hydrogen peroxide activates ion currents in rat mesangial cells

Kidney International, Vol. 56 (1999), pp. 181–189

Hydrogen peroxide activates ion currents in rat mesangial cells

JOACHIM GLOY, KARL-GEORG FISCHER, TOBIAS N. MEYER, PETER SCHOLLMEYER,RAINER GREGER, and HERMANN PAVENSTADT

Department of Medicine, Division of Nephrology, and Department of Physiology, Albert-Ludwigs-University, Freiburg, Germany

Hydrogen peroxide activates ion currents in rat mesangial cells. source for ROS, and particularly mesangial cells canBackground. Hydrogen peroxide (H2O2) is an important produce ROS independently from polymorphonuclear

mediator of glomerular injury, which induces proliferation and leukocytes [4]. It has also been assumed that the genera-cell contraction in mesangial cells. The aim of this study wastion of ROS by mesangial cells might not only directlyto investigate whether and which ion currents are activatedmediate toxic cellular effects but also amplify glomerularduring the early cellular responses to H2O2, and to study possi-

ble mechanisms of their activation. injury by the induction of Cox-2 gene expression in mes-Methods. The effect of H2O2 on membrane voltage of mesangial cells [5]. The reactive oxygen product hydrogen

angial cells in short-term culture was investigated with the peroxide (H2O2) is known to be a mediator of cellularpatch clamp technique in the fast whole cell configuration.injury. It acts either directly or serves as a source ofResults. H2O2 contracted mesangial cells and induced a con-

centration-dependent biphasic membrane voltage response. hypochloride, which is formed in the presence of chlorideOne hundred mmol/liter H2O2 led to a hyperpolarization of and of myeloperoxidase, an enzyme secreted by poly-mesangial cells from 245 6 1 to 255 6 1 mV, which was morphonuclear leukocytes [6]. The glomerular toxicityfollowed by a sustained depolarization to 220 6 3 mV. The

of H2O2 has been directly demonstrated in studies,hyperpolarization induced by H2O2 was completely blockedwherein intra-arterial infusion of H2O2 caused derange-by the K1 channel blocker Ba21. In the presence of a low

extracellular Cl2 concentration (32 mmol/liter), the depolariza- ments in glomerular permselectivity [7]. On the cellulartion induced by H2O2 was significantly increased. The H2O2- level, H2O2 is known to modulate different functions ininduced depolarization was inhibited by 100 mmol/liter of the mesangial cells, such as stimulation of the intracellulardisulfide-reducing agent dithiothreitol, whereas higher concen-

calcium activity ([Ca21]i), proliferation, and cell contrac-trations of dithiothreitol (1 mmol/liter) were required to par-tially inhibit the hyperpolarization. Protein kinase C inhibitors tion [8, 9]. Although the role for the contractility ofblocked the H2O2-induced depolarization, but not the hyperpo- mesangial cells in the regulation of the glomerular filtra-larization. tion rate (GFR) has been debated [10], H2O2-induced

Conclusions. The data indicate that H2O2 leads to a biphasiccontraction of mesangial cells might participate in themembrane voltage response in mesangial cells: an initial tran-H2O2-mediated reduction of the glomerular ultrafiltra-sient hyperpolarization, which is due to the activation of a K1

conductance, and a subsequent depolarization, which is, at tion coefficient. An activation of a Ca21-dependent Cl2

least in part, due to the activation of a Cl2 conductance. The conductance leading to cellular depolarization has beenoxidation of thiol groups by H2O2 is involved in the membrane implicated in initiating hormone-induced mesangial con-voltage response, and the depolarization may be regulated by

traction [11], but nothing is known about the regulationprotein kinase C.of ion channels by H2O2 in mesangial cells. The aimof this study was to investigate whether and which ioncurrents are activated during the early cellular responsesReactive oxygen species (ROS) are important media-to H2O2, and to examine the mechanisms of their activa-tors in pathological processes of a variety of glomerulartion. Changes of ion conductances are involved in manydiseases. For example, ROS have been considered to

be involved in neutrophil-dependent and -independent cellular events, such as contraction and proliferation, andglomerular injury, membranous glomerulopathy, and therefore, the regulation of H2O2-activated ion currentsminimal change disease [1–3]. Glomerular cells are a increases the knowledge about early cellular effects of

H2O2 in mesangial cells.Key words: membrane voltage, protein kinase C, dithiothreitol, dia-mide, glomerular injury.

METHODSReceived for publication March 3, 1998 Cell cultureand in revised form February 9, 1999Accepted for publication February 12, 1999 Mesangial cells were isolated and cultured as pre-

viously described [12]. In brief, rat glomeruli were ob- 1999 by the International Society of Nephrology

181

Gloy et al: H2O2 and ion currents in mesangial cells182

tained by the sieve technique and were incubated with in the voltage clamp mode (Vc) to Vm. Starting at thiscollagenase (1 g/liter; Sigma, Deisenhofen, Germany) value, the whole cell current (I) was measured by depo-for 15 minutes. Glomeruli were then suspended in RPMI larizing or hyperpolarizing Vc in steps of 10 mV to 6401640 medium supplemented with 170 g/liter fetal calf mV. Gm was calculated from the measured I, Ga, and Vc

serum (FCS), l-glutamine 2.5 mmol/liter, sodium pyr- using Kirchhoff’s and Ohm’s laws. Membrane conduc-uvate 0.1 mmol/liter, penicillin 105 U/liter, streptomycin tance (Gm) and the effect of H2O2 (0.1 mmol/liter) were100 mg/liter, nonessential amino acids 0.2 g/liter (all only examined if Ga was more than 50 nS (N 5 15).Seromed, Berlin, Germany), and an insulin-transferrin-

Mesangial cell contractionsodium selenite supplement 5 mg/liter (Boehringer-Mann-heim, Mannheim, Germany). Approximately 50 glome- Mesangial cells grown on collagen IV-coated glass

plates were mounted in a bath chamber on the table ofruli/cm2 were plated onto collagen-coated glass coverslips (Greiner, Nurtingen, Germany) and incubated at the inverted microscope and were continuously perfused

with a 378C Ringer-like solution. A camera (Contax,378C in an incubator with a water-saturated atmosphereof 5% CO2/95% air. Mesangial cells were morphologi- Zeiss, Germany) was connected to the microscope,

allowing the study of mesangial cells by interferencecally characterized by phase-contrast microscopy. Theystained positively for smooth muscle actin, desmin, and and phase contrast optics. While taking pictures, the

continuous bath flow was stopped for 10 seconds, andvimentin. Cytokeratin and factor VIII did not stain, thusexcluding the presence of glomerular epithelial and en- cells were photographed 0, 2, 4, 6, and 20 minutes after

the addition of 100 mmol/liter H2O2.dothelial cells. Mesangial cells contract in response to1027 mol/liter angiotensin II [11]. Most of the experi-

Chemicalsments were performed between the 13th and the 26thThe following agents were used: H2O2 (300 g/liter stockday of primary culture. Additional experiments were

solution), dimethyl sulfoxide (DMSO; Merck, Darmstadt,performed with subcultured mesangial cells (up to pas-Germany), DL-dithiothreitol (DTT), staurosporine, dia-sage 3).zenedicarboxylic acid bis 5N,N-dimethylamide (dia-

Patch clamp experiments mide), pyruvate (Sigma), bisindolylmaleimide I (BIM;Calbiochem, San Diego, CA, USA).The patch clamp method used in these experiments

has been described in detail [13, 14]. In brief, mesangialStatisticscells were mounted in a bath chamber on the stage of

The data are given as mean values 6 sem. N refers toan inverted microscope, were kept at 378C, and werethe number of experiments. A paired t-test was used tosuperperfused with a phosphate-buffered Ringer-like so-compare mean values within one experimental series. Alution. To vary free Ca21 concentration, the solutionsP value of less than 0.05 was accepted to indicate statisti-were prepared according to established techniques, withcal significance.egtazic acid (EGTA) and nitrilotriacetic acid (NTA) as

Ca21 buffers. The intracellular free Ca21 concentrationwas calculated from a standard equation and was deter- RESULTSmined with a Ca21 selective electrode (Radiometer,

H2O2 contracts mesangial cells and leads to a biphasicCopenhagen, Denmark). In ion replacement studies ex-membrane voltage responsetracellular Na1 or Cl2 were replaced by N-methyl-d-

In 138 experiments in the fast whole cell configuration,glucamine (NMDG1) or gluconate, respectively. Thea stable resting Vm of 245 6 1 mV was obtained inpatch pipettes were filled with a solution containing themesangial cells in primary culture. Figure 1 shows origi-following (in mmol/liter): K-gluconate 95, KCl 30,nal recordings of the effect of a long- (Fig. 1A) and short-Na2HPO4 4.8, NaH2PO4 1.2, CaCl2 0.73, MgCl2 1.03,term (Fig. 1B) application of H2O2 on the Vm of mesangialEGTA 1, d-glucose 5, pH 7.2; the intracellular free Ca21

cells. The addition of 100 mmol/liter H2O2 led to an imme-concentration was 1027 mol/liter. The patch pipettes haddiate and significant hyperpolarization of mesangial cellsan input resistance of 2 to 5 MV. A flowing (10 ml/hr)from 245 6 1 to 255 6 1 mV (N 5 86). In the continuousKCl (2 mol/liter) electrode was used as a reference. Thepresence of 100 mmol/liter H2O2, the hyperpolarizationdata were recorded using a patch clamp amplifier (Frobewas followed within two to three minutes by a long-and Busche, Physiologisches Institut, Freiburg, Ger-lasting and significant depolarization to 220 6 3 mVmany) and were continuously displayed on a pen re-(N 5 25). Short-term application of H2O2 (100 mmol/corder. The membrane voltage (Vm) of the cells wasliter) for less than two minutes caused a reversible hyper-continuously recorded using the current clamp mode ofpolarization, and no depolarization occurred (N 5 61).the patch clamp amplifier. The access conductance (Ga)The H2O2-induced hyperpolarization and depolarizationwas monitored in most of the experiments by a recentlywere concentration dependent, with an EC50 of approxi-described method [15]. To obtain the whole cell conduc-

tance (Gm), the voltage of the respective cell was clamped mately 60 mmol/liter (Fig. 2).

Gloy et al: H2O2 and ion currents in mesangial cells 183

Fig. 1. Original recordings of the effect of 100 mmol/liter hydrogenperoxide (H2O2) on membrane voltage (Vm) of mesangial cells. (A)Long-term application of H2O2 (more than 8 min): 100 mmol/liter H2O2 Fig. 2. Concentration response curves for the hyperpolarization andled to a hyperpolarization of cells that was followed within three minutes subsequent depolarization induced by H2O2. Mean 6 sem (N 5 numberby a sustained depolarization in the presence of H2O2. (B) Short-term of experiments). *Statistical significance compared with control.application (2 min): The hyperpolarization induced by 100 mmol/literH2O2 was reversible after withdrawal of H2O2 within two minutes. Notethat under these experimental conditions, no depolarization occurred.

H2O2 activates a K1 conductance in mesangial cellsAn increase of the extracellular K1 concentration

from 3.6 to 18.6 mmol/liter depolarized mesangial cellsDuring the H2O2 (100 mmol/liter)-induced hyperpolar-significantly by 7 6 1 mV (N 5 12). During the H2O2 (100

ization, the outward conductance slightly increased frommmol/liter)-induced hyperpolarization, the elevation of

8.15 6 1.51 nanoSiemens (nS) to 8.28 6 1.59 nS (P 5 the extracellular K1 concentration to 18.6 mmol/literNS), whereas the inward conductance decreased from caused a significantly enlarged depolarization of mesan-7.62 6 1.50 nS to 7.54 6 1.41 nS (P 5 NS). During the gial cells by 14 6 1 mV (N 5 12). Figure 4 shows andepolarization, both conductances decreased to 7.34 6 original recording and the summary of the paired experi-1.68 and 6.33 6 1.24 nS, respectively (N 5 15, P 5 ments.NS). In the presence of 100 mmol/liter H2O2, the input Figure 5 shows the effect of Ba21, a known potent

blocker of various types of K1 channels [16, 17] on theconductance (Ga) decreased significantly from 90.5 6hyperpolarization induced by H2O2. Two mmol/liter Ba214.91 to 72.1 6 6.16 nS. The Vm response to H2O2 was alsodepolarized mesangial cells from 247 6 5 to 221 6 3demonstrated in mesangial cells in short-term culture upmV, and it completely and reversibly inhibited the H2O2to passage 3. In these cells, H2O2 induced a hyperpolar-(100 mmol/liter)-induced hyperpolarization of the cellsization from 251 6 2 to 264 6 2 mV, which was followed(N 5 7).by a depolarization to 232 6 8 mV (N 5 5 to 13, data

not shown). Influence of a reduced extracellular chlorideDuring the superperfusion with 100 mmol/liter H2O2, concentration on the depolarization induced by H2O2

a contraction of more than 90% of mesangial cells within To test whether a Cl2 conductance may be activatedsix minutes was observed. The contraction of the cells during the H2O2-mediated depolarization, the influencewas accompanied by a retraction of the processes and a of a reduction of extracellular Cl2 in the presence ofshortening or rounding of their cell bodies. The effect of H2O2 was examined. Figure 6 shows the effect of a reduc-

tion of extracellular chloride concentration ([Cl2]e) onH2O2 on mesangial cell contraction is shown in Figure 3.


Fig. 3. H2O2 contracts mesangial cells. (A) Cells in primary culture (25th day). (B) The same cells six minutes after the addition of 100 mmol/literH2O2. Note the retraction and shortening of the processes and the rounding of the cell bodies during mesangial cell contraction.

H2O2-induced depolarization. A reduction of [Cl2]e from H2O2 also depolarized mesangial cells to 222 6 4 mV(N 5 5).147 to 32 mmol/liter did not alter the Vm of mesangial

cells (N 5 7). In the continuous presence of H2O2, theDiamide induces a biphasic membrane voltagereduction of [Cl2]e to 32 mmol/liter augmented the H2O2response in mesangial cells(100 mmol/liter)-induced depolarization significantly

from 219 6 2 to 25 6 2 mV, a level near the equilibrium To investigate whether or not the effect of H2O2 mightpotential for chloride under these experimental condi- be mediated by sulfhydryl modification, the influence oftions (N 5 7). the thiol-oxidizing agent diamide [18] was examined.

Figure 8 shows the effect of diamide on Vm of mesangialMembrane voltage response to H2O2 does not depend cells. Diamide (100 mmol/liter) induced a hyperpolariza-on the extracellular free Ca21 concentration tion from 248 6 7 to 265 6 5 mV, which was followed

by a repolarization to 255 6 7 mV (N 5 5). The VmFigure 7 shows the effect of a low extracellular freeresponse to 100 mmol/liter diamide was completely inhib-Ca21 ([Ca21]e) on H2O2-mediated Vm response. Loweringited by 1 mmol/liter DTT (N 5 3, data not shown).[Ca21]e from 1 mmol/liter to 1 mmol/liter or 1 nmol/

liter did not significantly influence the Vm response ofEffect of pyruvate and dithiothreitol on the membranemesangial cells. In a series of paired experiments, thevoltage response to H2O2H2O2 (100 mmol/liter)-induced hyperpolarization was not

significantly altered in the presence of low [Ca21]e (26 6 Preincubation of mesangial cells with 5 mmol/liter pyr-uvate, a H2O2 scavenger, for 7 to 30 minutes (N 5 5)3 vs. 26 6 2 mV, N 5 11). In the presence of low [Ca21]e,


Fig. 5. Effect of 2 mmol/liter Ba21 on the H2O2-induced hyperpolariza-tion. (A) Original recording. (B) Summary of the experiments. Abbrevi-ation is: C, control. Data are mean 6 sem (N 5 number of experiments).Fig. 4. Influence of an increased extracellular K1 concentration (from*Statistical significance compared with control. Note that in the presence3.6 to 18.6 mmol/liter) on membrane voltage (Vm) of mesangial cellsof Ba21, the hyperpolarization induced by 100 mmol/liter H2O2 wasin the absence and presence of 100 mmol/liter H2O2. (A) Original re-completely inhibited.cording. (B) Summary of 12 experiments. C, control. Note that the

depolarization caused by an elevated extracellular K1 concentrationwas significantly augmented during the H2O2-induced hyperpolariza-tion. Data are mean 6 sem (N 5 number of experiments). *Statisticalsignificance compared with control; 1significance between the effect of Effect of inhibitors of protein kinase C on the H2O2-an increased extracellular K1 concentration under control conditionsand in the presence of H2O2. induced membrane voltage change in mesangial cells

Figure 10 shows the effect of staurosporine on theH2O2-induced hyperpolarization and depolarization inmesangial cells. In the presence of staurosporine (0.1

did not significantly change Vm. In the presence of pyr-mmol/liter) or BIM (0.1 mmol/liter), the hyperpolariza-

uvate, the effect of H2O2 (100 mmol/liter) was completely tion induced by 100 mmol/liter H2O2 was not changed,inhibited (data not shown). Figure 9 shows the influence whereas the depolarization was inhibited (N 5 3 to 5).of the disulfide-reducing agent DTT (1 mmol/liter) onthe H2O2-mediated hyperpolarization and depolariza-

DISCUSSIONtion. Pretreatment of mesangial cells with the disulfide-reducing agent DTT (0.1 to 1 mmol/liter, N 5 10) for The regulation of ion channels plays a crucial role inthree to five minutes had no effect on Vm. In the presence many physiological and pathophysiological processes. Inof 100 mmol/liter DTT, the hyperpolarization induced mesangial cells, it has been assumed that an angiotensinby H2O2 (100 mmol/liter) was not changed (28 6 2 mV, II-mediated activation of a Ca21-dependent Cl2 conduc-N 5 5), whereas the H2O2-induced depolarization was tance is associated with cell contraction [11, 19]. Contrac-completely inhibited; that is, in the presence of DTT and tion of mesangial cells might lead to a reduction of filtra-H2O2, mesangial cells stayed hyperpolarized for more tion surface area, thereby decreasing the ultrafiltrationthan 10 minutes. After the removal of DTT and in the coefficient [19]. Furthermore, it has been speculated thatcontinued presence of H2O2, mesangial cells depolarized the permeability for Cl2 in mesangial cells plays a roleirreversibly to 222 6 8 mV (N 5 4). Preincubation of for the signaling systems involved in tubuloglomerularmesangial cells with higher concentrations of DTT (1 feedback [20]. Dysregulation of mesangial cell Cl2 chan-mmol/liter) also inhibited the H2O2 (100 mm)-induced nels by high glucose and abnormal insulin levels seems

to be involved in the development of glomerular hyper-hyperpolarization by 78 6 8% (N 5 5).


Fig. 6. Original recording (A) and summary (B) of seven paired experi-ments with reduction of the extracellular Cl2 concentration ([Cl2]e;from 147 to 32 mmol/liter) in the absence and presence of 100 mmol/liter H2O2 [C, control (147 mmol/liter Cl2)]. Data are mean 6 sem

Fig. 7. Original recording (A) and summary (B) of the effect of 100(N 5 number of experiments). 1Significance between the effect of H2O2

mmol/liter H2O2 on the membrane voltage of mesangial cells in theunder control conditions and H2O2 in the presence of 32 mmol/presence of a reduced extracellular Ca21 activity (low Ca21, 1 mmol/liter Cl2.liter or 1 nmol/liter). Data are mean 6 sem (N 5 number of experi-ments). The biphasic membrane voltage response induced by H2O2 stilloccurred in the presence of a low extracellular Ca21 concentration.

filtration in diabetic nephropathy [21]. K1 conductances,which repolarize Vm, have also been identified in mesan-gial cells [13, 22, 23]. liter H2O2 for one hour did not induce cytotoxicity [26],

This study demonstrates that H2O2, which seems to whereas Varani et al observed that H2O2 alone couldplay a crucial role in glomerular diseases [24], influences partially kill mesangial cells within 1.5 hours [27]. WeVm and ion conductances of mesangial cells. Short-term could not detect LDH release from mesangial cells inapplication of H2O2 caused a reversible hyperpolariza- primary culture exposed to 100 mmol/liter H2O2 for uption of mesangial cells. During longer application of to 30 minutes (data not shown). This fits well to observa-H2O2, the hyperpolarization was followed by a sustained tions in which mesangial cells excluded trypan blue evendepolarization. The Vm response to H2O2 was accompa- after an incubation with 1 mmol/liter H2O2 for up to 30nied by a mesangial cell contraction, which has been also minutes [25].reported by others [25]. The hyperpolarization induced by H2O2 seems to be

We cannot completely exclude that the effects of H2O2 due to the activation of a K1 conductance because it wason ion currents are partially due to the cytotoxic effects completely inhibited in the presence of the K1 channelof H2O2. However, relatively low concentrations of H2O2 blocker Ba21 [16, 17]. Moreover, in the presence of nor-(5 mmol/liter threshold concentration) influenced Vm, mal and elevated extracellular K1 concentrations, H2O2

and the hyperpolarization and depolarization induced shifted the Vm toward the equilibrium potential for K1.by H2O2 were completely and partially reversible. The The depolarization observed in these experiments isdata in the literature on the influence of H2O2 on mesan- most likely induced by the elevated K1 concentrationgial cell cytotoxicity are inconsistent. Johnson et al re- and not by the depolarizing effect of H2O2 because the

depolarization in the presence of a high K1 concentrationported that incubation of mesangial cells with 100 mmol/


Fig. 9. Original recording (A) and summary of five paired experimentsFig. 8. Effect of diamide (100 mmol/liter) on membrane voltage of (B) of the influence of dithiothreitol (DTT; 1 mmol/liter) on the H2O2mesangial cells. (A) Original recording. (B) Summary. Data are mean 6 (100 mmol/liter)-induced membrane voltage response in mesangial cells.sem (N 5 number of experiments). *Statistical significance compared Data are mean 6 sem (N 5 number of experiments). Preincubationwith control. with 1 mmol/liter DTT inhibited the membrane voltage response to

H2O2. *Statistical significance compared with control.

occurred rapidly. Conversely, the H2O2-mediated depolar-ization had a slow time course, and after readdition of a gial cell depolarization, Ca21 entry, and contraction [28,

29]. Chronic stimulation of mesangial cell Cl2 channelssolution with a physiological K1 concentration and inthe continuous presence of H2O2 (Fig. 4, bar 6), cells still by H2O2 might thus stimulate sustained Ca21 influx, re-

sulting in mesangial cell contraction and proliferation,were hyperpolarized and almost reached the Vm inducedafter the first addition of H2O2. K1 channels in mesangial and contribute to a progressive decrease of GFR. Sus-

tained elevation of the cytosolic free Ca21 concentrationcells have been implicated to induce membrane repolar-ization and to modulate the contractile response to vaso- has been also associated with apoptosis and cell death

[30]. Although we did not observe a lactate dehydroge-active agonists [23]. The early H2O2-induced hyperpolar-ization of mesangial cells might therefore initially nase release in mesangial cells that were treated with

H2O2 for a short time period, the dysregulation of thecounteract the cellular events leading to H2O2-induceddepolarization and contraction. cytosolic-free Ca21 concentration by a long-term expo-

sure to H2O2 may participate in H2O2 toxicity to mesan-Because the depolarization induced by H2O2 was aug-mented by a low extracellular Cl2 concentration, it may, gial cells.

It is known that mesangial cells possess Ca21-activatedat least in part, be due to an activation of a Cl2 conduc-tance. The activation of a Cl2 conductance in mesangial K1 and Cl2 channels [23]. Recently, we have reported

that H2O2 induces a delayed and sustained increase ofcells has been implicated to control mesangial cell con-traction [11, 19]. The activation of Cl2 channels by H2O2 the free cytosolic Ca21 concentration ([Ca21]i) in mesan-

gial cells [9]. The major part of this increase was due tomight thus induce mesangial cell contraction, decreasethe glomerular ultrafiltration coefficient (Kf), and thereby a Ca21 influx from the extracellular space. In contrast to

the H2O2-mediated increase of [Ca21]i, the effect of H2O2decrease GFR. The activation of a Cl2 conductance byH2O2 may be therefore linked to an H2O2-mediated re- on Vm of mesangial cells occurred immediately. In addi-

tion, a reduction of the extracellular Ca21 concentrationduction of GFR. It has been shown that an activationof a Cl2 conductance in mesangial cells leads to mesan- did not influence the H2O2-mediated hyperpolarization.


though the time course of the depolarization was verysimilar to the (influx-mediated) increase in [Ca21]i in-duced by H2O2, the depolarization still occurred in anominally Ca21-free extracellular solution, indicatingthat [Ca21]i does not play a major role in activating H2O2-induced Cl2 conductance.

The oxidative state of sulfhydryl groups is known toregulate the activity of different ion channels, such asK1-, Ca21-, and nonselective ion channels [35–37]. Toinvestigate whether Vm response to H2O2 in mesangialcells was due to a sulfhydryl modification, experimentswith the oxidizing agent diamide [18] and with the disul-fide-reducing agent DTT were performed. Diamide in-duced a hyperpolarization, which was followed by a repo-larization, compatible with the interpretation that theH2O2-mediated Vm response was due in part to a pertur-bation of the thiol status of membrane proteins. A long-lasting depolarization of mesangial cells was not inducedby diamide, suggesting that the depolarization inducedby H2O2 is regulated differently. It is known that althoughboth diamide and H2O2 induce oxidative stress, theirintracellular effects are not identical. Diamide depletesglutathione pools and oxidizes thiol groups [18], whereasH2O2 induces several different effects such as lipid perox-idation, protein oxidation, and DNA damage [38]. It has

Fig. 10. Influence of the protein kinase C (PKC) inhibitor stauro-also been reported that diamide and H2O2 regulate AP-1sporine (stauro, 0.1 mmol/liter) on the effect of 100 mmol/liter H2O2 on

membrane voltage (Vm). (A) Original recording. (B) Summary of the protein, a key mediator of oxidative stress, in a differentexperiments. Data are mean 6 sem (N 5 number of experiments). manner [39]. A possible involvement of redox mecha-*Statistical significance compared with control. Preincubation with

nisms in the activation of the ion conductances by H2O2staurosporine could not prevent the H2O2-mediated hyperpolarization,but inhibited a subsequent depolarization. was further supported by the inhibition of the H2O2-

mediated Vm response by DTT. Low concentrations (100or 500 mmol/liter) of DTT completely inhibited the H2O2-induced depolarization, whereas higher concentrations

In addition, it has been reported that a Ca21 ionophor (1 mmol/liter) of DTT were required to inhibit the hyper-depolarized rather than hyperpolarized mesangial cells polarization elicited by H2O2. The mechanisms for the[12]. Therefore, it is unlikely that the hyperpolarization different sensitivities of the DTT-induced inhibition ofinduced by H2O2 is mediated by an increase of [Ca21]i. the hyperpolarization and depolarization remain unclearSimilar to mesangial cells, the H2O2-induced activation and need to be clarified in future studies. The moreof a K1 conductance followed by hyperpolarization and potent effect of DTT in inhibiting the H2O2-induced de-relaxation has also been reported for smooth muscle cells polarization might be explained by a higher efficiency[31, 32]. However, in smooth muscle cells, the removal of of DTT in inhibiting the signal transduction pathwaysextracellular Ca21 prevented the H2O2-mediated hyper- that activate the Cl2 conductance in mesangial cells; thatpolarization, suggesting that H2O2 causes Ca21 influx and is, DTT might more efficiently inhibit the PKC-mediatedactivates Ca21-dependent K1 channels [32]. Therefore, activation of the Cl2 conductance.the regulation of the H2O2-activated K1 conductance In summary, H2O2 leads to a biphasic Vm response inappears to be different in mesangial cells. mesangial cells: (a) an initial transient hyperpolarization

H2O2 is known to activate protein kinase C (PKC) in that is due to the activation of a K1 conductance, anddifferent cell types [33]. To investigate whether PKC (b) a subsequent depolarization that is due, at least ininfluences the Vm response to H2O2, the effect of H2O2 part, to the activation of a PKC-activated Cl2 conduc-was examined in the presence of PKC inhibitors. PKC tance. The depolarization and the hyperpolarization caninhibitors completely prevented the depolarization in- be abolished by high concentrations of DTT. Althoughduced by H2O2, whereas the hyperpolarization was not it is controversial whether or not the contractile state ofaffected. Thus, a PKC-stimulated Cl2 conductance, mesangial cells influences Kf [10], it has been shown thatwhich has been reported for mesangial cells [34], may the integrity of the glomerular tuft and the counteraction

of the distending forces of the capillaries are arrangedparticipate in the depolarizing response to H2O2. Al-


of HT 29 colon carcinoma and CF-PAC cells. Pflugers Archby the mesangial cells [10]. The Vm response to H2O2, 419:209–211, 1991especially the enhanced Cl2 conductance and cell depo- 16. Greger R, Gogelein H: K1 conductive pathways in the nephron.

Kidney Int 31:1055–1064, 1987larization, might therefore reduce GFR and have a more17. Nagel W: Inhibition of potassium conductance by barium in frogchronic effect leading to the destruction of mesangial

skin epithelium. Biochim Biophys Acta 52:346–357, 1979cells, which would further compromise filtration. 18. Kosower NS, Kosower EM: Diamide: An oxidant probe for thiols.

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cell. Physiol Rev 69:1347–1424, 198920. Kurokawa K: Chloride conductance of mesangial cells: InsightsWe thank Mr. Temel Kilic and Ms. Monika v. Hofer for excellent

into the transcellular signaling of tubuloglomerular feedback andtechnical assistance. This work was supported by the Deutscheits physiological significance. Renal Physiol Biochem 16:15–20, 1993Forschungsgemeinschaft Fi 691/1-1.

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