The human proximal tubular HK-2 cell line is an immortalized cell line commonly used for studying proximal tubular toxicity. Even as their use is presently increasing, there unfortunately are no studies focused on functional changes in HK-2 cells associated with passaging. The aim of the present study, therefore, was to evaluate the functional stability of HK-2 cells during 13 weeks of continuous passaging after 6 and 24 h of treatment with model nephrotoxic compounds (i.e., acetaminophen, cisplatin, CdCl(2)). Short tandem repeat profile, the doubling time, cell diameter, glutathione concentration, and intracellular dehydrogenase activity were measured in HK-2 cells at each tested passage. The results showed that HK-2 cells exhibit stable morphology, cell size, and cell renewal during passaging. Mean doubling time was determined to be 54 h. On the other hand, we observed a significant effect of passaging on the susceptibility of HK-2 cells to toxic compounds. The largest difference in results was found in both cadmium and cisplatin treated cells across passages. We conclude that the outcomes of scientific studies on HK-2 cells can be affected by the number of passages even after medium-term cultivation and passaging for 13 weeks.

Department of Biological and Biochemical Sciences Faculty of Chemical Technology University of Pardubice Pardubice Czech Republic

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AMARAL M, GIRARD M, ÁLVAREZ R, PATON A, PATON J, REPETTO H, SACERDOTI F, IBARRA C. Ouabain protects human renal cells against the cytotoxic effects of shiga toxin type 2 and subtilase cytotoxin. Toxins. 2017;9:1–14. doi: 10.3390/toxins9070226. PubMed DOI PMC

BEN-DAVID U, SIRANOSIAN B, HA G, TANG H, OREN Y, HINOHARA K, STRATHDEE CA, DEMPSTER J, LYONS NJ, BURNS R, NAG A, KUGENER G, CIMINI B, TSVETKOV P, MARUVKA YE, O’ROURKE R, GARRITY A, TUBELLI AA, BANDOPADHAYAY P, TSHERNIAK A, VAZQUEZ F, WONG B, BIRGER C, GHANDI M, THORNER AR, BITTKER JA, MEYERSON M, GETZ G, BEROUKHIM R, GOLUB TR. Genetic and transcriptional evolution alters cancer cell line drug response. Nature. 2018;560:325–330. doi: 10.1038/s41586-018-0409-3. PubMed DOI PMC

BUŠEK P, STREMEŇOVÁ J, KŘEPELA E, ŠEDO A. Modulation of substance P signaling by dipeptidyl peptidase-IV enzymatic activity in human glioma cell lines. Physiol Res. 2008;57:443–449. PubMed

CAMPOS MAA, de ALMEIDA LA, GROSSI MF, TAGLIATI CA. In vitro evaluation of biomarkers of nephrotoxicity through gene expression using gentamicin. J Biochem Mol Toxicol. 2018;32:e22189. doi: 10.1002/jbt.22189. PubMed DOI

CLYNES M. Animal cell culture techniques. Springer-Verlag; Berlin and Heidelberg GmbH & Co KG, New York: 1998. p. 618. DOI

ČAPEK J, HAUSCHKE M, BRŮČKOVÁ L, ROUŠAR T. Comparison of glutathione levels measured using optimized monochlorobimane assay with those from ortho-phthalaldehyde assay in intact cells. J Pharmacol Toxicol Methods. 2017;88:40–45. doi: 10.1016/j.vascn.2017.06.001. PubMed DOI

DEVOCELLE A, LECRU L, FRANÇOIS H, DESTERKE C, GALLERNE C, EID P, ESTELLE O, AZZARONE B, GIRON-MICHEL J. Inhibition of TGF-β1 signaling by IL-15: A novel role for IL-15 in the control of renal epithelial-mesenchymal transition: IL-15 counteracts TGF-β1-induced EMT in renal fibrosis. Int J Cell Biol. 2019;2019:1–15. doi: 10.1155/2019/9151394. PubMed DOI PMC

DU B, MA L-M, HUANG M-B, ZHOU H, HUANG H-L, SHAO P, CHEN Y-Q, QU L-H. High glucose down-regulates miR-29a to increase collagen IV production in HK-2 cells. FEBS Lett. 2010;584:811–816. doi: 10.1016/j.febslet.2009.12.053. PubMed DOI

FRESHNEY RI. Culture of animal cells: A manual of basic techniques. John Wiley & Sons; 2005. p. 672. DOI

FUJIKI K, INAMURA H, MATSUOKA M. PI3K signaling mediates diverse regulation of ATF4 expression for the survival of HK-2 cells exposed to cadmium. Arch Toxicol. 2013;88:403–414. doi: 10.1007/s00204-013-1129-y. PubMed DOI

FUJIKI K, INAMURA H, SUGAYA T, MATSUOKA M. Blockade of ALK4/5 signaling suppresses cadmium- and erastin-induced cell death in renal proximal tubular epithelial cells via distinct signaling mechanisms. Cell Death Differ. 2019;26:2371–2385. doi: 10.1038/s41418-019-0307-8. PubMed DOI PMC

GAO S, CHEN T, CHOI M-Y, LIANG Y, XUE J, WONG Y-S. Cyanidin reverses cisplatin-induced apoptosis in HK-2 proximal tubular cells through inhibition of ROS-mediated DNA damage and modulation of the ERK and AKT pathways. Cancer Lett. 2013;333:36–46. doi: 10.1016/j.canlet.2012.12.029. PubMed DOI

GAO Z, ZHU W, ZHANG H, LI Z, CUI T. The influence of fasudil on renal proximal tubular cell epithelial-mesenchymal transition induced by parathormone. Ren Fail. 2017;39:575–581. doi: 10.1080/0886022X.2017.1349677. PubMed DOI PMC

GARCÍA-PASTOR C, BLÁZQUEZ-SERRA R, BOSH RJ, LUCIO-CAZAÑA FJ, FERNÁNDEZ-MARTÍNEZ AB. Apoptosis and cell proliferation in proximal tubular cells exposed to apoptotic bodies Novel pathophysiological implications in cisplatin-induced renal injury. Biochim Biophys Acta - Mol Basis Dis. 2019;1865:2504–2515. doi: 10.1016/j.bbadis.2019.06.008. PubMed DOI

GE Z, DIAO H, JI X, LIU Q, ZHANG X, WU Q. Gap junctional intercellular communication and endoplasmic reticulum stress regulate chronic cadmium exposure induced apoptosis in HK-2 cells. Toxicol Lett. 2018;288:35–43. doi: 10.1016/j.toxlet.2018.02.013. PubMed DOI

GENC G, KILINC V, BEDIR A, OZKAYA O. Effect of creatine and pioglitazone on HK-2 cell line cisplatin nephrotoxicity. Ren Fail. 2014;36:1104–1107. doi: 10.3109/0886022X.2014.926755. PubMed DOI

GRAHAM FL, SMILEY J, RUSSELL WC, NAIRN R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977;36:59–72. doi: 10.1099/0022-1317-36-1-59. PubMed DOI

HAN M, LI Y, WEN D, LIU M, MA Y, CONG B. NGAL protects against endotoxin-induced renal tubular cell damage by suppressing apoptosis. BMC Nephrol. 2018;19:1–10. doi: 10.1186/s12882-018-0977-3. PubMed DOI PMC

HANDL J, ČAPEK J, MAJTNEROVÁ P, PETIRA F, HAUSCHKE M, ROUŠAROVÁ E, ROUŠAR T. Transient increase in cellular dehydrogenase activity after cadmium treatment precedes enhanced production of reactive oxygen species in human proximal tubular kidney cells. Physiol Res. 2019;68:481–490. doi: 10.33549/physiolres.934121. PubMed DOI

HAUSCHKE M, ROUŠAROVÁ E, FLÍDR P, ČAPEK J, LIBRA A, ROUŠAR T. Neutrophil gelatinase-associated lipocalin production negatively correlates with HK-2 cell impairment: Evaluation of NGAL as a marker of toxicity in HK-2 cells. Toxicol In Vitro. 2017;39:52–57. doi: 10.1016/j.tiv.2016.11.012. PubMed DOI

HUANG F, ZHAO Y, WANG Q, HILLEBRANDS J-L, BORN JVD, JI L, AN T, QIN G. Dapagliflozin attenuates renal tubulointerstitial fibrosis associated with type 1 diabetes by regulating STAT1/TGFβ1 signaling. Front Endocrinol. 2019;10:1–13. doi: 10.3389/fendo.2019.00441. PubMed DOI PMC

HUANG H, ZHENG F, DONG X, WU F, WU T, LI H. Allicin inhibits tubular epithelial-myofibroblast transdifferentiation under high glucose conditions in vitro. Exp Ther Med. 2017;13:254–262. doi: 10.3892/etm.2016.3913. PubMed DOI PMC

HUANG JX, KAESLIN G, RANALL MV, BLASKOVICH MA, BECKER B, BUTLER MS, LITTLE MH, LASH LH, COOPER MA. Evaluation of biomarkers for in vitro prediction of drug-induced nephrotoxicity: comparison of HK-2, immortalized human proximal tubule epithelial, and primary cultures of human proximal tubular cells. Pharmacol Res Perspect. 2015;3:1–14. doi: 10.1002/prp2.148. PubMed DOI PMC

HUGHES P, MARSHALL D, REID Y, PARKES H, GELBER C. The costs of using unauthenticated, over-passaged cell lines: how much more data do we need? Biotechniques. 2007;43:575–584. doi: 10.2144/000112598. PubMed DOI

CHANG Y-W, SINGH KP. Nicotine-induced oxidative stress contributes to EMT and stemness during neoplastic transformation through epigenetic modifications in human kidney epithelial cells. Toxicol Appl Pharmacol. 2019;374:65–76. doi: 10.1016/j.taap.2019.04.023. PubMed DOI

CHOU X, DING F, ZHANG X, DING X, GAO H, WU Q. Sirtuin-1 ameliorates cadmium-induced endoplasmic reticulum stress and pyroptosis through XBP-1s deacetylation in human renal tubular epithelial cells. Arch Toxicol. 2019;93:965–986. doi: 10.1007/s00204-019-02415-8. PubMed DOI

JIN W, PENINGTON CJ, McCUE SW, SIMPSON MJ. A computational modelling framework to quantify the effects of passaging cell lines. Plos One. 2017;12:1–16. doi: 10.1371/journal.pone.0181941. PubMed DOI PMC

KIM J. Poly(ADP-Ribose) polymerase activation induces high mobility group box 1 release from proximal tubular cells during cisplatin nephrotoxicity. Physiol Res. 2016;65:333–340. doi: 10.33549/physiolres.932948. PubMed DOI

KIM SY, SOHN S-J, WON AJ, KIM HS, MOON A. Identification of noninvasive biomarkers for nephrotoxicity using HK-2 human kidney epithelial cells. Toxicol Sci. 2014;140:247–258. doi: 10.1093/toxsci/kfu096. PubMed DOI

KWIST K, BRIDGES WC, BURG KJL. The effect of cell passage number on osteogenic and adipogenic characteristics of D1 cells. Cytotechnology. 2015;68:1661–1667. doi: 10.1007/s10616-015-9883-8. PubMed DOI PMC

L’AZOU B, DUBUS I, OHAYON-COURTÈS C, CAMBAR J. Human glomerular mesangial IP15 cell line as a suitable model for in vitro cadmium cytotoxicity studies. Cell Biol Toxicol. 2006;23:267–278. doi: 10.1007/s10565-006-0888-0. PubMed DOI

LEE CP, NITHIYANANTHAM S, HSU HT, YEH KT, KUO TM, KO YC. ALPK1 regulates streptozotocin induced nephropathy through CCL2 and CCL5 expressions. J Cell Mol Med. 2019a;23:7699–7708. doi: 10.1111/jcmm.14643. PubMed DOI PMC

LEE YP, CHO Y, KIM EJ, LEE H, CHOI HY, WANG HJ, KANG ES, KIM YS, KIM MS, KIM BS. Reduced expression of pyruvate kinase in kidney proximal tubule cells is a potential mechanism of pravastatin altered glucose metabolism. Sci Rep. 2019b;9:1–8. doi: 10.1038/s41598-019-39461-2. PubMed DOI PMC

LU Y-T, MA X-L, XU Y-H, HU J, WANG F, QIN W-Y, XIONG W-Y. A fluorescent glucose transport assay for screening SGLT2 inhibitors in endogenous SGLT2-expressing HK-2 cells. Nat Prod Bioprospect. 2018;9:13–21. doi: 10.1007/s13659-018-0188-4. PubMed DOI PMC

MEDINA-NAVARRO R, TORRES-RAMOS YD, GUZMÁN-GRENFELL AM, DÍAZ-FLORES M, LEÓN-REYES G, HICKS GJJ. Lysosomal dysfunction induced by changes in albumin’s tertiary structure: Potential key factor in protein toxicity during diabetic nephropathy. Life Sci. 2019;230:197–207. doi: 10.1016/j.lfs.2019.05.069. PubMed DOI

NHO J-H, JUNG H-K, LEE M-J, JANG J-H, SIM M-O, JEONG D-E, CHO H-W, KIM J-C. Beneficial effects of cynaroside on cisplatin-induced kidney injury in vitro and in vivo. Toxicol Res. 2018;34:133–141. doi: 10.5487/TR.2018.34.2.133. PubMed DOI PMC

O’DRISCOLL L, GAMMELL P, McKIERNAN E, RYAN E, JEPPESEN PB, RANI S, CLYNES M. Phenotypic and global gene expression profile changes between low passage and high passage MIN-6 cells. J Endocrinol. 2006;191:665–676. doi: 10.1677/joe.1.06894. PubMed DOI

OH S-M, PARK G, LEE SH, SEO C-S, SHIN H-K, OH D-S. Assessing the recovery from prerenal and renal acute kidney injury after treatment with single herbal medicine via activity of the biomarkers HMGB1, NGAL and KIM-1 in kidney proximal tubular cells treated by cisplatin with different doses and exposure times. BMC Complement Altern Med. 2017;17:1–9. doi: 10.1186/s12906-017-2055-y. PubMed DOI PMC

POPELOVÁ A, KÁKONOVÁ A, HRUBÁ L, KUNEŠ J, MALETÍNSKÁ L, ŽELEZNÁ B. Potential neuroprotective and anti-apoptotic properties of a long-lasting stable analog of ghrelin: an in vitro study using SH-SY5Y cells. Physiol Res. 2018;67:339–346. doi: 10.33549/physiolres.933761. PubMed DOI

RACUSEN LC, MONTEIL C, SGRIGNOLI A, LUCSKAY M, MAROUILLAT S, RHIM JGS, MORIN J-P. Cell lines with extended in vitro growth potential from human renal proximal tubule: Characterization, response to inducers, and comparison with established cell lines. J Lab Clin Med. 1997;129:318–329. doi: 10.1016/S0022-2143(97)90180-3. PubMed DOI

REEVES SR, BARROW KA, WHITE MP, RICH LM, NAUSHAB M, DEBLEY JS. Stability of gene expression by primary bronchial epithelial cells over increasing passage number. BMC Pulmonar Med. 2018;18:1–11. doi: 10.1186/s12890-018-0652-2. PubMed DOI PMC

ROBERTS DD, TACIAK B, BIAŁASEK M, BRANIEWSKA A, SAS Z, SAWICKA P, KIRAGA Ł, RYGIEL T, KRÓL M. Evaluation of phenotypic and functional stability of RAW 264.7 cell line through serial passages. Plos One. 2018;13:e0198943. doi: 10.1371/journal.pone.0198943. PubMed DOI PMC

RUAN H, WANG L, WANG J, SUN H, HE X, LI W, ZHANG J. Sika deer antler protein against acetaminophen-induced oxidative stress and apoptosis in HK-2 cells via activating Nrf2/keap1/HO-1 pathway. J Food Biochem. 2019;00:e13067. doi: 10.1111/jfbc.13067. PubMed DOI

RYAN MJ, JOHNSON G, KIRK J, FUERSTENBERG SM, ZAGER RA, TOROK-STORB B. HK-2: An immortalized proximal tubule epithelial cell line from normal adult human kidney. Kidney Int. 1994;45:48–57. doi: 10.1038/ki.1994.6. PubMed DOI

SCHMIDT HHHW, CHO S, YU S-L, KANG J, JEONG BY, LEE HY, PARK CG, YU Y-B, JIN D-C, HWANG W-M, YUN S-R, SONG HS, PARK MH, YOON S-H. NADPH oxidase 4 mediates TGF-β1/Smad signaling pathway induced acute kidney injury in hypoxia. Plos One. 2019;14:e0219483. doi: 10.1371/journal.pone.0219483. PubMed DOI PMC

SONG M-F, YANG Y, YI Z-W, ZHANG Z-Q, SHEN X-D, HU G-H, ZHU Y-F. Sema 3A as a biomarker of the activated mTOR pathway during hexavalent chromium-induced acute kidney injury. Toxicol Lett. 2018;299:226–235. doi: 10.1016/j.toxlet.2018.09.005. PubMed DOI

TONG Z-B, HOGBERG H, KUO D, SAKAMURU S, XIA M, SMIRNOVA L, HARTUNG T, GERHOLD D. Characterization of three human cell line models for high-throughput neuronal cytotoxicity screening. J Appl Toxicol. 2017;37:167–180. doi: 10.1002/jat.3334. PubMed DOI PMC

VASILEVSKY NA, BRUSH MH, PADDOCK H, PONTING L, TRIPATHY SJ, LAROCCA GM, HAENDEL MA. On the reproducibility of science: unique identification of research resources in the biomedical literature. Peer J. 2013;1:e148. doi: 10.7717/peerj.148. PubMed DOI PMC

VOŠAHLÍKOVÁ M, SVOBODA P. The influence of monovalent cations on trimeric G protein Gi1α activity in HEK293 cells stably expressing DOR-Gi1α (Cys351-Ile351) fusion protein. Physiol Res. 2011;60:541–547. doi: 10.33549/physiolres.932096. PubMed DOI

VRBOVÁ M, ROUŠAROVÁ E, BRŮČKOVÁ L, ČESLA P, ROUŠAR T. Characterization of acetaminophen toxicity in human kidney HK-2 cells. Physiol Res. 2016;65:627–635. doi: 10.33549/physiolres.933158. PubMed DOI

WANG Q, LU Y, YUAN M, DARLING IM, REPASKY EA, MORRIS ME. Characterization of monocarboxylate transport in human kidney HK-2. Cells Mol Pharm. 2006;3:675–685. doi: 10.1021/mp060037b. PubMed DOI

WU Y, CONNORS D, BARBER L, JAYACHANDRA S, HANUMEGOWDA UM, ADAMS SP. Multiplexed assay panel of cytotoxicity in HK-2 cells for detection of renal proximal tubule injury potential of compounds. Toxicol in Vitro. 2009;23:1170–1178. doi: 10.1016/j.tiv.2009.06.003. PubMed DOI

XIA T, HAMILTON RF, BONNER JC, CRANDALL ED, ELDER A, FAZLOLLAHI F, GIRTSMAN TA, KIM K, MITRA S, NTIM SA, ORR G, TAGMOUNT M, TAYLOR AJ, TELESCA D, TOLIC A, VULPE CD, WALKER AJ, WANG X, WITZMANN FA, WU N, XIE Y, ZINK JI, NEL A, HOLIAN A. Interlaboratory evaluation of in vitro cytotoxicity and inflammatory responses to engineered nanomaterials: The NIEHS nano GO consortium. Environ Health Perspect. 2013;121:683–690. doi: 10.1289/ehp.1306561. PubMed DOI PMC

YANG A, LIU F, GUAN B, LUO Z, LIN J, FANG W, LIU L, ZUO W. p53 induces miR-199a-3p to suppress mechanistic target of rapamycin activation in cisplatin-induced acute kidney injury. J Cell Biochem. 2019a;120:17625–17634. doi: 10.1002/jcb.29030. PubMed DOI

YANG G, MA H, WU Y, ZHOU B, ZHANG C, CHAI C, CAO Z. Activation of TRPC6 channels contributes to (+)-conocarpan-induced apoptotic cell death in HK-2 cells. Food Chem Toxicol. 2019b;129:281–290. doi: 10.1016/j.fct.2019.04.061. PubMed DOI

YANG K-J, KIM JH, CHANG YK, PARK CW, KIM SY, HONG YA. Inhibition of xanthine oxidoreductase protects against contrast-induced renal tubular injury by activating adenosine monophosphate-activated protein kinase. Free Radic Biol Med. 2019c;145:209–220. doi: 10.1016/j.freeradbiomed.2019.09.027. PubMed DOI

YEH IJ, WANG T-Y, LIN J-C, LIN T-J, CHANG J-S, YEN M-C, LIU Y-H, WU P-L, CHEN F-W, SHIH Y-L, PENG C-Y. Optimal regimen of n-acetylcysteine on chromium-induced renal cell damage. Metabolites. 2019;9:172. doi: 10.3390/metabo9090172. PubMed DOI PMC

ZAZA G, MASOLA V, GRANATA S, BELLIN G, GASSA DA, ONISTO M, GAMBARO G, LUPO A. Sulodexide alone or in combination with low doses of everolimus inhibits the hypoxia-mediated epithelial to mesenchymal transition in human renal proximal tubular cells. J Nephrol. 2015;28:431–440. doi: 10.1007/s40620-015-0216-y. PubMed DOI

ZENG Y, WANG X, WANG J, YI R, LONG H, ZHOU M, LUO Q, ZHAI Z, SONG Y, QI S. The tumorgenicity of glioblastoma cell line U87MG decreased during serial in vitro passage. Cell Mol Neurobiol. 2018;38:1245–1252. doi: 10.1007/s10571-018-0592-7. PubMed DOI

ZHANG L, MU X, FU J, ZHOU Z. In vitro cytotoxicity assay with selected chemicals using human cells to predict target-organ toxicity of liver and kidney. Toxicol In Vitro. 2007;21:734–740. doi: 10.1016/j.tiv.2007.01.013. PubMed DOI

Quantitative spectrofluorometric assay detecting nuclear condensation and fragmentation in intact cells