Proteomics reveals novel oxidative and glycolytic mechanisms in type 1 diabetic patients' skin which are normalized by kidney-pancreas transplantation

Abstract

Background: In type 1 diabetes (T1D) vascular complications such as accelerated atherosclerosis and diffused macro-/microangiopathy are linked to chronic hyperglycemia with a mechanism that is not yet well understood. End-stage renal disease (ESRD) worsens most diabetic complications, particularly, the risk of morbidity and mortality from cardiovascular disease is increased several fold.

Methods and findings: We evaluated protein regulation and expression in skin biopsies obtained from T1D patients with and without ESRD, to identify pathways of persistent cellular changes linked to diabetic vascular disease. We therefore examined pathways that may be normalized by restoration of normoglycemia with kidney-pancreas (KP) transplantation. Using proteomic and ultrastructural approaches, multiple alterations in the expression of proteins involved in oxidative stress (catalase, superoxide dismutase 1, Hsp27, Hsp60, ATP synthase delta chain, and flavin reductase), aerobic and anaerobic glycolysis (ACBP, pyruvate kinase muscle isozyme, and phosphoglycerate kinase 1), and intracellular signaling (stratifin-14-3-3, S100-calcyclin, cathepsin, and PPI rotamase) as well as endothelial vascular abnormalities were identified in T1D and T1D+ESRD patients. These abnormalities were reversed after KP transplant. Increased plasma levels of malondialdehyde were observed in T1D and T1D+ESRD patients, confirming increased oxidative stress which was normalized after KP transplant.

Conclusions: Our data suggests persistent cellular changes of anti-oxidative machinery and of aerobic/anaerobic glycolysis are present in T1D and T1D+ESRD patients, and these abnormalities may play a key role in the pathogenesis of hyperglycemia-related vascular complications. Restoration of normoglycemia and removal of uremia with KP transplant can correct these abnormalities. Some of these identified pathways may become potential therapeutic targets for a new generation of drugs.

Figure 1. Typical silver-stained 2D electrophoresis pattern of proteins isolated from human skin biopsies in the broad nonlinear pH range 3–10.

Skin biopsies were obtained from controls, patients affected by type 1 diabetes (T1D), patients affected by T1D and end-stage renal disease (T1D+ESRD), and kidney-pancreas transplanted patients (KP).

Figure 2. Densitometric quantitation of differential protein expression in controls and type 1 diabetic patients (T1D).

Representative protein spots from 2D gels are inserted above each bar chart. SOD1 expression was increased by 2.5-fold and ATP synthase δ chain 2-fold in T1D compared with controls (p<0.01) (A). HSP27 and HSP60 were both up-regulated in T1D patients compared with controls (p<0.01) (A). Pyruvate kinase (10-fold) (p<0.001) and ACBP (2-fold) (p<0.01) expression in T1D patients was up-regulated compared with controls (B); while stratifin was not detectable in control patients and highly expressed in both T1D groups (p<0.001) (C). S100-calcyclin and rotamase expression was up-regulated by 2-fold in T1D compared with controls (p<0.05) (C). However, T1D showed a decrease in phosphoglycerate kinase and catalase expression compared with controls (p<0.05) (A, B). Flavin and Cathepsin are unchanged (A, C).

Figure 3. Densitometric quantitation of differential protein expression in controls, type 1 diabetic patients with end-stage renal disease (T1D+ESRD), and kidney-pancreas transplanted patients (KP).

Representative protein spots from 2D gels are inserted above each bar chart. SOD1 expression was increased by 5-fold in T1D+ESRD patients compared with controls (p<0.01). KP transplantation was shown to reduce by 2-fold this up-regulation (KP vs. T1D+ESRD and vs. controls, p<0.01 and p<0.05, respectively) (A). HSP27 expression was increased in T1D+ESRD patients compared with controls (p<0.01), and it was almost normalized in the KP group (KP vs. T1D+ESRD p<0.05). HSP60 expression was increased more than 2-fold in T1D+ESRD group compared with controls (p<0.01) and normalized in KP patients (KP vs. T1D+ESRD p<0.01) (A). ATP-synthase δ chain was 6-fold up-regulated in T1D+ESRD patients compared with controls (p<0.001) and was partially normalized in KP patients (KP vs. controls and T1D+ESRD p<0.05 and p<0.01) (A). The expression of pyruvate kinase (B) and stratifin (C) was increased in T1D+ESRD patients compared with controls (p<0.001). KP transplantation reduced the expression of pyruvate kinase (KP vs. T1D+ESRD p<0.05) and stratifin (KP vs. controls p<0.01 and vs. T1D+ESRD p<0.05) compared with T1D+ESRD. ACBP expression was increased by 3-fold in T1D+ESRD patients compared with controls (p<0.01) while in KP group the levels normalized (B). S100-calcyclin expression was up-regulated by 2-fold (p<0.01 vs. controls), while rotamase and cathepsin were unchanged (C). S100-calcyclin expression was normalized in KP group (p<0.05) (C). Catalase expression decreased 25-fold (p<0.001) (A) and phosphoglycerate kinase 1 expression decreased 4-fold (p<0.01) (A) in T1D+ESRD group compared with controls. A clear reversal of phosphoglycerate kinase 1 and partially of catalase expression abnormalities was observed after KP transplantation (p<0.05 and p<0.01 vs. T1D+ESRD for phosphoglycerate kinase 1 and partially of catalase, respectively), (B). Flavin reductase expression was halved in T1D+ESRD compared with controls (p<0.05), (A).

Figure 4. Immunohistochemical expression of Hsp27, Hsp60, ACBP, and S100 proteins in controls, patients affected by type 1 diabetes (T1D), type 1 diabetes+end-stage renal disease (T1D+ESRD), and kidney-pancreas transplanted patients (KP) skin specimens.

Hsp27 was strongly expressed in the cytoplasm of all epidermal cells without significant differences among the four categories of patients (A1–A4). Endothelial cells of control, T1D, and T1D+ESRD patients were intensely immunoreactive for Hsp27 (B1–B3), while in KP patients the endothelial Hsp27 immunoreactivity was weaker (B4). Hsp60 immunoreactivity was not found in the epidermal layer of control patients (C1), while it was expressed in the epidermal cells of T1D, T1D+ESRD, and KP patients (C2–C4), although the intensity of the reaction was lower than that of Hsp27. Hsp60 immunoreactivity was also found in endothelial cells of all patient groups, but the immunoreactivity was slightly more intense in control and KP patients (D1, D4) than in T1D and T1D+ESRD patients (D2, D3). ACBP immunoreactivity was intense and diffuse in epidermal epithelial cells of all four patient groups (E1–E4) without significant differences, while it was weaker in endothelial cells of the same patients (F1–F4). S100 protein immunoreactivity was intense in dendritic Langerhans cells distributed among epithelial cells of the epidermal layer (G1–G4) and in nerves (H1–H4) of all patients studied.

Figure 5. Ultrastructural features of skin tissues.

In the control group (A) the vessel lumen is correctly dilated and the endothelial cells are well preserved showing Weibel-Palade granules (arrow). The basal membrane is thin (asterisk). In skin specimens from patients affected by type 1 diabetes (T1D) (B), the vessel lumen is collapsed, endothelial cells show some degenerative markers, such as pre-apoptotic nuclei, dilated reticulum, ramificated microvilli, and rare Weibel-Palade granules (arrow). The thickness of the basal membrane is also remarkable (asterisk). Skin from patients with T1D and end-stage renal disease (T1D+ESRD) (C) showed endothelial cells with numerous signs of damage, including pre-apoptotic nuclei, marked bundles of intermediate filaments, dilated cysternae of reticulum, ramificated microvilli, and very rare Weibel-Palade granules (arrow). In addition, the basal membrane is very thick (asterisk). Skin from kidney-pancreas transplanted patients (KP) (D) showed reversal of almost all injury features: the basal membrane less thick (asterisk), lightly collapsed lumen, rare microvillar ramification, and presence of Weibel-Palade granules (arrow).

Figure 6. Peripheral levels of total, free and bound malondialdehyde.

Patients affected by type 1 diabetes (T1D) and T1D+ end-stage renal disease (T1D+ESRD) demonstrated an increase in total (p<0.01) (A), free (p<0.05 T1D vs. controls and p<0.01 T1D+ESRD vs. controls, respectively) (B), and bound malondialdehyde (MDA) (p<0.01) (C). The levels of total (ns), free (p<0.05), and bound MDA (p<0.05) in kidney-pancreas transplanted patients (KP) was comparable to or slightly increased the control group, indicating a profound effect of kidney-pancreas transplantation in correcting increased oxidative stress (A–C). In contrast, no significant differences were evident in the GSH/GSSG ratio among the four groups (D–F).

Figure 7. Proteins that were identified using MALDI-TOF MS analysis were examined using PathwayAssist.

The pathway was built by looking for direct interactions of the down-regulated and up-regulated proteins. The predominant cluster from this analysis is depicted in this figure. Briefly, peptidyl-prolyl cis-trans isomerase A (PPIA) and heat shock protein 27 kda (HSPB1) increase the expression of superoxide dismutase-1 (SOD1), which regulates catalase (CAT). SOD1 expression is under negative regulation by CAT.

Figure 8. Common regulators for the up-regulated and down-regulated proteins were analyzed with the help of PathwayAssist.

Heat shock protein 27 kda (HSPB1) and superoxide dismutase-1 (SOD1) are both central proteins in the pathway analysis performed with the up-regulated list (A). SOD1 is regulated by a number of cytokine molecules, and HSPB1 is directly associated with growth factors. Panel B shows the common regulators for the down-regulated proteins observed in this study. It is evident that CAT and crystal structures of mutant K206A, chain A (TF) is a key protein that interacts with a number of different signaling molecules in this pathway.