Evaluation of the Zucker diabetic fatty (ZDF) rat as a model for human disease based on urinary peptidomic profiles

Abstract

Representative animal models for diabetes-associated vascular complications are extremely relevant in assessing potential therapeutic drugs. While several rodent models for type 2 diabetes (T2D) are available, their relevance in recapitulating renal and cardiovascular features of diabetes in man is not entirely clear. Here we evaluate at the molecular level the similarity between Zucker diabetic fatty (ZDF) rats, as a model of T2D-associated vascular complications, and human disease by urinary proteome analysis. Urine analysis of ZDF rats at early and late stages of disease compared to age- matched LEAN rats identified 180 peptides as potentially associated with diabetes complications. Overlaps with human chronic kidney disease (CKD) and cardiovascular disease (CVD) biomarkers were observed, corresponding to proteins marking kidney damage (eg albumin, alpha-1 antitrypsin) or related to disease development (collagen). Concordance in regulation of these peptides in rats versus humans was more pronounced in the CVD compared to the CKD panels. In addition, disease-associated predicted protease activities in ZDF rats showed higher similarities to the predicted activities in human CVD. Based on urinary peptidomic analysis, the ZDF rat model displays similarity to human CVD but might not be the most appropriate model to display human CKD on a molecular level.

Figure 1. Study design.

For biomarker definition urine samples of ZDF and LEAN control rats were used. The defined ZDF rat biomarkers at the late stage of disease (8 months) were compared based on the orthology of amino acid sequences, to the human CKD and CVD biomarkers. The overlap in sequenced urine peptide markers between early (2 months) and late stage disease ZDF rat markers is given in the Venn-diagram.

Figure 2. ZDF rats develop renal damage and cardiac structural changes after the onset of diabetes.

A) Photomicrographs showing representative sections of kidneys from LEAN and ZDF rats at 2 and 8 months of age. No changes in renal morphology are observed in LEAN rats and in 2-month old ZDF rats. Sections from 8-month ZDF rats show glomerular sclerosis with thickening of the Bowman’s capsule and retraction of the tuft, tubular atrophy and dilation, and hyaline casts. PAS staining/200x; B) Time course of systolic blood pressure (SBP) in LEAN control and ZDF rats. C) Cardomyocyte area and D) Capillary density measured in cardiac tissue from LEAN and ZDF rats at 2 and 8 months of age. Values are mean±SD; *P<0.05; **P<0.01 vs. ZDF at 2 months; °P<0.05 vs. ZDF at 4 months.

Figure 3. Group specific contour plots of LEAN control rats at 2 months (n = 10) and 8 months (n = 7) of age and type 2 diabetes ZDF rats at 2 and 8 months of age (n = 10 at each time).

Each consisting of digitally compiled data sets of urine samples from all individual rats in a 3D depiction. Molecular mass of the analyzed polypeptides (0.8–25 kDa) in logarithmic scale is plotted against the CE migration time (18–60 min) with MS signal intensity in z-axis.

Figure 4. Similarity in amino acid sequences between rat and human biomarkers and predicted protease activities.

a) Amino acid sequence orthology between the 50 most significant human biomarkers for CKD and CVD and the defined 180 rodent markers for diabetes associated complications. To examine the orthology between human and rat markers we applied three criteria: first, we looked for identical fragments (both cleavage sites identical in human and rat); secondly, we looked for fragments with one identical cleavage site and third, we looked for peptides from the same protein area with a minimum overlap in two amino acids. b) Predicted protease activities related to rat and human COL1A1 biomarker fragments. The relative number of specific cleavage sites for MMPs, ADAMTS5, CTSK and F2 and direction of amplitude regulation for COL1A1 markers in rat and human models are given.