Roles of vacuolar H⁺-ATPase in mice treated with norepinephrine and acetylcholine

Abstract

Norepinephrine (NE) is widely used to treat cardiac arrest and profound hypotension. A prolonged vasoconstriction of blood vessel could cause ischemia and hypoxia which results in a decrease in intracellular pH. V-ATPases pump protons across the plasma membranes of numerous cell types. V-ATPases-mediated intracellular regulation in the ischemic kidney is incompletely studied; we sought to determine the roles of V-ATPases in mice treated with NE causing vasoconstriction or acetylcholine causing vasodilatation to enable comparison of its relative contributions to the affected mice. Mice were divided into 5 groups. Histology and immunohistochemistry were performed to examine pathologic changes in nephron segments. The expression of V-ATPases B1, B2 subunits were examined by Q-PCR and western blotting correlated with the transcription and translation of V-ATPase. All NE treated mice exhibited pronounced renal tubular degradation. However, the tubular pathologies were reversed by ACh. In immunohistochemical studies, NE treated mice showed a higher density of staining in the collecting ducts. These changes were gradually diminished by the treatment with Ach after NE. In Q-PCR, V-ATPase B1 subunit showed a fair expression in all subsets. Western blotting analysis has shown V-ATPase B1 statistical significance in multiple groups treated by NE alone or ACh post to NE. The overdosage of norepinephrine in clinical treatment is harmful to the kidney by vasoconstriction caused hypoxia and acidosis. Our data demonstrated that acetylcholine as a vasodilating agent could aid the cells recovery from hypoxic condition. V-ATPase plays a role by removing H⁺ allowing cells to recover from cellular acidosis. These findings also help us understand the pathophysiology of renal tubular disorders.

Introduction

Norepinephrine (NE) is commonly used to treat cardiac arrest and profound hypotension. Its receptor actions are dose-dependent and cause vasoconstriction and an increase in cardiac output by increasing systemic vascular resistance (SVR) and mean arterial pressure (MAP). This effect is strong in the skin, digestive tract, and kidneys. NE causes a greater response than epinephrine (E) when activating alpha 1 receptor.

It is known that the muscarinic receptors present on vascular endothelial cells are associated with acetylcholine (ACh)-dependent vasodilatation. Binding of ACh to endothelial muscarinic receptors in arteries activates both nitric oxide (NO)-dependent and independent vasodilatory pathways. ACh binding to the muscarinic receptor triggers an increase in intracellular calcium within endothelial cells. This activates endothelial nitric oxide synthase (eNOS) to produce the potent endogenous vasodilator nitric oxide. It has been shown in many arterial vessels that M3 receptors located on the vascular endothelium are coupled to the formation of nitric oxide (NO), which causes vasodilation [1-3].

Endothelial M3 has been also reported to mediate ACh-induced vasodilatation in murine retinal arterioles via the activation of NO synthase. This is also found in cutaneous tissue, skeletal muscle, and renal interlobar arteries [4]. Takenaga et al. [5] reported interesting findings that ACh induced an initial transient endothelium-dependent vasodilatation followed by a secondary long-lasting endothelium-independent vasodilatation in rat mesenteric vascular beds.

Ischemic tubular injury can result from acute vasoconstriction [6]. Prolonged vasoconstriction can lead to tubular dysfunction, tubular acidosis and necrosis, as well as inflammation of the surrounding interstitium.

Recent study has shown that administration of norepinephrine to mice has triggered adverse effects relating predominantly to the urogenital tract associated with norepinephrine-induced vasoconstriction, urinary retention, and renal impairment [7]. Ischemia/hypoxia generally leads to decreases in both intracellular pH (pHi) and extracellular pH (pHo) in brain cells [8].

Vacuolar-type H⁺-ATPases (V-ATPases), which are found within the membranes of many organelles, such as endosomes, lysosomes, and secretory vesicles in eukaryotic cells, catalyze ATP hydrolysis to proton transport across intracellular and plasma membranes [9].

Our previous data have shown that the contribution of bafilomycin-sensitive H⁺-ATPase to net acid secretion in the kidney tubules under low-K+ diet. H⁺-ATPase plays an important role in acid-base homeostasis during chronic hypokalemia, which leads to metabolic alkalosis [10-15].

Since the mechanism of H⁺-ATPase-mediated intracellular regulation in the ischemic/hypoxic kidney is incompletely studied in the mouse, we sought to determine the roles of vacuolar H⁺-ATPase in mice treated with norepinephrine causing vasoconstriction or/and acetylcholine causing vasodilatation to enable comparison of its relative contributions to those described for mice (Figure 1).

Figure 1.

The role of V-ATPase in intracellular pH homeostasis. Along with the regulatory mechanism of NE or ACh within the blood vessels. This diagram illustrates how vacuolar H⁺-ATPase (V-ATPase) plays an important role in the maintenance of intracellular pH (pHi) homeostasis. Prolonged vasoconstriction of the blood vessel could cause ischemia and result in a fall in pHi. Acetylcholine (ACh) is a muscarinic agonist that causes receptor-mediated, endothelium-dependent vasodilatation. It is known that acetylcholine (ACh), whether administered intravascularly or released by cholinergic autonomic (parasympathetic) nerves, binds to acetylcholine muscarinic M3 receptors (CHRM3) located on the vascular endothelium, which stimulates the formation and release of NO to produce vasodilation.

In this study, we observed histological and immunohistological changes in mice treated with norepinephrine or/and acetylcholine. We examined H⁺-ATPase transcript and protein level under various circumstances. To complement the current investigations on norepinephrine induced ischemia/hypoxia, we pre-treated or post-treated acetylcholine to see if H⁺-ATPase made a critical contribution to rescue the renal cells. A direct assessment of the effect of ischemic intracellular acidosis on the distribution of the proton pump in mice was made by using specific antibodies of vacuolar H⁺-ATPase B1-B2, B1, or B2.

Materials and methods

Animals

Seven-week old male C57BL6/J mice (20-25 g) were obtained from Shanghai Jiexijie laboratory animal co. LTD (Minhang, Shanghai, China). All mice were fed normal diets and had unrestricted access to water at 25°C under a 14-hour light, 10-hour dark photo period. After 3 days of adapting to the environment, the mice were divided into 5 groups with different treatments for 2 weeks. Control group (NS) of mice were administered 0.9% NaCl intraperitoneally (4 mice). The following four experimental groups (NE, ACh, AN, NA) were all administrated equal volumes of solutions compared to the control group, by using Acetylcholine independently in “ACh”, Norepinephrine independently in “NE”, Pre-Acetylcholine treatment in “AN”, and Post Acetylcholine treatment in “NA”. Both Norepinephrine and Acetylcholine chloride were dissolved in 0.9% NaCl (pH=5.5). NE group of mice were administered Norepinephrine (NE) (Sigma-Aldrich) intraperitoneally at a concentration of 1 mg/kg twice a day (6 mice). ACh group of mice were administered Acetylcholine chloride (ACh) (Santa Cruz) intraperitoneally at a concentration of 10 mg/kg twice a day (4 mice). AN group of mice were administered Acetylcholine chloride for only 1 week then Norepinephrine (NE) alone for 1 week (5 mice). NA group of mice were given Norepinephrine (NE) for 1 week then the mice were only given Acetylcholine chloride for another week (5 mice) [7].

Mice were anesthetized with methoxyflurane and euthanized by cervical dislocation [7]. The kidneys were removed. Then, each kidney was cut transversely into three thick slices about 2 to 3 mm. One of the tissue pieces was fixed in paraformaldehyde for histology and immunohistochemistry studies. One piece was frozen at -80°C for western blotting analysis and the final piece was treated with TRIzol (Thermofisher) for RT-PCR analysis. All experiments were performed in accordance with the NIH guidelines for the care and use of laboratory animals and approved by the animal ethics committee of SUMHS.

Histology

Slides were cut transversely through the kidney on a Vibratome at a thickness of 8 µm and stained with hematoxylin-eosin (Sigma-Aldrich) as suggested protocol gram (Sigma-Aldrich) to assess NE induced histological changes. Briefly, Hematoxylin for 3 min, rinsed in tap water for 10 min (to allow stain to develop), 1% Acid ethanol (to destain) for 30 sec, tap water for 5 min, Eosin staining 30 sec, dehydration 3 × 5 min in 95% ethanol and 3 × 5 min 100% ethanol, clear 3 × 10 min in Xylene, mount using Permount. Micrographs were taken using PRECICE 500 Microscope scanner (Youna technology co. LTD, Beijing, China).

Immunohistochemistry

Slides were processed for immunohistochemistry using an indirect immunoperoxidase method, according to the manufacturer’s protocol (Santa Cruz, USA). For antigen retrieval, all slices were washed with PBS (1.4 mM NaCl, 0.2 mM KCl, 0.1 mM Na₂HPO₄, and 0.002 mM KH₂PO₄; pH7.4) solution 3 times for 15 min. We pre-heated the staining dish containing 10 mM sodium citrate buffer (pH 6.0) until the temperature reached 95-100°C, immersed the slides into the staining dish, boiled them for 8 minutes in microwave oven, allowed the slides to cool in the buffer for approximately 20 minutes at room temperature. We washed them in deionized H₂O 3 times for 2 min each. After being preincubated in 3% H₂O₂ PBS 30 minutes and washed 3 times for 15 min at room temperature, all slides were incubated in 1% BSA-PBS 60 minutes at room temperature. Liquid was removed from slides.

Slides were then incubated overnight at 4°C in a mixture of mouse monoclonal antibody V-ATPase B1/2 Antibody (F-6) (sc-55544, Santa Cruz) diluted 1:500 in PBS containing 1% BSA. After 3 washes (10 min/wash) with PBS, the slides were then incubated with mouse IgG kappa binding protein conjugated to Horseradish Peroxidase (m-IgGκ BP-HRP) (sc-516102, Santa Cruz), and diluted 1:1000 in PBS at room temperature for 1 hour.

Slides were incubated in 0.01% hydrogen peroxide (sc-203336, Santa Cruz) and 2% DAB (sc-24982, Santa Cruz) mixed in 0.01 M PBS about 2 min, until desired stain intensity developed. Optimal time for staining was monitored to determine the proper development time. Sections were washed in deionized H₂O 3 times for 15 min. They were counterstain with Hematoxylin Solution (MHS16, Sigma-Aldric), dehydrated in graded alcohols to xylene, and mounted using xylene-based limonene (sc-45087, Santa Cruz).

Real-time quantitative PCR (Q-PCR)

Mouse kidney samples were homogenized in Trizol (Thermo-fisher, USA) to extract total RNA. Total RNA samples (1.3 ug) were treated with Rt enzyme mix1 (Takara) and reverse transcribed into cDNA using TB Green Premix Ex Taq TB (Takara). Specific primers for V-ATPase subunits B1 and B2, Foxi1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed using Beacon Designer 2.0 (Premier Biosoft International, Palo Alto, CA) and are summarized in Table 1.

Table 1.

Primers used for real-time PCR

Primer Pair	Forward	Reverse	Length (bp)
*V-ATPase
Atp6v1B1	5’-TGGACCAGGTCAAGTTTGC-3’	5’-CCCTGAAGTCCCTTCAAACA-3’	134
Atp6v1B2	5’-TGAGCCGGAACTACCTATCCCA-3’	5’-GTGCCATCTGGTAATGTCAAGTGG-3’	136
*Foxi1	5’-CCTCTCCACCATGACAGCAT-3’	5’-TCCCATGGCTACTGAGGTTG-3’	155
*GAPDH	5’-TGCACCACCAACTGCTTAGC-3’	5’-GGATGCAGGGATGATGTTCT-3’	176

^*V-ATPase, vacuolar H⁺-ATPase;

^*GAPDH, glyceraldehyde-3-phosphate dehydrogenase;

^*Foxi1, hypoxanthine guaninephosphoribosyl transferase.

Total RNA was purified following the manufacturer’s protocol (Thermo-fisher, USA), briefly, kidney tissues about 100 mg was homogenized in 1 ml TRIzol reagent on ice for 20 min, shook with 200 ul Chloroform in 1.5 ml RNase-free tube for 5 min, centrifuged at 12,000 rpm in phase lock gel tube for 15 min at 4°C, transferred upper aqueous phase to a new 1.5 ml RNase-free tube, precipitated the RNA by mixing with 500 ul isopropyl alcohol for 10 min at room temperature, centrifuged at 12,000 rpm for 15 min at 4°C, The supernatant was removed completely, The RNA pellet was washed twice with 1 ml 75% ethanol and centrifuged at 12,000 rpm for 15 min at 4°C, The RNA pellet was washed once with 1 ml absolute ethanol and centrifuged at 12,000 rpm for 15 min at 4°C, air-dried RNA pellet for 5-10 min, dissolved RNA in 50 ul DEPC-treated water. The concentration and purity of sample RNA were determined at 260 and 280 nm by Nano Drop Micro-UV/Vis Spectrophotometers (ND-one, Thermo-fisher), adjusted the concentration of sample RNA to 50 ng/ul with RNase-free water at appropriate dilution. All RNA pellets were stored at -20°C [16].

Following PrimeScript™ RT reagent Kit guide (RR037A, Takara), 26 ul (1.3 ug) of total RNA was reverse transcribed in a final volume of 40 ul containing 8 ul 5 X PrimeScript Buffer, 2 ul PrimeScript RT Enzyme Mix, 2 ul Oligo dT Primer and 2 ul Random 6 mers, for 15 min at 37°C, followed by for 5 sec at 85°C, and store at 4°C.

Real-time PCR analyses were performed in triplicate with a LightCycler 480 real time PCR system (Roche). According to the guide of TB Green® Premix Ex Taq (RR420A, Takara), each PCR consisted of TB Green Premix Ex Taq (TliRNaseH Plus) (2X) 12.5 ul, PCR Forward Primer (10 uM) 0.5 ul, PCR Reverse Primer (10 uM) 0.5 ul, Template (<100 ng) 2 ul and sterile purified water 9.5 ul in a final volume of 25 ul. PCR conditions were settled as incubation at 95°C for 30 sec followed by 40 cycles of 5 sec at 95°C, 30 sec at 60°C, melting curves analysis were settled as incubation at 1 cycle of 95°C 5 sec, 60°C 1 min, 95°C (5 per °C), and cooling 50°C 30 sec. PCR products were subjected to a melting-curve analysis, and representative samples were electrophoresed to verify that only a single product was present. Control reactions were conducted with sterile water to determine background levels and detect genomic DNA contamination. The standard curve of each gene was confirmed to be in a linear range, with GAPDH as an internal control.

Western blotting

The protein expression of V-ATPase was detected by western blot method, according to the suggested protocol (Santa Cruz, USA). Briefly, kidney samples were washed three times with ice-cold PBS, then lysed in RIPA lysis buffer [10 μl PMSF solution, 10 μl sodium orthovanadate solution and 10-20 μl protease inhibitor cocktail solution per ml] (sc-24948, Santa Cruz) for 30 min at ice. The lysate was centrifuged at 1000 rpm for 15 min at 4°C, and the resulting supernatant was centrifuged at 120,000 rpm for 15 min at 4°C. The pellet was suspended in homogenization buffer and stored at -80°C, before determination of protein concentration according Coomassie (Bradford) Protein Assay. 50 μg of each lysate sample was boiled for 5 min in sample buffer and were separated by 10% SDS-PAGE (20 ug/well) and transferred onto nitrocellulose membrane. After nonspecific reactivity was blocked in 5% nonfat dry milk in TBST (10 mM Tris-HCl, pH7.5, 150 mM NaCl, 0.05% Tween-20) for 1 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies, which included V-ATPase B1/2 Antibody (F-6) (sc-55544, Santa Cruz), V-ATPase B2 Antibody (D-11) (sc-166045, Santa Cruz), V-ATPase B1 Polyclonal Antibody (PA575580, Thermo-Fisher) and β-Actin Antibody (sc-517582, Santa Cruz) at dilute 1:1000 in TBST. Washed 3 times for 15 min, incubated separately for 1 h at room temperature with appropriate peroxidase-labeled second antibodies which included m-IgGκ BP-HRP (sc-516102, Santa Cruz) and mouse anti-rabbit IgG-HRP (sc-2357, Santa Cruz), washed 3 times for 15 min again, and visualized with enhanced chemiluminescence (ECL). Normalization for β-actin was obtained after stripping the blots and reprobing with the anti-β-actin antibody [17]. Immunoblots were scanned and exported to JPEG files, and the band intensities were analyzed by using Image J. All immunoblots were performed at least in duplicate.

Statistical analysis

Data are presented as means ± SD. Analysis of one-way ANOVA and t test were used where appropriate to determine statistical significance. P<0.05 was considered statistically significant.

Results

Phenotype evaluation and morphology

All NE treated mice that were detrimentally affected, exhibited physically unwell, displaying ruffled coat, slow response to touch, eating less, lower body temperature, and shivering. NE affected mice had pronounced bladder distension. The urinary bladder was over accumulated with urine and it appeared as a structural abnormity (Figure 2). NE treated mice often displayed pale kidneys (Figure 2). In comparison to NS, bladders appeared normal from ACh treated mice. Mice treated by ACh prior to NE exhibited similar changes to NE alone group. Whereas, mice treated by ACh after NE showed less severe changes.

Figure 2.

Phenotype evaluation of kidneys and bladders. Mice were administered for 2 week durations in the following groups: NS: Control; normal saline for 2 weeks. ACh: Mice under administration of Acetylcholine for 2 weeks. AN: Acetylcholine for 1 week, Norepinephrine for 1 week. NA: Norepinephrine for 1 week, Acetylcholine for 1 week. NE: Norepinephrine for 2 weeks. All NE treated mice were detrimentally affected; NE-affected mice had pronounced bladder distension. The urinary bladder was over-filled with urine and it appeared as a structural abnormity. Also, NE treated mice displayed pale kidneys (red arrows). Mice treated by ACh prior to NE exhibited similar changes compared to NE alone. In contrast, mice treated by ACh after NE showed less severe changes. In mice treated with Ach alone, the bladders, and the kidneys appeared normal compared to NS.

Representative images of hematoxylin-eosin stained kidney demonstrate that kidneys from NE affected mice exhibited tubular degradation, glomeruli destruction to some extent, increased inflammatory cell infiltration compared with kidneys from control mice (Figure 3). Renal tissue sections of different subsets were compared by segments, such as renal cortex (Figure 3A), outer stripe of outer medulla (OSOM) (Figure 3B), inner stripe of outer medulla (ISOM) (Figure 3C), and inner medulla (Figure 3D). The collecting ducts in NS and ACh treated mice show clear lumen, cell contour, and thin tubular wall indicated by the blue arrow. In contrast, the collecting ducts from NE treated mice were characterized by cell swelling, blurred contour, and stenosis as shown by the red arrow in AN, NA and NE subsets. Notably, these adverse changes were mitigated to some extent by treatment with ACh after NE, but not significantly improved by treatment with ACh prior to NE.

Figure 3.

Representative image (400x) of hematoxylin-eosin (H&E) stained renal tissue from mice treated by norepinephrine (NE) and/or acetylcholine (ACh) and normal saline (NS). The collecting ducts in NS or ACh treated mice shows clear lumen and thin tube wall (blue arrow). In contrast, the collecting ducts from NE treated mice were characterized by cell swelling, blurred contour, and stenosis (red arrow). (A) shows Cortex collecting ducts, (B) shows collecting ducts in outer stripe of medulla (OSOM), (C) shows collecting ducts in inner stripe of medulla (ISOM), (D) shows collecting ducts in inner medulla.

Immunohistochemistry analysis

To investigate the effects of norepinephrine on renal collecting duct cells, especially on the intercalate cells associated with acid secretion; we conducted immunohistochemical studies with anti-V-ATPase B1/B2 antibody to compare the staining patterns in each of the nephron segments. The results in the collecting ducts in NS or ACh treated mice show a small number of stained cells with a lower density indicated by the blue arrow (Figure 4). In contrast, the collecting ducts from NE treated mice showed a higher density of staining in a greater population of the cells indicated by the red arrow (Figure 4). These changes were gradually diminished by the treatment with Ach after NE. These changes can be observed in each of the nephron segments.

Figure 4.

Representative image (400x) of immunohistochemical stained renal tissue slices. Mice were treated by norepinephrine (NE) and/or acetylcholine (ACh) and normal saline (NS), V-ATPase B1/2 antibody was used. The collecting ducts in NS or ACh treated mice shows a small number of cells with a lower density of staining as shown by blue arrow. Whereas, the collecting ducts from NE treated mice show a higher density of staining in a greater population of cells, as shown by the red arrow. These changes were gradually diminished by the treatment with ACh after NE, but not significantly mitigated by treatment with ACh prior to NE. All nephron segments of collecting ducts with similar patterns of V-ATPase B1/2 antibody staining were observed. (A) shows cortex collecting ducts, (B) shows collecting ducts in OSOM, (C) shows collecting ducts in ISOM, (D) shows collecting ducts in inner medulla.

RT-PCRT analysis

The expression of V-ATPase B1, B2 subunits were investigated using real-time RT-PCR analyses (Figure 5), repeat experiments were performed, n was recorded as experiment times/samples (Table 2). V-ATPase B1 subunit showed relatively higher expressions in ACh subset (105%) and AN subset (118%). Whereas, the expression in NA subset (82.6%) and NE subset (97.6%). V-ATPase B2 subunit in all subsets showed a lower expression, from 98.3% to 85.8%. The expressions of B1, B2 subunits, and FOXI1 (the forkhead transcription factor Foxi1, which is a member of the HFH/winged helix family) showed a fair expression in different subsets. No Significant difference was detected between any two subsets (Table 2).

Figure 5.

Relative levels of transcripts in kidneys of all subsets. Real-time RT-PCR quantification for mRNA expression of B1 and B2 subunits, as well as Foxi1 in all subsets. The mRNA levels were first adjusted to GAPDH at every subset, then normalized to NS subset at 100% using the following formula: [Ratio= (Efficiency _target)^{ΔCt (subset - NS)}/(Efficiency _GAPDH)^{ΔCt (subset - NS)}]. The B1 (A), B2 (B) subunits and FOXI1 (C) showed a fair expression in different subsets, no significant difference between any two groups.

Table 2.

Relative transcript levels of V-ATPase B1, B2, and FOXI1^a

	ACh	AN	NA	NE	NS
V-ATPase B1	1.19±0.31	1.18±0.35	0.83±0.30	0.98±0.49	1
V-ATPase B2	1.02±0.34	0.86±0.40	0.67±0.28	0.92±0.47	1
FOXI1	0.80±0.42	1.25±0.62	0.68±0.65	0.81±0.53	1
n	7/3	7/4	6/4	7/5	8/3

^aReal-time PCR quantification for mRNA expression of B1 and B2 subunits, as well as FOXI1 in all subsets.

Data are mean ± SD. The mRNA levels were first adjusted to GAPDH as internal reference at every subset, then normalized to NS group at ratio using the following formula: [Ratio= (Efficiency _target)^{ΔCt (subset - NS)}/(Efficiency _GAPDH)^{ΔCt (subset - NS)}]. The B1, B2, subunits and FOXI1 showed a stable expression in different subsets. No significance between any two subsets. n, the number of experiences/animals. Statistical analysis was performed by t-test assuming equal variances, and one-way ANOVA. No significance between any two subsets.

Western blotting analysis

To quantitatively analyze the effect of norepinephrine (NE) as well as acetylcholine on V-ATPase, western blotting analysis was performed, and the B1 and B2 subunits of V-ATPase in the samples were relative quantified separately with β-actin as internal reference (Figure 6A-E).

Figure 6.

Expression of V-ATPase subunits B1 and B2. A. Relative expression of B1 in mouse kidney in all subsets. Densitometry analysis show 1.50 to 2.95 times expression of B1 subunit. Significant differences were revealed in the following groups: NS vs. NE (P<0.01). NE vs. NA and AN vs. NE (P<0.05). B. The expression of B2 in all subsets. Significant differences were revealed ACh vs. NA (P<0.05). C. Representative immunoblots for subunits B1 and B2, we observed 2 bands at 58 kd and 56 kd by using V-ATPase B1/B2, the bands are too close to be analyzed. D. Representative immunoblots for subunits B1. E. Representative immunoblots for subunits B2. A total of 20 ug of protein was loaded in each lane. Blots were probed with antibodies against B1 (1:1000), B2 (1:1000), β-actin (1:1,000).

In our study, we initially observed two bands at 58 kd and 56 kd by using anti-V-ATPase B1/2, according to their molecular weight, we believed the two bands were subunit B1 and subunit B2 of V-ATPase (Figure 6C). Since the two bands were too close to analyze, in the follow-up experiment, we used V-ATPase B2 antibody (D-11) (sc-166045) and V-ATPase B1 Polyclonal Antibody (PA575580) to stain membrane separately (Figure 6D and 6E). The experiment was repeated for the tissue samples, n was presented as experiment times/samples (Table 3). Immunoblots were analyzed by ImageJ.

Table 3.

Relative protein levels of V-ATPase B1 and B2^a

	ACh	AN	NA	NE	NS
V-ATPase B1c	2.34±1.21	1.53±0.74	1.57±0.93	2.95±1.13**/*/*	1.50±0.47
V-ATPase B2d	1.63±1.62	0.66±0.40	0.45±0.31*	0.89±0.32	0.89±0.56
n b	14/4	10/4	11/4	12/5	11/4

^aData are mean ± SD, expressed in the ratio of the intensity of each protein band to the internal reference β-actin.

The western blotting bands were measured by Image J. ACh, Acetylcholine; AN, Acetylcholine prior to Norepinephrine; NA, Norepinephrine before Acetylcholine; NE, Norepinephrine; NS, Control; normal saline.

^b n: shows the experiences/animals in western blotting.

^cStatistical significance of V-ATPase B1: **P<0.01 in NS vs. NE. *P<0.05 in the following four groups: NA vs. NE, AN vs. NE.

^dStatistical significance of V-ATPase B2: *P<0.05 in ACh vs. NA.

Relative protein levels of V-ATPase B1 demonstrated varying ranges from 1.50 to 2.95 times (Table 3). Significant differences were revealed in following groups: NS vs. NE (P<0.01). NE vs. NA and AN vs. NE (P<0.05). V-ATPase B2 showed varying degrees from 0.45 to 1.63 times (Table 3). Statistically significant levels of V-ATPase B2 was only observed between ACh vs. NA (P<0.05).

Discussion

The current experiment has examined the effects of norepinephrine and acetylcholine on the urinary system. We have examined the kidney and urinary bladder by histology and immunohistochemistry. We observed the morphological and histological changes in the kidney by the drug effects. According to previous studies conducted by Schlaich et al., 2018, we used norepinephrine for seven days and fourteen days and observed that the kidney exhibits hypoxic changes under this treatment [7]. We have seen that increased dosage and extended dates of administration of norepinephrine, the kidney has displayed significant swelling due to vasoconstriction within the nephron. These results are consistent with previous studies and their findings. In our histological studies, we have specifically examined in the renal cortex, the outer strip of the outer medulla (OSOM), as wells as the inner strip of the outer medulla (ISOM) and the inner medulla. We have found norepinephrine causes the collecting ducts to appear narrow in the lumen due to swelling of intercalated cells. Anatomically the glomerular afferent arteriole controls GFR through vasoconstriction or vasodilation. In contrast, the peritubular capillaries and vasa recta have a larger impact on the secretion and reabsorption mechanism to the nephron. As we know, norepinephrine has an effect on GFR through vasoconstriction [18-20]. However prolonged vasoconstriction leads to reduction of the circulation in peritubular capillaries and vasa recta [21,22]. This mechanism could cause hypoxic conditions in tubular cells and lead to hypoxic-ischemic cell injury. Hypoxia also causes a switch from oxidative to glycolytic energy generation, with increased lactic acid production and lower intracellular pH, which reflects the degree of anaerobic metabolism. This pathological progression leads to metabolic acidosis [23-26].

As the results show, the cell swelling and degradation are more prevalent in the collecting ducts in both the outer and inner stripes of the outer medulla. This indicates norepinephrine causes vasoconstriction related to cell damage more prevalent in the juxtamedullary nephron which is 15% of nephron population. As we know, the juxtamedullary nephron is highly related to urinary concentration mechanism. Because of this, we see the urine accumulate in the urinary bladder in our animal group under administration of norepinephrine. The cell swelling is caused by vasoconstriction under norepinephrine administration. However, these changes have been reversed or rescued by administration of acetylcholine, a vasodilating agent. These antagonistic factors could relieve oxygen deficiency caused by norepinephrine on the cell, as well as tissue degradation. In this study the acetylcholine affect was observed post norepinephrine treatment. However, it seems this has no significant antagonistic effect to norepinephrine with initial pre-treatment of acetylcholine to norepinephrine.

H⁺ V-ATPase is widely spread throughout the body, and is responsible for acid secretion. V-ATPases are ubiquitous in endomembrane of acidic organelles, ‘vacuoles’ such as lysosomes, but they are also found in the plasma membranes of ion-transporting epithelia [27-32]. Our previous study has shown V-ATPase is actively involved in acid transport under acidosis or electrolyte imbalance and other pathological conditions. We have shown the contribution of bafilomycin-sensitive H⁺-ATPase to net acid secretion in the kidney tubules under low-K+ diet. H⁺-ATPase plays an important role in acid-base homeostasis during chronic hypokalemia, which leads to metabolic alkalosis [10-15].

In this study we used the V-ATPase antibody to examine the expression of this transporter to see whether it plays a critical role for acid secretion (H⁺) in intracellular acidosis caused by hypoxic damage to the cells. Since the vasoconstriction causes hypoxia, further leading to acidosis in the cell, we anticipated that under these conditions, the expression of V-ATPase would increase. Our results show that V-ATPase is an active participant in hypoxic cells, due to administration of norepinephrine causing vasoconstriction. We have also used a V-ATPase antibody with B1/B2 subunits to localize the V-ATPase in the specific nephron segments under the circumstances of vasoconstriction causing cell degradation in hypoxic condition. We have observed that V-ATPase staining accumulates more in the apical membrane of the intercalated cells. This observation is consistent with our previous publications on V-ATPase assembly in the apical membrane for acid secretion [10-12]. We interpret that the V-ATPase transporter is activated for acid secretion under the circumstances. We have seen a higher V-ATPase staining in the renal medulla particularly in the collecting ducts in the outer strip of outer medulla and the inner strip of outer medulla. The localization of V-ATPase in the intercalated cells of the collecting duct is consistent with our previous publications [10-15].

As current results, we have found the mice under the administration of acetylcholine post to norepinephrine has shown less V-ATPase staining in the collecting ducts compared to using norepinephrine alone. The immunostaining of V-ATPase is similar to that of the control group of mice. This indicates V-ATPase activation is associated with the severity of the cell under acidosis caused by hypoxic conditions through vasoconstriction. However, acetylcholine by itself has not shown to increase V-ATPase staining in acetylcholine treated mice. These results indicate acetylcholine causing vasodilation has a significant effect on the hypoxic cells that was induced by norepinephrine through vasoconstriction.

It is understood B1 (58 KD) is more dominantly expressed in the kidney, whereas B2 (56 KD) is more dominantly expressed in the brain [33-35]. B1 subunit is in V1 cytoplasm domain of V-ATPases [36-38]. The V1 domain contains tissue-specific subunit isoforms including B, C, E, and G. Mutations to the B1 isoform result in the human diseases distal renal tubular acidosis and sensorineural deafness [39-42]. Our speculation was the result from the western blot by using a combination of V-ATPase B1/B2 could be one band of B1 subunit since the experiment was conducted in the kidney. However, we have observed two closed bands, their molecular weights were 58 KD and 56 KD. Therefore, we believe B2 subunit is also expressed in the kidney. In order to confirm B2 subunit of V-ATPase in our study, we have used B1 or B2 antibody separately to quantify the individual subunits of B1 or B2. Our results have shown that the expression of V-ATPase B1 is enhanced in the norepinephrine treated group labeled as NE group. This indicates V-ATPase is upgraded in norepinephrine, causing cell degradation secondary to acidosis. In the animal groups treated with acetylcholine post-norepinephrine, labeled in figures as NA, we observed significant changes compared to norepinephrine alone in the V-ATPase expression. The V-ATPase expression in the NA group is almost similar to that in the control group. This indicates acetylcholine does play a significant role to help cell recovery from acidosis. It also convinced us that norepinephrine caused cell acidosis or degradation due to vasoconstriction, which can be relieved by acetylcholine through vasodilation. In addition, the acetylcholine itself caused a higher mean value of V-ATPase expression, this may be due to the experimental variation, as shown in the table. The expression of B2 shows a similar pattern that we found in B1. We observed that the group treated with acetylcholine post-norepinephrine has the rescue factor, compared to the control group. However, the results in the B2 subunit are not statistically different between NE vs post-norepinephrine acetylcholine treated group. Although the quantity of V-ATPase expression is relatively lower in the B2 subunit compared to the B1 subunit, this can be attributed to the fact that B1 subunit is primarily found in the kidney, whereas B2 subunit is found primarily in the brain [38-40].

In our PCR results, the mice in the preacetylcholine norepinephrine treated group (AN) showed a higher expression of V-ATPase compared to the post-acetylcholine norepinephrine treated group (NA). Acetylcholine reduced V-ATPase expression in the post-acetylcholine norepinephrine (NA) treated group but our statistical testing did not show significance between these two; AN vs. NA.

Similar results are also observed in the NE vs. NA groups, in which there is, again, no statistical significance. In the transcriptional level we believe acetylcholine itself did not stimulate V-ATPase expression. Our results on Foxi1 have also shown no statistical differences between any two groups. The forkhead transcription factor Foxi1 is a master regulator of vacuolar H⁺-ATPase proton pump subunits in the inner ear, kidney and epididymis [16,43,44]. This finding could be an additional indication that neither norepinephrine nor acetylcholine changes V-ATPase expression on the transcription level.

We have noticed that there are some discrepancies between our western blot and PCR results. This is possibly due to differences between transcription and translation levels in V-ATPase expression. The effect of acetylcholine at the translational level, as seen on the western blot, is possibly due to an alteration of a V-ATPase assembly mechanism under intracellular acidification.

There are two possible V-ATPase assembly mechanisms. Mutational analysis and in vitro assays have shown that preassembled Vo and V1 domains can combine to form one complex in a process called independent assembly. Support for independent assembly includes the findings that the assembled Vo domain can be found at the vacuole in the absence of the V1 domain, whereas free V1 domains can be found in the cytoplasm and not at the vacuole [45-47]. In contrast, in vivo experiments have revealed early interactions between Vo and V1 subunits, the A and B subunits, suggesting that subunits are added in a step-wise fashion to form a single complex in a concerted assembly process [47]. Brown et al., 2003 has reported that B1 is not an absolute requirement for plasma membrane targeting of the V-ATPase [27]. Also, the result from Enerback et al. 2009 [43], suggests that B1 along with other V-ATPase subunit genes are regulated indirectly at the transcription level. In this study, we have detected an increase of V-ATPase immunostaining as well as higher expression levels of protein quantification by western blot. In contrast, the expression of V-ATPase in PCR has no statistical significances between NE and NA.

We speculate that B1 subunit with no protein steric structure could not be recognized and failed to be detected by the specific antibody, as described by the “key fits lock model” theory. The differences in detection levels between transcription and translation may possibly represent the B1 subunit alone, prior to forming a protein steric structure with the V1 domain, as opposed to being assembled in a V1/V0 complex.

In conclusion, the over-dosage of norepinephrine in the clinical treatment setting is harmful to the kidney on the cellular level, causing hypoxia and acidosis, secondary to vasoconstriction. Our data demonstrates that Acetylcholine as a vasodilating agent could play a role as “extracellular rescue” to aid the cells’ recovery from hypoxic conditions. Vacuolar H⁺-ATPase (V-ATPase)-mediated intracellular regulation is actively involved in the ischemic/hypoxic kidney. V-ATPase could play a role of “intracellular rescue” by removing H⁺ from the cell, providing recovery or partial recovery from cellular acidosis. These findings also help us understand the pathophysiology of renal tubular disorders.

Disclosure of conflict of interest

None.