Publications by authors named "Giuseppe Procino"

56 Publications

A Novel Formulation of Glucose-Sparing Peritoneal Dialysis Solutions with l-Carnitine Improves Biocompatibility on Human Mesothelial Cells.

Int J Mol Sci 2020 Dec 24;22(1). Epub 2020 Dec 24.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, 70125 Bari, Italy.

The main reason why peritoneal dialysis (PD) still has limited use in the management of patients with end-stage renal disease (ESRD) lies in the fact that the currently used glucose-based PD solutions are not completely biocompatible and determine, over time, the degeneration of the peritoneal membrane (PM) and consequent loss of ultrafiltration (UF). Here we evaluated the biocompatibility of a novel formulation of dialytic solutions, in which a substantial amount of glucose is replaced by two osmometabolic agents, xylitol and l-carnitine. The effect of this novel formulation on cell viability, the integrity of the mesothelial barrier and secretion of pro-inflammatory cytokines was evaluated on human mesothelial cells grown on cell culture inserts and exposed to the PD solution only at the apical side, mimicking the condition of a PD dwell. The results were compared to those obtained after exposure to a panel of dialytic solutions commonly used in clinical practice. We report here compelling evidence that this novel formulation shows better performance in terms of higher cell viability, better preservation of the integrity of the mesothelial layer and reduced release of pro-inflammatory cytokines. This new formulation could represent a step forward towards obtaining PD solutions with high biocompatibility.
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http://dx.doi.org/10.3390/ijms22010123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7795315PMC
December 2020

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

Sci Rep 2020 Sep 30;10(1):16469. Epub 2020 Sep 30.

Department of Sciences, University of Basilicata, Potenza, IT, Italy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-74040-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7525443PMC
September 2020

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

Sci Rep 2020 06 24;10(1):10268. Epub 2020 Jun 24.

Department of Sciences, University of Basilicata, Potenza, IT, Italy.

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 Ca 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.
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http://dx.doi.org/10.1038/s41598-020-67219-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314819PMC
June 2020

Functional Study of Novel Bartter's Syndrome Mutations in ClC-Kb and Rescue by the Accessory Subunit Barttin Toward Personalized Medicine.

Front Pharmacol 2020 17;11:327. Epub 2020 Mar 17.

Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", Bari, Italy.

Type III and IV Bartter syndromes (BS) are rare kidney tubulopathies caused by loss-of-function mutations in the and genes coding respectively for the ClC-Kb chloride channels and accessory subunit barttin. ClC-K channels are expressed in the Henle's loop, distal convoluted tubule, and cortical collecting ducts of the kidney and contribute to chloride absorption and urine concentration. In our Italian cohort, we identified two new mutations in , G167V and G289R, in children affected by BS and previously reported genetic variants, A242E, a chimeric gene and the deletion of the whole . All the patients had hypokalemia and metabolic alkalosis, increased serum renin and aldosterone levels and were treated with a symptomatic therapy. In order to define the molecular mechanisms responsible for BS, we co-expressed ClC-Kb wild type and channels with point mutations with barttin in HEK 293 cells and characterized chloride currents through the patch-clamp technique. In addition, we attempted to revert the functional defect caused by BS mutations through barttin overexpression. G167V and A242E channels showed a drastic current reduction compared to wild type, likely suggesting compromised expression of mutant channels at the plasma membrane. Conversely, G289R channel was similar to wild type raising the doubt that an additional mutation in another gene or other mechanisms could account for the clinical phenotype. Interestingly, increasing ClC-K/barttin ratio augmented G167V and A242E mutants' chloride current amplitudes towards wild type levels. These results confirm a genotype-phenotype correlation in BS and represent a preliminary proof of concept that molecules functioning as molecular chaperones can restore channel function in expression-defective ClC-Kb mutants.
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http://dx.doi.org/10.3389/fphar.2020.00327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7092721PMC
March 2020

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

Data Brief 2020 Feb 7;28:105096. Epub 2020 Jan 7.

Department of Chemistry, University of Bari "Aldo Moro", via E. Orabona 4, Bari, 70126, Italy.

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.
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http://dx.doi.org/10.1016/j.dib.2019.105096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961066PMC
February 2020

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

Mater Sci Eng C Mater Biol Appl 2020 Mar 18;108:110444. Epub 2019 Nov 18.

Department of Chemistry, University of Bari "Aldo Moro", via E. Orabona 4, Bari 70126, Italy. Electronic address:

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, sodium‑calcium 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.
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http://dx.doi.org/10.1016/j.msec.2019.110444DOI Listing
March 2020

Functional study of a KCNH2 mutant: Novel insights on the pathogenesis of the LQT2 syndrome.

J Cell Mol Med 2019 09 30;23(9):6331-6342. Epub 2019 Jul 30.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy.

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.
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http://dx.doi.org/10.1111/jcmm.14521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6714209PMC
September 2019

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

Cells 2019 04 9;8(4). Epub 2019 Apr 9.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, 70125 Bari, Italy.

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.
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http://dx.doi.org/10.3390/cells8040330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6523141PMC
April 2019

Inhibiting the urokinase-type plasminogen activator receptor system recovers STZ-induced diabetic nephropathy.

J Cell Mol Med 2019 02 13;23(2):1034-1049. Epub 2018 Nov 13.

Department of Biology, University of Pisa, Pisa, Italy.

The urokinase-type plasminogen activator (uPA) receptor (uPAR) participates to the mechanisms causing renal damage in response to hyperglycaemia. The main function of uPAR in podocytes (as well as soluble uPAR -(s)uPAR- from circulation) is to regulate podocyte function through αvβ3 integrin/Rac-1. We addressed the question of whether blocking the uPAR pathway with the small peptide UPARANT, which inhibits uPAR binding to the formyl peptide receptors (FPRs) can improve kidney lesions in a rat model of streptozotocin (STZ)-induced diabetes. The concentration of systemically administered UPARANT was measured in the plasma, in kidney and liver extracts and UPARANT effects on dysregulated uPAR pathway, αvβ3 integrin/Rac-1 activity, renal fibrosis and kidney morphology were determined. UPARANT was found to revert STZ-induced up-regulation of uPA levels and activity, while uPAR on podocytes and (s)uPAR were unaffected. In glomeruli, UPARANT inhibited FPR2 expression suggesting that the drug may act downstream uPAR, and recovered the increased activity of the αvβ3 integrin/Rac-1 pathway indicating a major role of uPAR in regulating podocyte function. At the functional level, UPARANT was shown to ameliorate: (a) the standard renal parameters, (b) the vascular permeability, (c) the renal inflammation, (d) the renal fibrosis including dysregulated plasminogen-plasmin system, extracellular matrix accumulation and glomerular fibrotic areas and (e) morphological alterations of the glomerulus including diseased filtration barrier. These results provide the first demonstration that blocking the uPAR pathway can improve diabetic kidney lesion in the STZ model, thus suggesting the uPA/uPAR system as a promising target for the development of novel uPAR-targeting approaches.
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http://dx.doi.org/10.1111/jcmm.14004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349167PMC
February 2019

Role of Lamin A/C Gene Mutations in the Signaling Defects Leading to Cardiomyopathies.

Front Physiol 2018 25;9:1356. Epub 2018 Sep 25.

Department of Sciences, University of Basilicata, Potenza, Italy.

Nuclear lamin A/C are crucial components of the intricate protein mesh that underlies the inner nuclear membrane and confers mainly nuclear and cytosolic rigidity. However, throughout the years a number of other key physiological processes have been associated with lamins such as modulation of both genes expression and the activity of signaling mediators. To further solidify its importance in cell physiology, mutations in the lamin A/C gene () have been associated to diverse pathological phenotypes with skeletal muscles and the heart being the most affected systems. When affected, the heart develops a wide array of phenotypes spanning from dilated cardiomyopathy with conduction defects to arrhythmogenic right ventricular cardiomyopathy. The surprising large number of cardiac phenotypes reflects the equally large number of specific mutations identified in the gene. In this review, we underlie how mutations in can impact the activity and the spatial/temporal organization of signaling mediators and transcription factors. We analyzed the ever-increasing amount of findings collected in Lmna mice whose cardiomyopathy resemble the most important features of the disease in humans and a number of key evidences from other experimental models. With this mini review, we attempt to combine the newest insights regarding both the pathogenic effects of mutations in terms of signaling abnormalities and cardiac laminopathies.
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http://dx.doi.org/10.3389/fphys.2018.01356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167438PMC
September 2018

Data on Manuka Honey/Gellan Gum composite hydrogels for cartilage repair.

Data Brief 2018 Oct 30;20:831-839. Epub 2018 Aug 30.

Department of Chemistry, University of Bari "Aldo Moro", via Orabona 4, 70126 Bari, Italy.

This work contains original data supporting our research paper "Antibacterial effectiveness meets improved mechanical properties: Manuka Honey/Gellan Gum composite hydrogels for cartilage repair", Bonifacio et al., in press [1], in which innovative composite hydrogels, based on Gellan Gum/Manuka honey/Halloysite nanotubes were described as biomaterials for cartilage regeneration. Here the composites were further examined by means of Fourier Transform Infrared Spectroscopy, in Attenuated Total Reflectance mode (FT-IR/ATR). Materials devoted to cartilage replacement must possess adequate fluid permeability and lubricating capability, therefore, a deeper investigation on water uptake kinetics of freeze-dried specimens up to 21 days in PBS was carried out. Moreover, since the degradation rate of a biomaterial plays a pivotal role in tissue engineering, weight loss measurements of the prepared hydrogels were performed in simulated synovial fluid, in phosphate buffer solution (PBS) and in lysozyme. Scanning Electron Microscopy images provide insight into the morphology of the freeze-dried samples. Finally, additional information on and ability to adhere onto the prepared hydrogel composites in short times were obtained, as well as the chondrogenic potential of the composites assessed by SDS-PAGE followed by Coomassie blue gel staining.
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http://dx.doi.org/10.1016/j.dib.2018.08.155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6134162PMC
October 2018

Antibacterial effectiveness meets improved mechanical properties: Manuka honey/gellan gum composite hydrogels for cartilage repair.

Carbohydr Polym 2018 Oct 28;198:462-472. Epub 2018 Jun 28.

Department of Chemistry, University of Bari "Aldo Moro", Italy. Electronic address:

Biomaterials for cartilage repair are still far from clinical requirements, even if several studies recently focused on this topic. In this respect, Nature-derived hydrogels are a promising class of scaffolds for cartilage tissue engineering, mimicking the native cellular microenvironment. However, they frequently lack mechanical features required for cartilage applications and are commonly subjected to infection threat. This work describes the innovative use of Manuka honey as molecular spacer for preparing gellan gum-based composites with intrinsic antibacterial properties and superior compressive Young's modulus in respect of several Nature-derived gels based on chitosan, hyaluronic acid or alginate. The addition of Manuka honey made hydrogels able to inhibit the proliferation of S. aureus and S. epidermidis clinical isolates. Furthermore, no cytotoxic effects were detected on human mesenchymal stem cells seeded on the hydrogels. Moreover, chondrogenesis experiments showed a consistent expression of collagen II and high synthesis of GAGs and proteoglycans, thus indicating the formation of cartilage matrix. Overall, these data suggest that the developed smart composites have a great potential as tools for cartilage tissue engineering.
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http://dx.doi.org/10.1016/j.carbpol.2018.06.115DOI Listing
October 2018

Human β3-Adrenoreceptor is Resistant to Agonist-Induced Desensitization in Renal Epithelial Cells.

Cell Physiol Biochem 2018 20;48(2):847-862. Epub 2018 Jul 20.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy.

Background/aims: We recently showed that the β3-adrenoreceptor (β3AR) is expressed in mouse kidney collecting ducts (CD) cells along with the type-2 vasopressin receptor (AVPR2). Interestingly, a single injection of a β3AR selective agonist promotes a potent antidiuretic effect in mice. Before considering the feasibility of chronic β3AR agonism to induce antidiuresis in vivo, we aimed to evaluate in vitro the signaling and desensitization profiles of human β3AR.

Methods: Human β3AR desensitization was compared with that of human AVPR2 in cultured renal cells. Video imaging and FRET experiments were performed to dissect β3AR signaling under acute and chronic stimulation. Plasma membrane localization of β3AR, 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 β3AR 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 β3AR 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 β3AR 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 β3AR to promote antidiuresis overcoming in vivo renal concentrating defects caused by inactivating mutations of the AVPR2.
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http://dx.doi.org/10.1159/000491916DOI Listing
September 2018

Dandelion Root Extract Induces Intracellular Ca Increases in HEK293 Cells.

Int J Mol Sci 2018 Apr 7;19(4). Epub 2018 Apr 7.

Department of Sciences, University of Basilicata, 85100 Potenza, Italy.

Dandelion Weber ex F.H.Wigg.) has been used for centuries as an ethnomedical remedy. Nonetheless, the extensive use of different kinds of dandelion extracts and preparations is based on empirical findings. Some of the tissue-specific effects reported for diverse dandelion extracts may result from their action on intracellular signaling cascades. Therefore, the aim of this study was to evaluate the effects of an ethanolic dandelion root extract (DRE) on Ca signaling in human embryonic kidney (HEK) 293 cells. The cytotoxicity of increasing doses of crude DRE was determined by the Calcein viability assay. Fura-2 and the fluorescence resonance energy transfer (FRET)-based probe ERD1 were used to measure cytoplasmic and intraluminal endoplasmic reticulum (ER) Ca levels, respectively. Furthermore, a green fluorescent protein (GFP)-based probe was used to monitor phospholipase C (PLC) activation (pleckstrin homology [PH]-PLCδ-GFP). DRE (10-400 µg/mL) exposure, in the presence of external Ca, dose-dependently increased intracellular Ca levels. The DRE-induced Ca increase was significantly reduced in the absence of extracellular Ca. In addition, DRE caused a significant Ca release from the ER of intact cells and a concomitant translocation of PH-PLCδ-GFP. In conclusion, DRE directly activates both the release of Ca from internal stores and a significant Ca influx at the plasma membrane. The resulting high Ca levels within the cell seem to directly stimulate PLC activity.
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http://dx.doi.org/10.3390/ijms19041112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979456PMC
April 2018

Functional Characterization of a Novel Truncating Mutation in Lamin A/C Gene in a Family with a Severe Cardiomyopathy with Conduction Defects.

Cell Physiol Biochem 2017 4;44(4):1559-1577. Epub 2017 Dec 4.

Department of Sciences, University of Basilicata, Via dell'Ateneo Lucano, Potenza, Italy.

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/β-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 β-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.
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http://dx.doi.org/10.1159/000485651DOI Listing
January 2018

Hereditary Nephrogenic Diabetes Insipidus: Pathophysiology and Possible Treatment. An Update.

Int J Mol Sci 2017 Nov 10;18(11). Epub 2017 Nov 10.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, 70126 Bari, Italy.

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.
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http://dx.doi.org/10.3390/ijms18112385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5713354PMC
November 2017

Urinary Excretion of Kidney Aquaporins as Possible Diagnostic Biomarker of Diabetic Nephropathy.

J Diabetes Res 2017 26;2017:4360357. Epub 2017 Jan 26.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy.

Diabetic nephropathy (DN) is a microangiopathic complication of diabetes mellitus (DM) affecting one-third of diabetic patients. The large variability in the clinical presentation of renal involvement in patients with DM makes kidney biopsy a prerequisite for a correct diagnosis. However, renal biopsy is an invasive procedure associated with risk of major complications. Numerous studies aimed to identify a noninvasive biomarker of DN but, so far, none of these is considered to be sufficiently specific and sensitive. Water channel aquaporins (AQPs), expressed at the plasma membrane of epithelial tubular cells, are often dysregulated during DN. In this work, we analyzed the urine excretion of AQP5 and AQP2 (uAQP5 and uAQP2), exosomes, in 35 diabetic patients: 12 normoalbuminuric with normal renal function (DM), 11 with proteinuric nondiabetic nephropathy (NDN), and 12 with histological diagnosis and classification of DN. ELISA and WB analysis independently showed that uAQP5 was significantly increased in DN patients. Interestingly, linear regression analysis showed a positive correlation between uAQP5 and the histological class of DN. The same analysis, focusing on uAQP2, showed comparable results. Taken together, these data suggest a possible use of AQP5 and AQP2 as novel noninvasive biomarkers to help in classifying the clinical stage of DN.
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http://dx.doi.org/10.1155/2017/4360357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5299189PMC
June 2017

The expression of Lamin A mutant R321X leads to endoplasmic reticulum stress with aberrant Ca handling.

J Cell Mol Med 2016 11 15;20(11):2194-2207. Epub 2016 Jul 15.

Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari, Bari, Italy.

Mutations in the Lamin A/C gene (LMNA), which encodes A-type nuclear Lamins, represent the most frequent genetic cause of dilated cardiomyopathy (DCM). This study is focused on a LMNA nonsense mutation (R321X) identified in several members of an Italian family that produces a truncated protein isoform, which co-segregates with a severe form of cardiomyopathy with poor prognosis. However, no molecular mechanisms other than nonsense mediated decay of the messenger and possible haploinsufficiency were proposed to explain DCM. Aim of this study was to gain more insights into the disease-causing mechanisms induced by the expression of R321X at cellular level. We detected the expression of R321X by Western blotting from whole lysate of a mutation carrier heart biopsy. When expressed in HEK293 cells, GFP- (or mCherry)-tagged R321X mislocalized in the endoplasmic reticulum (ER) inducing the PERK-CHOP axis of the ER stress response. Of note, confocal microscopy showed phosphorylation of PERK in sections of the mutation carrier heart biopsy. ER mislocalization of mCherry-R321X also induced impaired ER Ca handling, reduced capacitative Ca entry at the plasma membrane and abnormal nuclear Ca dynamics. In addition, expression of R321X by itself increased the apoptosis rate. In conclusion, R321X is the first LMNA mutant identified to date, which mislocalizes into the ER affecting cellular homeostasis mechanisms not strictly related to nuclear functions.
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http://dx.doi.org/10.1111/jcmm.12926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5082401PMC
November 2016

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

PLoS One 2016 23;11(5):e0156021. Epub 2016 May 23.

Department of Sciences, University of Basilicata, Viale dell'Ateneo Lucano 10, 85100, Potenza, Italy.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0156021PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4877099PMC
July 2017

β3 adrenergic receptor in the kidney may be a new player in sympathetic regulation of renal function.

Kidney Int 2016 09 17;90(3):555-67. Epub 2016 May 17.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy; Institute of Biomembranes and Bioenergetics, National Research Council, Bari, Italy; National Institute of Biostructures and Biosystems (INBB), Rome, Italy.

To date, the study of the sympathetic regulation of renal function has been restricted to the important contribution of β1- and β2-adrenergic receptors (ARs). Here we investigate the expression and the possible physiologic role of β3-adrenergic receptor (β3-AR) in mouse kidney. The β3-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 β3-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 β3-AR antagonist L748,337 or by the protein kinase A inhibitor H89. Interestingly, genetic inactivation of β3-AR in mice was associated with significantly increased urine excretion of water, sodium, potassium, and chloride. Stimulation of β3-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 β3-AR stimulation in the kidney. Hence, β3-AR agonism might be useful to bypass AVPR2-inactivating mutations.
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http://dx.doi.org/10.1016/j.kint.2016.03.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4996630PMC
September 2016

Clinical and functional characterization of a novel mutation in lamin a/c gene in a multigenerational family with arrhythmogenic cardiac laminopathy.

PLoS One 2015 2;10(4):e0121723. Epub 2015 Apr 2.

Cardiology Unit, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy.

Mutations in the lamin A/C gene (LMNA) were associated with dilated cardiomyopathy (DCM) and, recently, were related to severe forms of arrhythmogenic right ventricular cardiomyopathy (ARVC). Both genetic and phenotypic overlap between DCM and ARVC was observed; molecular pathomechanisms leading to the cardiac phenotypes caused by LMNA mutations are not yet fully elucidated. This study involved a large Italian family, spanning 4 generations, with arrhythmogenic cardiomyopathy of different phenotypes, including ARVC, DCM, system conduction defects, ventricular arrhythmias, and sudden cardiac death. Mutation screening of LMNA and ARVC-related genes PKP2, DSP, DSG2, DSC2, JUP, and CTNNA3 was performed. We identified a novel heterozygous mutation (c.418_438dup) in LMNA gene exon 2, occurring in a highly conserved protein domain across several species. This newly identified variant was not found in 250 ethnically-matched control subjects. Genotype-phenotype correlation studies suggested a co-segregation of the LMNA mutation with the disease phenotype and an incomplete and age-related penetrance. Based on clinical, pedigree, and molecular genetic data, this mutation was considered likely disease-causing. To clarify its potential pathophysiologic impact, functional characterization of this LMNA mutant was performed in cultured cardiomyocytes expressing EGFP-tagged wild-type and mutated LMNA constructs, and indicated an increased nuclear envelope fragility, leading to stress-induced apoptosis as the main pathogenetic mechanism. This study further expands the role of the LMNA gene in the pathogenesis of cardiac laminopathies, suggesting that LMNA should be included in mutation screening of patients with suspected arrhythmogenic cardiomyopathy, particularly when they have ECG evidence for conduction defects. The combination of clinical, genetic, and functional data contribute insights into the pathogenesis of this form of life-threatening arrhythmogenic cardiac laminopathy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0121723PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383583PMC
March 2016

The authors reply.

Kidney Int 2015 Apr;87(4):862-3

Department of Biosciences, Biotechnologies and Biofarmaceutics, University of Bari, Bari, Italy.

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http://dx.doi.org/10.1038/ki.2014.412DOI Listing
April 2015

Rosiglitazone promotes AQP2 plasma membrane expression in renal cells via a Ca-dependent/cAMP-independent mechanism.

Cell Physiol Biochem 2015 2;35(3):1070-85. Epub 2015 Feb 2.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy.

Background/aims: Thiazolidinediones are highly beneficial in the treatment of type II diabetes. However, they are also associated with edema and increased risk of congestive heart failure. Several studies demonstrated that rosiglitazone (RGZ) increases the abundance of aquaporin-2 (AQP2) at the plasma membrane of renal cells. The aim of this study was to investigate whether RGZ might activate a transduction pathway facilitating AQP2 membrane accumulation in renal cells.

Methods: We analyzed the effect of RGZ on renal AQP2 intracellular trafficking in MCD4 renal cells by confocal microscopy and apical surface biotinylation. Cytosolic Ca(2+) dynamics were measured by a video-imaging approach in single cell. Transient Receptor Potential (TRP) channels expression was determined by RT-PCR.

Results: We showed that in MCD4 cells, short-term exposure to RGZ dramatically increases the amount of apically expressed AQP2 independently on cAMP production, PKA activation and AQP2 phosphorylation. RGZ elicited a cytosolic Ca(2+) transient due to Ca(2+) influx prevented by ruthenium red, suggesting the involvement of TRP plasma membrane channels. We identified TRPV6 as the possible candidate mediating this effect.

Conclusions: Taken together these results provide a possible molecular mechanism explaining the increased AQP2 membrane expression under RGZ treatment: in renal cells RGZ elicits Ca(2+) transients facilitating AQP2 exposure at the apical plasma membrane, thus increasing collecting duct water permeability. Importantly, this effect suggests an unexplored application of RGZ in the treatment of pathological states characterized by impaired AQP2 trafficking at the plasma membrane.
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http://dx.doi.org/10.1159/000373933DOI Listing
November 2015

NKCC2 activity is inhibited by the Bartter's syndrome type 5 gain-of-function CaR-A843E mutant in renal cells.

Biol Cell 2015 Apr 12;107(4):98-110. Epub 2015 Mar 12.

Department of Biosciences, Biotechnologies, and Biopharmaceutics, University of Bari, 70125 Bari, Italy; Department of Sciences, University of Basilicata, 85100 Potenza, Italy.

Background Information: The gain-of-function A843E mutation of the calcium sensing receptor (CaR) causes Bartter syndrome type 5. Patients carrying this CaR variant show a remarkably reduced renal NaCl reabsorption in the thick ascending limb (TAL) of Henle's loop resulting in renal loss of NaCl in the absence of mutations in renal Na(+) and Cl(-) ion transporters. The molecular mechanisms underlying this clinical phenotype are incompletely understood. We investigated, in human embryonic kidney 293 (HEK 293) cells and porcine kidney epithelial (LLC-PK1) cells, the functional cross-talk of CaR-A843E with the Na(+):K(+):2Cl(-) co-transporter, NKCC2, which provides NaCl reabsorption in the TAL.

Results: The expression of the CaR mutant did not alter the apical localisation of NKCC2 in LLC-PK1 cells. However, the steady-state NKCC2 phosphorylation and activity were decreased in cells transfected with CaR-A843E compared with the control wild-type CaR (CaR WT)-transfected cells. Of note, low-Cl(-)-dependent NKCC2 activation was also strongly inhibited upon the expression of CaR-A843E mutant. The use of either P450 ω-hydroxylase (CYP4)- or phospholipase A2 (PLA2)-blockers suggests that this effect is likely mediated by arachidonic acid (AA) metabolites.

Conclusions: The data suggested that the activated CaR affects intracellular pathways modulating NKCC2 activity rather than NKCC2 intracellular trafficking in renal cells, and throw further light on the pathological role played by active CaR mutants in Bartter syndrome type 5.
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http://dx.doi.org/10.1111/boc.201400069DOI Listing
April 2015

A novel therapeutic effect of statins on nephrogenic diabetes insipidus.

J Cell Mol Med 2015 Feb 16;19(2):265-82. Epub 2015 Jan 16.

Department of Biomedical Sciences and Human Oncology, Internal Medicine, University Medical School, Bari, Italy.

Statins competitively inhibit hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase, resulting in reduced plasma total and low-density lipoprotein cholesterol levels. Recently, it has been shown that statins exert additional 'pleiotropic' effects by increasing expression levels of the membrane water channels aquaporin 2 (AQP2). AQP2 is localized mainly in the kidney and plays a critical role in determining cellular water content. This additional effect is independent of cholesterol homoeostasis, and depends on depletion of mevalonate-derived intermediates of sterol synthetic pathways, i.e. farnesylpyrophosphate and geranylgeranylpyrophosphate. By up-regulating the expression levels of AQP2, statins increase water reabsorption by the kidney, thus opening up a new avenue in treating patients with nephrogenic diabetes insipidus (NDI), a hereditary disease that yet lacks high-powered and limited side effects therapy. Aspects related to water balance determined by AQP2 in the kidney, as well as standard and novel therapeutic strategies of NDI are discussed.
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http://dx.doi.org/10.1111/jcmm.12422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407600PMC
February 2015

Role of nuclear Lamin A/C in cardiomyocyte functions.

Biol Cell 2014 Oct 20;106(10):346-58. Epub 2014 Aug 20.

Department of Sciences, University of Basilicata, Potenza, Italy; Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy.

Lamin A/C is a structural protein of the nuclear envelope (NE) and cardiac involvement in Lamin A/C mutations was one of the first phenotypes to be reported in humans, suggesting a crucial role of this protein in the cardiomyocytes function. Mutations in LMNA gene cause a class of pathologies generically named 'Lamanopathies' mainly involving heart and skeletal muscles. Moreover, the well-known disease called Hutchinson-Gilford Progeria Syndrome due to extensive mutations in LMNA gene, in addition to the systemic phenotype of premature aging, is characterised by the death of patients at around 13 typically for a heart attack or stroke, suggesting again the heart as the main site sensitive to Lamin A/C disfunction. Indeed, the identification of the roles of the Lamin A/C in cardiomyocytes function is a key area of exploration. One of the primary biological roles recently conferred to Lamin A/C is to affect contractile cells lineage determination and senescence. Then, in differentiated adult cardiomyocytes both the 'structural' and 'gene expression hypothesis' could explain the role of Lamin A in the function of cardiomyocytes. In fact, recent advances in the field propose that the structural weakness/stiffness of the NE, regulated by Lamin A/C amount in NE, can 'consequently' alter gene expression.
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http://dx.doi.org/10.1111/boc.201400033DOI Listing
October 2014

Na+/K+-ATPase β1-subunit is recruited in Na-K-2Cl co-transporter isoform 2 multiprotein complexes in rat kidneys: possible role in blood pressure regulation.

J Hypertens 2014 Sep;32(9):1842-53

aDepartment of Sciences, University of Basilicata, Potenza bDepartment of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari cDepartment of Environmental Sciences, Tuscia University, Viterbo dConsiglio Nazionale delle Ricerche, Bari, Italy.

Objective: The progression from prehypertensive to hypertensive state in spontaneous hypertensive rats (SHRs) is accompanied by a significant increase in membrane expression of Na-K-2Cl co-transporter isoform 2 (NKCC2), suggesting that the altered NKCC2 trafficking and activity are directly related with the development of hypertension in this strain. The aim of this work is to gain insights on the molecular mechanism that underlies this phenomenon.

Methods: We performed a comparative analysis of NKCC2 multiprotein complexes (MPCs) in the kidney of SHRs versus Wistar Kyoto rats by Blue Native difference gel electrophoresis combined with mass spectrometry.

Results: We found that the recruitment of the β-subunit isoform 1 of the Na(+)-K(+)-ATPase (β1NK) in NKCC2 MPCs was significantly increased in the kidneys of SHR compared with Wistar Kyoto rat control strain. Co-immunoprecipitation experiments showed that β1NK actually interacts with NKCC2 in the native tissue. The analysis of the physiological role of β1NK-NKCC2 interaction in human embryonic kidney cells showed that β1NK increased the steady-state membrane expression and activity of NKCC2 enhancing NKCC2 trafficking toward the plasma membrane.

Conclusion: We identify a new NKCC2-interacting partner involved in the modulation of NKCC2 intracellular trafficking and possibly involved in the regulation of blood pressure.
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http://dx.doi.org/10.1097/HJH.0000000000000258DOI Listing
September 2014

Extracellular GTP is a potent water-transport regulator via aquaporin 5 plasma-membrane insertion in M1-CCD epithelial cortical collecting duct cells.

Cell Physiol Biochem 2014 7;33(3):731-46. Epub 2014 Mar 7.

Department of Neurosciences and Imaging (DNI), 'G. d'Annunzio' University of Chieti-Pescara, Chieti, Italy.

Background/aims: Extracellular GTP is able to modulate some specific functions in neuron, glia and muscle cell models as it has been demonstrated over the last two decades. In fact, extracellular GTP binds its specific plasma membrane binding sites and induces signal transduction via [Ca(2+)]i increase. We demonstrate, for the first time, that extracellular GTP is able to modulate cell swelling in M1-CCD cortical collecting duct epithelial cells via upregulation of aquaporin 5 (AQP5) expression.

Methods: We used videoimaging, immunocitochemistry, flow cytometry, confocal techniques, Western blotting and RT-PCR for protein and gene expression analysis, respectively.

Results: We demonstrate that AQP5 mRNA is up-regulated 7 h after the GTP exposure in the cell culture medium, and its protein level is increased after 12-24 h. We show that AQP5 is targeted to the plasma membrane of M1-CCD cells, where it facilitates cell swelling, and that the GTP-dependent AQP5 up-regulation occurs via [Ca(2+)]i increase. Indeed, GTP induces both oscillating and transient [Ca(2+)]i increase, and specifically the oscillating kinetic appears to be responsible for blocking cell cycle in the S-phase while the [Ca(2+)]i influx, with whatever kinetic, seems to be responsible for inducing AQP5 expression.

Conclusion: The role of GTP as a regulator of AQP5-mediated water transport in renal cells is of great importance in the physiology of renal epithelia, due to its possible physiopathological implications. GTP-dependent AQP5 expression could act as osmosensor. In addition, the data presented here suggest that GTP might play the same role in other tissues where rapid water transport is required for cell volume regulation and maintenance of the homeostasis.
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http://dx.doi.org/10.1159/000358648DOI Listing
August 2016

Combination of secretin and fluvastatin ameliorates the polyuria associated with X-linked nephrogenic diabetes insipidus in mice.

Kidney Int 2014 Jul 12;86(1):127-38. Epub 2014 Feb 12.

1] Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy [2] Centro di Eccellenza di Genomica in Campo Biomedico ed Agrario (CEGBA), Bari, Italy.

X-linked nephrogenic diabetes insipidus (X-NDI) is a disease caused by inactivating mutations of the vasopressin (AVP) type 2 receptor (V2R) gene. Loss of V2R function prevents plasma membrane expression of the AQP2 water channel in the kidney collecting duct cells and impairs the kidney concentration ability. In an attempt to develop strategies to bypass V2R signaling in X-NDI, we evaluated the effects of secretin and fluvastatin, either alone or in combination, on kidney function in a mouse model of X-NDI. The secretin receptor was found to be functionally expressed in the kidney collecting duct cells. Based on this, X-NDI mice were infused with secretin for 14 days but urinary parameters were not altered by the infusion. Interestingly, secretin significantly increased AQP2 levels in the collecting duct but the protein primarily accumulated in the cytosol. Since we previously reported that fluvastatin treatment increased AQP2 plasma membrane expression in wild-type mice, secretin-infused X-NDI mice received a single injection of fluvastatin. Interestingly, urine production by X-NDI mice treated with secretin plus fluvastatin was reduced by nearly 90% and the urine osmolality was doubled. Immunostaining showed that secretin increased intracellular stores of AQP2 and the addition of fluvastatin promoted AQP2 trafficking to the plasma membrane. Taken together, these findings open new perspectives for the pharmacological treatment of X-NDI.
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http://dx.doi.org/10.1038/ki.2014.10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4080339PMC
July 2014

Co-regulated pendrin and aquaporin 5 expression and trafficking in Type-B intercalated cells under potassium depletion.

Cell Physiol Biochem 2013 18;32(7):184-99. Epub 2013 Dec 18.

Dept. of Biosciences Biotechnologies and Biopharmaceutics, University of Bari, Italy.

Background: We recently reported that aquaporin 5 (AQP5), a water channel never identified in the kidney before, co-localizes with pendrin at the apical membrane of type-B intercalated cells in the kidney cortex. Since co-expression of AQP5 and pendrin in the apical membrane domain is a common feature of several other epithelia such as cochlear and bronchial epithelial cells, we evaluated here whether this strict membrane association may reflect a co-regulation of the two proteins. To investigate this possibility, we analyzed AQP5 and pendrin expression and trafficking in mice under chronic K(+) depletion, a condition that results in an increased ability of renal tubule to reabsorb bicarbonate, often leads to metabolic alkalosis and is known to strongly reduce pendrin expression.

Methods: Mice were housed in metabolic cages and pair-fed with either a standard laboratory chow or a K(+)-deficient diet. AQP5 abundance was assessed by western blot in whole kidney homogenates and AQP5 and pendrin were localized by confocal microscopy in kidney sections from those mice. In addition, the short-term effect of changes in external pH on pendrin trafficking was evaluated by fluorescence resonance energy transfer (FRET) in MDCK cells, and the functional activity of pendrin was tested in the presence and absence of AQP5 in HEK 293 Phoenix cells.

Results: Chronic K(+) depletion caused a strong reduction in pendrin and AQP5 expression. Moreover, both proteins shifted from the apical cell membrane to an intracellular compartment. An acute pH shift from 7.4 to 7.0 caused pendrin internalization from the plasma membrane. Conversely, a pH shift from 7.4 to 7.8 caused a significant increase in the cell surface expression of pendrin. Finally, pendrin ion transport activity was not affected by co-expression with AQP5.

Conclusions: The co-regulation of pendrin and AQP5 membrane expression under chronic K(+)-deficiency indicates that these two molecules could cooperate as an osmosensor to rapidly detect and respond to alterations in luminal fluid osmolality.
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http://dx.doi.org/10.1159/000356638DOI Listing
September 2014