Urinary levels of regenerating islet-derived protein III β and gelsolin differentiate gentamicin from cisplatin-induced acute kidney injury in rats
2010; Elsevier BV; Volume: 79; Issue: 5 Linguagem: Inglês
10.1038/ki.2010.439
ISSN1523-1755
AutoresLaura Ferreira, Yaremi Quirós, Sandra M. Sancho‐Martínez, Omar García-Sánchez, César Raposo, José M. López‐Novoa, J.M. González-Buitrago, Francisco J. López‐Hernández,
Tópico(s)Biomedical Research and Pathophysiology
ResumoA key aspect for the clinical handling of acute kidney injury is an early diagnosis, for which a new generation of urine biomarkers is currently under development including kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin. A further diagnostic refinement is needed where one specific cause among several potentially nephrotoxic insults can be identified during the administration of multidrug therapies. In this study we identified increases in regenerating islet-derived protein III beta (reg IIIb) and gelsolin as potential differential urinary markers of gentamicin's nephrotoxicity. Indeed, urinary levels of both reg IIIb and gelsolin distinguish between the nephrotoxicity caused by gentamicin from that caused by cisplatin where these markers were not increased by the latter. Reg IIIb was found to be overexpressed in the kidneys of gentamicin-treated rats and excreted into the urine, whereas urinary gelsolin originated from the blood by glomerular filtration. Our results illustrate an etiological diagnosis of acute kidney injury through analysis of urine. Thus, our results raise the possibility of identifying the actual nephrotoxin in critically ill patients who are often treated with several nephrotoxic agents at the same time, thereby providing the potential for tailoring therapy to an individual patient, which is the aim of personalized medicine. A key aspect for the clinical handling of acute kidney injury is an early diagnosis, for which a new generation of urine biomarkers is currently under development including kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin. A further diagnostic refinement is needed where one specific cause among several potentially nephrotoxic insults can be identified during the administration of multidrug therapies. In this study we identified increases in regenerating islet-derived protein III beta (reg IIIb) and gelsolin as potential differential urinary markers of gentamicin's nephrotoxicity. Indeed, urinary levels of both reg IIIb and gelsolin distinguish between the nephrotoxicity caused by gentamicin from that caused by cisplatin where these markers were not increased by the latter. Reg IIIb was found to be overexpressed in the kidneys of gentamicin-treated rats and excreted into the urine, whereas urinary gelsolin originated from the blood by glomerular filtration. Our results illustrate an etiological diagnosis of acute kidney injury through analysis of urine. Thus, our results raise the possibility of identifying the actual nephrotoxin in critically ill patients who are often treated with several nephrotoxic agents at the same time, thereby providing the potential for tailoring therapy to an individual patient, which is the aim of personalized medicine. 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New, early urine markers are currently in an advanced degree of validation for the diagnosis and prognosis of ARF, including kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and others.40.Vaidya V.S. Ferguson M.A. Bonventre J.V. Biomarkers of acute kidney injury.Annu Rev Pharmacol Toxicol. 2008; 48: 463-493Crossref PubMed Scopus (458) Google Scholar, 42.Ferguson M.A. Vaidya V.S. Bonventre J.V. Biomarkers of nephrotoxic acute kidney injury.Toxicology. 2008; 245: 182-193Crossref PubMed Scopus (209) Google Scholar The next step in the refinement of ARF diagnosis will be the capability to differentiate the renal damage inflicted by a determined drug or insult from that exerted by others.43.Cataldi L. Mussap M. 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In this article, we identify regenerating islet-derived protein III β (reg IIIb) and gelsolin as new urinary markers to further profile and differentiate the AKI inflicted by gentamicin from that induced by cisplatin. As expected, after 6 days of treatment, gentamicin caused a marked ARF with an associated mortality of ∼50% (Figure 1b). Surviving animals coursed with a small but significant weight loss and polyuria. ARF was further characterized by a dramatic increase in plasma creatinine and urea concentration, suggesting a reduction of glomerular filtration rate (Figure 1a). NAG (Figure 1a), KIM-1, and bone morphogenetic protein 7 (Figure 1c) urinary excretion also increased, indicating tubular damage. Proteinuria was also evident in the urine of animals treated with gentamicin (Figure 1a). Hematoxylin–eosin-stained renal sections (Figure 1d) revealed a clear tubular necrosis in gentamicin-treated rats. No gross modification of the glomeruli was evident. At the papillary level, obstruction of collecting tubuli with hyaline material was widespread in gentamicin-treated animals. A representative image of two-dimensional (2D) gels (pH range 4–7) of urine samples from control and gentamicin-treated rats is shown in the upper panels of Figure 2. Many proteins concentrate in the range of pH 4.5–5.5. For that reason, 2D separations in this pH range were also done with the same urine samples. A representative image of the latter is shown in the lower panels of Figure 2. A great similarity was observed between samples from animals in the same group, and high reproducibility was obtained when repeating the 2D separation with the same sample, for quality assurance. However, the urine proteome of both groups is substantially different. Statistically significant, differentially present spots between control and gentamicin groups were recognized and numbered for chemical identification. Mass spectrometric analysis revealed the identity of two proteins increased in the urine of gentamicin-treated rats, which showed potential interest after discarding most of the other proteins, normally found in different proteinuric conditions. They were identified as reg IIIb and gelsolin (Figure 2). The increased urinary level of these proteins in the urine of gentamicin-treated rats was confirmed by western blot analysis. Moreover, the urine from rats treated with a nephrotoxic regime of cisplatin was also analyzed. Figure 3b shows data on plasma creatinine concentration and blood urea nitrogen from 6 control rats, 6 rats treated with gentamicin, and 6 rats treated with cisplatin. It demonstrates that animals treated with gentamicin or cisplatin developed an overt renal failure as demonstrated by the increment in plasma blood urea nitrogen and creatinine, increased NAG excretion, and the decrease in creatinine clearance. These urine samples were also analyzed for their content in reg IIIb and gelsolin. Figure 3a clearly shows that the urinary level of reg IIIb is markedly increased only in gentamicin-treated animals, despite undergoing a similar degree of renal damage than cisplatin-treated rats. Western blot of gelsolin revealed two reactive bands. The higher one corresponds to the full-length protein, whereas the lower one corresponds to a fragment thereof. The presence of gelsolin within the reactive bands was further re-confirmed by tandem mass spectra. Treatment with gentamicin induces the appearance in the urine of both the full-length gelsolin and the ∼43 kDa fragment. However, the full-length band was absent in the urine of all rats treated with cisplatin, except for one of them. Extensive analysis of the urine from other cisplatin-treated rats shows no presence of the full-length band (data not shown). We further analyzed the time course evolution of the urinary excretion of these proteins in rats treated with gentamicin or cisplatin. Figure 4 shows the temporal profile of the renal damage inflicted by gentamicin. Significant damage only occurs after 4 days of treatment, as revealed by the evolution of plasma creatinine, NAG excretion, proteinuria, and the urinary level of three sensitive markers of kidney injury, such as KIM-1, NGAL, and PAI-1 (plasminogen activator inhibitor 1). Congruently with the accumulated knowledge,44.Dennen P. Parikh C.R. Biomarkers of acute kidney injury: can we replace serum creatinine?.Clin Nephrol. 2007; 68: 269-278Crossref PubMed Google Scholar plasma creatinine is the least sensitive of all the markers tested. Furthermore, histological analysis of renal sections after 3 days of treatment reveals no findings of tubular damage. 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Inhibition of in vitro endosomal vesicle fusion activity by aminoglycoside antibiotics.J Biol Chem. 1998; 273: 25301-25309Crossref PubMed Scopus (12) Google Scholar In this scenario, western blot analysis showed that reg IIIb appears in the urine along with most other sensitive markers of renal injury, starting on day 4. Interestingly, urinary gelsolin (the ∼43 kDa fragment) appears as early as on day 1 and stays high through the treatment, long before all other sensitive markers do, including KIM-1, PAI-1, NGAL, and NAG. In the case of rats treated with cisplatin, reg IIIb and full-length gelsolin are not significantly increased in the urine during the same period of 6 days (Figure 5a), despite that rats treated with cisplatin develop a similar degree of renal injury to that observed in gentamicin-treated rats. This is evidenced by the evolution of parameters of renal dysfunction and damage, such as plasma creatinine, urinary excretion of NAG and KIM-1, and by the histological examination of renal tissue after 6 days of treatment with both drugs (Figure 5a–c, and also Figures 3 and 4). Yet, the level of the 43 kDa fragment of gelsolin increases in the urine of rats treated with cisplatin more pronouncedly than and before KIM-1 (Figure 5a), which also occurs in the case of the treatment with gentamicin (Figure 4). This can be potentially exploited for an early diagnosis of AKI.Figure 5Time course evolution of urinary regenerating islet-derived protein III β (reg IIIb) and gelsolin in rats treated with cisplatin. (a) Representative images of western blot analysis of urinary reg IIIb, gelsolin (full-length and the t fragment, indicated by the arrows), and kidney injury molecule 1 (KIM-1), and densitometric quantification of three independent experiments. (b) Evolution of plasma creatinine concentration and N-acetyl-β-D-glucosaminidase (NAG) excretion; n=6. (c) Representative images of the cortex and medulla in renal sections stained with hematoxylin and eosin from rats treated with cisplatin (day 6 after cisplatin injection; original magnification × 1000; n=3). Data represent the average±s.e.m. *P<0.05 vs time 0; AU, arbitrary units; C+, positive control (that is, urine from a gentamicin-treated, nephrotoxic rat).View Large Image Figure ViewerDownload (PPT) Western blot analysis of albumin-depleted plasma from control and gentamicin-treated rats indicated that reg IIIb is absent (to the detection limit of this technique), whereas gelsolin is normally found in the blood compartment. Even more, gentamicin treatment slightly increases the plasmatic level of the latter (Figure 6a). Gene expression analysis carried out on renal tissue by reverse transcriptase-PCR showed that these two proteins are normally expressed in the kidneys. Treatment of rats with gentamicin does not modify the renal expression pattern of gelsolin, but induces an increase in reg IIIb gene expression as early as on day 3 (Figure 6b), when no detectable kidney injury has occurred yet (Figure 4). On day 6, reg IIIb expression is highest. In order to study whether the origin of these urinary proteins was the blood, which would shed them to the urine through the GFB, we perfused the kidneys of rats treated for 6 days with gentamicin with Krebs solution (containing dextran to compensate for the oncotic pressure). We found that immediately before substituting the renal blood flow with Krebs, we could still detect reg IIIb and gelsolin in the urine (Figure 6c). However, once the renal blood flow was substituted with Krebs flow, gelsolin disappeared from the urine; yet, the upper band of reg IIIb was still detected, whereas the lower one disappeared. Nephrotoxicity poses a considerable health and economic problem worldwide. 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