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 β 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

History of Diabetes Insipidus

Under physiological conditions, fluid and electrolyte homoeostasis is maintained by the kidney adjusting urine volume and composition according to body needs. Diabetes Insipidus is a complex and heterogeneous clinical syndrome affecting water balance and characterized by constant diuresis, resulting in large volumes of dilute urine. With respect to the similarly named Diabetes Mellitus, a disease already known in ancient Egypt, Greece and Asia, Diabetes Insipidus has been described several thousand years later. In 1670s Thomas Willis, noted the difference in taste of urine from polyuric subjects compared with healthy individuals and started the differentiation of Diabetes Mellitus from the more rare entity of Diabetes Insipidus. In 1794, Johann Peter Frank described polyuric patients excreting nonsaccharine urine and introduced the term of Diabetes Insipidus. An hystorical milestone was the in 1913, when Farini successfully used posterior pituitary extracts to treat Diabetes Insipidus. Until 1920s the available evidence indicated Diabetes Insipidus as a disorder of the pituitary gland. In the early 1928, De Lange first observed that some patients with Diabetes Insipidus did not respond to posterior pituitary extracts and subsequently Forssman and Waring in 1945 established that the kidney had a critical role for these forms of Diabetes Insipidus resistant to this treatment. In 1947 Williams and Henry introduced the term Nephrogenic Diabetes Insipidus for the congenital syndrome characterized by polyuria and renal concentrating defect resistant to vasopressin. In 1955, du Vigneaud received the 1955 Nobel Prize in chemistry for the first synthesis of the hormone vasopressin representing a milestone for the treatment of Central Diabetes Insipidus

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

EGF stimulates IClswell by a redistribution of proteins involved in cell volume regulation.

Background: ICln is a multifunctional protein involved in the generation of chloride currents activated during regulatory volume decrease (RVD) after cell swelling (IClswell). Growth factor receptors play a key role in different cellular processes and epidermal growth factor (EGF) regulates swelling-activated chloride permeability. Aim: We set out to investigate if the EGF-induced upregulation of IClswell could be explained by a rearrangement of ICln subcellular distribution and interaction with its molecular partners. Methods: NIH-3T3 fibroblasts were serum-deprived for 24 hours and stimulated with EGF (40 ng/ml) for 30 minutes. IClswell activation, ICln distribution and interaction with its molecular partner HSPC038 were assessed by whole cell patch clamp and fluorescence resonance energy transfer (FRET). Results: EGF treatment significantly enhanced the direct molecular interaction between ICln and HSPC038 and also resulted in an increase of ICln and HSPC038 association with the plasma membrane. Importantly, these events are associated with a significant increase of IClswell. Conclusions: The present data indicate that EGF might exert its role in the modulation of volume-sensitive chloride currents in part through activation and translocation of ICln and HSPC038 to the plasma membrane

Integrin signaling modulates AQP2 trafficking via Arg-Gly-Asp (RGD) motif.

Aquaporin-2 (AQP2) increases the water permeability of renal collecting ducts in response to vasopressin. Vasopressin stimulation is accompanied by a profound remodeling of actin cytoskeleton whose dynamics are regulated by crosstalk between intracellular and extracellular signals. Here, we report that AQP2 contains a conserved RGD domain in its external C-loop. Co-immunoprecipitation experiments demonstrated that AQP2 binds integrin β1 in renal tissue and in MCD4 cells. To investigate the role of this interaction on AQP2 trafficking, cells were exposed to synthetic RGD-containing peptides, GRGDNP or GRGDSP, able to bind certain integrins. Incubation with these peptides increased the membrane expression of AQP2 in the absence of hormonal stimulation as assessed by confocal analysis and cell surface biotinylation. To identify the signals underlying the effects of peptides on AQP2 trafficking, some possible intracellular messengers were evaluated. Exposure of MCD4 cells to GRGDNP increased intracellular cAMP as assessed by FRET studies while GRGDSP increased intracellular calcium concentration. Taken together, these data propose integrins as new players controlling the cellular localization of AQP2, via two distinct signal transduction pathways dependent on cAMP and calcium respectively

Rho inhibits cAMP-induced translocation of aquaporin-2 into the apical membrane of renal cells

We have recently demonstrated that actin depolymerization is a prerequisite for cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) into the apical membrane in AQP2-transfected renal CD8 cells (29). The Rho family of small GTPases, including Cdc42, Rac, and Rho, regulates the actin cytoskeleton. In AQP2-transfected CD8 cells, inhibition of Rho GTPases with Clostridium difficile toxin B or with C. limosum C3 fusion toxin, as well as incubation with the Rho kinase inhibitor, Y-27632, caused actin depolymerization and translocation of AQP2 in the absence of the cAMP- elevating agent forskolin. Both forskolin and C3 fusion toxin-induced AQP2 translocation were associated with a similar increase in the osmotic water permeability coefficient. Expression of constitutively active RhoA induced formation of stress fibers and abolished AQP2 translocation in response to forskolin. Cytochalasin D induced both depolymerization of F-actin and AQP2 translocation, suggesting that depolymerization of F-actin is sufficient to induce AQP2 translocation. Together, these data indicate that Rho inhibits cAMP-dependent translocation of AQP2 into the apical membrane of renal principal cells by controlling the organization of the actin cytoskeleton

Mitochondrial cAMP prevents apoptosis modulating Sirt3 protein level and OPA1 processing in cardiac myoblast cells

Mitochondria, responding to a wide variety of signals, including oxidative stress, are critical in regulating apoptosis that plays a key role in the pathogenesis of a variety of cardiovascular diseases. A number of mitochondrial proteins and pathways have been found to be involved in the mitochondrial dependent apoptosis mechanism, such as optic atrophy 1 (OPA1), sirtuin 3 (Sirt3), deacetylase enzyme and cAMP signal. In the present work we report a network among OPA1, Sirt3 and cAMP in ROS-dependent apoptosis. Rat myoblastic H9c2 cell lines, were treated with tert-butyl hydroperoxide (t-BHP) to induce oxidative stress-dependent apoptosis. FRET analysis revealed a selective decrease of mitochondrial cAMP in response to t-BHP treatment. This was associated with a decrease of Sirt3 protein level and proteolytic processing of OPA1. Pretreatment of cells with permeant analogous of cAMP (8-Br-cAMP) protected the cell from apoptosis preventing all these events. Using H89, inhibitor of the protein kinase A (PKA), and protease inhibitors, evidences have been obtained that ROS-dependent apoptosis is associated with an alteration of mitochondrial cAMP/PKA signal that causes degradation/proteolysis of Sirt3 that, in turn, promotes acetylation and proteolytic processing of OPA1

Desmopressin Stimulates Nitric Oxide Production in Human Lung Microvascular Endothelial Cells

Desmopressin (dDAVP) is the best characterized analogue of vasopressin, the endocrine regulator of water balance endowed with potent vasoconstrictive effects. Despite the use of dDAVP in clinical practice, ranging from the treatment of nephrogenic diabetes insipidus to bleeding disorders, much remains to be understood about the impact of the drug on endothelial phenotype. The aim of this study was, thus, to evaluate the effects of desmopressin on the viability and function of human pulmonary microvascular endothelial cells (HLMVECs). The results obtained demonstrate that the vasopressor had no cytotoxic effect on the endothelium; similarly, no sign of endothelial activation was induced by dDAVP, indicated by the lack of effect on the expression of inflammatory cytokines and adhesion molecules. Conversely, the drug significantly stimulated the production of nitric oxide (NO) and the expression of the inducible isoform of nitric oxide synthase, NOS2/iNOS. Since the intracellular level of cAMP also increased, we can hypothesize that NO release is consequent to the activation of the vasopressin receptor 2 (V2R)/guanylate cyclase (Gs)/cAMP axis. Given the multifaceted role of NOS2-deriving NO for many physio-pathological conditions, the meanings of these findings in HLMVECs appears intriguing and deserves to be further address