Post on 21-Feb-2021
Accepted Manuscript
Title: The stress response of human proximal tubule cells tocadmium involves up-regulation of haemoxygenase 1 andmetallothionein but not cytochrome P450 enzymes
Author: Kanyarat Boonprasert Soisungwan Satarug ChrisMorais Glenda C. Gobe David W. Johnson KesaraNa-Bangchang David A. Vesey
PII: S0378-4274(16)30034-0DOI: http://dx.doi.org/doi:10.1016/j.toxlet.2016.02.016Reference: TOXLET 9330
To appear in: Toxicology Letters
Received date: 31-8-2015Revised date: 28-1-2016Accepted date: 26-2-2016
Please cite this article as: Boonprasert, Kanyarat, Satarug, Soisungwan, Morais, Chris,Gobe, Glenda C., Johnson, David W., Na-Bangchang, Kesara, Vesey, David A., Thestress response of human proximal tubule cells to cadmium involves up-regulation ofhaemoxygenase 1 and metallothionein but not cytochrome P450 enzymes.ToxicologyLetters http://dx.doi.org/10.1016/j.toxlet.2016.02.016
This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.
1
The stress response of human proximal tubule cells to cadmium involves up-
regulation of haemoxygenase 1 and metallothionein but not cytochrome P450
enzymes
Running title: Cytochrome P450 and Cd-induced stress in human kidney cells
Kanyarat Boonprasert1,2, Soisungwan Satarug2, Chris Morais2, Glenda C Gobe2,
David W Johnson2,3, Kesara Na-Bangchang1 and David A
Vesey2,3*
david.vesey@health.qld.gov.au
1Chulabhorn International College of Medicine, Thammasat University, Pathumthani, Thailand.
2Centre for Kidney Disease Research, The University of Queensland School of Medicine
Translational Research Institute, Brisbane, Australia.
3Department of Renal Medicine, Princess Alexandra Hospital, Brisbane, Australia.
*Corresponding author at: Department of Renal Medicine, Level 2, Ambulatory Renal and
Transplant Services Building Princess Alexandra Hospital, Ipswich Road, Woolloongabba
Brisbane Qld 4102 AUSTRALIA, Tel: +61 7 34438013, Fax: +61 7 3176 5480.
2
Highlights
Cytochrome P450 (CYP P450) enzymes 2B6, 4A11, 4F2, 2E1 and 3A4 are expressed by the human
kidney cortex.
Primary culture of human proximal tubule cells (PTC) express CYP P450 enzymes 2E1, 3A4 and
4F2.
Exposure of PTC to cadmium induces expression of the stress proteins metallothionein (MT) and
haemoxygenase 1 (HO-1) but not CYP P450 enzymes.
3
Abstract
Enzymes of the cytochrome P450 (CYP) super-family are implicated in cadmium (Cd) -induced
nephrotoxicity, however, direct evidence is lacking. This study investigated the endogenous
expression of various CYP proteins together with the stress-response proteins, heme oxygenase-1
(HO-1) and metallothionein (MT) in human kidney sections and in cadmium-exposed primary
cultures of human proximal tubular epithelial cells (PTC). By immunohistochemistry, the CYP
members 2B6, 4A11 and 4F2 were prominently expressed in the cortical proximal tubular cells and
to a lesser extent in distal tubular cells. Low levels of CYPs 2E1 and 3A4 were also detected. In
PTC, in the absence of Cd, CYP2E1, CYP3A4, CYP4F2 and MT were expressed, but HO-1,
CYP2B6 and CYP4A11 were not detected. A range of cadmium concentrations (0-100µM) were
utilized to induce stress conditions. MT protein was further induced by as little as 0.5µM cadmium,
reaching a 6-fold induction at 20µM, whereas for HO-1, a 5µM cadmium concentration was
required for initial induction and at 20µM cadmium reached a 15-fold induction. The expression of
CYP2E1, CYP3A4, and CYP4F2 were not altered by any cadmium concentrations tested at 48h.
Cadmium caused a reduction in cell viability at concentrations above 10µM. In conclusion although
cultured PTC, do express CYP proteins, (CYP2E1, CYP3A4, and CYP4F2), Cd-induced cell stress as
indicted by induction of HO-1 and MT does not alter expression of these CYP proteins at48h.
Abbreviations
CYP: Cytochrome P450
GGT: Gamma glutamyl transpeptidase 20-
HETE 20-hydroxyeicosatetraenoic acid
HO-1: Heme oxygenase-1
MT1/2: Metallothionein isoforms 1 and 2
PTC: Proximal Tubular Epithelial Cells
TNF-α: Tumor necrosis factor-alpha
Keywords: Cadmium; cytochrome P450 (CYP); drug-metabolizing enzymes; heme oxygenase-1;
HK2 cells; metallothionein; proximal tubular cells; stress response
4
Introduction
The kidney is particularly at risk of injury from long-term use of drugs and chronic exposure to a
range of environmental toxicants, including heavy metals [1-5]. The increased risk of kidney injury
has been attributable in part to the expression of cytochrome P450 (CYP) enzymes in this organ [6-8]
together with various transporters of drugs and metals, large blood flow and exposure to high
concentrations of solutes [1-5]. To-date, expression of various CYPs in human kidneys has not
been well characterized although their potential involvement in drug- and toxicant-induced kidney
injury and systemic blood pressure regulation has become apparent [9-16]. Specifically, kidney
CYP4A11 and CYP4F2 catalyse ω-hydroxylation of unsaturated fatty acids and one of the products,
20-hydroxyeicosatetraenoic acid (20-HETE) is known to be involved in regulation of salt balance in
the kidney and to have direct pressor functions [6;13-16]. Urinary 20-HETE excretion correlated
positively with blood pressure in salt-sensitive hypertension patients, when placed on salt loading
conditions, while it showed a positive correlation with salt excretion in salt-resistant hypertension
patients [17]. Likewise, 20-HETE and blood pressure correlation was seen in women, who had
greater urine 20-HETE levels than normotensive subjects [18]. In addition to blood pressure, urine
20-HETE correlated with F2-isoprostanes and serum gamma glutamyl transpeptidase (GGT) [19].
Elevated urinary 20-HETE excretion and blood pressure have been associated with certain CYP4F2
and CYP4A11 genetic variants [20-22].
Cadmium is a toxic heavy metal that accumulates in liver and kidney cortex and it is well
established that urine and tissue cadmium levels can be used to assess environmental exposure to
this metal [23-25]. One of the most frequently reported cadmium toxicities in the general
population is injury to kidney, where cadmium is re-absorbed and accumulates in the renal tubular
epithelial cells [25-27]. Tubular injury occurs, when cadmium accumulation exceeds the protective
capacity of two key stress-response proteins, heme oxygenase-1 (HO-1) and metallothionein (MT)
[12;28-32]. Indeed, cadmium exposure has been associated with chronic kidney disease [33-35],
kidney stones [36], end-stage kidney disease [37]and hypertension [38-41].
Evidence for the potential influence of tissue cadmium accumulation on CYP protein
expression levels in human kidneys comes from our previous study on CYP4F2 and CYP4A11
protein abundance in post-mortem Australian kidney samples [9-12], which showed a positive
correlation between CYP4F2 abundance and donors’ age and kidney cadmium content [12].
Conversely, kidney CYP4A11 protein abundance was inversely correlated with cadmium burden;
low CYP4A11 protein levels were detected in the kidney cortex samples with high cadmium
content (up to 60 µg/g kidney wet weight) [9;10]. However, direct evidence for the role of
cadmium on these CYPs is lacking. We thus undertook the present study to gain further insight
into endogenous expression levels and localization of CYP4A11 and CYP4F2 in human kidneys,
using archived human kidney tissue blocks, held by the Princess Alexandra Hospital Tissue Bank.
5
In addition, we examined expression of three other drug-metabolizing CYP enzymes (2B6, 2E1,
and 3A4) together with stress-response proteins, MT and HO-1, known to be induced by cadmium
[12;28-32]. We also examined the expression of these CYP and stress-response proteins in primary
cultures of human proximal tubular cells (PTC) and explored their ex vivo response to cadmium.
For comparison purposes, the HK2 immortalized human kidney cell line and the HepG2 human
liver cancer cell line were examined, concurrently
6
Materials and Methods
2.1 Antibodies
Rabbit anti-human CYP2B6, CYP2E1, CYP3A4, and CYP4A11 antibodies were provided by Dr.
Robert Edwards, Imperial College London, UK [42]. The rabbit anti-human CYP4F2 antibody
was from Dr. Jerome Lasker [43;44]. Mouse anti-MT 1/2 and mouse anti-HO-1 antibody
preparations were purchased from Dako Ltd (Carpinteria, CA, USA), and/or Abcam Ltd
(Cambridge, MA, USA) respectively. The mouse anti-alpha smooth muscle actin (ASMA), the
rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and mouse anti-CD 68 where
from Sigma-Aldrich Corp (St. Louis, MO, USA).
2.2 Immunohistochemistry
Seven formalin-fixed, paraffin embedded blocks used in this study were archived human kidney
tissues blocks held by the Princess Alexandra Hospital Tissue Bank, Brisbane, Queensland,
Australia. Sections were cut and stained with antibodies specific to the five CYP proteins, HO-1,
and MT1/2 as well as ASMA and CD68. The tissue sections were dewaxed in xylene and then
rehydrated with decreasing concentrations of ethanol. Sections stained for the CYPS were treated
with antigen retrieval solution (0.01M Tris/0.05M EDTA buffer pH 8.8) at 105°C for 15 min. Non-
specific binding was blocked with 10% donkey serum/1% bovine serum albumin (BSA) in Tris
buffered saline (TBS), and the tissue slides incubated overnight at room temperature with primary
antibodies CYP4A11, CYP4F2 and CYP3A4 (1:500), or CYP2B6 and CYP2E1 (1:350) in 2.5%
horse serum. Endogenous peroxidase activity was blocked with 1% hydrogen peroxide (H2O2) in
TBS for 10 min. The sections were incubated with Vector Impress horseradish peroxidase (HRP)
conjugated anti-rabbit secondary for 30 min. Colour was developed in 3, 3'-diaminobenzidine
(DAB) with H2O2 as substrate for 5 min. After colour development, the sections were lightly
counterstained in Mayers hematoxylin. For negative controls, the sections were incubated with 10%
donkey serum instead of primary antibody.
2.3 Cell cultures
PTCs were isolated aseptically from the renal cortex of human nephrectomy specimens using
previously published methods [45;46]. Prior to each operative procedure, written informed consent
was obtained from each patient. The study protocol was approved by the Princess Alexandra
Hospital Research Ethics Committee, (12/QPAH/125). Cells were seeded into 25 cm2 tissue-
culture flasks (NuncTM, Roskilde, Denmark), and cultured in serum free, hormonally-defined
DMEM/F12 media (Sigma-Aldrich Corp, St. Louis, MO, USA) as previously described [46]. In
all experiments primary PTC cultures were used when fully confluent at passage two. The
human proximal tubule cell line, HK2, and hepatoblastoma cell line, HepG2 were cultured in
DMEM/F12 media containing 10% foetal bovine serum and antibiotics (100 U/ml penicillin and
7
100µg/ml streptomycin). All cultures were maintained at 37°C under 5% CO2 and 95% air
atmosphere. Medium was replenished every 48h. At confluence cells were serum starved for 24h
in basic DMEM medium (PTC) containing 1% FCS (HepG2 & HK2 cells) before treatment
as indicated in the figures. They were then treated with the indicated concentrations of Cd
for 48h.
2.4 Western blot
Cells were grown to confluence in 6-well plates or 100mm petri dishes before being transferred to
basic culture medium: DMEM/F12, antibiotics and 1% FCS for HK2 and HepG2 cells and
DMEM/F12 and antibiotics alone for PTCs for 24 h. They were then treated with CdCl2 in basic
culture medium for 48 h. Cd dilutions were freshly prepared in basic medium from a 0.5M stock
solution of CdCl2. The final concentrations of CdCl2 were between 0.01-100 µM. Whole cell lysate
samples were prepared using the RIPA (Radio-Immunoprecipitation Assay) buffer containing 1%
of 200 mM phenylmethylsulfonyl fluoride (PMSF) and 2% of proteinase inhibitor cocktail (Sigma-
Aldrich Corp, St. Louis, MO, USA). Cell debris was removed by centrifugation at 13,000×g for 20
min (4°C). The protein concentrations of whole cell lysates were measured using a Pierce BCA
protein assay kit (Thermo Scientific, Rockford, IL, USA). A 40 µg protein aliquot of whole cell
lysate was separated on a NuPAGE 4-12% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) and
transferred to a polyvinylidene fluoride (PVDF) membrane (Pall Corporation, East Hills, NY,
USA) using a NOVEX XCell II blotting apparatus (Invitrogen, Carlsbad, CA, USA). The PVDF
membranes were incubated in 2% skimmed milk in PBS-T (PBS buffer with 0.05% Tween20) for 1
h with shaking. Membranes were then incubated at room temperature for 2 h with primary
antibodies against individual CYP isoforms, mouse anti- MT-1/2 and rabbit anti-glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) diluted 1:6,000 in 2% skimmed milk/PBS-T or at 4°C
overnight with mouse anti-HO-1 diluted 1:1,000. The horseradish peroxidase-conjugated goat anti-
rabbit or mouse IgG (Sigma-Aldrich Corp, St. Louis, MO, USA) at the dilution 1:12,000 was used
as secondary antibody and the membrane was incubated at room temperature with shaking for 1 h.
The blots were incubated for 1 min with Calbiochem RapidStepTM enhanced chemiluminescence
(ECL) reagent (EMD Millipore, Billerica, MA, USA). Immunoreactive proteins were detected by
exposure to x-ray film. The protein band intensity was analysed by Scion Image software (Scion
Corporation, Frederick, MD, USA).
2.5 Cytotoxicity assessment
2.5.1 DNA synthesis
DNA synthesis was assessed by measuring [3H]-thymidine incorporation into newly synthesized
DNA. For this assay, cells were seeded into 48-well plates (2×104 cells/well) and incubated with
incubation period, 8 µCi/ml [methyl-3H]-CdCl2 for 48 h. Six hours before the end of the
8
thymidine was added (Amersham, Arlington Heights, IL, USA). Thereafter, DNA was precipitated
thrice with 10% trichloro acetic acid (TCA) for 10 min and fixed with methanol. After being dried,
the precipitate was dissolved in 0.3 M NaOH/1% SDS at 37ºC. A 50 µl aliquot of the resultant
suspension from each well was transferred to liquid scintillant (Packard BioScience, Meriden, CT,
USA) for counting in beta scintillation counter (WallacMicroBeta TriLux, Perkin-Elmer, Wallac,
Turku, Finland).
2.5.2 Cell integrity
Membrane damage was assessed by measuring the leakage of the cytoplasmic enzyme lactate
dehydrogenase (LDH) in to the growth medium. For this assay, cells were grown in 48-well plates
and exposed to CdCl2 for 48 h. At the end of exposure period, the activity of LDH in the medium
was determined using commercial cell cytotoxicity kit from Roche (Roche, Mannheim, Germany).
The colour intensity development is proportional to the number of cells with damaged cell
membranes. Absorbance was measured at 492 nm using a microplate reader (LabsystemsiEMS
Reader MF, Helsinki, Finland).
2.5.3 Cell viability
Cell viability was determined, using water soluble tetrazolium-1dye (WST-1) (Roche, Mannheim,
Germany). Cells were seeded in 96-well plates (5×103 cells/well). Cells were grown to confluence
and then treated with CdCl2 for 48 h. Ten µ L of WST-1 reagent was added to each well and cells
were incubated for additional 2h. The colour intensity was measured at 450 nm against a
background control (only medium) using a microplate reader (LabsystemsiEMS Reader MF,
Helsinki, Finland). Results are presented as percentage of control (untreated) cells.
2.6 Statistical analysis
All experiments were repeated at least three times with different batches of cells, and results were
presented as mean ± SEM values. Statistical analysis was performed using the SPSS statistical
package (version 12.0). Mann-Whitney and Kruskall-Wallis tests were used to compare sample
groups. P values ≤ 0.05 were considered to represent statistically significant differences.
9
3. Results
3.1 Endogenous expression and localization of CYP enzymes and stress-response proteins in
human kidneys
Figure 1 shows expression levels of various proteins and their localization in kidney cortex of
humans. MT was predominantly localised to the cytoplasm and nuclei of proximal tubular cells (Fig.
1A). No MT staining was seen in the glomeruli, distal tubules, or associated interstitial and vascular
elements. Moderate-to-strong cytoplasmic HO-1 staining was seen within the proximal and distal
tubules of renal tubular epithelial cells (Fig. 1B). No HO-1 staining was seen in nuclei. Of the five
CYP proteins studied, 2B6 (C), 2E1 (D), 3A4 (E), 4A11 (F) showed a weak expression in proximal
and distal tubules, whereas 4F2 (G), showed a strong tubular and glomerular expression.
Representative negative control without primary antibody is shown in Figure 1 J. To assess if there
was significant underlying pathology in the archived kidneys samples, each kidney section was also
stained for alpha smooth muscle actin (ASMA, indicative of fibrosis) and CD68
(macrophage/monocyte specific marker, inductive of inflammation). Panel H is an example of
ASMA staining whereas panel I show CD68 staining. No dramatic extensive fibrosis or
inflammatory infiltrate was observed in any sample.
3.2 MT, HO-1 and CYP protein expression in PTC
Figure 2 shows Western blot results, indicating expression of MT (A) and HO-1 (B) in PTC.
Similar to tissue samples, PTC had a basal level expression of MT. Cadmium, 0.5 to 20 µM,
induced a dose-dependent increase in the expression of MT (A). HO-1, although not detected at
basal level, was induced in response to higher concentrations of cadmium (5 to 20 µM).
Figure 3 shows the expression of CYP3A4 (A), CYP2E1 (B), and CYP4F2 (C) in PTC remained
unchanged 48h after exposure to cadmium (range 0−20 µM). No expression of CYP4A and
CYPB26 was detectable in PTC (data not shown).
Figure 4 shows Western blot results, indicating CYP4F2 proteins were detected in both HK2 and
HepG2 cells under basal conditions. CYP4F2 protein levels in HK2 and HepG2 cells were not
altered by 5µM cadmium. As with PTCs, CYP4A11 protein was not detected in either HK2 or
HepG2 cells (data not shown). MT and HO-1 protein levels in HK2 cells were increased by 5µM
cadmium (Fig. 4A), while MT in HepG2 cells was induced by 5µM cadmium (Fig. 4B).
3.3 Cadmium cytotoxicity profiling
Figure 5 shows cadmium cytotoxicity test results, based on inhibition of DNA synthesis (A),
membrane damage (B) and cell viability (C) in PTCs in comparison with HK2 and HepG2 cells.
Exposure to cadmium (range 0-100 µM) caused differentiatial concentration-dependent inhibition
10
of DNA synthesis among the cell types tested, with HepG2 cells being the most sensitive. DNA
synthesis in HepG2 cells was reduced by 40%, 80% and 100% after exposure to 5, 10, and 50 µM
cadmium, respectively (Fig. 5A). DNA synthesis in PTCs and HK2 cells were unaffected by 5 or 10
µM cadmium, while 50 µM cadmium reduced DNA synthesis by 60% and 80% in PTCs and HK2
cells, respectively. Cell integrity test with LDH release assay gave the results in parallel to the DNA
synthesis (Fig. 5B). Exposure to 5 µM cadmium caused membrane damage in HepG2 cells, while
exposure to 50 µM cadmium caused membrane damage in PTCs and HK2 cells.
HepG2 cells were also found to be the most sensitive in cell viability assay; an approximate of 50
µM cadmium caused HepG2 cells a complete loss of cell viability (cell death), while it caused
PTCs and HK2 cells only a 50% loss of cell viability (Fig. 5C). A cadmium concentration causing
HepG2, PTCs and HK2 cells a 50% reduction in cell viability (termed 48h LC50 dose) was 10.7
µM, 47.6 µM and 52.5 µM, respectively. Cadmium toxicity profiles obtained for HepG2 cells were
in line with literature reports [47-50] and were consistent with markedly lower cadmium
accumulation in liver than kidney [9-11;23].
Discussion
Immunochemical staining of the sections from archived kidneys showed expression of five CYP
isoforms CYP2B6, CYP2E1, CYP3A4, CYP4A11 and CYP4F2 in the proximal and distal tubular
epithelial cells, as was HO-1. MT expression was not detected in distal tubules, but was prominent in
the proximal tubular epithelial cells. Consistent with a previous study, MT was present in both
cytoplasm and nuclei [27]. MT and HO-1 expression levels are known to be induced by a wide
variety of stimuli, including inflammation, oxidative stress and exposure to heavy metals such as
cadmium [1;12;29-31]. Thus the prominent MT expression in the proximal tubular epithelial cells
may relate to any of these factors. We noted varying expression levels of CYP, MT, and HO-1
proteins among the kidney samples examined. One possible source of the variation was the
presence of pre-existing pathology of individual kidneys as evidenced from the markers of
macrophages (Fig. 1I) and activated fibroblasts (myofibroblast) (Fig. 1H). These markers would be
suggestive of kidney disease [51].
Another potential source of variability in kidneys’ expression of CYP, MT and HO-1 proteins may
relate variation in cadmium accumulation. We previously showed differential expression of
CYP4A11 and CYP4F2 that could be related to kidneys’ cadmium accumulation levels [9], where
we examined CYP protein expression in microsomal samples, prepared from 31 Australian kidney
cortex samples with an approximate 32-fold difference in cadmium levels (range 2−63 µg/g wet
kidney cortex weight). Likewise, in a recent Swedish study, a nearly 37-fold difference in kidney
cadmium accumulation (range 1.5-55 µg/g wet weight) was seen among the kidney cortex biopsies
from 109 donors [24]. In the absence of data on cadmium content in the Australian kidneys, used in
the present study, any conclusions regarding cadmium effects on kidneys’ CYP expression could not
11
11
be made.
A few studies have examined CYP expression in primary culture of human PTCs. Two studies
showed expression of CYP3A4 protein in human PTCs [52;53], while another study reported
cadmium induction of CYP2E1 mRNA expression in PTCs [54]. Of interest, we found
expression of MT, CYP3A4 CYP2E1, and CYP4F2 proteins to be well preserved in PTCs. However,
we, did not detect expression of CYP2B6, CYP4A11 or HO-1 in PTCs used in this study. The reasons
for loss of CYP2B6, CYP4A11 and HO-1 remain unclear. CYP4A11 protein expression in PTCs was
lost four days after maintenance in in vitro culture conditions, despite only a modest reduction in this
CYP protein in two days culture period [52]. Endogenous expression of HO-1 and all five CYP
forms were localized to proximal tubules and distal tubules (Fig. 1). In the previous studies [6-8],
CYP4A11 protein was prominently expressed in the S2 and S3 segment of the tubules of human
kidneys and in the ascending loop of Henle, where salt re- uptake from glomerular filtrate back into
the systemic blood circulation. A lack of CYPB6 and CYP4A11 expression in primary culture of
PTCs could thus be expected if they were preferentially expressed in distal tubules. However a small
sample size and CYP genetic heterogeneity meant that relative expression CYPB6 and CYP4A11 in
the proximal vs. the distal tubules could not be determined with certainty.
Cadmium at the highest test level did not cause CYP4F2 induction in PTCs although a positive
correlation was noted between kidneys’ cadmium content and CYP4F2 protein levels in our previous
study of post-mortem specimens [7-9]. Many genetic polymorphisms now have been found in the
regulatory and non-coding regions of the both CYP4A11 and CYP4F2 gene [20-22]. These genetic
determinants may in part underlie the discrepancy observed in our studies that involved different
kidney donors. As expected, however, MT protein expression was up-regulated in PTCs upon
cadmium exposure as it was for the expression of HO-1 protein, despite a lack of HO-1 expression in
the absence of cadmium which acts as a stressor (Fig. 2). The active stress-response mechanism
indicated by the induction of HO-1 protein suggests that a recovery of CYP2B6 and CYP4A11
expression could potentially be achievable with the use of appropriate inducers and culture
conditions. Pro- inflammatory cytokines, notably TNF-α was found to induce CYP1B1 expression
in the WB-F344 rat liver epithelial cells [55]. Further, recovery of CYP1A1 expression was
observed in the primary cultures of rat kidney tubules six days after maintenance as a suspension by
a gyratory culture method and the CYP1A1 activity was maintained for additional twelve days [56].
In this study it was shown that PTCs partially mimick the expression of CYPs seen in kidneys
sections. Our rationale was thus that PTCs could be used as an in vitro model to investigate the
potential involvement of CYPs in drug- and toxicant-induced renal injury. Contrary what was
anticipated, cadmium did not alter the expression status of the CYPs and the upregulation of the
stress-proteins appeared to be independent of the CYPs here examined. Also like primary
hepatocytes which rapidly loose expression of CYP2B6 and CYP4A11 when placed in culture it
appears these enzymes are also lost by primary PTC in culture [52;57]. Thus to establish a role for
12
12
these enszymes in Cd toxicity in the kidney, primary PTC cultures may not be the ideal candidate
cell model.
The ability of cadmium to induce both MT and HO-1 expression in its target cells is known to
modulate cadmium toxicity in humans and experimental animals. The cytoprotective effects of HO-1
including antioxidant, anti-inflammation, and apoptosis resistance, are all attributed to the products,
namely carbon monoxide and biliverdin/bilirubin, derived from heme degradation, catalyzed by HO-
1 [30;31]. The role played by HO-1 in mitigation of oxidative stress has been strengthened further in
a recent study showing a new mechanism, independent of heme degradation products. Such
mechanism involves the interaction between HO-1 (localized to the nucleus) and the nuclear factor
erythroid 2-related factor 2 (Nrf2), leading to increased expression of phase II enzymes and glucose-
6-phosphate dehydrogenase [58]. Further mechanistic study using PTCs is warranted.
Acknowledgements
This study was supported in part by the Royal Jubilee Ph.D. scholarship (KB) and by the Thailand
Reverse Brain Drain Award (SS) from the Commission for Higher Education, Thailand’s Ministry
of Education. SS, KNB, DV and KB conceptualised and designed the research. GG, KB & DV
performed all the experimentation. SS, DV, GG, CM and KB wrote manuscript. DJ contributed
reagents and editorial assistance.
13
13
Reference List
1. Gobe G, Crane D: Mitochondria, reactive oxygen species and cadmium toxicity in the kidney.
Toxicol Lett 2010;198:49-55.
2. Soderland P, Lovekar S, Weiner DE, Brooks DR, Kaufman JS: Chronic kidney disease
associated with environmental toxins and exposures. Adv Chronic Kidney Dis 2010;17:254-
264.
3. Fassett RG, Venuthurupalli SK, Gobe GC, Coombes JS, Cooper MA, Hoy WE: Biomarkers in
chronic kidney disease: a review. Kidney Int 2011;80:806-821.
4. Nakhoul N, Batuman V: Role of proximal tubules in the pathogenesis of kidney disease.
Contrib Nephrol 2011;169:37-50.
5. Waring WS, Moonie A: Earlier recognition of nephrotoxicity using novel biomarkers of acute
kidney injury. Clin Toxicol (Phila) 2011;49:720-728.
6. Lasker JM, Chen WB, Wolf I, Bloswick BP, Wilson PD, Powell PK: Formation of 20-
hydroxyeicosatetraenoic acid, a vasoactive and natriuretic eicosanoid, in human kidney. Role
of Cyp4F2 and Cyp4A11. J Biol Chem 2000;275:4118-4126.
7. Moreno C, Maier KG, Hoagland KM, Yu M, Roman RJ: Abnormal pressure-natriuresis in
hypertension: role of cytochrome P450 metabolites of arachidonic acid. Am J Hypertens
2001;14:90S-97S.
8. Bellamine A, Wang Y, Waterman MR, Gainer JV, III, Dawson EP, Brown NJ, Capdevila JH:
Characterization of the CYP4A11 gene, a second CYP4A gene in humans. Arch Biochem
Biophys 2003;409:221-227.
9. Baker JR, Satarug S, Urbenjapol S, Edwards RJ, Williams DJ, Moore MR, Reilly PE:
Associations between human liver and kidney cadmium content and immunochemically
detected CYP4A11 apoprotein. Biochem Pharmacol 2002;63:693-696.
10. Baker JR, Satarug S, Edwards RJ, Moore MR, Williams DJ, Reilly PE: Potential for early
involvement of CYP isoforms in aspects of human cadmium toxicity. Toxicol Lett
2003;137:85-93.
11. Baker JR, Edwards RJ, Lasker JM, Moore MR, Satarug S: Renal and hepatic accumulation of
cadmium and lead in the expression of CYP4F2 and CYP2E1. Toxicol Lett 2005;159:182-191.
12. Satarug S, Nishijo M, Lasker JM, Edwards RJ, Moore MR: Kidney dysfunction and
hypertension: role for cadmium, p450 and heme oxygenases? Tohoku J Exp Med
2006;208:179-202.
13. Edson KZ, Rettie AE: CYP4 Enzymes as potential drug targets: focus on enzyme multiplicity,
inducers and inhibitors, and therapeutic modulation of 20-hydroxyeicosatetraenoic acid (20-
HETE) synthase and fatty acid omega-hydroxylase activities. Curr Top Med Chem
2013;13:1429-1440.
14. Williams JM, Murphy S, Burke M, Roman RJ: 20-hydroxyeicosatetraeonic acid: a new target
for the treatment of hypertension. J Cardiovasc Pharmacol 2010;56:336-344.
15. Wu CC, Gupta T, Garcia V, Ding Y, Schwartzman ML: 20-HETE and blood pressure
regulation: clinical implications. Cardiol Rev 2014;22:1-12.
16. Imig JD: Epoxyeicosatrienoic acids, 20- hydroxyeicosatetraenoic acid, and renal
14
14
microvascular function. Prostaglandins Other Lipid Mediat 2013;104-105:2-7.
17. Laffer CL, Laniado-Schwartzman M, Wang MH, Nasjletti A, Elijovich F: Differential
regulation of natriuresis by 20-hydroxyeicosatetraenoic Acid in human salt-sensitive versus
salt-resistant hypertension. Circulation 2003;107:574-578.
18. Ward NC, Rivera J, Hodgson J, Puddey IB, Beilin LJ, Falck JR, Croft KD: Urinary 20-
hydroxyeicosatetraenoic acid is associated with endothelial dysfunction in humans. Circulation
2004;110:438-443.
19. Ward NC, Puddey IB, Hodgson JM, Beilin LJ, Croft KD: Urinary 20-hydroxyeicosatetraenoic
acid excretion is associated with oxidative stress in hypertensive subjects. Free Radic Biol
Med 2005;38:1032-1036.
20. Gainer JV, Bellamine A, Dawson EP, Womble KE, Grant SW, Wang Y, Cupples LA, Guo
CY, Demissie S, O'Donnell CJ, Brown NJ, Waterman MR, Capdevila JH: Functional variant
of CYP4A11 20-hydroxyeicosatetraenoic acid synthase is associated with essential
hypertension. Circulation 2005;111:63-69.
21. Laffer CL, Gainer JV, Waterman MR, Capdevila JH, Laniado-Schwartzman M, Nasjletti A,
Brown NJ, Elijovich F: The T8590C polymorphism of CYP4A11 and 20-
hydroxyeicosatetraenoic acid in essential hypertension. Hypertension 2008;51:767-772.
22. Ward NC, Tsai IJ, Barden A, van Bockxmeer FM, Puddey IB, Hodgson JM, Croft KD: A
single nucleotide polymorphism in the CYP4F2 but not CYP4A11 gene is associated with
increased 20-HETE excretion and blood pressure. Hypertension 2008;51:1393-1398.
23. Satarug S, Baker JR, Reilly PE, Moore MR, Williams DJ: Cadmium levels in the lung, liver,
kidney cortex, and urine samples from Australians without occupational exposure to metals.
Arch Environ Health 2002;57:69-77.
24. Wallin M, Sallsten G, Fabricius-Lagging E, Ohrn C, Lundh T, Barregard L: Kidney cadmium
levels and associations with urinary calcium and bone mineral density: a cross-sectional study
in Sweden. Environ Health 2013;12:22.
25. Thevenod F: Nephrotoxicity and the proximal tubule. Insights from cadmium. Nephron
Physiol 2003;93:87-93.
26. Vesey DA: Transport pathways for cadmium in the intestine and kidney proximal tubule:
focus on the interaction with essential metals. Toxicol Lett 2010;198:13-19.
27. Sabolic I, Breljak D, Skarica M, Herak-Kramberger CM: Role of metallothionein in cadmium
traffic and toxicity in kidneys and other mammalian organs. Biometals 2010;23:897-926.
28. Bylander JE, Li SL, Sens MA, Sens DA: Exposure of human proximal tubule cells to
cytotoxic levels of CdCl2 induces the additional expression of metallothionein 1A mRNA.
Toxicol Lett 1995;76:209-217.
29. Garrett SH, Somji S, Todd JH, Sens DA: Exposure of human proximal tubule cells to cd2+,
zn2+, and Cu2+ induces metallothionein protein accumulation but not metallothionein isoform
2 mRNA. Environ Health Perspect 1998;106:587-595.
30. Kensler TW, Wakabayashi N, Biswal S: Cell survival responses to environmental stresses via
the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol 2007;47:89-116.
31. Gozzelino R, Jeney V, Soares MP: Mechanisms of cell protection by heme
15
15
oxygenase-1. Annu Rev Pharmacol Toxicol 2010;50:323-354.
32. Boonprasert K, Ruengweerayut R, Aunpad R, Satarug S, Na-Bangchang K: Expression of
metallothionein isoforms in peripheral blood leukocytes from Thai population residing in
cadmium-contaminated areas. Environ Toxicol Pharmacol 2012;34:935-940.
33. Hwangbo Y, Weaver VM, Tellez-Plaza M, Guallar E, Lee BK, Navas-Acien A: Blood
cadmium and estimated glomerular filtration rate in Korean adults. Environ Health Perspect
2011;119:1800-1805.
34. Ferraro PM, Costanzi S, Naticchia A, Sturniolo A, Gambaro G: Low level exposure to
cadmium increases the risk of chronic kidney disease: analysis of the NHANES 1999-2006.
BMC Public Health 2010;10:304.
35. Ginsberg GL: Cadmium risk assessment in relation to background risk of chronic kidney
disease. J Toxicol Environ Health A 2012;75:374-390.
36. Ferraro PM, Bonello M, Frigo AC, D'Addessi A, Sturniolo A, Gambaro G: Cadmium exposure
and kidney stone formation in the general population--an analysis of the National Health and
Nutrition Examination Survey III data. J Endourol 2011;25:875-880.
37. Hellstrom L, Elinder CG, Dahlberg B, Lundberg M, Jarup L, Persson B, Axelson O: Cadmium
exposure and end-stage renal disease. Am J Kidney Dis 2001;38:1001-1008.
38. Satarug S, Nishijo M, Ujjin P, Vanavanitkun Y, Moore MR: Cadmium-induced nephropathy
in the development of high blood pressure. Toxicol Lett 2005;157:57-68.
39. Eum KD, Lee MS, Paek D: Cadmium in blood and hypertension. Sci Total Environ
2008;407:147-153.
40. Tellez-Plaza M, Navas-Acien A, Crainiceanu CM, Guallar E: Cadmium exposure and
hypertension in the 1999-2004 National Health and Nutrition Examination Survey
(NHANES). Environ Health Perspect 2008;116:51-56.
41. Lee BK, Kim Y: Association of blood cadmium with hypertension in the Korean general
population: analysis of the 2008-2010 Korean National Health and Nutrition Examination
Survey data. Am J Ind Med 2012;55:1060-1067.
42. Edwards RJ, Adams DA, Watts PS, Davies DS, Boobis AR: Development of a comprehensive
panel of antibodies against the major xenobiotic metabolising forms of cytochrome P450 in
humans. Biochem Pharmacol 1998;56:377-387.
43. Jin R, Koop DR, Raucy JL, Lasker JM: Role of human CYP4F2 in hepatic catabolism of the
proinflammatory agent leukotriene B4. Arch Biochem Biophys 1998;359:89-98.
44. Powell PK, Wolf I, Jin R, Lasker JM: Metabolism of arachidonic acid to 20-hydroxy-5,8,11,
14-eicosatetraenoic acid by P450 enzymes in human liver: involvement of CYP4F2 and
CYP4A11. J Pharmacol Exp Ther 1998;285:1327-1336.
45. Vesey DA, Qi W, Chen X, Pollock CA, Johnson DW: Isolation and primary culture of human
proximal tubule cells. Methods Mol Biol 2009;466:19-24.
46. Qi W, Johnson DW, Vesey DA, Pollock CA, Chen X: Isolation, propagation and
characterization of primary tubule cell culture from human kidney. Nephrology (Carlton )
2007;12:155-159.
47. Dehn PF, White CM, Conners DE, Shipkey G, Cumbo TA: Characterization of the human
hepatocellular carcinoma (hepg2) cell line as an in vitro model for cadmium toxicity
16
16
studies. In Vitro Cell Dev Biol Anim 2004;40:172-182.
48. Souza V, Escobar Md MC, Gomez-Quiroz L, Bucio L, Hernandez E, Cossio EC, Gutierrez-
Ruiz MC: Acute cadmium exposure enhances AP-1 DNA binding and induces cytokines
expression and heat shock protein 70 in HepG2 cells. Toxicology 2004;197:213-228.
49. Fotakis G, Cemeli E, Anderson D, Timbrell JA: Cadmium chloride-induced DNA and
lysosomal damage in a hepatoma cell line. Toxicol In Vitro 2005;19:481-489.
50. Fotakis G, Timbrell JA: In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT
and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicol Lett
2006;160:171-177.
51. Meran S, Steadman R: Fibroblasts and myofibroblasts in renal fibrosis. Int J Exp Pathol
2011;92:158-167.
52. Cummings BS, Lasker JM, Lash LH: Expression of glutathione-dependent enzymes and
cytochrome P450s in freshly isolated and primary cultures of proximal tubular cells from
human kidney. J Pharmacol Exp Ther 2000;293:677-685.
53. Lash LH, Putt DA, Cai H: Drug metabolism enzyme expression and activity in primary
cultures of human proximal tubular cells. Toxicology 2008;244:56-65.
54. Garrett SH, Somji S, Sens MA, Zhang K, Sens DA: Microarray analysis of gene expression
patterns in human proximal tubule cells over a short and long time course of cadmium
exposure. J Toxicol Environ Health A 2011;74:24-42.
55. Umannova L, Machala M, Topinka J, Novakova Z, Milcova A, Kozubik A, Vondracek J:
Tumor necrosis factor-alpha potentiates genotoxic effects of benzo[a]pyrene in rat liver
epithelial cells through upregulation of cytochrome P450 1B1 expression. Mutat Res
2008;640:162-169.
56. Xu J, Patton D, Jackson SK, Purcell WM: In-vitro maintenance and functionality of primary
renal tubules and their application in the study of relative renal toxicity of nephrotoxic drugs. J
Pharmacol Toxicol Methods 2013;68:269-274.
57. Vanhaecke T, Rogiers V: Hepatocyte cultures in drug metabolism and toxicological research
and testing. Methods Mol Biol 2006;320:209-227.
58. Biswas C, Shah N, Muthu M, La P, Fernando AP, Sengupta S, Yang G, Dennery PA: Nuclear
heme oxygenase-1 (HO-1) modulates subcellular distribution and activation of Nrf2,
impacting metabolic and anti-oxidant defenses. J Biol Chem 2014;289:26882-26894.
17
17
Figure captions
Figure 1. Endogenous expression of CYPs and stress-response proteins in human kidney
sections
(A) MT1/2, (B) HO-1, (C) CYP2B6, (D) CYP2E1, (E) CYP3A4, (F) CYP4A11, (G) CYP4F2, (H) α-
SMA, (I) CD68 and (J) negative control. Arrows indicate positive CD68 cells. Abbreviations: PT,
proximal tubule; DT, distal tubule; G, glomerulus and V, vessel.
Figure 2. Cadmium-induced up-regulation of the stress-response proteins, MT and HO-1 in
PTC
PTC were grown to defined DMEM/F12 medium until they reached confluence, at which time they
changed to basic DMED/F12 medium (DMEM medium with serum or growth factors). The
abundance of MT1/2 protein (A) and HO-1 protein (B) were normalized to GAPDH and were
expressed as fold change, relative to unexposed (0µM cadmium) control for MT1/2 or relative to 5µM
cadmium-exposed sample for HO-1. Bar heights are mean ± SE values of the fold change, derived
from at least five independent experiments. *P < 0.05, compared with a respective control.
Figure 3. Expression of CYP proteins in PTCs in the absence or presence of cadmium
Abundance of CYP3A4 (A), CYP2E1 (B), and CYP4F2 (C) were normalized to GAPDH and were
expressed as fold change, relative to unexposed (0µM cadmium) control. Liver microsomal sample
(the first lane) served as a positive control for a respective CYP protein. Bar heights are mean ± SE
values of the fold change, derived from at least five independent experiments.
Figure 4. Cadmium-induced up-regulation of stress-response proteins in HK2 and HepG2 cells
Levels of MT, HO-1 and CYP4F2 proteins in HK2 (A) HepG2 cells (B) were normalized to GAPDH.
Bar heights are mean ± SE values of GADPH normalized levels, derived from at least five
independent experiments. *P < 0.05, compared with an unexposed (0µM Cd) control.
18
18
Figure 5. Cytotoxicity profiles of cadmium in PTC, HK2 and HepG2 cells.
(A) DNA synthesis. (B) Lactate dehydrogenase (LDH) leakage. (C) Cell viability. Results are mean ±
SE values from at least three independent experiments, run in triplicate for each cadmium
concentration. *P < 0.05 and **P < 0.001, compared with an unexposed (0µM Cd) control.
Figure 1
Figure 3
µM Cd
A.
GAPDH
CYP3A4
0 0.5 1 5 10 20
B.
C.
CYP2E1
CYP4F2
GAPDH
GAPDH
0 0.5 1 5 10 20
0 0.5 1 5 10 20
µM Cd
µM Cd
0.0
0.5
1.0
1.5
0.5 1 5 10 20CY
P3
A4
pro
tein
ab
und
ance
(Fo
ld c
hnag
e)
µM Cd
0.0
0.5
1.0
1.5
0.5 1 5 10 20CY
P2
E1
pro
tein
ab
und
ance
(Fo
ld c
han
ge)
µM Cd
0.0
0.5
1.0
1.5
0.5 1 5 10 20CY
P4
F2
pro
tein
ab
und
ance
(Fo
ld c
han
ge)
µM Cd
Figure 4
MT1/2
HO-1
CYP4F2
GAPDH
HK2 Cells
µM Cd 0 5 0 5 0 5
A.
B.
MT1/2
CYP4F2
GAPDH
µM Cd 0 5 0 5 0 5
HepG2 cells
0
0.5
1
1.5
CYP4F2 HO-1 MT-1/2 G
AD
PH
no
rmal
ized
lev
els
Control Cd 5 µM
*
*
0
1
2
3
CYP4F2 MT-1/2
GA
PD
H n
orm
aliz
ed l
evel
s
Control Cd 5 µM
*
19
19
Fig. 5