Impaired Hearing in Mice Lacking Aquaporin-4 Water Channels

A role for aquaporins (AQPs) in hearing has been suggested from the specific expression of aquaporins in inner ear and the need for precise volume regulation in epithelial cells involved in acoustic signal transduction. Using mice deficient in selected aquaporins as controls, we localized AQP1 in fibrocytes in the spiral ligament and AQP4 in supporting epithelial cells (Hensen's, Claudius, and inner sulcus cells) in the organ of Corti. To determine whether aquaporins play a role in hearing, auditory brain stem response (ABR) thresholds were compared in wild-type mice and transgenic null mice lacking (individually) AQP1, AQP3, AQP4, and AQP5. In 4–5-week-old mice in a CD1 genetic background, ABR thresholds in response to a click stimulus were remarkably increased by >12 db in AQP4 null mice compared with wild-type mice (p 12 db in AQP4 null mice compared with wild-type mice (p 8 back-crosses. For ABR measurements the investigators were blinded to genotype information until completion of the analysis. Protocols were approved by the University of California, San Francisco Committee on Animal Research. ABR measurements were performed using a Biopac MP100 work station equipped with differential amplifier (ERS100B) and stimulator (STM100A) modules. Mice were anesthetized with ketamine (80 mg/kg) and xylazine (14 mg/kg) by intraperitoneal injection. Rectal temperature was monitored continuously using a digital thermistor and maintained at 36–38 °C using a heating block. Mice were placed in a grounded Faraday cage contained in a light-proof sound isolation box. The three recording electrodes and speaker input cables entered a small hole in the box. Biaural sound stimuli were produced by a broad band speaker (Realistic, Model 40-1310B) positioned 30 cm from the mice. Click stimuli were generated by a square pulse of 0.1-ms duration and specified amplitude. Tone stimuli were generated using a frequency synthesizer and home-built waveform modulator to give a trapezoidal waveform with 1-ms rise/fall times and 2-ms flat segment. Sound intensity calibrations were done using a model C550H measurement microphone (Josephon Engineering) positioned at the location of the mouse head. For recording, subdermal stainless steel needle electrodes were placed at the vertex and ventrolateral to the left and right ears. ABR waveforms were recorded for 10 ms at a sampling rate of 45,000 Hz using 100–3000 Hz bandpass filter settings. Generally, waveforms from 200 stimuli at a frequency of 12 Hz were averaged. ABR waveforms were recorded in 10-db intervals down from a maximum amplitude of 90 db (for click stimuli) until no waveform could be visualized. Waveforms were stored for off-line analysis. Samples were fixed by intracardiac perfusion with 4% paraformaldehyde in PBS (pH 7.4). The temporal bone was removed, and the cochlea was post-fixed overnight in the same fixative solution. After decalcification, the cochlea was dehydrated and embedded in Tissue-Tek OCT compound for cryostat sections and in glycol methacrylate for plastic sections. For histological examination, the cochlea was infiltrated with JB-4 monomer (Polyscience Inc.), embedded under vacuum at room temperature, sectioned on a microtome (Sorvall), and stained with toluidine blue. For immunocytochemistry, 3–4-µm thick cryostat sections were incubated for 30 min with PBS containing 1% bovine serum albumin and then with affinity-purified aquaporin antibodies (dilution 1:400–1500) for 2 h at 23 °C in PBS containing 1% bovine serum albumin. Slides were rinsed with 2.7% NaC1 and then with PBS and incubated with a secondary Cy3-conjugated sheep anti-rabbit F(ab)2fragment (1:200) for visualization by fluorescence microscopy. Data are reported as the mean ± S.E. with p values determined by analysis of variance. Aquaporin localization in the organ of Corti in the inner ears of four sets of mice was done by immunostaining using specific antibodies (Fig. 1). The AQP1 antibody strongly labeled non-epithelial cells (fibrocytes) in the spiral ligament of wild-type mice (top left) with no labeling in AQP1 null mice (top right). The AQP4 antibody labeled supporting Hensen's cells, inner sulcus cells, and Claudius cells in wild-type mice (middle left) but not in AQP4 null mice (middle right). No immunostaining with AQP5 antibody was found (bottom left and right), despite strong label of other tissues known to express AQP5 such as salivary gland (inset). Immunostaining of AQP2 and AQP3 was negative, with strongly positive controls (mouse kidney, not shown). Hearing was evaluated functionally in wild-type and aquaporin null mice of age 4–5 weeks by ABR analysis. Fig. 2shows representative ABR waveforms in response to click stimuli of different intensities. As reported in other ABR studies in mice (19Zheng Q.Y. Johnson K.R. Erway L.C. Hear. Res. 1999; 130: 94-107Crossref PubMed Scopus (679) Google Scholar), at least four distinct peaks were identified corresponding to cochlear nerve activity (wave I) and downstream neural activity (waves II–IV). Decreasing click intensities resulted in a decrease in wave amplitudes. As generally defined, the ABR threshold was identified in each series of ABR waveforms as the lowest click intensity that produced at least two clearly visible waves. ABR thresholds for the data in Fig. 2 were 40 db (wild-type mouse) and 50 db (AQP4 null mouse). Control studies indicated that ABR thresholds were very reproducible in the same mice measured on different days, with identical ABR thresholds in of mice and db in mice. Fig. ABR thresholds measured in a large series of wild-type and aquaporin null mice. Although was found in different mice in the CD1 genetic background, there was a increased ABR threshold in AQP4 null mice by >12 db (p < ABR thresholds in mice lacking AQP1, AQP3, or AQP5 not from that in wild-type mice. studies were done in C57/bl6 inbred mice which the AQP4 null genotype was Fig. that AQP4 null mice were deaf, whereas ABR waveforms could be elicited in wild-type mice. The results indicated remarkably hearing in AQP4 null mice. ABR waveforms from wild-type and AQP4 null mice in the CD1 background were Fig. wave measured at and 50 db click intensities. each click the of wave were for wild-type for AQP4 null mice. amplitude of wave and wave for each was no different in amplitude in wild-type versus AQP4 null mice. these results that the hearing impairment from defective cochlear downstream neural Tone ABR analysis was done to determine whether the hearing impairment in AQP4 null mice was tone bursts were using a frequency Fig. waveforms with frequency by analysis of and tone representative ABR waveforms in response to tone ABR thresholds were increased in AQP4 null mice at all Fig. click and tone ABR thresholds for a series of wild-type and AQP4 null that the hearing impairment in AQP4 null mice is not of ABR thresholds for click and tone stimuli in wild-type and AQP4 null CD1 mice of age 4–5 data ± are shown for click and tone compared with wild-type p < p < of plastic sections of inner ear was done to determine whether anatomical differences could for the hearing in AQP4 null mice. Fig. representative sections from two wild-type and two AQP4 null mice. The plastic sections clearly an organ of Corti with hair cells and cells. No differences were in sections of inner ear evaluated from four wild-type and four AQP4 null mice. a of the mouse cochlea on the and studies The of this study was to determine whether aquaporins play a functional role in hearing. there was disagreement in the aquaporin expression in mammalian inner and no information was to on mouse inner we determined by the expression of aquaporins in mouse inner mice lacking aquaporins as controls. We found AQP1 in non-epithelial cells in spiral ligament and AQP4 in the basolateral plasma membranes of Hensen's cells and inner sulcus cells and the basal plasma membrane of Claudius cells in with in guinea pig We not specific immunostaining of AQP2, AQP3, or despite positive controls using mouse tissues known to express these aquaporins. the of antibody of ABR analysis was done on wild-type mice and knockout mice lacking AQP1, AQP3, AQP4, and AQP5. Although an AQP2 mouse model of autosomal nephrogenic diabetes insipidus was B. Gillespie A. Carlson E.J. Epstein C.J. Verkman A.S. J. Biol. Chem. 2001; 276: 2775-2779Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), these mice were not for ABR analysis they generally not the first of We found remarkable hearing impairment in AQP4 null with no of of AQP1, AQP3, or AQP5. The AQP4 null mice not in cochlear morphology at the These results suggest that water transport in epithelial cells in the organ of Corti is for hearing. ABR analysis in mice is an to hearing impairment from a of genetic and L.C. R.R. Hear. Res. PubMed Scopus Google Scholar, K.R. L.A. E.A. PubMed Scopus Google Scholar, K.R. Erway L.C. Q.Y. Hear. Res. 1997; PubMed Scopus Google Scholar, Erway L.C. Hear. Res. 1995; PubMed Scopus (94) Google Scholar). The measurements were done in mice to the hearing impairment that in mouse (19Zheng Q.Y. Johnson K.R. Erway L.C. Hear. Res. 1999; 130: 94-107Crossref PubMed Scopus (679) Google Scholar, L.C. Hear. Res. PubMed Scopus Google Scholar), which could the of Hearing impairment in AQP4 null mice was found in an and inbred genetic The ABR several the first cochlear and auditory nerve and the from activity in central auditory J. Clin. 1994; PubMed Scopus Google Scholar, A.R. J. Clin. 1994; PubMed Scopus Google Scholar). The AQP4 null mice with ABR thresholds not have ABR wave as by the amplitude of wave and wave that the hearing impairment in AQP4 null mice is caused by in the central of the hearing Tone pulse ABR measurements indicated the hearing impairment was not on the of AQP4, we its role in hearing. AQP4 is an that does not or other small B. Verkman A.S. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). AQP4 was from rat H. Ma T. M. Verkman A.S. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), and in brain G.M. J.M. Agre P. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar), stomach T. K. K. Y. M. K. Y. S. PubMed Scopus Google Scholar) and other have been mice lacking AQP4 manifest a urinary concentrating T. Yang B. Gillespie A. Carlson E.J. Epstein C.J. Verkman A.S. J. Clin. Invest. 1997; 100: 957-962Crossref PubMed Scopus (403) Google Scholar) of water transport in the inner duct Ma T. Yang B. M.A. Verkman A.S. Am. J. Physiol. 1998; 274: PubMed Google Scholar). However, AQP4 is strongly expressed in the nervous system, in the brain and and in cells A. M. Verkman A.S. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, E.A. M.L. R. Nielsen S. Agre P. Ottersen O.P. J. Neurosci. 1998; PubMed Google Scholar). AQP4 null mice showed remarkably altered water balance with from brain in a model of and an model of (5Manley G.T. Fujimura M. Ma T. Noshita N. Filiz F. Bollen A. Chan P. Verkman A.S. Nat. Med. 2000; 6: 159-163Crossref PubMed Scopus (1311) Google Scholar). A of AQP4 is its in membranes in square of these are by microscopy in cells B. Brown D. Verkman A.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and are found in the kidney, and muscle of wild-type but not AQP4 null mice J.M. Ma T. R. Verkman A.S. J. Sci. 1997; PubMed Google Scholar). The cochlea hair cells that are for sound on the of hair cells a with fluid (Fig. stimuli the inner ear the in K+ channel K+ from the hair cells, hair cell membrane and the of an signal by the auditory nerve to the The K+ in is and the is in a The of in after acoustic signal transduction rapid of K+ the N. J.J. B.A. Hear. Res. 1998; PubMed Scopus Google Scholar, T. B. A. S. T. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). at least of the proteins associated with in humans and mice are involved in K+ Science. 1999; PubMed Scopus Google Scholar), including the K+ which is in one of hearing T. B. A. S. T. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The K+ are then by the supporting hair cells, a of that from the epithelial supporting cells to the fibrocytes that form the spiral and are by epithelial cells of the AQP4 may be involved in osmotic balance during K+ AQP4 is expressed in supporting hair cells in the basolateral plasma membranes of Hensen's cells and in the basal plasma membrane of Claudius cells. We that the of K+ by Hensen's cells water and that water the supporting cell basal membranes of Claudius cells that the of the spiral The hearing in AQP4 null mice may thus from altered basal of outer hair cell The of AQP4 in K+ in supporting cells may be a in the of In the central nervous system, AQP4 in is proposed to facilitate K+ fluxes associated with R. J. PubMed Scopus Google Scholar, Neurosci. Full Text Full Text PDF PubMed Scopus Google Scholar, S. Nagelhus E.A. M. Agre P. O.P. J. Neurosci. 1997; PubMed Google Scholar), and in the AQP4 in cells is proposed to facilitate K+ fluxes associated with cells E.A. Y. A. A. Nielsen S. Y. Ottersen O.P. 1999; PubMed Scopus Google Scholar). A of AQP4 in of with K+ has been proposed J.E. T. Agre P. Nielsen S. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). The be to the and by which AQP4 K+ and water in We for transgenic mouse and genotype analysis.