RhoB plays a central role in hyperosmolarity-induced cell shrinkage in renal cells.

The small Rho GTP-binding proteins are important cell morphology, function, and apoptosis regulators. Unlike other Rho proteins, RhoB can be subjected to either geranylgeranylation (RhoB-GG) or farnesylation (RhoB-F), making that the only target of the farnesyltransferase inhibitor (FTI). Fluorescence resonance energy transfer experiments revealed that RhoB is activated by hyperosmolarity. By contrast, hyposmolarity did not affect RhoB activity. Interestingly, treatment with farnesyltransferase inhibitor-277 (FTI-277) decreased the cell size. To evaluate whether RhoB plays a role in volume reduction, renal collecting duct MCD4 cells and Human Kidney, HK-2 were transiently transfected with RhoB-wildtype-Enhance Green Fluorescence Protein (RhoB-wt-EGFP) and RhoB-CLLL-EGFP which cannot undergo farnesylation. A calcein-based fluorescent assay revealed that hyperosmolarity caused a significant reduction of cell volume in mock and RhoB-wt-EGFP-expressing cells. By contrast, cells treated with FTI-277 or expressing the RhoB-CLLL-EGFP mutant did not properly respond to hyperosmolarity with respect to mock and RhoB-wt-EGFP expressing cells. These findings were further confirmed by 3D-LSCM showing that RhoB-CLLL-EGFP cells displayed a significant reduction in cell size compared to cells expressing RhoB-wt-EGFP. Moreover, flow cytometry analysis revealed that RhoB-CLLL-EGFP expressing cells as well as FTI-277-treated cells showed a significant increase in cell apoptosis. Together, these data suggested that: (i) RhoB is sensitive to hyperosmolarity and not to hyposmolarity; (ii) inhibition of RhoB farnesylation associates with an increase in cell apoptosis, likely suggesting that RhoB might be a paramount player controlling apoptosis by interfering with responses to cell volume change.

In vivo treatment with calcilytic of CaSR knock-in mice ameliorates renal phenotype reversing downregulation of the vasopressin-AQP2 pathway.

High concentrations of urinary calcium counteract vasopressin action via the activation of the Calcium-Sensing Receptor (CaSR) expressed in the luminal membrane of the collecting duct cells, which impairs the trafficking of aquaporin-2 (AQP2). In line with these findings, we provide evidence that, with respect to wild-type mice, CaSR knock-in (KI) mice mimicking autosomal dominant hypocalcaemia, display a significant decrease in the total content of AQP2 associated with significantly higher levels of AQP2 phosphorylation at Ser261, a phosphorylation site involved in AQP2 degradation. Interestingly, KI mice also had significantly higher levels of phosphorylated p38MAPK, a downstream effector of CaSR and known to phosphorylate AQP2 at Ser261. Moreover, ATF1 phosphorylated at Ser63, a transcription factor downstream of p38MAPK, was significantly higher in KI. In addition, KI mice had significantly higher levels of AQP2-targeting miRNA137 consistent with a post-transcriptional downregulation of AQP2. In vivo treatment of KI mice with the calcilytic JTT-305, a CaSR antagonist, increased AQP2 expression and reduced AQP2-targeting miRNA137 levels in KI mice. Together, these results provide direct evidence for a critical role of CaSR in impairing both short-term vasopressin response by increasing AQP2-pS261, as well as AQP2 abundance, via the p38MAPK-ATF1-miR137 pathway. KEY POINTS: Calcium-Sensing Receptor (CaSR) activating mutations are the main cause of autosomal dominant hypocalcaemia (ADH) characterized by inappropriate renal calcium excretion leading to hypocalcaemia and hypercalciuria. Current treatments of ADH patients with parathyroid hormone, although improving hypocalcaemia, do not improve hypercalciuria or nephrocalcinosis. In vivo treatment with calcilytic JTT-305/MK-5442 ameliorates most of the ADH phenotypes of the CaSR knock-in mice including hypercalciuria or nephrocalcinosis and reverses the downregulation of the vasopressin-sensitive aquaporin-2 (AQP2) expression, providing direct evidence for a critical role of CaSR in impairing vasopressin response. The beneficial effect of calcilytic in reducing the risk of renal calcification may occur in a parathyroid hormone-independent action through vasopressin-dependent inhibition of cAMP synthesis in the thick ascending limb and in the collecting duct. The amelioration of most of the abnormalities in calcium metabolism including hypercalciuria, renal calcification, and AQP2-mediated osmotic water reabsorption makes calcilytic a good candidate as a novel therapeutic agent for ADH.

Novel signalling pathways in nephrogenic syndrome of inappropriate antidiuresis: functional implication of site-specific AQP2 phosphorylation.

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a rare X-linked disease caused by gain-of-function mutations of arginine vasopressin receptor 2 (V2R). Patients with NSIAD are characterized by the inability to excrete a free water load and by inappropriately increased urinary osmolality despite very low levels of plasma vasopressin, resulting in euvolaemic hyponatraemia. To dissect the signalling downstream V2R constitutively active variants, Flp-In T-REx Madin-Darby canine kidney (FTM) cells, stably transfected with V2R mutants (R137L, R137C and F229V) and AQP2-wt or non-phosphorylatable AQP2-S269A/AQP2-S256A, were used as cellular models. All three activating V2R mutations presented constitutive plasma membrane expression of AQP2-wt and significantly higher basal water permeability. In addition, V2R-R137L/C showed significantly higher activity of Rho-associated kinase (ROCK), a serine/threonine kinase previously suggested to be involved in S269-AQP2 phosphorylation downstream of these V2R mutants. Interestingly, FTM cells expressing V2R-R137L/C mutants and AQP2-S269A showed a significant reduction in AQP2 membrane abundance and a significant reduction in ROCK activity, indicating the crucial importance of S269-AQP2 phosphorylation in the gain-of-function phenotype. Conversely, V2R-R137L/C mutants retained the gain-of-function phenotype when AQP2-S256A was co-expressed. In contrast, cells expressing the F229V mutant and the non-phosphorylatable AQP2-S256A had a significant reduction in AQP2 membrane abundance along with a significant reduction in basal osmotic water permeability, indicating a crucial role of Ser256 for this mutant. These data indicate that the constitutive AQP2 trafficking associated with the gain-of-function V2R-R137L/C mutants causing NSIAD is protein kinase A independent and requires an intact Ser269 in AQP2 under the control of ROCK phosphorylation. KEY POINTS: Nephrogenic syndrome of inappropriate antidiuresis is caused by two constitutively active variant phenotypes of AVPR2, one sensitive to vaptans (V2R-F229V) and the other vaptan resistant (V2R-R137C/L). In renal cells, all three activating arginine vasopressin receptor 2 (V2R) variants display constitutive AQP2 plasma membrane expression and high basal water permeability. In cells expressing V2R-R137L/C mutants, disruption of the AQP2-S269 phosphorylation site caused the loss of the gain-of-function phenotype, which, in contrast, was retained in V2R-F229V-expressing cells. Cells expressing the V2R-F229V mutant were instead sensitive to disruption of the AQP2-S256 phosphorylation site. The serine/threonine kinase Rho-associated kinase (ROCK) was found to be involved in AQP2-S269 phosphorylation downstream of the V2R-R137L/C mutants. These findings might have clinical relevance for patients with nephrogenic syndrome of inappropriate antidiuresis.

Role of ryanodine receptor 2 and FK506-binding protein 12.6 dissociation in pulmonary hypertension.

Pulmonary hypertension (PH) is a devastating disease characterized by a progressive increase in pulmonary arterial pressure leading to right ventricular failure and death. A major cellular response in this disease is the contraction of smooth muscle cells (SMCs) of the pulmonary vasculature. Cell contraction is determined by the increase in intracellular Ca2+ concentration ([Ca2+]i), which is generated and regulated by various ion channels. Several studies by us and others have shown that ryanodine receptor 2 (RyR2), a Ca2+-releasing channel in the sarcoplasmic reticulum (SR), is an essential ion channel for the control of [Ca2+]i in pulmonary artery SMCs (PASMCs), thereby mediating the sustained vasoconstriction seen in PH. FK506-binding protein 12.6 (FKBP12.6) strongly associates with RyR2 to stabilize its functional activity. FKBP12.6 can be dissociated from RyR2 by a hypoxic stimulus to increase channel function and Ca2+ release, leading to pulmonary vasoconstriction and PH. More specifically, dissociation of the RyR2-FKBP12.6 complex is a consequence of increased mitochondrial ROS generation mediated by the Rieske iron-sulfur protein (RISP) at the mitochondrial complex III after hypoxia. Overall, RyR2/FKBP12.6 dissociation and the corresponding signaling pathway may be an important factor in the development of PH. Novel drugs and biologics targeting RyR2, FKBP12.6, and related molecules may become unique effective therapeutics for PH.

Mitochondrial Dynamics in Pulmonary Hypertension.

Mitochondria are essential organelles for energy production, calcium homeostasis, redox signaling, and other cellular responses involved in pulmonary vascular biology and disease processes. Mitochondrial homeostasis depends on a balance in mitochondrial fusion and fission (dynamics). Mitochondrial dynamics are regulated by a viable circadian clock. Hypoxia and nicotine exposure can cause dysfunctions in mitochondrial dynamics, increases in mitochondrial reactive oxygen species generation and calcium concentration, and decreases in ATP production. These mitochondrial changes contribute significantly to pulmonary vascular oxidative stress, inflammatory responses, contractile dysfunction, pathologic remodeling, and eventually pulmonary hypertension. In this review article, therefore, we primarily summarize recent advances in basic, translational, and clinical studies of circadian roles in mitochondrial metabolism in the pulmonary vasculature. This knowledge may not only be crucial to fully understanding the development of pulmonary hypertension, but also greatly help to create new therapeutic strategies for treating this devastating disease and other related pulmonary disorders.

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.

AQP2 trafficking in health and diseases: an updated overview.

Renal collecting duct principal cells play a key role in controlling body water balance. Principal cells express the water channels AQP2, AQP3, and AQP4 that mediate renal water reabsorption. AQP3 and AQP4 are expressed at the basolateral membrane constitutively. Conversely, AQP2 is localized in intracellular vesicles and translocates to the plasma membrane under vasopressin action. Stimulation with vasopressin activates the cAMP/PKA signal transduction pathway that induces the redistribution of AQP2 from an intracellular pool to the apical plasma membrane. AQP2 trafficking and function depend on multiple post-translational modifications. Moreover, several proteins control different steps activated by the vasopressin stimulation that triggers the redistribution of the AQP2 vesicles. A-kinase anchoring proteins (AKAPs) together with phosphodiesterases and adenylate cyclases play crucial roles in modulating local changes of cAMP. Soluble N-ethylmaleimide sensitive fusion factor attachment protein receptors (SNARE), cytoskeletal proteins, and the small GTPases of the Rho family regulate the fusion and the endocytotic retrieval of AQP2 vesicles. Abnormal vasopressin signaling and altered AQP2 expression or trafficking can lead to disorders characterized by deregulated mechanisms controlling water homeostasis. This review provides updated data on the molecular signals regulating vasopressin-induced AQP2 trafficking in health and disease.

Early Biomarkers of Altered Renal Function and Orthostatic Intolerance During 10-day Bedrest.

Exposure to actual or simulated microgravity results in alterations of renal function, fluid redistribution, and bone loss, which is coupled to a rise of urinary calcium excretion. We provided evidence that high calcium delivery to the collecting duct reduces local Aquaporin 2 (AQP2)-mediated water reabsorption under vasopressin action, thus limiting the maximal urinary concentration to reduce calcium saturation. To investigate early renal adaptation into simulated microgravity, we investigated the effects of 10 days of strict bedrest in 10 healthy volunteers. We report here that 10 days of inactivity are associated with a transient, significant decrease (day 5) in vasopressin (copeptin) paralleled by a decrease in AQP2 excretion, consistent with an increased central volume to the heart, resulting in reduced water reabsorption. Moreover, bedrest caused a significant increase in calciuria secondary to bone demineralization paralleled by a decrease in PTH. Urinary osteopontin, a glycoprotein exerting a protective effect on stone formation, was significantly reduced during bedrest. Moreover, a significant increase in adrenomedullin (day 5), a peptide with vasodepressor properties, was observed at day 5, which may contribute to the known reduced orthostatic capacity post-bedrest. We conclude that renal function is altered in simulated microgravity and is associated with an early increase in the risk of stone formation and reduced orthostatic capacity post-bedrest within a few days of inactivity.

Important Functions and Molecular Mechanisms of Mitochondrial Redox Signaling in Pulmonary Hypertension.

Mitochondria are important organelles that act as a primary site to produce reactive oxygen species (ROS). Additionally, mitochondria play a pivotal role in the regulation of Ca2+ signaling, fatty acid oxidation, and ketone synthesis. Dysfunction of these signaling molecules leads to the development of pulmonary hypertension (PH), atherosclerosis, and other vascular diseases. Features of PH include vasoconstriction and pulmonary artery (PA) remodeling, which can result from abnormal proliferation, apoptosis, and migration of PA smooth muscle cells (PASMCs). These responses are mediated by increased Rieske iron-sulfur protein (RISP)-dependent mitochondrial ROS production and increased mitochondrial Ca2+ levels. Mitochondrial ROS and Ca2+ can both synergistically activate nuclear factor κB (NF-κB) to trigger inflammatory responses leading to PH, right ventricular failure, and death. Evidence suggests that increased mitochondrial ROS and Ca2+ signaling leads to abnormal synthesis of ketones, which play a critical role in the development of PH. In this review, we discuss some of the recent findings on the important interactive role and molecular mechanisms of mitochondrial ROS and Ca2+ in the development and progression of PH. We also address the contributions of NF-κB-dependent inflammatory responses and ketone-mediated oxidative stress due to abnormal regulation of mitochondrial ROS and Ca2+ signaling in PH.

Pre-clinical evaluation of dual targeting of the GPCRs CaSR and V2R as therapeutic strategy for autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations of PKD1 or PKD2 genes, is characterized by development and growth of cysts causing progressive kidney enlargement. Reduced resting cytosolic calcium and increased cAMP levels associated with the tonic action of vasopressin are two central biochemical defects in ADPKD. Here we show that co-targeting two GPCRs, the vasopressin V2 receptor (V2R) and the calcium sensing receptor, using the novel V2R antagonist lixivaptan in combination with the calcimimetic R-568, reduced cyst progression in two animal models of human PKD. Lixivaptan is expected to have a safer liver profile compared to tolvaptan, the only drug approved to delay PKD progression, based on computational model results and initial clinical evidence. PCK rat and Pkd1RC/RC mouse littermates were fed without or with lixivaptan (0.5%) and R-568 (0.025% for rats and 0.04% for mice), alone or in combination, for 7 (rats) or 13 (mice) weeks. In PCK rats, the combined treatment strongly decreased kidney weight, cyst and fibrosis volumes by 20%, 49%, and 73%, respectively, compared to untreated animals. In Pkd1RC/RC mice, the same parameters were reduced by 20%, 56%, and 69%, respectively. In both cases the combined treatment appeared nominally more effective than the individual drugs used alone. These data point to an intriguing new application for two existing drugs in PKD treatment. The potential for synergy between these two compounds suggested in these animal studies, if confirmed in appropriate clinical investigations, would represent a welcome advancement in the treatment of ADPKD.

Reciprocal Correlations of Inflammatory and Calcium Signaling in Asthma Pathogenesis.

Asthma is a chronic disease characterized by airway hyperresponsiveness, which can be caused by exposure to an allergen, spasmogen, or be induced by exercise. Despite its prevalence, the exact mechanisms by which the airway becomes hyperresponsive in asthma are not fully understood. There is evidence that myosin light-chain kinase is overexpressed, with a concomitant downregulation of myosin light-chain phosphatase in the airway smooth muscle, leading to sustained contraction. Additionally, the sarco/endoplasmic reticulum ATPase may be affected by inflammatory cytokines, such as IL-4, IL-5, IL-13, and TNF-α, which are all associated with asthmatic airway inflammation. IL-13 and TNF-α seem to promote sodium/calcium exchanger 1 overexpression as well. Anyhow, the exact mechanisms beyond these dysregulations need to be clarified. Of note, multiple studies show an association between asthma and the ORMLD3 gene, opening new perspectives to future potential gene therapies. Currently, several treatments are available for asthma, although many of them have systemic side effects, or are not effective in patients with severe asthma. Furthering our knowledge on the molecular and pathophysiological mechanisms of asthma plays a pivotal role for the development of new and more targeted treatments for patients who cannot totally benefit from the current therapies.

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.

Calcium sensing receptor exerts a negative regulatory action toward vasopressin-induced aquaporin-2 expression and trafficking in renal collecting duct.

Vasopressin (AVP) plays a major role in the regulation of water homeostasis by its antidiuretic action on the kidney, mediated by V2 receptors. An increase in plasma sodium concentration stimulates AVP release, which in turn promotes water reabsorption. Upon binding to the V2 receptors in the renal collecting duct, AVP induces the expression and apical membrane insertion of the aquaporin-2 (AQP2) water channels and subsequent water reabsorption. AVP regulates two independent mechanisms: the short-term regulation of AQP2 trafficking and long-term regulation of the total abundance of the AQP2 protein in the cells. On the other hand, several hormones, acting through specific receptors, have been reported to antagonize AVP-mediated water transport in kidney. In this respect, we previously described that high luminal Ca2+ in the renal collecting duct attenuates short-term AVP-induced AQP2 trafficking through activation of the Ca2+-sensing receptor (CaSR). This effect is due to reduction of AVP-dependent cAMP generation and possibly hydrolysis. Moreover, CaSR signaling reduces AQP2 abundance both via AQP2-targeting miRNA-137 and the proteasomal degradation pathway. This chapter summarizes recent data elucidating the molecular mechanisms underlying the physiological role of the CaSR-dependent regulation of AQP2 expression and trafficking.

Gain-of-function mutations of the V2 vasopressin receptor in nephrogenic syndrome of inappropriate antidiuresis (NSIAD): a cell-based assay to assess constitutive water reabsorption.

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a recently identified chromosome X-linked disease associated with gain-of-function mutations of the V2 vasopressin receptor (V2R), a G-protein-coupled receptor. It is characterized by inability to excrete a free water load, hyponatremia, and undetectable vasopressin-circulating levels. Hyponatremia can be quite severe in affected male children. To gain a deeper insight into the functional properties of the V2R active mutants and how they might translate into the pathological outcome of NSIAD, in this study, we have expressed the wild-type V2R and three constitutively active V2R mutants associated with NSIAD (R137L, R137C, and the F229V) in MCD4 cells, a cell line derived from renal mouse collecting duct, stably expressing the vasopressin-sensitive water channel aquaporin-2 (AQP2). Our findings indicate that in cells expressing each active mutant, AQP2 was constitutively localized to the apical plasma membrane in the absence of vasopressin stimulation. In line with these observations, under basal conditions, osmotic water permeability in cells expressing the constitutively active mutants was significantly higher compared to that of cells expressing the wild-type V2R. Our findings demonstrate a direct link between activating mutations of the V2R and the perturbation of water balance in NSIAD. In addition, this study provides a useful cell-based assay system to assess the functional consequences of newly discovered activating mutations of the V2R on water permeability in kidney cells and to screen the effect of drugs on the mutated receptors.

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.