Segmental expression of the bradykinin type 2 receptor in rat efferent ducts and epididymis and its role in the regulation of aquaporin 9

Abstract

Water and solute transport in the efferent ducts and epididymis are important for the establishment of the appropriate luminal environment for sperm maturation and storage. Aquaporin 9 (AQP9) is the main water channel in the epididymis, but its regulation is still poorly understood. Components of the kinin-kallikrein system (KKS), leading to the production of bradykinin (BK), are highly expressed in the lumen of the male reproductive tract. We report here that the epididymal luminal fluid contains a significant amount of BK (2 nM). RT-PCR performed on epididymal epithelial cells isolated by laser capture microdissection (LCM) showed abundant BK type 2 receptor (Bdkrb2) mRNA expression but no type 1 receptor (Bdkrb1). Double-immunofluorescence staining for BDKRB2 and the anion exchanger AE2 (a marker of efferent duct ciliated cells) or the V-ATPase E subunit, official symbol ATP6V1E1 (a marker of epididymal clear cells), showed that BDKRB2 is expressed in the apical pole of nonciliated cells (efferent ducts) and principal cells (epididymis). Triple labeling for BDKRB2, AQP9, and ATP6V1E1 showed that BDKRB2 and AQP9 colocalize in the apical stereocilia of principal cells in the cauda epididymidis. While uniform Bdkrb2 mRNA expression was detected in the efferent ducts and along the epididymal tubule, marked variations were detected at the protein level. BDKRB2 was highest in the efferent ducts and cauda epididymidis, intermediate in the distal initial segment, moderate in the corpus, and undetectable in the proximal initial segment and the caput. Functional assays on tubules isolated from the distal initial segments showed that BK significantly increased AQP9-dependent glycerol apical membrane permeability. This effect was inhibited by BAPTA-AM, demonstrating the participation of calcium in this process. This study, therefore, identifies BK as an important regulator of AQP9.

FIG. 1.

RT-PCR analysis of Bdkrb1 and Bdkrb2 expression in epithelial cells of adult rat epididymis. Top panel: Epithelial cells were isolated by LCM and used as templates for conventional RT-PCR analysis. A strong signal was obtained for the Bdkrb2 transcript, but no Bdkrb1 was detected. Bottom panel: Strong Bdkrb2 and Bdkrb1 signals were obtained in a whole epididymis mRNA extract. NTC, No template control.

FIG. 2.

Detection of BDKRB2 in rat epididymis. A) Western blot with anti-BDKRB2 antibody in whole epididymis protein extract (50 μg per lane). A single band at about 45 kDa was detected using the native antibody (-P) and was abolished when the antibody was preabsorbed with the immunizing peptide (+P). B, C) Immunofluorescence staining for BDKRB2 on rat cauda epididymidis sections. Intense labeling was detected in the apical pole of many epithelial cells (B, arrows). Smooth-muscle cell labeling was also visible (arrowheads). The staining was abolished after preincubation of the antibody with the BDKRB2 peptide (C). Images were acquired at the same exposure time. Bars = 20 μm.

FIG. 3.

Cellular localization of BDKRB2 in the cauda region of rat epididymis. Double labeling for ATP6V1E1 (A, red) and BDKRB2 (B, green) revealed that principal cells (negative for ATP6V1E1; arrows) express BDKRB2 in their apical pole, and that clear cells (positive for ATP6V1E1) are negative for BDKRB2. C) Merged panel. Smooth-muscle cells are also labeled (arrowheads). Nuclei are labeled blue with DAPI. Bars = 20 μm.

FIG. 4.

Localization of BDKRB2 in efferent ducts and throughout the epididymis. Digital images were acquired with identical parameters to allow comparison of the levels of BDKRB2 expression among different regions. A) Distal efferent ducts (ED). B) Transition zone between distal efferent ducts and proximal initial segment (p-IS). C) Distal initial segment (d-IS). D) Caput. E) Corpus. F) Cauda. Stronger staining patterns are seen in the efferent ducts and cauda epididymidis. Intermediate staining is detected in the distal initial segments. Moderate staining is seen in the corpus epididymidis, and no significant labeling is detected in the proximal initial segment and caput epididymidis. Bars = 30 μm (A), 60 μm (B), 20 μm (C), and 25 μm (D–F).

FIG. 5.

RT-PCR analysis of Bdkrb2 expression in the efferent ducts (ED) and in different epididymal regions: initial segment (IS), caput (CAP), corpus (COR) and cauda (CAU). Analysis was performed both by conventional end-point (A) and quantitative (B) PCR. Bdkrb2 expression normalized for Gapdh was relatively constant in the ED and the different epididymal regions. Gapdh, Loading controls using primer sets specific for GAPDH; NTC, no template control.

FIG. 6.

Confocal image of cellular localization of BDKRB2 in the efferent ducts. Immunostaining for BDKRB2 was performed in the proximal (A; p-ED) and distal efferent ducts (B; d-ED). Pictures were acquired using the same parameters. Similar staining intensity was detected in both segments. BDKRB2 is localized in the apical membrane of most epithelial cells. Some cells are negative for BDKRB2 (arrows). C) Double labeling for BDKRB2 (red) and the ciliated cell marker AE2 (green) was performed in the d-ED. Intense BDKRB2 labeling was detected in the apical membrane of nonciliated cells. No staining was detected in ciliated cells identified by their basolateral staining for AE2 (arrow). Bars = 30 μm (A, B) and 3 μm (C).

FIG. 7.

Triple immunostaining for AQP9 (B; green), BDKRB2 (C; red), and the ATP6V1E1 (D; blue). A) Merged panel showing that BDKRB2 colocalizes with AQP9 in the apical membrane of principal cells (yellow) and is absent from clear cells (blue). Nuclei were counterstained with DAPI and are shown in pink by including them in the red and blue channels simultaneously using Photoshop. Bars = 30 μm.

FIG. 8.

Effect of BK on the initial rate of cell swelling induced by luminal isotonic addition of glycerol on tubules isolated from the distal initial segment. A) Global profile of cell volume variations induced upon replacement of 60 mM raffinose with glycerol (Gly). After a postcontrol period of 5 min in the absence of glycerol (Control), BK was added into the luminal perfusate (BK) for a period of 5 min, followed by a second pulse of glycerol still in the presence of BK (BK + Gly). Inset: Initial rates of cell swelling measured during the first minute of both glycerol pulses (highlighted by the dashed lines). Data are mean ± SEM (n = 7). B) Left bars: Initial rates of glycerol-induced cell swelling measured under control conditions (GLY) and in the presence of 1 μM BK (GLY + BK; n = 7). Middle bars: Initial rates of cell swelling under control condition (GLY) or in the presence of BK and 5 μM BAPTA-AM (GLY + BK + BAPTA-AM; n = 9). Right bars: Control (GLY) or BK plus phloretin (GLY + BK + Phloretin; n = 7). Data are mean ± SEM. *P < 0.001; **P < 0.0005. Student t-test was used for paired experiments.