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

Results

Samples from 22 patients included in the IRMA2 trial, where urine was collected at baseline before treatment (visit 2) and after two years treatment (visit 9) with Irbesartan or placebo, were analyzed. All available samples were included in the study, and analyzed using CE-MS, no additional specimens that fit the criteria (2 years follow up, placebo or 300 mg Irbesartan daily) are available from the IRMA2 trial. All samples analyzed passed the threshold of the quality control criteria given in the Methods section, no significant deterioration of peptides due to storage could be observed. The data from all analyses are presented in the Additional file 1. As shown in figure 1, the compiled data of these 4 groups disclosed first insights into changes of the urinary proteome, where high concentrations of some peptides decreased with Irbesartan intake. To assess the relevance of any proteomics changes with respect to diabetic nephropathy, we applied already established polypeptide patterns onto these data.

Figure 1.

Polypeptide patterns of patients with diabetes type 2 before and after 2-year treatment (Irbesartan and placebo) examined in the 'IRMA2' study. Shown are compiled patterns consisting of all samples from each of the four groups. The molecular mass (0.7 to 15 kDa, on a logarithmic scale) is plotted against normalized migration time (17 to 47 min). Signal intensity is encoded by peak height and color.

First, data from patients that received ARB treatment were evaluated applying a biomarker pattern indicative for diabetic nephropathy.[19] This analysis revealed no significant differences (p = 0.175) between these two groups of patients (visit 2 and visit 9) using Wilcoxon-test for paired samples (data not shown). However, the DN pattern was developed employing samples from diabetes type 1 patients treated with ARB,[19] hence may not be applicable for type 2 diabetic patients, and may further be inappropriate to reflect drug-induced changes.

We therefore also employed a polypeptide pattern indicative of chronic kidney disease (CKD), that consist of 273 known peptides[20] for the classification of the urine samples from the 'IRMA2' study. This model is based on the CE-MS analysis of urine samples from 340 patients with CKD of different etiologies (including focal segmental glomerulosclerosis, membranous glomerulonephritis, minimal change disease, IgA nephropathy, systemic lupus erythematosus, ANCA-associated vasculitis, and diabetic nephropathy) and 550 controls (healthy individuals as well as patients without any evidence for renal diseases). Figure 2 demonstrates the changes of these 273 peptides of the CKD model before and after treatment with Irbesartan and placebo, respectively. While the peptide pattern of the ARB treated patients is similar to that observed for diabetic nephropathy (compare Figure 1 in[19]) at the beginning of the study (prior treatment), it changed towards higher similarity to normalbuminuric subjects after 2 years of Irbesartan treatment. As depicted in the Box-and-Whisker plot in figure 3A, this classification resulted in a significant (p = 0.0244) decline of the median classification factor (indicating an improvement of the kidney physiology), which was reduced (from 0.721 at visit 2 to 0.277 at visit 9) below the established cut-off (0.343) of the CKD model. Irrespective of the values before Irbesartan intake, the classification factors were decreasing during Irbesartan treatment in all patients except one (see figure 3B). This patient progressed to DN several years after the end of the study, none of the eleven patients developed macroalbuminuria during the two year study period. In the urine samples of the eleven patients treated with placebo, a non significant (p = 0.1016) increase (indicating a change towards "chronic kidney disease") of the median classification factor (see figure 3C) from -0.104 at visit 2 to 0.188 at visit 9 could be observed. Although many patients of the placebo-group scored lower than those of the Irbesartan-group at baseline before treatment (see figure 3B and 3D), the classification factor of most placebo-treated patients was higher after two years, as expected for progressing disease.

Figure 2.

Peptide patterns of 273 CKD marker used for the proteomic analysis of patients from the 'IRMA2' subgroup. The compiled data sets of urine samples from patients derived from the 'IRMA2 study' before and after 2-year treatment of Irbesartan (upper panel) as well as placebo (lower panel) are shown. Normalized molecular mass (y-axis) is plotted against normalized CE-migration time (x-axis). The mean signal intensity is represented in 3D-depiction.

Figure 3.

Classification results of the 'IRMA2' patient samples, classified with the CKD model [20]. A) Box-and-Whisker plot of microalbuminuric patients before (visit 2) and after two years (visit 9) treatment with 300 mg Irbesartan. The red line indicates the cut-off of the CKD model (classification factors above this cut-off are suffering from renal disease). B) Dot-and-line diagram of microalbuminuric patients before (visit 2) and after two years (visit 9) treatment with 300 mg Irbesartan. Classification factors of all patients, excepting patient no. 34, declined after Irbesartan intake. C) Box-and-Whisker plot of microalbuminuric patients before (visit 2) and after two years (visit 9) treatment with placebo. D) Dot-and-line diagram of microalbuminuric patients before (visit 2) and after two years (visit 9) placebo administration.

We subsequently investigated which of the 273 biomarkers that were found significantly associated with CKD undergo significant changes upon Irbesartan treatment. Eighteen of these CKD markers showed significant differences (p < 0.05) in urine of patients before and after 2-year treatment with Irbesartan (see Table 1 ). Of these 18, 11 changed towards "normal controls", indicating possible benefit of therapy. Seven changes towards "chronic kidney disease", possibly indicating progression of pathophysiological changes over time that is not affected by therapy. We also investigated the 273 biomarkers in the placebo group. Here, we found 7 CKD markers which show significant differences within the 2-year treatment. Of these 7 peptides, all changed toward "disease". In total, 23 urinary CKD markers showed significant changes over the period of two years, either in the patients of the Irbesartan group or in the placebo group, 2 were significant in both groups. These two CKD markers, both collagen alpha-1 fragments (see Table 1 , bold letters), showed significant change towards "healthy" in the Irbesartan group and opposite regulation in the placebo group over the period of two years. While the amount of these two collagen fragments increased significantly in the ARB group (indicating an improvement towards "healthy"), their abundance was significantly decreased after 2 years of placebo treatment, indicating further progression of chronic kidney disease.

To obtain information on additional changes in the urinary proteome associated with Irbesatan treatment beyond those observed for the previously defined CKD biomarkers, we examined the data on all sequenced peptides[40,52] for significant changes between baseline and 2-year treatment (in each group; Irbesartan and placebo). We could not identify additional biomarkers, which revealed significant changes between baseline and 2-year treatment.

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