EVALUATION OF RENAL EPITHELIAL CELL PROTEIN UNDER STRESS CONDITION
Keywords:Oxidative stress, Kidney, Protein, Calcium phosphate, Crystal
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 Â°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 4.84Â±0.004Âµg/ml. Intracellular protein of CaP injured and H2O2 injured cells were 10.59Â±0.003 Âµg/ml and 10.78Â±0.011Âµg/ml respectively. While extracellular protein of injured cells was nearly 4 Âµg/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.
Luptak J, Bek-Jensen H, Fornander AM. Crystallization of calcium oxalate and calcium phosphate of supersaturation levels corresponding to those in different parts of the nephron. Scanning Microsc 1994;8:47â€“62.
Tiselius HG. Solution chemistry of supersaturation. In kidney stones-medical and Surgical Management. ed. FL Coe, MJ Flavus, CYC Pak, JH Parks, GM Preminger. Philadelphia: Lippincott-Raven Publishers; 1996. p. 33â€“64.
Asplin JR, Mandel NS, Coe FL. Evidence for calcium phosphate supersaturation in the loop of Henle. Am J Physiol 1996;270:F604â€“13.
HÃ¶jgaard I, Tiselius HG. Crystallization in the nephron. Urol Res 1999;27:397â€“403.
Khan SR. Reactive oxygen species, inflammation and calcium oxalate nephrolithiasis. Transl Androl Urol 2014;3:256-76.
Katz A, Orellana O. Protein Synthesis and the Stress Response, Cell-Free Protein Synthesis, Prof. Manish Biyani. Ed. InTech; 2012.
Kabra SG, Kabra V, Banerji P, Jain LK, Bhargava A, Chaturvedi RP. In vitro calculogenesis: methods to develop concretions of desired chemical composition. Indian J Exp Biol 1978;16:212-7.
Pathak P, Singh SK, Tandon C. Effect of Biomolecules from the human renal matrix of calcium oxalate monohydrate (CaOx) stones on in vitro calcium phosphate crystallization. Int Brazilian J Urol 2010;36:621-8.
Priyadarshini, Jain K. Cytoprotective effect of Ocimum extract on injured renal epithelial cells. Int J Pharmacol Pharm Sci 2015;7:15-8.
Ouyang JM, Yao XQ, Tan J, Wang FX. Renal epithelial cell injury and its promoting role in the formation of calcium oxalate monohydrate. JBIC J Biol Inorg Chem 2011;16:405-16.
Bradford MM. A rapid and sensitive for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54.
Singh A, Puri D, Kumari B, Singh SK. Heat shock proteins: knowledge so far and its future prospects. Asian J Pharm Clin Res 2016;9:17-24.
Kumar V, Yu S, Farell G, Toback FG, Lieske JC. Renal epithelial cells constitutively produce a protein that blocks adhesion of crystals to their surface. Am J Physiol Renal Physiol 2004;287:F373â€“83.
Salas PJ, Ponce MI, Brignoni M, RodrÃguez ML. Attachment of madin-darby canine kidney cells to extracellular matrix: the role of a laminin-binding protein related to the 37/67 kDa laminin receptor in the development of plasma membrane polarization. Biol Cell 1992;75:197-210.
Lisanti MP, Sargiacomo M, Greave L, Saltiel AR, Boulan ER. Polarized apical distribution of glycosyl-phosphatidyl inositol anchored proteins in a renal epithelial cell line. Proc Natl Acad Sci USA 1988;85:9557-61.
Cavaloc Y, Popielarz M, Fuchs JP, Gattoni R, Stvenin J. Characterization and cloning of the human splicing factor 9G8: a novel 35 kDa factor of the serine/arginine protein family. EMBO J 1994;13:2639â€“49.