Objective: Urine is an ideal source of biomarkers, but data on cat urine proteome is still very limited. The aim of our study is to apply proteomic techniques to identify putative biomarkers of nephropathy in cats affected by chronic kidney disease (CKD). Design and Methods: Urine samples were collected by cystocentesis from 4 healthy and 4 cats affected by CKD and analyzed by two-dimensional electrophoresis (2DE). The first dimension was performed by isoelectric focusing on 17 cm long IPG strips (pH 3-10); the second dimension was performed on 10% SDS-PAGE and stained with colloidal Coomassie. Spots were excised from the gel, reduced, alkylated and digested with trypsin and identified using ESI-Q-TOF mass spectrometry. Results: 2DE allowed the separation of 66 spots in the urine proteome of healthy and CKD cats. Twenty-one spots were successfully identified. In CKD samples, Uromodulin and Carboxylesterase-5A showed a significant reduction, supporting the tubular involvement in the pathology; Perlecan, a negatively charged proteoglycan showed no visible expression, suggesting its role in maintaining the integrity of the glomerular filtration barrier. On the other hand, 3 low molecular weight proteins, Retinol binding protein, Immunoglobulin K light chain and Complement factor D, increased significantly in CKD cats, confirming tubulo-interstitial involvement in cats affected by this disease. Conclusions: 2DE was successfully applied to the separation of cat urine proteome leading to the identification of 6 differentially expressed proteins; our data suggest their use as putative biomarker of nephropathy in cats and possibly in other veterinary species.

A PROTEOMICS STUDY IN CATS AFFECTED BY CHRONIC KIDNEY DISEASE – IDENTIFICATION OF PUTATIVE BIOMARKERS

FERLIZZA, ENEA;DONDI, FRANCESCO;ISANI, GLORIA
2014

Abstract

Objective: Urine is an ideal source of biomarkers, but data on cat urine proteome is still very limited. The aim of our study is to apply proteomic techniques to identify putative biomarkers of nephropathy in cats affected by chronic kidney disease (CKD). Design and Methods: Urine samples were collected by cystocentesis from 4 healthy and 4 cats affected by CKD and analyzed by two-dimensional electrophoresis (2DE). The first dimension was performed by isoelectric focusing on 17 cm long IPG strips (pH 3-10); the second dimension was performed on 10% SDS-PAGE and stained with colloidal Coomassie. Spots were excised from the gel, reduced, alkylated and digested with trypsin and identified using ESI-Q-TOF mass spectrometry. Results: 2DE allowed the separation of 66 spots in the urine proteome of healthy and CKD cats. Twenty-one spots were successfully identified. In CKD samples, Uromodulin and Carboxylesterase-5A showed a significant reduction, supporting the tubular involvement in the pathology; Perlecan, a negatively charged proteoglycan showed no visible expression, suggesting its role in maintaining the integrity of the glomerular filtration barrier. On the other hand, 3 low molecular weight proteins, Retinol binding protein, Immunoglobulin K light chain and Complement factor D, increased significantly in CKD cats, confirming tubulo-interstitial involvement in cats affected by this disease. Conclusions: 2DE was successfully applied to the separation of cat urine proteome leading to the identification of 6 differentially expressed proteins; our data suggest their use as putative biomarker of nephropathy in cats and possibly in other veterinary species.
E. Ferlizza; A. Campos; A. Cuoghi; E. Bellei; E. Monari; F. Dondi; A. M. Almeida; G. Isani
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/372516
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