Immunohistochemical localization of megalin and cubilin in the human inner ear

Abstract

Megalin and cubilin are endocytic receptors expressed in many absorptive polarized epithelia. These receptors have been implicated in the transport of gentamicin in the inner ear as possible contributors to ototoxic damage. Megalin and cubilin have been characterized in detail in the mouse and rat inner ear, but not in the human inner ear. In this study, megalin and cubilin were localized by immunohistochemistry using affinity-purified antibodies in formalin fixed frozen cryostat and celloidin embedded sections of the human inner ear. In the cochlea megalin and cubilin were localized in marginal cells of the stria vascularis, epithelial cells of the spiral prominence and the Reissner's membrane. In the macula utricle and cristae ampullaris, megalin and cubilin were localized in transitional and dark cells, but not in vestibular hair cells and supporting cells. In the endolymphatic duct megalin and cubilin were localized in the epithelial cells. The localization of megalin and cubilin in the human inner ear is consistent with previous reports in the inner ear of animal models and suggest that these receptors may play an important role in the inner ear endocytic transport, and maybe potential targets for prevention of ototoxic damage or the delivery of medications.

Keywords: Cochlea; Cubilin; Human inner ear; Megalin; Reissner’s membrane; Vestibular end organs.

Fig 1.

Megalin and cubilin immunofluorescence (-IF) in a formalin fixed frozen cryostat section of the Reissner’s membrane. (a) Megalin-IF in epithelial cells of the Reissner’s membrane (green color), (b) cubilin-IF was also detected in the epithelial cells of the Reissner’s membrane (red color). (c) This micrograph is a merge image from (a) and (b) showing the colocalization of megalin and cubilin (yellow color). (d) Higher magnification view from (c). DAPI stains cell nuclei (blue color). Scale bar in a and b = 40 μm, c= 20 μm, d= 20 μm.

Fig 2.

Megalin-IF and cubilin–IF in the stria vascularis (sv) and spiral prominence (sp). (a) Megalin-IF (green), thin arrows point to the marginal cells in the sv, thick arrowhead point to epithelial cells of the sp, the spiral ligament (sl) was no immunoreactive for both proteins. (b) Cubilin-IF (red), shows a near identical distribution. (c) Merged image from (a) and (b). (d) High magnification view of the sp shows the specific localization of megalin and cubilin. Cell nuclei in blue (DAPI). Sl: Scale bar in a, b and c = 60 μm, d= 25 μm.

Fig 3.

Megalin and cubilin immunoreactivity (-IR) in the human cochlea (celloidin embedded temporal bone section). (a) Megalin-IR was seen in the Reissner’s membrane (arrows), the stria vascularis and the spiral prominence (arrows). (b) Cubilin-IR showed a similar distribution to megalin. Scale bar = 70 μm.

Fig 4.

Megalin-IF and cubilin-IF in formalin fixed cryostat section of the human crista ampullaris and macula utricle. (a) Megalin-IF (green color, thick arrow) is present in transitional (t) and dark cells (d). (a1) Cubilin-IF (red color, long arrow) followed a similar pattern to megalin-IF. (a2) Image merged from (a) and (b) to show colocalization of megalin and cubilin. (a3) High magnification view of the transitional and dark cells from another specimen, showing colocalization of both megalin and cubilin. (b) Megalin-IF and (b1) cubilin-IF in transitional and dark cells of the macula utricle, (b2) Merged image from (b) and (b1). (b3) High magnification view of the transitional cells from another specimen, showing colocalization of both megalin and cubilin, lu: lumen. Scale bar in a,a1 and a2 and b, b1 and b2 = 50 μm, bar in a3 and b3 =10 μm.

Fig 5.

Megalin and cubilin -IR in celloidin sections of vestibular end organs. (a) megalin-IR was seen in transitional cells and dark cells (arrows) in the crista ampullaris, (a1) and macula utricle. The sensory epithelia (se) and stroma (st) was not immunoreactive. (b) cubilin-IR in the crista ampullaris and (b1) macula utricle (arrows) were found also in the transitional and dark cells of the macula utricle. Scale bar = 40 μm.

Fig 6.

Megalin-IR and cubilin-IR in the endolymphatic sac (formalin fixed celloidin embedded sections). (a) Megalin-IR and (b) cubilin-IR was seen in epithelial cells (ec) of the endolymphatic duct (dark amber color, arrowheads). Hematoxylin counterstained (blue color in cell nuclei). Scale bar = 40 μm.

Fig 7.

Megalin-IR and cubilin-IR in positive and negative controls. (a) Megalin-IF (green) in vestibular dark cells flanking the mouse crista ampullaris (arrow). (b) Cubilin-IF (red) in epithelial cells of the mouse Reissner’s membrane (arrow), (c) Megalin-IR in epithelial cells (arrow) of a human kidney section (positive control). (d) Negative control in a celloidin embedded section of the human cochlea, primary antibody was omitted, no immunoreactivity was detected. Celloidin embedded sections were incubated with (d1) Antibodies against megalin (sv: stria vascularis) and (d2) cubilin (rm: Reissner’s membrane) pre-absorbed with their corresponding antigen. No specific immunoreactivity was seen in both cases. oc: organ of Corti. Scale bar in a and b = 15 μm, c = 60 μm, d = 40 μm, d1 and d2= 50 μm.