Gentamicin induces Jun-AP1 expression and JNK activation in renal glomeruli and cultured mesangial cells

Life Sciences 77 (2005) 2285 – 2298 www.elsevier.com/locate/lifescie

Gentamicin induces Jun-AP1 expression and JNK activation in renal glomeruli and cultured mesangial cells Carlos Martı´nez-Salgadoa,T,1, Alicia Rodrı´guez-Barberob,1, Ne´lida Elenob, Jose´ M. Lo´pez-Novoab a

Unidad de Investigacio´n, Hospital Universitario de Salamanca, Paseo de San Vicente 58-132, 37007 Salamanca, Spain b Departamento de Fisiologı´a y Farmacologı´a and Instituto bReina Sofı´aQ de Investigacio´n Nefrolo´gica, Universidad de Salamanca, 37007 Salamanca, Spain Received 23 December 2004; accepted 2 February 2005

Abstract Reactive oxygen species (ROS) mediate MC contraction, proliferation and apoptosis induced by gentamicin (G) in vitro and in vivo. Sustained increases in cytosolic free calcium, increased iNOS expression and elevated nitric oxide (NO) production are associated with MC apoptosis in vitro. As NO strongly activated c-Jun Nterminal kinase (JNK) and increased AP1 expression, and these two factors are involved in MC proliferation in vitro, we have measured Jun-AP1 expression in rat glomeruli from G-treated rats, and the effect of G on JunAP1 expression and JNK activity in cultured MC. Moreover, we studied the expression of inducible (iNOS) and constitutive (cNOS) NO synthases in rat glomeruli. Glomeruli were obtained from rats treated with G (100 mg/ kg body weight/day) along 6 days, and MC primary cultures were evaluated after 24, 48 and 72 h incubation with 10 5 M G. G induced an increase in the expression of iNOS, cNOS and Jun-AP1 in rat glomeruli and in MC cultures. Moreover, G activated JNK; JNK activation was reduced by co-incubation with the calcium channel blocker verapamil and with the ROS scavengers superoxide dismutase and catalase. These results strongly suggest a role for reactive oxygen/nitrogen species produced by increased NOS activity in G-induced MC activation. These reactive oxygen molecules and increased intracellular free

* Corresponding author. Tel.: +34 923 294472; fax: +34 923 294669. E-mail address: [email protected] (C. Martı´nez-Salgado). 1 Authors with equal contributions to the study. 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.02.021

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calcium may mediate the increase in Jun-AP1 expression and JNK activation induced by G treatment in MC. D 2005 Elsevier Inc. All rights reserved. Keywords: Mesangial cells; Gentamicin; Jun kinase; Jun-AP1; NO; Proliferation; Apoptosis

Introduction Nephrotoxicity is the main side effect of long-term gentamicin (G) treatments. The most studied effect of this aminoglycoside in the kidney is tubular cell toxicity, but a chronic treatment with G also modifies glomerular haemodynamics, i.e. reduces renal blood flow (RBF) and glomerular filtration rate (GFR) without apparent glomerular damage (Rodrı´guez-Barbero et al., 1997). G-induced GFR reduction has been attributed to a decline either in glomerular plasma flow or ultrafiltration coefficient (Kf), or both (Baylis, 1980; Santos et al., 1989; Schor et al., 1981; Rodrı´guez-Barbero et al., 2000). Kf regulation depends mainly on the intraglomerular mesangial cells (MC) activity because they possess the ability to contract or relax, thus modifying the ultrafiltration surface (Mene´, 2001). Kf reduction observed after G treatment has been attributed to mesangial contraction (Baylis, 1980; Schor et al., 1981). In previous studies from our laboratory we have demonstrated that G induces a dose-dependent MC contraction and proliferation (Martı´nez-Salgado et al., 1997; Rivas-Caban˜ero et al., 1997; Rodrı´guezBarbero et al., 1995). Our group also demonstrated G-induced mesangial apoptosis both in vivo and in vitro. The simultaneous occurrence of cellular proliferation and apoptosis may be a mechanism regulating glomerular cell number after acute treatment with G (Martı´nez-Salgado et al., 2004). We also reported that reactive oxygen species (ROS) generated in cultured MCs exposed to G or in glomeruli of rats treated with G, mediate mesangial contraction, proliferation (Martı´nez-Salgado et al., 2002) and apoptosis (Martı´nez-Salgado et al., 2004). Moreover, we have measured sustained increased levels of O2 in MC incubated with G (Martı´nez-Salgado et al., 2002). A possible source of superoxide anion (O2 ) could be NO synthase (NOS) activity. Superoxide generation can be generated by the activation of either endothelial constitutive NOS (cNOS) (Xia et al., 1998a) or inducible NOS (iNOS) (Xia et al., 1998b). In addition, O2 also mediates iNOS expression in rat cultured MC treated with IL-1ß (Beck et al., 1998), and we have already demonstrated that incubation of rat MC with G induces both iNOS expression and NO synthesis (Rivas-Caban˜ero et al., 1997). There are divergent opinions concerning the effects of NO on MC apoptosis, although there is increasing evidence of an antiproliferative and proapoptotic effect of NO (Albina et al., 1993; Bruene et al., 1995; Okuda et al., 1996; Bonfoco et al., 1996; Sandau et al., 1997; Rupprecht et al., 2000; Schaefer et al., 2003). Among the major redox-inducible transcription factors, Jun-AP1 seems to be crucial for oxidative activation in MC (Bohler et al., 2000; Ishikawa et al., 1997; Ishikawa and Kitamura, 2000; MorenoManzano et al., 1999). The transcriptional protein AP1 plays a role in cell proliferation mediated by vasoconstrictor hormones. Thus, it has been described that endothelin-1 induces the transitory expression of c-fos Montero et al., 1993), and the sustained expression of fra-1, c-jun and c-myc (Simonson et al., 1992). AP1 is a crucial mediator of apoptosis induced by oxidizing agents in MC (Ishikawa et al., 1997; Yokoo and Kitamura, 1997). Nevertheless, AP1 may act as an inducer or inhibitor of apoptosis depending on the stimulus or cell type (Ishikawa et al., 1997).

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The purpose of this study is to assess the expression of Jun-AP1 protein in both glomeruli and MC after G treatment. c-Jun is phosphorylated and activated by the mitogen-activated protein kinase c-Jun N-terminal kinase (JNK) (Whitmarsh and Davis, 1996), one of the major signal transduction molecules responsible for cell proliferation, apoptosis and a variety of genes expression (Kyriakis and Avruch, 2001; Bokemeyer et al., 1996). As JNK activation appears to be a prerequisite for the oxidative activation of MCs (Bohler et al., 2000; Ishikawa et al., 1997; Ishikawa and Kitamura, 2000; MorenoManzano et al., 1999), we examined the possibility that JNK is activated by G in MC. Moreover, as JNK is strongly activated by NO, and the pro-apoptotic action of NO in MC is mediated by JNK (Bruene, 2002), we studied the expression of both inducible and constitutive NOS in glomeruli from rats after an acute treatment with G.

Materials and methods Chemicals and reagents G sulphate was a kind gift of Shering Plough SA (Madrid, Spain). The sterile plastic material used in cell culture was obtained from Nunc (Roskilde, Denmark). Superoxide dismutase (SOD), catalase (CAT), phenyl-methyl-sulphonyl-fluoride (PMSF), Nonidet-P40 (NP40), sodium dodecyl sulfate (SDS) and ethylene-diamine-tetra-acetic acid (EDTA) were obtained from Sigma Quı´mica (Madrid, Spain). [3H]-methyl thymidine was from New England Nuclear (Bad Homburg, Germany). Crystal violet was obtained from Fluka (Buchs, Switzerland). Super Sensitive Immunodetection System and levamisol were purchased by Biogenex Laboratories (California, USA). Culture medium RPMI 1640 was from Gibco Labs (Barcelona, Spain); fetal calf serum (FCS) and trypsin solution were from Whittaker Labs (Barcelona, Spain). Rabbit anti-mouse Jun/AP1 and rabbit anti-rat JNK1 antibodies were from Santa Cruz Biotechnology (CA, USA). Rabbit anti-mouse iNOS and mouse anti-human cNOS antibodies were from Transduction Laboratories (Lexington, KY, USA). Goat anti-rabbit IgG (H + L) and goat antimouse horseradish peroxidase (HRP) conjugated antibodies were from BioRad (Madrid, Spain). Hyperfilm MP was obtained from Amersham (Buckinghamshire, UK). [g-32P]ATP was purchased from Perkin Elmer (Boston, MA, USA). Verapamil was a kind gift of Knoll AG (Ludwigshafen, Germany). All other reagents used were of analytical grade and obtained from Sigma Quı´mica (Madrid, Spain), Probus (Madrid, Spain) and Merck (Madrid, Spain). G-induced nephrotoxicity in rats Experiments were made in Wistar rats bred in the animal house of the Edificio Departamental (University of Salamanca, Spain). In vivo experiments were carried out in 250 g rats placed into metabolic cages in a temperature (20 8C), light and humidity-controlled animal house. Daily subcutaneous injections of G sulfate in saline solution (100 mg/kg body weight) were administered; studies were performed in rats treated with G during 2, 4 and 6 days, and 2 days after finishing the treatment with G during 6 days, as previously described (Martı´nez-Salgado et al., 2004, 2002). Animals were treated following the Recommendations from the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals stated in the international regulations and in the following European and national institutions: Conseil de ´lEurope (published in the Official Daily N.

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L358/1-358/6, 18th December 1986), and Spanish Government (published in Boletı´n Oficial del Estado N. 67, pp. 8509–8512, 18th March 1988, and Boletı´n Oficial del Estado N. 256, pp. 31349–31362, 28th October 1990). Glomerular isolation and mesangial cell culture Glomeruli were isolated by successive mechanical sieving of kidneys from 150 g Wistar rats, and primary cultures of MC were obtained as previously described (Rodrı´guez-Puyol et al., 1989). The identity of the cells was confirmed by morphological and functional criteria (Rodrı´guez-Puyol et al., 1989). Cells were incubated in 0.5% FCS for 24 h, thus reaching a quiescent state (Martı´nez-Salgado et al., 2002). Mesangial cell proliferation and apoptosis Cell proliferation was measured by both [3H-methyl]thymidine incorporation into DNA and counting the number of viable cells using the crystal violet method (Martı´nez-Salgado et al., 2002). For this purpose, cells were subcultured by treatment with 0.05% trypsin and 0.02% EDTA, and plated in 6  4 well plates. Experiments were performed on cells approaching confluence from the first passage in order to avoid cell dedifferentiation.

100 mg gentamicin / kg body weight day 0 day 2 day 4 day 6 day 6+2 cNOS 200

*

cNOS, % vs. control

100

0 day 0 day 2

day 4 day 6 day 6+2

100 mg gentamicin / kg body weight

Fig. 1. cNOS protein expression in glomeruli from gentamicin-treated rats, analyzed by western blot. Upper panel shows a representative blot of three different experiments, performed in similar conditions. Lower panel shows cNOS expression with respect to control glomeruli, day 0 (100%); data are expressed as mean F standard error of the mean (S.E.M.). *Statistically significant difference ( P b 0.01) vs. day 0.

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DNA fragmentation associated with apoptosis was detected by the terminal desoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) method and by nuclear staining with propidium iodide. For propidium iodide staining, renal slices (fixed as previously described for histological techniques) or monolayers of mesangial cells (fixed in 4% buffered formalin) were incubated for 45 min at 37 8C in PBS (pH 7.4) containing propidium iodide (0.1 Ag/mL) plus RNAse A (1 Ag/mL). TUNEL labeling was performed as previously described (Rodrı´guez-Lo´pez et al., 1998). Quantification of apoptotic nuclei in the glomeruli of renal slices was performed as previously described for the PCNA. Cell suspensions and membrane enriched fractions MCs were harvested from the surface of culture bottles by treatment with 0.05% trypsin and 0.02% EDTA, washed twice with phosphate buffered saline (PBS: 2.6 mM PO4H2K, 4.1 mM PO4HNa2, 0.81% NaCl, pH 7.4) and suspended in an appropriate buffer solution. Membrane-enriched protein fractions were obtained from MCs suspensions. Cells were lysed in 140 mM NaCl, 10 mM EDTA, 10% glycerol, 20 mM Tris pH 8, 100 U/mL aprotinin, 2 mM PMSF, 60 Ag/mL soya bean trypsin inhibitor and 1% NP40 at 4 8C for 15 min. The cell lysates were centrifuged at 5000 g for 18 min at 4 8C. The supernatants were centrifuged again at 18 000 g for 45 min at 4 8C. The pellets were collected and suspended in an appropriate buffer for analytical determinations. Protein content was determined by Bradford’s method (Bradford, 1976). 100 mg gentamicin / kg body weight day 0 day 2

day 4

day 6

day 6+2

iNOS 3000

iNOS, % vs. Control

* * *

2000

1000

*

0 day 0

day 2

day 4

day 6 day 6+2

100 mg gentamicin / kg body weight

Fig. 2. iNOS protein expression in glomeruli from gentamicin-treated rats, analyzed by western blot. Upper panel shows a representative blot of three different experiments, performed in similar conditions. Lower panel shows iNOS expression with respect to control glomeruli, day 0 (100%); data are expressed as mean F S.E.M. *Statistically significant difference ( P b 0.01) vs. day 0.

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Western blot analysis Immunoblots of MC or glomeruli were carried out as previously described (Valdivielso et al., 2001). Proteins (80 Ag in each lane) were separated in 8% (for cNOS, iNOS) and 15% (for Jun/AP1) SDSpolyacrylamide gel. Primary antibodies (anti-Jun/AP1, anti-cNOS, and anti-iNOS) and secondary antirabbit IgG or anti-mouse HRP conjugated antibodies were used at final concentrations between 0.1–1 Ag/ml and 0.01–1 Ag/ml, respectively. For quantification, films were digitalized with the Adobe Photoshop 3.0, and relative optical densities for each lane were measured using an image analysis program (MacBAS 2.2). Immunoprecipitation kinase assay JNK kinase activity was measured in immune complexes using c-Jun as the substrate. MCs plated in 100 mm dishes (2  105 cells/plate) were cultured for two days. Cells were placed in serum-free media overnight, and then treated with 10 5 M G for different periods. After treatment cells were washed twice with PBS before being lysed on ice in RIPA buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS) containing protease inhibitors (1 mM PMSF, 1 mM EDTA, 1 Ag/mL leupeptin, 1 Ag/mL pepstatin, 1 Ag/mL aprotinin, 25 mM h-glycerophosphate, 200 AM sodium orthovanadate, 10 mM NaF). Insoluble material was removed by centrifugation at 12 000 g for 10 min at 4 8C. Cell extracts were 100 mg gentamicin / kg body weight day 0 day 2 day 4 day 6 day 6+2 AP-1

*

2000

AP-1, % vs. control 1500

* 1000

* 500

0

* day 0 day 2

day 4 day 6 day 6+2

100 mg gentamicin / kg body weight Fig. 3. Jun-AP1 protein expression in glomeruli from gentamicin-treated rats, analyzed by western blot. Upper panel shows a representative blot of three different experiments, performed in similar conditions. Lower panel shows AP1 expression percentage with respect to control glomeruli, day 0 (100%); data are expressed as mean F S.E.M. *Statistically significant difference ( P b 0.01) vs. day 0.

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immunoprecipitated for 2 h at 4 8C with 2 Al of polyclonal anti-JNK1 antibody and 60 AL protein A-Sepharose. For measurement of JNK1 activity, the respective immunocomplexes were collected by centrifugation, washed 4 times with a washing buffer (50 mM Tris/HCl, pH 7,5, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1mM EDTA) and once with a kinase buffer (20 mM HEPES, pH 7,6, 20 mM MgCl2, 25 mM h-glycerophosphate) and resuspended in a final volume of 50 Al of kinase buffer containing 60 Ag/mL cJun, 10 AM ATP, 100 AM sodium orthovanadate, and 1 ACi [g-32P]ATP. The reaction was initiated by incubation at 30 8C and was continued for 30 min. Thereafter 30 Al of 2x Laemmli sample buffer were added to terminate the reaction. Samples were then boiled for 3 min and subjected to electrophoresis in 10% SDS-PAGE. The gels were dried and exposed for 24 to 48 h to Hyperfilm MP at 70 8C using an intensifying screen. Kinase activity was visualized and quantified by densitometry of the exposed autoradiographic film. A brief shock of UV light (100 mJul, ~5 s) was used as positive stimulus to activate JNK activity in MCs. Statistical methods The Kolmogorov–Smirnov test was used to assess normality of the data distribution. One-way analysis of variances and Scheffe´’s test were used for normally distributed data. A bPQ value lower than 0.05 was considered statistically significant.

10-5M gentamicin 0h

48 h

24 h

72 h

AP-1 200

*

AP-1, % vs. control (0 h)

* 100

0 0h

48 h

24 h

72 h

-5

10 M gentamicin

Fig. 4. Jun-AP1 protein expression in mesangial cells in culture, analyzed by western blot. Upper panel shows a representative blot of three different experiments, performed in similar conditions. Lower panel shows AP1 expression percentage with respect to cells incubated during 0 h (0.5% FCS, 100%); data are expressed as mean F S.E.M. *Statistically significant difference ( P b 0.01) vs. 0 h.

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Results NOS expression Treatment of rats with G induced an increased expression of endothelial type cNOS and iNOS in isolated renal glomeruli. The cNOS highest expression was observed in glomeruli from rats treated with G during 4 days, and 2 days after the end of G treatment (Fig. 1). Moreover, the maximal expression of iNOS was observed in glomeruli from rats treated with G during 4, 6 days, and 2 days after 6 days of G treatment (Fig. 2). Jun-AP1 expression Fig. 3 shows that G treatment in rats induced the expression of the 39 KDa protein Jun-AP1 in renal glomeruli. This expression was maximal in glomeruli from rats treated with G during 4 days, and 2 days after the end of G treatment. G also induced AP1 expression in quiescent cultured MC. The highest expression was observed after 24 h of incubation, and this expression was slightly lower after 48 and 72 h of G incubation (Fig. 4).

10-5M gentamicin 0 min 5 min 15 min 30 min C+ JNK activation 300

JNK activity, % vs. control

200

100

0 min 5 min 15 min 30 min -5 10 M gentamicin

Fig. 5. Time-course (5–30 min) of the effect of 10 5 M gentamicin on Jun kinase (JNK) activation in cultured MCs. Histogram represents the mean F S.E.M. of the optical density of the bands, expressed as % over the control (time 0), of 2 experiments. Abbreviations: C+: positive control (see Materials and methods text).

C. Martı´nez-Salgado et al. / Life Sciences 77 (2005) 2285–2298 10-5M gentamicin -5 10 M v erapamil SOD + CAT

-

+ -

+ + -

+ -

+ +

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+ C+

JNK activation

130

JNK activity, % vs. control

125 120 115 110 105 100

10-5M -5

gentamicin 10 M v erapamil SOD + CAT

-

+ -

+ + -

+ -

+ +

+

Fig. 6. Effect of calcium channel blocker verapamil and reactive oxygen species scavengers superoxide dismutase and catalase on Jun kinase (JNK) activation induced by 30 min administration of 10 5 M gentamicin in cultured mesangial cells. Verapamil (10 5 M) is added 30 min prior to gentamicin administration, and superoxide dismutase and catalase are added 10 min before gentamicin administration. Histogram represents the mean F S.E.M. of the optical density of the bands, expressed as % over the control (time 0), of 2 experiments. Abbreviations: SOD + CAT: 15 U/ml superoxide dismutase + 80 U/ml catalase; C+: positive control (see Materials and methods text).

JNK activation We observed that G (10 5 M) activated JNK in cultured MC, with maximal activity occurring after 5 min (~3-fold over control) and a progressive decrease afterwards (Fig. 5). Moreover, both the calcium Table 1 Effect of ROS scavengers superoxide dismutase and catalase, and calcium antagonist verapamil on mesangial cell proliferation induced by G 0.5% FCS G G + SOD + CAT SOD + CAT G+V

DNA synthesis (cpm/well)

Cells/well (% vs. 0.5% FCS)

200.12 F 12.31 1642.43 F 95.12a 331.75 F 34.35b 368.87 F 29.55b 717.11 F 98.44b

100.00 F 0.00 182.34 F 19.23a 122.12 F 9.44b 114.34 F 9.28b 110.66 F 11.30b

Values are mean + S.E.M. of 8–12 experiments done in triplicate. G: 10 CAT: 80 U/ml catalase; V: 10 5 M Verapamil. a P b 0.01 significantly different from 0.5% FCS. b P b 0.01 significantly different from G.

5

M gentamicin; SOD: 15 U/ml superoxide dismutase;

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Table 2 Effect of ROS scavengers superoxide dismutase and catalase on mesangial cell apoptosis induced by G G G + SOD + CAT SOD + CAT

Tunel (% apoptotic cells)

Propidium iodide staining (% apoptotic cells)

32.05 F 5.58 12.46 F 0.83a 12.23 F 0.54a

24.13 F 1.39 12.38 F 0.53a 11.16 F 0.35a

Values are mean + S.E.M. of 8–12 experiments done in triplicate. G: 10 CAT: 80 U/ml catalase. a P b 0.01 significantly different from G.

5

M gentamicin; SOD: 15 U/ml superoxide dismutase;

channel blocker verapamil, and the ROS scavengers superoxide dismutase and catalase reduced the Ginduced increase in JNK activation (Fig. 6). Cell proliferation and apoptosis Incubation with G induced a marked increase in MC proliferation (Table 1) and apoptosis (Table 2). G-induced MC proliferation was prevented by incubation with the ROS scavengers SOD + CAT, and by calcium channel blocker verapamil (Table 1). In addition, SOD + CAT also inhibited G-induced apoptosis (Table 2).

Discussion In the present study, we demonstrate that G treatment induces the expression of Jun-AP1 both in vivo (in glomeruli isolated from G-treated rats) and in vitro (in primary cultures of MC). G also induced an increased JNK activation in MC in vitro. Moreover, iNOS and cNOS expression was increased in glomeruli of rats treated in vivo with G. Calcium channel blocker verapamil and ROS scavengers superoxide dismutase and catalase, prevented G-induced mesangial proliferation and apoptosis, and also reduced the G-induced increase in JNK activation. In addition, blockade of NOS with L-NAME also prevented G-induced apoptosis. These results strongly suggest the role of increased NOS and JNK activity and Jun-AP1 expression in G-induced MC activation. Previous experiments from our laboratory showed that in cultured MC, G directly induces superoxide generation that mediated MC proliferation (Martı´nez-Salgado et al., 2002) and apoptosis (Martı´nezSalgado et al., 2004) either in vitro and in vivo. Jun-AP1, one of the main redox-inducible transcription factors, is crucial for MC oxidative activation (Bohler et al., 2000; Ishikawa et al., 1997; Ishikawa and Kitamura, 2000; Moreno-Manzano et al., 1999). Here we report that G stimulates AP1 expression either in vitro or in vivo. c-Jun is phosphorylated and activated by JNK (Whitmarsh and Davis, 1996). The increase in JNK activity is probably responsible for the elevation in AP1 expression in vitro (Kawano et al., 2003). Our data also show that G-induced JNK activation in cultured MC was reduced by coincubation with the ROS scavengers superoxide dismutase and catalase; thus, ROS seems to mediate the increase in JNK induced by G treatment. We have previously reported that G directly raises intracellular Ca2+; the increase in cytosolic free Ca2+ mediates G-induced MC contraction and proliferation (Mene´, 2001; Rodrı´guez-Barbero et al., 1995; Martı´nez-Salgado et al., 2000). The present data shows that G-induced JNK activation was

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reduced by verapamil, suggesting that increases in intracellular Ca2+ also mediates JNK activation induced by G. Published data about the involvement of transcription factors in MC (and other cell types) apoptosis and proliferation are conflictive. ROS induce the transcription of c-jun, c-fos and c-myc protooncogenes (Pinkus et al., 1996), which produce factors involved in differentiation and cell growth processes (Amstad et al., 1992). Recent data by Kawano et al. (2003) showed that AP1 activation is partially due to JNK activation, and is involved in MC proliferation; however, it is also known that AP1 is a crucial mediator of oxidizing agents-induced apoptosis in MC (Ishikawa et al., 1997; Yokoo and Kitamura, 1997). The transcriptional protein AP1 may participate in apoptosis regulation as an inductor or an inhibitor (Ishikawa et al., 1997); in our case, the time-course of AP1 expression in glomeruli of rats treated with G is similar to the expression of proliferating cell nuclear antigen (PCNA) that we have shown recently (Martı´nez-Salgado et al., 2004), suggesting that Jun-AP1 may be involved in MC proliferation or apoptosis inhibition. We can also suspect that Jun-AP1 participates in the transcription of genes involved in MC proliferation in vitro, since we found the lowest AP1 expression in quiescent cells, and AP-1 expression increased when MC were incubated with 10% FCS, which induces the maximal proliferative state (data not shown). G increases Jun-AP1 expression in quiescent MC in a time-dependent way being highest at 24 h, and decreases at 48 and 72 h. The time-course of AP1 expression is also similar to the expression of the proapoptotic protein Bax that we have previously described (Martı´nez-Salgado et al., 2004). Our data show increases in the expression of both inducible and constitutive NOS isoforms in glomeruli from rats treated with G. cNOS is expressed constitutively in glomeruli from untreated animals, whereas iNOS expression was induced as a result of G treatment. In both cases, we found the highest expression in the 4th day of G treatment. The increase in NOS expression probably increases NO production, as previous studies from our laboratory showed that glomeruli from Gtreated animals had increased NO production and cGMP levels (Rivas-Caban˜ero et al., 1997). Moreover, NOS activity can also be responsible of ROS production observed after G treatment (Martı´nez-Salgado et al., 2004, 2002). It has been shown that the pro-apoptotic action of NO in MC is transmitted at least in part by activation of JNK1/2, and that NO itself promoted strong JNK1/2 activation and apoptosis (Bruene, 2002). Moreover, G was able to induce both cNOS and iNOS in cultured MC (Rivas-Caban˜ero et al., 1997). Thus, we have assessed cNOS and iNOS expression in glomeruli of rats treated with G. NO shows an unusual divergence of action, working as a physiologic signaling molecule or as a toxic mediator. NO-mediated cellular damage occurs by different mechanisms: uncoupling of mitochondrial respiration, enzyme inhibition, lipid peroxidation and genetic mutations, through mediators as N2O3 and peroxinitrites (originated by the reaction between NO and O2 ); these processes may happen when high NO concentrations are generated by iNOS induction (Gordge, 1998). With the present data we can suggest that the increase in NOS expression and the increase in NO synthesis are involved in the increase in MC apoptosis in vivo. According with this, in vivo studies have demonstrated that G increases iNOS mRNA synthesis in MC, together with an increase in NO synthesis, and this NO seems to play an antiproliferative role in G-treated MC (Rivas-Caban˜ero et al., 1997). Moreover, in the apoptotic process induced in MC by increases in intracellular calcium concentrations, NO acts as an apoptotic-activating factor (Rodrı´guez-Lo´pez et al., 1999). In addition, Bruene (2002) showed that the pro-apoptotic action of NO in MC is transmitted at least in part by activation of JNK1/2,

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and NO itself promoted strong JNK1/2 activation and apoptosis. Although NO production seems to be more related with increased apoptosis than with cell proliferation, O2 released from increased NOS activity could also induce MC proliferation (Martı´nez-Salgado et al., 2002). In summary, we have shown that Jun-AP1 may participate in G-induced MC proliferation and/or apoptosis. On the other hand, NOS-produced increased ROS levels and increased intracellular calcium may mediate G-induced MC proliferation and apoptosis. We propose that the increase in NOS expression together with the elevation in intracellular Ca2+ activate NOS activities, and therefore ROS synthesis, which act as an apoptotic stimulus; moreover, increased ROS production and increased cytosolic free calcium also stimulate MC proliferation. The final balance of all these processes is what we can observe after either in vivo or in vitro treatment with G, the simultaneous occurrence of MC proliferation and apoptosis (Martı´nez-Salgado et al., 2004).

Acknowledgments This work was partially supported by a grant from Ministerio de Ciencia y Tecnologı´a (SAF2001/ 1701). We thank to Shering Plough, S.A., Madrid, Spain, for the kind gift of the gentamicin sulfate used in this experimental work.

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