Proteome profiling in the aorta and kidney of type 1 diabetic rats

Abstract

Diabetes is associated with a number of metabolic and cardiovascular risk factors that contribute to a high rate of microvascular and macrovascular complications. The risk factors and mechanisms that contribute to the development of micro- and macrovascular disease in diabetes are not fully explained. In this study, we employed mass spectrometric analysis using tandem LC-MS/MS to generate a proteomic profile of protein abundance and post-translational modifications (PTM) in the aorta and kidney of diabetic rats. In addition, systems biology analyses were employed to identify key protein markers that can provide insights into molecular pathways and processes that are differentially regulated in the aorta and kidney of type 1 diabetic rats. Our results indicated that 188 (111 downregulated and 77 upregulated) proteins were significantly identified in the aorta of diabetic rats compared to normal controls. A total of 223 (109 downregulated and 114 upregulated) proteins were significantly identified in the kidney of diabetic rats compared to normal controls. When the protein profiles from the kidney and aorta of diabetic and control rats were analyzed by principal component analysis, a distinct separation of the groups was observed. In addition, diabetes resulted in a significant increase in PTM (oxidation, phosphorylation, and acetylation) of proteins in the kidney and aorta and this effect was partially reversed by insulin treatment. Ingenuity pathway analysis performed on the list of differentially expressed proteins depicted mitochondrial dysfunction, oxidative phosphorylation and acute phase response signaling to be among the altered canonical pathways by diabetes in both tissues. The findings of the present study provide a global proteomics view of markers that highlight the mechanisms and putative processes that modulate renal and vascular injury in diabetes.

Fig 1. Plasma glucose levels (A) and body weights (B) in control, diabetic and insulin-treated diabetic rats.

(A) Plasma glucose levels were significantly increased one day after STZ injection in both diabetic groups compared to controls. (B) Initial body weights were not significantly different between diabetic and control rats. The blue line is for Control rats, the red line for Diabetic rats, and the green line is for Insulin-treated diabetic rats. Cross-sectional analysis using non-parametric Kruskal-Wallis followed by Bonferroni correction was employed to assess the difference between the groups at each time point. * represents a significant difference between diabetes and control groups with p<0.03, and # represents a significant difference between insulin-treated diabetes and diabetes groups with p = 0.02.

Fig 2. Hierarchical clustering (heat maps) of protein expression profiles in the aorta (A) and kidney (B) among the three groups of rats.

The compared groups are Diabetic vs. Control samples, Insulin-treated diabetic vs. Control samples, and Insulin-treated diabetic vs. Diabetic samples. Green color represents downregulation of protein expression, whereas the red color represents upregulation of protein expression. Color intensity reflects the expression level of the proteins. The label on the right-hand side of the heat maps represents the accession number of the proteins.

Fig 3. Principal component analysis (PCA) of the aorta (A) and the kidney (B) samples.

The total normalized expression data of the proteins was used to depict the scatter plots of the first (X) and the second (Y) principal components. The numbering of the nodes is an identification of the samples. Abbreviation A (Control), B (Diabetic), and C (Insulin-treated Diabetic). Blue oval encircles the control samples, green oval encircles the diabetic samples, and red oval encircles the Insulin-treated diabetic samples.

Fig 4. Scatter plot of the mean intensities of the three PTMs in the aorta (A, B, and C) and the kidney (D, E, and F) samples.

A and C show the scatter of the oxidized proteins among the 3 different groups. B and E show the scatter of the phosphorylated proteins among the 3 different groups. C and F show the scatter of the acetylated proteins among the 3 different groups (* p<0.05).

Fig 5. Canonical pathways of the comparative groups in the aorta and the kidney samples.

A: top Canonical pathways related to proteins altered in the aorta of Diabetes vs. control samples. B: Top Canonical Pathways related to proteins altered in the aorta of Insulin-treated Diabetic vs Diabetic samples C: Top Canonical Pathways related to proteins altered in the kidney of Diabetic vs. Control samples. D: Top Canonical Pathways related to proteins altered in the kidney of Insulin-treated Diabetic vs. Diabetic samples. Bars show the total number of proteins identified in each pathway. The green color represents the downregulated proteins, and the red color represents the upregulated proteins.

Fig 6. IPA network analysis of the modified proteins in the aorta of diabetic relative to control rats.

A: The top diseases and functions related to the modified proteins are Lipid Metabolism, Molecular Transport, and Small Molecule Biochemistry. The top Toxicity and functions predicted by IPA related to the modified proteins are Cardiac Fibrosis, Cardiac Hypertrophy, Cardiac Necrosis/Cell Death, Increased Cardiac Dysfunction, Increased Cardiac Proliferation, Increased Cardiac Dilatation, Mitochondrial Dysfunction, and Oxidative Stress. The main regulated proteins connected in this network were Kng1, AKT, and Leptin. B: Diseases and Biological Functions predicted by IPA related to the differentially expressed proteins in this comparative group. The diseases related to these proteins are apoptosis of endothelial cells, formation of thrombus, necrosis of epithelial tissue, and occlusion of blood vessel. The color intensity of the nodes reflects the expression of the proteins. Green nodes are downregulated proteins, red nodes are upregulated proteins, white nodes are IPA predicted proteins with non-consistent activation pattern, blue nodes are IPA predicted proteins to be inhibited, and orange nodes are IPA predicted proteins to be activated.

Fig 7. IPA network analysis of the modified proteins in the aorta of insulin-treated diabetic relative to diabetes rats.

The top Toxicity and functions predicted by IPA related to the modified proteins are Cardiac Fibrosis, Hypertrophy, Necrosis/Cell Death, Oxidative Stress, Increased Cardiac Proliferation, Dilation, and Dysfunction. The main regulated proteins connected in this network are ACE, AGT, and TNF. B: Diseases and Biological Functions predicted by IPA related to the differentially expressed proteins in this comparative group. The main biological functions altered in this network are apoptosis, metabolism of hydrogen peroxide, and release of reactive oxygen species. The diseases related to these proteins are damage of blood vessel, and hypertrophy of thoracic aorta. The color intensity of the nodes reflects the expression of the proteins. Green nodes are downregulated proteins, red nodes are upregulated proteins, white nodes are IPA predicted proteins with non-consistent activation pattern, blue nodes are IPA predicted proteins to be inhibited, and orange nodes are IPA predicted proteins to be activated.

Fig 8. IPA network analysis of the modified proteins in the kidney of diabetic relative to control rats.

A: The top Toxicity and functions predicted by IPA related to the modified proteins are Increase Renal Damage, Nephritis, Proliferation, Necrosis/Cell Death, Oxidative Stress, Acute Renal Failure Panel (Rat), and Mitochondrial Dysfunction. The main regulated proteins in this network are EGFR, LEP, LEPR, and TNF. B: Diseases and Biological Functions predicted by IPA related to the differentially expressed proteins in this comparative group. The main biological functions altered in this network are adhesion of glomerular cells, generation of reactive oxygen species, the growth of epithelial tissue, and metabolism of hydrogen peroxide and reactive oxygen species. The diseases related to these proteins are nephrosis, and passive Heymann nephritis. The color intensity of the nodes reflects the expression of the proteins. Green nodes are downregulated proteins, red nodes are upregulated proteins, white nodes are IPA predicted proteins with non-consistent activation pattern, blue nodes are IPA predicted proteins to be inhibited, and orange nodes are IPA predicted proteins to be activated.

Fig 9. IPA network analysis of the modified proteins in the kidney of insulin-treated diabetic relative to diabetic rats.

A: The top Toxicity and functions predicted by IPA related to the modified proteins are Increase Damage to Mitochondria, Glomerular Injury, Renal Proliferation, Necrosis/Cell Death, Oxidative Stress, Acute Renal Failure Panel (Rat), and Mitochondrial Dysfunction. The main regulated proteins in this network are NFkB (complex), PRDX5, and SOD2. B: Diseases and Biological Functions predicted by IPA related to the differentially expressed proteins in this comparative group. The main biological functions altered in this network are adhesion of glomerular cells and metabolism and synthesis of reactive oxygen species. The diseases related to these proteins are nephrosis, passive Heymann nephritis. The color intensity of the nodes reflects the expression of the proteins. Green nodes are downregulated proteins, red nodes are upregulated proteins, white nodes are IPA predicted proteins with non-consistent activation pattern, and orange nodes are IPA predicted proteins to be activated.