Aquaporin-1 Deletion Research Articles

Abstract 14736: Aquaporin 1 Modulates H 2 o 2 -mediated Endothelial Inflammaging

Background: Aquaporin 1 (AQP1), a key regulator of endothelial cell function, transports hydrogen peroxide (H 2 O 2 ) into the cells. H 2 O 2 -mediated inflammation has been implicated in endothelial dysfunction, yet specific roles of AQP1-mediated signaling pathways underlying age-dependent endothelial dysfunction remains incompletely understood. Methods: In these studies, we dissected AQP1-regulated inflammation modulation of eNOS signaling pathways in human aortic endothelial cells (HAEC) from passages 5 to 15 by exploiting siRNA approaches, live cell fluorescence imaging with genetically-encoded H 2 O 2 biosensor HyPer, biochemical and in vitro endothelial function assays. Results: We discovered that AQP1 expression remarkably increases in senescent HAEC (P.15) in association with significant increased SA-βgal activity compared to young cells (P.5). H 2 O 2 levels were increased in senescent cell cytosol revealed by fluorescence HyPer imaging. We found that senescence-associated increase in AQP1-mediated H 2 O 2 led to enhanced TNF-α, unlike HO-1, transcription (p<0.01). Moreover, immunofluorescence assay documented that AQP1 gene knock-down significantly ameliorated senescence-associated elevation of adhesion molecule ICAM-1. Immunoblot analyses demonstrated that increased AQP1 protein levels in senescent HAEC leads to significant increase in caveolin-1 phosphorylation (2-fold) and decreases in phosphorylation of AMPK (Thr172; 4-fold) and eNOS (Ser1177; 2-fold) compared to young ECs (for each, p<0.05, n>6). We discovered that AQP1 knock-down improved the reduced angiogenesis and wound healing capacity in association with eNOS down-regulation in senescent endothelial cells. Conclusion: These results establish that AQP1 plays a crucial role in the regulation of H 2 O 2 -mediated inflammation-associated endothelial senescence, and AQP1 deletion improves endothelial dysfunction by ameliorating ROS-modulated inflammaging.

Down-regulation of Aquaporin-1 mediates a microglial phenotype switch affecting glioma growth

Aquaporin 1 (AQP1), a transmembrane protein that forms water channels, has previously been shown to facilitate growth and progression of many types of tumors by modulating tumor cell migration, proliferation and angiogenesis. Here, we determined the impact of AQP1 expression in the tumor environment on the progression of brain tumors. Primary microglia from wild type(WT) and AQP1 knockout(KO) mice were used to test AQP1 effect on microglia function by using Western blot, quantative PCR, in an experimental in vivo mouse glioma model and organotypic brain slice culture. Deletion of AQP1 in the host tissue significantly reduced the survival of the mice implanted with GL261 glioma cells. The density of glioma-associated microglia/macrophages was almost doubled in AQP1KO mice. We found that factors secreted from GL261 cells decrease microglial AQP1 expression via the MEK/ERK pathway, and that inhibition of this pathway with Trametinib reduced tumor growth and prolonged the survival of tumor bearing mice, an effect which required the presence of microglia. Deletion of AQP1 in cultured microglia resulted in an increase in migratory activity and a decrease in TLR4-dependent innate immune responses. Our study demonstrates a functional relevance of AQP1 expression in microglia and hints to AQP1 as a potential novel target for glioma therapy.

Effect of inhibitors on oxygen permeability of wild type and knockout mouse red blood cells

We now recognize that the paradigm that all gases cross red blood cell (RBC) membranes by dissolving in and diffusing through the lipid phase is not correct. Work on human RBCs genetically deficient in aquaporin‐1 (AQP1) or the Rh complex (mainly RhAG) showed that these two proteins account for ~90% of membrane CO2 permeability (PM,CO2), and that 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonate (DIDS) reduces PM,CO2. We used stopped‐flow (SF) analysis of hemoglobin (Hb) absorbance spectra as we mixed oxygenated RBCs with an extracellular O2 scavenger, and determined the rate constant of Hb deoxygenation (kHbO2). Because kHbO2 depends on Hb kinetics as well as diffusion of O2 to the extracellular space, we used a mathematical model to estimate effects on membrane O2 permeability (PM,O2). We examined the effects of DIDS and chloromercuribenzenesulfonate (pCMBS), agents that decrease PM,CO2. We find that, in RBCs from wild type (WT) mice, pCMBS reduces kHbO2 by 64% (est. PM,O2 by ~84%) and DIDS, by 41% (est. PM,O2 by ~67%). Because pCMBS and DIDS likely act by targeting membrane proteins (rather than lipids), we hypothesized that O2, at least in part, crosses RBC membranes via the same membrane proteins that conduct CO2. Therefore, we examined RBCs from mice genetically deficient in AQP1, RhAG, or both, ± inhibitors. Deletion of AQP1 lowers kHbO2 by 11% (est. PM,O2 by ~27%); RhAG, by 16% (est. PM,O2 by ~35%); the double knockout (dKO), by 33% (est. PM,O2 by ~59%). The combination dKO + pCMBS lowers kHbO2 (vs. WT without inhibitors) by 77% (est. PM,O2 by ~91%), whereas dKO + DIDS lowers kHbO2 by 52% (est. PM,O2 by ~76%). The effects of pCMBS on estimated PM,O2 are very similar when we compare dKO vs. RhAG−/−, or when we compare WT vs. AQP1−/−. Thus, the deletion of AQP1 may have little effect on the sensitivity of PM,O2 to pCMBS—consistent with the hypothesis that AQP1 conducts O2 only via pathways other than the mercury‐sensitive monomeric pores (e.g., via the hydrophobic central pore). In summary, our data suggest that AQP1 and RhAG account for nearly 60% of RBC O2 permeability, and point towards the existence of other O2 channels that account for at least 30%.Support or Funding InformationSupported by ONR N00014‐15‐1‐2060, ONR N00014‐16‐1‐2535 to WFB and NIH K01‐DK107787 to ROThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Aqp-1 Gene Knockout Attenuates Hypoxic Pulmonary Hypertension of Mice.

Objective- Hypoxic pulmonary hypertension (HPH) is characterized by proliferative vascular remodeling. Abnormal pulmonary artery smooth muscle cells proliferation and endothelial dysfunction are the primary cellular bases of vascular remodeling. AQP1 (aquaporin-1) is regulated by oxygen level and has been observed to play a role in the proliferation and migration of pulmonary artery smooth muscle cells. The role of AQP1 in HPH pathogenesis has not been directly determined to date. To determine the possible roles of AQP1 in the pathogenesis of HPH and explore its possible mechanisms. Approach and Results- Aqp1 knockout mice were used, and HPH model was established in this study. Primary pulmonary artery smooth muscle cells, primary mouse lung endothelial cells, and lung tissue sections from HPH model were used. Immunohistochemistry, immunofluorescence and Western blot, cell cycle, apoptosis, and migration analysis were performed in this study. AQP1 expression was upregulated by chronic hypoxia exposure, both in pulmonary artery endothelia and medial smooth muscle layer of mice. Aqp1 deficiency attenuated the elevation of right ventricular systolic pressures and mitigated pulmonary vascular structure remodeling. AQP1 deletion reduced abnormal cell proliferation in pulmonary artery and accompanied with accumulation of HIF (hypoxia-inducible factor). In vitro, Aqp1 deletion reduced hypoxia-induced proliferation, apoptosis resistance, and migration ability of primary cultured pulmonary artery smooth muscle cells and repressed HIF-1α protein stability. Furthermore, Aqp1 deficiency protected lung endothelial cells from apoptosis in response to hypoxic injury. Conclusions- Our data showed that Aqp1 deficiency could attenuate hypoxia-induced vascular remodeling in the development of HPH. AQP1 may be a potential target for pulmonary hypertension treatment.

Mechanisms of Crystalloid versus Colloid Osmosis across the Peritoneal Membrane.

Background Osmosis drives transcapillary ultrafiltration and water removal in patients treated with peritoneal dialysis. Crystalloid osmosis, typically induced by glucose, relies on dialysate tonicity and occurs through endothelial aquaporin-1 water channels and interendothelial clefts. In contrast, the mechanisms mediating water flow driven by colloidal agents, such as icodextrin, and combinations of osmotic agents have not been evaluated.Methods We used experimental models of peritoneal dialysis in mouse and biophysical studies combined with mathematical modeling to evaluate the mechanisms of colloid versus crystalloid osmosis across the peritoneal membrane and to investigate the pathways mediating water flow generated by the glucose polymer icodextrin.ResultsIn silico modeling and in vivo studies showed that deletion of aquaporin-1 did not influence osmotic water transport induced by icodextrin but did affect that induced by crystalloid agents. Water flow induced by icodextrin was dependent upon the presence of large, colloidal fractions, with a reflection coefficient close to unity, a low diffusion capacity, and a minimal effect on dialysate osmolality. Combining crystalloid and colloid osmotic agents in the same dialysis solution strikingly enhanced water and sodium transport across the peritoneal membrane, improving ultrafiltration efficiency over that obtained with either type of agent alone.Conclusions These data cast light on the molecular mechanisms involved in colloid versus crystalloid osmosis and characterize novel osmotic agents. Dialysis solutions combining crystalloid and colloid particles may help restore fluid balance in patients treated with peritoneal dialysis.

Aquaporin-1 regulates platelet procoagulant membrane dynamics and in vivo thrombosis.

In response to collagen stimulation, platelets use a coordinated system of fluid entry to undergo membrane ballooning, procoagulant spreading, and microvesiculation. We hypothesized that water entry was mediated by the water channel aquaporin-1 (AQP1) and aimed to determine its role in the platelet procoagulant response and thrombosis. We established that human and mouse platelets express AQP1 and localize to internal tubular membrane structures. However, deletion of AQP1 had minimal effects on collagen-induced platelet granule secretion, aggregation, or membrane ballooning. Conversely, procoagulant spreading, microvesiculation, phosphatidylserine exposure, and clot formation time were significantly diminished. Furthermore, in vivo thrombus formation after FeCl3 injury to carotid arteries was also markedly suppressed in AQP1-null mice, but hemostasis after tail bleeding remained normal. The mechanism involves an AQP1-mediated rapid membrane stretching during procoagulant spreading but not ballooning, leading to calcium entry through mechanosensitive cation channels and a full procoagulant response. We conclude that AQP1 is a major regulator of the platelet procoagulant response, able to modulate coagulation after injury or pathologic stimuli without affecting other platelet functional responses or normal hemostasis. Clinically effective AQP1 inhibitors may therefore represent a novel class of antiprocoagulant antithrombotics.

Mechanisms of acid-base regulation in peritoneal dialysis.

Peritoneal dialysis (PD) contributes to restore acid-base homeostasis in patients with end-stage renal disease. The transport pathways for buffers and carbon dioxide (CO2) across the peritoneal membrane remain poorly understood. Combining well-established PD protocols, whole-body plethysmography and renal function studies in mice, we investigated molecular mechanisms of acid-base regulation in PD, including the potential role of the water channel aquaporin-1 (AQP1). After instillation in peritoneal cavity, the pH of acidic dialysis solutions increased within minutes to rapidly equilibrate with blood pH, whereas the neutral pH of biocompatible solutions remained constant. Predictions from the three-pore model of peritoneal transport suggested that local production of HCO3- accounts at least in part for the changes in intraperitoneal pH observed with acidic solutions. Carbonic anhydrase (CA) isoforms were evidenced in the peritoneal membrane and their inhibition with acetazolamide significantly decreased local production of HCO3- and delayed changes in intraperitoneal pH. On the contrary, genetic deletion of AQP1 had no effect on peritoneal transport of buffers and diffusion of CO2. Besides intraperitoneal modifications, the use of acidic dialysis solutions enhanced acid excretion both at pulmonary and renal levels. These findings suggest that changes in intraperitoneal pH during PD are mediated by bidirectional buffer transport and by CA-mediated production of HCO3- in the membrane. The use of acidic solutions enhances acid excretion through respiratory and renal responses, which should be considered in patients with renal failure.

Novel Endothelial Cell-Specific AQP1 Knockout Mice Confirm the Crucial Role of Endothelial AQP1 in Ultrafiltration during Peritoneal Dialysis.

The water channel aquaporin-1 (AQP1) mediates about 50% ultrafiltration during a 2-hour hypertonic dwell in global AQP1 knockout (AQP1-/-) mice. Although AQP1 is widely expressed in various cell types including mesothelial cells, the ultrafiltration has been assumed to be mediated via endothelial AQP1 of the peritoneum. The partial embryonic lethality and reduced body weight in AQP1-/- mice may reflect potential confounding phenotypic effects evoked by ubiquitous AQP1 deletion, which may interfere with functional analysis of endothelial AQP1. Using a Cre/loxP approach, we generated and characterised endothelial cell- and time-specific AQP1 knockout (AQP1fl/fl; Cdh5-Cre+) mice. Compared to controls, AQP1fl/fl; Cdh5-Cre+ mice showed no difference in an initial clinical and biological analysis at baseline, including body weight and survival. During a 1-hour 3.86% mini-peritoneal equilibration test (mini-PET), AQP1fl/fl; Cdh5-Cre+ mice exhibited strongly decreased indices for AQP1-related transcellular water transport (43.0% in net ultrafiltration, 93.0% in sodium sieving and 57.9% in free water transport) compared to controls. The transport rates for small solutes of urea and glucose were not significantly altered. Our data provide the first direct experimental evidence for the functional relevance of endothelial AQP1 to the fluid transport in peritoneal dialysis and thereby further validate essential predictions of the three-pore model of peritoneal transport.

Aquaporin‐1 retards renal cyst development in polycystic kidney disease by inhibition of Wnt signaling

ObjectiveAutosomal dominant polycystic kidney disease (ADPKD) is one of the most common human monogenic diseases. Aquaporin‐1 (AQP1) is expressed mainly at epithelial cell plasma membranes in renal proximal tubules, as well as in epithelial cells lining the cysts in ADPKD kidneys, postulating that AQP1 may be involved in cytogenesis. Here we investigated the role of AQP1 on ADPKD development.Methods: We used Aqp1/Pkd1 double knockout mice, embryonic kidney organ cultures, and matrix‐grown MDCK cells in this study to investigate the role of AQP1 in ADPKD. Expression of Wnt signaling‐related proteins were detected in MDCK and AQP1‐MDCK cells. Co‐immunoprecipitation was used to determine the interaction between AQP1 and β‐catenin,GSK3β,LRP6 and Axin1.Results: Kidney size and cyst number were significantly greater in AQP1‐null PKD mice than in AQP1‐expressing PKD mice. In embryonic kidney cultures, AQP1 deletion increased cyst development. In matrix‐grown MDCK cells, AQP1 inhibited cystogenesis and promoted tubulogenesis. AQP1 decreased β‐catenin and cyclinD1 expression, suggesting down‐regulation of Wnt signaling. Co‐immunoprecipitation showed AQP1 interaction with β‐catenin, GSK3β, LRP6 and Axin1. Biochemical analysis revealed decreased p‐β‐catenin and increased β‐catenin expression in Aqp1/Pkd1 double knockout mice, suggesting enhanced Wnt signaling.ConclusionsThese results suggest that AQP1 may be involved in maintaining the stability of the “destruction complex” to promote β‐catenin phosphorylation and suppress the Wnt signaling. This study reveals a previously unrecognized role of AQP1 in ADPKD and hence may provide new therapeutic targets.

Aquaporin-1 retards renal cyst development in polycystic kidney disease by inhibition of Wnt signaling.

Water channel aquaporin-1 (AQP1) is expressed at epithelial cell plasma membranes in renal proximal tubules and thin descending limb of Henle. Recently, AQP1 was reported to interact with β-catenin. Here we investigated the relationship between AQP1 and Wnt signaling in in vitro and in vivo models of autosomal dominant polycystic kidney disease (PKD). AQP1 overexpression decreased β-catenin and cyclinD1 expression, suggesting down-regulation of Wnt signaling, and coimmunoprecipitation showed AQP1 interaction with β-catenin, glycogen synthase kinase 3β, LRP6, and Axin1. AQP1 inhibited cyst development and promoted branching in matrix-grown MDCK cells. In embryonic kidney cultures, AQP1 deletion increased cyst development by up to ∼ 40%. Kidney size and cyst number were significantly greater in AQP1-null PKD mice than in AQP1-expressing PKD mice, with the difference mainly attributed to a greater number of proximal tubule cysts. Biochemical analysis revealed decreased β-catenin phosphorylation and increased β-catenin expression in AQP1-null PKD mice, suggesting enhanced Wnt signaling. These results implicate AQP1 as a novel determinant in renal cyst development that may involve inhibition of Wnt signaling by an AQP1-macromolecular signaling complex.

Water transport across the peritoneal membrane

Peritoneal dialysis involves diffusive and convective transports and osmosis through the highly vascularized peritoneal membrane. The capillary endothelium offers the rate-limiting hindrance for solute and water transport. It can be functionally described in terms of a three-pore model including transcellular, ultrasmall pores responsible for free-water transport during crystalloid osmosis. Several lines of evidence have demonstrated that the water channel aquaporin-1 (AQP1) corresponds to the ultrasmall pore located in endothelial cells. Studies in Aqp1 mice have shown that deletion of AQP1 is reflected by a 50% decrease in ultrafiltration and a disappearance of the sodium sieving. Haploinsufficiency in AQP1 is also reflected by a significant attenuation of water transport. Conversely, studies in a rat model and in PD patients have shown that the induction of AQP1 in peritoneal capillaries by corticosteroids is reflected by increased water transport and ultrafiltration, without affecting the osmotic gradient and small-solute transport. Recent data have demonstrated that a novel agonist of AQP1, predicted to stabilize the open-state conformation of the channel, modulates water transport and improves ultrafiltration. Whether increasing the expression of AQP1 or gating the already existing channels would be clinically useful in PD patients remains to be investigated.

Aquaporin 1 does not contribute to the progression of abdominal aortic aneurysm in mice

Abdominal aortic aneurysm (AAA) is an asymptomatic chronic dilatation of the abdominal aorta observed predominantly in elderly males. The disease is usually first discovered at the time of rupture and often has a lethal outcome. The mechanism of aneurysm progression is not well characterized, but includes extensive vascular remodeling. Aquaporin 1 (AQP1) is necessary for endothelial‐ and vascular smooth muscle cell migration in tumors and in hypoxic conditions. Thus, we hypothesized that deletion of AQP1 attenuates the formation and progression of abdominal aortic aneurysms. 10–12 weeks old male ApoE KO and AQP1‐ApoE dKO mice were fed western diet and given angiotensin II (Ang II, 1 μg/kg/min) via subcutaneous osmotic mini pumps for 28 days in order to increase blood pressure and induce AAA formation. At 28 days mice were euthanized and aorta dissected. As expected plasma Ang II concentration did not differ between ApoE KO and AQP1‐ApoE dKO mice (403 ± 96 pg/ml vs. 370 ± 74 pg/ml, n=10–11, p=0.8). There was no difference in the incidence of AAA formation (64% vs. 61%). AAA sizes were similar in ApoE KO and AQP1‐ApoE dKO mice, as measured by outer abdominal diameter (1.45 ± 0.18 mm vs. 1.25 ± 0.18 mm, n = 10–11, p=0.4), and by wet weight of the abdominal aorta (15.4 ± 3.7 mg vs. 14.7 ± 4.3 mg, n= 10–11, p=0.9). It is concluded that in ApoE KO mice on western diet, AQP1 does not contribute significantly to incidence and progression of abdominal aorta aneurysm.Supported by Alfred Andersen grant and Director Jacob Madsen & Olga Madsen's grant

Aquaporin-1 Promotes Angiogenesis, Fibrosis, and Portal Hypertension Through Mechanisms Dependent on Osmotically Sensitive MicroRNAs

Changes in hepatic vasculature accompany fibrogenesis, and targeting angiogenic molecules often attenuates fibrosis in animals. Aquaporin-1 (AQP1) is a water channel, overexpressed in cirrhosis, that promotes angiogenesis by enhancing endothelial invasion. The effect of AQP1 on fibrogenesis in vivo and the mechanisms driving AQP1 expression during cirrhosis remain unclear. The purpose of this study was to test the effect of AQP1 deletion in cirrhosis and explore mechanisms regulating AQP1. After bile duct ligation, wild-type mice overexpress AQP1 that colocalizes with vascular markers and sites of robust angiogenesis. AQP1 knockout mice demonstrated reduced angiogenesis compared with wild-type mice, as evidenced by immunostaining and endothelial invasion/proliferation in vitro. Fibrosis and portal hypertension were attenuated based on immunostaining, portal pressure, and spleen/body weight ratio. AQP1 protein, but not mRNA, was induced by hyperosmolality in vitro, suggesting post-transcriptional regulation. Endothelial cells from normal or cirrhotic mice were screened for microRNA (miR) expression using an array and a quantitative PCR. miR-666 and miR-708 targeted AQP1 mRNA and were decreased in cirrhosis and in cells exposed to hyperosmolality, suggesting that these miRs mediate osmolar changes via AQP1. Binding of the miRs to the untranslated region of AQP1 was assessed using luciferase assays. In conclusion, AQP1 promotes angiogenesis, fibrosis, and portal hypertension after bile duct ligation and is regulated by osmotically sensitive miRs.

Aquaporins at a glance

With a concentration of 55,000 mM, water is by far the most prevalent molecule in biological systems. For many years it was assumed that biological membranes are freely water permeable, so that the existence of a family of water channels would not have been predicted. However, biophysical studies in

WATER CHANNEL ACTIVITY OF PLASMA MEMBRANE AFFECTS CHONDROCYTE MIGRATION AND ADHESION

1. Recent studies indicate that the aquaporin-1 (AQP1) water channel is expressed in human and equine articular chondrocytes. The role of AQP1 in chondrocyte function has not been characterized. In the present study, we investigated the expression of the AQP1 water channel in cultured articular chondrocytes from wild-type (AQP1(+/+)) and AQP1-knockout (AQP1(-/-)) mice and characterized its function in chondrocyte proliferation, migration and adhesion. 2. Expression of AQP1 mRNA and protein was identified in freshly isolated neonatal AQP(+/+) chondrocytes. Immunofluorescence localized the AQP1 protein to the plasma membrane of AQP(+/+) chondrocytes in primary cultures. Relative plasma membrane water permeability of AQP1(+/+) chondrocytes was approximately 1.6-fold higher than that of AQP1(-/-) chondrocytes. 3. The chondrocyte proliferation rate was not affected by AQP1 deletion. However, the serum-induced transwell migration rate of AQP1(-/-) chondrocytes was markedly reduced compared with AQP1(+/+) chondrocytes (16.2 +/- 0.2 vs 27.1 +/- 0.3%, respectively; P < 0.01). Cell adhesion to type II collagen-coated plates was also significantly reduced in AQP1(-/-) chondrocytes compared with AQP1(+/+) chondrocytes (38.1 +/- 0.3 vs 51 +/- 1%, respectively; P < 0.01). 4. The results provided direct evidence that AQP1-mediated plasma membrane water permeability plays an important role in chondrocyte migration and adhesion.

Aquaporin-1 plays an essential role in water permeability and ultrafiltration during peritoneal dialysis

The water channel aquaporin-1 (AQP1) is considered as the molecular counterpart of the ultrasmall pore predicted by the three-pore model of fluid transport across the peritoneal membrane. However, the definitive proof of the implication of AQP1 in solute-free water transport, sodium sieving, and ultrafiltration (UF) during peritoneal dialysis (PD) is lacking, and the effects of its deletion on the structure of the membrane are unknown. Using real-time reverse transcriptase-polymerase chain reaction and immunogold electron microscopy, we showed that AQP1 is the most abundant member of the AQP gene family expressed in the mouse peritoneum, and the only one located in the capillary endothelium. Transport studies during a 2-h dwell demonstrated that, in comparison with Aqp1(+/+) littermates, Aqp1(-/-) mice had no sodium sieving; an approximately 70% decrease in the initial, solute-free UF; and an approximately 50% decrease in cumulative UF. These modifications occurred despite unchanged osmotic gradient and transport of small solutes in the Aqp1(-/-) mice. Heterozygous Aqp1(+/-) mice showed intermediate values in sodium sieving and initial UF, whereas cumulative UF was similar to Aqp1(+/+) mice. The deletion of AQP1 had no effect on the expression of other AQPs and on the density, structure, or diameter of peritoneal capillaries. These data provide direct evidence for the role of AQP1 during PD. They validate essential predictions of the three-pore model: (i) the ultrasmall pores account for the sodium sieving, and (ii) they mediate 50% of UF during a hypertonic dwell.

Aquaporins in endothelia

Aquaporin-1 (AQP1) water channels are expressed widely in microvascular endothelia outside of the central nervous system, including renal vasa recta and tumor microvessels, as well as in non-vascular endothelia in pleura, peritoneum, cornea, and lymphatics. In kidney, AQP1-facilitated water transport in outer medullary descending vasa recta is likely an important component of the urinary concentrating mechanism. However, in most vascular endothelia outside of kidney, it remains uncertain whether AQP1 expression and high water permeability are physiologically important. AQP1 in non-vascular endothelia at the inner corneal surface is involved in the maintenance of corneal transparency. Recently, a new role of AQP1 in endothelial cell migration was discovered in analyzing the cause of defective tumor angiogenesis in AQP1-deficient mice. AQP1 facilitates endothelial cell migration by a mechanism that may involve facilitated water transport across cell protrusions (lamellipodia). AQP1 inhibitors may thus have aquaretic and antiangiogenic activity.

Slowed diffusion in tumors revealed by microfiberoptic epifluorescence photobleaching

It has not been possible to measure diffusion deep in solid tissues such as tumors because of the limited light penetration of conventional optical techniques. Here we report a microfiberoptic epifluorescence photobleaching (MFEP) method in which photobleaching is done by laser epi-illumination through a multimode fiberoptic whose micron-sized tip can be introduced deep into tissues. We applied MFEP to measure the diffusion of fluorescent macromolecules in tumors in living mice, at depths well beyond those accessible by surface optical measurements. Macromolecule diffusion was slowed about twofold within 200 microm of the surface of a solid tumor, but was slowed greater than tenfold beyond 500 microm. Our results reveal a remarkable and previously unrecognized slowing of diffusion deep in tumors, which correlated with the differing tissue architectures of tumor periphery versus core, and with altered tumor vasculature produced by aquaporin-1 deletion. MFEP should have wide applications for measuring diffusion in organs, solid tumors and other light-inaccessible tissue masses.

Urea and urine concentrating ability in mice lacking AQP1 and AQP3

Aquaporin-1 (AQP1) and aquaporin-3 (AQP3) water channels expressed in the kidney play a critical role in the urine concentrating mechanism. Mice with AQP1 or AQP3 deletion have a urinary concentrating defect. To better characterize this defect, we studied the influence of an acute urea load (300 mumol ip) in conscious AQP1-null, AQP3-null, and wild-type mice. Urine was collected and assayed every 2 h, from 2 h before (baseline) to 8 h after the urea load. Mice of all genotypes excreted the urea load in approximately 4 h with the same time course. Interestingly, despite their low baseline, the AQP3-null mice raised their urine osmolality and urea concentration progressively after the urea load to values almost equal to those in wild-type mice at 8 h. In contrast, urine non-urea solute concentration did not change. Urine volume fell in the last 4 h to about one-fourth of basal values. AQP1-null mice increased their urine flow rate much more than AQP3-null mice and showed no change in urine osmolality and urea concentration. The urea load strongly upregulated urea transporter UT-A3 expression in all three genotypes. These observations show that the lack of AQP3 does not interfere with the ability of the kidney to concentrate urea but impairs its ability to concentrate other solutes. This solute-selective response could result from the capacity of AQP3 to transport not only water but also urea. The results suggest a novel role for AQP3 in non-urea solute concentration in the urine.

Reduced cerebrospinal fluid production and intracranial pressure in mice lacking choroid plexus water channel Aquaporin‐1

Aquaporin-1 (AQP1) is a water channel expressed strongly at the ventricular-facing surface of choroid plexus epithelium. We developed novel methods to compare water permeability in isolated choroid plexus of wild-type vs. AQP1 null mice, as well as intracranial pressure (ICP) and cerebrospinal fluid (CSF) production and absorption. Osmotically induced water transport was rapid in choroid plexus from wild-type mice and reduced by fivefold by AQP1 deletion. AQP1 deletion did not affect choroid plexus size or structure. By stereotaxic puncture of the lateral ventricle with a microneedle, ICP was 9.5 +/- 1.4 cm H2O in wild-type mice and 4.2 +/- 0.4 cm H2O in AQP1 null mice. CSF production, an isosmolar fluid secretion process, was measured by a dye dilution method involving fluid collections using a second microneedle introduced into the cisterna magna. CSF production in wild-type mice was (in microl min(-1)) 0.37 +/- 0.04 (control), 0.16 +/- 0.03 (acetazolamide-treated), and 1.14 +/- 0.15 (forskolin-treated), and reduced by approximately 25% in AQP1 null mice. Pressure-dependent CSF outflow, measured from steady-state ICP at different ventricular infusion rates, was not affected by AQP1 deletion. In a model of focal brain injury, AQP1 null mice had remarkably reduced ICP and improved survival compared with wild-type mice. The reduced ICP and CSF production in AQP1 null mice provides direct functional evidence for the involvement of AQP1 in CSF dynamics, suggesting AQP1 inhibition as a novel option for therapy of elevated ICP.