An experimental in vitro study was carried out on a culture of bovine renal epithelial cells (ATCC bovine kidney) after more than 8 days of growth. This investigation was based on cytotoxicity analysis on the recombinant protein ApoL1, in bovine kidney cells, to evaluate an adverse renal effect that had previously been described by some authors (O’Toole et al. 2018). For this, an experiment was designed by means of the Minitab® software, version 21 (“Arend, Dominic N. (1993). Choices (Version 4.0) [Computer software]. Champaign, IL: U.S. Army Corps of Engineers Research Laboratory. (CERL Report No.CH7-22510)”, s/f). In this, the final response variable was the percentage viability, which was expected to be higher than 90% and was derived from analysis on absorbance, using spectrophotometry. This was done in order to determine whether the ApoL1 protein would cause damage to treated cells. The running time used for the assay was 8 days, not counting the incubation time of the cells. Four factors were considered: ApoL1 protein concentration (levels of 0.1, 1 and 10 ug/ml), positive control for cell death (levels of 200 and 250 ng/ml), negative control (with and without PBS) and negative cell control (with and without cells). The trial summary is described below:
Factors: 4 Replications: 3
Running base: 48 Total runs: 144
Obtaining the recombinant ApoL1-like protein
The protein came in a presentation of 100 µg from the company ABCAM, Cambrige, UK. It is a 52 kDa protein, with a total of 238 amino acids, which is produced in wheat germ. Its sequence is: (MEGAALLRVSVLCIWMSALFLGVGVRAEEAGARVQQNVPSGTDTGDPQSKPL
YGKKWWTQA) and its access number in the Uniprot database is O14791.
Kidney cells were obtained from the company ATCC®, Virginia, USA. with the reference MDBK (NBL-1), CCL-22. These cells originated from the kidneys of an adult male Bos Taurus animal and had been characterized as epithelial cells. For culturing, the cells were removed from the liquid nitrogen tank where they had been kept at -130 °C since purchase. Eagle's essential medium (catalog no. 30-2003) and fetal equine serum (Sigma-Aldrich) were added at a final concentration of 10%.
Once the vial of cells had been removed, it was continually shaken at 37 °C in a water bath for 2 hours. The contents of the vial were then resuspended in a 25 cm2 growth vessel. This was placed in an incubator at 37 °C for 24 hours with 5% CO2. The medium was exchanged after 8 days of growth, in order to make the evaluation.
Cell subculture: 0.25% trypsin and 0.03% EDTA were added and then the solution was stirred. It was left at room temperature for 10 minutes, dry and added to a new medium. Subculturing was performed at a concentration of approximately 10e+5 cells per ml.
Preparation of MTT solution: A stock solution of 12 mM was prepared by adding 1 ml of PBS to a vial of 5 mg of MTT. This was then vortexed for 30 seconds. If did not was filtered until could be dissolved. The solution was immediately covered to avoid the effect of light.
Preparation of HCL-SDS: 10 ml of Hydrochloric acid solution (HCL) was added to with 1 g of SDS in a tube and inverted several times.
Addition of treatments: Serial dilutions of the protein were made, with the original concentration of 100 ug/ml. The cells were then added at a concentration of 10e+5 cells per ml, in a 96-well plate. Lane 1 consisted of distilled water; Lane 2, cells at a concentration of 10e+5, with 10 ug/ml of the protein; Lane 3, cells at a concentration of 10e+5, with 1 ug/ml of protein; Lane 4, cells at a concentration of 10e+5, with 0.1 ug/ml protein; Lane 5, blank with cells; Lane 6, MM8 (methyl methane sulfonate) at 200 ug/ml; Lane 7, MM8 at 250 ug/ml; Lane 8, blank with cells; Lane 9, blank without cells; Lane 10, culture medium; and Lane 11, PBS.
MTT analysis: The medium was exchanged again as mentioned above, 100 ul of medium, and 10 ul of the MTT stock solution was added. This mixture was incubated for 4 hours and the assay was viewed using a Multiskan Sky ELISA reader (Thermo fisher Scientific®), and the readings were stored until analysis. Three replicates were made on each plate and the assay was done three times.
The cells were cultured in accordance with the manufacturer's recommendations and were kept at a standard concentration (1-2 x 10e+5). These cells were then seeded in a 96-well plate with a final volume of 10 µl, with concentrations of 0.1, 1 and 10 mg/ml of ApoL1r. There was also one xxxxx lane without protein administration, as a negative control, but under the same conditions. After 12 hours of growth, the cells were treated with 50 ng/ml of tetracycline in complete Dulbecco's modified Eagle’s medium (DMEM). Cytotoxicity and viability were measured at 24 hours, using a cell proliferation kit (I-MTT; Roche®).
The technique MTT is a colorimetric assay for non-radioactive quantification of cell proliferation, viability and cytotoxicity. The sample material was adherent cells in 96-well microplates. Through the colorimetric assay, the number of viable cells was analyzed according to the cleavage of tetrazolium salts that were added to the culture medium. This technique requires neither washing nor cell harvesting, and the entire assay, from microculture to data analysis using an ELISA reader, is performed on the same microplate (van Meerloo, Kaspers, y Cloos 2011).
Each treatment was run in three replications and three repetitions of this, and the data obtained were averaged to establish the point values.