Sub-nephrotoxic doses of gentamicin predispose animals to developing acute kidney injury and to excrete ganglioside M2 activator protein
2010; Elsevier BV; Volume: 78; Issue: 10 Linguagem: Inglês
10.1038/ki.2010.267
ISSN1523-1755
AutoresYaremi Quirós, Laura Ferreira, Sandra M. Sancho‐Martínez, J.M. González-Buitrago, José M. López‐Novoa, Francisco J. López‐Hernández,
Tópico(s)Antibiotics Pharmacokinetics and Efficacy
ResumoWe studied whether nephrotoxic drug administration sensitizes to acute renal failure (ARF) by administering a sub-nephrotoxic dose of gentamicin. This pre-treatment sensitized animals with no sign of renal injury to develop ARF when exposed to a second potential nephrotoxic drug, also given at sub-nephrotoxic doses that would be otherwise harmless to non-sensitized animals. We identified urinary ganglioside M2 activator protein (GM2AP) as a biomarker of an enhanced sensitivity to suffer ARF following sub-nephrotoxic treatment with gentamicin. Sub-nephrotoxic gentamicin did not alter renal GM2AP gene expression or protein levels, determined by reverse transcriptase-PCR, western blot, and immunostaining, nor was its serum level modified. The origin of increased GM2AP in the urine is thought to be a defective tubular handling of this protein as a consequence of gentamicin action. Hence, markers of acquired sensitivity may improve the prevention of ARF by enhancing our capacity to monitor for this condition, in a preemptive manner. We studied whether nephrotoxic drug administration sensitizes to acute renal failure (ARF) by administering a sub-nephrotoxic dose of gentamicin. This pre-treatment sensitized animals with no sign of renal injury to develop ARF when exposed to a second potential nephrotoxic drug, also given at sub-nephrotoxic doses that would be otherwise harmless to non-sensitized animals. We identified urinary ganglioside M2 activator protein (GM2AP) as a biomarker of an enhanced sensitivity to suffer ARF following sub-nephrotoxic treatment with gentamicin. Sub-nephrotoxic gentamicin did not alter renal GM2AP gene expression or protein levels, determined by reverse transcriptase-PCR, western blot, and immunostaining, nor was its serum level modified. The origin of increased GM2AP in the urine is thought to be a defective tubular handling of this protein as a consequence of gentamicin action. Hence, markers of acquired sensitivity may improve the prevention of ARF by enhancing our capacity to monitor for this condition, in a preemptive manner. Acute renal failure (ARF) is an extremely serious condition in which the renal excretory function abruptly falls within a few hours or days after an insult to the kidneys.1.Kellum J.A. Levin N. Bouman C. et al.Developing a consensus classification system for acute renal failure.Curr Opin Crit Care. 2002; 8: 509-514Crossref PubMed Scopus (360) Google Scholar, 2.Bellomo R. Kellum J.A. Ronco C. Defining and classifying acute renal failure: from advocacy to consensus and validation of the RIFLE criteria.Intensive Care Med. 2007; 33: 409-413Crossref PubMed Scopus (306) Google Scholar ARF still leads to death in 50% of the cases, a number that grows to 80% if multiorgan damage occurs.3.Neild G.H. 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Acute renal failure: changing causes?.Kidney Blood Press Res. 1997; 20: 163Crossref PubMed Scopus (6) Google Scholar A key determinant for a successful clinical handling of ARF is an early diagnosis, which significantly improves therapeutic intervention and outcome.8.Devarajan P. Neutrophil gelatinase-associated lipocalin (NGAL): a new marker of kidney disease.Scand J Clin Lab Invest Suppl. 2008; 241: 89-94Crossref PubMed Scopus (266) Google Scholar, 9.Vaidya V.S. Ferguson M.A. Bonventre J.V. Biomarkers of acute kidney injury.Annu Rev Pharmacol Toxicol. 2008; 48: 463-493Crossref PubMed Scopus (520) Google Scholar Traditionally, ARF has been diagnosed through measurable symptoms of renal dysfunction, such as the increase in serum creatinine and blood urea nitrogen (BUN) concentrations, or changes in the fractional excretion of sodium.9.Vaidya V.S. Ferguson M.A. Bonventre J.V. Biomarkers of acute kidney injury.Annu Rev Pharmacol Toxicol. 2008; 48: 463-493Crossref PubMed Scopus (520) Google Scholar However, owing to compensatory adaptation, renal dysfunction only appears after an extensive loss of functional nephrons occurs.10.Mueller P.W. Price R.G. Finn W.F. New approaches for detecting thresholds of human nephrotoxicity using cadmium as an example.Environ Health Perspect. 1998; 106: 227-230Crossref PubMed Scopus (57) Google Scholar Consequently, a new generation of biomarkers (mostly urine biomarkers) is under development, associated with early pathophysiological events underlying the incipient acute kidney injury (AKI), before it turns into an overt ARF. They, most significantly, include kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin, and others.9.Vaidya V.S. Ferguson M.A. Bonventre J.V. Biomarkers of acute kidney injury.Annu Rev Pharmacol Toxicol. 2008; 48: 463-493Crossref PubMed Scopus (520) Google Scholar Gentamicin is an aminoglycoside antibiotic widely used against Gram-negative infections. The most important side effect of this drug is its nephrotoxicity,11.Mingeot-Leclerq M.P. Tulkens P. Aminoglycosides: nephrotoxicity.Antimicrob Agents Chemother. 1999; 43: 1003-1012PubMed Google Scholar, 12.Martínez-Salgado C. López-Hernández F.J. López-Novoa J.M. Glomerular nephrotoxicity of aminoglycosides.Toxicol Appl Pharmacol. 2007; 223: 86-98Crossref PubMed Scopus (206) Google Scholar which occurs in ∼10–25% of therapeutic courses, despite correct dosage and hydration status monitoring.13.Kacew S. Bergeron M.G. Pathogenic factors in aminoglycoside-induced nephrotoxicity.Toxicol Lett. 1990; 51: 241-259Crossref PubMed Scopus (51) Google Scholar, 14.Laurent G. Kishore B.K. Tulkens P.M. 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Sun D.F. et al.Plasma protein extravasation and vascular endothelial growth factor expression with endothelial nitric oxide synthase induction in gentamicin-induced acute renal failure in rats.Virchows Arch. 2004; 444: 362-374Crossref PubMed Scopus (16) Google Scholar, 25.Secilmis M.A. Karatas Y. Yorulmaz O. et al.Protective effect of L-arginine intake on the impaired renal vascular responses in the gentamicin-treated rats.Nephron Physiol. 2005; 100: 13-20Crossref PubMed Scopus (22) Google Scholar that, depending on the dose, contribute to a larger or lesser extent of renal dysfunction.26.Hishida A. Nakajima T. Yamada M. et al.Roles of hemodynamic and tubular factors in gentamicin-mediated nephropathy.Ren Fail. 1994; 16: 109-116Crossref PubMed Scopus (19) Google Scholar However, it is not yet well characterized to what extent a subtoxic treatment with gentamicin sensitizes individuals to ARF, such as the ARF induced by subsequent sub-nephrotoxic exposure to another potentially nephrotoxic agent. A typical clinical situation exists, where a patient treated with gentamicin showing no signs of renal disease is thereupon given another potentially nephrotoxic agent, such as another drug, or a diagnostic contrast medium, also within a theoretically subtoxic regime. These scenarios pose relevant clinical situations of special importance for its hidden nature, which should be addressed from the diagnostic and therapeutic perspectives. In this study, we demonstrate that a sub-nephrotoxic regime of gentamicin primes the rats to develop an ARF induced by a subsequent or concomitant exposure to sub-nephrotoxic doses of a second nephrotoxicant. We also show that the urinary level of ganglioside M2 activator protein (GM2AP) may be used to identify this condition. Urinary levels of GM2AP could also serve for the early diagnosis of gentamicin-induced ARF. Detection of the increased risk enables a preemptive handling of drug toxicity by anticipating situations that can result in injury, before the slightest alterations that are usually observed arise. A sub-nephrotoxic regime of gentamicin was identified to test whether it would induce sensitisation to ARF in the absence of a direct deleterious effect on the kidneys. After pilot studies, a regime of six daily consecutive doses of 50 mg/kg/day gentamicin (G-50 group) was observed to exert no obvious renal injury symptoms. It was further characterized to ensure the absence of nephrotoxicity. Survival during the whole treatment was identical to that of control rats (100%), whereas in rats treated with a nephrotoxic regime of gentamicin (150 mg/kg/day; G-150 group), which developed a clear ARF, survival decreased to 50%. Similarly, body weight increased by a 3–4% in control and G-50 animals, whereas it was reduced by 4–5% in G-150 rats. As shown in Figure 1, we were unable to find a single marker of renal damage or dysfunction in G-50 rats, when compared with controls. On the contrary, G-150 rats underwent a typical and overt ARF characterized by an increase in plasma creatinine concentration (Crpl) and BUN, proteinuria, increased fractional excretion of sodium, and the presence of urinary (i.e., increased N-acetyl-glucosaminidase (NAG) excretion) and renal tissue (KIM-1, plasminogen activator inhibitor 1, and vimentin) markers of tubular lesion (Figure 1a–f). A gross morphological examination of renal slices showed that renal parenchyma in G-50 was indistinguishable from that of control rats, whereas a clear tubular necrosis and obstruction was evident in G-150 rats (Figure 1g). These results indicate that the G-50 regime exerts no apparent deleterious action on the kidneys, as evaluated by the finest diagnostic methods available. Under these sub-nephrotoxic circumstances, we tested whether the G-50 regime sensitizes rats to ARF, e.g., by reducing the nephrotoxicity threshold of another potential nephrotoxicant. We first used uranyl nitrate (UN), which we titrated for dose-nephrotoxic effect in pilot studies. A single dose of 0.5 mg/kg UN was found to lack nephrotoxic effects, which was confirmed in further experiments (Figure 2). However, when this dose of UN was administered to rats previously treated with G-50, a clear ARF ensued, which was not observed in control rats or in that treated with UN or G-50 alone. This ARF was characterized (Figure 2a–d) by an increase in Crpl, BUN and NAG excretion, proteinuria, and a decrease in creatinine clearance. This sensitization appears along with the first sub-nephrotoxic dose of gentamicin and lasts at least 1 week after gentamicin withdrawal. This is evidenced by the increase in serum creatinine in rats that were given the single dose of UN at the onset of the gentamicin regime, as well as in rats in which UN is administered 1 week after cessation of the gentamicin treatment (data not shown). Interestingly, the sensitization produced by gentamicin is also effective on other potentially nephrotoxic drugs, such as the antineoplastic cisplatin or the iodinated contrast medium iohexol. Figure 2e–f shows how rats, previously exposed to G-50 for 6 days, suffered a renal damage when subsequently exposed to iohexol (in 24 h) and cisplatin (in 2 days), both used at sub-nephrotoxic doses (titrated in previous pilot studies). This is evidenced by increased serum creatinine and BUN, and elevated NAG excretion. Next, we performed a differential proteomic analysis comparing the urine of control rats and G-50 rats at the end of the treatment, before the administration of the second nephrotoxicant. The objective of this study was to identify whether proteins increased or decreased in the urine of G-50 (sensitized to AKI) compared with control (non-sensitized to AKI) rats, which might prospectively serve as biomarkers of gentamicin-induced sensitization to AKI. As shown in Figure 3a, both urinary proteomes were almost identical. However, a protein was clearly increased in the urinary proteome of G-50, which was unambiguously identified using liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as GM2AP. A polyclonal antibody raised in rabbits against an epitope found in rat and human GM2AP further confirmed the increased levels of GM2AP in the urine of G-50 using western blot analysis (Figure 3b). Furthermore, the urine of eight patients treated with gentamicin for at least 2 days was analyzed using western blot for the level of GM2AP, and compared with that of eight sex and age matching untreated individuals. A total of seven of eight gentamicin-treated patients, whereas only two of eight untreated controls, showed increased levels of GM2AP in the urine, also determined using western blot analysis (Figure 3c). We also decided to study the early diagnostic capacity of urinary GM2AP on an animal model of gentamicin-induced ARF. A time course experiment revealed that, in this model, GM2AP appears in the urine from the first day of treatment with overtly nephrotoxic doses of gentamicin, largely preceding not only classical markers such as serum creatinine, BUN, NAG excretion, or proteinuria, but also the new, earlier, and more sensitive urinary markers of AKI, KIM-1, and plasminogen activator inhibitor 1 (Figure 4a). Furthermore, the level of GM2AP in the urine progressively increased with time, which makes it potentially suitable for monitoring AKI evolution induced by gentamicin in a much more specific manner than other novel markers. GM2AP also appears in the urine of overtly nephrotoxic rats as a consequence of cisplatin or UN administration at toxic doses (Figure 4b). In the case of cisplatin, GM2AP appears in the urine in parallel or after KIM-1 (Figure 4c). These results indicate that urinary GM2AP, in the absence of damage markers, likely reflects the increased risk of ARF induced by gentamicin, because further exposure to the drug produces an overt ARF. To unravel the origin of the urinary GM2AP, we studied the effect of gentamicin on the presence and production of GM2AP in renal tissue, the urine, and the blood. The immunohistochemical analysis of GM2AP distribution in renal tissue shows (Figure 5a and b) that this protein is mainly located in the renal cortex, with great selectivity, within the proximal tubules. The latter is evidenced by a perfect co-staining of GM2AP with the proximal tubule-restricted protein megalin, and the absence of co-staining with the distal tubule-borne protein calbindin. Sub-nephrotoxic gentamicin (G-50) did not modify the histological distribution or apparent expression level of GM2AP (Figure 5a and b). Western blot analysis of total GM2AP protein level in renal tissue homogenates (Figure 5c), as well as GM2AP gene expression (by reverse transcriptase-PCR; Figure 5d), confirmed the lack of effect of gentamicin on the renal expression of the protein. GM2AP was detected in the serum and its serum levels were not altered by G-50. However, G-150 treatment significantly increased serum GM2AP (Figure 5e). In acute experiments with anesthetized rats, a bolus administration of a high dose of gentamicin induced a rapid increase in the urinary excretion of GM2AP (Figure 5g). The increased excretion declined after 3 h, probably correlating with the bioavailability of gentamicin. Interestingly, when, under similar circumstances, kidneys were perfused in situ with Krebs solution (instead of blood) by means of an extracorporeal circuit, gentamicin did not produce an increase in urinary GM2AP (Figure 5h). All together, these results indicate that the increase in urinary GM2AP produced by gentamicin is an acute effect which is tightly dependent on the presence of gentamicin and, most importantly, that the urinary GM2AP comes from the blood and not from renal tissues. These results can be explained by an altered renal handling of GM2AP (e.g., reduced reuptake) as the mechanism responsible for its increase in the urine. In fact, GM2AP appears in the urine shortly after treatment with maleate (Figure 5f), indicating that it is transported by the megalin complex (see discussion). This is further supported by the colocalization of GM2AP and megalin in proximal tubule cells within subcellular structures, probably being endocytosis vesicles, as revealed by confocal microscopy (Figure 5b; lower panel). Our experiments show that gentamicin-induced sensitization to ARF, a condition hitherto largely underestimated, is distinctly differentiated from early and mild renal injury. It has a potentially high clinical relevance because it poses an unnoticed risk of ARF. The recognition of acquired sensitization to ARF as an existing and relevant pathological state makes obvious the necessity to identify tools to create a level of diagnosis for its detection and appropriate clinical handling. Our results also show that the increased urinary level of GM2AP is associated with the sensitization to AKI induced by sub-nephrotoxic gentamicin. They further show that an increased level of GM2AP also appears in the urine of rats undergoing an overt ARF and, in the case of gentamicin-induced AKI, urinary GM2AP appears earlier than other sensitive markers such as KIM-1 or plasminogen activator inhibitor 1. However, in the case of other nephrotoxicants, such as cisplatin, urinary GM2AP is elevated in parallel or even after the appearance early damage marker KIM-1. This fact has special importance because GM2AP might be exploited also for an etiological and selective diagnosis of AKI within the very early stages of the disease. The sub-nephrotoxic and the early nephrotoxic situations related to gentamicin treatments show the common characteristic of lacking the markers of tissue damage, while showing increased urinary levels of GM2AP. As such, monitoring the progressive increment of the urinary level of GM2AP from the onset of a gentamicin regime will provide means of detecting the increasing risk of an AKI burst as a consequence of further gentamicin administration or treatment with another potential nephrotoxicant. In the case of the sensitization to AKI, GM2AP urinary excretion would serve as a diagnostic tool to discern which patients have acquired an increased risk as a consequence of a gentamicin regime, when contemplating the need of subjecting them to additional potentially nephrotoxic circumstances like the administration of other drugs. A typical and relevant case, from the clinical and socioeconomic point of views, is posed by the fact that 0.6–2.3% of not-at-risk patients undergoing a contrast radiography, with no previous history of renal disease, develop some degree of AKI.27.Mehran R. Nikolsky E. Contrast-induced nephropathy: definition, epidemiology, and patients at risk.Kidney Int Suppl. 2006; 100: S11-S15Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar We propose that a part of this patient group might be silently coursing with an increased risk to AKI owing to a previous treatment or exposure to an environmental agent that has induced no clinical symptoms of renal lesions, such as a treatment with gentamicin. The urinary level of GM2AP could be used as a marker to detect this risk. Very interestingly, urinary GM2AP is found to be increased (to a variable degree) in most patients treated with gentamicin for at least 3 days, and whose estimated glomerular filtration rate and urinary levels of sensitive renal damage markers (e.g., neutrophil gelatinase-associated lipocalin, KIM-1) remain normal during analysis. 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