Sub-chronic testosterone treatment increases the levels of epithelial sodium channel (ENaC)-α, β and γ in the kidney of orchidectomized adult male Sprague–Dawley rats

Animal preparation and hormonal treatment

Eight weeks old male Sprague–Dawley (SD) rats were housed in a clean and well ventilated environment under light-dark cycle (12/12 h). The animals were fed with standard rat diet (Harlan, Germany) and tap water ad libitum. All procedures were approved by Institutional Animal Care and Use Committee (IACUC), University of Malaya (ethics number: 2014-05-07/physio/R/NS). Sham operation and orchidectomies were performed under ketamine/xylazine anesthesia. Two (2) weeks following recovery, hormonal treatments were initiated, which coincide with the age of the rats at 10 weeks old. Rats were divided into eight (8) treatment groups with eight (8) rats per group and were given the following treatment for seven (7) consecutive days.

Group 1: Intact, sham-operated receiving peanut oil only—S

Group 2: Orchiectomized, receiving peanut oil only—O

Group 3: Orchiectomized, receiving 125 µg/kg/day testosterone—T125

Group 4: Orchiectomized, receiving 250 µg/kg/day testosterone—T250

Group 5: Orchiectomized, receiving 125 µg/kg/day testosterone plus flutamide (8 mg/kg/day)—T125+FU

Group 6: Orchiectomized, receiving 250 µg/kg/day testosterone plus flutamide (8 mg/kg/day)—T250+FU

Group 7: Orchiectomized, receiving 125 µg/kg/day testosterone plus finasteride (5 mg/kg/day)—T125+FN

Group 8: Orchiectomized, receiving 250 µg/kg/day testosterone plus finasteride (5 mg/kg/day)—T250+FN

At the end of the treatment, rats were sacrificed by cervical dislocation and kidneys were removed for molecular biological and histological analyses.

Quantification of Enac mRNA levels in kidneys by high throughput qPCR (Fluidigm)

Immediately following removal, the kidneys from n = 4 rats per group were stored in RNAlater solution (Ambion, Foster City, CA, USA) in order to preserve the RNA integrity. Tissues were then weighted and disrupted in a tissue lysis buffer by using a rotor-stator homogenizer (Heidolph DIAX 600). RNA extraction was performed by using a Macherey-Nagel Nucleo Spin RNA kit (Düren, Germany) according to the manufacturer’s guidelines. cDNA synthesis was then carried out by using a Bio-Rad iScript Reverse Transcription Supermix for RT-qPCR (Biorad, Hercules, CA, USA). A high throughput qPCR-based microfluid dynamic array technology (Fluidigm) was performed to evaluate the changes in gene expression. The converted cDNA was pre-amplified by using Applied Biosystems PreAmp Master Mix, first, one cycle 95°C for 10 min, then 10 cycles, 95°C for 15 s and finally one cycle, 60°C for 4 min. The pre-amplified cDNA was diluted accordingly and was then used to perform the qPCR in Fluidigm dynamic array chip following the manufacturer’s protocol with the aid of applicant specialists. The chip was run in the BioMark Instrument for qPCR at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and lastly at 60°C for 1 min. Gapdh was selected as a house-keeping gene as its expression were the most stable in kidneys. Taqman primers and probes for Gapdh, Scnn1a (Enac-α), Scnn1b (Enac-β) and Scnn1g (Enac-γ) were purchased from Applied Biosystems, CA, USA. All experiments were carried out in triplicates and the data were analyzed by using a Fluidigm Real-Time PCR analysis software.

Quantification of ENaC protein levels in kidneys by Western blotting

The frozen kidneys, obtained from n = 4 rats per group were weighted, cut and the tissues submerged in RIPA buffer solution (BioVision, CA, USA) containing a protease inhibitor (BioVision, CA, USA), then disrupted using a rotor-stator homogenizer (Heidolph DIAX 600). The total proteins were obtained by centrifugation at 14,000× g for 15 min at 4°C and protein concentration was quantified by a Thermo Scientific Micro BCA Protein Assay Kit (Rockford, IL, USA) following the manufacturer guidelines. Equal amount of proteins were added into a loading dye, boiled for 5 min and loaded onto a 10% SDS-PAGE gel. Following separations, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, USA), then blocked in 5% bovine serum albumin (BSA) (Sigma-Aldrich, USA) for 60 min at room temperature. The membranes were then probed with primary antibodies against α-ENaC, β-ENaC, γ-ENaC (sc-22239, sc-25354, sc-21014, Santa Cruz Biotechnology, CA, USA respectively) for 90 min at room temperature. This was followed by incubation in horseradish peroxidase (HRP) conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 60 min at room temperature. The blots were then developed by using a Thermo Scientific Super Signal West Pico Chemiluminescent Substrate (Rockford, USA) according to the manufacturer’s manual. Chemiluminescent signals were captured by using a highly sensitive CCD camera-based imager (BioSpectrum Imaging System). The band intensity of each target was analyzed by using Image J software. GAPDH (Santa Cruz Biotechnology) was used as endogenous control. The experiment was performed in triplicate and the average ratios of target protein/GAPDH band intensity were then determined.

Whole body perfusion fixation and detection of ENaC distribution in kidneys by immunofluorescence

Another set of animals (n = 4 rats per group), which were duplicate of those used for Western blotting and qPCR analyses, were anaesthetized with ketamine/xylazine anesthesia and a transcardial perfusion was performed with injection of 4% paraformaldehyde (PFA) in phosphate buffer saline (PBS). The animals were then killed and the kidneys were removed and fixed in 30% sucrose for three days. The tissues were then processed and embedded in a paraffin wax. The kidneys were sectioned coronally into 5 µm by using a microtome. Sections were then mounted onto poly-L-lysine coated glass slides. Sections were then de-parafinized and antigens were retrieved by boiling the sections in 1mM EDTA (BioVision InC.), pH 8.0 containing 0.05% (v/v) Tween20 (Sigma Aldrich). Sections were then blocked with appropriate 10% (v/v) normal serum (Santa Cruz Biotechnology) in PBS for 1 h at room temperature and were then incubated with target primary antibodies (as above) at the ratio of 1:100 at 4°C, overnight. After three times of washing with PBS, sections were then incubated in appropriate fluorophore conjugated secondary antibodies at a dilution of 1:200, for 1 h at room temperature and then sealed with UltraCruz mounting medium (Santa Crus Biotechnology). Secondary antibodies used in this experiment were goat anti-mouse IgG FITC (sc-2010, Santa Crus Biotechnology, CA, USA), donkey anti-goat lgG (H+L) conjugated with DyLight 550 and donkey anti-rabbit lgG (H+L) conjugated with DyLight 488 (SA5-10087, SA5-10038, Thermo Scientific, Rockford, IL, USA). The images were viewed and captured by using a confocal laser scanning microscope (Leica TCS SP5 II) at a fixed exposure time. All experiments were carried out in four (4) replicates and the representative images were selected. All images were taken at 63x magnification and the slides without tissue were used as white balancer.

Measurement of serum testosterone level by enzyme-linked immunoassay (ELISA)

Immediately following sacrifice and prior to transcardial perfusion, the blood from n = 8 rats per group was collected via heart puncture and the serum was separated by centrifugation at 3,500× g, 25°C for 10 min. Levels of testosterone in serum were analysed by enzyme-linked immunosorbent assay (ELISA) kit (BioSource International, Inc., Camarillo, CA, USA), according to the manufacturer guideline.

Statistical analysis

All data were analyzed by SPSS software with mean ± standard error of mean (S.E.M) obtained. Statistical significant between groups was evaluated by Student t-test followed by one-way analysis of variance (ANOVA). Statistically significant difference were denoted as p < 0.05. Tukey’s post-hoc test was used to examine adequacy of the samples and all values were >0.08 which indicate adequate sample size.

Testosterone increases α-ENaC protein and mRNA levels in kidneys

In Fig. 1A, the levels of α-Enac mRNA were highest following testosterone-only treatment. Co-treatment with flutamide or finasteride caused α-Enac mRNA levels to decrease (p < 0.05). The effect of flutamide was greater in rats which received 125 µg/kg/day testosterone when compared to 250 µg/kg/day testosterone. However, the effect of finasteride in rats treated with 125 µg/kg/day testosterone- was not significantly different when compared to its effect in rats treated with 250 µg/kg/day testosterone.

In Figs. 1B and 1C, expression levels of α-ENaC protein was highest in rats which received 250 µg/kg/day testosterone. In these rats, co-administration of flutamide or finasteride caused α-ENaC protein expression level to decrease (p < 0.05). Flutamide effect was greater in rats treated with 125 µg/kg/day testosterone than in rat treated with 250 µg/kg/day testosterone. However, following co-administration of finasteride, no significant difference in α-ENaC protein expression level was observed between rats which received 125 mg/kg/day testosterone and rats which received 250 µg/kg/day testosterone.

Testosterone increases β-ENaC protein and mRNA levels in kidneys

In Fig. 2A, the levels of β-Enac mRNA were highest in rats which received testosterone-only treatment. In these rats, co-administration of flutamide or finasteride resulted in β-Enac mRNA levels to decrease (p < 0.05). The effect of flutamide was greater in rats which received 125 µg/kg/day testosterone when compared to rats which received 250 µg/kg/day testosterone. However, co-administration of finasteride did not cause β-Enac mRNA levels in rats which received 125 µg/kg/day testosterone to be difference from rats which received 250 µg/kg/day testosterone.

In Figs. 2B and 2C, the levels of expression of β-ENaC protein were highest in rats which received 250 µg/kg/day testosterone. In these rats, co-administration of flutamide or finasteride resulted in β-ENaC protein expression level to decrease (p < 0.05). The effects of flutamide were greater in rats which received 125 µg/kg/day testosterone when compared to rats which received 250 µg/kg/day testosterone. However, co-administration of finasteride did not cause β-ENaC protein expression level in rats which received 125 µg/kg/day testosterone to be different from rats which received 250 µg/kg/day testosterone.

Testosterone increases γ-ENaC protein and mRNA levels in kidneys

In Fig. 3A, the levels of γ-Enac mRNA were highest in rats which received 250 µg/kg/day testosterone. In these rats, co-administration of flutamide or finasteride resulted in γ-Enac mRNA levels to decrease (p < 0.05). The effect of flutamide was not significantly different between rats which received 125 µg/kg/day testosterone and rats which received 250 µg/kg/day testosterone. Similarly, the effect of finasteride in rats which received 125 µg/kg/day testosterone was not significantly different from its effect in rats which received 250 mg/kg/day testosterone.

In Figs. 3B and 3C, the levels of γ-ENaC protein was highest in rats which received 250 µg/kg/day testosterone. In these rats, co-administration of flutamide or finasteride resulted in γ-ENaC protein expression level to decrease (p < 0.05). Co-administration of flutamide with testosterone did not cause γ-ENaC protein expression level in rats which received 125 µg/kg/day testosterone to be different from rats which received 250 µg/kg/day testosterone. In contrast, co-administration of finasteride with testosterone caused greater decrease in the level of this protein in rats which received 250 µg/kg/day testosterone when compared to rats which received 125 µg/kg/day testosterone.

Distribution of α-ENaC protein in nephrons

In Fig. 4, α-ENaC protein was seen to be highly distributed in distal tubules and collecting ducts of rats which received 125 and 250 µg/kg/day testosterone. In these rats, co-administration of flutamide or finasteride resulted in α-ENaC protein distribution level to be relatively lower (Fig. 5).

Distribution of β-ENaC protein in nephrons

In Fig. 4, β-ENaC protein was highly distributed in distal tubules and collecting ducts in rats which received 250 µg/kg/day testosterone. However, co-administration of flutamide or finasteride with testosterone resulted in relatively lower β-ENaC protein distribution level when compared to rats which received testosterone-only treatment (Fig. 6).

Distribution of γ-ENaC protein in nephrons

In Fig. 4, γ-ENaC protein was highly distributed in distal tubule and collecting ducts in rats which received 125 and 250 µg/kg/day testosterone. However, co-administration of flutamide with testosterone resulted in relatively lower γ-ENaC protein distribution when compared to testosterone-only treatment (Fig. 7). Similar effects could be seen in rats following concomitant co-administration of finasteride with testosterone.

Serum testosterone level

In Table 1, administration of 125 µg/kg/day testosterone resulted in serum level of this hormone to be 13.5 fold greater than its level in non-testosterone-treated orchiectomised rats. Treatment with 250 µg/kg/day testosterone resulted in serum testosterone level to be 16.5 fold greater than in non-testosterone treated orchiectomized rats.