Aquaporin-2 downregulation in kidney medulla of aging rats is posttranscriptional and is abolished by water deprivation.

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 Wip1 phosphatase (PPM1D) antagonizes activation of the Chk2 tumour suppressor kinase.

We previously demonstrated that type 2C protein phosphatases (PP2C) Ptc2 and Ptc3 are required for DNA checkpoint inactivation after DNA double-strand break repair or adaptation in Saccharomyces cerevisiae. Here, we show the conservation of this pathway in mammalian cells. In response to DNA damage, ataxia telangiectasia mutated (ATM) phosphorylates the Chk2 tumour suppressor kinase at threonine 68 (Thr68), allowing Chk2 kinase dimerization and activation by autophosphorylations in the T-loop. The oncogenic protein Wip1, a PP2C phosphatase, binds Chk2 and dephosphorylates phospho-Thr68. Consequently, Wip1 opposes Chk2 activation by ATM after ionizing irradiation of cells. In HCT15 colorectal cancer cells corrected for functional Chk2 activity, Wip1 overexpression suppressed the contribution of Chk2 to the G2/M DNA damage checkpoint. These results indicate that Wip1 is one of the phosphatases regulating the activity of Chk2 in response to DNA damage.

Downregulation of aquaporin-2 and -3 in aging kidney is independent of V(2) vasopressin receptor.

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.

Localization of the FA-CHIP water channel in frog urinary bladder.

Like mammalian kidney collecting duct, the water permeability of frog urinary bladder epithelial cells is antidiuretic hormone (ADH)-sensitive. In kidney, this permeability is mediated by water channels named aquaporins. We recently reported the cloning of the frog aquaporin CHIP (FA-CHIP), a water channel from frog urinary bladder. FA-CHIP has 79% identity with rat Aquaporin 1 (AQP1) and only 42% identity with the kidney collecting duct Aquaporin 2 (AQP2). The purpose of this study was to examine the localization of FA-CHIP in frog urinary bladder. We raised antibodies against peptides of 15 to 17 residues, encompassing the N-ter and C-ter regions of FA-CHIP. Anti-FA-CHIP antibodies were used for Western blotting, indirect immunofluorescence microscopy and gold labeling electron microscopy in urinary bladder and other frog tissues. By Western blotting of frog urinary bladder total homogenate, the antibodies recognized a band of 29 kDa and glycosylated forms of the protein between 40 and 70 kDa. No signal was found on membrane preparations from epithelial cell homogenate. FA-CHIP was also found in frog skin, brain, gall bladder, and lung. In immunofluorescence microscopy on urinary bladder sections, FA-CHIP was localized to endothelial cells of blood capillaries and on mesothelial cells of the serosal face. Red blood cells, epithelial and basal cells were unstained. The localization of FA-CHIP in cell plasma membranes was confirmed by gold labeling electron microscopy. In other positive tissues, FA-CHIP was also localized to capillaries. In brain, plasma membranes of epithelial cells were also stained. In conclusion, like its mammalian homologue AQP1, FA-CHIP appears to be localized to constitutively water permeable cells of frog. Therefore, it belongs to the AQP1 family of proteins although unlike AQP1, FA-CHIP is absent from red blood cells and kidney. In frog urinary bladder and skin, FA-CHIP probably plays an important role in water transport across the barriers in series with the ADH-sensitive epithelial cells.

Glycerol permeability of mutant aquaporin 1 and other AQP-MIP proteins: inhibition studies.

In a recent work, we showed that the aquaporins 1 (AQP1) are permeable to certain small solutes such as glycerol. Here, we have further investigated the permeation pathway of glycerol through human AQP1 (hAQP1) by the use of mutants (C189S, H180A, H209A) and inhibitors such as P-chloromercuribenzene sulphonate (pCMBS), CuSO4 or phloretin, in comparison with other AQP-MIP (where MIP denotes major intrinsic protein) proteins: hAQP2, plant water channel gammaTIP and bacterial glycerol permease facilitator, GlpF. Glycerol movements were measured in Xenopus laevis oocytes. Apparent glycerol permeability coefficients (P'gly) were calculated from the rates of oocyte swelling upon exposure to an isoosmotic medium containing an inwardly directed gradient of glycerol and from [3H]glycerol uptake measurements. Similar P'gly values were obtained for hAQP1 and hAQP2 6 to 8 times greater than control indicating that hAQP2 also transports glycerol. P'gly of hAQP2-injected oocytes was pCMBS and CuSO4 sensitive. In contrast, the P'gly value of gammaTIP was close to that of control, indicating that gammaTIP does not transport glycerol. The hAQP1-C189S, -H180A and -H209A mutants gave P'gly values similar to those obtained for wild hAQP1, indicating that these mutations did not affect glycerol movements. However, the H209A mutant has an osmotic water permeability coefficient (Pf) value decreased by 50%. The inhibitory effect pCMBS on P'gly was maintained for the 2 His mutants and, more interestingly, was also conserved for the C189S mutant. CuSO4 significantly inhibited P'gly of oocytes expressing hAQP1, hAQP1-C189S, -H180A, and -H209A mutants and had no effect on P'gly of GlpF-injected oocytes. Phloretin was shown to inhibit by around 80% the glycerol fluxes of wild and mutant hAQP1, hAQP2 and to fully inhibit glycerol uptake in GlpF-injected oocytes.

Functional expression of the human CHIP28 water channel in a yeast secretory mutant.

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.

Sequence and functional expression of an amphibian water channel, FA-CHIP: a new member of the MIP family.

A new member of the family of water channel proteins (aquaporin-CHIP) related to the major intrinsic protein (MIP) family is described. The cDNA coding for this amphibian CHIP was cloned from frog (Rana esculenta) urinary bladder, a model for the kidney collecting duct, using a RT-PCR cloning strategy. The encoded protein, designated FA-CHIP (frog aquaporin-CHIP), shows 77.4%, 42.4% and 35.6% identity with the three proteins now referred to as the aquaporins of the MIP family, i.e., human CHIP28, WCH-CD and gamma-TIP, respectively. Xenopus leavis injected with FA-CHIP cRNA exhibited a marked increase of the osmotic water permeability.

Neutral aminopeptidase: a potential marker enzyme of the amphibian urinary bladder epithelial cell apical membrane.

Antidiuretic hormone induces, in the apical plasma membrane of amphibian urinary bladder epithelial cells, the exocytotic insertion of intramembranous particle aggregates that probably contain water channels. Purification of the apical membrane is a way to characterize the aggregates. The isolation of such purified membranous fractions involves the use of specific exogenous or endogenous markers. One of them could be the neutral aminopeptidase (AP), whose activity was detected in urinary bladder. Enrichment in AP activity was observed in plasma membrane preparations compared to cell homogenates (X2.7). However, a large part of the enzyme activity was also recovered in the soluble fraction of the preparation, suggesting large proteolysis of the protein. The enzyme presents a low optimal pH (6.4) and a high specificity for proline-p-nitroanilide as compared to the AP present in kidneys and intestines. To localize the protein in the amphibian bladder epithelium, an immunological approach was necessary due to the low activity of the enzyme in this tissue. The low enzymatic activity also prevented the purification of sufficient amounts of the urinary bladder AP as antigen, and we prepared antibodies against purified AP from frog or toad kidneys where the activity is 60 times higher than in the bladder. The serum specificity was verified by spot immunodetection, Western blot, inhibition capacity of antibodies, and immunoadsorption on a solid support with the renal enzyme. The sera were found to be able to react with native as well as denatured forms of the kidney enzyme. Antibodies cross-reacted with several peptides of low molecular weight (40-60 kDa) from urinary bladder plasma membrane proteins (Western blot).(ABSTRACT TRUNCATED AT 250 WORDS)

Apical material extracted from amphibian urinary bladder epithelium by enzymes and detergent treatment.

The apical plasma membrane of epithelial cells of frog and toad urinary bladder is subject to large modifications during the induction of water permeability by the antidiuretic hormone. A better characterization of the apical membrane is necessary for a clear understanding of the mechanisms of hormone action. Towards this end, apical material was extracted by enzymatic treatment and by incubation with detergent. Proteolytic enzyme alone had little effect under our conditions. A pretreatment with several glycosidases (alpha-mannosidase or endo-beta-N-acetylglucosaminidase H) increased the hydrolytic action of papain, elastase, proteinase K or Staphylococcus aureus V8 protease and allowed the detection of a major 76 kD in SDS gel electrophoresis. The n-octyl-beta-D-glucopyranoside (0.2%) led to the extraction after 150 mn of 1 to 5 micrograms proteins per cm2 of amphibian urinary bladder apical surface. The extracted proteins migrated as several bands on SDS gels. One of them probably corresponds to the 76 kD fragment obtained after proteolysis. The absence of alteration of the water permeability after extraction and the good preservation of the ultrastructure are evidence for the localisation of the 76 kD at the apical membrane surface. This protein may be the best candidate as antigen to raise antibodies against the apical surface of amphibian urinary bladder epithelial cells.