In this study we employed several different techniques such as proteomics, clinical biochemistry, electron microscopy, and immunohistochemistry to identify pathways of persistent cellular changes in skin biopsies of T1D patients. The effect of a KP transplant on cellular pathways, protein expression, and ultrastructural features was evaluated. We focused on the altered expression of several proteins involved in oxidative stress, aerobic and anaerobic glycolysis, and intracellular signaling normalized by KP transplant and combined them in molecular/ultrastructural studies.
T1D patients showed an up-regulation of HSP60, HSP27, MnSOD, and ATP synthase δ chain with a further increase in those patients with ESRD associated with T1D. This suggests that HSP and anti-oxidative machinery is entirely altered and thereafter restored by KP transplantation. These data are consistent with previous studies indicating that transient exposure of pancreatic islets to high glucose increases the activities of antioxidant enzyme, such as Cu/Zn-SOD 
. HSP60 and HSP27 are synthesized in large amounts when cells are exposed to stressful stimuli such as inflammation, infection, and exposure to oxidizing agents 
. We also identified down-regulation of catalase, which has important antioxidant functions. Consequently, there is decreased ability to counteract increased oxidative stress in long-standing T1D. It has been reported that high levels of glucose can produce permanent chemical alterations in proteins, increase lipid peroxidation and production of free radicals in several experimental models of hyperglycemia 
. In addition to the above-mentioned group of proteins, proteomics data showed that long-standing T1D when associated with ESRD also regulates cytoplasmic proteins involved in aerobic and anaerobic glycolysis, gluconeogenesis, and mitochondrial electron transport. Interestingly, T1D+ESRD patients who were hyperglycemic and hyperinsulinemic also had increased triglycerides, which are produced by anaerobic glycolysis and up-regulated pyruvate kinase, while KP-transplanted patients were less dyslipidemic and presented lower pyruvate kinase levels than T1D+ESRD patients comparable with controls 
. T1D patients were analogous to T1D+ESRD patients as far as the expression of these three proteins, suggesting effect of T1D on these pathways. The last identified altered expressed proteins (stratifin, rotamase, S100 calcyclin) were involved in the intracellular signaling pathway. Lee and co-workers suggested 
that rotamase may play a role in the folding of SOD-1 and in its dimerization, possibly explaining the up-regulation of rotamase in parallel to the up-regulation of SOD-1. In particular, this association was related to a calcium-dependent pro-apoptotic mechanism, and ultrastructural analysis emphasized the presence of apoptotic nuclei both in T1D patients and in T1D+ESRD patients.
Aiming to address if a parallel increase of redox state took place in the periphery, we evaluated malondialdehyde (MDA), a terminal compound derived from lipid peroxidation and from eicosanoid biosynthesis, widely used to monitor oxidative stress 
. We measured both the free and the total MDA forms, the first being considered an index of recent damage and the second an index of prior damage. Therefore, we evaluated the levels of endogenous antioxidants such as GSH and GSSG. In T1D patients, increased MDA levels in plasma were evident consistently with the increased oxidative status than the controls, while KP-transplanted patients presented lower MDA levels and were comparable to the controls. We did not observe any differences in GSH levels, which counteract the effect of free radicals. This is consistent with a previous study from our group, which showed that KP transplantation can reduce the levels of MDA 
The ultrastructural alterations found in T1D and T1D+ESRD skin biopsies included thickening of the capillary basal membrane, collapse of vessel lumen, and microvillar ramification. We observed that basal membranes were thicker in T1D and T1D+ESRD groups compared with controls, and that these alterations were corrected in patients who had a KP transplant for at least five years. We note that basal membrane thickening was particularly evident in T1D+ESRD patients. The lumen of the vessels was collapsed and microvilli were more branched in the same group. Moreover, the T1D+ESRD group had an apoptotic pattern of endothelial cells consistent with previous studies that describe a role of hyperglycemia in inducing apoptosis in endothelial cells 
. All of these alterations were somehow more evident in T1D+ESRD compared with T1D patients, possibly due to the coexistence of two “toxic” situations, i.e., uremia and hyperglycemia that may act additively. Skin biopsies from KP-transplanted patients presented an impressive improvement of ultrastructural alterations (basal membrane thickening, collapse of vessel lumen, microvillar ramifications), as previously described in kidney-transplanted patients who received islet transplantation 
. It is well known that hyperglycemia and diabetes induce oxidative stress responses in animal models and cell culture systems 
. However, few studies have employed human tissues to study the biochemistry of diabetic complications 
. The improvement of ultrastructural abnormalities is consistent with what has been reported in the literature by Eberl and co-workers, who showed that long-term blood glucose normalization achieved by pancreas transplantation improved most skin microcirculation parameters with a positive effect on functionality of the skin 
These findings are consistent with the hypothesis that hyperglycemia and uremia, through different mechanisms, determine persistent cellular changes of the oxidative status and pathways and that restoration of normoglycemia with KP transplantation can correct most of these biochemical abnormalities. To a lesser extent, T1D not associated with ESRD is also characterized by an increase of oxidative stress. It is not clear if the alterations of these pathways may determine alteration at chromatin levels and altered DNA repairing. The next logical step will be to evaluate the status of DNA damage during the normalization of these pathways after kidney-pancreas transplantation.
Some of these proteins or pathways addressed in our study may become either biomarkers of oxidative stress in vivo or could be potential therapeutic targets of a new class of drugs aimed at correcting persistent cellular changes when normoglycemia cannot be restored.