title: Kidney tissue proteomics reveals regucalcin downregulation in response to diabetic nephropathy with reflection in urinary exosomes.
abstract: Diabetic nephropathy (DN) is a major complication of diabetes mellitus and the most frequent cause of end-stage renal disease. DN progresses silently and without clinical symptoms at early stages. Current noninvasive available markers as albuminuria account with severe limitations (late response, unpredictable prognosis, and limited sensitivity). Thus, it urges the discovery of novel markers to help in diagnosis and outcome prediction. Tissue proteomics allows zooming-in where pathophysiological changes are taking place. We performed a differential analysis of renal tissue proteome in a rat model of early DN by 2-dimensional differential gel electrophoresis and mass spectrometry. Confirmation was performed by Western blot, immunohistochemistry (IHC), and selected reaction monitoring (SRM). Rat urine samples were collected and exosomes were isolated from urine to evaluate if these microvesicles reflect changes directly occurring at tissue level. The protein showing maximum altered expression in rat tissue in response to DN was further analyzed in human kidney tissue and urinary exosomes. Regucalcin protein or senescence marker protein-30 (SMP30) (Swiss-Prot Q03336) was found to be strongly downregulated in DN kidney tissue compared with healthy controls. The same trend was observed in exosomes isolated from urine of control and DN rats. These data were further confirmed in a pilot study with human samples. IHC revealed a significant decrease of regucalcin in human kidney disease tissue vs control kidney tissue, and regucalcin was detected in exosomes isolated from healthy donors' urine but not from kidney disease patients. In conclusion, regucalcin protein expression is reduced in DN kidney tissue and this significant change is reflected in exosomes isolated from urine. Urinary exosomal regucalcin represents a novel tool, which should be explored for early diagnosis and progression monitoring of diabetic kidney disease.
authors.affiliation: Department of Immunology, IIS-Fundación Jiménez Díaz, UAM, REDINREN, Madrid, Spain.
authors.forename: Irene
authors.initials: I
authors.lastname: Zubiri
authors.affiliation: Department of Immunology, IIS-Fundación Jiménez Díaz, UAM, REDINREN, Madrid, Spain.
authors.forename: Maria
authors.initials: M
authors.lastname: Posada-Ayala
authors.affiliation: Department of Nephrology/UAM/IRSIN, IIS-Fundación Jiménez Díaz, REDINREN, Madrid, Spain.
authors.forename: Alberto
authors.initials: A
authors.lastname: Benito-Martin
authors.affiliation: Department of Immunology, IIS-Fundación Jiménez Díaz, UAM, REDINREN, Madrid, Spain.
authors.forename: Aroa S
authors.initials: AS
authors.lastname: Maroto
authors.affiliation: Department of Immunology, IIS-Fundación Jiménez Díaz, UAM, REDINREN, Madrid, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Martin-Lorenzo
authors.affiliation: Department of Pathology, IIS-Fundación Jiménez Díaz, Madrid, Spain.
authors.forename: Pablo
authors.initials: P
authors.lastname: Cannata-Ortiz
authors.affiliation: Department of Vascular Physiopathology, Hospital Nacional de Parapléjicos, SESCAM, Toledo, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: de la Cuesta
authors.affiliation: Department of Immunology, IIS-Fundación Jiménez Díaz, UAM, REDINREN, Madrid, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Gonzalez-Calero
authors.affiliation: Department of Vascular Physiopathology, Hospital Nacional de Parapléjicos, SESCAM, Toledo, Spain.
authors.forename: Maria G
authors.initials: MG
authors.lastname: Barderas
authors.affiliation: Department of Nephrology/UAM/IRSIN, IIS-Fundación Jiménez Díaz, REDINREN, Madrid, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Fernandez-Fernandez
authors.affiliation: Department of Nephrology/UAM/IRSIN, IIS-Fundación Jiménez Díaz, REDINREN, Madrid, Spain.
authors.forename: Alberto
authors.initials: A
authors.lastname: Ortiz
authors.affiliation: Department of Immunology, IIS-Fundación Jiménez Díaz, UAM, REDINREN, Madrid, Spain; Department of Biochemistry and Molecular Biology I, Universidad Complutense, Madrid, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: Vivanco
authors.affiliation: Department of Immunology, IIS-Fundación Jiménez Díaz, UAM, REDINREN, Madrid, Spain. Electronic address: galvarez@fjd.es.
authors.forename: Gloria
authors.initials: G
authors.lastname: Alvarez-Llamas
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases; D016022:Case-Control Studies; D003928:Diabetic Nephropathies / Q000378:metabolism* / Q000652:urine; D015536:Down-Regulation; D055354:Exosomes / Q000378:metabolism*; D005260:Female; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D040901:Proteomics; D051381:Rats; D011921:Rats, Inbred WKY
chemical_list: D002135:Calcium-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C513323:RGN protein, human; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat