Proteomica di fibroblasti cutanei coltivati in vitro, ottenuti da biopsie di soggetti normali e di pazienti diabetici di tipo 1, caratterizzati dalla presenza o assenza di complicanze microvascolari renali

The target of this research project was to identify protein markers of diabetic nephropathy (DN), a microvascular complication which develops in about 30% of subject with type 1 diabetes and which is often associated with an increase risk of developing cardiovascular diseases and premature death in comparison with non nephropatic subjects. Many studies indicate that hyperglycemia is a necessary although not sufficient condition to determine the diabetes associated renal damage. Indeed, the incidence of nephropathy reaches a maximum after 15-20 years of disease and thereafter it decreases. Such a behaviour is compatible with the existence of an individual susceptibility (genetic) to renal damage, induced by factors other than hyperglycaemia, thus highlighting the need for an early detection of the type 1 diabetic subjects with high risk of developing DN, and to clarify the pathophysiological mechanisms Human fibroblasts represent an ideal experimental model, also because easily accessible, for an ample investigation on the genetic predisposition to diabetic disease, as well as to study of the mechanisms leading to cellular and metabolic damages occurring in diabetic complications. We have established an experimental protocol for the extraction of proteins from cultured cells and we have defined the optimal experimental conditions to obtain reproducible 2-D gels. So we compared protein profiles of cultured fibroblasts, harvested from skin biopsies of type 1 diabetic patients with long disease duration and with or without DN, and normal healthy subjects. This research has allowed, firstly, a more and deeper knowledge about human fibloblast proteome, useful data as a reference for subsequent investigations applied to the study of various diseases including non-diabetic. Secondly, the comparison among groups showed significant changes in the amounts of some proteins including cytoskeletal proteins and proteins involved in energy metabolism and protein turnover. Some of these results have been validated with more depth analysis as western blot and enzymatic activity test. These data, entirely original at the international level, represent a starting point for further investigations for the early identification of subjects with a genetic predisposition to DN and revealed new clues to understand physiopathological mechanisms that lead to the development of this pathology. Finally we made the comparison of protein levels after exposure to high levels of glucose, within each group and among the three groups, obtaining information about proteins involved in cellular response to hyperglycemia.