H. W. Harris

Phosphorylation of Aquaporin-2 Does Not Alter the Membrane Water Permeability of Rat Papillary Water Channel-containing Vesicles

Antidiuretic hormone modulates the water permeability (P) of epithelial cells in the rat kidney by vesicle-mediated insertion and removal of the aquaporin-2 (AQP-2) water channel. AQP-2 possesses a single consensus cAMP-dependent protein kinase A (PKA) phosphorylation site (Ser-256) hypothesized to regulate channel P(Kuwahara, M., Fushimi, K., Terada, Y., Bai, L., Sasaki, S., and Marumo, F.(1995) J. Biol. Chem. 270, 10384-10387). To test whether PKA phosphorylation of AQP-2 alters channel P, we compared the P values of purified AQP-2 endosomes after incubation with either PKA or alkaline phosphatase. Studies using [-P]ATP reveal that AQP-2 endosomes contain endogenous PKA and phosphatase activities that add and remove P label from AQP-2. However, the P (0.16 ± 0.06 cm/s) of endosomes containing phosphorylated AQP-2 (0.7 ± 0.3 mol of PO/mol of protein) is not significantly different from the same AQP-2 endosomes where 95 ± 8% of the phosphate has been removed (P 0.14 ± 0.06 cm/s). These data do not support a role for PKA phosphorylation in alteration of AQP-2s P. Instead, AQP-2 phosphorylation by PKA may modulate AQP-2s distribution between plasma membrane and intracellular vesicle compartments.

Transport defects of rabbit medullary thick ascending limb cells in obstructive nephropathy.

To characterize the sodium transport defect responsible for salt wasting in obstructive nephropathy, the major sodium transporters in the medullary thick ascending limb (mTAL), the apical Na-K-2Cl cotransporter and the basolateral Na-K-ATPase, were studied in fresh suspensions of mTAL cells and outer medulla plasma membranes prepared from obstructed and untreated kidneys. Oxygen consumption (QO2) studies in intact cells revealed marked reductions in the inhibitory effects of both furosemide and ouabain on QO2 in cells from obstructed, as compared with control animals, indicating a reduction in activities of both the Na-K-2Cl cotransporter and the Na-K-ATPase. Saturable [3H]bumetanide binding was reduced in membranes isolated from obstructed kidneys, but the Kd for [3H]bumetanide was unchanged, indicating a decrease in the number of functional luminal Na-K-2Cl cotransporters in obstructed mTAL. Ouabain sensitive Na-K-ATPase activity in plasma membranes was also reduced, and immunoblots using specific monoclonal antibodies directed against the alpha and beta subunits of rabbit Na-K-ATPase showed decreased amounts of both subunits in outer medullas of obstructed kidney. A significant decrease in [3H]bumetanide binding was detected after 4 h of ureteral obstruction, whereas Na-K-ATPase activity at this time was still not different from control. We conclude that ureteral obstruction reduces the amounts of both luminal Na-K-2Cl cotransporter and basolateral Na-K-ATPase in mTAL of obstructed kidney and that these reductions contribute to the salt wasting observed after release of obstruction.

The molecular structure of the antidiuretic hormone elicited water channel

Measurements of osmotic water permeability (Pf) have shown that the plasma membranes of human red cells and certain epithelial cells possess specialized water channels. Although these water channels have been characterized extensively using biophysical techniques, the proteins that compose these unique channels have only recently been identified. Antidiuretic hormone (ADH) stimulation rapidly increases collecting duct epithelial cell Pf by fusion of water channel-containing vesicles (WCV) with their apical membranes. The proteins of WCV from toad bladder and rodent kidney have been characterized. The principal proteins in toad bladder WCV are 55,000 daltons (55 kDa) and 53 kDa and span the lipid bilayer of these vesicles. Polyclonal antisera raised against the 55-kDa and 53-kDa proteins inhibit ADH-stimulated toad bladder Pf by 80% and recognize protein bands of 46, 38 and 30 kDa in mouse kidney. Purification of WCV from rat kidney reveals enrichment of the 46-kDa protein. Recently, a 28-kDa integral membrane protein (called CHIP-28) has been isolated from human red cells. It forms functional water channels inXenopus oocytes and when reconstituted into proteoliposomes. Large amounts of CHIP-28 protein are present in epithelial cells of the proximal tubule and descending thin limb of Henles loop. Molecular cloning efforts are underway to elucidate the structure and function of these candidate water channel proteins.

Molecular aspects of water transport

Due to its fundamental importance, the movement of water across cell membranes has been an active area of research for more than 100 years. This subject is central to consideration of normal water metabolism by terrestrial animals, as well as derangements in overall water balance that are frequently encountered by nephrologists in the care of their patients. The objective of this review is to discuss the most basic aspects of cell membrane water permeability and provide a framework for these data in the context of the care of pediatric patients with renal disease. While the water permeability of most cell membranes can be accounted for by the diffusion of water across the lipid bilayer, other cells, including the red blood cell and certain epithelial cells that line the proximal and collecting tubules of the kidney and the urinary bladder of amphibians, possess specialized water channels. Water channels are composed of specialized proteins that create aqueous pores across cell membrane. Currently, there are active research efforts to isolate and characterize water channel proteins from these cell types. Data concerning the distribution, permeability and function of these various water channels will greatly enhance our knowledge of how water is transported across cell membranes.Due to its fundamental importance, the movement of water across cell membranes has been an active area of research for more than 100 years. This subject is central to consideration of normal water metabolism by terrestrial animals, as well as derangements in overall water balance that are frequently encountered by nephrologists in the care of their patients. The objective of this review is to discuss the most basic aspects of cell membrane water permeability and provide a framework for these data in the context of the care of pediatric patients with renal disease. While the water permeability of most cell membranes can be accounted for by the diffusion of water across the lipid bilayer, other cells, including the red blood cell and certain epithelial cells that line the proximal and collecting tubules of the kidney and the urinary bladder of amphibians, possess specialized water channels. Water channels are composed of specialized proteins that create aqueous pores across cell membrane. Currently, there are active research efforts to isolate and characterize water channel proteins from these cell types. Data concerning the distribution, permeability and function of these various water channels will greatly enhance our knowledge of how water is transported across cell membranes.