Resveratrol inhibits gentamicin-induced mesangial cell contraction

Life Sciences 78 (2006) 2373 – 2377 www.elsevier.com/locate/lifescie

Resveratrol inhibits gentamicin-induced mesangial cell contraction Ana I. Morales, Alicia Rodrı´guez-Barbero, Cesa´reo Vicente-Sa´nchez, Paula Mayoral, Jose´ M. Lo´pez-Novoa, Fernando Pe´rez-Barriocanal * Instituto ‘‘Reina Sofı´a’’ de Investigacio´n Nefrolo´gica, Departamento de Fisiologı´a y Farmacologı´a, Universidad de Salamanca, Salamanca, Spain Received 3 June 2005; accepted 24 September 2005

Abstract Gentamicin is an aminoglycoside antibiotic that is very effective in treating different gram negative infections, however, one of its main side effects is nephrotoxicity. Gentamicin-induced decreases in glomerular filtration rate could be mediated by mesangial cell contraction. Resveratrol, a natural hydroxystilbene, has been identified to be a potent antioxidant with many biological activities including protection against kidney diseases. As we have previously demonstrated that gentamicin induced a reduction of planar surface area of cultured rat mesangial cells, and that resveratrol has a protective effect on gentamicin-induced nephrotoxicity in vivo, the aim of this study was to investigate the effect of resveratrol on gentamicin-induced mesangial cell contraction. This study demonstrates that the contractile effect of gentamicin on mesangial cells can be prevented by incubation with resveratrol at an optimal dose of 10 AM, as it blunted the gentamicininduced reduction in planar cell surface area and the number of contracted cells. Besides, the preincubation with 10 5M diphenylene iodinium (DPI), an inhibitor of the NADP(H) oxidase, also blunted gentamicin-induced cell contraction. This preventive effect was higher when cells were incubated with both substances together. These results strongly suggest that the protective effect resveratrol against gentamicin-induced reduction in renal function in vivo could be mediated by inhibiting gentamicin-induced mesangial cells contraction. D 2005 Elsevier Inc. All rights reserved. Keywords: Gentamicin; Mesangial cells; Nephrotoxicity; Reactive oxygen species; Resveratrol

Introduction Gentamicin is an aminoglycoside antibiotic that is very effective in treating different gram negative infections (Ho and Barza, 1987). However, one of its main side effects is nephrotoxicity (Kaloyanides, 1991). In addition of its tubular effects, gentamicin induces decreases in glomerular filtration rate (GFR) (Rodrı´guez-Barbero et al., 1997) that have been associated with a marked decline in the ultrafiltration coefficient (Kf) which could be mediated by mesangial cell contraction (Avasthi et al., 1981; Baylis, 1980; Schor et al., 1981). Mesangial cells are perivascular pericytes located within the central portion of the glomerular tuft between capillary loops. Most authors agree that mesangial cell contraction plays a major role in regulation of filtration surface area and Kf. We previously * Corresponding author. Departamento de Fisiologı´a y Farmacologı´a, Edificio Departamental, Campus Miguel de Unamuno, Universidad de Salamanca, 37007 Salamanca, Spain. Tel.: +34 923294472; fax: +34 923294669. E-mail address: [email protected] (F. Pe´rez-Barriocanal). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.09.045

have demonstrated that gentamicin induces a contraction of cultured rat mesangial cells (Rodrı´guez-Barbero et al., 1995; Martı´nez-Salgado et al., 1997), and we have suggested that mesangial cell contraction is involved in gentamicin-induced renal failure (Rodrı´guez-Barbero et al., 2000). Increased superoxide production seems to play a major role in gentamicin-induced mesangial cell contraction (Martı´nez-Salgado et al., 2002) and proliferation (Martı´nez-Salgado et al., 2005). Trans-resveratrol (trans-3,5,4V-hydroxystilbene) is an important natural antioxidant that possesses many biological activities including protection against kidney diseases, such as ischemic –reperfusion injury (Giovannini et al., 2001; Bertelli et al., 2002) and gentamicin-induced nephrotoxicity (Morales et al., 2002). The aim of this study was to test the hypothesis that resveratrol protection against gentamicin nephrotoxicity is mediated, at least in part, by preventing mesangial cell contraction. In the present study, we demonstrate that resveratrol inhibits gentamicin-induced mesangial cell contraction. Thus, this study

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Materials and methods Materials and reagents The sterile plastic material used in cell culture was obtained from Nunc (Denmark). Resveratrol and diphenylene iodinium (DPI) were obtained from Sigma-Aldrich Quı´mica (Spain). Gentamicin was a gift from Schering-Plough Laboratories (Spain). Culture medium RPMI 1640 was from Gibco (Spain) and fetal calf serum (FCS) from Whittaker (Spain). All other reagents used were of analytical grade and obtained from SigmaAldrich Quı´mica (Spain), Probus (Spain) and Merck (Spain). Mesangial cell culture Primary cultures of mesangial cells were obtained from 150 g Wistar rat glomeruli isolated by successive mechanical sieving as we previously described (Rodrı´guez-Barbero et al., 1995; Martı´nez-Salgado et al., 1997). Rats were bred in the animal house of the Edificio Departamental (University of Salamanca, Spain). 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 l’Europe (published in the Official Daily N. 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). The culture medium consisted of RPMI 1640 supplemented with 10% heat-inactivated FCS (Bio Whittaker, Barcelona, Spain), l-glutamine, penicillin/streptomycin, insulin, transferrin, selenium and amphotericin B and was buffered with 5% CO2 (pH 7.2), as previously described (Rodrı´guez-Barbero et al., 1995). The culture medium was changed every 2 days. Cells were confirmed as mesangial by morphological and functional criteria previously described (Rodrı´guez-Puyol et al., 1989; Olivera et al., 1992). Studies were performed using growing cells in passages 5 –7. Mesangial cell contraction Direct observation of the mesangial cells grown in conventional plastic culture plates was carried out at 30 -C under phase contrast with an inverted Nikon photomicroscope using a CCD video camera (Hitachi KP 110) and a Hitachi monitor. Cells were left for 1 h to stabilize, then photographs were taken before and after experimental treatments using an on-line video printer (Sony UP-910; Sony Corp., Tokyo, Japan) (Rodrı´guez-Barbero et al., 1995). The cells were treated with 10 5 mol/L gentamicin for 1 h or resveratrol (5, 10, 20 and 50 Amol/L) and/or DPI (10 5 mol/L) for 30 min prior gentamicin addition. Planar cell surface area (PCSA) was

determined by computerized image analysis using the scion image system from NIH (version Beta 4.0.2). Actual area was calculated after correction for microscope and photographic magnification. Each individual cell served as its own control. Five to ten cells were analyzed per photograph. In every experimental set cells were from a single culture. Experiments were performed, at least, by triplicate. Mesangial cell contraction was calculated in two different ways: 1) decreased PCSA at the end of the treatment, expressed as the percentage of the initial area, and 2) number of contracted cells at the end of the treatment, considering contraction is 10% or has a higher reduction in PCSA. Statistical analysis The Kolmogorov – Smirnov test was used to assess the normality of data distribution. Results are expressed as mean T standard error of the mean (X T SEM) for parametric data. Statistical analysis was performed by one-way (ANOVA 1) analysis of variance for group comparisons followed by Scheffe’s test when the data were normally distributed and by the Kruskal – Wallis test when they were not normally distributed. Statistical tests were performed using the Number Cruncher Statistical System (NCSS), version 6.0.10 for Windows. A P value < 0.05 and a z value > 1.96 were considered significant.

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provides important new insights into the pathways that may contribute to the beneficial effects of resveratrol in kidney disease.

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Fig. 1. Effect of resveratrol (5, 10, 20 and 50 AM) on gentamicin-induced mesangial cell contraction (gentamicin 10 5M). Mesangial cell contraction was calculated as planar cell surface area (PCSA) reduction in cultured mesangial cells during 60 min (A) and as percentage (%) of contracted cells in the same groups during 60 min (B). Data are means T SEM of, at least, 3 experiments with 5 to 10 cells measured in each one. Statistically significant differences are: + P < 0.05, vs. cells incubated in control conditions. *P < 0.05, vs. cells incubated with gentamicin 10 5M.

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Results

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The addition of gentamicin to the incubation medium reduced mesangial PCSA and increased the number of contracted cells with respect to cells incubated in control conditions (Fig. 1A and B). Resveratrol at the concentration of 10 and 20 AM reduced significantly the contractile response to gentamicin 10 5 M, while the 50 AM resveratrol on gentamicin-induced reduction in PCSA was not statistically significant (Fig. 1A). The percentage of gentamicin-induced contracted cells decreased significantly in the presence of resveratrol, mainly at the concentration of 10 AM (Fig. 1B). No changes in planar cell surface area (PCSA) could be observed when mesangial cells were incubated in control conditions or with resveratrol alone at doses of 10 and 20 AM. However, PCSA % was decreased at a dose of 50 AM (Fig. 2A). The number of contracted cells treated with resveratrol at doses of 10, 20 and 50 AM was not significantly different to that of cells incubated with the culture medium (Fig. 2B). We next assessed the effect of NADP(H) inhibition on gentamicin-stimulated mesangial cell contraction. Pretreatment of mesangial cells with diphenylene iodinium (DPI) (10 5M), an inhibitor of the NADP(H) oxidase, reduced the gentamicininduced cell contraction (Fig. 3A). The number of contracted cells decreased significantly when cells treated with gentamicin were preincubated with DPI (Fig. 3B). Cells pretreated with

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Fig. 3. Effect of diphenylene iodinium (DPI) 10 M on gentamicin-induced mesangial cell contraction (gentamicin 10 5M). Mesangial cell contraction was calculated as planar cell surface area (PCSA) reduction in cultured mesangial cells during 60 min (A) and as percentage (%) of contracted cells in the same groups during 60 min (B). Data are means T SEM of, at least, 3 experiments with 5 to 10 cells measured in each one. Statistically significant differences are: + P < 0.05, vs. cells incubated in control conditions. *P < 0.05, vs. cells incubated with gentamicin 10 5M.

resveratrol 10 AM and DPI 10 5 M showed a gentamicininduced reduction in PCSA lower that those receiving DPI or resveratrol alone although the difference did not reach statistical significance (Fig. 4A). The percentage of contracted cells, when mesangial cells were incubated both with gentamicin (10 5 M) and resveratrol (10 AM) plus DPI (10 5 M), was lower than when cells were incubated with these substances separately (Fig. 4B), although the differences did not reach statistical significance. Discussion

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Fig. 2. Effect of resveratrol (5, 10, 20 and 50 AM) on mesangial cell contraction. Mesangial cell contraction was calculated as planar cell surface area (PCSA) reduction in cultured mesangial cells during 60 min (A) and as percentage (%) of contracted cells in the same groups during 60 min (B). Data are means T SEM of, at least, 3 experiments with 5 to 10 cells measured in each one. Statistically significant differences are: +P < 0.05, vs. cells incubated in control conditions.

Despite its nephrotoxic potential, the aminoglycoside antibiotic gentamicin is still considered to be an important agent against life-threatening infections (Ali, 2003). The goal of protecting against gentamicin nephrotoxicity has attracted much research effort during the last decade. Potential therapeutic approaches to protect or reverse renal gentamicin damage would have very important clinical consequences in increasing the safety of the drug. The major finding of this study is that resveratrol inhibits gentamicin-induced mesangial cell contraction in primary cultured rat mesangial cells. This result agrees with the observation that treatment with resveratrol protects the kidney against gentamicin-induced nephrotoxicity (Morales et al., 2002). Although the best known effect of this aminoglycoside

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Fig. 4. Effect of resveratrol (10 AM) and diphenylene iodinium (DPI) 10 5M on gentamicin-induced mesangial cell contraction (gentamicin 10 5M). Mesangial cell contraction was calculated as planar cell surface area (PCSA) reduction in cultured mesangial cells during 60 min (A) and as percentage (%) of contracted cells in the same groups during 60 min (B). Data are means T SEM of, at least, 3 experiments with 5 to 10 cells measured in each one. Statistically significant differences are: +P < 0.05, vs. cells incubated in control conditions. *P < 0.05, vs. cells incubated with gentamicin 10 5M.

in the kidney is tubular cell toxicity, a chronic treatment with gentamicin also modifies glomerular haemodynamics, i.e. reduces renal blood flow (RBF) and GFR without apparent morphological glomerular damage (Rodrı´guez-Barbero et al., 1997). Gentamicin-induced GFR reduction has been attributed to a decline either in RBF or in Kf, or both (Avasthi et al., 1981; Baylis, 1980; Schor et al., 1981). Kf regulation depends mainly on the activity of intraglomerular mesangial cells because they possess the capacity to contract or relax, thus modifying the ultrafiltration surface; this dynamic phenomenon is highly regulated by numerous vasoactive substances (Mene´, 2001). The reduction of the Kf observed after gentamicin treatment in vivo has been attributed to a mesangial contractile response (Baylis, 1980; Rodrı´guez-Barbero et al., 2000; Schor et al., 1981). Resveratrol is a major component of the polyphenols from grapes and red wine. A number of studies have demonstrated the antioxidant effects of resveratrol (Martı´nez and Moreno, 2000; Olas et al., 1999). This amphipathic molecule is capable of scavenging lipid hydroperoxyl free radicals as well as hydroxyl and superoxide anion radicals (Das et al., 1999; Ray et al., 1999). Resveratrol was found to protect the kidney, heart and brain from ischemic –reperfusion injury via its antioxidant ability (Bastianetto et al., 2000; Das et al., 1999; Ray et al., 1999).

There is increasing evidence suggesting that gentamicininduced glomerular dysfunction in vivo is mediated by reactive oxygen species (ROS) since administration of antioxidants attenuated the reduction in GFR (Walker et al., 1999; Ali, 1995; Nakajima et al., 1994; Zurovsky and Habe, 1995). In vitro experiments showed that ROS production is enhanced by gentamicin and that renal cortical mitochondria were the source of ROS (Yang et al., 1995). Besides, an elevated production of ROS may augment renal susceptibility to gentamicin observed in obstructive jaundice (Tajiri et al., 1995). In previous studies from our laboratory we have demonstrated that gentamicin induces a dose-dependent mesangial cells contraction and proliferation (Martı´nez-Salgado et al., 1997; Rivas-Caban˜ero et al., 1997; Rodrı´guez-Barbero et al., 1995) and that gentamicininduced mesangial cells activation occurs, at least in part, via the generation of O2 (Martı´nez-Salgado et al., 2002, 2005). Superoxide dismutase (SOD) administration in rats treated with gentamicin was associated with a marked increase in RBF, suggesting that O2 must be responsible for renal vasoconstriction induced by gentamicin in vivo (Nakajima et al., 1994). Indeed, studies from our laboratory demonstrate that protective effect of resveratrol on the impairment of renal function induced by gentamicin was associated with its ability to prevent the increase in lipoperoxidation (Morales et al., 2002). The present study demonstrates that the effect of gentamicin on mesangial cell contraction can be prevented by incubation with resveratrol at an optimal dose of 10 AM. High doses of resveratrol (50 AM) did not show these protective effects, and when mesangial cells are incubated with these high resveratrol doses alone, cell contraction can be observed. This cell contraction can be explained by the toxic effect of reveratrol at high concentrations as it has been reported that resveratrol and other antioxidants can also have cytotoxic properties (Tedesco et al., 2005). Previous studies from our laboratory demonstrate that DPI, an inhibitor of NADP(H) oxidase, inhibited almost completely gentamicin-induced O2 generation by mesangial cells suggesting that this enzyme could be one of the sources of O2 in gentamicin-stimulated mesangial cells (Martı´nez-Salgado et al., 2002). In the present study, we also report that the preincubation with DPI blunted the gentamicin-induced reduction in PCSA and the increase in the number of contracted cells. The prevention of the contractile effects of gentamicin on mesangial cells was not significantly higher when cells were incubated with resveratrol and DPI than when the cells were incubated with resveratrol or DPI alone, thus suggesting that the effects of resveratrol are dependent mainly on its ability to block O2 synthesis. Conclusion We have shown the inhibition by resveratrol of gentamicininduced mesangial cells contraction in vitro. This inhibition might explain the improvement of glomerular function produced by the administration of antioxidants in animals treated with gentamicin (Ali, 1995; Nakajima et al., 1994; Nakamura et al., 1998; Morales et al., 2002). In addition, our

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results suggest that the protective effects of resveratrol on gentamicin-induced mesangial cells contraction could be due to either its properties as ROS scavengers or by inhibiting O2 generation by NADP(H) oxidase. Acknowledgements This work was partially supported by grants from Comisio´n Interministerial de Ciencia y Tecnologı´a (CICYT, SAF 20011701) to J.M. L-N. and from the Instituto Nacional de Investigacio´n y Tecnologı´a Agraria y Alimentaria (INIA, VIN02019) to A.M. We thank Shering Plough, S.A., Madrid, Spain, for the kind gift of gentamicin sulfate used in this experimental work. References Ali, B.H., 1995. Gentamicin nephrotoxicity in humans and animals: some recent research. General Pharmacology 26, 1477 – 1487. Ali, B.H., 2003. Agents ameliorating or augmenting experimental gentamicin nephrotoxicity: some recent research. Food and Chemical Toxicology 41 (11), 1447 – 1452. Avasthi, P.S., Evan, A.P., Huser, J.W., Luft, F.C., 1981. Effect of gentamicin on glomerular ultrastructure. Journal of Laboratory and Clinical Medicine 98 (3), 444 – 454. Bastianetto, S., Zheng, W.H., Quirion, R., 2000. Neuroprotective abilities of resveratrol and other red wine constituents against nitric oxide-related toxicity in cultured hippocampal neurons. British Journal of Pharmacology 131 (4), 711 – 720. Baylis, C., 1980. The mechanism of the decline in glomerular filtration rate in gentamicin-induced acute renal failure in the rat. Journal of Antimicrobial Chemotherapy 6, 381 – 386. Bertelli, A.A., Migliori, M., Panichi, V., Origlia, N., Filippi, C., Das, D.K., Giovannini, L., 2002. Resveratrol, a component of wine and grapes, in the prevention of kidney disease. Annals of the New York Academy of Sciences 957, 230 – 238. Das, D.K., Sato, M., Ray, P.S., Maulik, G., Engelman, R.M., Bertelli, A.A., Bertelli, A., 1999. Cardioprotection of red wine: role of polyphenolic antioxidants. Drugs Under Experimental and Clinical Research 25 (2 – 3), 115 – 120. Giovannini, L., Migliori, M., Longoni, B.M., Das, D.K., Bertelli, A.A., Panichi, V., Filippi, C., Bertelli, A., 2001. Resveratrol, a polyphenol found in wine, reduces ischemia reperfusion injury in rat kidneys. Journal of Cardiovascular Pharmacology 37 (3), 262 – 270. Ho, J.L., Barza, M., 1987. Role of aminoglycoside antibiotics in the treatment of intra abdominal infections. Antimicrobial Agents and Chemotherapy 31, 485 – 491. Kaloyanides, G.J., 1991. Metabolic interactions between drugs and renal tubulointerstitial cell: role in nephrotoxicity. Kidney International 39, 531 – 540. Martı´nez, J., Moreno, J.J., 2000. Effect of resveratrol, a natural polyphenolic compound, on reactive oxygen species and prostaglandin production. Biochemical Pharmacology 59 (7), 865 – 870. Martı´nez-Salgado, C., Rodrı´guez-Barbero, A., Rodrı´guez-Puyol, D., Pe´rez de Lema, G., Lo´pez-Novoa, J.M., 1997. Involvement of phospholipase A2 in gentamicin-induced rat mesangial cell activation. American Journal of Physiology 273, F60 – F66. Martı´nez-Salgado, C., Eleno, N., Tavares, P., Rodrı´guez-Barbero, A., Garcı´aCriado, J., Bolan˜os, J.P., Lo´pez-Novoa, J.M., 2002. Involvement of reactive

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