Aging kidney is associated in humans and rodents with polyuria and reduced urine concentrating ability. In senescent female WAG/Rij rats, this defect is independent of arginine-vasopressin (AVP)/V(2) receptor/cAMP pathway. It has been attributed to underexpression and mistargeting of aquaporin-2 (AQP2) water channel in the inner medullary collecting duct (IMCD). We showed previously that dDAVP administration could partially correct this defect. Since AQP2 can also be regulated by AVP-independent pathways in water deprivation (WD), we investigated AQP2 and phosphorylated AQP2 (p-AQP2) regulation in thirsted adult (10 mo old) and senescent (30 mo old) female WAG/Rij rats. Following 2-day WD, urine flow rate decreased and urine osmolality increased in both groups. However, in agreement with significantly lower cortico-papillary osmotic gradient with aging, urine osmolality remained lower in senescent animals. WD induced sixfold increase of plasma AVP in all animals which, interestingly, did not result in higher papillary cAMP level. Following WD, AQP2 and p-AQP2 expression increased hugely in 10- and 30-mo-old rats and their mistargeting in old animals was corrected. Moreover, the age-related difference in AQP2 regulation was abolished after WD. To further investigate the mechanism of AQP2 underexpression with aging, AQP2 mRNA was quantified by real-time RT-PCR. In the outer medulla, preservation of AQP2 protein expression was achieved through increased AQP2 mRNA level in senescent rats. In the IMCD, no change in AQP2 mRNA was detected with aging but AQP2 protein expression was markedly lower in 30-mo-old animals. In conclusion, there is a posttranscriptional downregulation of AQP2 with aging, which is abolished by WD.

The thermophilic cyanobacterium, Thermosynechococcus elongatus, has been grown in the presence of Sr2+ instead of Ca2+ with the aim of biosynthetically replacing the Ca2+ of the oxygen-evolving enzyme with Sr2+. Not only were the cells able to grow normally with Sr2+, they actively accumulated the ion to levels higher than those of Ca2+ in the normal cultures. A protocol was developed to purify a fully active Sr2+-containing photosystem II (PSII). The modified enzyme contained a normal polypeptide profile and 1 strontium/4 manganese, indicating that the normal enzyme contains 1 calcium/4 manganese. The Sr2+- and Ca2+-containing enzymes were compared using EPR spectroscopy, UV-visible absorption spectroscopy, and O2 polarography. The Ca2+/Sr2+ exchange resulted in the modification of the EPR spectrum of the manganese cluster and a slower turnover of the redox cycle (the so-called S-state cycle), resulting in diminished O2 evolution activity under continuous saturating light: all features reported previously by biochemical Ca2+/Sr2+ exchange in plant PSII. This allays doubts that these changes could be because of secondary effects induced by the biochemical treatments themselves. In addition, the Sr2+-containing PSII has other kinetics modifications: 1) it has an increased stability of the S3 redox state; 2) it shows an increase in the rate of electron donation from TyrD, the redox-active tyrosine of the D2 protein, to the oxygen-evolving complex in the S3-state forming S2; 3) the rate of oxidation of the S0-state to the S1-state by TyrD. is increased; and 4) the release of O2 is slowed down to an extent similar to that seen for the slowdown of the S3TyrZ. to S0TyrZ transition, consistent with the latter constituting the limiting step of the water oxidation mechanism in Sr2+-substituted enzyme as well as in the normal enzyme. The replacement of Ca2+ by Sr2+ appears to have multiple effects on kinetics properties of the enzyme that may be explained by S-state-dependent shifts in the redox properties of both the manganese complex and TyrZ as well as structural effects.

Biochemical and biophysical studies have shown that the strictly water-permeable aquaporins have a tetrameric structure, whereas results concerning the oligomeric state of GlpF, the glycerol facilitator of Escherichia coli, are dependent upon the analytical technique used. Here, we analyzed the oligomerization of the AQP3 aquaglyceroporin, which presents a mixed selectivity for water, glycerol, and urea. At first, based on transcript detection by reverse transcription-PCR from human erythroid tissues and membrane expression detected by flow cytometry analysis, we demonstrated that AQP3 is expressed on human and rat but not on mouse red blood cells. Then, the quaternary structure of AQP3 was determined using as models human red blood cell membranes, which carry both AQP1 and AQP3, and two heterologous expression systems: Xenopus laevis oocyte, for density and size estimation of aquaporins, and Saccharomyces cerevisiae yeast, which expressed a non-glycosylated form of AQP3. By velocity sedimentation in sucrose gradient after non-denaturing detergent solubilization, AQP3 was essentially found as mono- and dimeric species in conditions under which AQP1 preserved its tetrameric structure. Freeze-fracture studies on oocyte plasma membranes gave a size of AQP3 particles in favor of a dimeric or trimeric structure. Finally, by cross-linking experiments with red blood cell membranes, AQP3 is visible as different oligomeric structures, including a tetrameric one.

The mechanisms underlying the prevention of age-related polyuria by chronic food restriction were investigated in female WAG/Rij rats. The decreased osmolality of renal papilla observed in senescent rats was not corrected by food restriction. A reduced urea content in the inner medulla of senescent rats, fed ad libitum or food-restricted, was suggested by the marked decrease in expression of UT-A1 and UT-B1 urea transporters. Aquaporin-2 (AQP2) downregulation in the inner medulla of senescent rats was partially prevented by food restriction. Both AQP2 and the phosphorylated form of AQP2 (p-AQP2), the presence of which was diffuse within the cytoplasm of collecting duct principal cells in normally fed senescent rats, were preferentially targeted at the apical region of the cells in food-restricted senescent animals. Plasma vasopressin (AVP) was similar in 10- and 30-mo-old rats fed ad libitum, but was doubled in food-restricted 30-mo-old rats. This study indicates that 1) kidney aging is associated with a marked decrease in AQP2, UT-A1, and UT-B1 expression in the inner medulla and a reduced papillary osmolality; and 2) the prevention of age-related polyuria by chronic food restriction occurs through an improved recruitment of AQP2 and p-AQP2 to the apical membrane in inner medulla principal cells, permitted by increased plasma AVP concentration.

The Saccharomyces cerevisiae strain Sigma1278b possesses two putative aquaporins, Aqy1-1p and Aqy2-1p. Previous work demonstrated that Aqy1-1p functions as a water channel in Xenopus oocyte. However, no function could be attributed to Aqy2-1p in this system. Specific antibodies were used to follow the expression of Aqy1-1p and Aqy2-1p in the yeast. Aqy1-1p was never detected whatever the growth phase and culture conditions tested. In contrast, Aqy2-1p was detected only during the exponential growth phase in rich medium containing glucose. Aqy2-1p expression was repressed by hyper-osmotic culture conditions. Both immunocytochemistry and biochemical subcellular fractionation demonstrated that Aqy2-1p is located on the endoplasmic reticulum (ER) as well as on the plasma membrane. In microsomal vesicles enriched in ER, a water channel activity due to Aqy2-1p was detected by stopped-flow analysis. Our results show that the expression of aquaporins is tightly controlled. The physiological relevance of aquaporin-mediated water transport in yeast is discussed.

To test the involvement of the water channel aquaporin (AQP)-4 in gastric acid physiology, the human gastric cell line (HGT)-1 was stably transfected with rat AQP4. AQP4 was immunolocalized to the basolateral membrane of transfected HGT-1 cells, like in native parietal cells. Expression of AQP4 in transfected cells increased the osmotic water permeability coefficient (Pf) from 2.02 ± 0.3 × 10−4 to 16.37 ± 0.5 × 10−4 cm/s at 20°C. Freeze-fracture EM showed distinct orthogonal arrays of particles (OAPs), the morphological signature of AQP4, on the plasma membrane of AQP4-expressing cells. Quantitative morphometry showed that the density of OAPs was 2.5 ± 0.3% under basal condition and decreased by 50% to 1.2 ± 0.3% after 20 min of histamine stimulation, mainly due to a significant decrease of the OAPs number. Concomitantly, Pf decreased by ∼35% in 20-min histamine-stimulated cells. Both Pf and OAPs density were not modified after 10 min of histamine exposure, time at which the maximal hormonal response is observed. Cell surface biotinylation experiments confirmed that AQP4 is internalized after 20 min of histamine exposure, which may account for the downregulation of water transport. This is the first evidence for short term rearrangement of OAPs in an established AQP4-expressing cell line.

The mechanisms underlying age-related polyuria were investigated in 10- and 30-mo-old female WAG/Rij rats. Urinary volume and osmolality were 3.9 +/- 0.3 ml/24 h and 2,511 +/- 54 mosmol/kgH(2)O in adult rats and 12.8 +/- 0.8 ml/24 h and 1,042 +/- 44 mosmol/kgH(2)O in senescent animals. Vasopressin V(2) receptor mRNA did not significantly differ between 10 and 30 mo, and [(3)H]vasopressin binding sites in membrane papilla were reduced by 30%. The cAMP content of the papilla was unchanged with age, whereas papillary osmolality was significantly lowered in senescent animals. The expression of aquaporin-1 (AQP1) and -4 was mostly unaltered from 10 to 30 mo. In contrast, aquaporin-2 (AQP2) and -3 (AQP3) expression was downregulated by 80 and 50%, respectively, and AQP2 was markedly redistributed into the intracellular compartment, in inner medulla of senescent animals, but not in renal cortex. These results indicate that age-related polyuria is associated with a downregulation of AQP2 and AQP3 expression in the medullary collecting duct, which is independent of vasopressin-mediated cAMP accumulation.

The Kidd (JK) blood group locus encodes a urea transporter that is expressed on human red cells and on endothelial cells of the vasa recta in the kidney. Here, we report the identification in human erythroblasts of a novel cDNA, designated HUT11A, which encodes a protein identical to the previously reported erythroid HUT11 urea transporter, except for a Lys(44) --> Glu substitution and a Val-Gly dipeptide deletion after proline 227, which leads to a polypeptide of 389 residues versus 391 in HUT11. Genomic typing by polymerase chain reaction and transcript analysis by ribonuclease protection assay demonstrated that HUT11A encodes the true Kidd blood group/urea transporter protein, which carries only 2 Val-Gly motifs. Upon expression at high levels in Xenopus oocytes, the physiological Kidd/urea transporter HUT11A conferred a rapid transfer of urea (which was insensitive to p-chloromercuribenzene sulfonate or phloretin), a high water permeability, and a selective uptake of small solutes including amides and diols, but not glycerol and meso-erythritol. However, at plasma membrane expression levels close to the level observed in the red cell membrane, HUT11A-mediated water transport and small solutes uptake were absent and the urea transport was poorly inhibited by p-chloromercuribenzene sulfonate, but strongly inhibited by phloretin. These findings show that, at physiological expression levels, the HUT11A transporter confers urea permeability but not water permeability, and that the observed water permeability is a feature of the red cell urea transporter when expressed at unphysiological high levels.

The yeast YPR192w gene, which encodes a protein (Aqy1p) with strong homology to aquaporins (AQPs), was cloned from nine S. cerevisiae strains. The osmotic water permeability coefficient (Pf) of X. laevis oocytes expressing the gene cloned from the Sigma1278b strain (AQY1-1) was 5.7 times higher than the Pf of oocytes expressing the gene cloned from other strains (AQY1-2). Aqy1-1p, initially cloned without its C-terminus (Aqy1-1DeltaCp), mediated an approximately 3 times higher water permeability than the full-length protein. This corresponds to a 3-fold higher protein density in the oocyte plasma membrane, as shown by freeze-fracture electron microscopy. Pf measurements in yeast spheroplasts confirmed the presence of functional water channels in Sigma1278b and a pharmacological study indicated that this strain contains at least a second functional aquaporin.

Freeze-fracture electron microscopy (FFEM) of kidney collecting duct, muscle, astrocytes in brain, and other mammalian tissues has revealed regular square arrays of intramembrane particles called orthogonal arrays of particles (OAPs). Their possible role in membrane structure and transport have been proposed, and their absence or decrease has been noted in a variety of hereditary and acquired diseases. A transgenic mouse lacking water channel AQP4 was used to show that AQP4 is the OAP protein. FFEM was done on kidney, skeletal muscle, and brain from AQP4 wild-type [+/+], heterozygous [+/-] and knock-out [-/-] mice. The [-/-] mice did not express detectable AQP4 protein, but were grossly indistinguishable from [+/+] mice. FFEM was done on blinded samples of kidney, brain and muscle from 9 mice. In all 6 kidney samples from [+/+] and [+/-] mice, OAPs similar to those in AQP4-transfected CHO cells were found in basolateral membranes of collecting duct principal cells. In all muscle and brain samples from [+/+] and [+/-] mice, OAPs of identical ultrastructure to those in kidney were seen, but in smaller patch sizes. OAPs were not seen in any sample from [-/-] mice. Label-fracture analysis using a peptide-derived AQP4 polyclonal antibody showed immunogold labeling of OAPs in AQP4-expressing CHO cells. These studies provide direct evidence that AQP4 is required for formation of OAPs and is a component of OAPs, thus establishing the identity and function of OAPs.

Caco-2 cells, originated in a human colonic cancer, are currently used as model systems to study transepithelial transports. To further characterize their water permeability properties, clone P1 Caco-2 cells were cultured on permeable supports. At confluence, the transepithelial net water movement (JW), mannitol permeability (Ps), and electrical resistance (R) were simultaneously measured. The observed results were correlated with transmission and freeze-fracture electron microscopy studies and compared with those obtained, in similar experimental conditions, in a typical mammalian epithelial barrier: the rabbit rectum. When the serosal solution was made hypertonic (50 mm polyethylene glycol-PEG), the spontaneously observed secretory Jw rapidly reversed, became absorptive and then stabilized. Simultaneously, the R values dropped and Ps went up. In the case of the rabbit rectal epithelium, a similar treatment did not elicit significant changes in the water permeability during the first 20 min following the osmotic challenge while there was a significant increase in the transepithelial resistance. After exposure to serosal hypertonicity, several morphological modifications developed in the Caco-2 cells: Localized dilations in the intercellular spaces and vacuoles in the cytoplasm appeared. Nevertheless, most cells remained in contact and no evidence of cell shrinking was observed. Simultaneously, the tight-junction structure was more or less disorganized. The filament network lost its sharpness and “omega” figures appeared, bordering the intercellular spaces. In some cases the tight-junction network was completely disrupted. In the case of the rabbit rectum the structural modifications were completely different: Serosal hypertonicity rapidly induced cell shrinking and the opening of the intercellular spaces, with no noticeable change in the tight-junction structure. These results suggest that Caco-2-P1 cell membranes, contrary to the case of the basolateral membrane of rabbit rectal cells, have no water channels and that a paracellular route could play a central role in the water movements across this epithelial barrier.

The temperature-sensitive Saccharomyces cerevisiae mutant strain NY17, deficient in the secretory pathway (sec6-4 mutation), is used for the heterologous expression of the human CHIP28 water channel. After a heat-shock, the protein is present in partially purified post-golgi secretory vesicles. Immunodetection and water transport studies, directly made on the vesicles, showed that CHIP28 is highly expressed and active in the yeast membranes.

The urinary bladder of the aquatic toad Xenopus laevis is known to exhibit a low permeability to water and a poor sensitivity to antidiuretic hormone. In order to precise the characteristics and the specific cellular mechanisms of this reduced hydroosmotic response we used a sensitive volumetric technique to monitor net water flow and studied the correlation between the anti-diuretic hormone (ADH)-induced net water flow and the fine ultrastructural appearence of the urinary bladder epithelium. Transmural net water flow was entirely dependent on the osmotic gradient across the preparation and not on the hydrostatic pressure difference. We observed the existence of a low but significant hydro-osmotic response to arginine vasopressin. Freeze-fracture electron microscopy demonstrated the presence of typical aggrephores in the subapical cytoplasm. The response to the hormone was accompanied by the appearance of typical intramembrane aggregates into the apical plasma membrane. Water permeability increase and apical aggregate insertion were both slowly but fully reversible. Except for the multilayered structure of the epithelium and the particularly low response to antidiuretic hormone, all the studied permeability and ultrastructural characteristics of the bladder were thus very similar to those observed in other sensitive epithelia such as the amphibian bladder and skin and the mammalian collecting duct which exhibit a high hydro-osmotic response to the hormone.

1. In salt-acclimated frogs, water and urea bladder permeabilities are markedly higher than in tap water-acclimated animals. 2. Intra-membrane particle aggregates (IMPA) cover an unusually large surface area of the salt-acclimated frog bladder apical plasma membrane. 3. In saline-adapted animals, proteins extracted from the apical plasma membrane contain additional species of 19, 26, 31 and 53-61 kDa. These proteins might be related to the water channels contained by IMPA.