Abstract
Protein expression profiles in rat bladder smooth muscle were compared between animal models of streptozotocin-induced diabetes mellitus (STZ-DM) and age-matched controls at 1 week and 2 months after induction of hyperglycemia with STZ treatment. At each time point, protein samples from four STZ-DM and four age-matched control rat bladder tissues were prepared independently and analyzed together across multiple DIGE gels using a pooled internal standard sample to quantify expression changes with statistical confidence. A total of 100 spots were determined to be significantly changing among the four experimental groups. A subsequent mass spectrometry analysis of the 100 spots identified a total of 56 unique proteins. Of the proteins identified by two-dimensional DIGE/MS, 10 exhibited significant changes 1 week after STZ-induced hyperglycemia, whereas the rest showed differential expression after 2 months. A network analysis of these proteins using MetaCore suggested induction of transcriptional factors that are too low to be detected by two-dimensional DIGE and identified an enriched cluster of down-regulated proteins that are involved in cell adhesion, cell shape control, and motility, including vinculin, intermediate filaments, Ppp2r1a, and extracellular matrix proteins. The proteins that were up-regulated include proteins involved in muscle contraction (e.g. Mrlcb and Ly-GDI), in glycolysis (e.g. alpha-enolase and Taldo1), in mRNA processing (e.g. heterogeneous nuclear ribonucleoprotein A2/B1), in inflammatory response (e.g. S100A9, Annexin 1, and apoA-I), and in chromosome segregation and migration (e.g. Tuba1 and Vil2). Our results suggest that the development of diabetes-related complications in this model involves the down-regulation of structural and extracellular matrix proteins in smooth muscle that are essential for normal muscle contraction and relaxation but also induces proteins that are associated with cell proliferation and inflammation that may account for some of the functional deficits known to occur in diabetic complications of bladder.
Highlights
Protein expression profiles in rat bladder smooth muscle were compared between animal models of streptozotocin-induced diabetes mellitus (STZ-DM) and age-matched controls at 1 week and 2 months after induction of hyperglycemia with STZ treatment
To probe protein-protein interaction networks that can predict the signaling pathways that are activated or deactivated during the initiation and development of the diabetic bladder dysfunction and to identify transcriptional factors and relatively low abundance proteins that cannot be identified by 2D DIGE/MS, we further analyzed the 2D DIGE data using MetaCoreTM pathway analysis tools [30, 31] along with validation of specific expression changes by Western blotting. With this integrated approach we identified a number of differentially expressed proteins that may provide valuable mechanistic insight into the molecular changes that are precursors for and are associated with DM-induced bladder dysfunction
To monitor hyperglycemia-associated protein expression changes in rat bladder at two specific time points compared with age-matched controls, total soluble proteins from smooth muscle strips at 1 week and 2 months after the onset of STZ-induced diabetes were analyzed using 2D DIGE/MS [27,28,29]
Summary
Protein expression profiles in rat bladder smooth muscle were compared between animal models of streptozotocin-induced diabetes mellitus (STZ-DM) and age-matched controls at 1 week and 2 months after induction of hyperglycemia with STZ treatment. Our results suggest that the development of diabetes-related complications in this model involves the down-regulation of structural and extracellular matrix proteins in smooth muscle that are essential for normal muscle contraction and relaxation and induces proteins that are associated with cell proliferation and inflammation that may account for some. As far as we are aware, there are no studies that have attempted to characterize diabetes-related changes in bladder smooth muscle at the level of the proteome This highlights the fact that there are uncertainties about the time course, magnitude, and mechanism of diabetes-related changes in bladder function both in this animal model and in humans
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