High levels of tissue advanced glycation end products (AGEs) that result from the spontaneous modification of proteins by glucose occur In diabetes and aging. To address the potential pathogenic role of AGEs in the glomerulosclerosis of diabetes or nephrosclerosls of aging, doses of AGE-modified rat albumin (25 mg per kg per day, i.v.) sufficient to elevate circulating AGE levels to the range of diabetic serum were admired daily to healthy rats alone or in combination with the AGE inhibitor ae. After 5 months, the AGE content of renal tissues in AGE-treated rats rose to 50% above controls (P < 0.025), whereas serum contained 2.8-fold greater AGE levels (P < 0.025). Light and electron microscopy of kidneys from AGE-treated rats revealed a more than 50% increase in glomerular volume compared to controls (P < 0.001), I t periodic add/Schiff reagent-positive deposits, basement membrane widening, and mesanga extraceflular matrix increase and indicated significant glomerulosclerosis compared to untreated (P < 0.002) or albumin-treated controls (P < 0.002). These changes were associated with si nt loss of protein (P < 0.005) and albumin (P < 0.002) in the urine of AGE-treated rats compared to controls. Cotreatment with aminoguanidine markedly limited both the structural and fuctional defects. These in vivo data demonstrate that AGEs influence glomerular structure and function in a manner leading to glomerulosclerosis. (8), while in normal SJL mice AGEs enhance the expression of alIV collagen and laminin B1 mRNAs within the glomerulus (9). However, the long-term impact of cumulative AGE deposition on the structure and function of the kidney has remained obscure.In this report, we present evidence indicating that prolonged in vivo exposure of normal renal tissues to AGEmodified homologous serum albumin induces marked renal lesions and that certain aspects ofthis pathology are inhibited by the coadministration of aminoguanidine. METHODSPreparation of Advanced Glycation Products. Rat serum albumin (RSA) (Sigma) was passed over an Affi-Gel Blue column (Bio-Rad), a heparin-Sepharose CL-6B column (Pharmacia), and an endotoxin-binding affinity column (Detoxigel, Pierce) to remove possible contaminants (8, 9). RSA modified by AGEs was prepared as described (8,9). AGE levels were measured by an AGE-specific ELISA (10) (AGE-rat albumin, 62 AGE units/mg of protein; unmodified rat albumin, 1.2 units/mg). Each reagent contained endotoxin (E-Toxate, Sigma) at <0.2 ng/ml. Animal Studies. Male Sprague-Dawley rats (150 g) aged 3 months (n = 50) (Charles River Breeding Laboratories) were used in these studies, which were conducted in accordance with The Picower Institute Laboratory Animal Center guidelines. After a 2-week adaptation period, rats were given tail vein injections with AGE-modified or native RSA (25 mg per kg per day) or with AGE-RSA followed by infusions of aminoguanidine hydrochloride (100 mg per kg per day, i.v.) for 5 months. Serum samples were collected at the end ofthe treatment period from all groups for seru...

Glucose and other reducing sugars react with proteins by a nonenzymatic, post-translational modification process called nonenzymatic glycosylation or glycation. The sugar-derived carbonyl group adds to a free amine, forming a reversible adduct which over time rearranges to produce a class of products termed advanced-glycation end-products (AGEs). These remain irreversibly bound to macromolecules and can covalently crosslink proximate amino groups. The formation of AGEs on long-lived connective tissue and matrix components accounts largely for the increase in collagen crosslinking that accompanies normal ageing and which occurs at an accelerated rate in diabetes. AGEs can activate cellular receptors and initiate a variety of pathophysiological responses. They modify an appreciable fraction of circulating low-density lipoproteins preventing uptake of these particles by their high-affinity tissue receptors. Advanced glycation has also been implicated in the pathology of Alzheimer's disease. Because AGEs may form by a pathway involving reactive alpha-dicarbonyl intermediates, we investigated a potential pharmacological strategy for selectively cleaving the resultant glucose-derived protein crosslinks. We now describe a prototypic AGE crosslink 'breaker', N-phenacylthiazolium bromide (PTB), which reacts with and cleaves covalent, AGE-derived protein crosslinks. The ability of PTB to break AGE crosslinks in vivo points to the importance of an alpha-dicarbonyl intermediate in the advanced glycation pathway and offers a potential therapeutic approach for the removal of established AGE crosslinks.

Rationale: The ubiquitin proteasome system (UPS) becomes dysfunctional as a result of ischemia/reperfusion (I/R), which may lead to dysregulation of signaling pathways. Ischemic preconditioning (IPC) may prevent dysregulation by preventing UPS dysfunction through inhibition of oxidative damage. Objective: Examine the hypothesis that early IPC preserves postischemic UPS function thus facilitating prosurvival signaling events. Methods and Results: I/R decreased proteasome chymotryptic activity by 50% in isolated rat heart and an in vivo murine left anterior descending coronary artery occlusion model. Following IPC, proteasome activity was decreased 25% (P<0.05) in isolated heart and not different from baseline in the murine model. Enriched 26S proteasome was prepared and analyzed for protein carbonyl content. Increased (P<0.05) carbonylation in a 53-kDa band following I/R was diminished by IPC. Immunoprecipitation studies indicated that the 53-kDa carbonylation signal was of proteasomal origin. Two-dimensional gel electrophoresis resolved the 53-kDa band into spots analyzed by liquid chromatography/tandem mass spectrometry containing Rpt3/Rpt5 both of which could be immunoprecipitated conjugated to dinitrophenylhydrazine (DNPH). Higher amounts of DNPH-tagged Rpt5 were immunoprecipitated from the I/R samples and less from the IPC samples. I/R increased Bax levels by 63% (P<0.05) which was decreased by IPC. Lactacystin (lac) pretreatment of preconditioned hearts increased Bax by 140% (P<0.05) and also increased ubiquitinated proteins. Pretreatment of hearts with a proteasome inhibitor reversed the effects of IPC on postischemic Rpt5 carbonylation, cardiac function, morphology and morphometry, and ubiquitinated and signaling proteins. Conclusions: These studies suggest that IPC protects function of the UPS by diminishing oxidative damage to 19S regulatory particle subunits allowing this complex to facilitate degradation of proapoptotic proteins. (Circ Res. 2010;106:1829-1838.)Key Words: ischemia Ⅲ reperfusion Ⅲ preconditioning Ⅲ ubiquitin-proteasome system I schemic preconditioning (IPC) decreases the vulnerability of the myocardium to longer durations of ischemia as a result of preischemic exposure to short ischemic bursts as evidenced by improved postischemic hemodynamic function and reduced markers of injury. 1 The mechanisms involved with IPC has been the subject of intense research and the earlier steps appear to involve signaling changes resulting in opening of the inward mitochondrial K ATP channels 2 and prevention of opening of the mitochondrial permeability transition pore. 3 These early changes have been linked to decreased release of cytochrome c with diminished cellular apoptosis, 3 as well as decreased production of oxidative species. 4 We have shown that a downstream effect of preconditioning is diminished myocardial oxidative injury. 5 One later effect of IPC is decreased levels of certain proapoptotic proteins, such as Bax. 6 Earlier studies focused on diminished upregulation of these proteins t...