title: Glutamyl aminopeptidase in microvesicular and exosomal fractions of urine is related with renal dysfunction in cisplatin-treated rats.
abstract: OBJECTIVE
  The 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.
  
  
  METHODS
  Urine 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.
  
  
  RESULTS
  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. 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.
  
  
  CONCLUSIONS
  Determination 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.
authors.affiliation: Unidad de Nefrología, Hospital Virgen de las Nieves, FIBAO, Granada, Spain.
authors.forename: Andrés
authors.initials: A
authors.lastname: Quesada
authors.affiliation: Área de Fisiología, Departamento de Ciencias de la Salud, Universidad de Jaén, Jaén, Spain.
authors.forename: Ana Belén
authors.initials: AB
authors.lastname: Segarra
authors.affiliation: Área de Fisiología, Departamento de Ciencias de la Salud, Universidad de Jaén, Jaén, Spain.
authors.forename: Sebastián
authors.initials: S
authors.lastname: Montoro-Molina
authors.affiliation: Unidad de Nefrología, Hospital Virgen de las Nieves, FIBAO, Granada, Spain.
authors.forename: María Del Carmen
authors.initials: MD
authors.lastname: de Gracia
authors.affiliation: Unidad de Nefrología, Hospital Virgen de las Nieves, FIBAO, Granada, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Osuna
authors.affiliation: Departamento de Anatomía Patológica, IBIMER, Universidad de Granada, Granada, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: O'Valle
authors.affiliation: Área de Fisiología, Departamento de Ciencias de la Salud, Universidad de Jaén, Jaén, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Gómez-Guzmán
authors.affiliation: Departamento de Fisiología, Facultad de Medicina, Universidad de Granada, Granada, Spain.
authors.forename: Félix
authors.initials: F
authors.lastname: Vargas
authors.affiliation: Área de Fisiología, Departamento de Ciencias de la Salud, Universidad de Jaén, Jaén, Spain.
authors.forename: Rosemary
authors.identifier: http://orcid.org/0000-0001-7161-9925
authors.initials: R
authors.lastname: Wangensteen
mesh_terms: D000818:Animals; D002945:Cisplatin / Q000008:administration & dosage* / Q000633:toxicity; D055354:Exosomes / Q000201:enzymology*; D043384:Glutamyl Aminopeptidase / Q000652:urine*; D007668:Kidney / Q000187:drug effects* / Q000201:enzymology / Q000503:physiopathology; D008297:Male; D051381:Rats; D017208:Rats, Wistar
chemical_list: D043384:Glutamyl Aminopeptidase; D002945:Cisplatin