Expression Of Aquaporin Water Channels Research Articles

Altered expression of aquaporin water channels in a rat model of chronic diarrhea due to bile acid malabsorption

BackgroundBile acid malabsorption (BAM) is a condition where excess amounts of bile acids (BA) in the colon cause chronic watery diarrhea. BAM occur following resection or inflammation of the terminal ileum, cholecystectomy or as a primary condition, where the feedback regulation of bile acid (BA) synthesis is defective. The mechanisms by which BAs in the large intestine cause diarrhea are largely unknown. My aim is to determine if aquaporin water channels (AQPs) in the colonic epithelium are involved in the development of diarrhea during BAM.MethodsSix rats kept in metabolic cages were fed a diet containing 1% BA for 10 days as a model for BAM. Six other rats fed standard chow served as controls. Colonic epithelial cells were isolated by Ca2+‐chelation. Cells were processed for RNA and protein analyses. Colonic tissue was also prepared for histology.ResultsBA‐fed rats had greater feces dry weight and feces water content compared to rats fed standard chow. BA rats also had a higher water intake than controls. There were no differences in urine output or urine osmolality between groups. In contrast to controls, BA rats did not gain weight over time. The difference in weight was significant at the end of the experiment. Semi‐quantitative real‐time PCR of epithelial cells showed that organic solute transporter β (Slc51b) mRNA was increased in the colonic epithelium from the BA‐treated group compared to the control group indicating activation of the nuclear bile acid receptor (FXR). Aqp3 mRNA was increased in the distal colonic epithelium in the BA‐treated group, while mRNA for Aqp8 was increased in both the proximal and the distal colonic epithelium. mRNA for Aqp1, ‐4 and ‐7 was not different between groups. AQP3 was detected as a non‐glycosylated and a glycosylated form by western blotting. The glycosylated form was significantly reduced in the distal colonic epithelium of BA rats compared to controls. AQP8 was significantly increased in the distal colonic epithelium of BA rats compared to controls.PAS‐staining showed no clear differences in the histology of the colons from BA rats compared to colons from control rats. The expression pattern of AQP3 and ‐8 as shown with immunohistochemistry was not different between BA rats and controls.ConclusionIn a rat model of BAM we find increased expression of Aqp3 and Aqp8 mRNA and alterations of protein expression of AQP3 and ‐8 in the distal colon indicating a role for AQPs in the development of diarrhea during BAM.Support or Funding InformationSupport for my PhD‐study comes from the Faculty of Health's Research training supplement as well as from my main supervisor, Hanne Moeller, who received a Starting grant from Aarhus University Research Foundation, when she was hired as associate professor.

Vasopressin regulation of sodium transport in the distal nephron and collecting duct.

Arginine vasopressin (AVP) is released from the posterior pituitary gland during states of hyperosmolality or hypovolemia. AVP is a peptide hormone, with antidiuretic and antinatriuretic properties. It allows the kidneys to increase body water retention predominantly by increasing the cell surface expression of aquaporin water channels in the collecting duct alongside increasing the osmotic driving forces for water reabsorption. The antinatriuretic effects of AVP are mediated by the regulation of sodium transport throughout the distal nephron, from the thick ascending limb through to the collecting duct, which in turn partially facilitates osmotic movement of water. In this review, we will discuss the regulatory role of AVP in sodium transport and summarize the effects of AVP on various molecular targets, including the sodium-potassium-chloride cotransporter NKCC2, the thiazide-sensitive sodium-chloride cotransporter NCC, and the epithelial sodium channel ENaC.

Expression of aquaporin water channels in human urothelial carcinoma: correlation of AQP3 expression with tumour grade and stage

To study the expression, localization and potential clinical significance of aquaporin water channels both in well-established urothelial cancer (UC) cell lines and in human bladder carcinoma specimens of different stages and grades and to discuss the clinical relevance of the findings. AQP transcript and protein expression by RT4, RT112 and T24 UC cell lines was investigated using reverse transcriptase polymerase chain reaction and immunofluorescence labelling. Immunohistochemistry (IHC) was used to assess AQP protein expression in 94 UC specimens of various grades and stages. AQP3 and 9 transcripts were expressed in low-grade RT4 and RT112, but not in high-grade T24 cells. By contrast, AQP4 mRNA was absent in RT4, but expressed by RT112 and T24. Transcripts for AQP7 and 11 were detected in all three UC cell lines. Immunofluorescence microscopy confirmed the expression of AQP3, 4 and 7 at the protein level. By IHC, AQP3 was shown to be intensely expressed by 86 %, 66 % and 33 % of specimens of stage pTa, pT1 and pT2 tumours, respectively (p < 0.001). Whereas 100 % of G1 tumours were positive, only 73 % and 55 % of G2 and G3 tumours were found to express AQP3 (p = 0.004). This is the first study to demonstrate that several AQPs are expressed in UC. Our results indicate that there is a correlation between AQP3 protein expression and tumour stage and grade, with AQP3 expression being reduced or lost in tumours of higher grade and stage. Taken together with the available evidence from other studies, we conclude that AQPs may play a role in the progression of UC and, in particular, that this could be of prognostic value.

Expression of Aquaporin Water Channels in Equine Endometrium is Differentially Regulated During the Oestrous Cycle and Early Pregnancy

The expression of 12 different aquaporin subtypes in equine endometrium was examined at the mRNA and protein level. Endometrial samples were obtained during anoestrus, oestrus, 8, and 14days after ovulation in non-pregnant mares, and 14days after ovulation in pregnant mares. Quantitative PCR revealed a time-dependent pattern for all aquaporin subtypes examined except for AQP10 and 12. AQP3, 5 and 7 showed highest mRNA abundance 8days after ovulation, while AQP0 and 2 were most abundant at Day 14 of the cycle in non-pregnant mares. At 14days of pregnancy, AQP1, 4, 8, 9 and 11 displayed highest expression levels. Western blot analysis confirmed protein expression of AQP0, 2 and 5. Immunohistochemistry localized protein expression to luminal and glandular epithelial and stromal cells. AQP0 staining intensity was highest in samples obtained on Day 14 of the oestrous cycle. AQP2 immunoreactivity seemed to be stronger in samples collected 14days after ovulation from non-pregnant animals, in particular luminal epithelial staining. Samples collected 8days after ovulation from cyclic animals were characterized by intense AQP5 staining of glandular epithelium, predominantly in the deeper glands. Progesterone treatment of anoestrous mares did not enhance expression of AQPs, indicating that factors other than progesterone are required for the up-regulation of certain AQP subtypes during dioestrus. In conclusion, it seems that an equine-specific collaboration of aquaporin subtypes contributes to changes in endometrial fluid content occurring throughout the oestrous cycle and contributes to endometrial receptivity during early pregnancy in the mare.

Expression of aquaporin water channels in canine fetal adnexa in respect to the regulation of amniotic fluid production and absorption

Amniotic fluid (AF) is created by the flow of fluid from the fetal lung and bladder and reabsorbed in part by fetal swallowing and partly by the transfer across the amnion to the fetal circulation. Placental water flux is an important factor in determining AF volume and fetal hydration. In addition the fetal membranes might be involved in the regulation of fluid composition. To understand the mechanisms responsible for maintaining a correct balance of AF volume we evaluated the expression of aquaporins (AQPs) in canine fetal adnexa. AQPs are a family of integral membrane proteins permitting passive but physiologically rapid transcellular water movement. The presence of AQP1, 3, 5, 8 and −9 was immunohistochemically assessed in canine fetal adnexa, collected in early, middle and late-gestation during ovario-hysterectomies performed with fully informed owners' consent. Changes in AF volume and biochemical composition were also evaluated throughout pregnancy. Our results show distinct aquaporin expression patterns in maternal and extraembryonic tissues in relation to pregnancy period. AQP1 was localized in placental endothelia, allantochorion, amnion, allantois and yolk sac. AQP3 was present in the placental labyrinth, amnion, allantois and yolk sac. AQP8 was especially evident on the epithelia lining the glandular chambers, the amniotic and allantois sacs. AQP9, a channel highly permeable to water and urea, was observed in epithelia of amnion, allantois and yolk sac. In summary, AQP1, 3, 5, 8 and −9 have distinct expression patterns in canine fetal membranes and placenta in relation to pregnancy period, suggesting an involvement in mediating the AF changes during gestation.

Expression of Aquaporin Water Channels in the Vagina in Premenopausal Women

Aquaporins (AQPs) are membrane proteins that facilitate water movement across biological membranes. This study builds on a previous report on the distinct localization of AQPs in the rat vagina. The purposes of this study were to investigate the localization and expression of the AQPs in the vaginal tissue of premenopausal women. Anterior vaginal tissue was collected during transvaginal uterine myomectomy or hysterectomy from 10 premenopausal women (mean age, 40 years) for Western blot and immunohistochemistry. The expression and cellular localization of AQP1-9 were determined in the human vagina by Western blot and immunohistochemistry. Immunolabeling showed that AQP1 was mainly expressed in the capillaries and venules of the vagina, AQP2 was expressed in the cytoplasm of the epithelium, AQP3 was mainly associated with the plasma membrane of the vaginal epithelium, and both AQP5 and AQP6 were expressed in the cytoplasm throughout all vaginal epithelium. Western blot analysis revealed bands at 28 kDa for AQP1, 2, 3, 5, and 6 proteins. However, AQP4, 7, 8, and 9 were not detected. The distinct localization of AQPs in the human vagina suggests that AQP1, 2, 3, 5, and 6 may play an important role in vaginal lubrication in women.

Undernutrition during foetal to prepubertal life affects aquaporin 9 but not aquaporins 1 and 2 expression in the male genital tract of adult rats

Expression of aquaporin water channels (AQPs) in the male excurrent ducts, is of major importance for local water movements. To study the influence of pre- and postnatal undernutrition on AQP-expression in the adult male genital tract, 4 pregnant female rats were fed ad libitum (control group) and 4 with 33.5% of gestational feed requirements (underfed group). Feeding restriction of underfed group pups continued up to weaning (25 days of age), then all pups were fed ad libitum until slaughtered at 100 days of age. Epididymides were sampled and processed for aquaporin immunohistochemistry. Expression of AQP1 was similar either in the control and underfed groups of rats, strongly evidenced at the apical and lateral plasma membrane of the efferent ducts non-ciliated cells, in the smooth muscle cells surrounding epididymal duct and in blood vessel endothelium throughout the epididymis. AQP2-immunoreactivity was present in the corpus and cauda regions, strongly expressed in the principal cells of both groups of rats. In contrast, AQP9 expression was modified by early life undernourishment, as it was weakly evidenced at the microvilli in the principal cells and strongly diminished or completely lacked in the clear cells of the cauda, in underfed group epididymides. Since it is known that clear cells are involved in luminal fluid acidification, this function might be altered in adult animals, which were underfed during early life.

Altered Renal Expression of Aquaporin Water Channels and Sodium Transporters in Rats with Two-Kidney, One-Clip Hypertension

Aims: To determine whether the renal regulation of aquaporin (AQP) water channels and sodium transporters are altered in 2-kidney, 1-clip (2K1C) hypertension. Methods: Male Sprague-Dawley rats were used. They were made 2K1C hypertensive for 1 week. The renal expression of AQPs and sodium transporters was determined by semiquantitative immunoblotting and immunohistochemistry. The activity of adenylyl cyclase was measured by stimulated generation of cAMP. Results: Systolic blood pressure was increased in 2K1C rats. Experimental rats revealed impaired urinary concentration in association with increased urine volume. Urinary sodium excretion also increased. The expression of AQP1–3 was decreased in the clipped kidney compared with the control kidney, whereas it was unchanged in the non-clipped kidney. The adenylyl cyclase activity provoked by arginine vasopressin, sodium fluoride or forskolin was blunted in the clipped kidney, but remained unaltered in the contralateral kidney. The expressions of the Na,K-ATPase α1-subunit, type 3 Na⁺/H⁺ exchanger, Na-K-2Cl cotransporter and epithelial sodium channels were decreased in the clipped kidney, while remaining unchanged in the non-clipped kidney. Conclusion: The downregulation of AQPs and major sodium transporters/channels in the clipped kidney may play a role in the urinary concentration defect and impaired sodium reabsorption in 2K1C hypertension.

O.114 Ex vivo gene therapy of hydrocephalus: Intrathecal implantation of mesenchymal stem cells producing the neuroprotective peptide Glucagon like peptide-1

Urothelium is generally considered to be impermeable to water and constituents of urine. The possibility that human urothelium expresses aquaporin (AQP) water channels as the basis for water and solute transport has not previously been investigated.To investigate the expression of AQP water channels by human urothelium in situ, in proliferating urothelial cell cultures and in differentiated tissue constructs.AQP expression by human urothelium in situ and cultured urothelial cells was assessed by reverse transcriptase–polymerase chain reaction (RT-PCR) and immunolabelling. Expression screening was carried out on samples of freshly isolated urothelia from multiple surgical (bladder and ureteric) specimens and on proliferating and differentiated normal human urothelial (NHU) cells in culture. Urothelial tissue constructs were established and investigated for expression of urothelial differentiation markers and AQPs.Qualitative study.Transcripts for AQP3, AQP4, AQP7, AQP9, and AQP11 were expressed consistently by freshly isolated urothelia as well as by cultured NHU cells. AQP0, AQP1, AQP2, AQP5, AQP6, AQP8, AQP10, and AQP12 were not expressed. Immunochemistry confirmed expression of AQP3, AQP4, AQP7, and AQP9 at the protein level. AQP3 was shown to be intensely expressed at cell borders in the basal and intermediate layers in both urothelium in situ and differentiated tissue constructs in vitro.This is the first study to demonstrate that AQPs are expressed by human urothelium, suggesting a potential role in transurothelial water and solute transport. Our findings challenge the traditional concept of the urinary tract as an impermeable transit and storage unit and provide a versatile platform for further investigations into the biological and clinical relevance of AQPs in human urothelium.

Expression and Localisation of Aquaporin Water Channels in Human Urothelium In Situ and In Vitro

BackgroundUrothelium is generally considered to be impermeable to water and constituents of urine. The possibility that human urothelium expresses aquaporin (AQP) water channels as the basis for water and solute transport has not previously been investigated. ObjectiveTo investigate the expression of AQP water channels by human urothelium in situ, in proliferating urothelial cell cultures and in differentiated tissue constructs. Design, setting, and participantsAQP expression by human urothelium in situ and cultured urothelial cells was assessed by reverse transcriptase–polymerase chain reaction (RT-PCR) and immunolabelling. Expression screening was carried out on samples of freshly isolated urothelia from multiple surgical (bladder and ureteric) specimens and on proliferating and differentiated normal human urothelial (NHU) cells in culture. Urothelial tissue constructs were established and investigated for expression of urothelial differentiation markers and AQPs. MeasurementsQualitative study. Results and limitationsTranscripts for AQP3, AQP4, AQP7, AQP9, and AQP11 were expressed consistently by freshly isolated urothelia as well as by cultured NHU cells. AQP0, AQP1, AQP2, AQP5, AQP6, AQP8, AQP10, and AQP12 were not expressed. Immunochemistry confirmed expression of AQP3, AQP4, AQP7, and AQP9 at the protein level. AQP3 was shown to be intensely expressed at cell borders in the basal and intermediate layers in both urothelium in situ and differentiated tissue constructs in vitro. ConclusionsThis is the first study to demonstrate that AQPs are expressed by human urothelium, suggesting a potential role in transurothelial water and solute transport. Our findings challenge the traditional concept of the urinary tract as an impermeable transit and storage unit and provide a versatile platform for further investigations into the biological and clinical relevance of AQPs in human urothelium.

Analysis of Urinary and Plasma Electrolytes in a Rat Model of Unilateral Ureteric Obstruction (UUO)

Following unilateral ureteric obstruction (UUO), hydronephrosis is associated with long term reductions in renal blood flow (RBF) and glomerular filtration rate (GFR), tubular resistance to arginine-vasopressin (AVP), and reduced expression of aquaporin water channels and sodium (Na + ) and urea transporters. During UUO, renal function is contingent upon adaptive changes in the contralateral kidney. In the short term this response is associated with an increased GFR and a reduced expression of Na + transporters. We aimed to assess global renal function in UUO, including a comparison between the obstructed and non-obstructed kidneys. Adult male rats were subjected to UUO of either 3 or 10 days duration. Control rats underwent sham surgery. Before release of the ureteric ligature, blood and both bladder and obstructed renal pelvis urine was sampled, then both kidneys harvested. Urinary and plasma electrolytes and osmolality, and kidney weights, were measured. We observed i) mild uraemia in the obstructed animals, ii) extreme dilution of urine from the pelvis of the obstructed kidney in the absence of noteworthy alterations in plasma ions, and iii) evidence indicative of a contralateral diuresis/natriuresis. To the best of our knowledge, this is the first study simultaneously examining such an extensive range of urinary (bladder and obstructed renal pelvis) and plasma markers of renal function in UUO. The results demonstrate that UUO is associated with a small deterioration in overall renal function and marked changes in renal electrolyte handling, leading to changes in the composition of urinary output from both the non-obstructed and obstructed kidneys.

ORIGINAL RESEARCH–BASIC SCIENCE: Expression of Aquaporin Water Channels in Rat Vagina: Potential Role in Vaginal Lubrication

Aquaporins (AQPs) are membrane proteins that facilitate water movement across biological membranes. There has been little research on the role of AQPs in the female sexual arousal response. The purposes of this study were to investigate the localization and functional roles of AQP1, AQP2, and AQP3 in rat vagina. Female Sprague-Dawley rats (230-240 g, N = 20) were anesthetized. The vaginal branch of the pelvic nerve was stimulated for 60 seconds (10 V, 16 Hz, 0.8 ms), and the animals were sacrificed either immediately or 5 minutes later. The expression and cellular localization of AQP1, 2, and 3 were determined by Western blot and immunohistochemistry of the vagina. The intracellular membrane and plasma membrane fractions of the proteins in vaginal tissue were studied by immunoblot analysis with the differential centrifugation. The expression and cellular localization of AQPs, and pelvic nerve stimulation induced translocation of AQPs in rat vaginal tissue. Immunolabeling showed that AQP1 was mainly expressed in the capillaries and venules of the vagina. AQP2 was expressed in the cytoplasm of the epithelium, and AQP3 was mainly associated with the plasma membrane of the vaginal epithelium. AQPs were found to be present primarily in the cytosolic fraction of untreated tissues. The translocation of AQP1 and 2 isoforms from the cytosolic compartment to the membrane compartment was observed immediately after nerve stimulation and had declined at 5 minutes after nerve stimulation, while the subcellular localization of AQP3 was not changed by nerve stimulation. These results showed a distinct localization of AQPs in the rat vagina. Pelvic nerve stimulation modulated short-term translocation of AQP1 and 2. These results imply that AQPs may play an important role in vaginal lubrication.

Cloning and expression of aquaporin water channels in the euryhaline bull shark, Carcharhinus leucas

Cloning and expression of aquaporin water channels in the euryhaline bull shark, Carcharhinus leucas

Expression of Aquaporin Water Channels in Rat Taste Buds

In order to gain insight into the molecular mechanisms that allow taste cells to respond to changes in their osmotic environment, we have used primarily immunocytochemical and molecular approaches to look for evidence of the presence of aquaporin-like water channels in taste cells. Labeling of isolated taste buds from the fungiform, foliate, and vallate papillae in rat tongue with antibodies against several of the aquaporins (AQPs) revealed the presence of AQP1, AQP2, and AQP5 in taste cells from these areas. AQP3 antibodies failed to label isolated taste buds from any of the papillae. There was an apparent difference in the regional localization of AQP labeling within the taste bud. Antibodies against AQP1 and AQP2 labeled predominantly the basolateral membrane, whereas the AQP5 label was clearly evident on both the apical and basolateral membranes of cells within the taste bud. Double labeling revealed that AQP1 and AQP2 labeled many, but not all, of the same taste cells. Similar double-labeling experiments with anti-AQP2 and anti-AQP5 clearly showed that AQP5 was expressed on or near the apical membranes whereas AQP2 was absent from this area. The presence of these 3 types of AQPs in taste buds but not in non-taste bud-containing epithelia was confirmed using reverse transcription-polymerase chain reaction. Experiments using patch clamp recording showed that the AQP inhibitor, tetraethylammonium, significantly reduced hypoosmotic-induced currents in rat taste cells. We hypothesize that the AQPs may play roles both in the water movement underlying compensatory mechanisms for changes in extracellular osmolarity and, in the case of AQP5 in particular, in the gustatory response to water.

Regulation of Amniotic Fluid Volume

Water arrives in the mammalian gestation from the maternal circulation across the placenta. It then circulates between the fetal water compartments, including the fetal body compartments, the placenta and the amniotic fluid. Amniotic fluid is created by the flow of fluid from the fetal lung and bladder. A major pathway for amniotic fluid resorption is fetal swallowing; however, in many cases the amounts of fluid produced and absorbed do not balance. A second resorption pathway, the intramembranous pathway (across the amnion to the fetal circulation), has been proposed to explain the maintenance of normal amniotic fluid volume. Amniotic fluid volume is thus a function both of the amount of water transferred to the gestation across the placental membrane, and the flux of water across the amnion. Water flux across biologic membranes may be driven by osmotic or hydrostatic forces; existing data suggest that intramembranous flow in humans is driven by the osmotic difference between the amniotic fluid and the fetal serum. The driving force for placental flow is more controversial, and both forces may be in effect. The mechanism(s) responsible for regulating water flow to and from the amniotic fluid is unknown. In other parts of the body, notably the kidney, water flux is regulated by the expression of aquaporin water channels on the cell membrane. We hypothesize that aquaporins have a role in regulating water flux across both the amnion and the placenta, and present evidence in support of this theory. Current knowledge of gestational water flow is sufficient to allow prediction of fetal outcome when water flow is abnormal, as in twin–twin transfusion syndrome. Further insight into these mechanisms may allow novel treatments for amniotic fluid volume abnormalities with resultant improvement in clinical outcome.

Expression of Aquaporin Water Channels in the Rat Pituitary Gland

We investigated the expression of aquaporin (AQP) subtypes (AQP1-AQP9) in the rat pituitary gland by the reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Specific amplification by RT-PCR was observed in AQP1, AQP3, AQP4 and AQP5 but not in the others. AQP1 immunoreactivity was detected in the endothelial cells of blood vessels. No positive reaction to AQP3 was observed by immunohistochemistry, in spite of the detection of mRNA. AQP4 was localized in the supporting cells of the adenohypophysis and neurohypophysis. AQP5 was expressed in parts of the marginal cells in Rathke's residual pouch. These observations suggest that several subtypes of the AQP family are involved in regulation of water homeostasis in the rat pituitary gland.

Decreased Expression of Aquaporin Water Channels in Denervated Rat Kidney

Aims: A neural mechanism regulating aquaporin (AQP) water channels in the kidney was investigated. Methods: Male Sprague-Dawley rats were used. Renal denervation was induced by painting the renal vessels with 10% phenol. The expression of AQP1–4 proteins was determined in the denervated and contralateral kidneys. The expression was also examined in rats which were renally denervated and subjected to water restriction or deoxycorticosterone acetate (DOCA)-salt treatment. Results: Following the unilateral denervation, tissue contents of norepinephrine were significantly decreased in the denervated kidney, while increased in the contralateral kidney. Accordingly, the expression of AQP1–4 proteins was decreased by 15–40% in the denervated kidney, and increased by 30–50% in the contralateral kidney. Immunohistochemistry of AQP2 confirmed its decreases in the denervated kidney and increases in the contralateral kidney. In bilaterally denervated rats, the urine flow increased along with decreased osmolarity. The water restriction increased the expression of AQP channels, however, the magnitude of which was lower in the denervated than in the contralateral kidney. Renal denervation decreased the degree of DOCA-salt hypertension, along with lower expression of AQP channels. Conclusion: It is suggested that the sympathetic nerve should play a specific excitatory role in the regulation of AQP channels in the kidney.

Expression of aquaporin water channels in mouse spinal cord

Aquaporins (AQPs) are membrane proteins involved in water transport in many fluid-transporting tissues. Aquaporins AQP1, AQP4, and AQP9 have been identified in brain and hypothesized to participate in brain water homeostasis. Here we use reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry to describe the expression and immunolocalization of AQPs in adult mouse spinal cord. AQP4 was expressed in glial cells, predominantly in gray matter, and in astrocytic end-feet surrounding capillaries in spinal cord white matter. AQP9 expression extensively co-localized with glial fibrillary acidic protein-immunoreactive astrocytes, located predominantly in the white matter. AQP5 was detected by RT-PCR but not by immunohistochemical analysis. Interestingly, AQP8 was detected primarily in ependymal cells lining the fluid-filled central canal. The aquaporin expression pattern in spinal cord suggests involvement in water homeostasis and diseases associated with abnormal water fluxes such as spinal cord injury and syringomyelia.

Diminished expression of aquaporin water channels in ureteral-obstructed kidney in rats.

We investigated whether regulation of aquaporin (AQP) water channels is altered in the ureteral-obstructed kidney. Male Sprague-Dawley rats were unilaterally obstructed of their left proximal ureters for 48 h. The protein expression of AQP1-3 channels was determined in the kidney by Western blot analysis. The expression of AQP2 was also determined by immunohistochemistry. In order to specifically determine primary impairment of the pathway leading to an altered regulation of AQP channels stimulated by the arginine vasopressin (AVP)/cyclic adenosine monophosphate (cAMP) pathway, the catalytic activity and protein expression of different parts of the adenylyl cyclase complex were separately determined. In the previously obstructed kidney, urinary osmolality and free water reabsorption were greatly decreased. The expression of AQP2 proteins was decreased in the cortex, outer medulla and inner medulla. Immunohistochemistry also showed a marked decrease in AQP2 expression. The expression of AQP1 and AQP3 was decreased in the outer medulla and inner medulla. cAMP generation in response to AVP, sodium fluoride or forskolin was greatly decreased. The expression of Gsalpha and adenylyl cyclase VI proteins was decreased. The contralateral kidney showed minimal or no changes in these parameters. The reduced abundance of AQP water channels may at least partly account for the urinary concentration defect in the ureteral-obstructed kidney. The primary point of impairment of AQP channels regulated by the AVP/cAMP pathway may lie at the level of the catalytic unit of adenylyl cyclase.

Diminished renal expression of aquaporin water channels in rats with experimental bilateral ureteral obstruction.

Whether postobstructive diuresis could be related to altered regulation of aquaporin (AQP) water channels in the kidney was investigated. Male Sprague-Dawley rats underwent bilateral obstruction of the proximal ureters for 48 h. The renal expression of AQP1 to AQP4 proteins was then determined by Western blot and immunohistochemical analyses. For elucidation of the primary impairment in the upstream pathway leading to the expression of cAMP-mediated AQP channels, the expression of G(salpha) and that of adenylyl cyclase were also determined. For some rats, the obstruction was released for collection of urine samples. After the ureteral obstruction, the urinary flow rate was increased and free water reabsorption was decreased. In the obstructed kidneys, the expression of AQP1 to AQP3 was decreased in the cortex, outer medulla, and inner medulla, whereas that of AQP4 was decreased in the inner medulla. Immunoreactivities for AQP1 to AQP4 were also decreased in the obstructed kidneys. The protein expression of G(salpha) was decreased in the cortex, outer medulla, and inner medulla, whereas that of adenylyl cyclase VI was decreased in the outer and inner medullae. cAMP generation stimulated by arginine vasopressin was decreased in the cortex, outer medulla, and inner medulla. cAMP generation in response to forskolin was decreased in the outer and inner medullae, whereas that in response to sodium fluoride was decreased in the cortex, outer medulla, and inner medulla. These results suggest that a reduced abundance of AQP water channels in the kidney accounts in part for postobstructive diuresis. The primary impairment of AQP channels that are regulated via the arginine vasopressin/cAMP pathway may lie at the level of G proteins and adenylyl cyclase itself.