Urinary Proteome Analysis Enables Assessment of Renoprotective Treatment in Type 2 Diabetic Patients with Microalbuminuria

Sten Andersen; Harald Mischak; Petra Zürbig; Hans-Henrik Parving; Peter Rossing

Disclosures

BMC Nephrology. 2010;11 

In This Article

Methods

Patient Characteristics

Spontaneously voided urine samples were collected from type 2 diabetic patients followed at Steno Diabetes Center as a subset of the 'IRMA2' study described previously.[45] The study was in compliance with the Helsinki Declaration and all patients gave written informed consent. The study was approved by the ethics committee of Copenhagen County KA 97015 gms. Samples from all patients included in the study receiving either Irbesartan in a dose of 300 mg once daily or placebo were employed for CE-MS analysis, if samples were available from both, baseline and after two years of treatment. As the effect of a dose of 150 mg once daily was not significant on UAER, in the IRMA2 study, we only used 300 mg daily and compared with placebo. In total, samples from 22 patients (11 irbesartan and 11 placebo) were available. At baseline 2 patients in the placebo and 4 in the irbesartan group were treated with insulin, after 2 years it was 5 and 4. Unchanged throughout the study, 8 patients in the placebo and 6 in the irbesartan group were treated with oral hypoglycemic agents at baseline, 3 patients in each group were treated with a statin at baseline, 8 patients in the placebo and 5 in the irbesartan group were treated with aspirin for cardiovascular protection at baseline. Demographic data of the patients included are shown in Additional file 1, spreadsheet: 'patient data'.

Sample Preparation

Samples consisted of overnight urines, stored in aliquots at -20°C for 8-12 years, which were prepared essentially as described.[46] A 0.7 mL aliquot was thawed immediately before use and diluted with 0.7 mL 2 M urea, 10 mM NH4OH containing 0.02 % SDS. In order to remove high molecular weight polypeptides, samples were filtered using Centrisart ultracentrifugation filter devices (20 kDa molecular weight cut-off; Sartorius, Goettingen, Germany) at 3,000 g until 1.1 mL of filtrate was obtained. Subsequently, filtrate was desalted using PD-10 column (GE Healthcare, Sweden) equilibrated in 0.01% NH4OH in HPLC-grade water. Finally, samples were lyophilized and stored at 4°C. This procedure results in an average recovery of sample in the preparation procedure ~85%.[21] Shortly before CE-MS analysis, lyophilisates were resuspended in HPLC-grade water to a final protein concentration of 0.8 μg/μL checked by BCA assay (Interchim, Montlucon, France).

CE-MS Analysis

CE-MS analysis was performed as previously described.[37,47] The limit of detection was ~1 fmol, mass resolution was above 8000 enabling resolution of monoisotopic mass signals for z≤ 6. After charge deconvolution, mass deviation was < 25 ppm for monoisotopic resolution and < 100 ppm for unresolved peaks (z > 6). The analytical precision of the platform was assessed by (a) reproducibility achieved for repeated measurement of the same replicate and (b) by the reproducibility achieved for repeated preparation and measurement of the same urine sample; details on analytical precision were reported recently.[21] To ensure high data consistency, a minimum of 950 peptides/proteins had to be detected with a minimal MS resolution of 8,000 in a minimal migration time interval of 10 minutes.

Data Processing

Mass spectral ion peaks representing identical molecules at different charge states were deconvoluted into single masses using MosaiquesVisu software.[48] Both CE-migration time and ion signal intensity (amplitude) show variability, mostly due to different concentration of ions in the sample, and are consequently normalized. Reference signals of 1770 urinary polypeptides are used for CE-time calibration by local regression. For normalization of analytical and urine dilution variances, MS signal intensities are normalized relative to 29 "housekeeping" peptides generally present in at least 90% of all urine samples with small relative standard deviation. For calibration, local regression is performed.[49] The obtained peak lists characterize each polypeptide by its molecular mass [Da], normalized CE migration time [min] and normalized signal intensity. All detected peptides were deposited, matched, and annotated in a Microsoft SQL database allowing further statistical analysis.

Data Analysis

The datasets were examined either with respect to significant changes in single, predefined peptides and with respect to scoring in biomarker models (see Additional file 1, spreadsheet: 'classification factor'). These biomarker models consist of 65 or 273 biomarkers respectively, which were previously found to be significantly associates with diabetic nephropathy[19] or chronic kidney disease.[20]

For the application of the previously established biomarker patterns, Wilcoxon test (for paired samples) was performed to receive Box-and-Whisker plots and dot-and-line diagrams[50] (MedCalc version 8.1.1.0, MedCalc Software, Belgium, https://www.medcalc.be).

For multiple testing corrections, p-values were corrected using the false discovery rate procedure introduced by Benjamini and Hochberg.[51] To eliminate sporadic findings, only proteins that were detected in a diagnostic group of patients in at least 50% of samples were considered.

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