Cardioprotection by postconditioning is lost in WOKW rats with metabolic syndrome: role of glycogen synthase kinase 3beta.

BACKGROUND AND OBJECTIVE: Postconditioning by repetitive I/R cycles immediately after onset of reperfusion protects the heart. Metabolic disorders undermine the protection of preconditioning. The present study tested whether postconditioning protects hearts from rats with established metabolic syndrome [Wistar-Ottawa-Karlsburg W rats (WOKW)]. METHODS AND RESULTS: After 28 weeks of age, WOKW rats were much heavier than DA (Dark Agouti) and Wistar control rats and showed the pattern of the metabolic syndrome. Postconditioning was performed by 3 30-second cycles of reperfusion/ischemia immediately after the regional ischemia (30 minutes). Infarct size was comparable in all control hearts from DA, Wistar, and WOKW rats (58 +/- 2%, 49 +/- 3%; 49 +/- 2%, respectively). Postconditioning significantly reduced the infarct size in DA rats (39 +/- 5%) and Wistar rats (29 +/- 3%). In WOKW rats, the infarct sparing effect of postconditioning was lost (43 +/- 4%).GSK-3beta and Erk are involved in the signaling of postconditioning. Therefore, the phosphorylation of these proteins was determined by Western blot analysis. Postconditioning significantly increased the phosphorylation of GSK-3beta in DA and Wistar rats (1.6-fold in DA rats, 2.3-fold in Wistar rats, P < 0.05) but failed to do so in WOKW rats. Similarly, a trend for an increased phosphorylation of Erk was found in DA rats but not in WOKW rats. Thus the inefficacy of postconditioning in reducing infarct size in rats with metabolic syndrome is paralleled by a lack of phosphorylation of GSK-3beta and Erk. CONCLUSION: The metabolic syndrome, as shown in this animal model, completely abrogates the postconditioning. This blockade involves the phosphorylation of GSK-3beta. Further studies have to evaluate whether this block of postconditioning makes patients with a metabolic syndrome more susceptible to myocardial damage after infarction.

Activation of tubular epithelial cells in diabetic nephropathy.

Previous studies have shown that renal function in type 2 diabetes correlates better with tubular changes than with glomerular pathology. Since advanced glycation end products (AGEs; AGE-albumin) and in particular carboxymethyllysine (CML) are known to play a central role in diabetic nephropathy, we studied the activation of nuclear factor kappaB (NF-kappaB) in tubular epithelial cells in vivo and in vitro by AGE-albumin and CML. Urine samples from healthy control subjects (n = 50) and type 2 diabetic patients (n = 100) were collected and tested for excretion of CML and the presence of proximal tubular epithelial cells (pTECs). CML excretion was significantly higher in diabetic patients than in healthy control subjects (P < 0.0001) and correlated with the degree of albuminuria (r = 0.7, P < 0.0001), while there was no correlation between CML excretion and HbA(1c) (r = 0.03, P = 0.76). Urine sediments from 20 of 100 patients contained pTECs, evidenced by cytokeratin 18 positivity, while healthy control subjects (n = 50) showed none (P < 0.0001). Activated NF-kappaB could be detected in the nuclear region of excreted pTECs in 8 of 20 patients with pTECs in the urine sediment (40%). Five of eight NF-kappaBp65 antigen-positive cells stained positive for interleukin-6 (IL-6) antigen (62%), while only one of the NF-kappaB-negative cells showed IL-6 positivity. pTECs in the urine sediment correlated positively with albuminuria (r = 0.57, P < 0.0001) and CML excretion (r = 0.55, P < 0.0001). Immunohistochemistry in diabetic rat kidneys and a human diabetic kidney confirmed strong expression of NF-kappaB in tubular cells. To further prove an AGE/CML-induced NF-kappaB activation in pTECs, NF-kappaB activation was studied in cultured human pTECs by electrophoretic mobility shift assays (EMSAs) and Western blot. Stimulation of NF-kappaB binding activity was dose dependent and was one-half maximal at 250 nmol/l AGE-albumin or CML and time dependent at a maximum of activation after 4 days. Functional relevance of the observed NF-kappaB activation was demonstrated in pTECs transfected with a NF-kappaB-driven luciferase reporter plasmid and was associated with an increased release of IL-6 into the supernatant. The AGE- and CML-dependent activation of NF-kappaBp65 and NF-kappaB-dependent IL-6 expression could be inhibited using the soluble form of the receptor for AGEs (RAGE) (soluble RAGE [sRAGE]), RAGE-specific antibody, or the antioxidant thioctic acid. In addition transcriptional activity and IL-6 release from transfected cells could be inhibited by overexpression of the NF-kappaB-specific inhibitor kappaBalpha. The findings that excreted pTECs demonstrate activated NF-kappaB and IL-6 antigen and that AGE-albumin and CML lead to a perpetuated activation of NF-kappaB in vitro infer that a perpetuated increase in proinflammtory gene products, such as IL-6, plays a role in damaging the renal tubule.