Publications by authors named "Lisa Mastrofrancesco"

11 Publications

  • Page 1 of 1

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

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

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

A protein kinase A-independent pathway controlling aquaporin 2 trafficking as a possible cause for the syndrome of inappropriate antidiuresis associated with polycystic kidney disease 1 haploinsufficiency.

J Am Soc Nephrol 2014 Oct 3;25(10):2241-53. Epub 2014 Apr 3.

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

Renal water reabsorption is controlled by arginine vasopressin (AVP), which binds to V2 receptors, resulting in protein kinase A (PKA) activation, phosphorylation of aquaporin 2 (AQP2) at serine 256, and translocation of AQP2 to the plasma membrane. However, AVP also causes dephosphorylation of AQP2 at S261. Recent studies showed that cyclin-dependent kinases (cdks) can phosphorylate AQP2 peptides at S261 in vitro. We investigated the possible role of cdks in the phosphorylation of AQP2 and identified a new PKA-independent pathway regulating AQP2 trafficking. In ex vivo kidney slices and MDCK-AQP2 cells, R-roscovitine, a specific inhibitor of cdks, increased pS256 levels and decreased pS261 levels. The changes in AQP2 phosphorylation status were paralleled by increases in cell surface expression of AQP2 and osmotic water permeability in the absence of forskolin stimulation. R-Roscovitine did not alter cAMP-dependent PKA activity but specifically reduced protein phosphatase 2A (PP2A) expression and activity in MDCK cells. Notably, we found reduced PP2A expression and activity and reduced pS261 levels in Pkd1(+/-) mice displaying a syndrome of inappropriate antidiuresis with high levels of pS256, despite unchanged AVP and cAMP. Similar to previous findings in Pkd1(+/-) mice, R-roscovitine treatment caused a significant decrease in intracellular calcium in MDCK cells. Our data indicate that reduced activity of PP2A, secondary to reduced intracellular Ca(2+) levels, promotes AQP2 trafficking independent of the AVP-PKA axis. This pathway may be relevant for explaining pathologic states characterized by inappropriate AVP secretion and positive water balance.
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http://dx.doi.org/10.1681/ASN.2013111234DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178447PMC
October 2014

Calcium-sensing receptor and aquaporin 2 interplay in hypercalciuria-associated renal concentrating defect in humans. An in vivo and in vitro study.

PLoS One 2012 5;7(3):e33145. Epub 2012 Mar 5.

Department of Biosciences, Biotechnologies and Pharmacological Sciences and Center of Excellence in Comparative Genomics, University of Bari, Bari, Italy.

One mechanism proposed for reducing the risk of calcium renal stones is activation of the calcium-sensing receptor (CaR) on the apical membranes of collecting duct principal cells by high luminal calcium. This would reduce the abundance of aquaporin-2 (AQP2) and in turn the rate of water reabsorption. While evidence in cells and in hypercalciuric animal models supports this hypothesis, the relevance of the interplay between the CaR and AQP2 in humans is not clear. This paper reports for the first time a detailed correlation between urinary AQP2 excretion under acute vasopressin action (DDAVP treatment) in hypercalciuric subjects and in parallel analyzes AQP2-CaR crosstalk in a mouse collecting duct cell line (MCD4) expressing endogenous and functional CaR. In normocalciurics, DDAVP administration resulted in a significant increase in AQP2 excretion paralleled by an increase in urinary osmolality indicating a physiological response to DDAVP. In contrast, in hypercalciurics, baseline AQP2 excretion was high and did not significantly increase after DDAVP. Moreover DDAVP treatment was accompanied by a less pronounced increase in urinary osmolality. These data indicate reduced urinary concentrating ability in response to vasopressin in hypercalciurics. Consistent with these results, biotinylation experiments in MCD4 cells revealed that membrane AQP2 expression in unstimulated cells exposed to CaR agonists was higher than in control cells and did not increase significantly in response to short term exposure to forskolin (FK). Interestingly, we found that CaR activation by specific agonists reduced the increase in cAMP and prevented any reduction in Rho activity in response to FK, two crucial pathways for AQP2 translocation. These data support the hypothesis that CaR-AQP2 interplay represents an internal renal defense to mitigate the effects of hypercalciuria on the risk of calcium precipitation during antidiuresis. This mechanism and possibly reduced medulla tonicity may explain the lower concentrating ability observed in hypercalciuric patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033145PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3293925PMC
July 2012

AQP5 is expressed in type-B intercalated cells in the collecting duct system of the rat, mouse and human kidney.

Cell Physiol Biochem 2011 14;28(4):683-92. Epub 2011 Dec 14.

Department of General and Environmental Physiology, University of Bari, Via Amendola 165/A, Bari, Italy.

We screened human kidney-derived multipotent CD133+/CD24+ ARPCs for the possible expression of all 13 aquaporin isoforms cloned in humans. Interestingly, we found that ARPCs expressed both AQP5 mRNA and mature protein. This novel finding prompted us to investigate the presence of AQP5 in situ in kidney. We report here the novel finding that AQP5 is expressed in human, rat and mouse kidney at the apical membrane of type-B intercalated cells. AQP5 is expressed in the renal cortex and completely absent from the medulla. Immunocytochemical analysis using segment- and cell type-specific markers unambiguously indicated that AQP5 is expressed throughout the collecting system at the apical membrane of type-B intercalated cells, where it co-localizes with pendrin. No basolateral AQPs were detected in type-B intercalated cells, suggesting that AQP5 is unlikely to be involved in the net trans-epithelial water reabsorption occurring in the distal tubule. An intriguing hypothesis is that AQP5 may serve an osmosensor for the composition of the fluid coming from the thick ascending limb. Future studies will unravel the physiological role of AQP5 in the kidney.
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http://dx.doi.org/10.1159/000335762DOI Listing
April 2012

In-vivo administration of CLC-K kidney chloride channels inhibitors increases water diuresis in rats: a new drug target for hypertension?

J Hypertens 2012 Jan;30(1):153-67

Section of Pharmacology, Department of Pharmacobiology, Faculty of Pharmacy, University of Bari, Bari, Italy.

Objective: The human kidney-specific chloride channels ClC-Ka (rodent ClC-K1) and ClC-Kb (rodent ClC-K2) are important determinants of renal function, participating to urine concentration and blood pressure regulation mechanisms. Here we tested the hypothesis that these chloride channels could represent new drug targets for inducing diuretic and antihypertensive effects.

Methods: To this purpose, the CLC-K blockers benzofuran derivatives MT-189 and RT-93 (10, 50, 100 mg/kg), were acutely administered by gavage in Wistar rats, and pharmacodynamic and pharmacokinetic parameters determined by functional, bioanalytical, biochemical and molecular biology assays.

Results: Plasma concentration values for MT-189 and RT-93 were indicative of good bioavailability. Both MT-189 and RT-93 dose-dependently increased urine volume without affecting electrolyte balance. A comparable reduction of SBP was observed in rats after MT-189, RT-93 or furosemide administration. Benzofuran derivatives treatment did not affect kidney CLC-K mRNA level or inner medulla osmolality, whereas a significant vasopressin-independent down-regulation of aquaporin water channel type 2 was observed at protein and transcriptional levels. In rats treated with benzofuran derivatives, the observed polyuria was mainly water diuresis; this finding indirectly supports a cross-talk between chloride and water transport in nephron. Moreover, preliminary in-vitro evaluation of the drugs capability to cross the blood-inner ear barrier suggests that these compounds have a limited ability to induce potential auditory side effects.

Conclusion: CLC-K blockers may represent a new class of drugs for the treatment of conditions associated with expanded extracellular volume, with a hopeful high therapeutic potential for hypertensive patients carrying ClC-K gain-of-function polymorphisms.
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http://dx.doi.org/10.1097/HJH.0b013e32834d9eb9DOI Listing
January 2012

Altered urinary excretion of aquaporin 2 in IgA nephropathy.

Eur J Endocrinol 2011 Oct 8;165(4):657-64. Epub 2011 Aug 8.

Section of Nephrology and Bioagromed, Department of Biomedical Sciences, University of Foggia, 71122 Foggia, Italy.

Objective: The intrarenal renin-angiotensin system (RAS) activation plays a pivotal role in immunoglobulin A nephropathy (IgAN) pathogenesis, which is still largely undefined. Recently, vasopressin (AVP) has been advocated to contribute to the genesis and progression of chronic kidney diseases (CKD) directly, and indirectly, via RAS activation. Our aim is to explore the intrarenal activity of AVP, its relationship with RAS activity, as well as its modulation by therapies in IgAN.

Design: In this observational study, we measured plasma copeptin, a surrogate marker of AVP, the urine excretion of aquaporin 2 (AQP2), a protein reflecting renal AVP action, and angiotensinogen (AGT), a parameter of renal RAS activation, and their relationship with renal function in 44 IgAN patients at the time of renal biopsy, without any drug therapy, and after 6-month treatment with ACEi or steroid+ACEi. Twenty-one patients with other CKD and 40 healthy subjects were recruited as controls.

Methods: ELISAs were used to measure all variables of interest.

Results: At baseline, IgAN patients showed higher urinary levels of AQP2, compared with controls and patients with other CKD. Urinary AQP2 and AGT levels strongly correlated with the presence of arterial hypertension. Steroids+ACEi caused the decrease of all the variables examined. The fall of urinary AQP2 and AGT following drug treatments was associated with the decrease of daily proteinuria.

Conclusion: Our findings would support the involvement of AVP-AQP2 axis, interacting with the RAS, in the progression of IgAN and candidate AQP2 as a possible novel marker of the disease.
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http://dx.doi.org/10.1530/EJE-11-0512DOI Listing
October 2011

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

Cell Physiol Biochem 2011 17;27(6):739-48. Epub 2011 Jun 17.

Department of General and Environmental Physiology, University of Bari, Via Amendola 165/A, 70125 Bari, Italy.

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.
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http://dx.doi.org/10.1159/000330082DOI Listing
October 2011

Differential modulation of intracellular Ca2+ responses associated with calcium-sensing receptor activation in renal collecting duct cells.

Cell Physiol Biochem 2010 4;26(6):901-12. Epub 2011 Jan 4.

Department of General and Environmental Physiology, University of Bari, Bari, Italy.

In this work, we studied G protein-coupled Extracellular Calcium Sensing Receptor (CaR) signaling in mouse cortical collecting duct cells (MCD4) expressing endogenous CaR. Intracellular [Ca(2+)] measurements performed with real time video imaging revealed that CaR stimulation with 5 mM Ca(2+), 300 μM Gd(3+) and with 10 μM of specific allosteric modulator NPS-R 568, all resulted in an increase in [Ca(2+)](i) although displaying different features. Specifically, Ca(2+) as well as stimulation with NPS-R 568 induced a rapid peak of [Ca(2+)](i) while stimulation with Gd(3+) induced transient intracellular Ca(2+) oscillations. PLC inhibition completely abolished any [Ca(2+)](i) increase after stimulation with CaR agonists. Inhibition of Rho or Rho kinase (ROK) abolished [Ca(2+)](i) oscillations induced by Gd(3+), while the peak induced by high Ca(2+) was similar to control. Conversely, emptying the intracellular calcium stores abolished the response to Gd(3+). On the other hand, the inhibition of calcium influx did not alter calcium changes. We conclude that in our cell model, CaR stimulation with distinct agonists activates two distinct transduction pathways, both PLC-dependent. The transient cytosolic Ca(2+) oscillations produced by Gd(3+) are modulated by Rho-Rho kinase signaling, whereas the rapid peak of intracellular Ca(2+) in response to 5 mM [Ca(2+)](o) is mainly due to PLC/IP3 pathway activation.
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http://dx.doi.org/10.1159/000323999DOI Listing
April 2011

Disuse of rat muscle in vivo reduces protein kinase C activity controlling the sarcolemma chloride conductance.

J Physiol 2007 Nov 13;584(Pt 3):983-95. Epub 2007 Sep 13.

Section of Pharmacology, Department of Pharmacobiology, Faculty of Pharmacy, University of Bari, Via Orabona 4 - Campus, 70125, Bari, Italy.

Muscle disuse produced by hindlimb unloading (HU) induces severe atrophy and slow-to-fast fibre type transition of the slow-twitch soleus muscle (Sol). After 2 weeks HU, the resting ClC-1 chloride conductance (g(Cl)) of sarcolemma, which controls muscle excitability, increases in Sol toward a value typical of the fast-twitch EDL muscle. After 3 days of HU, the g(Cl) increases as well before initiation of fibre type transition. Since ClC-1 channels are acutely silenced by PKC-dependent phosphorylation, we studied the modulation of g(Cl) by PKC and serine-threonine phosphatase in Sol during HU, using a number of pharmacological tools. We show that a fraction of ClC-1 channels of control Sol are maintained in an inactive state by PKC basal activity, which contributes to the lower g(Cl) in control Sol compared to EDL. After 14 days of HU, PKC/phosphatase manipulation produces effects on Sol g(Cl) that corroborate the partial slow-to-fast transition. After 3 days of HU, the early increase of g(Cl) in Sol is entirely attributable to a reduction of PKC activity and/or activation of phosphatase, maintaining ClC-1 channels in a fully active state. Accordingly, we found that HU reduces expression of PKCalpha, epsilon, and isoenzymes in Sol and EDL muscles and reduces total PKC activity. Moreover, we show that the rheobase current is increased in Sol muscle fibres as soon as after 3 days of HU, most probably in relation to the increased g(Cl). In conclusion, Sol muscle disuse is characterized by a rapid reduction of PKC activity, which reduces muscle excitability and is likely to contribute to disuse-induced muscle impairment.
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http://dx.doi.org/10.1113/jphysiol.2007.141358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2276996PMC
November 2007