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.


Effects of OLE on vasopressin induced AQP2 function in MCD4 cells.
To investigate the possible involvement of OLE on vasopressin-dependent AQP2 function, renal collecting duct MCD4 cells, stably expressing the vasopressin receptor 2 (V2R) and human AQP2, were used as an experimental model 18 . Confocal studies (Fig. 1A) revealed that, compared with dDAVP treated cells, in which AQP2 staining localized to the apical plasma membrane, treatment with OLE prevented the membrane localization of AQP2 induced by stimulation with dDAVP. Consistent with these observations, OLE treatment impaired the dDAVP-induced increase of the temporal osmotic response (indicated as 1/τ in Fig. 1B) (OLE/dDAVP = 88.70 ± 1.86%, n = 292 cells vs dDAVP = 206.8 ± 5.49%, n = 186 cells; p < 0.001). Altogether these findings suggested that treatment with OLE reduced principal cell permeability by preventing AQP2 translocation from an intracellular vesicle pool to the apical plasma membrane. Vasopressin-induced AQP2 trafficking is controlled by intracellular cAMP, which stimulated the cAMP-dependent kinase (PKA) 19 . Further, to evaluate the functionality of the V2R in the presence of OLE in terms of cAMP production, permeable 8-Br-cAMP was used as an external source of cAMP. Treatment with 8-Br-cAMP abolished the inhibitory effect elicited by OLE on osmotic water permeability (OLE/8-Br-cAMP = 188.7 ± 3.56%, n = 293 cells vs OLE = 85.93 ± 2.27%, n = 238 cells; p < 0.001). Therefore, to evaluate whether treatment with OLE regulates AQP2 trafficking by fine-tuning intracellular cAMP level, fluorescence resonance energy transfer (FRET) technology was applied. Compared to untreated cells (CTR), normalized netFRET signals are reduced with dDAVP stimulation (dDAVP = 77.86 ± 3.13%, n = 173 cells vs CTR = 100.00 ± 3.56%, n = 276 cells; p < 0.001; Fig. 2A), consistent with a significant increase of intracellular cAMP. Conversely, treatment with OLE prevented the dDAVP dependent decrease of netFRET signals consistent with a decrease of the vasopressin dependent cAMP release (OLE/dDAVP = 92.97 ± 3.46%, n = 211 cells vs dDAVP = 77.86 ± 3.13%, n = 173 cells; p < 0.05; Fig. 2A). Treatment with OLE alone did not affect the intracellular cAMP level compared with cells left under basal conditions (OLE = 96.15 ± 3.80%, n = 184 cells vs CTR = 100.00 ± 3.56%, n = 276 cells; p < 0.05). Besides, Western Blotting studies (Fig. 2B) revealed that treatment with OLE significantly reduced the dDAVP induced increase of AQP2 phosphorylation at serine 256 (AQP2-pS256), indicating that AQP2 phosphorylation and trafficking, in response to OLE, are dependent on cAMP-PKA function. The phosphorylation level of AQP2-pS256 in response to OLE was not statistically different from the control even though it tended to be reduced. To gain insight into the molecular signals underlying the action of OLE, intracellular calcium dynamics were evaluated. MCD4 cells endogenously express a functional calciumsensing-receptor (CaSR) 20 , which plays an important role in controlling AQP2 expression and trafficking 14 . Long term incubation with OLE (0.1 mg/ml) slightly increased the intracellular calcium level compared with untreated cells (Fig. 3; OLE = 209.0 ± 13.45 nM, n = 128 cells vs CTR = 163.3 ± 8.957 nM, n = 97 cells; p < 0.05). Co-incubation with OLE (0.1 mg/ml) and the selective NPS2143 (1 µM) antagonist of the CaSR abolished the OLE induced intracellular calcium mobilization (OLE/NPS2143 = 168.8 ± 11.43 nM, n = 144 cells vs OLE = 209.0 ± 13.45 nM, n = 128 cells; p < 0.05). To dissect further intracellular calcium signals in response to OLE, functional experiments were also performed under acute stimulation with OLE and NPS2143. Short term treatment with OLE (1 mg/ml) evoked specific calcium oscillations (Fig. 4A)
To evaluate the effect of OLE on AQP2 phosphorylation and trafficking in the kidney, western blotting analysis, and confocal studies were performed ( Fig. 9). In OLE/dDAVP injected rats, phosphorylation of AQP2 at serine 256 was significantly reduced compared with renal tissues of dDAVP treated animals (OLE/dDAVP = 0.77 ± 0.16, n = 5 rats vs dDAVP = 2.16 ± 0.25, n = 5 rats; p < 0.001, Fig. 9A), thereby confirming the in vitro findings (Fig. 2B). Besides, confocal studies ( Fig. 9B) of kidney sections revealed that exposure to OLE prevented the AQP2 membrane localization that instead was observed in dDAVP treated rats. www.nature.com/scientificreports/

Discussion
Calcium is an important intracellular messenger that modulates a plethora of cellular functions 21 . Abnormal calcium signaling may lead to several diseases including heart failure, cancer, and hypertension 22,23 . Specific control of the expression and the activity of proteins controlling multiple intracellular calcium signals has been proven successful to face numerous disorders and is therefore attractive for drug development. This study provides novel insights into the mechanism of action of OLE by showing its ability to modulate intracellular calcium signaling through the stimulation of the CaSR that is involved in fine-tuning the vasopressin induced AQP2 trafficking and expression 12,24 . Under physiological conditions, AQP2 plays an important role in controlling body water homeostasis 25 . Stimulation of the intracellular machinery, leading to localization of AQP2 at the apical plasma membrane, results in abnormal water retention and consequent hyponatremia as in the syndrome of inappropriate antidiuretic hormone (SIADH) secretion, liver cirrhosis, and congestive heart failure [26][27][28] . In a mouse model of SIADH, associated with polycystic kidney disease 1 haploinsufficiency, the basal intracellular calcium level was significantly reduced compared to the level measured in the collecting ducts of wild type mice 29 . Low intracellular calcium downregulated the activity of certain enzymes including the protein phosphatase PP2A resulting in the upregulation of AQP2 phosphorylation at serine 256 30,31 . By contrast, activation of the CaSR with the calcimimetic NPS-R568 increased the intracellular calcium concentration and reduced the cAMP level in PKD1 deficient cells 32 . Importantly, cytosolic calcium can downregulate the calcium-inhibitable adenylated cyclase 33 thereby fine-tuning the intracellular level of cAMP. In MCD4 cells, indeed, stimulation of the CaSR with NPS-R568 decreased AQP2-pS256 through the inhibition of the adenylate cyclase 14 . In this study, treatment with OLE prevented the vasopressin dependent increase of the cAMP and AQP2-pS256, possibly secondary to an increase in the intracellular calcium concentration. Of note, exposure to an external source of cAMP, using permeable 8-Br-cAMP, significantly counteracted the inhibitory effect of OLE on the V2R pathway.  www.nature.com/scientificreports/ An elevated level of cytosolic calcium may lead to cell death and apoptosis. However, a sustained increase in intracellular calcium from 250 nM to greater than 600 nM promoted neuronal survival 34 . In MCD4 cells, treatment with OLE at 0.1 mg/ml does not display a cytotoxic effect 35 . At this concentration, OLE (0.1 mg/ml) slightly increased the intracellular calcium level. Calcium imaging revealed that acute stimulation with OLE caused a transient increase of intracellular calcium through the activation of the CaSR that is abolished when cells were preincubated with the selective CaSR antagonist NPS2143. In renal proximal tubule cells, allosteric activation of the CaSR with I-ornithine reduced the ROS production thereby downregulating the mitochondrial oxidative stress that caused cell apoptosis 36 . Moreover, our previous study showed that the    www.nature.com/scientificreports/ In the kidney, stimulation of the CaSR signaling is associated with a downregulation of AQP2 expression possibly through the miR-137 generation. miRNAs are pivotal posttranscriptional modulators. Several vasopressin-dependent miRNAs targeting AQP2 expression have also been described 43 . Transfection with miR-32 and miR-137 significantly reduced the expression level of AQP2 in mpkCCDc14 cells 43 . Interestingly, OLE can attenuate inflammatory signals and exert protective effects by modulating the expression level of several miRNA 44 . In particular, in glioblastoma multiforme cells, OLE promoted the expression of different miRNA including miR-137 45 that is involved in the downregulation of Akt/mTOR signaling pathway 46 . Of note, treatment with oleuropein prevents Akt/mTOR signaling through the activation of calcium-induced CAMK and AMPK 47 . Activation of CaSR with NPS-R568 activates AMPK and reduces mTOR activity in human proximal tubular cells 32 . Consistent with these findings, incubation with OLE increases cytosolic calcium, downregulates PKA activity, and reduces the cyst size in a 3D-cell culture model 48 . Together these findings suggest that OLE may be useful in treating disorders characterized by dysregulation of intracellular calcium dynamics related to the downregulation of CaSR signaling.
Olive leaf extract consists of several bioactive components, including polyphenols, fibers, and minerals 49 . At the moment, it is not clear if one compound or a synergic combination of phytocompounds in OLE may be beneficial in modulating intracellular responses. The extract used in this study has been applied as a possible food additive 5 and it is therefore important to define the physiological impact of the extract itself considering that several phenols, including hydroxytyrosol, may be filtered by the kidney and recovered in the urine 50 . Further studies will provide the efficacy of specific components that may actively regulate intracellular calcium signaling through the CaSR. To conclude, this study provides evidence that OLE may be considered a novel potential adjuvant useful to mitigate disorders characterized by a reduction of CaSR expression as well as signaling and affecting renal water permeability. Phenolics-rich extract production and chemical characterization. The production of a phenolicsrich extract from olive leaves was carried out as reported in Ranieri et al. 35 . After milling with a blender (Waring-Commercial, Torrington, CT, USA), ddH2O water was added (ratio 1/20, w/v) and then subjected to ultrasound (CEIA, Viciomaggio, Italy) three times, for 30 min at 35 ± 5 °C each time. Finally, the extracts were filtered through filter paper, lyophilized, and stored at -20 °C. The obtained extracts were filtered with nylon filters of 0.45 µm (Merck KGaA, Darmstadt, Germany) and used for chemical characterization. The total phenol content, the antioxidant activity, and the single phenolic compounds identification were performed according to Difonzo et al. 5 . The OLE showed a content of polyphenols, determined by Folin-Ciocalteu, equal to 195 mg·g -1 gallic acid equivalents (GAE) and an antioxidant activity, determined by ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt), accounting for 750 µmol TE (Trolox equivalents) g -1 .

Materials and methods
Cell culture and treatments. Mouse cortical collecting duct MCD4 cells, stably expressing human AQP2 and the chimeric V2R-Rluc were used as an experimental model 18 . Cells were grown in a 1:1 mixture of Dulbecco's modified Eagle's medium and F-12 supplemented with 5% (v/v) fetal bovine serum, 1% (v/v) L-glutamine and 1% penicillin/streptomycin, 5 µM dexamethasone, 400 µg/ml G418 (for AQP2 resistance) and 1 µg/ml puromycin (for V2R-Rluc resistance), in a humidified atmosphere of 5% CO 2 at 37 °C. MCD4 cells were left under basal condition (CTR) or long-term treated with OLE at 0.1 mg/ml O/N, dDAVP at 100 nM for 30 min or co-treated with OLE and dDAVP or NPS2143 at 1 µM O/N. Alternatively, cells were exposed to 8-Br-cAMP at 500 µM for 45 min. Short term experiments were carried out with OLE at 1 mg/ml for 5 min and NPS2143 at 10 µM for 15 min. Experiments were performed 3-5 independent times using cells from different passages. Five rats were treated with subcutaneous injection of 1 ng dDAVP (Sigma-Aldrich, Glostrup, Denmark) in 200 µl saline/animal, and five vehicle-treated rats served as controls. After 30 min, the rats were killed, and the kidneys were processed as described below. www.nature.com/scientificreports/ presence of proteases (1 mM PMSF, 2 mg/ml leupeptin, and 2 mg/ml pepstatin A) and phosphatases (10 mM NaF and 1 mM sodium orthovanadate) inhibitors. Alternatively, kidney sections of approximately 0.5 mm were prepared and equilibrated for 10 min in a buffer containing 118 mM NaCl, 16 mM Hepes, 17 mM Na-Hepes, 14 mM glucose, 3.2 mM KCl, 2.5 mM CaCl2, 1.8 mM MgSO4, and 1.8 mM KH2PO4 (pH 7.4). Renal papilla were minced with scissors in the same buffer in the presence of proteases (1 mM PMSF, 2 mg/ml leupeptin, and 2 mg/ml pepstatin A) and phosphatases (10 mM NaF and 1 mM sodium orthovanadate) inhibitors. After sonication (60 kHz for 5 s), lysates were centrifuged at 12,000 × g for 10 min. The supernatants were collected and used for immunoblotting studies 18 .

Confocal microscopy.
Confocal studies were performed as previously described 31 . Briefly, cells were grown on cell culture PET inserts and treated as described above. Alternatively, kidney sections obtained from control, OLE, dDAVP, and OLE with dDAVP treated rats were subjected to immunofluorescence experiments as previously described 31 . Images were obtained with a confocal laser-scanning fluorescence microscope Leica TCS SP2 (Leica Microsystems, Heerbrugg, Switzerland).
Water permeability assay. Osmotic water permeability was measured as previously described 14,53  . Measurements were performed using an inverted TE2000-S microscope (Nikon Eclipse microscope, Tokyo, Japan). The ratio of fluorescence intensities at 340 and 380 nm was plotted and calculated as the change in fluorescence. In particular, stimulation with OLE (1 mg/ml) or NPS2143 (10 µM) was compared in the same cell type to those obtained after stimulation with a maximal dose of the calcium-mediated agonist ATP (100 µM) that was used as an internal control (100%). Intracellular calcium level was measured at steady-state and calibrated as described by Grynkiewicz 57 . OLE and NPS2143 was used at long term at 0.1 mg/ml and 1 µM, respectively and each sample was calibrated by the addition of 5 µM ionomycin in presence of 1 mM EGTA (R min ) followed by 5 µM ionomycin in 5 mM CaCl 2 (R max ).

Real-time PCR analysis of AQP2 and CaSR mRNA in control and treated rats. Real-time PCR
experiments were performed to measure the relative expression of mRNA in inner medulla collecting duct (IMCD) isolated from control and treated rat kidney papillae. Kidney slices of about 0.5 mm were made and equilibrated for 10 min in a buffer containing 118 mM NaCl, 16  miRNA-137 evaluation in control and Treated rats. miRNA-137 content in control and treated rat inner medulla collecting duct was evaluated using TaqMan Advanced miRNA Assays (has-miR-137; Assay ID: 477904_mir; Thermo Fisher Scientific, Waltham, MA, U.S.A.), which enabled highly sensitive and specific quantification of mature miRNA using quantitative PCR. Kidney slices of about 0.5 mm were made and equilibrated for 10 min in a buffer containing 118 mM NaCl, 16  The authors confirm that all methods were carried out in accordance with relevant guidelines and regulations. Moreover, the authors confirm that the study was carried out in compliance with the ARRIVE guidelines.