Computational protocol: Urinary albumin and transferrin as early diagnostic markers of chronic kidney disease

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Protocol publication

[…] Animals: Urine samples collected from 31 cats with nephropathy (stage I CKD) brought to Maeda Veterinary Hospital between July 28, 2007 and July 10, 2008 (American Shorthair: 3, Chinchilla: 1, Mix: 27; 15 males and 16 females aged 3–14 years) were used in the study. Urine samples collected from 92 cats in IRIS stage I aged ≤2 years (Scottish Fold: 1, American Shorthair: 1, Mix: 90; 42 males and 50 females aged 4 months to 2 years) were used as normal controls. Regarding the diagnostic criteria, cats <2 years old clinically diagnosed as normal based on serum biochemistry test results were defined as normal cats, and cats aged ≥6 years showing clinical signs of CKD in blood chemistry were defined as those with stage I CKD. Criteria for normal urinalysis were USG >1.030, UPC <0.4 and a negative bacteriologic urine culture []. Diagnosis of CKD was made prior to inclusion in the study, based on clinical and laboratory (i.e., renal azotemia and USG <1.030) findings. Cats were classified into 4 stages according to the IRIS guidelines after stabilization [], with p-Cre of <1.6, 1.6–2.8 mg/dl and 2.9–5.0 mg/dl in stages I, II and III, respectively. Urine was collected by catheterization. Samples were centrifuged at 1,190 g for 10 min (Kubota, Tokyo, Japan) and then stored at −80°C for further use. All owners gave signed informed consent to participation of their animal in the study. Urine samples collected from 31 cats with nephropathy (stage I CKD) brought to Maeda Veterinary Hospital.SDS-PAGE analysis: Urine samples were dissolved in PAGE sample buffer (pH 6.8; 50 mM Tris-HCl, 50 mM dithiothreitol, 0.5% SDS and 10% glycerol), and the resulting solution was analyzed using SDS-PAGE (e-PAGEL, 10% acrylamide and 14 wells; Atto Corp., Tokyo, Japan). The gel was stained with Coomassie brilliant blue (CBB) (PhastGel Blue R; GE Healthcare, Little Chalfont, U.K.). The intensities of the albumin and transferrin bands were quantified using CS Analyzer ver. 3.0 (Atto Corp.) and used as indices of the level of protein expression. Bovine serum albumin and human transferrin (both from Sigma-Aldrich, St. Louis, MO, U.S.A.) were used as reference proteins.In-gel digestion of proteins: The gel was cut into small pieces, destained in 50% Acetonitril/50 mM NH4HCO3 and washed with deionized water. The gel pieces were dehydrated in 100% Acetonitril for 15 min and dried in a SpeedVac Evaporator (Wakenyaku, Kyoto, Japan) for 45 min. The gel pieces were rehydrated in 10–30 µl of 25 mM Tris-HCl/20% Acetonitril containing 25 ng/l trypsin (Trypsin sequence grade, Roche, Basel, Swiss) for 45 min. After removal of unabsorbed solution, the gel pieces were incubated in 10–20 µl of 50 mM Tris-HCl/20% Acetonitril for 20 hr at 37°C. The solution containing digested fragments of proteins was transferred to a new tube, and peptide fragments remaining in the gel were extracted in 5% formic acid/50% ACN for 20 min at room temperature [].Protein identification: In-gel digested peptides were injected into a 0.3 × 5 mm L-trap column (Chemicals Evaluation and Research Institute, Saitama, Japan) and a 0.1 × 50 mm analytical Monolith column (AMR, Tokyo, Japan) attached to a HPLC system (Nanospace SI-2; Shiseido Fine Chemicals, Tokyo, Japan). The flow rate of the mobile phase was 1 µl/min. The solvent composition of the mobile phase was programmed to change in 35-min cycles with varying mixing ratios of solvent A (2% v/v CH3CN and 0.1% v/v HCOOH) to solvent B (90% v/v CH3CN and 0.1% v/v HCOOH): 5–50% B for 20 min, 50 to 95% B for 1 min, 95% B for 3 min, 95 to 5% B for 1 min and 5% B for 10 min. Purified peptides were introduced from HPLC to an LTQ-XL ion trap mass spectrometer (Thermo Scientific, Waltham, CA, U.S.A.) via an attached Pico Tip (New Objective, Woburn, MA, U.S.A.). MS and MS/MS peptide spectra were measured in a data-dependent manner based on the manufacturer’s operating specifications. The Mascot search engine (Matrix Science, London, U.K.) was used to identify proteins from the mass and tandem mass spectra of peptides. Peptide mass data were matched by searching the International Protein Index database (European Bioinformatics Institute, Cambridge, U.K.) using the MASCOT engine. The minimum criterion for the probability-based MASCOT/MOWSE score was set at 5% as the significant threshold level.Other procedures: Creatinine was measured by an enzymatic method with creatinine deiminase using a Fuji DRI-CHEM Slide CRE-PIII kit (Fuji Film Medical, Tokyo, Japan). BUN was measured using a N-Assay BUN-L Nittobo D-Type kit (Nittobo Medical Co., Ltd., Tokyo, Japan). The specific gravity was measured using a pocket refractometer for urine specific gravity PAL-09S (Atago, Tokyo, Japan). Numerical data are presented as the mean ± standard deviation (SD). Statistical analysis was performed using IBM SPSS Statistics 19 software (SPSS Inc., Chicago, IL, U.S.A.) with P<0.05 considered to be significant.Histopathological examination: The u-Tf level was >1.5 mg/dl in one each of the normal and stage I CKD cats, and these animals were examined histopathologically. Renal biopsy was performed with a Tru-Cut needle (16G, 9 cm) []. Fixed tissues embedded in paraffin were sectioned and stained with hematoxylin and eosin (H&E) after appropriate standard treatments. []. The biopsy specimens were reviewed by experienced pathologists. […]

Pipeline specifications

Software tools Mascot Server, SPSS
Databases IPI
Applications Miscellaneous, MS-based untargeted proteomics
Organisms Felis catus
Diseases Kidney Diseases, Renal Insufficiency, Chronic
Chemicals Creatinine