Kidney International, Vol. 56 (1999), pp. 1223–1229

Oxidant-induced apoptosis of glomerular cells: Intracellularsignaling and its intervention by bioflavonoid

MASANORI KITAMURA and YOSHIHISA ISHIKAWA

Department of Medicine, University College London Medical School, London, England, United Kingdom

Oxidant-induced apoptosis of glomerular cells: Intracellular are ubiquitously present in nature. These constituentssignaling and its intervention by bioflavonoid. Oxidant stress of the diet are important in the maintenance of the body,plays a crucial role in the generation of a wide range of glomeru- especially to keep integrity of the cardiovascular system.lar disease. In the first part of this article, we describe intracellular

Clinically relevant functions ascribed to flavonoids in-signaling pathways involved in the oxidant-initiated apoptosisclude antihypertensive activity, anti-inflammatory prop-of mesangial cells, especially highlighting the tyrosine kinase–

c-Jun/AP-1 pathway. In the second part, we address a novel erties, hypocholesterolemic activity, and platelet stabili-potential of bioflavonoid quercetin as an inhibitor of apoptosis zation [11]. Quercetin (3,39,49,5,7-pentahydroxyflavone)in glomerular cells. Possible mechanisms for the antiapoptotic is one of the most widely distributed bioflavonoids in theaction of quercetin and its therapeutic utility are discussed.

plant kingdom. Like other members of the bioflavonoidfamily, this compound facilitates apoptosis of tumor cells[12], in part, through depression of an endogenous cyto-Apoptosis is an innate program of cell suicide that isprotective molecule, heat-shock protein 70 [13, 14]. How-required for the removal of unnecessary or damagedever, we unexpectedly found that in mesangial cells,cells from bodily structures. Apoptosis contributes toquercetin inhibited apoptotic cell death triggered by oxi-developmental processes, as well as maintenance of adultdative stress. In the second part of this article, we de-organs. However, its inappropriate activation leads toscribe how quercetin intervenes in the apoptotic pathwayintractable pathologies, including stroke, heart attack,triggered by oxidants in glomerular cells.and neurodegenerative disorders. In the renal glomeru-

lus, apoptosis of glomerular cells is observed in severaltypes of glomerular diseases and supposedly plays a role MOLECULAR MECHANISMS OF OXIDANT-in the initiation and progression of injury [1–4]. INITIATED APOPTOSIS

The molecular mechanisms involved in the apoptosis Reactive oxygen species (ROS) are the well-knownof resident cells during glomerular disease have not yet triggers for apoptosis in various cell types [15]. Severalbeen determined, but several possibilities have been pos-

oxidant-inducible molecules have been identified as in-tulated. During the initiation and progression of in-

tracellular mediators for apoptosis. The best-known ex-flammation, for example, toxic substances elaborated by

ample is c-Jun/activating protein-1 (AP-1), which is re-leukocytes may induce apoptosis of resident cells. These

garded as a redox-sensitive transcription factor. AP-1,triggers include cytokines, nitric oxide, and reactive oxy-

mainly composed of either homodimers of c-Jun or het-gen species (ROS) [5–8]. ROS play crucial roles in theerodimers of c-Jun and c-Fos, binds to a particular cisgeneration of a broad array of human and experimentalelement, the 12-O-tetradecanoylphorbol 13-acetate re-glomerular diseases [9]. Using hydrogen peroxide (H2O2) sponse element (TRE), and initiates transcription of targetas an inducer, recent studies have shown that oxidantgenes [16]. Accumulative data have suggested the impor-induces apoptosis of cultured mesangial cells [8, 10]. Thetance of c-Jun N-terminal kinase (JNK) and its substratefirst part of this article addresses the intracellular signal-c-Jun in the signaling pathways to apoptosis. For exam-ing pathways involved in the H2O2-initiated apoptosis ofple, the exposure of cells to apoptotic stimuli, includingglomerular cells.ultraviolet light, g-irradiation, tumor necrosis factor-a,Bioflavonoids are semiessential food components thatand ceramide triggers JNK activity [17–20]. Dominant-negative inactivation of either SEK1, JNK, or c-Jun canprevent certain apoptotic processes [17, 18, 20–22]. Fur-Key words: reactive oxygen intermediate, apoptosis, quercetin, tyro-

sine kinase, cell signaling. thermore, constitutive activation of the JNK–c-Jun path-way results in apoptotic cell death [22–24]. 1999 by the International Society of Nephrology

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Kitamura and Ishikawa: Glomerular cell apoptosis and bioflavonoids1224

Protein tyrosine kinases and phosphatases play keyroles in various cellular responses, including mitogenesis,adhesion, differentiation, transformation, and apoptosis.A body of literature suggests that tyrosine phosphoryla-tion/dephosphorylation-dependent signal transductioncan be modulated by the intracellular redox status [25].In general, tyrosine kinase activity is stimulated by ROSdirectly or indirectly. For example, H2O2 stimulates phos-phorylation of tyrosine residues of the insulin receptor[25]. In contrast, tyrosine phosphatase activity may beinhibited by ROS [26].

In this article, we highlight the roles of c-Jun/AP-1and tyrosine kinases during oxidant induced, glomerularcell apoptosis.

Role of the tyrosine kinase–c-Jun/AP-1 pathway inoxidant-induced apoptosis of mesangial cells

Cultured rat mesangial cells were exposed to H2O2,and microscopic analysis was performed. Mesangial cellstreated with H2O2 exhibited shrinkage of cytoplasm,membrane blebbing, and condensation of nuclei typicalof apoptosis (Fig. 1A). The nuclei of H2O2-exposed mes-angial cells were intensely positive for terminal deoxy-nucleotidyl transferase (TdT)-mediated dUTP nick-endlabeling (TUNEL) (Fig. 1B). Consistent with these re-sults, agarose gel electrophoresis of oligonucleosomalDNA detected DNA ladder formation (Fig. 1C). Thesedata evidenced induction of mesangial cell apoptosis byH2O2.

To explore the molecular mechanisms involved, weinvestigated the role of c-Jun/AP-1, an oxidant-induc-ible, putative mediator for apoptosis. Northern blot anal-ysis revealed that, after the stimulation with H2O2, tran-sient up-regulation of c-jun was observed with a peak attwo hours [10]. Immunoblot analysis using a phospho-specific antibody showed phosphorylation of JNK inH2O2-triggered mesangial cells (Note added in proof).Consistently, transient transfection studies using a TREreporter plasmid revealed that H2O2 stimulated the activ-ity of AP-1 [10].

To examine whether the activation of c-Jun/AP-1 isrequired for the induction of mesangial cell apoptosis,stably transfected mesangial cell clones JUNDN1,JUNDN2, and JUNAS2 were utilized. In these clones,the function of c-Jun/AP-1 is selectively attenuated viaan antisense or a dominant-negative mechanism [27–29].These transfectants and a control clone were exposed toH2O2, and cell survival was examined. Compared withmock-transfected cells, JUNDN1, JUNDN2, and JUNAS2cells showed resistance to the oxidant-induced cellulardeath [10]. Similarly, dominant-negative inhibition of Fig. 1. Induction of apoptosis by hydrogen peroxide (H2O2) in cultured

rat mesangial cells. Mesangial cells were exposed to 400 mm H2O2 for 16JNK attenuated the H2O2-induced apoptosis of mesan-hours and subjected to analyses. (A) Microscopic analysis. (B) Terminalgial cells (Note added in proof). To confirm the involve-deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling

ment of c-Jun/AP-1 further, mesangial cells were pre- (TUNEL). (C) Detection of oligonucleosomal DNA fragmentation byagarose gel electrophoresis.treated with or without a pharmacological inhibitor of

Kitamura and Ishikawa: Glomerular cell apoptosis and bioflavonoids 1225

the JNK/AP-1 pathway, curcumin, and subsequently, dependent manner [31]. This cytoprotective effect wasnot observed when the cells were first exposed to H2O2were exposed to H2O2. Compared with control, curcumin

selectively suppressed the expression of c-jun [29] and and then treated with quercetin. Microscopic analysisrevealed that quercetin protected the oxidant-exposedsignificantly improved the cell survival [10]. These data

suggest that c-Jun/AP-1 mediates the oxidant-induced cells from shrinkage of cytoplasm, membrane blebbing,and condensation of nuclei (Fig. 2A). Consistently, quer-apoptosis in mesangial cells.

It is known that ROS elicit tyrosine phosphorylation- cetin suppressed the H2O2-induced DNA fragmentationwhen examined by agarose gel electrophoresis (Fig. 2B).dependent signaling pathways [25]. In certain cell types,

the activation of tyrosine kinases is involved in apoptotic As mentioned earlier in this article, c-Jun/AP-1 plays acrucial role in the H2O2-triggered apoptosis of mesangialprocesses [30]. To examine the involvement of tyrosine

kinases in oxidant-induced apoptosis, mesangial cells cells. H2O2 induces c-jun expression as well as activationof c-Jun/AP-1, and either genetic or pharmacologicalwere treated with H2O2, and immunoblot analysis was

performed on phosphotyrosine. Following the exposure inhibition of this event attenuates apoptosis. To elucidatemechanisms involved in the antiapoptotic action of quer-to H2O2, dramatic protein phosphorylation at tyrosine

residues was detected within 5 minutes and peaked at 30 cetin, its effect on c-Jun/AP-1 was tested. Northern blotanalysis showed that quercetin dramatically suppressedminutes [31]. To explore whether activation of tyrosine

kinases is an essential event for the induction of apopto- the c-jun induction by H2O2 [31]. Reporter assays re-vealed that the activation of AP-1 by H2O2 was signifi-sis, mesangial cells were pretreated with tyrosine kinase

inhibitors, either genistein or herbimycin A, and then cantly depressed by quercetin [31]. These results sug-gested that quercetin inhibits apoptosis via interventionstimulated by H2O2. Both tyrosine kinase inhibitors sig-

nificantly improved mesangial cell survival [31]. in the c-Jun/AP-1 pathway.Protein phosphorylation at tyrosine residues is an es-To examine whether the tyrosine phosphorylation is

an event upstream of the activation of c-Jun/AP-1, mes- sential event upstream of the activation of c-Jun/AP-1during the oxidant-initiated mesangial cell apoptosis. Toangial cells were pretreated with genistein and stimu-

lated by H2O2. After two hours, the expression of c-jun examine whether or not quercetin inhibits the apoptosisvia suppression of tyrosine phosphorylation, mesangialwas tested. Northern blot analysis showed that genistein

significantly suppressed the induction of c-jun by H2O2 cells were pretreated with or without quercetin and thenexposed to H2O2. Immunoblot analysis showed that phos-[31]. A similar suppressive effect was observed by an-

other tyrosine kinase inhibitor, herbimycin A. Consistent phorylation of tyrosine residues by H2O2 was suppressedby quercetin dose dependently. Fifty micromolar quercetinwith these results, a reporter assay revealed that the

activation of AP-1 in response to H2O2 was abrogated completely abolished the tyrosine phosphorylation [31].These data demonstrate a novel potential of quercetinby genistein [31]. These findings suggested that oxidant

stress induces apoptosis of mesangial cells via the tyro- as an inhibitor of apoptosis in mesangial cells. The inhibi-tory action of this agent is due to the inactivation of c-Jun/sine kinase–c-Jun/AP-1 pathway.

Pfeilschifter and Huwiler recently reported that the AP-1, the crucial mediator for oxidant-induced apopto-sis. Furthermore, c-Jun/AP-1 may not be the primaryexposure of rat mesangial cells and bovine glomerular

endothelial cells to nitric oxide resulted in activation of target of quercetin; that is, tyrosine phosphorylation wasidentified to be a target upstream of c-Jun/AP-1.JNK preceding apoptosis [32]. Pretreatment of the cells

with a tyrosine kinase inhibitor attenuated the c-JunQuercetin as a general inhibitor of apoptosisphosphorylation. Furthermore, antioxidant N-acetylcys-

teine markedly reduced the activation of JNK in response To examine whether the cytoprotective action of quer-cetin is restricted to apoptosis triggered by oxidants, an-to nitric oxide. This result, together with our findings,

supports the idea that the tyrosine kinase–c-Jun/AP-1 other apoptosis inducer, staurosporine, was used. Mesan-gial cells were pretreated with or without quercetin andpathway is involved in the oxidant-mediated apoptosis

of glomerular cells. exposed to staurosporine. Compared with untreated con-trol, cell survival was significantly improved by the treat-ment with quercetin [31]. To investigate further whether

PHARMACOLOGICAL MANIPULATION OFthe cytoprotective action of quercetin is specific to mes-

APOPTOSIS BY BIOFLAVONOID QUERCETINangial cells, LLC-PK1 epithelial cells and NRK49F fi-

Intervention in the oxidant-induced apoptosis of broblasts were pretreated with or without quercetin andmesangial cells by quercetin were exposed to H2O2. Microscopic analysis showed that,

in both cell types, quercetin effectively prevented theTo examine the effect of quercetin on mesangial cellapoptosis, cells were pretreated with or without querce- H2O2-induced cellular death [31].

The anti-apoptotic effect of quercetin on glomerulartin and then exposed to H2O2. Pretreatment with querce-tin significantly improved the cell survival in a dose- cells is observed under a more physiologically relevant

Kitamura and Ishikawa: Glomerular cell apoptosis and bioflavonoids1226

Fig. 2. Suppression of glomerular cell apoptosis by quercetin. (A andB) Inhibition of oxidant-triggered mesangial cell apoptosis. Mesangialcells were pretreated with or without quercetin (50 mM) for 1 hour andexposed to H2O2 (400 mm) for 16 hours. Microscopic analysis (A) andladder detection assay (B). (C) The inhibition of spontaneous apoptosisin isolated glomeruli. Isolated normal rat glomeruli were incubated inthe presence of quercetin (0 to 100 mm) for two hours and subjectedto ladder detection assay.

situation. We recently found that in isolated rat glomer- a number of enzymes, including tyrosine kinases anduli, resident cells, especially podocytes, spontaneously phosphatases. We found that stimulation of mesangialundergo apoptosis immediately after explantation [33]. cells by H2O2 induced tyrosine phosphorylation of multi-A number of resident cells were positive for TUNEL ple proteins. Currently, however, it is undetermined (a)when examined two hours after explantation [33]. Lad- whether the tyrosine phosphorylation is via direct order detection assay showed that within 15 minutes, DNA indirect activation of tyrosine kinases by H2O2, and (b)fragmentation was induced in the cells of isolated glo- how inactivation of tyrosine phosphatases by H2O2 [26]meruli [33]. Ladder formation of oligonucleosomal DNA contributes to this process.was peaked at two hours and was attenuated thereafter. Genistein and herbimycin A prevented the oxidant-Quercetin inhibited this spontaneous apoptosis in a dose- initiated apoptosis, suggesting the crucial contributiondependent manner (Fig. 2C). These results further sup- of tyrosine kinases. An unanswered question is the sub-port the possibility that quercetin may act as a general type of tyrosine kinases involved in this process. Naka-inhibitor of apoptosis.

mura et al reported that in T cells, a member of the SrcThe findings described earlier in this article are sum-family of tyrosine kinase Lck is preferentially phosphory-marized in Figure 3.lated and activated following exposure of the cells toH2O2 [34]. Uckun et al reported that Bruton’s tyrosine

UNANSWERED ISSUES kinase (Btk), but not Lyn and Syk, mediates oxidant-Mechanisms of oxidant-induced induced apoptosis in lymphoma cells [35]. Currently itmesangial cell apoptosis is unknown whether these tyrosine kinases contribute to

the H2O2-initiated apoptosis in mesangial cells.From oxidant to tyrosine kinase. Alterations in theintracellular redox state may influence the activity of From tyrosine kinase to c-Jun/AP-1. The mechanism

Kitamura and Ishikawa: Glomerular cell apoptosis and bioflavonoids 1227

Fig. 3. The oxidant-induced apoptotic path-way in glomerular cells and its interventionby quercetin. Oxidant stress activates the tyro-sine kinase–c-Jun/AP-1 pathway that medi-ates the induction of apoptosis. Quercetin in-hibits this signaling cascade upstream of thetyrosine phosphorylation.

involved in the activation of c-Jun/AP-1 by tyrosine ki- might be the primary target of quercetin. Indeed, certainbioflavonoids have the ability to modulate the activitynases has been proposed by several investigators. One

possibility is that tyrosine kinases activate Ha-Ras and of tyrosine kinases and/or tyrosine phosphatases directly[46]. Alternatively, like other bioflavonoids, quercetinRaf-1 that induce phosphorylation of pre-existing c-Jun

and thereby augment the activity of c-Jun/AP-1 [36]. may function as a scavenger of ROS [46]. In this case,this agent may prevent the action of H2O2 at the initialTRE is located in the promoter region of the c-jun

gene, and expression of c-jun is subsequently evoked by step.activated AP-1 via positive autoregulation [37]. Alterna- Our data, however, showed that quercetin inhibitedtively, the tyrosine kinase-mediated activation of the spontaneous apoptosis in isolated glomeruli, which oc-Ras–Raf mitogen-activated protein kinase pathway may curs independently of oxidant stress or tyrosine kinaseinduce c-fos expression and thereby augment the activity activation (our unpublished data). Furthermore, apopto-of AP-1 [38]. These stimulatory mechanisms may be in- sis of mesangial cells in response to a different stimulus,volved in the activation of c-Jun/AP-1 via tyrosine kinases. staurosporine, was also prevented by quercetin. Of note,

Downstream of c-Jun/AP-1. The downstream mecha- staurosporine-induced apoptosis is oxidant independentnisms by which c-Jun/AP-1 elicits apoptosis have not [47]. These results imply a possibility that quercetin inter-been identified. Because c-Jun/AP-1 is a transcription feres with a common pathway required for apoptosis. Itfactor that triggers expression of a variety of genes, it has been reported that quercetin prevents the benzo(a)-may up-regulate expression of certain inducers of apo- pyrene-induced nuclear injury in vitro and in vivo, possi-ptosis via TRE. Alternatively, c-Jun/AP-1 could affect bly via inhibiting its binding to DNA [48]. This agentfunction of other transacting molecules [39] and thereby also directly interacts with and stabilizes DNA, leadingmodulate the activity of other cis elements required for to its resistance to S1 nuclease [49]. Quercetin mightthe induction of “death genes.” Extensive studies will therefore act as an inhibitor of apoptosis in the nucleusbe necessary to determine the events downstream of via direct interaction with DNA.c-Jun/AP-1. One of crucial regulators of apoptosis is p53 [50]. Low

Other pathways. Both genetic and pharmacological levels of p53 protein are found in normal cells, but itsinactivation of c-Jun/AP-1 achieved partial inhibition of level rises rapidly in response to stimuli, including oxi-apoptosis triggered by H2O2. H2O2 may evoke other sig- dant stress [51]. In mesangial cells, superoxide inducesnaling pathways in which c-Jun/AP-1 is not involved. For p53 during mesangial cell apoptosis. The induction ofexample, in certain cell types, H2O2 induces expression of p53 is required for the oxidant-initiated apoptosis in sev-c-myc [40] that facilitates apoptosis via the induction of eral cell types [52]. Avila et al reported that quercetinornithine decarboxylase or stabilization of p53 [41, 42]. selectively inhibited the induction of p53 in human can-Oxidant-induced c-Myc and c-Jun may cooperatively cer cells at the transcriptional level [53]. Quercetin mayparticipate in the induction of apoptosis. inhibit the oxidant-triggered apoptosis via suppression

Oxidants may directly damage DNA via depletion of of the p53-dependent apoptotic pathway. H2O2 also in-cellular NAD/NADH and ATP, leading to apoptosis duces c-myc [40], a well-known inducer of apoptosis. It[43]. Oxidative stress may also affect lipid layers in cell has been shown that in certain cell types, quercetin inhibitsmembranes, and oxygenated derivatives of arachidonic the expression of c-myc [12]. The suppression of c-mycacid could induce apoptosis [44, 45]. These direct actions might, in part, explain the anti-apoptotic action of quer-of oxidants might explain the incomplete suppression of cetin described here.apoptosis via inhibition of c-Jun/AP-1.

Mechanisms of anti-apoptotic action of quercetin PERSPECTIVE

Oxidant stress plays a crucial role in the generationHow does quercetin inhibit activation of tyrosine ki-nases? In mesangial cells, tyrosine phosphorylation is of a wide range of glomerular injury [9]. ROS generated

by infiltrating cells induce injury of resident cells anddetectable within five minutes following the exposure toH2O2 [31]. Based on this rapid response, tyrosine kinase glomerular basement membrane (GBM), facilitate glo-

Kitamura and Ishikawa: Glomerular cell apoptosis and bioflavonoids1228

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