In Vitro to In Vivo Concordance of Toxicity Using the Human Proximal Tubule Cell Line HK-2

2020 ◽  
Vol 39 (5) ◽  
pp. 452-464
Author(s):  
Miriam E. Mossoba ◽  
Robert L. Sprando
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Cell Injury ◽  
Culture Media ◽  
Sugar Transport ◽  
Molecular Characteristics ◽  
Proximal Tubule Cell ◽  
Tubule Cell

The renal proximal tubule cell line, human kidney 2 (HK-2), recapitulates many of the functional cellular and molecular characteristics of differentiated primary proximal tubule cells. These features include anchorage dependence, gluconeogenesis capability, and sodium-dependent sugar transport. In order to ascertain how well HK-2 cells can reliably reveal the toxicological profile of compounds having a potential to cause proximal tubule injury in vivo, we sought to evaluate the effects of known proximal tubule toxicants using the HK-2 cell line. We selected 20 pure nephrotoxic compounds that included chemotherapeutic drugs, antibiotics, and heavy metal-containing compounds and evaluated their ability to induce HK-2 cell injury relative to 10 innocuous pure compounds or cell culture media alone. We performed a comprehensive set of in vitro cellular toxicological assays to evaluate cell viability, oxidative stress, mitochondrial integrity, and a specific biomarker of renal injury, Kidney Injury Molecule 1. For each of our selected compounds, we were able to establish a reproducible profile of toxicological outcomes. We compared our results to those described in peer-reviewed publications to understand how well the HK-2 cellular model agrees with overall in vivo rat or human toxicological outcomes. This study begins to address the question of how well in vitro data generated with HK-2 cells can mirror in vivo animal and human outcomes.

scholarly journals Long-term in vitro effects of exposing the human HK-2 proximal tubule cell line to 3-monochloropropane-1,2-diol

2020 ◽  
Vol 45 (1) ◽  
pp. 45-56 ◽  
Author(s):  
Miriam E. Mossoba ◽  
Mapa S.T. Mapa ◽  
Magali Araujo ◽  
Yang Zhao ◽  
Brenna Flannery ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Proximal Tubule Cell ◽  
Tubule Cell

scholarly journals Expression and Role of Parathyroid Hormone-Related Protein in Human Renal Proximal Tubule Cells during Recovery from ATP Depletion

1999 ◽  
Vol 10 (2) ◽  
pp. 238-244
Author(s):  
ADOLFO GARCÍA-OCAÑA ◽  
SUSAN C. GALBRAITH ◽  
SCOTT K. VAN WHY ◽  
KAI YANG ◽  
LINA GOLOVYAN ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Recovery Period ◽  
Ischemic Injury ◽  
Atp Depletion ◽  
Proximal Tubule Cell ◽  
Pthrp Expression ◽  
Human Proximal Tubule Cell

Abstract. Parathyroid hormone (PTH)-related protein (PTHrP) is widely expressed in normal fetal and adult tissues and regulates growth and differentiation in a number of organ systems. Although various renal cell types produce PTHrP, and PTHrP expression in rat proximal renal tubules is upregulated in response to ischemic injury in vivo, the role of PTHrP in the kidney is unknown. To study the effects of injury on PTHrP expression and its consequences in more detail, the immortalized human proximal tubule cell line HK-2 was used in an in vitro model of ATP depletion to mimic in vivo renal ischemic injury. These cells secrete PTHrP into conditioned medium and express the type I PTH/PTHrP receptor. Treatment of confluent HK-2 cells for 2 h with substrate-free, glucose-free medium containing the mitochondrial inhibitor antimycin A (1 μM) resulted in 75% depletion of cellular ATP. After an additional 2 h in glucose-containing medium, cellular ATP levels recovered to approximately 75% of baseline levels. PTHrP mRNA levels, as measured in RNase protection assays, peaked at 2 h into the recovery period (at four times baseline expression). The increase in PTHrP mRNA expression was correlated with an increase in PTHrP protein content in HK-2 cells at 2 to 6 h into the recovery period. Heat shock protein-70 mRNA expression was not detectable under baseline conditions but likewise peaked at 2 h into the recovery period. Treatment of HK-2 cells during the recovery period after injury with an anti-PTHrP(1-36) antibody (at a dilution of 1:250) resulted in significant reductions in cell number and uptake of [3H]thymidine, compared with nonimmune serum at the same titer. Similar results were observed in uninjured HK-2 cells. It is concluded that this in vitro model of ATP depletion in a human proximal tubule cell line reproduces the pattern of gene expression previously observed in vivo in rat kidney after ischemic injury and that PTHrP plays a mitogenic role in the proliferative response after energy depletion.

scholarly journals The actin cytoskeleton and integrin expression in the recovery of cell adhesion after oxidant stress to a proximal tubule cell line (JTC-12).

1998 ◽  
Vol 9 (10) ◽  
pp. 1787-1797
Author(s):  
S Nigam ◽  
C E Weston ◽  
C H Liu ◽  
E E Simon
Keyword(s):  
Cell Adhesion ◽  
Actin Cytoskeleton ◽  
Proximal Tubule ◽  
Cell Line ◽  
Oxidant Stress ◽  
Proximal Tubule Cell ◽  
Integrin Expression ◽  
Tubule Cell ◽  
Essentially Normal ◽  
Atp Levels

This study examines the role of the actin cytoskeleton and integrin expression in the recovery of cell adhesion in the proximal tubule cell line JTC-12 after peroxide injury. The cells were exposed to 10, 20, or 50 mM hydrogen peroxide for 10 min and then allowed to recover. Viability measurements by trypan blue exclusion confirmed that the injury was largely nonlethal with 85% viability at 1 h even at 50 mM peroxide. ATP levels fell immediately after the peroxide incubation in all groups to approximately 10% of normal, but already showed some recovery by 1 h and full recovery in the 10 and 20 mM groups by 24 h. Cell adhesion to extracellular matrix immediately after injury was depressed at 20 and 50 mM peroxide, but by 12 h was abnormal only at 50 mM peroxide and at 24 h was essentially normal at all peroxide concentrations. Immediately after exposure to 10 mM peroxide, there were subtle abnormalities in the actin cytoskeleton (thickening of fibrils) as assessed by phalloidin staining, with more pronounced effects at 20 and 50 mM. At 1 h, many cells showed collapse of the actin cytoskeleton to the periphery. There was some recovery at 4 h; by 12 h, the actin cytoskeleton showed further recovery, although was still abnormal (coarsened microfilaments), especially at 20 and 50 mM peroxide. By 24 h, the actin cytoskeleton showed only subtle coarsening. Integrin surface expression was assessed by flow cytometry. The alpha6 subunit on cells exposed to 20 mM peroxide was unchanged at 1 h and 4 h, but by 12 h had increased to 118.5+/-4.5% and by 24 h to 146+/-13.4% of control levels. The expression of the beta1 and alphaVbeta3 integrins remained unchanged. Thus, despite coarsening of the actin cytoskeleton and depressed ATP levels, cell adhesion recovered from oxidant stress. Abnormal cell adhesion after injury was not a consequence of a decrease in integrin expression, and recovery of cell adhesion was not a consequence of the modest and selective increase in integrin expression.

Expression of xenobiotic transporters in the human renal proximal tubule cell line RPTEC/TERT1

2015 ◽  
Vol 30 (1) ◽  
pp. 95-105 ◽  
Author(s):  
Lydia Aschauer ◽  
Giada Carta ◽  
Nadine Vogelsang ◽  
Eberhard Schlatter ◽  
Paul Jennings
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cell ◽  
Tubule Cell

ER stress contributes to renal proximal tubule injury by increasing SREBP-2-mediated lipid accumulation and apoptotic cell death

2012 ◽  
Vol 303 (2) ◽  
pp. F266-F278 ◽  
Author(s):  
Šárka Lhoták ◽  
Sudesh Sood ◽  
Elise Brimble ◽  
Rachel E. Carlisle ◽  
Stephen M. Colgan ◽  
...  
Keyword(s):  
Er Stress ◽  
Proximal Tubule ◽  
Lipid Accumulation ◽  
Cell Injury ◽  
Apoptotic Cell ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
Proximal Tubule Cells ◽  
Tubule Cells

Renal proximal tubule injury is induced by agents/conditions known to cause endoplasmic reticulum (ER) stress, including cyclosporine A (CsA), an immunosuppressant drug with nephrotoxic effects. However, the underlying mechanism by which ER stress contributes to proximal tubule cell injury is not well understood. In this study, we report lipid accumulation, sterol regulatory element-binding protein-2 (SREBP-2) expression, and ER stress in proximal tubules of kidneys from mice treated with the classic ER stressor tunicamycin (Tm) or in human renal biopsy specimens showing CsA-induced nephrotoxicity. Colocalization of ER stress markers [78-kDa glucose regulated protein (GRP78), CHOP] with SREBP-2 expression and lipid accumulation was prominent within the proximal tubule cells exposed to Tm or CsA. Prolonged ER stress resulted in increased apoptotic cell death of lipid-enriched proximal tubule cells with colocalization of GRP78, SREBP-2, and Ca2+-independent phospholipase A2 (iPLA2β), an SREBP-2 inducible gene with proapoptotic characteristics. In cultured HK-2 human proximal tubule cells, CsA- and Tm-induced ER stress caused lipid accumulation and SREBP-2 activation. Furthermore, overexpression of SREBP-2 or activation of endogenous SREBP-2 in HK-2 cells stimulated apoptosis. Inhibition of SREBP-2 activation with the site-1-serine protease inhibitor AEBSF prevented ER stress-induced lipid accumulation and apoptosis. Overexpression of the ER-resident chaperone GRP78 attenuated ER stress and inhibited CsA-induced SREBP-2 expression and lipid accumulation. In summary, our findings suggest that ER stress-induced SREBP-2 activation contributes to renal proximal tubule cell injury by dysregulating lipid homeostasis.

Nitric oxide activates PKCα and inhibits Na+-K+-ATPase in opossum kidney cells

1999 ◽  
Vol 277 (6) ◽  
pp. F859-F865 ◽  
Author(s):  
Mingyu Liang ◽  
Franklyn G. Knox
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Proximal Tubule ◽  
Cell Line ◽  
Soluble Guanylate Cyclase ◽  
Inhibitory Effect ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
Opossum Kidney ◽  
Ok Cells

Nitric oxide (NO) reduces the molecular activity of Na+-K+-ATPase in opossum kidney (OK) cells, a proximal tubule cell line. In the present study, we investigated the cellular mechanisms for the inhibitory effect of NO on Na+-K+-ATPase. Sodium nitroprusside (SNP), a NO donor, inhibited Na+-K+-ATPase in OK cells, but not in LLC-PK1cells, another proximal tubule cell line. Similarly, phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, inhibited Na+-K+-ATPase in OK, but not in LLC-PK1, cells. PKC inhibitors staurosporine or calphostin C, but not the protein kinase G inhibitor KT-5823, abolished the inhibitory effect of NO on Na+-K+-ATPase in OK cells. Immunoblotting demonstrated that treatment with NO donors caused significant translocation of PKCα from cytosolic to particulate fractions in OK, but not in LLC-PK1, cells. Furthermore, the translocation of PKCα in OK cells was attenuated by either the phospholipase C inhibitor U-73122 or the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. U-73122 also blunted the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. The phospholipase A2inhibitor AACOCF3 did not blunt the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. AACOCF3 alone, however, also decreased Na+-K+-ATPase activity in OK cells. In conclusion, our results demonstrate that NO activates PKCα in OK, but not in LLC-PK1, cells. The activation of PKCα in OK cells by NO is associated with inhibition of Na+-K+-ATPase.

scholarly journals P-GLYCOPROTEIN IN HK-2 PROXIMAL TUBULE CELL LINE

Renal Failure ◽  
2001 ◽  
Vol 23 (3-4) ◽  
pp. 331-337 ◽  
Author(s):  
Gianfranco Tramonti ◽  
Nadia Romiti ◽  
Maria Norpoth ◽  
Elisabetta Chieli
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
P Glycoprotein

Contribution of actin cytoskeletal alterations to ATP depletion and calcium-induced proximal tubule cell injury

1996 ◽  
Vol 270 (1) ◽  
pp. F39-F52 ◽  
Author(s):  
S. Nurko ◽  
K. Sogabe ◽  
J. A. Davis ◽  
N. F. Roeser ◽  
M. Defrain ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Proximal Tubule ◽  
Laser Scanning ◽  
Structural Changes ◽  
Cell Injury ◽  
Membrane Damage ◽  
Atp Depletion ◽  
Similar Proportion ◽  
Proximal Tubule Cell ◽  
Tubule Cell

The actin cytoskeleton of rabbit proximal tubules was assessed by deoxyribonuclease (DNase) binding, sedimentability of detergent-insoluble actin, laser-scanning confocal microscopy, and ultrastructure during exposure to hypoxia, antimycin, or antimycin plus ionomycin. One-third of total actin was DNase reactive in control cells prior to deliberate depolymerization, and a similar proportion was unsedimentable from detergent lysates during 2.5 h at 100,000 g. Tubules injured by hypoxia or antimycin alone, without glycine, showed Ca(2+)-dependent pathology of the cytoskeleton, consisting of increases in DNase-reactive actin, redistribution of pelletable actin, and loss of microvilli concurrent with lethal membrane damage. In contrast, tubules similarly depleted of ATP and incubated with glycine showed no significant changes of DNase-reactive actin or actin sedimentability for up to 60 min, but, nevertheless, developed substantial loss of basal membrane-associated actin within 15 min and disruption of actin cores and clubbing of microvilli at durations > 30 min. These structural changes that occurred in the presence of glycine were not prevented by limiting Ca2+ availability or pH 6.9. Very rapid and extensive cytoskeletal disruption followed antimycin-plus-ionomycin treatment. In this setting, glycine and pH 6.9 decreased lethal membrane damage but did not ameliorate pathology in the cytoskeleton or microvilli; limiting Ca2+ availability partially protected the cytoskeleton but did not prevent lethal membrane damage. The data suggest that both ATP depletion-dependent but Ca(2+)-independent, as well as Ca(2+)-mediated, processes can disrupt the actin cytoskeleton during acute proximal tubule cell injury; that both types of change occur, despite protection afforded by glycine and reduced pH against lethal membrane damage; and that Ca(2+)-independent processes primarily account for prelethal actin cytoskeletal alterations during simple ATP depletion of proximal tubule cells.

Locally formed dopamine stimulates cAMP accumulation in LLC-PK1 cells via a DA1 dopamine receptor

1991 ◽  
Vol 260 (6) ◽  
pp. F906-F912 ◽  
Author(s):  
A. Grenader ◽  
D. P. Healy
Keyword(s):  
Cell Line ◽  
Sch 23390 ◽  
Acid Decarboxylase ◽  
Epithelial Cell Line ◽  
Proximal Tubule Cell ◽  
Dependent Manner ◽  
Camp Accumulation ◽  
Renal Epithelial Cell

Proximal tubules have been shown to produce dopamine (DA) from (-)-3-(3,4-dihydroxyphenyl)-L-alanine (L-dopa) and to express DA1 dopamine (DA1) receptors linked to inhibition of sodium transport. The LLC-PK1 renal epithelial cell line expresses proximal tubule cell-like properties in vitro. Here, we sought to determine whether the LLC-PK1 cell line would be a useful model system to study dopaminergic mechanisms in vitro. LLC-PK1 cells contained high levels of aromatic L-amino acid decarboxylase (AADC) (Km 0.19 +/- 0.08 mM, Vmax 3.69 +/- 0.57 nmol.mg-1.min-1) and converted L-dopa to DA in a nonsaturable fashion up to 1 mM L-dopa. DA production was blocked by the AADC inhibitor carbidopa. Dopamine stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in LLC-PK1 cells in a dose-dependent manner (50% effective concentration, 1.53 +/- 0.38 microM; maximal stimulation, 46.6 +/- 10.88 pmol/mg protein); this effect was blocked by addition of DA1-receptor antagonists. L-Dopa also stimulated cAMP accumulation, and this effect was attenuated by an equimolar concentration of carbidopa and blocked by the DA1 antagonist Sch 23390. These results indicate that LLC-PK1 cells convert L-dopa to DA, which then stimulates cAMP via a DA1 receptor coupled to activation of adenylate cyclase. Moreover, the demonstration that locally formed DA can act as an autocrine/paracrine substance in LLC-PK1 cells in vitro is consistent with a role for DA as an autocrine/paracrine substance in vivo.

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