Metformin prevents experimental gentamicin-induced nephropathy by a mitochondria-dependent pathway
2010; Elsevier BV; Volume: 77; Issue: 10 Linguagem: Inglês
10.1038/ki.2010.11
ISSN1523-1755
AutoresAna I. Morales, Dominique Détaille, Marta Prieto, Angel Puente, Elsa Briones, Miguel Arévalo, Xavier Leverve, José M. López‐Novoa, Mohamad‐Yehia El‐Mir,
Tópico(s)Hyperglycemia and glycemic control in critically ill and hospitalized patients
ResumoThe antidiabetic drug metformin can diminish apoptosis induced by oxidative stress in endothelial cells and prevent vascular dysfunction even in nondiabetic patients. Here we tested whether it has a beneficial effect in a rat model of gentamicin toxicity. Mitochondrial analysis, respiration intensity, levels of reactive oxygen species, permeability transition, and cytochrome c release were assessed 3 and 6 days after gentamicin administration. Metformin treatment fully blocked gentamicin-mediated acute renal failure. This was accompanied by a lower activity of N-acetyl-β-D-glucosaminidase, together with a decrease of lipid peroxidation and increase of antioxidant systems. Metformin also protected the kidney from histological damage 6 days after gentamicin administration. These in vivo markers of kidney dysfunction and their correction by metformin were complemented by in vitro studies of mitochondrial function. We found that gentamicin treatment depleted respiratory components (cytochrome c, NADH), probably due to the opening of mitochondrial transition pores. These injuries, partly mediated by a rise in reactive oxygen species from the electron transfer chain, were significantly decreased by metformin. Thus, our study suggests that pleiotropic effects of metformin can lessen gentamicin nephrotoxicity and improve mitochondrial homeostasis. The antidiabetic drug metformin can diminish apoptosis induced by oxidative stress in endothelial cells and prevent vascular dysfunction even in nondiabetic patients. Here we tested whether it has a beneficial effect in a rat model of gentamicin toxicity. Mitochondrial analysis, respiration intensity, levels of reactive oxygen species, permeability transition, and cytochrome c release were assessed 3 and 6 days after gentamicin administration. Metformin treatment fully blocked gentamicin-mediated acute renal failure. This was accompanied by a lower activity of N-acetyl-β-D-glucosaminidase, together with a decrease of lipid peroxidation and increase of antioxidant systems. Metformin also protected the kidney from histological damage 6 days after gentamicin administration. These in vivo markers of kidney dysfunction and their correction by metformin were complemented by in vitro studies of mitochondrial function. We found that gentamicin treatment depleted respiratory components (cytochrome c, NADH), probably due to the opening of mitochondrial transition pores. These injuries, partly mediated by a rise in reactive oxygen species from the electron transfer chain, were significantly decreased by metformin. Thus, our study suggests that pleiotropic effects of metformin can lessen gentamicin nephrotoxicity and improve mitochondrial homeostasis. The major clinical problem in the use of gentamicin, an aminoglycoside antibiotic extensively used in the treatment of Gram-negative bacterial infection,1.Singenthaler W. Bonetti A. Luthy R. Aminoglycoside antibiotics in infectious diseases.Am J Med. 1986; 80: 2-11Abstract Full Text PDF Scopus (52) Google Scholar is its nephrotoxicity even at the lowest therapeutic doses.2.Humes H.D. Aminoglycoside nephrotoxicity.Kidney Int. 1988; 33: 900-911Abstract Full Text PDF PubMed Scopus (184) Google Scholar Indeed, in addition to tubular toxicity, a part of gentamicin-induced renal damage is based on its glomerular effects, especially those altering the function of mesangial cells.3.Martinez-Salgado C. Lopez-Hernandez F.J. Lopez-Novoa J.M. Glomerular nephrotoxicity of aminoglycosides.Toxicol Appl Pharmacol. 2007; 223: 86-98Crossref PubMed Scopus (192) Google Scholar, 4.Rodriguez-Barbero A. L’Azou B. 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Sonoda K. et al.Reactive oxygen species from mitochondria induce cyclooxygenase-2 gene expression in human mesangial cells: potential role in diabetic nephropathy.Diabetes. 2003; 52: 2570-2577Crossref PubMed Scopus (272) Google Scholar,10.Plotnikov E.Y. Kazachenko A.V. Vyssokikh M.Y. et al.The role of mitochondria in oxidative and nitrosative stress during ischemia/reperfusion in the rat kidney.Kidney Int. 2007; 72: 1493-1502Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar In fact, numerous in vivo and in vitro studies showed that reactive oxygen species (ROS) were often involved in the onset and progression of these injuries.11.Baliga R. Ueda N. Walker P.D. et al.Oxidant mechanisms in toxic acute renal failure.Drug Metab Rev. 1999; 31: 971-997Crossref PubMed Scopus (296) Google Scholar,12.Forbes J.M. Coughlan M.T. Cooper M.E. Oxidative stress as a major culprit in kidney disease in diabetes.Diabetes. 2008; 57: 1446-1454Crossref PubMed Scopus (843) Google Scholar Similarly, ROS-induced cell death processes were proposed to have a relevant role in gentamicin-mediated acute renal failure, characterized by necrosis of proximal tubular cells13.Rodriguez-Barbero A. Lopez-Novoa J.M. Arevalo M. Involvement of platelet-activating factor in gentamicin nephrotoxicity in rats.Exp Nephrol. 1997; 5: 47-54PubMed Google Scholar,14.Pannu N. Nadim M.K. An overview of drug-induced acute kidney injury.Crit Care Med. 2008; 36: S216-S223Crossref PubMed Scopus (221) Google Scholar and simultaneous occurrence of glomerular cell proliferation and apoptosis.15.Martinez-Salgado C. Eleno N. Tavares P. et al.Involvement of oxygen reactive species on gentamicin-induced mesangial cell activation.Kidney Int. 2002; 62: 1682-1692Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar,16.Servais H. Ortiz A. Devuyst O. et al.Renal cell apoptosis induced by nephrotoxic drugs: cellular and molecular mechanisms, and potential approaches to modulation.Apoptosis. 2008; 13: 11-32Crossref PubMed Scopus (152) Google Scholar Interestingly enough, gentamicin enhanced ROS formation in isolated cortical mitochondria.17.Walker P.D. Shah S.V. Gentamicin enhanced production of hydrogen peroxide by renal cortical mitochondria.Am J Physiol. 1987; 253: C495-C499PubMed Google Scholar We previously reported that treatment with resveratrol, a natural antioxidant, modulated the toxic effects of gentamicin by preventing increase in oxidative stress.18.Morales A.I. Buitrago J.M. Santiago J.M. et al.Protective effect of trans-resveratrol on gentamicin-induced nephrotoxicity.Antioxid Redox Signal. 2002; 4: 893-898Crossref PubMed Scopus (63) Google Scholar Similar protective effects on renal function in response to gentamicin injection were found with other ROS scavengers.19.Maldonado P. Barrera D. Rivero I. et al.Antioxydant S-allylcysteine prevents gentamicin-induced oxidative stress and renal damage.Free Radic Biol Med. 2003; 35: 317-324Crossref PubMed Scopus (142) Google Scholar, 20.Bledsoe G. Crickman S. Mao J. et al.Kallikrein/kinin protects against gentamicin-nephro-toxicity by inhibition of inflammation and apoptosis.Nephrol Dial Transplant. 2006; 21: 624-633Crossref PubMed Scopus (84) Google Scholar, 21.Priyamvada S. Priyadarshini M. Arivarasu N.A. et al.Studies of the protective effect of dietary fish oil on gentamicin-induced nephrotoxicity and oxidative damage in rat kidney.Prostaglandin Leukot Essent Fatty Acids. 2008; 78: 369-381Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar However, the exact biochemical mechanisms involved in nephroprotection, as well the specific contribution of mitochondria in this event, were barely examined in most of these studies. On the contrary, data revealing the preventive actions of metformin, a reference medication for prime treatment of type-2 diabetes and its long-term complications,22.Wiernsperger N. 50 years later: is metformin a vascular drug with antidiabetic properties?.Br J Diabetes Vasc Dis. 2007; 7: 204-210Crossref Scopus (26) Google Scholar have accumulated over the past few years. Recent investigations strongly showed that this antidiabetic agent prevented oxidative stress-induced death in several cell types,23.Guigas B. Detaille D. Chauvin C. et al.Metformin inhibits mitochondrial permeability transition and cell death: a pharmacological in vitro study.Biochem J. 2004; 382: 877-884Crossref PubMed Scopus (120) Google Scholar,24.El-Mir M.Y. Detaille D. Villanueva G.R. et al.Neuroprotective role of antidiabetic drug metformin against apoptotic cell death in primary cortical neurons.J Mol Neurosci. 2008; 34: 77-87Crossref PubMed Scopus (158) Google Scholar including human endothelial cells,25.Detaille D. Guigas B. Chauvin C. et al.Metformin prevents high glucose-induced endothelial cell death through a mitochondrial permeability transition-dependent process.Diabetes. 2005; 54: 2179-2187Crossref PubMed Scopus (195) Google Scholar through a mechanism dependent on the mitochondrial permeability transition pore (PTP) opening. Importantly, a randomized comparative trial showed that, for an equivalent effect on glycemic control after years of treatment, metformin was largely superior to other therapeutic measures for reducing vessel diseases and all-cause-related mortality.26.UK Prospective Diabetes Study GroupIntensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33).Lancet. 1998; 352: 837-853Abstract Full Text Full Text PDF PubMed Scopus (17578) Google Scholar Therefore, the aim of this study was to examine the potential properties of metformin protecting the kidney from a nephrotoxicant insult, as well to investigate the main underlying mechanisms. Gentamicin exposure led to acute renal failure, as evidenced by decreased creatinine clearance and increased excretion of N-acetyl-β-D-glucosaminidase (Table 1). Treated animals also had a lower glomerular filtration rate (Figure 1a), reduced renal plasma flow (RPF) and blood flow (RBF) (Figure 1b and c), as well as higher renal vascular resistance (Figure 1d) than control rats. Metformin fully mitigated this nephrotoxic profile as rats receiving both metformin and gentamicin showed higher glomerular filtration rate, RPF, and RBF than gentamicin-treated rats, the end values even reaching or rising above those in the control group (Figure 1). Expectedly, lipid peroxidation substantially increased with gentamicin as compared with that in the control group (Figure 2a), whereas plasma total antioxidant status (TAS) simultaneously diminished (Figure 2b). Importantly, metformin, when administered before and together with gentamicin, not only normalized lipid peroxidation but also increased TAS (Figure 2).Table 1Urinary flow, NAG activity, proteinuria, and creatinine clearance in control rats, rats that received a daily intraperitoneal injection of gentamicin, rats that received metformin-supplemented drinking water, and rats that received metformin plus gentamicin (n=4, performed in duplicate)ControlGentamicinMetforminMetformin+gentamicinUrinary flow (ml day−1)12.5±4.47.6±1.518.7±10.959.8±19.4†,§NAG activity (AU day−1)0.94±0.1718.2±4.5†0.96±0.11.6±0.3§Proteinuria (mg day−1)20.7±5.839.9±7.6†26.3±6.915.6±7.9§Creatinine clearance (ml min−1)1.25±0.0150.096±0.04†1.4±0.031.3±0.2§Abbreviation: NAG, N-acetyl-β-D-glucosaminidase.†P<0.05 versus control; §P<0.05 versus gentamicin. Open table in a new tab Figure 2Effect of metformin on the oxidative stress in vivo related to gentamicin-induced nephrotoxicity in rats. (a) Lipid peroxidation, as estimated by production of thiobarbituric acid-reactive substances (TBARS) in kidney homogenates and (b) total antioxidant systems (TAS) of the plasma were accordingly assessed in the four groups of rats studied. #P<0.05 versus control; *P<0.05 versus gentamicin.View Large Image Figure ViewerDownload (PPT) Abbreviation: NAG, N-acetyl-β-D-glucosaminidase. †P<0.05 versus control; §P<0.05 versus gentamicin. Light-microscopic examination of kidneys from control and metformin-treated rats showed no structural alterations in renal tissues (Figure 3a and c). Massive and diffuse cell necrosis was observed in the proximal tubules of kidneys from rats injected with gentamicin. In addition, the tubular lumen was frequently filled with hyaline casts or heterogeneous cellular debris (Figure 3b). In contrast, in rats treated with gentamicin and metformin, most of the proximal tubules showed completely viable cells, and manifest necrosis was observed in less than 10% of cells, although most proximal tubular cells showed signs of initial cellular degeneration (Figure 3d). Paradoxically, such a protection afforded by metformin against anatomical and functional signs of gentamicin-induced nephrotoxicity was not accompanied by a reduction, but rather by an apparent increase in the renal accumulation of aminoglycoside. Indeed, metformin-treated rats incorporated two times more gentamicin in their renal cortices than did rats treated with only gentamicin (1235±192 versus 666±88 μg/g tissue, P<0.05), whereas low amounts of gentamicin present in the medulla did not significantly vary between both animal groups (103±13 versus 136±34 μg/g tissue). We propose that a profound mitochondrial dysfunction is at the origin of gentamicin-caused pathogenesis, leading ultimately to necrotic degradation of kidney cells. To test this theory, we addressed the questions of whether PTP opening and cytochrome c release were involved in gentamicin toxicity, and how metformin modulated these events in vivo. This experimental set was mainly conducted using rats receiving only three doses of gentamicin to assess the early occurrence of mitochondrial changes. Similar to that observed in the liver,27.Leverve X. Fontaine E. Role of substrates in the regulation of mitochondrial function in situ.IUBMB Life. 2001; 52: 221-229Crossref PubMed Scopus (33) Google Scholar kidney mitochondria that were energized with glutamate–malate or succinate absorbed and retained Ca2+ until the final increase in fluorescence (Figure 4a), indicative of mitochondrial permeability transition induced by calcium overload, and were also sensitive to cyclosporine-A (CsA), the reference PTP inhibitor. Metformin increased the Ca2+ amount required for PTP opening, irrespective of the nature of respiratory substrates. Importantly, addition of Ca2+ to mitochondria from rats treated with either three or six doses of gentamicin, stimulated a release of accumulated Ca2+ under all conditions, a phenomenon that was hindered by metformin and CsA (Figure 4b). We next studied cytochrome c compartmentalization during gentamicin treatment. For this purpose, the cytosolic and mitochondrial fractions were isolated from the renal cortex (Figure 5). After three doses of gentamicin, cytochrome c release into the cytosol was obvious. Nevertheless, the released part was <25% of the total cytochrome c and, hence, mitochondrial cytochrome c did not show a clear decrease. Metformin ameliorated this cytochrome c delocalization, indicating that PTP inhibition by metformin provides a potential means of reducing the nephrotoxicity of gentamicin.Figure 5Cytochrome c release during gentamicin nephrotoxicity and prevention by metformin. After 3 days of gentamicin treatment in the absence or presence of metformin, cortical tissues were freshly collected to isolate the cytosolic and mitochondrial fractions for cytochrome c immunoblotting. Total protein concentration is 50 μg per lane in all samples. Cytochrome c release depicts changes in mitochondrial membrane permeability. #P<0.05 versus control; *P<0.05 versus gentamicin.View Large Image Figure ViewerDownload (PPT) Table 2 summarizes all mitochondrial respiration data from groups receiving gentamicin for 3 or 6 days, either alone or with metformin, before administration to rats for 1 week. Compared with control values, gentamicin treatment resulted in alterations in respiratory chain function independent of the respiratory fuels used, after as few as three doses of this nephrotoxicant. Gentamicin mildly reduced the oxygen consumption rates (Jo2) under the phosphorylating condition, that is, in the presence of ADP (state-3), and in the uncoupling state, after DNP addition. Jo2 with TMPD-ascorbate, used to assess the maximal activity of cytochrome oxidase, was not altered by gentamicin. After six doses of this antibiotic, all respiratory values were significantly different from that of the control; respiration rate in state 4, that is, in the presence of ATP synthase inhibitor oligomycin, was also below normal, resulting in no significant change in the respiratory control index or RCR (state-3/state-4). Metformin, when administrated before gentamicin, prevented this progressive mitochondrial dysfunction almost completely (Table 2).Table 2Oxygen consumption rates (nmol O2/min/mg protein) in kidney mitochondria isolated from control rats or rats treated with gentamicin for 3 or 6 days, and subject or not to treatment with metformin (n=4–6)ControlGentamicin (3 days)Gentamicin (6 days)MetforminMetformin+ Genta (3 days)Metformin+ Genta (6 days)Glutamate/malate (GM)State-417.6±0.420.1±1.310.2±1.1†15.8±1.3†24.9±2.615.1±0.9†,§State-3148.9±7.2128.9±9.2†80.6±2.1†119.8±6.9†155.5±3.3§126.3±8.1§DNP150.3±11103.4±7.1†76.1±2.2†129.5±14.4148.9±15.7§111.8±7.2†,§RCR8.2±0.26.4±0.097.8±0.48.1±0.17.1±0.48.2±0.3Succinate+rotenoneState-448.4±2.752.2±6.128.1±1.5†44.6±3.352.6±14.140.9±2.3§State-3285.9±8.7235.7±9.2†125.6±18.2†286.6±6.7294.8±20.5§206.1±12.7†,§DNP290.9±16.8255.8±16.7†150.9±18.5†300±18.2297.3±17.5§252.8±21.4§RCR6.4±0.14.7±0.45.1±0.2†6.6±0.36.1±0.355.8±0.1TMPD-ascorbate514.8±42.7492.5±25.3330.6±48.6†503.9±27.1502.8±19.3484.5±61.9§GM+succinateState-440.8±2.243.7±3.624.6±2†40.1±458.7±3.239.2±1.4§State-3302.7±9.8231.3±27.3†145.1±5.3†314.4±7.7327.1±17.8§258.4±24.3§DNP296.4±10210±23132.7±11.4†312.1±13.9318.2±25§243.5±16.3§RCR7.1±0.15.05±0.346.75±0.37.3±0.45.8±0.26.85±0.2Abbreviations: DNP, dinitrophenol-uncoupled respiration; RCR, respiratory control ratio; State-3, ADP-stimulated respiration; State-4, basal respiration.†P<0.05 versus control; §P<0.05 versus gentamicin (3 or 6 days accordingly). Open table in a new tab Abbreviations: DNP, dinitrophenol-uncoupled respiration; RCR, respiratory control ratio; State-3, ADP-stimulated respiration; State-4, basal respiration. †P<0.05 versus control; §P<0.05 versus gentamicin (3 or 6 days accordingly). The fact that metformin weakly reduced glutamate/malate-dependent respiration may be consistent with a physiological inhibitory effect on the functionally isolated complex-I, which we previously reported in a quite different setting.28.El-Mir M.Y. Nogueira V. Fontaine E. et al.Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I.J Biol Chem. 2000; 275: 223-228Crossref PubMed Scopus (955) Google Scholar Here, complex-I activity from the control mitochondria was significantly reduced by metformin, and this inhibition was even preserved in gentamicin-treated rats. On the contrary, the fact that gentamicin itself drastically lowered complex-I activity after 6 days of treatment, would be linked more to molecular alterations (proteolysis, conformational change) in this huge complex, rather than to a generalized damage of mitochondria, as citrate synthase activity, an usual indicator of the respiratory chain content in tissues, was only poorly attenuated (Table 3).Table 3Measurement of functionally isolated respiratory chain complex-I and matrix CS activities in kidney mitochondria isolated from control or gentamicin-treated rats, and subject or not to treatment with metformin (n=6)ControlGentamicinMetforminMetformin+gentamicinCS activity (nmol CoA/min/mg protein)303.1±6.6275.2±6.8302.9±6.7294.1±6.2Rotenone-sensitive activity of complex-I (nmol NADH/min/mg protein)91.7±2.838±4.1†77.6±2.4†71.7±8.8§Complex-I/CS (nmol NADH/unit of CS)0.31±0.010.13±0.02†0.27±0.01†0.25±0.02§Abbreviation: CS, citrate synthase.†P<0.05 versus control; §P<0.05 versus gentamicin. Open table in a new tab Abbreviation: CS, citrate synthase. †P<0.05 versus control; §P<0.05 versus gentamicin. There is a trend toward considering that alterations in the respiratory chain are responsible for the formation of excessive amounts of ROS, thereby contributing to cell damage.29.Batandier C. Leverve X. Fontaine E. Opening of the mitochondrial permeability transition pore induces reactive oxygen species production at the level of the respiratory chain complex I.J Biol Chem. 2004; 279: 17197-17204Crossref PubMed Scopus (193) Google Scholar,30.Plotnikov E.Y. Kazachenko A.V. Vyssokikh M.Y. et al.The role of mitochondria in oxidative and nitrosative stress during ischemia/reperfusion in the rat kidney.Kidney Int. 2007; 72: 1493-1502Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar Although hydrogen peroxide (H2O2) levels were barely affected by gentamicin, when glutamate–malate or succinate was individually used without inhibitors (Figure 6c and d), a decrease in NADH fluorescence was found in both situations (Figure 6a and b). As the main NADH-oxidizing pathway was blocked by gentamicin (Table 3), an escape of NADH from mitochondria would therefore be envisaged, possibly through the open PTP. A gentamicin-related increase in ROS was especially sizeable in mitochondria respiring on both glutamate–malate and succinate (Figure 6e). Remarkably, metformin prevented the mitochondrion-driven oxidative stress by lowering or normalizing ROS production along the respiratory chain (Figure 6c–e). Moreover, gentamicin-induced depletion of mitochondrial NADH was highly recovered with metformin treatment under either glutamate–malate or succinate conditions (Figure 6a and b). Although gentamicin continues to be an irreplaceable treatment against life-threatening infections, its use remains seriously limited by its nephrotoxicity. Although various studies reported on the benefits of several agents in gentamicin-induced renal poisoning,31.Ali B.H. Agents ameliorating or augmenting experimental gentamicin nephrotoxicity: some recent research.Food Chem Toxicol. 2003; 41: 1447-1452Crossref PubMed Scopus (141) Google Scholar the basis of nephroprotection remains elusive. To the best of our knowledge, this report is the first to show that metformin, a drug widely used in the treatment of diabetes, prevents functional, histological, and biochemical kidney injuries in the setting of gentamicin insult. Of utmost importance, all these impressive effects, seen at a metformin dosage corresponding to the clinically evidenced therapeutic range,32.Wilcock C. Bailey C.J. Accumulation of metformin by tissues of the normal and diabetic mouse.Xenobiotica. 1994; 24: 49-57Crossref PubMed Scopus (307) Google Scholar partly proceed from a normalization of in vivo oxidative stress and restoration of mitochondrial functional integrity. Our findings corroborate those of earlier studies demonstrating that an enhanced endogenous oxidative stress has a major role in the severity of gentamicin-induced acute renal failure.3.Martinez-Salgado C. Lopez-Hernandez F.J. Lopez-Novoa J.M. Glomerular nephrotoxicity of aminoglycosides.Toxicol Appl Pharmacol. 2007; 223: 86-98Crossref PubMed Scopus (192) Google Scholar,17.Walker P.D. Shah S.V. Gentamicin enhanced production of hydrogen peroxide by renal cortical mitochondria.Am J Physiol. 1987; 253: C495-C499PubMed Google Scholar Although having no effect alone, metformin blunted alterations in the hemodynamics induced by the daily injection of a nephrotoxic dose of gentamicin. In addition, RPF, RBF, and urinary flow even increased significantly in metformin plus gentamicin-treated rats as compared with that under baseline conditions. Although these remarkable outcomes cannot be readily explained, one may conceive that such a protection afforded by metformin could be mediated, at least in part, by the in vivo antioxidant features of this antidiabetic agent recently found,33.Faure P. Rossini E. Wiernsperger N. et al.An insulin sensitizer improves the free radical defense system potential and insulin sensitivity in high fructose-fed rats.Diabetes. 1999; 48: 353-357Crossref PubMed Scopus (143) Google Scholar,34.Rösen P. Wiernsperger N. Metformin delays the manifestation of diabetes and vascular dysfunction in Goto–Kakizaki rats by reduction of mitochondrial oxidative stress.Diabetes Metab Res Rev. 2006; 22: 323-330Crossref PubMed Scopus (80) Google Scholar as well by its ability to prevent gentamicin-induced lipid peroxidation and to enhance antioxidant defenses in the control and treated animals (this study). One of the early sensitive markers of tubular injury after exposure to aminoglycosides is increased excretion of lysosomal enzymes. Our data showing that metformin considerably prevented the increase of gentamicin-induced urinary N-acetyl-β-D-glucosaminidase excretion, suggest evident protection against structural and functional tubular alterations. Strikingly, metformin-reduced gentamicin toxicity occurs in spite of larger intra-renal amounts of toxicant. This paradoxical result of high cortical concentrations of gentamicin with preservation of renal function was similarly reported by others using rats treated with a combination of gentamicin and polyaspartic acid.35.Gilbert D.N. Wood C. Kohlhepp S. et al.Polyaspartic acid prevents experimental amino-glycoside nephrotoxicity.J Infect Dis. 1989; 159: 945-953Crossref PubMed Scopus (95) Google Scholar As a cationic drug, metformin is known to be transported across the kidney through organic cation transporter-2.36.Kimura N. Masuda S. Tanihara Y. et al.Metformin is a superior substrate for renal organic cation transporter OCT-2 rather than hepatic OCT-1.Drug Metab Pharmacokinet. 2005; 20: 379-386Crossref PubMed Scopus (271) Google Scholar In the case of gentamicin, the endocytotic receptor megalin has been reported to be responsible for its tubular accumulation, which is directly related to its toxicity.37.Schmitz C. Hilpert J. Jacobsen C. et al.Megalin deficiency offers protection from renal aminoglycoside accumulation.J Biol Chem. 2002; 277: 618-622Crossref PubMed Scopus (153) Google Scholar The fact that metformin did not diminish gentamicin accumulation reflects a lack of interaction between both ways of transport, and suggests that protection conferred by biguanide occurs from within renal cells. Another potentially important finding of this study is that gentamicin induces drastic mitochondrial changes that largely precede overt cell necrosis and acute renal failure. After three doses of gentamicin, mitochondrial damage included PTP opening and leakage of cytochrome c into the cytosol, whereas no evident differences in the histology of treated rats as compared with that in the controls were found, except for cytoplasmic vacuolization in most of the proximal tubules (data not shown). It is noteworthy that these initial mitochondrial abnormalities were improved by metformin. A progressive deterioration in mitochondrial function and cellular integrity was observed with increasing gentamicin dosage. After six doses, PTP opening was higher than that with three doses and, expectedly, light microscopic examination revealed serious lesions and necrosis in a large number of proximal tubular cells. Thus, necroses and shedding of dead cells into the urine would account for the sharp decline in cortical gentamicin concentrations. As the harmful effect of ROS on tissues is widely appreciated, we provide enough evidence that these cytotoxic effects for gentamicin are partly associated with mitochondrial oxidative stress. Despite the presence of non-physiological substrate concentrations, specific conditions of dual-electron entry at both respiratory chain site-1 (glutamate/malate) and site-2 (succinate), as it is most likely the case in living cells, led to higher H2O2 contents after gentamicin treatment, indicating major derangements along all electron transfer chain segments and excess generation of ROS. This result correlates with the reported depletion in plasma antioxidant capacity. Among the various effects of ROS on cell metabolism, they are recognized to induce pore opening in either in vitro or in vivo settings.38.Bernardi P. Krausk
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