Estrogen stimulates water imbibition in the uterine endometrium. This water then crosses the epithelial cells into the lumen, leading to a decrease in viscosity of uterine luminal fluid. To gain insight into the mechanisms underlying this estrogen-stimulated water transport, we have explored the expression profile and functionality of water channels termed aquaporins (AQPs) in the ovariectomized mouse uterus treated with ovarian steroid hormones. Using immunocytochemical analysis and immunoprecipitation techniques, we have found that AQP-1, -3, and -8 were constitutively expressed. AQP-1 expression was restricted to the myometrium and may be slightly regulated by ovarian steroid hormones. AQP-3 was expressed at low levels in the epithelial cells and myometrium, whereas AQP-8 was found in both the stromal cells and myometrium. AQP-2 was absent in vehicle controls but strongly up-regulated by estrogen in the epithelial cells and myometrium of the uterus. This localization implicates all four isotypes in movement of water during uterine imbibition and, based on their localization to the luminal epithelial cells, AQP-2 and -3 in facilitating water movement into the lumen of the uterus. The analysis of the plasma membrane permeability of luminal epithelial cells by two separate cell swelling assays confirmed a highly increased water permeability of these cells in response to estrogen treatment. This finding suggests that estrogen decreases the luminal fluid viscosity, in part, by enhancing the water permeability of the epithelial layer, most likely by increasing the expression of AQP-2 and/or the availability of AQP-3. Together these results provide novel information concerning the mechanism by which estrogen controls water imbibition and luminal fluid viscosity in the mouse uterus.

Apoptosis is characterized by a conserved series of morphological events beginning with the apoptotic volume decrease (AVD). This study investigated a role for aquaporins (AQPs) during the AVD. Inhibition of AQPs blocked the AVD in ovarian granulosa cells undergoing growth factor withdrawal and blocked downstream apoptotic events such as cell shrinkage, changes in the mitochondrial membrane potential, DNA degradation, and caspase-3 activation. The effects of AQP inhibition on the AVD and DNA degradation were consistent in thymocytes and with two additional apoptotic signals, thapsigargin and C(6)-ceramide. Overexpression of AQP-1 in Chinese hamster ovary (CHO-AQP-1) cells enhanced their rate of apoptosis. The AVD is driven by loss of K(+) from the cell, and we hypothesize that after the AVD, AQPs become inactive, which halts further water loss and allows K(+) concentrations to decrease to levels necessary for apoptotic enzyme activation. Swelling assays on granulosa cells, thymocytes, and CHO-AQP-1 cells revealed that indeed, the shrunken (apoptotic) subpopulation has very low water permeability compared with the normal-sized (nonapoptotic) subpopulation. In thymocytes, AQP-1 is present and was shown to colocalize with the plasma membrane receptor tumor necrosis factor receptor-1 (TNF-R1) both before and after the AVD, which suggests that this protein is not proteolytically cleaved and remains on the cell membrane. Overall, these data indicate that AQP-mediated water loss is important for the AVD and downstream apoptotic events, that the water permeability of the plasma membrane can control the rate of apoptosis, and that inactivation after the AVD may help create the low K(+) concentration that is essential in apoptotic cells. Furthermore, inactivation of AQPs after the AVD does not appear to be through degradation or removal from the cell membrane.

Ovarian folliculogenesis is characterized, in part, by the formation and expansion of the fluid-filled antrum. Development of this cavity requires water influx, which may occur by transcellular or pericellular transport mechanisms. To assess the contribution of these mechanisms to the water permeability of an antral follicle, the rate of (3)H(2)O and (14)C-inulin (a complex sugar restricted to the extracellular compartment) uptake into isolated follicles was determined. The rate of H(2)O movement was 3.5-fold greater than that of inulin, suggesting that water enters a follicle primarily by transcellular pathways. Preincubation of the follicles with 50 microM HgCl(2) [a nonspecific aquaporin (Aqp) inhibitor] decreased H(2)O movement to levels seen with inulin, indicating that transcellular water movement is mediated through Aqp. To demonstrate the functional presence of Aqp in granulosa cells, we show that swelling in response to a hypotonic insult is attenuated by preincubation with 50 microM HgCl(2). Flow cytometry demonstrated the presence of Aqps-7, -8, and -9, thus identifying candidate Aqp potentially mediating water movement into antral follicles. These results suggest that water permeability of antral follicles occurs primarily through transcellular mechanisms, which may be mediated by Aqps -7, -8, and/or -9 in granulosa cells.

Objective To correlate renal calculi and other clinical factors with urinary biochemical analytes in patients with inflammatory bowel disease, and to investigate the relative importance of hyperoxaluria (associated with fat malabsorption) or reduced stone inhibitors in the development of calculi in these patients. Patients, subjects and methods Samples were obtained from 25 patients with Crohn's disease (CD), 15 with ulcerative colitis (UC) and 17 normal subjects (controls). Evidence for the presence of renal calculi was obtained from plain films, ultrasonography or intravenous urography. Urine oxalate and citrate were analysed using commercial enzymatic assays; magnesium was measured using atomic absorption and other analytes assayed using standard methods on automated analysers. Results Renal calculi were found in two patients with CD and in none with UC. Hyperoxaluria was present in 36% of patients with CD but was absent in those with UC. Analysis of covariance showed an association between low urinary citrate/creatinine ratio and renal stones (P=0.02), and between a combined urinary citrate and magnesium deficit relative to calcium, as expressed in the CMC index ((citrater magnesium)/calcium), and renal stones (P=0.017). Changes in urinary calcium, oxalate, urate, magnesium or the calcium oxalate index were not associated with the presence of stones. There was no independent relationship between any clinical factor and the presence of stones. Conclusion Lower urinary concentrations of magnesium and citrate (stone inhibitors), relative to calcium (stone promoter; the CMC index) may be more important in lithogenesis in inflammatory bowel disease than is hyperoxaluria. In patients with a functioning colon, a low CMC index may predict likely stone-formers; this requires a prospective evaluation. Avoiding low urinary levels of magnesium and citrate may aid in preventing and treating renal calculi.

Ovarian folliculogenesis is characterized, in part, by the formation and expansion of the fluid-filled antrum. Development of this cavity requires water influx, which may occur by transcellular or pericellular transport mechanisms. To assess the contribution of these mechanisms to the water permeability of an antral follicle, the rate of (3)H(2)O and (14)C-inulin (a complex sugar restricted to the extracellular compartment) uptake into isolated follicles was determined. The rate of H(2)O movement was 3.5-fold greater than that of inulin, suggesting that water enters a follicle primarily by transcellular pathways. Preincubation of the follicles with 50 microM HgCl(2) [a nonspecific aquaporin (Aqp) inhibitor] decreased H(2)O movement to levels seen with inulin, indicating that transcellular water movement is mediated through Aqp. To demonstrate the functional presence of Aqp in granulosa cells, we show that swelling in response to a hypotonic insult is attenuated by preincubation with 50 microM HgCl(2). Flow cytometry demonstrated the presence of Aqps-7, -8, and -9, thus identifying candidate Aqp potentially mediating water movement into antral follicles. These results suggest that water permeability of antral follicles occurs primarily through transcellular mechanisms, which may be mediated by Aqps -7, -8, and/or -9 in granulosa cells.

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