EVALUATION OF RENAL EPITHELIAL CELL PROTEIN UNDER STRESS CONDITION

Abstract

Objective: Proteins are an important component of cells which are involved in various cellular functions. Different kind of stressing conditions has different responses in the components of the protein synthesis system. Super saturation condition in kidney environment leads to crystallization process. Crystals thus formed injure the surrounding cells and result in reactive oxygen species (ROS) formation. There might be some changes in the protein synthesis when the kidney cells enter in oxidative stress. In the present study, kidney cell lines were exposed to oxidative stress and their proteins were analyzed using Bradford analysis and SDS-PAGE.Methods: Vero cells were obtained from NCCS Pune and cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) and maintained in a humidified incubator at 37 °C with 5% CO2. Calcium phosphate (CaP) crystals were prepared by the homogeneous system. After FTIR analysis crystals were used to injure Vero cell line. H2O2 was also used to injure the Vero cells. Intracellular protein was extracted from healthy cells and injured cells (with CaP crystals and H2O2). Ammonium sulfate precipitation method was used for the isolation of extracellular protein from the media of healthy and injured cells. Bradford method was used for the quantitative estimation of protein. Extracted proteins were analyzed by SDS-PAGE.Results: Amount of intracellular and extracellular protein of normal cells was 4.84±0.004µg/ml. Intracellular protein of CaP injured and H2O2 injured cells were 10.59±0.003 µg/ml and 10.78±0.011µg/ml respectively. While extracellular protein of injured cells was nearly 4 µg/ml. Intracellular protein bands ranging from 14.3 to 97.4 kDa was observed in healthy cells. Protein bands of ~40kDa and ~20kDa was absent in H2O2 and CaP injured intracellular protein extract. Two extracellular protein bands of 66kDa and ~60kDa were present in injured cells and healthy cells. Conclusion: When exposed to oxidative stress several proteins are oxidized decreasing the activity of many metabolic pathways. In the present study amount of intracellular protein increases when cells are injured with CaP or H2O2. While extracellular protein remains more or less same in both healthy and injured condition of cells. In SDS-PAGE analysis few bands were missing in the intracellular extract of injured cells. These results indicate that the amount of protein varies when cells are injured with CaP and H2O2.