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ORIGINAL ARTICLE
Year : 2020  |  Volume : 11  |  Issue : 1  |  Page : 30-34  

Effects of indoxyl sulfate on dopaminergic neurons and motor functions


1 BK21 PLUS Integrated Education and Research Center for Nature-Inspired Drug Development Targeting Healthy Aging, Kyung Hee University, Seoul, Republic of Korea
2 Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul, Republic of Korea
3 Department of Medical Science of Meridian, Graduate School, Kyung Hee University, Seoul, Republic of Korea
4 Department of Life and Nanopharmaceutical Sciences; Department of Oriental Pharmaceutical Science, College of Pharmacy and Kyung Hee East-West Pharmaceutical Research Institute, Kyung Hee University, Seoul, Republic of Korea

Date of Submission27-Nov-2019
Date of Decision29-Feb-2020
Date of Acceptance23-Jun-2020
Date of Web Publication20-Jul-2020

Correspondence Address:
Dr. Myung Sook Oh
Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University, 26, Kyungheedae-Ro, Dongdaemun-Gu, Seoul 02447; Department of Oriental Pharmaceutical Science and Kyung Hee East-West Pharmaceutical Research Institute, College of Pharmacy, Kyung Hee University, 26, Kyungheedae-Ro, Dongdaemun-Gu, Seoul 02447
Republic of Korea
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpnr.JPNR_23_19

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   Abstract 


Introduction: Increased levels of indoxyl sulfate (IS), a uremic toxin and a metabolite produced by gut bacteria, are reported in patients with Parkinson's disease (PD). However, the effects of IS on dopaminergic neurons and motor functions remain unexplored. In this study, we investigated whether IS treatment induces PD-related phenotypes such as cell death in dopaminergic neurons and motor function defects in vitro and in vivo. Materials and Methods: PC12 cells were treated with IS (100 μM) for 24 h, and tyrosine hydroxylase (TH) levels were measured. In vivo experiments in mice included: (1) short-term treatment with IS (200 mg/kg/d, i. p., or 400 mg/kg/d, p. o., once a day for 5 days) and (2) long-term treatment with IS (200 mg/kg/d, i. p., once a day for 28 days). Control mice received an equal volume of saline. Motor functions were evaluated using the open field test, pole test, and rotarod test. TH expression in the substantia nigra and striatum regions of the mouse brain was evaluated using immunohistochemistry and Western blotting. Results: No significant difference in the levels of TH was observed between the untreated and IS-treated PC12 cells. Further, in vivo studies in mice showed that both, acute and chronic IS treatment, failed to induce motor deficits and cell death in dopaminergic neurons. Conclusion: Taken together, our results suggest that IS has no effects on motor functions and dopaminergic neurons.

Keywords: Dopaminergic neuron, indoxyl sulfate, motor function, Parkinson's disease


How to cite this article:
Choi JG, Ju IG, Huh E, Noh D, Gu PS, Oh MS. Effects of indoxyl sulfate on dopaminergic neurons and motor functions. J Pharm Negative Results 2020;11:30-4

How to cite this URL:
Choi JG, Ju IG, Huh E, Noh D, Gu PS, Oh MS. Effects of indoxyl sulfate on dopaminergic neurons and motor functions. J Pharm Negative Results [serial online] 2020 [cited 2020 Aug 4];11:30-4. Available from: http://www.pnrjournal.com/text.asp?2020/11/1/30/290205




   Introduction Top


Parkinson's disease (PD) is the most common neurodegenerative disease clinically characterized by motor function defects and progressive loss of dopaminergic neurons in the brain.[1] Recent reports suggest dramatic changes in gut microbiota including gut bacterial overgrowth in PD patients.[2],[3],[4] These changes in the gut microbiota are closely associated with the release of metabolites produced by these microorganisms. Increasing evidence suggests alterations in the gut microbial metabolites in PD patients.[5] One of these metabolites, indoxyl sulfate (IS), a product of tryptophan metabolism, is a protein-bound uremic toxin that causes nephrotoxicity in kidney diseases.[6],[7]

It has been reported that IS, an inducer of oxidative stress, not only shows cardiovascular toxicity but also induces inflammatory reactions in the lipopolysaccharide-treated macrophages.[8],[9] Studies also suggest that it is a potential candidate that can trigger neurotoxicity in the brain.[10] IS also induces the production of reactive oxygen species in the glial cells and damages cortical and hippocampal neurons in a dose-dependent manner.[11] A clinical study showed that the levels of IS were significantly higher in the urine of PD patients compared to that of the healthy controls.[12] However, whether IS could induce PD-associated motor symptoms and dopaminergic neuronal loss in the brain remains unexplored.

This study aimed to investigate whether IS treatment affects motor functions and damages dopaminergic neurons. To address this, we first measured the levels of the enzyme tyrosine hydroxylase (TH) in the IS-treated PC12 cells. Next, we performed behavioral tests for motor functions and analyzed the levels of TH in the IS-treated mouse brain to address whether IS induces PD phenotypes such as motor function impairment and dopaminergic neuronal cell death.


   Materials and Methods Top


Reagents

Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), and penicillin–streptomycin (P/S) were purchased from Hyclone Laboratories Inc. Horse serum was purchased from Gibco Industries Inc. IS, paraformaldehyde (PFA), 3,3'-diaminobenzidine (DAB), sodium chloride, sucrose, DPX histomount medium, and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich. Tetramethylethylenediamine, protein assay reagent, Tween 20, ammonium persulfate, acrylamide, enzyme-linked chemiluminescence (ECL) reagent, and skimmed milk were purchased from Bio-Rad Laboratories. Rabbit anti-TH and immobilon-P transfer membranes were purchased from Merck Millipore. Horseradish peroxidase-conjugated (HRP) anti-rabbit secondary antibody was purchased from Enzo Life Sciences. Mouse anti-β-actin was purchased from Santa Cruz Biotechnology. Biotinylated goat anti-rabbit antibody, normal goat serum, and avidin–biotin complex (ABC) were purchased from Vector Labs.

Cell culture and treatment

PC12 cell line, a rat pheochromocytoma, was obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were maintained in RPMI 1640 medium supplemented with 5% heat-inactivated FBS, 10% heat-inactivated horse serum, and 1% P/S in a 5% CO2 incubation at 37°C. The culture medium was changed every 3 days, and cells were subcultured about twice a week. Cells were seeded at a density of 2 × 105 cells/mL on 96-well plates. After 24 h of incubation, cells were treated with IS (100 μM) for 24 h. Cell pellet was used for Western blotting.

Animals and treatment

Male ICR mice (8 weeks old) were purchased from Daehan Biolink (Eumseong, Korea). The animals were housed (n = 8 per cage) at an ambient temperature of 23°C ± 1°C and relative humidity 60% ± 10% under a 12-h light/dark cycle and were allowed free access to water and food. This study was carried out in accordance with the Principles of Laboratory Animal Care (NIH publication number 80–23, revised 1996). The protocol was approved by the Animal Care and Use Guidelines of Kyung Hee University, Seoul, Korea (Permit number: KHUASP (SE)-18-031). Mice were monitored for total schedule once daily. Mice were euthanized in case of 35% weight loss (humane endpoints) according to the approved protocol, but the euthanized mice were not detected in this study.

Two experiments were performed by the period of IS treatment. IS was dissolved in saline. (1) Short-term treatment with IS: intraperitoneal injection of IS (200 mg/kg/d, i. p.) or oral administration of IS (400 mg/kg/d, p. o.) was treated once a daily for 5 days. (2) Long-term treatment with IS: IS (200 mg/kg/d, i. p.) was treated once a daily for 28 days. Normal mice were treated with the equal volume of saline.

Open field test

The open field test was performed on the 16 days after last administration of IS (short-term treatment of IS set) or 1, 3, 7, 10, 14, 21, and 28 days after the treatment of IS (long-term treatment of IS set). This test was performed between 9 p. m. and 2 a. m. to avoid diurnal variation. The mice were placed in the testing chamber (40 cm × 25 cm × 18 cm) with white floors, followed by a 30-min recording period using a computerized automatic analysis system (Viewer; Biobserve, Bonn, Germany). The data collected by computer included the total distance traveled by tracking the center of the animal.

Rotarod test

The rotarod test was performed on the 16 days after last administration of IS (short-term treatment of IS set) or 1, 3, 7, 10, 14, 21, and 28 days after the treatment of IS (long-term treatment of IS set). The rotarod unit consists of a rotating spindle (7.3 cm diameter) and five individual compartments. After two or three times of training (8–10 rpm rotation speed), the rotation speed was increased to 16 rpm in a test session. The time each mouse remained on the rotating bar was recorded over three trials per mouse with a maximum length of 3 min per trial. Data are presented as the mean time on the rotating bar over the three test trials.

Pole test

We performed the pole test on the 16 days after last administration of IS. The mice were held on the top of the pole (diameter 8 mm, height 55 cm, with a rough surface). The time needed for the mice to climb down and place all four feet on the floor was recorded with a 30 s cutoff limit.

Brain tissue preparation

For immunohistochemical studies, at 24 h after behavioral tests, mice were perfused transcardially with 0.05 M PBS and then fixed with cold 4% PFA in a 0.1 M phosphate buffer. Brains were removed and postfixed in a 0.1 M phosphate buffer containing 4% PFA overnight at 4°C and then immersed in a solution containing 30% sucrose in 0.05 M PBS for cryoprotection. Serial 30-μm-thick coronal sections were cut on a freezing microtome (Leica, Germany) and stored in cryoprotectant (25% ethylene glycol, 25% glycerol, and 0.05 M phosphate buffer) at 4°C until use. For Western blot analysis, the mice were decapitated and the brain tissues were dissected with striatum (ST) and substantia nigra (SN), respectively. The tissues were stored at −80°C until use.

Immunohistochemistry

Brain sections were taken from the each brain region; between bregma-3.16 mm and bregma-3.64 mm for SN tissues, between bregma 0.98 mm and bregma 0.38 mm for ST tissues according to mouse brain atlas.[13] The brain sections were briefly rinsed in PBS and treated with 1% hydrogen peroxide for 15 min. The sections were incubated with a rabbit anti-TH antibody (1:1000) for SN and ST tissues overnight at 4°C in the presence of 0.3% Triton X-100. After rinsing in PBS, the sections were then incubated with biotinylated anti-rabbit IgG (1:200) for 1 h and with ABC mixture (1:100) for 1 h at room temperature. Peroxidase activity was visualized by incubating sections with DAB in 0.05 M Tris buffer. After several rinses with PBS, the sections were mounted on gelatin-coated slices, dehydrated, and cover-slipped using a DPX histomount medium. The images were acquired at ×200 or ×400 magnifications using an optical light microscope (BX51; Olympus, Japan) equipped with a ×20 objective lens. For measurement of the optical density of TH-positive area in the ST, the total region of interest was manually outlined and averaged optical densities were acquired in images with converted eight-bit indexed color. The number of TH-positive cells was quantified according to stereological counting.[14],[15] They were analyzed with Image J software (Bethesda, Maryland, USA).

Western blotting

The PC12 cell pellet or brain tissues were lysed with a triple-detergent lysis buffer. The lysates were separated by 10% SDS–polyacrylamide gel electrophoresis, and gels were transferred onto immobilon-P transfer membranes for 1 h 30 min. The membranes were blocked with 5% skim milk in Tris-buffered saline-0.01% Tween 20 (TBST) for 1 h and then were incubated overnight at 4°C with primary antibodies (TH 1:1500 and β-actin 1:3000). After that, the membranes were washed four times for 10 min with TBST, and the blots were incubated with respective HRP-conjugated secondary antibodies for 1 h at room temperature. Blots were detected using an ECL detection kit, and an LAS-4000 mini system (Fujifilm Corp., Japan) was used for visualization. The intensities of the bands were normalized to the β-actin band using Multi Gauge software (Fujifilm Corp., Japan).

Statistical analysis

All statistical parameters were calculated using GraphPad Prism 5.0 software (GraphPad Software Inc., La Jolla, CA, USA). Values were expressed as the mean ± standard error of the mean. The results were analyzed with the Student's t-test followed by an unpaired test. Differences with P < 0.05 were deemed to be statistically significant.


   Results Top


Indoxyl sulfate does not reduce the expression of tyrosine hydroxylase in PC12 cells

To investigate whether IS affects the expression of TH, a marker of dopaminergic neurons, we measured the levels of TH in PC12 cells after treatment with IS (100 μM). Prior to measuring the TH expression, we found the significant decrease of the cell viability at IS 100 μM in PC12 cells followed by the supplementary methods. Results showed no significant difference in the levels of TH between the untreated and IS-treated PC12 cells [Figure 1].
Figure 1: Effects of indoxyl sulfate on tyrosine hydroxylase expression in PC12 cells. Values are expressed as mean ± standard error of the mean (n = 5)

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Indoxyl sulfate does not induce motor deficits in mice

To evaluate the effects of IS on motor functions in mice, open field test, rotarod test, and pole test were performed. Short-term IS treatment (200 mg/kg/d, i. p., or 400 mg/kg/d, p. o. for 5 days) failed to induce motor impairments in mice [Figure 2]a-c]. Similar results were obtained in the long-term IS-treated mice (200 mg/kg [i. p.] for 28 days) [Figure 2]d and e].
Figure 2: Effects of indoxyl sulfate on motor functions in mice. We conducted (a) open field test, (b) rotarod test, and (c) pole test for the short-term indoxyl sulfate-treated mice. We also performed (d) open field test and (e) rotarod test for long-term indoxyl sulfate-treated mice on 1, 3, 7, 10, 14, 21, and 28 days of the administration. Values are expressed as mean ± standard error of the mean (n = 8)

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Indoxyl sulfate does not damage dopaminergic neurons in the mouse brain

Next, we explored whether IS induces cell death in the dopaminergic neurons in mice. Immunohistochemical analysis showed that short-term treatment with IS (200 mg/kg [i. p.] or 400 mg/kg [p. o.] for 5 days) did not damage the dopaminergic neurons, both in the ST and in the SN pars compacta (SNpc) regions of the mouse brain [Figure 3]a and [Figure 3]b. Moreover, no significant change in the levels of TH was observed in the ST and SNpc regions of the long-term IS-treated (200 mg/kg [i. p.] for 28 days) mouse brain compared to that of the control mice [Figure 3]c and [Figure 3]d.
Figure 3: Effects of indoxyl sulfate on tyrosine hydroxylase expression in mouse brains. We analyzed (a) the optical density of tyrosine hydroxylase in striatum and (b) number of tyrosine hydroxylase-positive cells in substantia nigra pars compacta of short-term indoxyl sulfate-treated mouse brains. We also measured the expression of tyrosine hydroxylase both in (c) striatum and in (d) substantia nigra of mouse brains with indoxyl sulfate treatment for 28 days. Scale bar is 100 μm. Values are expressed as mean ± standard error of the mean (n = 4)

Click here to view



   Discussion Top


In this study, we investigated whether IS could induce PD-associated pathological changes such as degeneration of dopaminergic neurons and movement impairments.

Several studies showed high levels of IS in patients with chronic kidney disease (CKD), which is also related to the severity of CKD.[16],[17] In CKD patients, the number of gut microbiota containing urease and indole-related enzymes is increased leading to an increase in the levels of intestinal bacteria-derived uremic toxins such as IS.[18] It has been reported that renal diseases such as CKD are significantly associated with the increased risk of PD.[19],[20] Moreover, oxidative stress involved in CKD conditions plays a key role in PD pathogenesis.[21],[22] Thus, we hypothesized that IS may be a potential metabolite that may cause PD-like pathology.

In the present study, we found no significant difference in TH expression between the IS-treated and untreated PC12 cells. Further, to investigate the effects of IS treatment on motor functions in mice, we performed motor behavior tests. Results of the three motor behavior tests (open field, rotarod, and pole tests) indicated that IS treatment could not induce PD-associated motor symptoms in mice regardless of the duration of treatment or route of administration. In particular, oral administration and intraperitoneal injection have different absorption and metabolic processes, so the final product of the administered substance may be different.[23] When administered orally, almost twice the intraperitoneal dose should be administered to have a similar level of bioavailability due to the first-pass metabolisms after the oral administration.[24] Therefore, we tried to explore the diversity of results through the difference in dosage according to the administration route of the IS, but there was no difference in the state change of dopaminergic neurons according to the two administration routes. Moreover, IS treatment did not induce any change in TH expression in the mouse brain.

Our results conflict with those of a previous study that showed that IS could trigger neuronal cell death in the hippocampus and cortex by inducing neuroinflammation and the release of reactive oxygen species.[11] The discrepancy between the two studies might be because a higher dose of IS (800 mg/kg, i. p.) was used in the previous study which might be responsible for neuronal cell death in the mouse brain. However, further studies regarding IS toxicity to dopaminergic neurons are required for more conclusive evidence.


   Conclusion Top


This study investigated the effects of IS, a representative uremic toxin, on dopaminergic neurons and motor functions. Our results indicate that IS treatment failed to damage dopaminergic neurons and could not induce motor deficits in mice.

Acknowledgments

We would like to thank Editage (www.editage.co.kr) for English language editing.

Financial support and sponsorship

This study was supported by Basic Science Research Program through the National Research Foundation funded by the Ministry of Education (NRF-2018R1D1A1B07048099).

Conflicts of interest

There are no conflicts of interest.



 
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