Abstract
PurposeThe aim of this work was to investigate if the content of glutamyl aminopeptidase (GluAp) in microvesicular and exosomal fractions of urine is related with renal dysfunction in cisplatin-treated rats.MethodsUrine samples were collected 24 hours after injection of cisplatin (7 mg/kg, n = 10) or saline serum (n = 10), and they were subjected to differential centrifugation at 1.000, 17.000 and 200.000 g to obtain microvesicular and exosomal fractions. GluAp was measured with a commercial ELISA kit in both fractions. Serum creatinine (SCr) and body weight were measured 15 days after treatment. We analyzed if early excretion of GluAp in microsomal and exosomal fractions was correlated with final SCr and body weight increase. In a second experiment, enzymatic activities of GluAp and alanyl aminopeptidase (AlaAp) in urine, microvesicular and exosomal fractions were measured three days after injection. We analyzed the correlation of both markers with SCr determined at this point. Finally, we studied the expression of GluAp and extracellular vesicles markers Alix and tumor susceptibility gene (TSG101) in both fractions by immunoblotting.ResultsGluAp excretion was increased in all fractions of urine after cisplatin treatment, even if data were normalized per mg of creatinine, per body weight or per total protein content of each fraction. We found significant predictive correlations with SCr concentration, and inverse correlations with body weight increase determined 15 days later. Three days after injection, aminopeptidasic activities were markedly increased in all fractions of urine in cisplatin-treated rats. The highest correlation coefficient with SCr was found for GluAp in microvesicular fraction. Increase of GluAp in microvesicular and exosomal fractions from cisplatin-treated rats was confirmed by immunoblotting. Alix and TSG101 showed different patterns of expression in each fraction.ConclusionsDetermination of GluAp content or its enzymatic activity in microvesicular and exosomal fractions of urine is an early and predictive biomarker of renal dysfunction in cisplatin-induced nephrotoxicity. Measurement of GluAp in these fractions can serve to detect proximal tubular damage independently of glomerular filtration status.
Highlights
Diagnosis of acute kidney injury remains to be a challenge nowadays
glutamyl aminopeptidase (GluAp) excretion was increased in all fractions of urine after cisplatin treatment, even if data were normalized per mg of creatinine, per body weight or per total protein content of each fraction
Alterations in glomerular filtration are accompanied with a low excretion of urinary creatinine that could falsely increase the levels of urinary markers
Summary
Diagnosis of acute kidney injury remains to be a challenge nowadays. Traditional biomarkers, like serum blood urea nitrogen (BUN) or creatinine (SCr) start to rise when kidney has lost at least a 50% of its function [1,2] and they have been considered by some authors as delayed biomarkers [3].several biomarkers such as N-acetyl-β-D-glucosaminidase (NAG), kidney injury molecule-1 (KIM-1) or neutrophil gelatinase-associated lipocalin (NGAL) have been proposed during the last years as early markers of renal dysfunction in different pathologies, and urine is the most promising source of substances that can be related with the extent of kidney injury [4].Cisplatin is an antineoplasic drug that induces nephrotoxicity as a collateral effect [5,6], and it has been widely used to evoke kidney injury in experimental models [7]. Traditional biomarkers, like serum blood urea nitrogen (BUN) or creatinine (SCr) start to rise when kidney has lost at least a 50% of its function [1,2] and they have been considered by some authors as delayed biomarkers [3]. Several biomarkers such as N-acetyl-β-D-glucosaminidase (NAG), kidney injury molecule-1 (KIM-1) or neutrophil gelatinase-associated lipocalin (NGAL) have been proposed during the last years as early markers of renal dysfunction in different pathologies, and urine is the most promising source of substances that can be related with the extent of kidney injury [4]. Amount and composition of exosomes excreted in urine might be important in diagnosis of renal dysfunction, because they can constitute a noninvasive source of multiple disease biomarkers that could provide clinically useful information [14,15,16]
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