Effect of cadmium on active ion transport and cytotoxicity in cultured renal epithelial cells (A6)

Toxicology

in Vitro 11 (1997) 717-722

Effect of Cadmium on Active Ion Transport and Cytotoxicity in Cultured Renal Epithelial Cells (A6) B. FAURSKOV* Department

of Life Sciences

and H. F. BJERREGAARD

and Chemistry,

Roskilde Denmark

University,

PO Box 260, 4000 Roskilde,

Abstract-A cultured epithelial cell line from toad kidney (A6) was used to study the mechanism by which cadmium (Cd) affects transepithelial resistance (TER) and active transepithelial ion transport measured as short-circuit current (SCC) in oi~ro. The influence of Cd on cell integrity was investigated by measuring time-dependent TER under controlled conditions and the half-maximal inhibition concentration (IC,,) 24 hr after exposure to 1 mM CdCl,. The data suggest that Cd deterioration of cell integrity is stronger when applied to the apical relative to the basolateral solution (IC,, = 173.9 and 147.8 PM, respectively). Also, the data demonstrate that addition of Cd to the basolateral solution results in a prompt and transient stimulation of the active ion transport from 2.6 f 0.4 to 8.7 + 1.1 PA/cm’. Use of the sodium channel blocker amiloride indicate that Na transport is not involved in Cd-stimulated SCC. Substitution of Cl with S04’-- in the basolateral solution and use of the Cl channel inhibitors, diphenylamine-2-carboxylase (DPC) and niflumic acid indicate strongly that Cd increases Cl secretion in A6 epithelium. Thapsigargin (TG), an intracellular Ca-ATPase blocker, inhibits Cd-stimulated active ion transport indicating that Ca-activated Cl channels are probably involved. Therefore, we suggest that Cd by interaction with the basolateral membrane, become internalized and increase Ca intracellularly. In a dose- and time-dependent way an increase in Ca activates specific Cl channels leading to an increased SCC. Thereafter, the increase in Ca leads to disruption of tight junctions thereby decreasing TER. This may lead to deterioration of cell integrity and perhaps even cell death. Q 1997 Published by Elseuier Science Ltd Abbreviations: concentration;

DPC = diphenylamine-2-carboxylase; Fur = furosemide; IC,, = half maximal inhibition SCC = short-circuit current; TER = transepithelial resistance; TG = thapsigargin.

Keywords: cadmium: active sodium-chloride (A6); cell integrity; in oirro toxicity.

transport:

INTRODUCTION

Cadmium is an important industrial and environmental pollutant that causes severe damage to a variety of organs, especially the liver and kidney (Friberg, 1986; Herber, 1994; Nordberg, 1984). Numerous studies have documented that the major site of Cd toxicity is the proximal tubule of the kidney (for review see Friberg, 1986) although the distal part of the kidney also accumulates Cd, but to a lesser degree than the proximal part (Girolami, 1989; Tohyama, 1988; Zhang, 1995). The mechanisms underlying Cd-induced nephrotoxicity are largely unknown. However, many overt toxic effects of Cd in uiuo seem to involve an increase in the permeability of various endothelial and epithelial surfaces (Nolan, 1986; Prozialeck, 1991). To gain further knowledge of the mechanism by which Cd acts on active transepithelial ion transport as well as to evaluate cell toxicity towards Cd, we examined the effects of basolateral Cd exposure on *Author for correspondence. 0887-2333/97/$17.00 + 0.00 0 SSDI 0887-2333(97)00045-3

1997 Published

transepithelial

resistance;

renal epithelial

cells

cells of the established renal epithelial cell line A6 originating from the toad Xenopus laevis. The toxicological effects of Cd have previously been examined in leaky proximal cell lines such as LLC-PK, (Bruggerman, 1990; Prozialeck, 1991, 1993). However, leaky cell lines are not suitable for measuring TER and transepithelial ion transport because of massive paracellular routes. The A6 cell line exhibits morphological and functional properties of the distal tubular epithelium (Perkins, 1981) and has been used as a model system for physiological and toxicological studies because of its tight epithelial nature (Bjerregaard, 1993 and 1995). When grown under optimal conditions A6 cells form a tight epithelia with a large transepithelial resistance (TER) excluding any paracellular transport that otherwise could complicate the interpretation of the attained results. Thus, any modification in measured active transport is due to intracellular effects only. Furthermore, A6 cells are sensitive to aldosterone and antidiuretic hormone and exhibit sodium and chloride transport similar to that observed in oivo

by Elsevier Science

Ltd. All rights

reserved.

Printed

in Great

Britain

718

B. Faurskov and H. F. Bjerregaard

(Bjerregaard, 1992; Chalfant, 1993; Perkins, 1981; Sariban-Sohraby, 1983). Therefore, A6 are suitable cells for the investigation of transepithelial transport using well-characterized inhibitors or stimulators of ion transport. In addition, the large TER provides a good model for determination of cell integrity since low levels of TER may indicate that tight junctions of the epithelial are leaky or that the cell membranes are being destroyed.

MATERIALS AND METHODS

Cell line and culture methods

dent potential drop between the potential-measuring calomel electrodes (saturated electrodes with agar bridges). To detect TER, the monolayer was clamped for 3 set at - IO mV at certain intervals. The clamping current was passed through Ag-AgCl electrodes connected to the solution via agar bridges. In experiments where effects on SCC were investigated, the snap wells were mounted in modified Ussing chambers, and bathed from both sides in aerated NaCl-Ringer solution containing I I5 mM Na+. 2.5 mM K’, 117 mM Cl’ 1 mM Cal+, 2.5 mM CO: , 5 mM glucose, pH 7.8.’ In Cl depletion, the chamber on the basolateral side contained I10 tIIM Na’, 2 IrIM K+, 1 mM Ca2+, 57 mM SOa’-, 73 mM sucrose, pH 7.8. The ion transport capacities were improved by preincubation of the epithelia with 1 pM aldosterone for 24 hr (Bjerregaard, 1992). Applications of the test drugs were performed within 40 min after Cd exposure (see results). The increase in SCC (-SCC) was calculated as the difference in SCC before addition of the agent and the steady or maximal SCC level reached after addition of the agent. The results were expressed as means f SE, and were analysed statistically using F-test to evaluate for differences in variance and then further by using Student t-test. P-values of less than 5% were considered to be significant.

A6 cells were purchased from the American Type Culture Collection (Rockville, MD, USA), at serial passage 67. The epithelial cell line was initially established from the kidney of the aquatic south African clawed toads Xenopus laez!is (Rafferty, 1969). A6 cells were cultured in plastic culture flasks (25 cm’, Costar, Northumbria Biologicals, Cramlington, Northumbria, UK) with 10 ml growth medium. The cells were grown at 26’C in a humidified atmosphere of 5% CO, in air. Dulbecco’s modified Eagle’s medium (cat. no. 041-01885; Gibco, Grand Island, NY, USA) adjusted with 20% distilled water to an osmolarity appropriate for amphibian cells (Perkins, 1981; Sariban-Sohraby, 1983) and supplemented with 100 IU/ml penicillin, 100 pg/ml streptomycin, and 10% foetal bovine serum (Gibco) was used as a growth medium. Cells were seeded at a density of S-10 x 10J cells/cm’ in culture flasks. Twice a week the growth medium was replaced with fresh medium. When cells became confluent and exhibited dome formation (5-10 days), they were subcultured by incubation in 2 ml Ca’+ -Mg’+-free amphibian salt solution with 0.25% trypsin containing I10 mM NaCI, 2.5 tnM NaHCO,, 3 mM KCl, I mM KH,PO,, I mM EGTA and 5 mM glucose (pH 7.6). On detachment from the culture flask (3-5 min), the subculture medium was decanted. After 5IO min, the action of trypsin was inhibited by adding 5 ml culture medium. The cells were resuspended and added to either culture flasks or six wells-multi disks with filter inserts (Nunc, Denmark). All experiments were conducted with cells at passages 70-80.

The integrity of cell-cell junctions was assessed by monitoring TER. Effect of Cd on short-term basiswithin 2 hr-was estimated by successive TER measurements. IC,, values (inhibiting concentration) for Cd were determined by increasing concentrations added to serum-free medium on either the basolateral or apical sides of 24 monolayer. The cells were not preincubated in aldosterone as for electrophysiological measurements. After 24 and 48 hr, TER was measured using an epithel-volt-ohmmeter (MillicellERS). The results were calculated as ohms x cm* of cell growing surfaces, after subtracting the values for the resistance measured in cell-free filter inserts. The results were expressed as percentages of the control values (no Cd exposure). IC,, values were calculated by linear regression as the concentration causing 50% inhibition of TER. The I&, tests were obtained from cells of the same passage.

Electrophysiological

Chemicals

measurements

To study active transepithelial ion transport and effects of various test drugs, cells were grown on filter inserts in snap wells without any extracellular matrix (Costar; 0.4 pm pore size, I cm’). Experiments were performed 8 days after inoculation. Potential difference and active ion transport measured as SCC across the monolayer were monitored according to the technique of Ussing and Zerah (Ussing, 1953) using a multichannel voltage/current clamp amplifier (WPI EVC-4000) that compensates for the current-depen-

Functional studies of rhe epithelial

Blockers: amiloride, furosemide, niflumic acid and DPC. Activators: amphibian ADH and TG. Amiloride (A-7410), furosemide (F-4381), niflumic acid (N-0630), thapsigargin (T-9033), CdC12 (C-2544) and amphibian ADH (AVT, arginin vasotocin, V-0130) were purchased from Sigma (USA), and DPC (I4 450-9) was purchased from Aldrich (Germany). Niflumic acid and DPC were dissolved in dimethyl sulfoxide. All drugs were of the highest purity obtainable.

Effect of cadmium in transepithelial resistance and ion transport RESULTS AND DISCUSSION

The cell toxicity towards Cd is determined by TER and IC,, measurements. Furthermore measurements of active ion transport were conducted by application of various well-characterized inhibitors and stimulators of ion transport to either the basolateral or apical solution of polarized A6 epithelial. The influence of Cd on cell integrity was estimated by measuring time dependent TER under controlled conditions and the half-maximal inhibition concentration (IQ. Successive determinations of TER in cells exposed to 1 tYIMCdCl, indicated that the time from Cd addition to the time when TER started to decrease dramatically was 39.8 + 5.7 min. In addition, the results demonstrated that TER almost reached 0 a in 109.2 + 29.3 min at the applied concentration (n = 5 in both cases). Therefore, to ensure that cell integrity during SCC experiments was not influenced by Cd, all experiments were terminated within 40 min after addition of CdCl*. If, however, TER was used as an endpoint, apical and basolateral additions of CdCl, for 24 hr resulted in I&, values of 173.9 and 147.8 FM, respectively (Fig. 1). A further 24-hr incubation period increased the I&, value for apical CdCI, addition to 247.0 PM, whereas the I&, value for basolateral exposure were unchanged. Within 24 hr of 200 PM CdC& exposure TER decreased dramatically to 1.2 and 32.9% for basolateral and apical solution, respectively. Above 300 PM the basolateral TER dropped further to 0% and the apical curve levelled off and never decreased below 20.3% at concentrations as high as 1000 pM. The results show that the effect of Cd on cell integrity is partly side-specific and far more pronounced at the

1

719

basolateral side (Fig. 1). Similar results have been observed in the other epithelial cell lines such as Caco-2 (Rossi, 1996) and LLC-PK, (Bruggerman, 1992), but with other endpoints than TER. However, the presented experiment did not reveal whether the Cd induced decrease in TER is caused by modulation of tight junctions or whether the decrease in TER is a direct cause of cytotoxic action of Cd on the apical cell membrane. Further studies using measurements of tight junctional resistance (Jovov, 1994) or determination of the released lactate dehydrogenase from the intracellular environment (Mitchell, 1991) are neccesary to address this question. The heterogeneous levels of basolateral or apical TER suggest different mechanisms by which Cd may interact with the cell membrane leading to time-dependent lowering of TER. Recent studies in other epithelial cell types have shown that basolateral uptake of ‘OPCd was higher than apical uptake and that Cd reached the intracellular compartment in its toxic, ionic form. Consequently, the cytotoxic action of Cd may be restricted to intracellular effects (Bruggerman, 1990 and 1992; Prozialeck, 1993). Furthermore, Cd uptake could evidently be described as a kinetic model of second order with an initial rapid internalization followed by a slow intracellular distribution (Foulkes, 1988; Templeton, 1990). Our results support the idea that Cd deterioration of cell integrity is stronger when applied to the basolateral than to the apical solution. As will be discussed below, it is possible that Cd increases Ca intracellularly when applied on the basolateral side, whereas Cd applied on the apical side does not promote an increase in intracellular Ca of the same order of magnitude. High intracellular Ca is known to destabilize tight

7

0

loo

200

300

400 Cd

500

600

700

800

Qoo

concentration (FM)

Fig. 1. Effect of apical and basolateral Cd exposure on transepithelial resistance. CdCI? was applied basolaterally or apically. A6 cells were exposed for 24 hr at the concentrations shown. Values are means f SE of two experiments.

1000

720

B. Faurskov 10

T

6 --

%4

and H. F. Bjerregaard

5-L

/Amiloride:

_

1

4 --

10

20

30

40

50

60

70

Time (min) Fig. 2. EtTect of amiloride on Cd-stimulated short-circuit current. Time courses of a typical experiment. A6 cells were preincubated for 24 hr in aldosterone (200 pM). Arrows indicate when 100 pM amiloride followed by I mM CdCl? was added to the basolateral and apical solution of exposed cells, respectively. Base line of control = - 3.7 /IA:cm’has been adjusted for all values. XC = 5.3 + 0.5 (n = 2) and 6.1 + 0.9 (n = 15) /tA’cm’ for amiloride exposed and control epithelial, respectively.

junctions leading to deterioration ofcell integrity and sometimes cell death (Jovov, 1994; Orrenius. 1989: Prozialeck, 1991). To examine the sensitivity of active ion transport to Cd an accumulated dose-response was performed by adding, in 5-min intervals. increasing concentrations of CdCIZ to the basolateral solution and measuring SCC. The results show that below 100 ELM CdCI, A6 cells respond marginally (data not presented). Above 100 FM A6 cells show a dosedependent increase in accumulated SCC from 2.5 k 0.1 to 8.7 5 0.7 PA/cm’ in 1000 ,UM CdCI, exposed cells. The half maximal stimulation concentration determined as mean of two experiments was 563 PM. A test concentration of I mM CdCI, was selected to study basolateral effects of Cd on active ion transport. Addition of I mM CdCl? to the basolateral solution increased SCC markedly within minutes after administration from 2.6 + 0.4 to 8.7 + I.1 ,rtA:‘cm’ (n = IS, P < 0.01). The activation was sustained for approximately 5 min before returning to base level (Figs 2 and 3). The data were pooled from two different cell preparations obtained between passages 70 and 80. To investigate the ionic nature behind the increase in SCC, inhibitors of Na and Cl transport were used. When 100 PM of the sodium channel blocker amiloride (Garty, 1988) was added to the apical solution, an immediate inhibition of SCC was observed. However, the characteristic SCC-peak observed after I mM CdCI, addition was still evident and undiminished (Fig. 2). Also, when amiloride

followed by furosemide (see below) were added to the solutions, the Cd-induced SCC stimulation compared with controls were 43.2 &- 5.7% whereas the corresponding value for furosemide exposure alone was 42.4 + 8.0%. This result indirectly shows that Na is not involved in Cd-stimulated SCC. Therefore, the results suggest that sodium is not involved in the observed Cd mediated activation of active transport. To test whether chloride was involved in Cd activated SCC, distinct blockers of chloride channels were used. In addition. the cells were depleted for chloride by using solutions where Cl was replaced by SOJ’ (see methods). The results are presented in Table I. Furosemide is known as a specific inhibitor of basolateral Na/CI co-transport (Kaplan, 1996) whereas the fairly specific inhibitor DPC acts on apical chloride channels (Chang, 1993; Stutts, 1990). As expected, both drugs reduced base level of SCC when applied to the solutions (data not shown). Table

I.

Etfects of Cl channels inhibitors and chloride depletion Cd-induced SCC

Chemicals Fur-Cd DPC-Cd Fur-DPC-Cd DPC-Fur-Cd Cl detAetion-Cd

% of control 81.4 92.9 16.6 62.5 6.1

f f f + +

30.1 (6) 20.2 (7) 7.5 (5) 14.4 (4) 2.6 (2)

on

P NS NS
NS = not significant *Based on results from the same cell preparation. The values are expressed as percentage of maximal Cd peak compared with control level. The data were pooled from two different cell batches. between cell passages 7C-80. Fur (0.5 IIIM. basolateral), DPC (350 PM. apical). Cl - free medium were added to the basolateral solution.

Effect of cadmium in transepithelial resistance and ion transport

721

20 -

0

10

20

30

40

50

60

70

80

90

100

Time (mln) Fig. 3. Effect of TG on Cd-stimulated short-circuit current (n = 2). Time courses of a typical experiment. A6 cells were preincubated in aldosterone (200 PM).Arrows indicate when 400 nMTG followed by I mM CdCI, was added to the basolateral at the solution. Base line of control = + 1.3 PA/cm’ has been adjusted for all values. TG-exposed cells; control cells.

However, pooled data from different cell preparations indicate that neither furosemide nor DPC reduced Cd-induced SCC significantly as the level of maximal Cd-stimulated SCC compared with control was 81.4 + 30.1% and 92.9 +_20.2%, respectively. However, if furosemide was applied before DPC, the Cd-induced SCC was reduced significantly (P < 0.001) to 16.6 + 7.5%. Interestingly, this phenomenon was not observed when the test drugs were added in reverse order, that is, application of DPC and then furosemide since the change in Cdstimulated SCC was not significant (62.5 &- 14.4%). A possible explanation could be that inhibition of Na/CI co-transport mechanisms by furosemide results in a blockage of intracellular Cl loading, whereas inhibition of apical Cl channels by DPC does not restrict loading of Cl. Therefore, the loading process of Cl takes place despite DPC addition. This could also explain why exposure to DPC followed by exposure to furosemide does not reduce Cd-induced SCC. However, in this situation, it can be expected that Cd-stimulated SCC is transient since the blockage of co-transport by furosemide means that Cl is being ‘trapped’ intracellularly. Further studies are clearly necessary to elucidate this aspect. Although the use of DPC as inhibitor of chloride channels does not indicate any involvement of Cl in Cd-induced SCC, it is still possible that Cl channels are involved because of the lack of specific blockers of Cl channels (Guggino, 1994). It seems likely that secretion of Cl is an important feature of Cd-stimulated SCC since Cl-depleted cells

demonstrate a significant (P -c0.02) reduction of SCC to 6.7 +_2.6% compared with control level (Table 1). Some authors report that Cl transport is affected by Cd, but these investigations were performed on cell lines with properties differing from the one presently investigated (Bohme, 1992; Lefevre, 1996). Among chloride channels two types of secretory Cl channels have been described, namely Caand CAMP-activated channels (Guggino, 1994; Rasmussen, 1990; Simmons, 1990). The instant Cd-induced increase of SCC indicates that calcium could be a possible second messenger because Ca-activated mechanisms normally respond within 0.5-l min, whereas CAMP activation usually takes place after 5-10 min (Rasmussen, 1990). To study whether Ca was involved in Cd-induced stimulation of Cl secretion, the well-characterized inhibitor TG was used (Fig. 3). TG is highly lipophilic and easily penetrates biomembranes. Intracellularly, TG acts as a potent inhibitor of Ca-ATPases in endoplasmatic and sarcoplasmatic reticulum. Many studies have shown a marked TG-mediated increase in intracellular Ca concentration in different tissues (Inesi, 1994; Thastrup, 1989) and in tight epithelia (Brodin, 1994). In the present study, addition of TG (400 nM>to the basolateral solution resulted in a nearly complete blockage of the Cd-induced SCC. In the reverse situation, in which TG was added before Cd, the characteristic response to TG was absent (Fig. 3). These results indicate that Ca could be an important second messenger in Cd-induced SCC, probably by

722

B. Faurskov and H. F. Bjerregaard

Ca-mediated stimulation of apical chloride channels. Also, preliminary studies with niflumic acid, a Cl-dependent inhibitor of apical chloride channels (White, 1990), indicate that Ca could be involved in Cd signal transduction because niflumic acid reduces Cd-stimulated SCC. In conclusion, the results presented show that the effect of CdCI, on TER depends on the side and time of exposure. In addition. the study shows that basolateral Cd administration on short-term basis (up to 40 min) does not affect the integrity of the epithelial cell because TER is unaffected. Use of distinct blockers and stimulators of ion transport indicates that Na transport is not involved in Cd-stimulated Ca-activated

SCC.

whereas

Cl channels.

Cl channels,

are involved.

probably

Therefore,

we

Cd in a dose-dependent way increases intracellular Ca that in the short term activates Cl-secreting channels leading to an increased SCC. In the long term, Cd-mediated high intracellular Ca provides disruption of tight junctions. thereby decreasing TER and eventually leads to cell death evoked by apoptosis or necrosis. suggest

that

REFERENCES

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in Clinical and Analytical Chemistry. Edited by H. G. Seiler, A. Sigel and H. Sigel. pp. 283-297. Marcel Dekker, New York. Inesi G. (1994) Specific inhibitors of intracellular Ca’+ transport ATPases. Journal of Membrane Biology l-6. Jovov B. (I 994) Role of intracellular Ca’+ in modulation of tight junction resistance in A6 cells. Renal FIuid Elwtrolyte Physiologys 35, F775-F784. Kaplan M. R. (1996) Molecular mechanisms of NaCl cotransport. (Abstract) Annual Review qf Physiology 58, 649-668. Lefevre T. (1996) Effects of chloride ion substitutes and chloride channel blockers on the transient outward current in rat ventricular myocytes (Abstract). Bba Biornergetics 1273, 31-43. Mitchell D. (1991) Evaluation of cytotoxicity in cultured cells by enzyme leakage. Journal of Tissue Culture Methods 6, I 13-l 16. Nolan C. (1986) The vascular endothelium as a target tissues in acute cadmium toxicity. L[/ti Sciences 39, 1403-1409. Nordberg G. F. (1984) Chelating agents and cadmium toxicity: problems and propects. Environmental Health Perspectives 54, 2 13-2 18. Orrenius S. (1989) Role of Ca’+ in toxic cell killing. Trend.7 in Pharmacological Science 10, 28 l-285. Perkins F. (1981) Transport properties of toad kidney epithelia in culture. American Journal of Ph_vsiology 41, Cl54-c159. Prozialeck W. (1991) Cadmium (Cd’ + ) disrupts intercellular junctions and actin filaments in LLC-PK, cells. To.uicolog~ and Applied Pharmacolog>j 107, 8 l-97. Prozialeck W. (1993) Surface binding and uptake of cadmium (Cd’+ ) by LLC-PK, cells on permeable membrane supports. k&ices of Toxicology 67; 113-l 19. Rafferty K. (1969) Mass Culture of Amphibian Cells; Methods and Observations Concerning Stability of Cell rvpe. pp. 52-81. Springer Verlag, New York. Rasmussen H. (1990) Interactions between Ca’+ and CAMP messenger system in regulation of airway smooth muscle contraction. Lung Cell Molecular Physiology 258, L279pL288. Rossi A. (1996) Heavy metal toxicity following apical and basolateral exposure in the human intestinal cell line taco-2. Toxicology in Vitro 10, 27-36. Sariban-Sohraby S. (1983) Apical sodium uptake in toad kidney epithelial cell line A6. American Journal of Physiology 245, C 167-C 17 I. Simmons N. L. (1993) Renal epithelial Cl- secretion. E?cperimental Physiology 78, I 17-l 37. Stutts M. J. (1990) Diphenylamine-2-carboxylase (DPC) inhibits both Cl conductance and cyclooxygenase of canine tracheal epithelium. European Journal of Physiology 61 I-616. Templeton D. (1990) Cadmium uptake by cells of renal origin. Journal of Biological Chemistry 21, 21764-21770. Thastrup 0. (1989) Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage. Agents and Actions 27, 17-23. Tohyama C. (1988) Immunohistochemical localization of metallothionein in the liver and kidney of cadmium- or zinc-treated rats. Acta Histochemica et Cytochemica 21, 91-102. Ussing H. H. (1953) Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiologica Scandinavica I lO--127. White M. M. (1990) Niflimic and flufenamic acids are potent reversible blockers of Ca’+ -activated Cl-channels in Xenopus oocytes. Molecular Pharmacology 37, 72& 724. Zhang B. (1995) Difference in cadmium cytotoxicity in two kidney cell lines (LLC-PKI and MDCK) with special reference to metallothionein. Toxicology in Virro 9, 765.