The detailed localization pattern of Na+/K+/2Cl- cotransporter type 2 and its related ion transport system in the rat endolymphatic sac

Abstract

The endolymphatic sac (ES) is a part of the membranous labyrinth. ES is believed to perform endolymph absorption, which is dependent on several ion transporters, including Na(+)/K(+)/2Cl(-) cotransporter type 2 (NKCC-2) and Na(+)/K(+)-ATPase. NKCC-2 is typically recognized as a kidney-specific ion transporter expressed in the apical membrane of the absorptive epithelium. NKCC-2 expression has been confirmed only in the rat and human ES other than the kidney, but the detailed localization features of NKCC-2 have not been investigated in the ES. Thus, we evaluated the specific site expressing NKCC-2 by immunohistochemical assessment. NKCC-2 expression was most frequently seen in the intermediate portion of the ES, where NKCC-2 is believed to play an important role in endolymph absorption. In addition, NKCC-2 expression was also observed on the apical membranes of ES epithelial cells, and Na(+)/K(+)-ATPase coexpression was observed on the basolateral membranes of ES epithelial cells. These results suggest that NKCC-2 performs an important role in endolymph absorption and that NKCC-2 in apical membranes and Na(+)/K(+)-ATPase in basolateral membranes work coordinately in the ES in a manner similar to that in renal tubules.

Figure 1

Serial sections of rat ES from PSP to DSP stained with hematoxylin. A, B, and C show PSP, ISP, and DSP, respectively. Each right upper inset is the high-power field of ES epithelium. ES, endolymphatic sac; PSP, proximal sac portion; ISP, intermediate sac portion; DSP, distal sac portion; SC, semicircular canal; O, operculum. Bar = 200 μm.

Figure 2

Immunofluorescent double staining with specific antibodies for anti-Na⁺/K⁺/2Cl⁻ cotransporter type 2 (anti-NKCC-2) and anti-Na⁺/K⁺-ATPase. Green and red signals indicate NKCC-2– and Na⁺/K⁺-ATPase–immunoreactive cells, respectively. NKCC-2 (1–3): apical membranes of ES epithelia are locally stained; positive cells are most frequently observed at ISP, weaker positive signals are observed at PSP, and almost no signal is observed at DSP. Na⁺/K⁺-ATPase (5–7): basolateral membranes of ES epithelia are locally stained; positive cells are observed in all ES individual parts, but the positive region at PSP is more partial than that at ISP, and DSP shows faint staining. Kidney is shown as a positive control (4 and 8). Merged images are also shown to indicate the coexpression pattern of NKCC-2 with Na⁺/K⁺-ATPase (dark field: 9–12 and bright field: 13–15). Right upper insets in 13–15 show the high-power field of ES epithelium. As a negative control, ISP merged image without primary antibodies is also shown, and no labeling was observed (16). Labeling conditions and microscope settings were identical for all ES sections. PSP, proximal sac portion; ISP, intermediate sac portion; DSP, distal sac portion; Kid, kidney; L, lumen of the ES; V, vein of vestibular aqueduct. Bar = 50 μm.