Reduced water permeability and altered ultrastructure in thin descending limb of Henle in aquaporin-1 null mice Chung-Lin Chou, … , Tonghui Ma, A.S. Verkman J Clin Invest. 1999;103(4):491-496. https://doi.org/10.1172/JCI5704. Article 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°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 μm–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 […] Find the latest version: https://jci.me/5704/pdf Reduced water permeability and altered ultrastructure in thin descending limb of Henle in aquaporin-1 null mice Chung-Lin Chou,1 Mark A. Knepper,1 Alfred N. van Hoek,2 Dennis Brown,2 Baoxue Yang,3 Tonghui Ma,3 and A.S. Verkman3 1Laboratory of Kidney and Electrolyte Metabolism, National Institutes of Health, Bethesda, Maryland 20892, USA 2Renal Unit and Program in Membrane Biology, Massachusetts General Hospital East, Boston, Massachusetts 02114, USA 3Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California–San Francisco, San Francisco, California 94143-0521, USA Address correspondence to: Alan S. Verkman, 1246 Health Sciences East Tower, Cardiovascular Research Institute, University of California–San Francisco, San Francisco, California 94143-0521, USA. Phone: (415) 476-8530; Fax: (415) 665-3847; E-mail: [email protected]; http://www.ucsf.edu/verklab Received for publication November 3, 1998, and accepted in revised form January 4, 1999. 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, transepithe- lial 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°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 µm–2, SD, 5–12 micrographs): 5,880 ± 238 (+/+); 5,780 ± 450 (+/–); and 877 ± 420 (–/–). IMP size distribution analy- sis 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 sim- ilar Pf and AQP1 expression in TDLH of (+/+) and (+/–) mice was an unexpected finding that probably accounts for the unimpaired urinary concentrating ability in (+/–) mice. J. Clin. Invest. 103:491-496 (1999). Introduction Water permeability is greatest in the proximal portion of The thin descending limb of Henle (TDLH) in kidney TDLH in long-loop nephrons and becomes low in deep has been proposed to have an important role in the for- medullary segments. mation of a concentrated urine by the countercurrent A possible molecular basis for the high water perme- multiplication mechanism. Countercurrent multiplica- ability in TDLH was suggested by freeze-fracture electron tion relies on active solute transport out of the lumen in microscopy (FFEM) studies showing higher densities of the thick ascending limb of Henle, rapid osmotic equili- intramembrane particles (IMPs) in plasma membranes of bration along the lumen of TDLH, and efficient uptake TDLH compared with other nephron segments (8, 9). of water by the renal microvasculature (1, 2). Measure- The majority of the IMPs in plasma membranes of rat ments from several laboratories indicate that transep- TDLH were subsequently shown to consist of tetramers ithelial osmotic water permeability (Pf) in TDLH is of aquaporin-1 (AQP1) water channels (10). AQP1 is a exceptionally high. Early split-oil droplet measurements water-selective transporting protein that is expressed showed rapid osmotically driven water movement into widely in fluid-transporting epithelia and endothelia the lumen of TDLH (3), and subsequent in vivo and in (reviewed in refs. 11, 12). In kidney, AQP1 is expressed at vitro microperfusion studies showed very high Pf values the apical and basolateral plasma membranes of epithe- of >0.2 cm/s (reviewed in ref. 4), substantially greater lial cells in proximal tubule and TDLH, and in endothe- than Pf in the proximal tubule and the vasopressin-stim- lial cells of descending vasa recta (13–16). Consistent with ulated collecting duct. The transport of water through these observations, measurement of AQP1 protein in aqueous pores in TDLH cell membranes was suggested microdissected tubule segments using a fluorescence- from the findings of a high ratio of osmotic-to-diffu- based enzyme-linked immunosorbent assay (ELISA) sional water permeability and of inhibition of water per- method showed that TDLH of long-looped nephrons meability by the mercurial p-chloromercuribenzene sul- have the highest AQP1 content among nephron seg- fonate (5). It was also shown that water permeability in ments (17). Recently, transgenic knockout mice deficient TDLH depends on nephron type (long-loop vs. short- in AQP1 were generated by targeted gene disruption and loop nephrons) and position along the tubule axis (6, 7). found to be polyuric and unable to concentrate their The Journal of Clinical Investigation | February 1999 | Volume 103 | Number 4 491 urine in response to water deprivation (18). The inability in which cortex and inner medulla were separated. Samples of vasopressin to increase urine osmolality in the knock- were homogenized in saline containing multiple protease out mice was taken as evidence for defective countercur- inhibitors, resolved on SDS-PAGE, and electrotransferred and rent multiplication. Analysis of proximal tubule function immunoblotted as described previously (18). Quantitative band integration was done by enhanced chemiluminescence and in AQP1 knockout mice by in vitro microperfusion and in densitometry. Multiple standards were included in all blots to vivo micropuncture indicated fivefold decreased water correct for film nonlinearity (see Results). ∼ permeability and 50% decreased isosmolar fluid absorp- Freeze-fracture electron microscopy. Kidneys were fixed in 2% glu- tion (19). However, the profound urinary concentrating taraldehyde for 2 h, washed, and stored in PBS containing defect in the AQP1 null mice is probably not the conse- 0.01% NaN3. Small tissue fragments from the base of the papil- quence of proximal tubule dysfunction, as distal fluid la were infiltrated with 30% glycerol in 0.1 M cacodylate buffer delivery was relatively unaffected because of tubu- (pH 7.4) overnight, placed on copper specimen holders, and loglomerular feedback and a compensatory decrease in frozen in N2-cooled Freon-22. Frozen samples were transferred glomerular filtration rate. It was proposed that defective into a freeze-fracture apparatus (Cressington Scientific Instru- TDLH and/or descending vasa recta function were pri- ments, Watford, United Kingdom). Sample temperature was raised to –120°C, and membranes were fractured under vacu- marily responsible for the urinary concentrating defect. um (10–7 torr). Fractured membranes were shadowed at 45 The purpose of this study was to characterize osmotic degrees with platinum, followed by a carbon coat perpendicu- water permeability and ultrastructure in TDLH of wild- lar to the fracture plane. Specimens were digested with sodium type (+/+), AQP1 heterozygous (+/–), and AQP1 null hypochlorite solution and then rinsed with distilled water. The (–/–) mice. Pf was measured by in vitro tubule microper- replicas were examined on nickel grids in a Philips CM10 elec- fusion, and membrane ultrastructure was studied by tron microscope (Mahwah, New Jersey, USA). FFEM. The principal finding was a profoundly decreased TDLH were identified based on established criteria (22): the Pf and IMP particle density in TDLH from the AQP1 null presence of