Vasopressin regulates water reabsorption in renal collecting duct principal cells by a cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the cell membrane. In the present work primary cultured inner medullary collecting duct cells were used to study the role of the proteins of the Rho family in the translocation of AQP2. Clostridium difficile toxin B, which inhibits all members of the Rho family, Clostridium limosum C3 toxin, which inactivates only Rho, and the Rho kinase inhibitor, Y-27632, induced both depolymerization of actin stress fibers and AQP2 translocation in the absence of vasopressin. The data suggest an inhibitory role of Rho in this process, whereby constitutive membrane localization is prevented in resting cells. Expression of constitutively active RhoA induced formation of actin stress fibers and abolished AQP2 translocation in response to elevation of intracellular cAMP, confirming the inhibitory role of Rho. Cytochalasin D induced both depolymerization of the F-actin cytoskeleton and AQP2 translocation, indicating that depolymerization of F-actin is sufficient to induce AQP2 translocation. Thus Rho is likely to control the intracellular localization of AQP2 via regulation of the F-actin cytoskeleton.The antidiuretic hormone arginine-vasopressin (AVP) 1 regulates water reabsorption in renal collecting duct principal cells by inducing the translocation of the water channel aquaporin-2 (AQP2) from intracellular vesicles primarily into the apical cell membrane (shuttle hypothesis; Refs. 1 and 2). The molecular targets of AVP on the surface of principal cells are heptahelical vasopressin V2 receptors coupled to the G s /adenylyl cyclase system. Activation of this system by the hormone raises the level of intracellular cAMP and results in the activation of protein kinase A (PKA) which then phosphorylates its substrates, one of which is AQP2.The phosphorylation of AQP2 by PKA and also the anchoring of PKA to subcellular compartments via protein kinase A anchoring proteins are prerequisites for AQP2 translocation to the cell membrane (2-5). In addition, the involvement of a heterotrimeric G protein of the G i family in the AQP2 translocation has been demonstrated in CD8 cells (6).The cytoskeleton consists of various components, including microtubules and F-actin, both of which are involved in AVPmediated changes of osmotic water permeability (2, 7-9). Microtubule-disrupting drugs like colchicine and nocodazole inhibit AVP-mediated increases in osmotic water permeability in renal collecting ducts by 65 and 72%, respectively (10 -13). Disruption of the F-actin cytoskeleton by cytochalasin B or dihydrocytochalasin B inhibits the AVP-induced increase in osmotic water permeability in toad bladder epithelium by 25-50% (13, 14). The F-actin cytoskeleton also undergoes rearrangements after stimulation of cells with cAMP-elevating agents. After stimulation with vasopressin, total F-actin decreases in toad bladders by 20 -30% (15) and apical F-actin in rat coll...

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Arginine-vasopressin (AVP) modulates the water channel aquaporin-2 (AQP2) in the renal collecting duct to maintain homeostasis of body water. AVP binds to vasopressin V2 receptors (V2R), increasing cAMP, which promotes the redistribution of AQP2 from intracellular vesicles into the plasma membrane. cAMP also increases AQP2 transcription, but whether altered degradation also modulates AQP2 protein levels is not well understood. Here, elevation of cAMP increased AQP2 protein levels within 30 minutes in primary inner medullary collecting duct (IMCD) cells, in human embryonic kidney (HEK) 293 cells ectopically expressing AQP2, and in mouse kidneys. Accelerated transcription or translation did not explain this increase in AQP2 abundance. In IMCD cells, cAMP inhibited p38-mitogen-activated protein kinase (p38-MAPK) via activation of protein kinase A (PKA). Inhibition of p38-MAPK associated with decreased phosphorylation (serine 261) and polyubiquitination of AQP2, preventing proteasomal degradation. Our results demonstrate that AVP enhances AQP2 protein abundance by altering its proteasomal degradation through a PKA-and p38-MAPK-dependent pathway. Aquaporin-2 (AQP2) is the water channel mediating arginine-vasopressin (AVP)-increases in water re-absorption in renal collecting duct principal cells. 1-4 AVP binds to plasma membrane-located vasopressin V2 receptors, thereby stimulating adenylyl cyclase and elevating cAMP. cAMP activates protein kinase A (PKA), which phosphorylates AQP2 at serine 256 (S256), inducing its redistribution from intracellular vesicles into the plasma membrane. 3,4 This short-term regulation of AQP2 occurs within seconds to minutes. In the case of long-term regulation, cAMP enhances AQP2 mRNA expression, followed by a rise in the AQP2 protein level within hours. 5,6 AQP2 can be degraded in proteasomes and lysosomes. 7,8 Ubiquitination directs proteins for degradation to both compartments. Monoubiquitination (mUb) is a signal for degradation in lysosomes, whereas polyubiquitination (pUb) is mainly linked to proteasomal degradation. 9 mUb of AQP2 is induced by FSK stimulation and occurs at the apical plasma membrane. 10 In WT5 cells, a model for AQP2 regulation, increased mUb of AQP2 persists

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 RhoGDI 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 translocatio

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.