Reduced water permeability and altered ultrastructure in thin descending limb of Henle in aquaporin-1 null mice.
(1/469)
It has been controversial whether high water permeability in the thin descending limb of Henle (TDLH) is required for formation of a concentrated urine by the kidney. Freeze-fracture electron microscopy (FFEM) of rat TDLH has shown an exceptionally high density of intramembrane particles (IMPs), which were proposed to consist of tetramers of aquaporin-1 (AQP1) water channels. In this study, transepithelial osmotic water permeability (Pf) was measured in isolated perfused segments (0.5-1 mm) of TDLH in wild-type (+/+), AQP1 heterozygous (+/-), and AQP1 null (-/-) mice. Pf was measured at 37 degrees C using a 100 mM bath-to-lumen osmotic gradient of raffinose, and fluorescein isothiocyanate (FITC)-dextran as the luminal volume marker. Pf was (in cm/s): 0.26 +/- 0.02 ([+/+]; SE, n = 9 tubules), 0.21 +/- 0.01 ([+/-]; n = 12), and 0.031 +/- 0.007 ([-/-]; n = 6) (P < 0.02, [+/+] vs. [+/-]; P < 0.0001, [+/+] vs. [-/-]). FFEM of kidney medulla showed remarkably fewer IMPs in TDLH from (-/-) vs. (+/+) and (+/-) mice. IMP densities were (in microm-2, SD, 5-12 micrographs): 5,880 +/- 238 (+/+); 5,780 +/- 450 (+/-); and 877 +/- 420 (-/-). IMP size distribution analysis revealed mean IMP diameters of 8.4 nm ([+/+] and [+/-]) and 5.2 nm ([-/-]). These results demonstrate that AQP1 is the principal water channel in TDLH and support the view that osmotic equilibration along TDLH by water transport plays a key role in the renal countercurrent concentrating mechanism. The similar Pf and AQP1 expression in TDLH of (+/+) and (+/-) mice was an unexpected finding that probably accounts for the unimpaired urinary concentrating ability in (+/-) mice. (+info)
Lung fluid transport in aquaporin-1 and aquaporin-4 knockout mice.
(2/469)
The mammalian lung expresses water channel aquaporin-1 (AQP1) in microvascular endothelia and aquaporin-4 (AQP4) in airway epithelia. To test whether these water channels facilitate fluid movement between airspace, interstitial, and capillary compartments, we measured passive and active fluid transport in AQP1 and AQP4 knockout mice. Airspace-capillary osmotic water permeability (Pf) was measured in isolated perfused lungs by a pleural surface fluorescence method. Pf was remarkably reduced in AQP1 (-/-) mice (measured in cm/s x 0.001, SE, n = 5-10: 17 +/- 2 [+/+]; 6.6 +/- 0.6 AQP1 [+/-]; 1.7 +/- 0.3 AQP1 [-/-]; 12 +/- 1 AQP4 [-/-]). Microvascular endothelial water permeability, measured by a related pleural surface fluorescence method in which the airspace was filled with inert perfluorocarbon, was reduced more than 10-fold in AQP1 (-/-) vs. (+/+) mice. Hydrostatically induced lung interstitial and alveolar edema was measured by a gravimetric method and by direct measurement of extravascular lung water. Both approaches indicated a more than twofold reduction in lung water accumulation in AQP1 (-/-) vs. (+/+) mice in response to a 5- to 10-cm H2O increase in pulmonary artery pressure for five minutes. Active, near-isosmolar alveolar fluid absorption (Jv) was measured in in situ perfused lungs using 125I-albumin as an airspace fluid volume marker. Jv (measured in percent fluid uptake at 30 min, n = 5) in (+/+) mice was 6.0 +/- 0.6 (37 degrees C), increased to 16 +/- 1 by beta-agonists, and inhibited to less than 2.0 by amiloride, ouabain, or cooling to 23 degrees C. Jv (with isoproterenol) was not affected by aquaporin deletion (18.9 +/- 2.2 [+/+]; 16.4 +/- 1.5 AQP1 [-/-]; 16.3 +/- 1.7 AQP4 [-/-]). These results indicate that osmotically driven water transport across microvessels in adult lung occurs by a transcellular route through AQP1 water channels and that the microvascular endothelium is a significant barrier for airspace-capillary osmotic water transport. AQP1 facilitates hydrostatically driven lung edema but is not required for active near-isosmolar absorption of alveolar fluid. (+info)
Developmental expression of aquaporin 1 in the rat renal vasculature.
(3/469)
Aquaporin 1 (AQP-1) is a water channel protein that is constitutively expressed in renal proximal tubule and descending thin limb cells as well as in endothelial cells of the descending vasa recta. Studies in the developing rat kidney have demonstrated that AQP-1 is expressed in renal tubules before birth. However, nothing is known about the expression of AQP-1 in the renal vasculature during kidney development. The purpose of this study was to establish the distribution of AQP-1 in the renal vasculature of the developing rat kidney and follow the differentiation of the vascular system during kidney development. Kidneys from 16-, 17-, 18-, and 20-day-old fetuses and 1-, 4-, 7-, 14-, 21-, and 28-day-old pups were preserved and processed for immunohistochemical studies using a preembedding immunoperoxidase procedure. AQP-1 immunoreactivity was detected using affinity-purified rabbit polyclonal antibodies to AQP-1. AQP-1 was expressed throughout the arterial portion of the renal vasculature of the fetal and neonatal kidney from gestational age 17 days to 1 wk after birth. AQP-1 immunoreactivity gradually disappeared from the renal vasculature between 1 and 2 wk of age and remained only in the descending vasa recta. In contrast, AQP-1 immunoreactivity was not observed in lymphatic vessels until 3 wk of age and persisted in the adult kidney. AQP-1 was also expressed in a population of interstitial cells in the terminal part of the renal papilla at 3 wk of age as well as in the adult kidney. The transient expression of AQP-1 in the arterial portion of the renal vasculature in the developing rat kidney suggests that AQP-1 is important for fluid equilibrium and/or drainage in the developing kidney or, alternatively, plays a role in the regulation of growth and/or branching of the vascular tree during kidney development. (+info)
Molecular identification and immunolocalization of the water channel protein aquaporin 1 in CBCECs.
(4/469)
PURPOSE: Water channel proteins are important pathways for water movements across cell membranes, including those in the corneal endothelium that contribute to the fluid transport mechanism essential in maintaining corneal transparency. This study was conducted to identify and locate the water channel protein(s) in cultured bovine corneal endothelial cells (CBCECs). METHODS: Poly(A)+ RNA was isolated from CBCECs, and MMLV reverse transcriptase and random hexamer primers were used to generate a cDNA pool by reverse transcription-polymerase chain reaction (RT-PCR). Two specific degenerate primers were synthesized based on consensus sequences from the major intrinsic lens protein superfamily; a "touchdown" PCR protocol accommodated the degeneracy. Immunolocalization was performed by incubating sections of CBCECs with an antibody against human aquaporin 1 (AQP1). Cryosections (0.85 microm) of CBCECs were used for light microscopy, and 800-A ultrathin cryosections were used for electron microscopy (EM). RESULTS: A 372-bp fragment was isolated. Its encoded amino acid sequence was 100% identical with that of bovine AQP1 (AQP2_bovin). CBCECs reacted strongly with the anti-AQP1 antibody, and the labeling was selectively localized to the plasma membrane by light microscopy. Subcellular localization by EM revealed immunoreactivity with the inner leaflets of the plasma membrane. CONCLUSIONS: The identity of the aquaporin, its abundance, and its membrane location suggest that it is a major pathway for fluid flow across endothelial cell membranes. This is consistent with transcellular endothelial fluid transport. (+info)
Novel method for evaluation of the oligomeric structure of membrane proteins.
(5/469)
Assessment of the quaternary structure of membrane proteins by PAGE has been problematic owing to their relatively poor solubility in non-dissociative detergents. Here we report that several membrane proteins can be readily solubilized in their native quaternary structure with the use of the detergent perfluoro-octanoic acid (PFO). Further, PFO can be used with PAGE, thereby providing a novel, accessible tool with which to assess the molecular mass of homo-multimeric protein complexes. (+info)
Regulation of aquaporin-1 and nitric oxide synthase isoforms in a rat model of acute peritonitis.
(6/469)
The loss of ultrafiltration (UF) that accompanies acute peritonitis is a common problem in peritoneal dialysis (PD). It has been suggested that changes in nitric oxide (NO)-mediated vascular tone and permeability might be involved in the loss of UF, whereas channel-mediated water permeability should not be affected. This study used a model of acute peritonitis in rats to characterize changes in PD parameters, in correlation with: (1) expression studies of water channel aquaporin-1 and NO synthase (NOS) isoforms and (2) enzymatic assays for NOS in the peritoneum. Compared with controls, rats with peritonitis had a higher removal of plasma urea, a faster glucose absorption, and a loss of UF. Additional changes, including high protein loss, elevated leukocyte counts in dialysate, positive bacterial cultures, edema, and mononuclear infiltrates, were similar to those observed in PD patients with acute peritonitis. Acute peritonitis in rats induced a major increase in total NOS activity, which was inversely correlated with free-water permeability. The increased NOS activity was mediated by both inducible (Ca2+-independent) and endothelial (Ca2+-dependent) NOS isoforms and was reflected by increased peritoneal staining for nitrotyrosine. In contrast, aquaporin-1 expression was unchanged in rats with peritonitis. These findings cast light on the pathophysiology of permeability changes and loss of UF that characterize acute peritonitis. In particular, these data suggest that a local production of NO, mediated by different NOS isoforms, might play a key role in these changes. (+info)
Micropuncture analysis of tubuloglomerular feedback regulation in transgenic mice.
(7/469)
Micropuncture methods have been used widely as a means to define the function of single tubules and study the functional connection between tubules and afferent arterioles (so-called tubuloglomerular feedback [TGF]). Transgenic mouse strains have become a new research tool with the potential of shedding new light on the role of specific gene products in renal tubular and vascular function. The micropuncture approach has therefore been adapted to studies in the mouse kidney. Although the data presented here support the feasibility of using this technique in the mouse, technical improvements are desirable in the areas of anesthesia, ureteral urine collections, blood collections, volume replacement, and functional stability for extended time periods. During ketamine/inactin anesthesia, TGF responses could regularly be elicited in wild-type mice. In contrast, changes in loop flow did not alter stop-flow pressure in angiotensin II type 1A receptor and angiotensin-converting enzyme knockout mice. Infusion of angiotensin II in subpressor doses partially restored TGF responsiveness in angiotensin-converting enzyme knockout animals. Normal TGF responses compared to wild type were found in nitric oxide synthase I and thromboxane receptor knockout mice. Using free-flow micropuncture techniques, the proximal-distal single-nephron GFR difference was found to be augmented in aquaporin-1 and Na/H exchanger-3 knockout mice, suggesting TGF activation in these strains of mice. These results support an essential role of angiotensin II in TGF regulation mediated through the angiotensin II type 1A receptor. Chronic nitric oxide synthase I and thromboxane receptor deficiency did not change TGF responsiveness. Aquaporin-1 and Na/H exchanger-3 deficiency enhances TGF suppression of TGF probably by volume depletion-mediated TGF sensitization. The use of micropuncture methodology in transgenic mice combines old and new research tools in a way that promises to yield important new insights into single-nephron function in physiologic and pathophysiologic conditions. (+info)
Safety and efficacy of adenovirus-mediated transfer of the human aquaporin-1 cDNA to irradiated parotid glands of non-human primates.
(8/469)
This study evaluated the safety and efficacy of a single administration of a recombinant adenovirus encoding human aquaporin-1 (AdhAQP1) to the parotid glands of adult rhesus monkeys. In anticipation of possible clinical use of this virus to correct irradiation damage to salivary glands, AdhAQP1 was administered (at either 2 x 10(9) or 1 x 10(8) plaque-forming units/gland) intraductally to irradiated glands and to their contralateral nonirradiated glands. Radiation (single dose, 10 Gy) significantly reduced salivary flow in exposed glands. Virus administration resulted in gene transfer to irradiated and nonirradiated glands and was without untoward local (salivary) or systemic (sera chemistry, complete blood count) effects in all animals. However, the effect of AdhAQP1 administration varied and did not result in a consistent positive effect on salivary flow rates for all animals under these experimental conditions. We conclude that a single adenoviral-mediated gene transfer to primate salivary glands is well-tolerated, although its functional utility in enhancing fluid secretion from irradiated parotid glands is inconsistent. (+info)