CHIP28 is an integral membrane protein that has been identified as the erythrocyte water channel and that is also expressed in the kidney. Antibodies against erythrocyte CHIP28 were used to localize this protein along the rat urinary tubule. By Western blotting, CHIP28 was detected in kidney plasma membrane and endosome fractions. With the use of immunocytochemistry, CHIP28 was located in brush-border and basolateral plasma membranes of the proximal tubule. The initial S1 segment was weakly stained, but the S2 and S3 segments were heavily labeled. Subapical vesicles were also positive. Apical and basolateral membranes of the long thin descending limb were strongly labeled, but ascending thin and thick limbs of Henle and distal convoluted tubules were negative. Some vasa recta profiles in the medulla were positive. CHIP28 is, therefore, present in membranes with a high constitutive water permeability, where it probably acts as a transmembrane water-conducting channel. Finally, a weak staining of apical and basolateral membranes of cortical collecting duct principal cells was detectable, suggesting a potential relationship of CHIP28 to the vasopressin-sensitive water channel.

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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In this study we analyzed the expression of aquaporin-4 (AQP4) in mammalian skeletal muscle. Immunohistochemical experiments revealed that affinity-purified AQP4 antibodies stained selectively the sarcolemma of fast-twitch fibers. By immunogold electron microscopy, little or no intracellular labeling was detected. Western blot analysis showed the presence of two immunopositive bands with apparent molecular masses of 30 and 32 kD specifically present in membrane fraction of a fast-twitch rat skeletal muscle (extensor digitorum longus, EDL) and not revealed in a slow-twitch muscle (soleus). PCR Southern blot experiments resulted in a selective amplification in EDL of a 960-bp cDNA fragment encoding for the full-length rat form of AQP4. Functional experiments carried out on isolated skeletal muscle bundle fibers demonstrated that the osmotic response is faster in EDL than in soleus fibers isolated from the same rat. These results provide for the first time evidence for the expression of an aquaporin in skeletal muscle correlated to a specific fiber-type metabolism.

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

. Rho inhibits cAMP-induced translocation of aquaporin-2 into the apical membrane of renal cells. Am J Physiol Renal Physiol 281: F1092-F1101, 2001. First published August 8, 2001; 10.1152/ajprenal.00091.2001We have recently demonstrated that actin depolymerization is a prerequisite for cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) into the apical membrane in AQP2-transfected renal CD8 cells (29). The Rho family of small GTPases, including Cdc42, Rac, and Rho, regulates the actin cytoskeleton. In AQP2-transfected CD8 cells, inhibition of Rho GTPases with Clostridium difficile toxin B or with C. limosum C3 fusion toxin, as well as incubation with the Rho kinase inhibitor, Y-27632, caused actin depolymerization and translocation of AQP2 in the absence of the cAMP-elevating agent forskolin. Both forskolin and C3 fusion toxin-induced AQP2 translocation were associated with a similar increase in the osmotic water permeability coefficient. Expression of constitutively active RhoA induced formation of stress fibers and abolished AQP2 translocation in response to forskolin. Cytochalasin D induced both depolymerization of F-actin and AQP2 translocation, suggesting that depolymerization of F-actin is sufficient to induce AQP2 translocation. Together, these data indicate that Rho inhibits cAMP-dependent translocation of AQP2 into the apical membrane of renal principal cells by controlling the organization of the actin cytoskeleton.aquaporin; C3 toxin; toxin B; actin cytoskeleton; G proteins; adenosine 3Ј,5Ј-cyclic monophosphate IN RENAL PRINCIPAL CELLS, the antidiuretic hormone arginine-vasopressin (AVP) regulates the shuttling of the water channel AQP2 from intracellular vesicles into the plasma membrane (8,14,34). AVP binds vasopressin V 2 receptors coupled to the G s -adenylyl cyclase system. Activation of this system results in the activation of protein kinase A (PKA), which induces phosphorylation of AQP2 at Ser 256 (10, 12). The phosphorylation of AQP2 by PKA and also the anchoring of PKA to subcellular compartments via protein kinase A anchoring proteins (AKAPs) are prerequisites for AQP2 translocation to the cell membrane (11, 12).F-actin has been demonstrated to be involved in AVP-mediated changes of osmotic water permeability (3,4,12,28). Depolymerization of cortical F-actin has been considered an important prerequisite for exocytosis. Calcium/ATP causes cortical F-actin disassembly (calmodulin dependent) and secretion (calmodulin independent) in permeabilized mast cells (25). Thus the cortical actin network may represent a cage that blocks the exocytotic process, and its disassembly constitutes an early stage of this reaction. In toad bladder, a renal-like epithelium, as well as in rat collecting duct epithelium, the total F-actin content decreases by 20-30 and 26%, respectively, after stimulation with vasopressin (5, 24). In AQP2-transfected renal collecting duct cells (CD8 cells), okadaic acid, an inhibitor of 1 and 2A phosphatases, induces actin depolymerization, leading to AQP2 transl...

Abstract. Channel forming integral protein of 28 kD (CHff'28) functions as a water channel in erythrocytes, kidney proximal tubule and thin descending limb of Henle. CHIP28 morphology was examined by freeze-fracture EM in proteoliposomes reconstituted with purified CHIP28, CHO cells stably transfected with CHIP28k cDNA, and rat kidney tubules. Liposomes reconstituted with HPLC-purified CHIP28 from human erythrocytes had a high osmotic water permeability (Pf 0.04 cm/s) that was inhibited by HgCl2. Freeze-fracture replicas showed a fairly uniform set of intramembrane particles (IMPs); no IMPs were observed in liposomes without incorporated protein. By rotary shadowing, the IMPs had a diameter of 8.5 + 1.3 nm (mean + SD); many IMPs consisted of a distinct arrangement of four smaller subunits surrounding a central depression. IMPs of similar size and appearance were seen on the P-face of plasma membranes from CHIP28k-transfected (but not mocktransfected) CHO cells, rat thin descending limb (TDL) of Henle, and S3 segment of proximal straight tubules. A distinctive network of complementary IMP imprints was observed on the E-face of CHIP28-contaming plasma membranes. The densities of IMPs in the size range of CHIP28 IMPs, determined by nonlinear regression, were (in IMPs/~m2): 2,494 in CHO cells, 5,785 in TDL, and 1,928 in proximal straight tubules; predicted Pf, based on the CHIlW28 single channel water permeability of 3.6 x 10 -14 cm3/s (10°C), was in good agreement with measured Pf of 0.027 cm/s, 0.075 cm/s, and 0.031 cm/s, respectively, in these cell types. Assuming that each CHIP28 monomer is a right cylindrical pore of length 5 nm and density 1.3 g/cm 3, the monomer diameter would be 3.2 nm; a symmetrical arrangement of four cylinders would have a greatest diameter of 7.2 nm, which after correction for the thickness of platinum deposit, is similar to the measured IMP diameter of ,,~8.5 nm. These results provide a morphological signature for CHIP28 water channels and evidence for a tetrameric assembly of CHIP28 monomers in reconstituted proteoliposomes and cell membranes.

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.