Methylguanidine cytotoxicity on HK-2 cells and protective effect of antioxidants against MG-induced apoptosis in renal proximal tubular cells in vitro

Abstract

Methylguanidine (MG), a small molecule among guanidine compounds, is a product of protein catabolism. The concentration of MG in the serum of uremic patients is nearly 80 times of that in the serum of normal people. The present study was designed to explore the toxic effect of MG on renal proximal tubular cells as well as the protective effect of antioxidants PGE1 and probucol against MG-induced apoptosis in renal proximal tubular cells. HK-2 cells were used as the subject. The cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. N-Acetyl-3-d-glucosaminidase (NAG) activity, malondialdehyde (MDA) content, and superoxide dismutase (SOD) activity were determined. Cell apoptosis was determined by flow cytometry (light scatter and propidium iodide/annexin V-FTC fluorescence) and by nuclear staining with Hoechst 33258. Cells were exposed to MG (0.25, 0.5, or 1 mmol/L), MG (0.5 mmol/L) + PGE1 (2 μg/L), and MG (0.5 mmol/L) + probucol (20 μmol/L) respectively for 24 h. MG induced a significant dose-dependent loss of cell viability. Both PGE1 and probucol improved the viability of MG-treated HK-2 cells. Cells showed apoptotic morphology (deepened stain, karyopyknosis, and apoptotic body) when exposed to 0.5 mmol/L MG for 24 h, and the apoptosis ratio was increased compared with the control. The presence of PGE1 or probucol significantly lowered the apoptotic ratio. Moreover, PGE1 or probucol notably decreased the MDA content and increased the SOD activity compared with when the cells were treated with MG only. The results of the present study clearly demonstrate that MG could promote apoptosis of renal proximal tubular cells in vitro. Both PGE1 and probucol could protect renal proximal tubular cells from MG-induced apoptosis.

Keywords:

• methylguanidine
• HK-2
• prostaglandin E1
• probucol
• apoptosis

INTRODUCTION

Methylguanidine (MG) is a metabolic product of creatinine (CTN) and some amino acid.^1–3 Studies revealed that the liver and the kidneys oxidize CTN into creatol, which is converted into MG.^4–7 Oxidation of CTN with active oxygen (e.g., hydrogen peroxide) was proven to generate MG, whereas scavengers of active oxygen (e.g., glutathione, sorbitol) considerably reduced MG synthesis.⁸ MG is expelled mainly from the kidney and is considered as not only a neurotoxin^9–11 but also a critical uremic toxin. Some experiments revealed that animals with high-level MG displayed a lot of functional and pathological characteristic features of uremia.^12,13 Another study showed significant reduction of the glomerular filtration rate, renal plasma flow, and renal blood flow after administration of MG to normal rats¹⁴ and rats with adenine-induced chronic renal failure.¹⁵ Thus, MG has apparent toxic effects on kidney and causes deterioration of renal function, but the pathogenesis is still not very well understood. Considering that apoptosis is an important mechanism that contributes to the progressive loss of glomerular and tubular cells during the course of experimental chronic renal failure (CRF),¹⁶ and our previous study showing that 1-methylhydantoin (another metabolic by-product of creatinine) has the ability to induce apoptosis in renal proximal tubular cells in vitro,¹⁷ we hypothesized that MG impaired the renal function partly due to the induction of apoptosis in renal tubular epithelial cells.

Prostaglandin E1 (PGE1), known as an antioxidant, has wide-ranging cytoprotective effects for gastrointestinal mucosa,¹⁸ liver,¹⁹ and kidneys.²⁰ Many animal experiments demonstrated that PGE1 has antiapoptotic effect on various cells, such as neuronal apoptosis in spinal cord induced by sciatic constriction injury,²¹ hepatocyte apoptosis in excessive hepatectomy,²² endothelial progenitor cell (EPC) apoptosis,²³ and renal cell apoptosis induced by cisplatin.²⁴ Probucol, a lipid-lowering drug, is a synthetic antioxidant²⁵ that has been shown to inhibit oxidative stress-induced apoptosis of endothelial cells.²⁶ It has also been reported that pretreatment with probucol attenuates cardiomyocyte apoptosis induced by adriamycin²⁷ and in heart failure rat model²⁸ and atrial apoptosis in prolonged pacing-induced atrial fibrillation dogs.²⁹ Research by Wang et al.³⁰ discovered that probucol protects the renal microvascular endothelial cells from advanced glycation end product-induced damage, but its protective effect against apoptosis in renal tubular epithelial cells has not been shown so far in any published document. On the basis of the aforementioned similarities, we hypothesized that PGE1 and probucol might have protective effects on MG-induced apoptosis in renal proximal tubular cells. Our in vitro model allowed us to assess the toxicity of MG to HK-2 cells and antiapoptotic effects of PGE1 and probucol on MG-induced HK-2 cell apoptosis at the cellular level.

MATERIALS AND METHODS

Cell culture and preparation

All reagents were obtained from Sigma-Aldrich Co. unless otherwise stated. HK-2 cells (American Type Culture Collection, Manassas, Virginia, USA), an immortalized human proximal tubule epithelial cell line, were grown and passaged in 75 cm² cell culture flasks that contained 1640 medium supplemented with 5% fetal calf serum (FCS) and antibiotics (100 U/mL penicillin G, 100 mg/mL streptomycin). Cells were maintained at 37°C in an atmosphere of 95% air and 5% CO₂. Near-confluent (80%) HK-2 cells were incubated with serum-free media for 24 h to arrest and synchronize cell growth. Then the media were exchanged for fresh serum-free media, treated with culture medium containing various concentrations of MG (0.25, 0.5, or 1 mmol/L), MG (0.5 mmol/L) + PGE1 (2 μg/L), and MG (0.5 mmol/L) + probucol (20 μmol/L). Cells incubated with culture medium without any drugs were set up as control group. After treatment for 24 h, the cells were harvested for detection.

Measurement of cell viability by MTT assay

The HK-2 cell was dispensed onto 96-well microplate at a concentration of 1 × 10⁴ cell well⁻¹. The multi-well plates were incubated at 37°C, 5% CO₂ in air for 24 h with MG (0.25, 0.5, or 1 mmol/L), MG (0.5 mmol/L) + PGE1 (2 μg/L), and MG (0.5 mmol/L) + probucol (20 μmol/L), in 5% bovine serum-supplemented 1640 medium. All experiments were repeated at least twice, and each experimental condition was repeated at least in quadruplicate wells in each experiment. At the end of each treatment, cells were incubated with 1 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in fresh media and kept in a dark environment for 4 h at 37°C. Dimethyl sulfoxide (DMSO), 150 μL per well, was added under vigorous pipetting to dissolve the formazan crystals, as described previously.³¹ Dye absorbance (A) in viable cells was measured at 570 nm, with 630 nm as a reference wavelength by the method of Sheerin et al.³²

NAG enzyme assay

N-Acetyl-3-d-glucosaminidase (NAG), a lysosomal enzyme that is a good marker of proximal tubular cell integrity, was evaluated. Cells were plated in 24-well plates to 70–80% confluence and then incubated for 24 h at 37°C. NAG was leaked into the supernatant and measured by Hitachi 707, as seen in the methods of Pugh et al.³³

Morphological observation of apoptotic cells

HK-2 cells were exposed for 24 h to MG (0.5 mmol/L) or media. To determine the cell morphologic changes, Hoechst staining was carried out according to the manufacturer's instructions (Beyotime, China). Briefly, cells were washed with phosphate-buffered saline (PBS), fixed with methanol/acetic acid (3:1) for 10 min, and then incubated with 0.5 mL Hoechst 33258 for 5 min. Observation was performed under a fluorescent microscope.

Apoptosis measurement by annexin V/PI staining

The HK-2 cell was dispensed onto a 6-well microplate at a concentration of 5 × 10⁴ cell well⁻¹. Eighty percent confluent HK-2 cells were incubated with serum-free media for 12 h to arrest and synchronize cell growth, supernatant was discarded, and annexin V/PI (propidium iodide, PI) staining (an apoptosis detection kit; Beijing Biosea Biotechnology Co., Ltd, China) was performed according to manufacturer's instructions, and then cells were analyzed using a flow cytometer (Becton Dickinson FACSAria, USA). Cells that showed annexin V-positive (with translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane) and PI-negative staining (with intact cellular membrane) were considered as early apoptotic cells.^34–36

Measurement of MDA content and SOD activity

Malondialdehyde (MDA) in HK-2 cell lysates was determined by the thiobarbituric acid (TBA) method and the activity of superoxide dismutase (SOD) was measured by the method of xanthine oxidase according to manufacturer's instructions. One SOD unit was defined as the amount of enzyme that inhibited formazan production by 50% at 37°C.

Statistical analysis

Data are presented as the mean ± standard error of the mean (SEM). Comparisons were made using a one-way analysis of variance (ANOVA) followed by a multiple comparison procedure (Fisher least significant difference test), which was provided by SPSS 13.0 statistical software (SPSS, Chicago, Illinois, USA). A value of p < 0.05 was considered statistically significant.

RESULTS

Measurement of cell viability by MTT assay

Evaluation of cell viability by the MTT assay demonstrated a significant (p < 0.01) dose-dependent reduction in MTT absorbance in HK-2 cells treated with MG for 24 h. Dye absorbance (A) of each concentration of MG was significantly (p < 0.01) lower than control. When PGE1 or probucol was added in MG group, the dye absorbance (A) increased significantly (p < 0.01), and the change level of PGE1 group was more obvious than that of the probucol group (p < 0.05). On the basis of the ability of viable cells to reduce MTT and the direct relationship between HK-2 cell number and MTT absorbance, MG induced a significant loss of cell viability, and the presence of PGE1 or probucol significantly lowered the MG-induced loss of cell viability (see Figure 1).

FIGURE 1. MG cytotoxicity and protective effects of antioxidants from cytotoxicity induced by MG in HK-2 cells. Six groups of HK-2 cells were treated with medium only, MG (0.25, 0.5, or 1 mmol/L), MG (0.5 mmol/L) + PGE1 (2 μg/L), and MG (0.5 mmol/L) + probucol (20 μmol/L), for 24 h. Cell viability was assayed by the MTT assay. Values are presented as mean ± SEM (n = 8). *p < 0.01 versus control, ^#p < 0.01 versus MG 0.5 mmol/L group, ^Δp < 0.05 versus MG (0.5 mMol/L) + PGE1 (2 μg/L) group.

NAG enzyme assay

The exponentially growing HK-2 cell line was treated with MG at different concentrations of 0.25, 0.5, or 1 mmol/L, MG (0.5 mmol/L) + PGE1 (2 μg/L), and MG (0.5 mmol/L) + probucol (20 μmol/L). HK-2 cells treated with MG for 24 h displayed a significant (p < 0.01) dose-dependent increase in the supernatant concentration of NAG. Each concentration of MG was significantly (p < 0.01) greater than control. The presence of PGE1 or probucol significantly lowered the concentration of NAG (see Figure 2).

FIGURE 2. MG cytotoxicity and protective effects of antioxidants from cytotoxicity induced by MG in HK-2 cells. Six groups of HK-2 cells were treated with medium only, MG (0.25, 0.5, or 1 mmol/L), MG (0.5 mmol/L) + PGE1 (2 μg/L), and MG (0.5 mmol/L) + probucol (20 μmol/L), for 24 h. NAG release in the supernatant was determined. Values are presented as mean ± SEM (n = 8). *p < 0.01 versus control, ^#p < 0.01 versus MG 0.5 mmol/L group, ^Δp < 0.05 versus MG (0.5 mmol/L) + PGE1 (2 μg/L) group.

Morphological observation of apoptotic cells

Hoechst staining showed that typical morphologic features of apoptosis, such as deepened stain, karyopyknosis, karyorrhexis, and apoptotic body (shown by the arrows in Figure 3), were observed after 24 h exposure to 0.5 mmol/L MG. No apoptotic morphology was observed in the control group (see Figure 3).

FIGURE 3. Epifluorescent micrographs showing Hoechst staining of DNA in HK-2 cells.

Note: Figure A stands for the HK-2 cells of normal control group; Figure B stands for the HK-2 cell of MG group, which were treated with 0.5mMol/L MG for 24 h. In figure A, shows normal nucleolus. Exposure to MG resulting in karyopyknosis and deepened stain () and the appearance of apoptotic bodies ( in figure B). Photographs taken at a magnification of × 400 (n = 4).

Effect of PGE1 and probucol on MG-induced HK-2 cell apoptosis by annexin V/PI staining

HK-2 cell apoptosis was assessed by annexin V/PI staining. As shown in Figure 4, there was a low level of apoptotic cells in the control condition (2.473 ± 0.321)%. Exposure to MG for 8 h induced a significant increase in HK-2 cell apoptosis to (18.590 ± 1.413)% from (2.473 ± 0.321)% (p < 0.01). PGE1 or probucol pretreatment exerted a significant antiapoptotic effect: the presence of PGE1 or probucol significantly lowered the apoptotic ratio (4.183 ± 0.403)% or (5.030% ± 0.372)% versus (18.590 ± 1.413)% (p < 0.01), respectively, and the change level of PGE1 group was more obvious than that of the probucol group (p < 0.05).

FIGURE 4. Effect of PGE1 and probucol on MG-induced HK-2 cells apoptosis using flow cytometric analysis. Apoptosis ratio (%): (apoptosis cells/total cells) × 100%. (I) Flow cytometric analysis. (II) Densitometric analysis. A: control; B: 0.5 mmol/L MG; C: 0.5 mmol/L MG + 2μg/L PGE1; D: 0.5 mmol/L MG + 20 μmol/L probucol. Population a (annexin V⁻/PI⁺) is considered necrotic cells; population b (annexin V⁺/PI⁺) is considered late stage apoptotic cells; population c (annexin V⁺/PI⁻) is considered early apoptotic cells; population d (annexin V⁻/PI⁻) is considered normal HK-2 cells. Apoptotic ratio: *p < 0.01 versus control; ^#p < 0.01 versus 0.5 mmol/L MG group (n = 4).

Effects of PGE1 and probucol on oxidation in HK-2 cells

MDA levels were evaluated to estimate the degree of lipid peroxidation on cell culture. As shown in Figure 5, MG caused significant increases in MDA levels. Meanwhile, PGE1 and probucol notably inhibited the increase in MDA content. SOD activity was used to detect the scavenging superoxide anion ability of cells. Total SOD activity diminished in the MG group compared to the control group at 24 h. PGE1 and probucol partially prevented the decrease in total SOD activity (see Figure 6).

FIGURE 5. Effects of PGE1 and probucol on MG-induced oxidation changes in HK-2 cells. Four groups of HK-2 cells were treated with medium only, MG 0.5 mmol/L, MG (0.5 mmol/L) + PGE1 (2 μg/L), and MG (0.5 mmol/L) + probucol (20 μmol/L), respectively, for 24 h. MDA content was determined. Values are presented as mean ± SEM (n = 4). *p < 0.01 versus control, ^#p < 0.01 versus MG 0.5 mMol/L group, ^Δp < 0.05 versus MG (0.5 mmol/L) + PGE1 (2 μg/L) group.

FIGURE 6. Effects of PGE1 and probucol on MG-induced oxidation changes in HK-2 cells. Four groups of HK-2 cells were treated with medium only, MG 0.5 mmol/L, MG (0.5 mmol/L) + PGE1 (2 μg/L), and MG (0.5 mmol/L) + probucol (20 μmol/L), for 24 h. SOD activity was determined. Values are presented as mean ± SEM (n = 4). *p < 0.01 versus control, ^#p < 0.01 versus MG 0.5 mmol/L group.

DISCUSSION

Animal experiments³⁷ demonstrated that renal cell apoptosis takes part in the progress of glomerulosclerosis, glomerular atrophy, and renal interstitial fibrosis. Renal intrinsic apoptosis is an important pathological characteristic of chronic renal progressivity disease, which is one of the important factors associated with the progressive deterioration of renal function. Apoptosis of renal cells induced by some substances such as radiocontrast agents, hypoxia, albumin, aristolochic acid, adriamycin, and gentamicin has been widely reported.^38–43 However, the role of uremic toxins in inducing renal intrinsic apoptosis during progressive nephron deprivation in chronic renal insufficiency has been reported less frequently. Our present study was to examine whether the increased level of MG in the plasma of CRF patients induces apoptosis in renal proximal tubular cells and investigate some pharmacological interventions of this cell apoptosis in vitro.

In our experiment, when MG at different concentrations was added in media, the dye absorbance (A) decreased significantly and concentration of NAG in the supernatant increased significantly, which demonstrates that MG is toxic to proximal tubular cells. NAG is a sort of intracellular lysosomal enzyme EC3.2.1.30, which mainly exists in the renal proximal tubule and is one of the most sensitive marker enzymes of tubular epithelia injury.^44,45 It is also discovered that after the MG effects on HK-2 cells, typical morphologic features of apoptosis, such as deepened stain, karyopyknosis, karyorrhexis, and apoptotic body, were observed by using Hoechst staining assay, which revealed that MG can promote HK-2 cells apoptosis in vitro. In addition, the annexin V/PI double staining method was employed to observe early apoptosis by flow cytometry⁴⁶ and revealed that cell apoptosis ratio in the MG group is significantly higher than in the normal control. This fact also confirms that MG has apoptotic effect on HK-2 cells. In fact, the increase in NAG in the supernatant is more compatible with the occurrence of tubular necrosis instead of apoptosis; thus, it suggests that both injuries could coexist in this model. Mori et al.^47,48 demonstrated that extremely high level of guanidino compounds, including MG, generated reactive oxygen species (ROS) using an electron spin resonance (ESR) spectrometry with spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). We also determined MDA content and SOD activity of HK-2 cells. MG increased MDA content and decreased SOD activity in vitro, which indicated that MG was effective in inducing oxidative process and inhibiting scavenging O^2–. MDA is an important end product of lipid peroxidation and a widely used indicator of ROS production. The increase of MDA activity implied that the mechanism of MG-induced apoptosis may be somewhat related to its ability to produce ROS. However, the exact mechanism by which MG induces apoptosis remains unclear.

This experiment showed a protective role of PGE1 and probucol on MG-induced apoptosis in HK-2 cells. Experiment by Hsu et al.⁴³ proves that PGE1 can protect renal tubular cells from gentamicin-induced apoptosis, and also shows that PGE1 can block the activity of caspase-3 and decrease the generation of ROS. Another report⁴⁹ shows that PGE1 can enhance the activity of SOD and stimulate adenylate cyclase (AC) system, thus triggering the activity of some key enzymes like Na, K, ATPase and blocking the activity of phosphodiesterase (PDE), which causes the opening of K⁺ channels in cell membranes, the increasing outflow of K⁺, and the decrease of intracellular Ca⁺ level, thus alleviating the overload of intracellular Ca⁺. In the meantime, PGE1 can protect the stability of cell membrane and cell organelle as well. In our study, when PGE1 was added in the media, the dye absorbance (A) increased significantly while the concentration of NAG significantly decreased in the supernatant, and annexin/PI assay also displayed a significant decrease of apoptosis ratio versus MG group, which reveals that PGE1 has a quite good antagonism effect on the apoptosis of renal proximal tubular cells induced by MG. What is more, after HK-2 cells were pretreated with PGE1, SOD activity increased and MDA content decreased compared with that of cells treated with MG only. SOD, as an important free oxygen radical scavenger in our body, eliminates superoxide anion and blocks lipid peroxidation chain reaction through disproportionating reaction. The finding suggested that the possible mechanism of the protective effect of PGE1 against MG-induced apoptosis in renal proximal tubular cells is related to its ability to elevate the oxidant scavenging ability and reduce the production of ROS.

Probucol is a hypolipidemic agent and has antioxidation effects. It has the ability of enhancing the activity of glutathione peroxidase (GSH-Px) and hydrogen peroxidase, inhibiting the generation of intracellular active oxygen and increasing its clearance.^50,51 As a matter of fact, huge imbalance in the redox status that enhances the concentration of intracellular active oxygen is exactly the major mechanism of inducing cell apoptosis. GSH, as the most important intracellular thiol antioxidant, can directly eliminate hydroxyl radical (–OH) and superoxide anion (oxygen-derived free radicals) and eliminate H₂O₂ as a cofactor of GSH-Px.^52,53 Our experiment reveals that after probucol co-treatment with MG on HK-2 cells, the dye absorbance (A) detected by MTT assay increased significantly while the concentration of NAG in the supernatant decreased significantly, Annexin/PI assay also displays a significant decrease of apoptosis ratio versus MG group; SOD activity increased and MDA content decreased compared with that of cells treated with MG only, which revealed that probucol can significantly attenuate the renal proximal tubular cells apoptosis induced by MG. The possible mechanism is related to its ability of direct clearance of free radical generated by MG.

In conclusion, cultured proximal tubular cells allowed us to observe and partly explain the toxic effect of MG on the kidney. MG-induced apoptosis in renal tubular epithelial cells is probably one of the important factors associated with the progressive deterioration of renal function. Antioxidants PGE1 and probucol can protect renal proximal tubular cells from MG-induced apoptosis in vitro partially because of their ability to attenuate the oxidative stress induced by MG, which lays the foundation for the further study of its precise mechanism and experiments in vivo. Our data suggested that PGE1 and probucol could be a potential therapeutic treatment for chronic progressive renal disease as there is still lack of an effective treatment today.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.