title: Neutrophil gelatinase-associated lipocalin production negatively correlates with HK-2 cell impairment: Evaluation of NGAL as a marker of toxicity in HK-2 cells.
abstract: Neutrophil gelatinase-associated lipocalin is an extracellular protein produced mostly in kidney. Recently, it has become a promising biomarker of renal damage in vivo. On the other hand, the validation of NGAL as a biomarker for nephrotoxicity estimation in vitro has not been characterized in detail yet. Since the HK-2 cells are frequently used human kidney cell line, we aimed to characterize the production of NGAL in these cells and to evaluate NGAL as a possible marker of cell impairment. We used heavy metals (mercury, cadmium), peroxide, drugs (acetaminophen, gentamicin) and cisplatin to mimic nephrotoxicity. HK-2 cells were incubated with selected compounds for 1-24h and cell viability was measured together with extracellular NGAL production. We proved that HK-2 cells possess a capacity to produce NGAL in amount of 2pg/ml/h. We found a change in cell viability after 24h incubation with all tested toxic compounds. The largest decrease of the viability was detected in mercury, acetaminophen, cisplatin and gentamicin. Unexpectedly, we found also a significant decrease in NGAL production in HK-2 cells treated with these toxins for 24h: to 11±5%, 54±5%, 57±6% and 76±9% respectively, compared with controls (=100%). Our results were followed with qPCR analysis when we found no significant increase in LCN2 gene expression after 24h incubation. We conclude that extracellular NGAL production negatively correlates with HK-2 cell impairment.
authors.affiliation: Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Hauschke
authors.affiliation: Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic.
authors.forename: Erika
authors.initials: E
authors.lastname: Roušarová
authors.affiliation: Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic; Generi-Biotech, Hradec Králové, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Flídr
authors.affiliation: Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Čapek
authors.affiliation: Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic; Generi-Biotech, Hradec Králové, Czech Republic.
authors.forename: Antonín
authors.initials: A
authors.lastname: Libra
authors.affiliation: Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic. Electronic address: Tomas.Rousar@upce.cz.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Roušar
mesh_terms: D000082:Acetaminophen / Q000633:toxicity; D058186:Acute Kidney Injury / Q000139:chemically induced / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D002104:Cadmium / Q000633:toxicity; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000633:toxicity; D005839:Gentamicins / Q000633:toxicity; D006801:Humans; D000071068:Lipocalin-2 / Q000235:genetics / Q000378:metabolism*; D008628:Mercury / Q000633:toxicity; D020122:tert-Butylhydroperoxide / Q000633:toxicity
chemical_list: D015415:Biomarkers; D005839:Gentamicins; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D002104:Cadmium; D000082:Acetaminophen; D020122:tert-Butylhydroperoxide; D008628:Mercury; D002945:Cisplatin
keywords: Cell viability; HK-2 cells; In vitro nephrotoxicity; NGAL; Nephrotoxicity markers