title: Diabetic nephropathy induces changes in the proteome of human urinary exosomes as revealed by label-free comparative analysis.
abstract: Diabetic nephropathy (DN) is a major complication of diabetes mellitus (DM), the most frequent cause of end-stage renal disease (ESRD). Exosomes isolated from urine are considered a rich non-invasive source of markers for renal events. Proteinuria associated with DN patients at advanced stages may result in "contamination" of exosomal fraction by co-precipitation of high abundance urine proteins, making it enormously difficult to obtain a reliable comparison of healthy individuals and DN patients and to detect minor proteins. We evaluated different protocols for urinary exosome isolation (ultracentrifugation-based and Exoquick® reagent-based) in combination with an easy and quick depletion procedure of contaminating high abundance proteins (albumin). The optimal methodology was then applied to investigate the proteome of human urinary exosomes in DN and controls using spectral counting LC-MS/MS analysis followed by selected reaction monitoring (SRM) confirmation. A panel of 3 proteins (AMBP, MLL3, and VDAC1) is differentially present in urinary exosomes from DN patients, opening a new field of research focused on improving diagnosis and follow-up of this pathology.
  
  
  CONCLUSIONS
  Diabetic nephropathy (DN) is a progressive proteinuric kidney disease, a major complication of diabetes mellitus, and the most frequent cause of end-stage renal disease. Current markers of disease (i.e. creatinine and urinary albumin excretion) have proven limitations (i.e. some patients regress to normoalbuminuria, kidney damage may be already present in recently diagnoses microalbuminuric patients and renal function may decrease in the absence of significant albuminuria). We show here the first study on human DN proteome of urinary exosomes. Proteinuria associated to DN patients resulting in contamination of exosomal fraction and the associated difficulty to reliably compare healthy and disease conditions, are here overcome. A combined methodology pointed to increase exosomal proteome recovery and depletion of high-abundance proteome was here set-up. A total of 352 proteins were here identified for the first time associated to human urinary exosomes. Label-free quantitative comparison of DN urinary exosomes vs control group and SRM further validation, resulted in the discovery of a panel of three proteins (AMBP, MLL3 and VDAC1) which changes in DN, opening a new field of research focused to improve diagnosis and follow-up of this pathology.
authors.affiliation: Departmento de Inmunologia, IIS-Fundacion Jimenez Diaz, Madrid, Spain.
authors.forename: Irene
authors.initials: I
authors.lastname: Zubiri
authors.affiliation: Departmento de Inmunologia, IIS-Fundacion Jimenez Diaz, Madrid, Spain.
authors.forename: Maria
authors.initials: M
authors.lastname: Posada-Ayala
authors.affiliation: Departmento de Inmunologia, IIS-Fundacion Jimenez Diaz, Madrid, Spain.
authors.forename: Aroa
authors.initials: A
authors.lastname: Sanz-Maroto
authors.affiliation: Unidad de Proteomica, CNIC, Madrid, Spain.
authors.forename: Enrique
authors.initials: E
authors.lastname: Calvo
authors.affiliation: Departmento de Inmunologia, IIS-Fundacion Jimenez Diaz, Madrid, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Martin-Lorenzo
authors.affiliation: Departmento de Inmunologia, IIS-Fundacion Jimenez Diaz, Madrid, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Gonzalez-Calero
authors.affiliation: Department of Vascular Physiopathology, Hospital Nacional de Paraplejicos SESCAM, Toledo, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: de la Cuesta
authors.affiliation: Unidad de Proteomica, CNIC, Madrid, Spain.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Lopez
authors.affiliation: Departamento de Nefrologia, IIS-Fundacion Jimenez Diaz-IRSIN-UAM, Madrid, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Fernandez-Fernandez
authors.affiliation: Departamento de Nefrologia, IIS-Fundacion Jimenez Diaz-IRSIN-UAM, Madrid, Spain.
authors.forename: Alberto
authors.initials: A
authors.lastname: Ortiz
authors.affiliation: Departmento de Inmunologia, IIS-Fundacion Jimenez Diaz, Madrid, Spain; Departamento de Bioquimica y Biologia Molecular I, Universidad Complutense, Madrid, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: Vivanco
authors.affiliation: Departmento de Inmunologia, IIS-Fundacion Jimenez Diaz, Madrid, Spain. Electronic address: galvarez@fjd.es.
authors.forename: Gloria
authors.initials: G
authors.lastname: Alvarez-Llamas
mesh_terms: D000328:Adult; D000368:Aged; D000419:Albuminuria / Q000652:urine; D000510:Alpha-Globulins / Q000652:urine*; D004268:DNA-Binding Proteins / Q000652:urine*; D003928:Diabetic Nephropathies / Q000652:urine*; D055354:Exosomes / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D020543:Proteome / Q000378:metabolism*; D050995:Voltage-Dependent Anion Channel 1 / Q000652:urine*
chemical_list: D000510:Alpha-Globulins; D004268:DNA-Binding Proteins; C452979:KMT2C protein, human; D020543:Proteome; C494762:VDAC1 protein, human; C016001:alpha-1-microglobulin; D050995:Voltage-Dependent Anion Channel 1
keywords: Depletion; Diabetic nephropathy; Exosomes; Label-free quantitation; Proteomics; Selected reaction monitoring