Knock-out models reveal new aquaporin functions

Abstract

Knockout mice have been informative in the discovery of unexpected biological functions of aquaporins. Knockout mice have confirmed the predicted roles of aquaporins in transepithelial fluid transport, as in the urinary concentrating mechanism and glandular fluid secretion. A less obvious, though predictable role of aquaporins is in tissue swelling under stress, as in the brain in stroke, tumor and infection. Phenotype analysis of aquaporin knockout mice has revealed several unexpected cellular roles of aquaporins whose mechanisms are being elucidated. Aquaporins facilitate cell migration, as seen in aquaporin-dependent tumor angiogenesis and tumor metastasis, by a mechanism that may involve facilitated water transport in lamellipodia of migrating cells. The ' aquaglyceroporins', aquaporins that transport both glycerol and water, regulate glycerol content in epidermis, fat and other tissues, and lead to a multiplicity of interesting consequences of gene disruption including dry skin, resistance to skin carcinogenesis, impaired cell proliferation and altered fat metabolism. An even more surprising role of a mammalian aquaporin is in neural signal transduction in the central nervous system. The many roles of aquaporins might be exploited for clinical benefit by modulation of aquaporin expression/function - as diuretics, and in the treatment of brain swelling, glaucoma, epilepsy, obesity and cancer.

Fig. 1

Impaired urinary concentrating ability in AQP null mice. (a) Sites of AQP expression in kidney. (b) Twenty-four hour urine collections showing polyuria in mice lacking AQP1 and AQP3, individually and together. (c) Urine osmolalities before and after 36 h water deprivation (S.E.). (d) Transepithelial osmotic water permeability (P_f) in microperfused proximal tubule, thin descending limb of Henle and inner medullary collecting from mice of indicated genotype (S.E.). Data summarized from Ma et al. (1997, 1998, 2000b) and Schnermann et al. (1998)

Fig. 2

AQP4 deletion reduces brain water accumulation in cytotoxic edema, but slows removal of excess brain water in vasogenic edema and hydrocephalus. (a) Water intoxication model of cytotoxic edema. Mouse survival after acute water intoxication produced by intraperitoneal water injection. (b) (top) Ischemic stroke model of cytotoxic edema. Brain sections of mice at 24 h after ischemic stroke produced by permanent middle cerebral artery occlusion. (bottom) Average hemispheric enlargement expressed as a percentage determined by image analysis of brain sections (S.E., ^*P < 0.01). (c) Mouse survival in a bacterial model of meningitis produced by cisternal injection of S. pneumoniae. (d) Reduced elevation in intracranial pressure (ICP, S.E., ^*P < 0.01) and brain water content (S.E., ^*P < 0.001) following continuous intraparenchymal infusion of artificial cerebrospinal fluid at 0.5μL min⁻¹. (e) Accelerated progression of hydrocephalus in AQP4 null mice. Coronal sections wildtype and AQP4 null mouse brain at 5 days after kaolin injection. Data from Manley et al. (2000), Papadopoulos et al. (2004), and Bloch et al. (2006)

Fig. 3

Involvement of AQP4 in brain neuroexcitation. (a) Increased seizure duration in AQP4 null mice. (top) Bipolar electrodes implanted in the right hippocampus were connected to a stimulator and electroencephalograph recording system. (bottom) Representative electroencephalographic recordings in freely moving mice following electrically induced generalized seizures. (b) Delayed K⁺ clearance in brain following electrically induced seizure-like neuroexcitation. Measurements done using K⁺-sensitive microelectrodes inserted into brain cortex in living mice. (c) Slowed K⁺ clearance in brain ECS during cortical spreading depression measured using TAC-Red, a K⁺-sensitive fluorescent probe. (top) Representative data. (bottom) Half-times (t_1/2) for K⁺ reuptake. (d) Expanded brain ECS in AQP4 null mice measured by cortical surface photobleaching. (left) Mouse brain surface exposed to FITC-dextran with dura intact following craniectomy and fluorescence imaging of cortical surface after loading with FITC-dextran (inset). (middle) Photobleaching apparatus. A laser beam is modulated by an acousto-optic modulator and directed onto the surface of the cortex using a dichroic mirror and objective lens. (right) In vivo fluorescence recovery in cortex of wildtype mouse shown in comparison to aCSF and 30% glycerol in a CSF. Taken from Binder et al. (2004b, 2006) and Padmawar et al. (2005)

Fig. 4

Ocular phenotype in AQP deficient mice. (a) Sites of AQP expression in the eye. (b) (top) Stained plastic sections of corneas of mice indicated genotype showing epithelium (upper surface), stroma and endothelium. (bottom) Photographs of wildtype and AQP1 null mice at 40 min after a 10 min exposure of the corneal surface to distilled water showing corneal opacification in the AQP1 null mouse (white arrow). (c) (top) Retinal morphology in an ischemia-reperfusion model of retinal injury. Hematoxylin and eosin-stained retinal sections before and at 12 and 96 h after 60 min retinal ischemia in wildtype and AQP4 null mice. Note retinal swelling at 12 h and degeneration at 96 h. (bottom) Functional analysis by electroretinography. Representative electroretinograms before and at 1, 2 and 4 days after 45 min retinal ischemia in wildtype and AQP4 null mice. From Thiagarajah and Verkman (2002) and Da and Verkman (2004)

Fig. 5

Impaired tumor growth and endothelial cell migration in AQP1 null mice. (a) (left) Tumor in a wildtype vs. AQP1 null mouse, 2 weeks after subcutaneous injection of 10⁶ B16F10 melanoma cells. (right) Tumor growth data (ten mice per group, S.E., P < 0.001). (b) (left) Wound healing of cultured endothelial cells (initial wound edge blue, after 24 h red). (right) Wound edge speed (n = 4 per group, S.E., ^*P < 0.01). (c) Tracks of six migrating CHO cells expressing AQP1 vs. six non-AQP expressing control CHO cells, tracked for 4 h. Initial cell positions indicated by arrows. (d) AQP1 protein (green) polarization to lamellipodia (arrows) in a migrating CHO cell. (e) Proposed mechanism of AQP-facilitated endothelial cell migration: Actin de-polymerization and ion movements increase osmolality at the anterior end of the cell. Water entry increases local hydrostatic pressure, producing cell membrane expansion to form a protrusion. Actin re-polymerizes stabilizing the protrusion. Adapted from Saadoun et al. (2005a)

Fig. 6

Reduced skin hydration in AQP3 deficiency. (a) Schematic showing stratum corneum and epidermal layers. (b) (left) Immunofluorescence showing AQP3 in mouse epidermal cells. E epidermis; D dermis; sc stratum corneum. (right) High-frequency superficial skin surface conductance in dorsal skin of hairless wildtype and AQP3 null mice (S.E., 20 mice per group). Skin conductance measured after 24 h exposure to relative humidity of 10, 40, or 90%; ‘occluded’ indicates a plastic occlusion dressing that prevents evaporative water loss (S.E., five mice per group). (c) Glycerol content measured in SC and epidermis (S.E., ^*P < 0.01). (d) Correlation between SC glycerol content and skin conductance for wildtype (filled circles) and AQP3 null (open circles) mice in a 90% humidity atmosphere for indicated times. Mice were given glycerol orally ad libitum as their only fluid source. From Hara et al. (2002), Ma et al. (2002) and Hara and Verkman (2003)

Fig. 7

AQP3 expression in human squamous cell carcinoma and protection against cutaneous papillomas in AQP3-null mice. (a) AQP3 immunostaining in human skin squamous cell carcinoma. Bar, 50μm. (b) (left) Dorsal skin of mice was treated with a single application of DMBA, followed by twice-weekly applications of TPA for 20 weeks. Representative photographs showing multiple papillomas in wildtype mouse but no papillomas in AQP3 null mouse. (right) Percentage of mice with papillomas. (c) Proposed cellular mechanism of AQP3-facilitated tumorigenesis. Adapted from Hara-Chikuma and Verkman (2008b)

Fig. 8

Progressive fat accumulation and adipocyte hypertrophy in AQP7 deficiency. (a) (top) Photographs of mice showing increased gonadal fat in AQP7 null mice at age 16 weeks (white arrows). (bottom) Histology of gonadal fat (stained with hematoxylin and eosin). Bar, 100μm. (b) Age-dependent epididymal fat mass (S.E., six mice per group). (c) Glycerol and triglyceride content in adipocytes from mice of age 16 weeks (S.E., ^*P < 0.01). (d) Proposed mechanism for adipocyte hypertrophy in AQP7 deficiency. See text for explanations. From Hara-Chikuma et al. (2005)