabolic profile and drug sensitivity. To disentangle their molecular features, we focused on the co-amplified PVT1, frequently overexpressed and originating circular (circRNA) and chimeric RNAs. We analyzed hsa_circ_0001821 (circPVT1) and PVT1/AKT3 (chimPVT1) as examples of such transcripts, respectively, to unveil their tumorigenic contribution to SCLC. In detail, circPVT1 activated a pro-proliferative and anti-apoptotic program when over-expressed in lung cells, and knockdown of chimPVT1 induced a decrease in cell growth and an increase of apoptosis in SCLC in vitro. Moreover, the investigated PVT1 transcripts underlined a functional connection between MYC and YAP1/POU2F3, suggesting that they contribute to the transcriptional landscape associated with MYC amplification. In conclusion, we have uncovered a functional role of circular and chimeric PVT1 transcripts in SCLC; these entities may prove useful as novel biomarkers in MYC-amplified tumors.

dDAVP Downregulates the AQP3-Mediated Glycerol Transport via V1aR in Human Colon HCT8 Cells.

Vasopressin (AVP) plays a key function in controlling body water and salt balance through the activation of the vasopressin receptors V1aR and V2R. Abnormal secretion of AVP can cause the syndrome of inappropriate antidiuresis that leads to hyponatremia, which is an electrolyte disorder often observed in the elderly hospitalized and oncologic patients. Beyond kidneys, the colonic epithelium modulates water and salt homeostasis. The water channel AQP3, expressed in villus epithelial cells is implicated in water absorption across human colonic surface cells. Here, the action of dDAVP, a stable vasopressin analog, was evaluated on the AQP3 expression and function using human colon HCT8 cells as an experimental model. Confocal and Western Blotting analysis revealed that HCT8 cells express both V1aR and V2R. Long-term (72 h) treatment with dDAVP reduced glycerol uptake and cell viability. These effects were prevented by SR49059, a synthetic antagonist of V1aR, but not by tolvaptan, a specific V2R antagonist. Of note, the SR49059 action was impaired by DFP00173, a selective inhibitor of AQP3. Interestingly, compared to the normal colonic mucosa, in the colon of patients with adenocarcinoma, the expression of V1aR was significantly decreased. These findings were confirmed by gene expression analysis with RNA-Seq data. Overall, data suggest that dDAVP, through the V1aR dependent pathway, reduces AQP3 mediated glycerol uptake, a process that is reversed in adenocarcinoma, suggesting that the AVP-dependent AQP3 pathway may represent a novel target in colon diseases associated with abnormal cell growth.

"Good Wine Makes Good Blood": An Integrated Approach to Characterize Autochthonous Apulian Grapevines as Promising Candidates for Healthy Wines.

Wine production represents an ancient human activity and one of the most economically important markets in Europe. Moreover, the health effects of grapes and related products have been largely demonstrated, and mostly depend on their richness in bioactive molecules such as flavonoid and non-flavonoid phenolic compounds. Italy has the highest global wine production and provides one of the richest grapevine germplasm in the Mediterranean area. In this paper, our attention was focused on the evaluation of the total phenol and anthocyanin content in five autochthonous Apulian grapevine cultivars, in both wines and their non-alcoholic extracts. Moreover, the potential antioxidant effects of the non-alcoholic wine extracts on the cell viability of Caco-2 and HeLa carcinoma cell lines were tested. Finally, for the most promising autochthonous selected cultivars (Negramaro, Nero di Troia and Susumaniello), comparative transcriptomic analysis in berries was performed using high-throughput sequencing technology.

Functional interplay between CFTR and pendrin: physiological and pathophysiological relevance.

The transport of chloride and bicarbonate across epithelia controls the pH and volume of the intracellular and luminal fluids, as well as the systemic pH and vascular volume. The anion exchanger pendrin (SLC26A4) and the cystic fibrosis transmembrane conductance regulator (CFTR) channel are expressed in the apical membrane of epithelial cells of various organs and tissues, including the airways, kidney, thyroid, and inner ear. While pendrin drives chloride reabsorption and bicarbonate, thiocyanate or iodide secretion within the apical compartment, CFTR represents a pathway for the apical efflux of chloride, bicarbonate, and possibly iodide. In the airways, pendrin and CFTR seems to be involved in alkalinization of the apical fluid via bicarbonate secretion, especially during inflammation, while CFTR also controls the volume of the apical fluid via a cAMP-dependent chloride secretion, which is stimulated by pendrin. In the kidney, pendrin is expressed in the cortical collecting duct and connecting tubule and co-localizes with CFTR in the apical membrane of ß intercalated cells. Bicarbonate secretion occurs via pendrin, which also drives chloride reabsorption. A functional CFTR is required for pendrin activity. Whether CFTR stimulates pendrin via a direct molecular interaction or other mechanisms, or simply provides a pathway for chloride recycling across the apical membrane remains to be established. In the thyroid, CFTR and pendrin might have overlapping functions in driving the apical flux of iodide within the follicular lumen. In other organs, including the inner ear, the possible functional interplay between pendrin and CFTR needs to be explored.

Olive Leaf Extract (OLE) impaired vasopressin-induced aquaporin-2 trafficking through the activation of the calcium-sensing receptor.

Vasopressin (AVP) increases water permeability in the renal collecting duct through the regulation of aquaporin-2 (AQP2) trafficking. Several disorders, including hypertension and inappropriate antidiuretic hormone secretion (SIADH), are associated with abnormalities in water homeostasis. It has been shown that certain phytocompounds are beneficial to human health. Here, the effects of the Olive Leaf Extract (OLE) have been evaluated using in vitro and in vivo models. Confocal studies showed that OLE prevents the vasopressin induced AQP2 translocation to the plasma membrane in MCD4 cells and rat kidneys. Incubation with OLE decreases the AVP-dependent increase of the osmotic water permeability coefficient (Pf). To elucidate the possible effectors of OLE, intracellular calcium was evaluated. OLE increases the intracellular calcium through the activation of the Calcium Sensing Receptor (CaSR). NPS2143, a selective CaSR inhibitor, abolished the inhibitory effect of OLE on AVP-dependent water permeability. In vivo experiments revealed that treatment with OLE increases the expression of the CaSR mRNA and decreases AQP2 mRNA paralleled by an increase of the AQP2-targeting miRNA-137. Together, these findings suggest that OLE antagonizes vasopressin action through stimulation of the CaSR indicating that this extract may be beneficial to attenuate disorders characterized by abnormal CaSR signaling and affecting renal water reabsorption.

Antioxidant Efficacy of Olive By-Product Extracts in Human Colon HCT8 Cells.

The production of olive oil is accompanied by the generation of a huge amount of waste and by-products including olive leaves, pomace, and wastewater. The latter represents a relevant environmental issue because they contain certain phytotoxic compounds that may need specific treatments before the expensive disposal. Therefore, reducing waste biomass and valorizing by-products would make olive oil production more sustainable. Here, we explore the biological actions of extracts deriving from olive by-products including olive pomace (OP), olive wastewater (OWW), and olive leaf (OLs) in human colorectal carcinoma HCT8 cells. Interestingly, with the same phenolic concentration, the extract obtained from the OWW showed higher antioxidant ability compared with the extracts derived from OP and OLs. These biological effects may be related to the differential phenolic composition of the extracts, as OWW extract contains the highest amount of hydroxytyrosol and tyrosol that are potent antioxidant compounds. Furthermore, OP extract that contains a higher level of vanillic acid than the other extracts displayed a cytotoxic action at the highest concentration. Together these findings revealed that phenols in the by-product extracts may interfere with signaling molecules that cross-link several intracellular pathways, raising the possibility to use them for beneficial health effects.

The Vasopressin Receptor 2 Mutant R137L Linked to the Nephrogenic Syndrome of Inappropriate Antidiuresis (NSIAD) Signals through an Alternative Pathway that Increases AQP2 Membrane Targeting Independently of S256 Phosphorylation.

NSIAD is a rare X-linked condition, caused by activating mutations in the AVPR2 gene coding for the vasopressin V2 receptor (V2R) associated with hyponatremia, despite undetectable plasma vasopressin levels. We have recently provided in vitro evidence that, compared to V2R-wt, expression of activating V2R mutations R137L, R137C and F229V cause a constitutive redistribution of the AQP2 water channel to the plasma membrane, higher basal water permeability and significantly higher basal levels of p256-AQP2 in the F229V mutant but not in R137L or R137C. In this study, V2R mutations were expressed in collecting duct principal cells and the associated signalling was dissected. V2R-R137L and R137C mutants had significantly higher basal pT269-AQP2 levels -independently of S256 and PKA-which were reduced to control by treatment with Rho kinase (ROCK) inhibitor. Interestingly, ROCK activity was found significantly higher in V2R-R137L along with activation of the Gα12/13-Rho-ROCK pathway. Of note, inhibition of ROCK reduced the basal elevated osmotic water permeability to control. To conclude, our data demonstrate for the first time that the gain-of-function mutation of the V2R, R137L causing NSIAD, signals through an alternative PKA-independent pathway that increases AQP2 membrane targeting through ROCK-induced phosphorylation at S/T269 independently of S256 of AQP2.

In Vitro and In Vivo Nutraceutical Characterization of Two Chickpea Accessions: Differential Effects on Hepatic Lipid Over-Accumulation.

Dietary habits are crucially important to prevent the development of lifestyle-associated diseases. Diets supplemented with chickpeas have numerous benefits and are known to improve body fat composition. The present study was undertaken to characterize two genetically and phenotypically distinct accessions, MG_13 and PI358934, selected from a global chickpea collection. Rat hepatoma FaO cells treated with a mixture of free fatty acids (FFAs) (O/P) were used as an in vitro model of hepatic steatosis. In parallel, a high-fat diet (HFD) animal model was also established. In vitro and in vivo studies revealed that both chickpea accessions showed a significant antioxidant ability. However, only MG_13 reduced the lipid over-accumulation in steatotic FaO cells and in the liver of HFD fed mice. Moreover, mice fed with HFD + MG_13 displayed a lower level of glycemia and aspartate aminotransferase (AST) than HFD mice. Interestingly, exposure to MG_13 prevented the phosphorylation of the inflammatory nuclear factor kappa beta (NF-kB) which is upregulated during HFD and known to be linked to obesity. To conclude, the comparison of the two distinct chickpea accessions revealed a beneficial effect only for the MG_13. These findings highlight the importance of studies addressing the functional characterization of chickpea biodiversity and nutraceutical properties.

Green olive leaf extract (OLE) provides cytoprotection in renal cells exposed to low doses of cadmium.

Cadmium (Cd) is a heavy and highly toxic metal that contaminates air, food and water. Cadmium accumulates in several organs altering normal functions. The kidney is the major organ at risk of damage from chronic exposure to cadmium as a contaminant in food and water. This study aims to investigate the beneficial effects of OLE in renal collecting duct MCD4 cells exposed to a low dose cadmium (1 µM). In MCD4 cells cadmium caused an increase in ROS production, as well as generation of lipid droplets and reduced cell viability. Moreover, cadmium exposure led to a remarkable increase in the frequency of micronuclei and DNA double-strand breaks, assessed using the alkaline comet assay. In addition, cadmium dramatically altered cell cytoskeleton architecture and caused S-glutathionylation of actin. Notably, all cadmium-induced cellular deregulations were prevented by co-treatment with OLE, possibly due to its antioxidant action and to the presence of bioactive phytocompounds. Indeed, OLE treatment attenuated Cd-induced actin S-glutathionylation, thereby stabilizing actin filaments. Taken together, these observations provide a novel insight into the biological action of OLE in renal cells and support the notion that OLE may serve as a potential adjuvant against cadmium-induced nephrotoxicity.

Modulation of Aquaporins by Dietary Patterns and Plant Bioactive Compounds.

Healthful dietary patterns and bioactive compounds supplementation can be adopted as simple and easy intervention to prevent, attenuate or cure clinical disorders, especially when it comes to degenerative and chronic diseases. In the recent years, a growing body of evidence indicates Aquaporins (AQPs), a family of membrane channel proteins widely expressed in the human body, among the targets underlying the beneficial action played by some food nutrients and phytochemical compounds. Here, we provide an overview of what is known regarding the AQP modulation exerted by healthful dietary patterns and plant polyphenols.

Activation of the Calcium-Sensing Receptor Corrects the Impaired Mitochondrial Energy Status Observed in Renal Polycystin-1 Knockdown Cells Modeling Autosomal Dominant Polycystic Kidney Disease.

Autosomal Dominant Polycistic kidney Disease (ADPKD) is a renal channelopathy due to loss-of-function mutations in the PKD1 or PKD2 genes, encoding polycystin-1 (PC1) or polycystin-2 (PC2), respectively. PC1 is a large protein found predominantly on the plasma membrane where interacts with different proteins, including PC2. PC2 is a smaller integral membrane protein also expressed in intracellular organelles, acting as a non-selective cation channel permeable to calcium. Both PC1 and PC2 are also localized to the primary cilium of renal epithelial cells serving as mechanosensor that controls calcium influx through the plasma membrane and regulates intracellular calcium release from the endoplasmic reticulum. The mechanisms by which PC1/2 dysfunction leads to ADPKD needs still to be clarified. We have recently reported that selective Calcium-Sensing Receptor (CaSR) activation in human conditionally immortalized Proximal Tubular Epithelial cells deficient for PC1 (ciPTEC-PC1KD), deriving from urine sediments reduces intracellular cAMP and mTOR activity, and increases intracellular calcium reversing the principal ADPKD dysregulations. Reduced cellular free calcium found in ADPKD can, on the other hand, affect mitochondrial function and ATP production and, interestingly, a relationship between mitochondria and renal polycystic diseases have been suggested. By using ciPTEC-PC1KD as experimental tool modeling of ADPKD, we show here that, compared with wild type cells, ciPTEC-PC1KD have significantly lower mitochondrial calcium levels associated with a severe deficit in mitochondrial ATP production, secondary to a multilevel impairment of oxidative phosphorylation. Notably, selective CaSR activation with the calcimimetic NPS-R568 increases mitochondrial calcium content close to the levels found in resting wild type cells, and fully recovers the cell energy deficit associated to the PC1 channel disruption. Treatment of ciPTEC-PC1KD with 2-APB, an IP3R inhibitor, prevented the rescue of bioenergetics deficit induced by CaSR activation supporting a critical role of IP3Rs in driving ER-to-mitochondria Ca2+ shuttle. Together these data indicate that, besides reversing the principal dysregulations considered the most proximal events in ADPKD pathogenesis, selective CaSR activation in PKD1 deficient cells restores altered mitochondrial function that, in ADPKD, is known to facilitate cyst formation. These findings identify CaSR as a potential therapeutic target.

Activation of Calcium-Sensing Receptor increases intracellular calcium and decreases cAMP and mTOR in PKD1 deficient cells.

Clinical and fundamental research suggest that altered calcium and cAMP signaling might be the most proximal events in ADPKD pathogenesis. Cells from ADPKD cysts have a reduced resting cytosolic calcium [Ca2+]i and increased cAMP levels. CaSR plays an essential role in regulating calcium homeostasis. Its activation is associated with [Ca2+]i increase and cAMP decrease, making CaSR a possible therapeutic target. Human conditionally immortalized Proximal Tubular Epithelial cells (ciPTEC) with stable knockdown of PKD1 (ciPTEC-PC1KD) and ciPTEC generated from an ADPKD1 patient (ciPTEC-PC1Pt) were used as experimental tools. CaSR functional expression was confirmed by studies showing that the calcimimetic NPS-R568 induced a significant increase in [Ca2+]i in ciPTEC-PC1KD and ciPTEC-PC1Pt. Resting [Ca2+]i were significantly lower in ciPTEC-PC1KD with respect to ciPTECwt, confirming calcium dysregulation. As in native cyst cells, significantly higher cAMP levels and mTOR activity were found in ciPTEC-PC1KD compared to ciPTECwt. Of note, NPS-R568 treatment significantly reduced intracellular cAMP and mTOR activity in ciPTEC-PC1KD and ciPTEC-PC1Pt. To conclude, we demonstrated that selective CaSR activation in human ciPTEC carrying PKD1 mutation increases [Ca2+]i, reduces intracellular cAMP and mTOR activity, reversing the principal dysregulations considered the most proximal events in ADPKD pathogenesis, making CaSR a possible candidate as therapeutic target.

Impact of atypical mitochondrial cyclic-AMP level in nephropathic cystinosis.

Nephropathic cystinosis (NC) is a rare disease caused by mutations in the CTNS gene encoding for cystinosin, a lysosomal transmembrane cystine/H+ symporter, which promotes the efflux of cystine from lysosomes to cytosol. NC is the most frequent cause of Fanconi syndrome (FS) in young children, the molecular basis of which is not well established. Proximal tubular cells have very high metabolic rate due to the active transport of many solutes. Not surprisingly, mitochondrial disorders are often characterized by FS. A similar mechanism may also apply to NC. Because cAMP has regulatory properties on mitochondrial function, we have analyzed cAMP levels and mitochondrial targets in CTNS-/- conditionally immortalized proximal tubular epithelial cells (ciPTEC) carrying the classical homozygous 57-kb deletion (delCTNS-/-) or with compound heterozygous loss-of-function mutations (mutCTNS-/-). Compared to wild-type cells, cystinotic cells had significantly lower mitochondrial cAMP levels (delCTNS-/- ciPTEC by 56% ± 10.5, P < 0.0001; mutCTNS-/- by 26% ± 4.3, P < 0.001), complex I and V activities, mitochondrial membrane potential, and SIRT3 protein levels, which were associated with increased mitochondrial fragmentation. Reduction of complex I and V activities was associated with lower expression of part of their subunits. Treatment with the non-hydrolysable cAMP analog 8-Br-cAMP restored mitochondrial potential and corrected mitochondria morphology. Treatment with cysteamine, which reduces the intra-lysosomal cystine, was able to restore mitochondrial cAMP levels, as well as most other abnormal mitochondrial findings. These observations were validated in CTNS-silenced HK-2 cells, indicating a pivotal role of mitochondrial cAMP in the proximal tubular dysfunction observed in NC.

AQP2 Abundance is Regulated by the E3-Ligase CHIP Via HSP70.

BACKGROUND/AIMS: AQP2 expression is mainly controlled by vasopressin-dependent changes in protein abundance which is in turn regulated by AQP2 ubiquitylation and degradation, however the proteins involved in these processes are largely unknown. Here, we investigated the potential role of the CHIP E3 ligase in AQP2 regulation. METHODS: MCD4 cells and kidney slices were used to study the involvement of the E3 ligase CHIP on AQP2 protein abundance by cell homogenization and immunoprecipitation followed by immunoblotting. RESULTS: We found that AQP2 complexes with CHIP in renal tissue. Expression of CHIP increased proteasomal degradation of AQP2 and HSP70 abundance, a molecular signature of HSP90 inhibition. Increased HSP70 level, secondary to CHIP expression, promoted ERK signaling resulting in increased AQP2 phosphorylation at S261. Phosphorylation of AQP2 at S256 and T269 were instead downregulated. Next, we investigated HSP70 interaction with AQP2, which is important for endocytosis. Compared with AQP2-wt, HSP70 binding decreased in AQP2-S256D and AQP2-S256D-S261D, while increased in AQP2-S256D-S261A. Surprisingly, expression of CHIP-delUbox, displaying a loss of E3 ligase activity, still induced AQP2 degradation, indicating that CHIP does not ubiquitylate and degrade AQP2 itself. Conversely, the AQP2 half-life was increased upon the expression of CHIP-delTPR a domain which binds Hsc70/HSP70 and HSP90. HSP70 has been reported to bind other E3 ligases such as MDM2. Notably, we found that co-expression of CHIP and MDM2 increased AQP2 degradation, whereas co-expression of CHIP with MDM2-delRING, an inactive form of MDM2, impaired AQP2 degradation. CONCLUSION: Our findings indicate CHIP as a master regulator of AQP2 degradation via HSP70 that has dual functions: (1) as chaperone for AQP2 and (2) as an anchoring protein for MDM2 E3 ligase, which is likely to be involved in AQP2 degradation.

Interleukin-13 increases pendrin abundance to the cell surface in bronchial NCI-H292 cells via Rho/actin signaling.

Interleukin-13 (IL13) is a major player in the development of airway hyperresponsiveness in several respiratory disorders. Emerging data suggest that an increased expression of pendrin in airway epithelia is associated with elevated airway hyperreactivity in asthma. Here, we investigate the effect of IL13 on pendrin localization and function using bronchiolar NCI-H292 cells. The data obtained revealed that IL13 increases the cell surface expression of pendrin. This effect was paralleled by a significant increase in the intracellular pH, possibly via indirect stimulation of NHE. IL13 effect on pendrin localization and intracellular pH was reversed by theophylline, a bronchodilator compound used to treat asthma. IL13 upregulated RhoA activity, a crucial protein controlling actin dynamics, via G-alpha-13. Specifically, IL13 stabilized actin cytoskeleton and promoted co-localization and a direct molecular interaction between pendrin and F-actin in the plasma membrane region. These effects were reversed following exposure of cells to theophylline. Selective inhibition of Rho kinase, a downstream effector of Rho, reduced the IL13-dependent cell surface expression of pendrin. Together, these data indicate that IL13 increases pendrin abundance to the cell surface via Rho/actin signaling, an effect reversed by theophylline.

The V2 receptor antagonist tolvaptan raises cytosolic calcium and prevents AQP2 trafficking and function: an in vitro and in vivo assessment.

Tolvaptan, a selective vasopressin V2 receptor antagonist, is a new generation diuretic. Its clinical efficacy is in principle due to impaired vasopressin-regulated water reabsorption via aquaporin-2 (AQP2). Nevertheless, no direct in vitro evidence that tolvaptan prevents AQP2-mediated water transport, nor that this pathway is targeted in vivo in patients with syndrome of inappropriate antidiuresis (SIAD) has been provided. The effects of tolvaptan on the vasopressin-cAMP/PKA signalling cascade were investigated in MDCK cells expressing endogenous V2R and in mouse kidney. In MDCK, tolvaptan prevented dDAVP-induced increase in ser256-AQP2 and osmotic water permeability. A similar effect on ser256-AQP2 was found in V1aR -/- mice, thus confirming the V2R selectively. Of note, calcium calibration in MDCK showed that tolvaptan per se caused calcium mobilization from the endoplasmic reticulum resulting in a significant increase in basal intracellular calcium. This effect was only observed in cells expressing the V2R, indicating that it requires the tolvaptan-V2R interaction. Consistent with this finding, tolvaptan partially reduced the increase in ser256-AQP2 and the water permeability in response to forskolin, a direct activator of adenylyl cyclase (AC), suggesting that the increase in intracellular calcium is associated with an inhibition of the calcium-inhibitable AC type VI. Furthermore, tolvaptan treatment reduced AQP2 excretion in two SIAD patients and normalized plasma sodium concentration. These data represent the first detailed demonstration of the central role of AQP2 blockade in the aquaretic effect of tolvaptan and underscore a novel effect in raising intracellular calcium that can be of significant clinical relevance.