Indoxyl sulfate potassium salt (IS), RIPA buffer, secondary antibodies conjugated with alkaline phosphatase, 5,5′,6,6′-Tetrachloro-1,1′,3,3′tetraethylbenzimidazolocarbocyanine iodide (JC-1), 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI), 3-Amino-7-dimethylamino-2-methylphenazine hydrochloride (Neutral red), LMP agarose type XI, NMP agarose type I were purchased from Sigma-Aldrich (Sigma-Aldrich Sigma, St. Louis, MO, USA). Mouse polyclonal antibodies for Bax, Bcl-2, caspase-3, caspase-9 and β-actin were obtained from Santa Cruz Biotechnology (USA). PARP Degradation ELISA—PARP (Cleaved) [214/215] Human ELISA Kit was purchased from Thermo Fisher Scientific (MA USA). Unless otherwise indicated, all other chemicals were purchased from POCH S.A. (Gliwice, Poland). All dishes necessary for cell culture were obtained from NUNC.
Isolation of mononuclear blood cells (MNCs)
The research was conducted on mononuclear blood cells (MNCs) isolated from the human blood buffy coat obtained from the Regional Center for Blood Donation and Haemotherapy (RCKiK) in Lodz, Poland. Every single experiment was performed at least in duplicate (3–4 times) on cells from one donor, and n-numbers of presented results represent cells from different individuals.
Cell isolation was performed by density gradient centrifugation of blood buffy coat with Histopaque®-1077 (Sigma-Aldrich Sigma, St. Louis, MO, USA). The isolated MNCs were washed two times with phosphate-buffered saline pH 7.4 (PBS).
Cells treatment conditions
Freshly isolated mononuclear blood cells (MNCs) were placed in RPMI medium (Corning, Mediatech, Inc, Manassas, VA, USA) supplemented with 10% heat-inactivated foetal bovine serum (Capricorn Scientific, GmbH) and antibiotics 10 U/mL penicillin and 50 μg/mL streptomycin (Corning, Mediatech, Inc, Manassas, VA, USA).
Cells were seeded at suitable density for the performed assay into 96-well plates (5 × 104) or Petri dishes (4 × 106) and incubated for 24 h with indoxyl sulfate (IS) at a final concentration of 0.2 mM, 1 mM 2 mM in standard culture conditions (37 °C, 100% relative humidity, 5% CO2 incubator). Indoxyl sulfate was dissolved in PBS. Control cells were treated with a corresponding volume of PBS. After incubation, cells were washed with PBS and used for the following measurements.
The cytotoxicity test
The Neutral Red Uptake (NRU) method is a cytotoxicity test based on the uptake and accumulation of neutral red inside organelles with an acidic pH. The transport of the dye takes place in a passive way. Neutral red penetrates a living cell with an intact membrane cell and accumulates inside lysosomes and endosomes. The amount of neutral red accumulated in the organelles is proportional to the number of living cells41.
After incubation, the cells were placed for 2 h in a medium containing neutral red. Subsequently, the cells were washed, the dye was extracted in each well, and the absorbance was measured at a wavelength of λ = 540 nm on a microplate reader (BioTek). The obtained absorbance values of the reaction product were presented as a percentage, taking the absorbance value of the control as 100%.
Caspase activity test
The activities of caspases were determined using Life Technologies™ ApoTarge™ caspase colourimetric protease assay sampler kit (caspases-2, -3, -6, -8, and -9). According to the information provided by the manufacturer, the assay contains the substrates VDVAD (for caspase-2), DEVD (for caspase-3), VEID (for caspase-6), IETD (for caspase-8) and LEHD (for caspase-9. The substrates for caspase activity measurement are labelled at their C-termini with para-nitroaniline (pNA). The light absorption by free pNA can be measured at 400 or 405 nm. Comparing the absorbance of pNA from samples allows for determining changes in caspase activity. The experiment was performed according to the manufacturer’s protocol and as described before42. The results are presented as a percentage of change in absorbance at 405 nm relative to control calculated as 100%.
Measurement of mitochondrial membrane potential
The mitochondrial potential in MNCs was measured using a JC-1 fluorescent probe43. This fluorescent carbocyanine dye accumulates in the mitochondrial membrane in two forms (monomers and dimers). The negative potential of the inner mitochondrial membrane facilitates the formation of dye aggregates. Measuring the ratio of JC-1 dimer to monomer fluorescence is a suitable method for estimating changes in mitochondrial membrane potential.
After the treatment, the cells were placed in Hank’s buffered salt solution (HBSS) containing 5 µmol/L JC-1 fluorescent probe and incubated in total darkness for 30 min at 37 °C. Before fluorescence measurements, cells were washed to remove the dye and suspended in fresh HBSS solution. The fluorescence intensity of the monomer and dimer was measured on a spectrofluorometer Cary Eclipse (Varian, Inc.) using filter pairs of 535 nm/590 nm (dimers) and 475 nm/530 nm (monomers). Subsequently, the ratio of the fluorescence intensities at 530 nm/590 nm was calculated42. The mitochondrial potential in MNCs was measured immediately after incubation with indoxyl sulfate. Results are expressed as a percentage of the control, which was taken as 100%.
Measurement of DNA fragmentation
DNA damage was determined by comet assay (single-cell electrophoresis), a rapid and sensitive method for detecting single-stranded and double-stranded DNA breaks.
After 24 h of treatment with indoxyl sulfate, the cells were suspended in PBS 2.5 × 105 cells/ml. Low melting point agarose (LMP type XI) was added to the cell suspension, and the mixture was applied to a glass slide previously coated with standard melting point agarose (NMP type I). After solidification of the agarose, glass slides were incubated in lysis buffer containing (2,5 mol/L NaCl, 100 mmol/L Na2-EDTA, 10 mmol/L TRIS, 1% Triton X-100) for 2 h. At the end of lysis, the electrophoresis was conducted in a buffer containing (1 mmol/L Na2-EDTA, 300 mmol/L NaOH) and (29 V, 30 mA). Before analysing, the slides were stained with DAPI solution (2 ng/ml)42. The analysis was performed under alkaline conditions according to the procedure of Singh et al.44 with slight modifications. The slides were analysed using a fluorescence microscope Zeiss Axio Scope A1 (Carl Zeiss, Germany) with a video camera connected to the image-analysis system Lucia-Comet v. 7.60 (Laboratory Imaging, Praha, Czech Republic). The values are the mean ± SD of 50 cells from glass slide in 6 independent experiments.
Cell lysate and immunoblotting
In order to assess the expression of proteins (caspase-3, caspase-9, Bax, Bcl-2 and β-actin), MNCs after incubation cells were lysed (4 °C, 20 min) in a RIPA buffer containing 150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0 with protease inhibitors/EDTA (final concentration 10 µM). After centrifugation, the supernatants were collected11. About 30 µg of proteins were loaded into each lane. The probes were electrophoretically separated by 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon P as described by Towbin et al.45. Protein concentration in MNCs lysates was determined using the method of Lowry et al.46. Subsequently, the membranes were blocked in 5% nonfat dry milk in TBST buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20) for 1 h at room temperature. After blocking, the membranes were incubated overnight with antibodies specific to Bax, Bcl-2, caspase-3, caspase-9 and β-actin (1:500 dilution) in a TBST buffer in a cold room. After 24 h incubation with the primary antibody, the membranes were washed thrice with TBST and incubated with appropriate secondary anti-mouse antibodies conjugated with alkaline phosphatase in TBST for 2 h at room temperature. The membranes were then washed several times with TBST. The proteins were visualized by incubation with the substrate solution (0.33 mg/mL of nitro blue tetrazolium, 0.17 mg/mL of 5-bromo-4-chloro-3-indolyl phosphate in 100 mM Tris–HCl, pH 9.5, 100 mM NaCl and 5 mM MgCl2), prepared according to Leary et al.11,47. Analysis of the relative change in the expression of the tested proteins was performed on the basis of signal intensity and densitometric analysis in ImageJ 1.48v software (National Institutes of Health, Bethesda, MD). The results are presented as the ratio of the densitometric values of the band intensity measurements for an investigated protein to the actin reference protein for each well.
Measurement of cleaved PARP levels
After incubation, the cells were centrifuged (1500 rpm; 3 min; 4 °C) and the supernatant was removed. The collected cell pellet was lysed in RIPA buffer (50 mM Tris–HCL, pH 8.0, with 150 mM sodium chloride, 1.0% IGEPAL® CA-630 (NP-40), 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate) with protease inhibitor (phenylmethylsulfonyl fluoride) (Sigma-Aldrich Sigma, St. Louis, MO, USA). Cleaved Poly (ADP-ribose) polymerase (PARP) levels were assessed using the PARP Cleaved [214/215] ELISA kit (Thermo Fisher Scientific, MA USA) according to the protocol described in the manufacturer’s instructions and, as described before48.
The normality of data was tested using the Shapiro–Wilk test, and variance homogeneity was verified with Levene’s test. The significance of the differences between the groups was estimated by a one-way ANOVA using Tukey’s post hoc multiple comparisons test for the data with normal distribution. For the data with a departure from normality, the non-parametric Kruskal–Wallis test was used. Statistical significance was accepted at p < 0.05. The sample size was estimated for type I and type II statistical errors of 0.05 and 0.8, respectively. Accurate conclusions were not formulated for data with statistical power below 80%. The statistical analysis was performed using Statistica v. 13.3 (StatSoft Polska, Krakow, Poland).