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.

ß3-Adrenoceptor as a new player in the sympathetic regulation of the renal acid-base homeostasis.

Efferent sympathetic nerve fibers regulate several renal functions activating norepinephrine receptors on tubular epithelial cells. Of the beta-adrenoceptors (ß-ARs), we previously demonstrated the renal expression of ß3-AR in the thick ascending limb (TAL), the distal convoluted tubule (DCT), and the collecting duct (CD), where it participates in salt and water reabsorption. Here, for the first time, we reported ß3-AR expression in the CD intercalated cells (ICCs), where it regulates acid-base homeostasis. Co-localization of ß3-AR with either proton pump H+-ATPase or Cl-/HCO3 - exchanger pendrin revealed ß3-AR expression in type A, type B, non-A, and non-B ICCs in the mouse kidney. We aimed to unveil the possible regulatory role of ß3-AR in renal acid-base homeostasis, in particular in modulating the expression, subcellular localization, and activity of the renal H+-ATPase, a key player in this process. The abundance of H+-ATPase was significantly decreased in the kidneys of ß3-AR-/- compared with those of ß3-AR+/+ mice. In particular, H+-ATPase reduction was observed not only in the CD but also in the TAL and DCT, which contribute to acid-base transport in the kidney. Interestingly, we found that in in vivo, the absence of ß3-AR reduced the kidneys' ability to excrete excess proton in the urine during an acid challenge. Using ex vivo stimulation of mouse kidney slices, we proved that the ß3-AR activation promoted H+-ATPase apical expression in the epithelial cells of ß3-AR-expressing nephron segments, and this was prevented by ß3-AR antagonism or PKA inhibition. Moreover, we assessed the effect of ß3-AR stimulation on H+-ATPase activity by measuring the intracellular pH recovery after an acid load in ß3-AR-expressing mouse renal cells. Importantly, ß3-AR agonism induced a 2.5-fold increase in H+-ATPase activity, and this effect was effectively prevented by ß3-AR antagonism or by inhibiting either H+-ATPase or PKA. Of note, in urine samples from patients treated with a ß3-AR agonist, we found that ß3-AR stimulation increased the urinary excretion of H+-ATPase, likely indicating its apical accumulation in tubular cells. These findings demonstrate that ß3-AR activity positively regulates the expression, plasma membrane localization, and activity of H+-ATPase, elucidating a novel physiological role of ß3-AR in the sympathetic control of renal acid-base homeostasis.

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.

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.

Role of Nuclear Lamin A/C in the Regulation of Nav1.5 Channel and Microtubules: Lesson From the Pathogenic Lamin A/C Variant Q517X.

In this work, we studied an lmna nonsense mutation encoding for the C-terminally truncated Lamin A/C (LMNA) variant Q517X, which was described in patients affected by a severe arrhythmogenic cardiomyopathy with history of sudden death. We found that LMNA Q517X stably expressed in HL-1 cardiomyocytes abnormally aggregates at the nuclear envelope and within the nucleoplasm. Whole-cell patch clamp experiments showed that LMNA Q517X-expressing cardiomyocytes generated action potentials with reduced amplitude, overshoot, upstroke velocity and diastolic potential compared with LMNA WT-expressing cardiomyocytes. Moreover, the unique features of these cardiomyocytes were 1) hyper-polymerized tubulin network, 2) upregulated acetylated α-tubulin, and 3) cell surface Nav1.5 downregulation. These findings pointed the light on the role of tubulin and Nav1.5 channel in the abnormal electrical properties of LMNA Q517X-expressing cardiomyocytes. When expressed in HEK293 with Nav1.5 and its ß1 subunit, LMNA Q517X reduced the peak Na+ current (INa) up to 63% with a shift toward positive potentials in the activation curve of the channel. Of note, both AP properties in cardiomyocytes and Nav1.5 kinetics in HEK293 cells were rescued in LMNA Q517X-expressing cells upon treatment with colchicine, an FDA-approved inhibitor of tubulin assembly. In conclusion, LMNA Q517X expression is associated with hyper-polymerization and hyper-acetylation of tubulin network with concomitant downregulation of Nav1.5 cell expression and activity, thus revealing 1) new mechanisms by which LMNA may regulate channels at the cell surface in cardiomyocytes and 2) new pathomechanisms and therapeutic targets in cardiac laminopathies.

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.

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.

Activation of the Thiazide-Sensitive Sodium-Chloride Cotransporter by Beta3-Adrenoreceptor in the Distal Convoluted Tubule.

We previously showed that the beta-3 adrenergic receptor (BAR3) is expressed in most segments of the nephron where its agonism promotes a potent antidiuretic effect. We localized BAR3 in distal convoluted tubule (DCT) cells expressing the thiazide-sensitive sodium-chloride cotransporter (NCC). Aim of this study is to investigate the possible functional role of BAR3 on NCC modulation in DCT cells. Here, we found that, in mice, the knockout of BAR3 was paralleled by a significant attenuation of NCC phosphorylation, paralleled by reduced expression and activation of STE-20/SPS1-related proline-alanine-rich kinase (SPAK) and WNKs the main kinases involved in NCC activation. Conversely, in BAR1/2 knockout mice, we found reduced NCC abundance with no changes in the phosphorylation state of NCC. Moreover, selective BAR3 agonism promotes both SPAK and NCC activation in wild-type mouse kidney slices. In conclusion, our findings suggest a novel role for BAR3 in the regulation of NCC in DCT.

Publisher Correction: Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka.

The physiological role of the renal ClC-Ka/ClC-K1 channels is to confer a high Cl- permeability to the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla that drives water reabsorption from collecting ducts. Here, we investigated by whole-cell patch-clamp measurements on HEK293 cells co-expressing ClC-Ka (tagged with GFP) and the accessory subunit barttin (tagged with m-Cherry) the effect of a natural diuretic extract from roots of Dandelion (DRE), and other compounds activating PKC, such as ATP, on ClC-Ka activity and its membrane localization. Treatment with 400 µg/ml DRE significantly inhibited Cl- currents time-dependently within several minutes. Of note, the same effect on Cl- currents was obtained upon treatment with 100 µM ATP. Pretreatment of cells with either the intracellular Ca2+ chelator BAPTA-AM (30 µM) or the PKC inhibitor Calphostin C (100 nM) reduced the inhibitory effect of DRE. Conversely, 1 µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of DRE on ClC-Ka. Finally, we found that pretreatment with 30 µM Heclin, an E3 ubiquitin ligase inhibitor, did not revert DRE-induced Cl- current inhibition. In agreement with this, live-cell confocal analysis showed that DRE treatment did not induce ClC-Ka internalization. In conclusion, we demonstrate for the first time that the activity of ClC-Ka in renal cells could be significantly inhibited by the activation of PKC elicited by classical maneuvers, such as activation of purinergic receptors, or by exposure to herbal extracts that activates a PKC-dependent pathway. Overall, we provide both new information regarding the regulation of ClC-Ka and a proof-of-concept study for the use of DRE as new diuretic.

Data on the influence of inorganic clays to improve mechanical and healing properties of antibacterial Gellan gum-Manuka honey hydrogels.

This work contains original data supporting our research paper "Advances in cartilage repair: the influence of inorganic clays to improve mechanical and healing properties of antibacterial Gellan gum-Manuka honey hydrogels", by Maria A. Bonifacio, Andrea Cochis, Stefania Cometa, Annachiara Scalzone, Piergiorgio Gentile, Giuseppe Procino, Serena Milano, Alessandro C. Scalia, Lia Rimondini, Elvira De Giglio [1]. The main paper describes how four different clays (i.e., mesoporous silica, bentonite and halloysite nanotubes, coded as MS, BE and HNT) as cheap, abundant and versatile feed materials can be used for the preparation of highly performant hydrogels as cartilage substitutes, based on Gellan Gum (GG) and Manuka Honey (MH). Here the composites were further examined by means of Thermogravimetric Analysis (TGA), histological analysis (Alcian blue and Safranin-O) and static compression tests. This set of data strengthens the evidence that these hydrogels possess biological and physicochemical characteristics suitable for their application as reinforcing inorganic fillers in composite materials designed for cartilage regeneration.

Advances in cartilage repair: The influence of inorganic clays to improve mechanical and healing properties of antibacterial Gellan gum-Manuka honey hydrogels.

Effective treatment of cartilage defects represents a challenging problem, mainly due to the tissue's limited intrinsic self-repair capacity; the use of polymeric scaffolds as tissue substitute is rapidly increasing, but it is still limited by poor mechanical properties. Moreover, the onset of an infection can irreversibly affect the healing process. Accordingly, in this work we describe, for the first time, the preparation of composite scaffolds based on gellan gum, antibacterial Manuka honey and an inorganic clay (mesoporous silica, sodium­calcium bentonite or halloysite nanotubes). The surface composition, morphology, mechanical and biological features of such composites are herein assessed, aiming to optimize the composition of a superior scaffold for cartilage repair. Results demonstrated that after 45 days of in vitro incubation with human mesenchymal stem cells, the mesoporous silica-composite hydrogels exhibited significant changes in peak elastic and dynamic moduli over time thus demonstrating superior mechanical properties. Moreover, mesoporous silica provided the best performances in terms of in vitro cytocompatibility and antibacterial preventive activity in protection of cells in a co-culture model. Therefore, this selected composition was exploited for subcutaneous implantation in mice to investigate materials biocompatibility and infection prevention. Results demonstrated that composites did not cause severe immune response as well as they were able to restrain the infection. Accordingly, GG-MH-MS composites represent a very promising tool for cartilage tissue engineering.

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.

AQP1-Containing Exosomes in Peritoneal Dialysis Effluent As Biomarker of Dialysis Efficiency.

The water channel Aquaporin 1 (AQP1) plays a fundamental role in water ultrafiltration during peritoneal dialysis (PD) and its reduced expression or function may be responsible for ultrafiltration failure (UFF). In humans, AQP1 is expressed in the endothelium of the peritoneal capillaries but its expression in mesothelial cells (MC) and its functional role in PD is still being debated. Here, we studied a cohort of 30 patients using PD in order to determine the presence of AQP1 in peritoneal biopsies, AQP1 release in the PD effluent through exosomes and the correlation of AQP1 abundance with the efficiency of peritoneal ultrafiltration. The experiments using immunofluorescence showed a strong expression of AQP1 in MCs. Immunoblotting analysis on vesicles isolated from PD effluents showed a consistent presence of AQP1, mesothelin and Alix and the absence of the CD31. Thus, this suggests that they have an exclusive mesothelial origin. The immunoTEM analysis showed a homogeneous population of nanovesicles and confirmed the immunoblotting results. Interestingly, the quantitative analysis by ELISA showed a positive correlation between AQP1 in the PD effluent and ultrafiltration (UF), free water transport (FWT) and Na-sieving. This evidence opens the discussion on the functional role of mesothelial AQP1 during PD and suggests that it may represent a potential non-invasive biomarker of peritoneal barrier integrity, with predictive potential of UFF in PD patients.

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.

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.

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.

Spilanthol from Acmella Oleracea Lowers the Intracellular Levels of cAMP Impairing NKCC2 Phosphorylation and Water Channel AQP2 Membrane Expression in Mouse Kidney.

Acmella oleracea is well recognized in Brazilian traditional medicine as diuretic, although few scientific data have been published to support this effect. Aim of this study was to determine the molecular effect of Acmella oleracea extract and its main alkylamide spilanthol on two major processes involved in the urine concentrating mechanism: Na-K-2Cl symporter (NKCC2) activity in the thick ascending limb and water channel aquaporin 2 accumulation at the apical plasma membrane of collecting duct cells. Phosphorylation of NKCC2 was evaluated as index of its activation by Western blotting. Rate of aquaporin 2 apical expression was analyzed by confocal laser microscopy. Spilanthol-induced intracellular signalling events were dissected by video-imaging experiments. Exposure to spilanthol reduced the basal phosphorylation level of NKCC2 both in freshly isolated mouse kidney slices and in NKCC2-expresing HEK293 cells. In addition, exposure to spilanthol strongly reduced both desmopressin and low Cl--dependent increase in NKCC2 phosphorylation in mouse kidney slices and NKCC2-expressing HEK293 cells, respectively. Similarly, spilanthol reduced both desmopressin- and forskolin-stimulated aquaporin 2 accumulation at the apical plasma membrane of collecting duct in mouse kidney slice and MCD4 cells, respectively. Of note, when orally administered, spilanthol induced a significant increase in both urine output and salt urinary excretion associated with a markedly reduced urine osmolality compared with control mice. Finally, at cellular level, spilanthol rapidly reduced or reversed basal and agonist-increased cAMP levels through a mechanism involving increases in intracellular [Ca2+]. In conclusion, spilanthol-induced inhibition of cAMP production negatively modulates urine-concentrating mechanisms thus holding great promise for its use as diuretic.

ß3 adrenergic receptor in the kidney may be a new player in sympathetic regulation of renal function.

To date, the study of the sympathetic regulation of renal function has been restricted to the important contribution of ß1- and ß2-adrenergic receptors (ARs). Here we investigate the expression and the possible physiologic role of ß3-adrenergic receptor (ß3-AR) in mouse kidney. The ß3-AR is expressed in most of the nephron segments that also express the type 2 vasopressin receptor (AVPR2), including the thick ascending limb and the cortical and outer medullary collecting duct. Ex vivo experiments in mouse kidney tubules showed that ß3-AR stimulation with the selective agonist BRL37344 increased intracellular cAMP levels and promoted 2 key processes in the urine concentrating mechanism. These are accumulation of the water channel aquaporin 2 at the apical plasma membrane in the collecting duct and activation of the Na-K-2Cl symporter in the thick ascending limb. Both effects were prevented by the ß3-AR antagonist L748,337 or by the protein kinase A inhibitor H89. Interestingly, genetic inactivation of ß3-AR in mice was associated with significantly increased urine excretion of water, sodium, potassium, and chloride. Stimulation of ß3-AR significantly reduced urine excretion of water and the same electrolytes. Moreover, BRL37344 promoted a potent antidiuretic effect in AVPR2-null mice. Thus, our findings are of potential physiologic importance as they uncover the antidiuretic effect of ß3-AR stimulation in the kidney. Hence, ß3-AR agonism might be useful to bypass AVPR2-inactivating mutations.