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

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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.

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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.

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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

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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.

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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

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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

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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

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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.

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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

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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

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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

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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.

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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.

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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