Biomedical subjects

Giuseppe Procino

Publications and source records attributed to Giuseppe Procino.

7 recordsLinked to original sources

PubMed2005

Minireview: aquaporin 2 trafficking.

In the kidney aquaporin-2 (AQP2) provides a target for hormonal regulation of water transport by vasopressin. Short-term control of water permeability occurs via vesicular trafficking of AQP2 and long-term control through changes in the abundance of AQP2 and AQP3 water channels. Defective AQP2 trafficking causes nephrogenic diabetes insipidus, a condition characterized by the kidney inability to produce concentrated urine because of the insensitivity of the distal nephron to vasopressin. AQP2 is redistributed to the apical membrane of collecting duct cells through activation of a cAMP signaling cascade initiated by the binding of vasopressin to its V2-receptor. Protein kinase A-mediated phosphorylation of AQP2 has been proposed to be essential in regulating AQP2-containing vesicle exocytosis. Cessation of the stimulus is followed by endocytosis of the AQP2 proteins exposed on the plasma membrane and their recycling to the original stores, in which they are retained. Soluble N-ethylmaleimide sensitive fusion factor attachment protein receptors (SNARE) and actin cytoskeleton organization regulated by small GTPase of the Rho family were also proved to be essential for AQP2 trafficking. Data for functional involvement of the SNARE vesicle-associated membrane protein 2 in AQP2 targeting has recently been provided. Changes in AQP2 expression/trafficking are of particular importance in pathological conditions characterized by both dilutional and concentrating defects. One of these conditions, hypercalciuria, has shown to be associated with alteration of AQP2 urinary excretion. More precisely, recent data support the hypothesis that, in vivo external calcium, through activation of calcium-sensing receptors, modulates the expression/trafficking of AQP2. Together these findings underscore the importance of AQP2 in kidney pathophysiology.

Animals↗

PubMed2004

Role of the p66Shc isoform in insulin-like growth factor I receptor signaling through MEK/Erk and regulation of actin cytoskeleton in rat myoblasts.

To investigate the role of Shc in IGF action and signaling in skeletal muscle cells, Shc protein levels were reduced in rat L6 myoblasts by stably overexpressing a Shc cDNA fragment in antisense orientation (L6/Shcas). L6/Shcas myoblasts showed marked reduction of the p66Shc protein isoform and no change in p52Shc or p46Shc proteins compared with control myoblasts transfected with the empty vector (L6/Neo). When compared with control, L6/Shcas myoblasts demonstrated 3-fold increase in Erk-1/2 phosphorylation under basal conditions and blunted Erk-1/2 stimulation by insulin-like growth factor I (IGF-I), in the absence of changes in total Erk-1/2 protein levels. Increased basal Erk-1/2 activation was paralleled by a greater proportion of phosphorylated Erk-1/2 in the nucleus of L6/Shcas myoblasts in the absence of IGF-I stimulation. The reduction of p66Shc in L6/Shcas myoblasts resulted in marked phenotypic abnormalities, such as rounded cell shape and clustering in islets or finger-like structures, and was associated with impaired DNA synthesis in response to IGF-I and lack of terminal differentiation into myotubes. In addition, L6/Shcas myoblasts were characterized by complete disruption of actin filaments and cell cytoskeleton. Treatment of L6/Shcas myoblasts with the MEK inhibitor PD98059 reduced the abnormal increase in Erk-1/2 activation to control levels and restored the actin cytoskeleton, re-establishing the normal cell morphology. Thus, the p66Shc isoform exerts an inhibitory effect on the mitogen-activated protein kinase signaling pathway in rodent myoblasts, which is necessary for maintenance of IGF responsiveness of the MEK/Erk pathway and normal cell phenotype.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Selective decrease in urinary aquaporin 2 and increase in prostaglandin E2 excretion is associated with postobstructive polyuria in human congenital hydronephrosis.

This study was undertaken to determine the role of aquaporin 2 (AQP2) in the impaired urinary concentrating capacity observed in patients who underwent pyeloplasty because of congenital unilateral hydronephrosis as a result of pyeloureteral junction disease. Twelve children (mean age, 8 +/- 2 mo) were examined in the study. From day 1 to day 5 after surgery, the urine was collected separately from pyelostomy draining only from the postobstructed kidney and from the bladder catheter draining mostly from the contralateral kidney used as internal control. After pyeloplasty, the postobstructed kidney was characterized by a reduced urinary excretion of AQP2 (approximately 54%) associated with polyuria that persisted from day 1 to day 5 (433 +/- 58 versus 310 +/- 74 ml/24 h at day 1; 326 +/- 44 versus 227 +/- 26 ml/24 h at day 5). In parallel, urine osmolality from the postobstructed kidney was significantly reduced compared with the contralateral kidney (111 +/- 12 versus 206 +/- 49 at day 1; 136 +/- 24 versus 235 +/- 65 mOsm/kg at day 5). Creatinine clearance from the postobstructed kidney was not significantly different compared with the contralateral kidney throughout the 4 d after surgery. However, on day 5, creatinine clearance from the postobstructed kidney became significantly lower. Prostaglandin E2 in the urine from postobstructed kidneys was found to be twofold higher than in the contralateral samples (26.0 +/- 6.7 versus 13.5 +/- 2.5 at day 5). It is concluded that (1) the selective downregulation of AQP2 in postobstructed kidney may account for the higher excretion of hypotonic urine, and (2) the local increase in prostaglandin E2 synthesis in postobstructed kidney may be involved in AQP2 downregulation and in maintaining a GFR similar to that of the contralateral kidney.

Adolescent↗

PubMed2004

Extracellular calcium antagonizes forskolin-induced aquaporin 2 trafficking in collecting duct cells.

BACKGROUND: Urinary concentrating defects and polyuria are the most important renal manifestations of hypercalcemia and the resulting hypercalciuria. In this study, we tested the hypothesis that hypercalciuria-associated polyuria in kidney collecting duct occurs through an impairment of the vasopressin-dependent aquaporin 2 (AQP2) water channel targeting to the apical membrane possibly involving calcium-sensing receptor (CaR) signaling. METHODS: AQP2-transfected collecting duct CD8 cells were used as experimental model. Quantitation of cell surface AQP2 immunoreactivity was performed using an antibody recognizing the extracellular AQP2 C loop. Intracellular cyclic adenosine monophosphate (cAMP) accumulation was measured in CD8 cells using a cAMP enzyme immunoassay kit. To study the translocation of protein kinase C (PKC), membranes or cytosol fractions from CD8 cells were subjected to Western blotting using anti-PKC isozymes antibodies. The amount of F-actin was determined by spectrofluorometric techniques. Intracellular calcium measurements were performed by spectrofluorometric analysis with Fura-2/AM. RESULTS: We demonstrated that extracellular calcium (Ca2+ o) (5 mmol/L) strongly inhibited forskolin-stimulated increase in AQP2 expression in the apical plasma membrane. At least three intracellular pathways activated by extracellular calcium were found to contribute to this effect. Firstly, the increase in cAMP levels in response to forskolin stimulation was drastically reduced in cells pretreated with Ca2+ o compared to untreated cells. Second, Ca2+ o activated PKC, known to counteract vasopressin response. Third, quantification of F-actin demonstrated that Ca2+ o caused a nearly twofold increase in F-actin content compared with basal conditions. All these effects were mimicked by a nonmembrane permeable agonist of the extracellular CaR, Gd3+. CONCLUSION: Together, these data demonstrate that extracellular calcium, possibly acting through the endogenous CaR, antagonizes forskolin-induced AQP2 translocation to the apical plasma membrane in CD8 cells. In hypercalciuria, this mechanism might blunt water reabsorption and prevent further calcium concentration, thus protecting against a potential risk of urinary calcium-containing stone formation.

Actins↗

PubMed2003

Ser-256 phosphorylation dynamics of Aquaporin 2 during maturation from the ER to the vesicular compartment in renal cells.

Aquaporin 2 (AQP2) phosphorylation at Ser-256 by protein kinase A (PKA) is a key signal for vasopressin-stimulated AQP2 insertion into the plasma membrane in renal cells. This study underscores the possible role of phosphorylation at Ser-256 in regulating AQP2 maturation. AQP2-transfected renal CD8 cells were incubated with brefeldin A (BFA) to accumulate newly synthesized AQP2 in the endoplasmic reticulum (ER), and AQP2 flow from ER to the vesicular compartment was analyzed after BFA washout. We found that a) in the ER, AQP2 is weakly phosphorylated; b) the amount of phosphorylated AQP2 (p-AQP2) at Ser-256 increased significantly during transit in the Golgi, even in the presence of the PKA inhibitor H89; and c) AQP2 transport from the Golgi to the vasopressin-regulated vesicular compartment occurred with a concomitant decrease in p-AQP2 at Ser-256. These results support the hypothesis that AQP2 transition in the Golgi apparatus is associated with a PKA-independent increase in AQP2 phosphorylation at Ser-256. Conversely, impaired constitutive phosphorylation in a Golgi-associated compartment occurring in cells expressing mutated S256A-AQP2 or E258K-AQP2 causes phosphorylation-defective AQP2 routing to lysosomes. This result might explain the molecular basis of the dominant form of nephrogenic diabetes insipidus caused by the mutation E258K-AQP2, in which the phenotype is caused by an impaired routing of AQP2.

Aquaporin 2↗

PubMed2003

cAMP-induced AQP2 translocation is associated with RhoA inhibition through RhoA phosphorylation and interaction with RhoGDI.

We have recently demonstrated that inhibition of Rho GTPase with Clostridium difficile toxin B, or with Clostridium botulinum C3 toxin, causes actin depolymerization and translocation of aquaporin 2 (AQP2) in renal CD8 cells in the absence of hormonal stimulation. Here we demonstrate that Rho inhibition is part of the signal transduction cascade activated by vasopressin leading to AQP2 insertion into the apical membrane. Quantitation of active RhoA (GTP-bound) by selective pull down experiments demonstrated that the amount of active RhoA decreased upon stimulation of CD8 cells with the cAMP-elevating agent forskolin. Consistent with this observation, forskolin treatment resulted in a decreased expression of membrane-associated (active) Rho, as assessed by cell fractionation followed by western blotting analysis. In addition, the abundance of the endogenous Rho GDP dissociation inhibitor (Rho-GDI) was found to have decreased in the membrane fraction after forskolin stimulation. Co-immunoprecipitation experiments revealed that, after forskolin stimulation, the amount of Rho-GDI complexed with RhoA increased, suggesting that Rho GTPase inhibition occurs through association of RhoA with Rho-GDI. Finally, forskolin stimulation was associated with an increase in Rho phosphorylation on a serine residue, a protein modification known to stabilize the inactive form of RhoA and to increase its interaction with Rho-GDI. Taken together, these data demonstrate that RhoA inhibition through Rho phosphorylation and interaction with Rho-GDI is a key event for cytoskeletal dynamics controlling cAMP-induced AQP2 translocation.

Animals↗

PubMed2002

Functional involvement of VAMP/synaptobrevin-2 in cAMP-stimulated aquaporin 2 translocation in renal collecting duct cells.

The involvement of soluble N-ethylmaleimide sensitive factor-attachment protein receptor (SNARE) proteins in the cAMP-induced exocytosis of aquaporin 2 (AQP2)-containing vesicles was investigated in AQP2-transfected renal CD8 cells. RT-PCR and western blot analysis confirmed the presence of the SNARE homologs VAMP/synaptobrevin-2, syntaxin-1, syntaxin-4 and SNAP-23 in CD8 cells. Tetanus neurotoxin (TeNT) was efficient in cleaving synaptobrevin-like protein both in vitro and in intact CD8 cells incubated with the toxin. TeNT treatment in intact CD8 cells completely abolished cAMP-stimulated AQP2 targeting to the plasma membrane, as assessed by quantification of cell-surface immunoreactivity to an anti-AQP2 antibody raised against a peptide reproducing the extracellular AQP2 C-loop. These results represent the first evidence for the functional involvement of VAMP-2 in cAMP-induced AQP2 exocytosis in renal cells.

Animals↗