Cisplatin decreases the abundance of aquaporin water channels in rat kidney.
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The present study examined whether the cisplatin induced urinary concentration defect can be related to an altered regulation of aquaporin (AQP) water channels in the kidney. Cisplatin (8 mg/kg) was injected intraperitoneally into male Sprague-Dawley rats. The control group was without cisplatin treatment. Four d later, the expression of AQP1, AQP2, and AQP3 proteins was determined in the kidney. To specify further the primary point of derangement in the pathway that activates the arginine vasopressin-mediated AQP channels, different components of adenylyl cyclase complex were examined separately. The cisplatin treatment caused a polyuric renal failure in association with decreases of free water reabsorption. The expression of AQP1 and AQP2 was decreased in the cortex, the outer medulla, and the inner medulla, whereas that of AQP3 was decreased in the outer medulla and the inner medulla. The expression of AQP2 proteins in the apical membrane-enriched fraction decreased in parallel with that in the subapical vesicle-enriched fraction, indicating a preserved targeting. Immunohistochemistry of the outer medulla also revealed that cisplatin decreased immunoreactivity for AQP1, AQP2, and AQP3. The arginine vasopressin-evoked generation of cyclic adenosine monophosphate was attenuated by cisplatin, being most prominent in the outer medulla. However, the cyclic adenosine monophosphate generation in response to forskolin was not affected, whereas that to sodium fluoride was diminished significantly. Cisplatin also decreased the expression of Gsalpha proteins in the outer medulla and the inner medulla. These results suggest that a reduced expression of AQP water channels accounts at least in part for the cisplatin-induced urinary concentration defect. (+info)
Expression of aquaporin-1 in human peritoneal mesothelial cells and its upregulation by glucose in vitro.
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Aquaporin (AQP) is a family of water channels that are highly selective for the passage of water and occasionally glycerol. In previous studies, only AQP1 was found in human peritoneal endothelial cells in both control subjects and patients on peritoneal dialysis. As human peritoneal mesothelial cells (HPMC) play an important role in dialysis adequacy and fluid balance in continuous ambulatory peritoneal dialysis patients, this study examined whether AQP1 is present in HPMC. It was found that AQP1 mRNA and protein are present in HPMC constitutively. The localization of AQP1 protein in peritoneal mesothelial cells was confirmed by double immunohistochemical staining of the mesothelial lining of human peritoneal membrane. More important, the expression of AQP1 in HPMC is not constitutive and the transcription and biosynthesis of AQP1 in HPMC is inducible by osmotic agents such as glucose and mannitol. There was significant enhancement of AQP1 biosynthesis upon exposure to glucose in a time- and dose-dependent manner (P < 0.0001). Similar findings were observed in the AQP1 biosynthesis by an endothelial cell line, EA.hy 926. Of particular interest, the upregulation in AQP1 mRNA or biosynthesis in mesothelial cells was always significantly higher than that of endothelial cells when the experiments were conducted under identical settings (P < 0.001). AQP1 expression in HPMC was demonstrated for the first time. Osmotic agents upregulate both mRNA and protein expression of this aquaporin. The role of AQP1 in HPMC in maintaining the ultrafiltration of the peritoneal membrane is potentially of clinical interest. (+info)
Gentamicin decreases the abundance of aquaporin water channels in rat kidney.
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The present study was performed to examine whether the gentamicin-induced urinary concentration defect is related to an altered regulation of aquaporin (AQP) water channels in the kidney. Male Sprague-Dawley rats were subcutaneously injected with gentamicin (20, 50 or 100 mg/kg per day) for 6 days. The protein expression of AQP1-3 channels and the catalytic activity of adenylyl cyclase were determined in the kidney. Gentamicin treatment resulted in renal failure associated with decreased tubular free water reabsorption and increased urinary flow rate. The expression of AQP2 proteins was significantly decreased in the kidney, in which the cortex was most susceptible, followed by the outer medulla and inner medulla in order. Gentamicin treatment also decreased the shuttling of AQP2, as evidenced by a decrease of its expression in the membrane fraction in proportion to that in the cytoplasmic fraction. The protein expression of AQP1 as well as that of AQP3 was also decreased in the cortex by treatment with the highest dose of gentamicin. The cAMP generation in response to arginine vasopressin or sodium fluoride was decreased by gentamicin, while that to forskolin was not significantly altered. These findings suggest that the primary impairment in the pathway leading to the generation of cAMP lies at the level of G proteins, resulting in a decreased expression of cAMP-mediated AQP channels. The gentamicin-induced urinary concentration defect may in part be accounted for by a reduced abundance of AQP water channels in the kidney. (+info)
Downregulation of AQP1, -2, and -3 after ureteral obstruction is associated with a long-term urine-concentrating defect.
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Previously, we demonstrated that 24 h of bilateral ureteral obstruction (BUO) and short-term release of BUO was associated with a decrease in the expression of aquaporin-2 (AQP2), polyuria, and a reduced urinary concentrating capacity (10). The purposes of the present study were to examine whether BUO and the long-term release of BUO (BUO-R) for 3, 14, and 30 days were associated with changes in the expression of renal AQP1, AQP2, and AQP3 and whether such changes were associated with parallel changes in urinary output and urinary concentrating capacity. Rats (n = 4-7 in each group) were kept in metabolic cages for measurements of urinary output. Kidneys were removed to determine the expression levels of AQP1, AQP2, and AQP3 by semiquantitative immunoblotting. AQP2 was downregulated after 24 h of BUO (42 +/- 3%). Downregulation of AQP2 persisted 3 (43 +/- 14%; P < 0.01) and 15 days after BUO-R (48 +/- 11%; P < 0.01) but was normalized 30 days after BUO-R. AQP3 showed a similar pattern. Moreover, AQP1 was downregulated in response to BUO (65 +/- 7%) and remained downregulated 3 days after BUO-R (41 +/- 5%), 14 days after BUO-R (57 +/- 8%), and 30 days after BUO-R (59 +/- 5%). BUO-R resulted in a significant polyuria that gradually decreased, although it remained significant at day 30. Urinary concentrating capacity remained significantly impaired when determined 3, 14, and 30 days after BUO-R in response to a 24-h period of thirst (1,712 +/- 270 vs. 2,880 +/- 91 mosmol/kgH2O at day 30, P < 0.05). In conclusion, the expression of AQP1, AQP2, and AQP3 were long-term downregulated after BUO-R, suggesting that dysregulation of aquaporins located at the proximal tubule, thin descending limb of the loop of Henle, and the collecting duct may contribute to the long-term polyuria and impairment of urinary concentrating capacity associated with obstructive nephropathy. (+info)
Impaired hearing in mice lacking aquaporin-4 water channels.
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A role for aquaporins (AQPs) in hearing has been suggested from the specific expression of aquaporins in inner ear and the need for precise volume regulation in epithelial cells involved in acoustic signal transduction. Using mice deficient in selected aquaporins as controls, we localized AQP1 in fibrocytes in the spiral ligament and AQP4 in supporting epithelial cells (Hensen's, Claudius, and inner sulcus cells) in the organ of Corti. To determine whether aquaporins play a role in hearing, auditory brain stem response (ABR) thresholds were compared in wild-type mice and transgenic null mice lacking (individually) AQP1, AQP3, AQP4, and AQP5. In 4-5-week-old mice in a CD1 genetic background, ABR thresholds in response to a click stimulus were remarkably increased by >12 db in AQP4 null mice compared with wild-type mice (p < 0.001), whereas ABR thresholds were not affected by AQP1, AQP3, or AQP5 deletion. In a C57/bl6 background, nearly all AQP4 null mice were deaf, whereas ABRs could be elicited in wild-type controls. ABRs in AQP4 null CD1 mice measured in response to tone bursts (4-20 kHz) indicated a frequency-independent hearing deficit. Light microscopy showed no differences in cochlear morphology of wild-type versus AQP4 null mice. These results provide the first direct evidence that an aquaporin water channel plays a role in hearing. AQP4 may facilitate rapid osmotic equilibration in epithelial cells in the organ of Corti, which are subject to large K(+) fluxes during mechano-electric signal transduction. (+info)
Identification and localization of aquaporin water channels in human salivary glands.
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Aquaporin (AQP) water channels are expressed in a variety of fluid-transporting epithelia and are likely to play a significant role in salivary secretion. Our aim was to identify and localize the aquaporins expressed in human salivary glands. Total RNA was extracted from human parotid, submandibular, sublingual, and labial glands and from human brain. Expression of aquaporin mRNA was assessed by RT-PCR using specific primers for human AQP1, AQP3, AQP4, and AQP5. All four aquaporins were detected by RT-PCR in all of the glands, and the sequences were confirmed after further amplification with nested primers. Cleaned PCR products were then used as (32)P-labeled cDNA probes in a semiquantitative Northern blot analysis using glyceraldehyde-3-phosphate dehydrogenase as reference. Only AQP1, AQP3, and AQP5 mRNAs were present at significant levels. AQP localization was determined by immunohistochemistry on paraffin sections using affinity-purified primary antibodies and peroxidase-linked secondary antibodies. Each salivary gland type showed a broadly similar staining pattern: AQP1 was localized to the capillary endothelium and myoepithelial cells; AQP3 was present in the basolateral membranes of both mucous and serous acinar cells; AQP4 was not detected; and AQP5 was expressed in the luminal and canalicular membranes of both types of acinar cell. We conclude that AQP3 and AQP5 together may provide a pathway for transcellular osmotic water flow in the formation of the primary saliva. (+info)
Water and ion permeation of aquaporin-1 in planar lipid bilayers. Major differences in structural determinants and stoichiometry.
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The aquaporin-1 (AQP1) water channel protein is known to facilitate the rapid movement of water across cell membranes, but a proposed secondary role as an ion channel is still unsettled. Here we describe a method to simultaneously measure water permeability and ion conductance of purified human AQP1 after reconstitution into planar lipid bilayers. Water permeability was determined by measuring Na(+) concentrations adjacent to the membrane. Comparisons with the known single channel water permeability of AQP1 indicate that the planar lipid bilayers contain from 10(6) to 10(7) water channels. Addition of cGMP induced ion conductance in planar bilayers containing AQP1, whereas cAMP was without effect. The number of water channels exceeded the number of active ion channels by approximately 1 million-fold, yet p-chloromethylbenzenesulfonate inhibited the water permeability but not ion conductance. Identical ion channel parameters were achieved with AQP1 purified from human red blood cells or AQP1 heterologously expressed in Saccharomyces cerevisae and affinity purified with either N- or C-terminal poly-histidine tags. Rp-8-Br-cGMP inhibited all of the observed conductance levels of the cation selective channel (2, 6, and 10 pS in 100 mm Na(+) or K(+)). Deletion of the putative cGMP binding motif at the C terminus by introduction of a stop codon at position 237 yielded a truncated AQP1 protein that was still permeated by water but not by ions. Our studies demonstrate a method for simultaneously measuring water permeability and ion conductance of AQP1 reconstituted into planar lipid bilayers. The ion conductance occurs (i) through a pathway distinct from the aqueous pathway, (ii) when stimulated directly by cGMP, and (iii) in only an exceedingly small fraction of AQP1 molecules. (+info)
Expression of aquaporin-1 in human trabecular meshwork cells: role in resting cell volume.
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PURPOSE: Drainage of aqueous humor from the human eye appears dependent on intracellular volume of trabecular meshwork (TM) cells, the predominant cell type of the human outflow pathway. Thus, the modulation of water and solute flux across the plasma membrane of TM cells is predicted to be an important factor in regulating outflow facility. Aquaporin (AQP)-1 is a hexahelical integral membrane protein that functions as a regulated channel for water and cations in fluid-secreting and -absorbing tissues. AQP1 is present in many tissues of the human eye, including the TM; however, its role in outflow facility is unknown. The purpose of the present study was twofold: to evaluate the prospect of manipulating AQP1 protein levels in TM cells using sense and antisense mRNA and to investigate the functional role of AQP1 in TM cells. METHODS: An adenovirus (AV) expression system was used to alter AQP1 protein levels. AQP1 protein expression was monitored using immunoblot analysis, and resting cell volume was measured by forward light scatter, electronic cell sizing, and [(14)C]-sucrose/urea equilibration. Permeability of TM monolayers to [(14)C]-sucrose was also assessed as an indirect evaluation of cell volume. RESULTS: AV-mediated gene transfer of AQP1 cDNA to TM cells resulted in a titer-dependent increase in recombinant AQP1, whereas transfer of antisense cDNA decreased native AQP1 protein by 71.7% +/- 5.5% (P < 0.01) after 5 days. A novel finding of this study is that mean resting volumes of AQP1(s) AV-infected TM cells in suspension were 8.7% +/- 3.0% greater (P < 0.05) than control cells. Conversely, AQP1 antisense (as) AV-infected cells had resting volumes 7.8% +/- 2.9% less than control cells (P < 0.05). Similar effects of AQP1 expression on resting cell volume were observed in TM monolayers. Consistent with this finding, paracellular permeability of AQP1(s) AV-infected TM monolayers to [(14)C]-sucrose decreased by 8.0% +/- 1.4% (P < 0.001). CONCLUSIONS: In addition to influencing the osmotic permeability of TM plasma membranes, the level of AQP1 protein expression influences resting intracellular volume and thus paracellular permeability of TM cell monolayers in vitro. These data suggest that AQP1 expression may affect outflow facility in vivo. (+info)