Nosirudeen Quadri

OBJECTIVE: Previous studies showed that genetic deletion or pharmacological blockade of the receptor for advanced glycation end products (RAGE) prevents the early structural changes in the glomerulus associated with diabetic nephropathy. To overcome limitations of mouse models that lack the progressive glomerulosclerosis observed in humans, we studied the contribution of RAGE to diabetic nephropathy in the OVE26 type 1 mouse, a model of progressive glomerulosclerosis and decline of renal function. RESEARCH DESIGN AND METHODS: We bred OVE26 mice with homozygous RAGE knockout (RKO) mice and examined structural changes associated with diabetic nephropathy and used inulin clearance studies and albumin:creatinine measurements to assess renal function. Transcriptional changes in the Tgf-beta1 and plasminogen activator inhibitor 1 gene products were measured to investigate mechanisms underlying accumulation of mesangial matrix in OVE26 mice. RESULTS: Deletion of RAGE in OVE26 mice reduced nephromegaly, mesangial sclerosis, cast formation, glomerular basement membrane thickening, podocyte effacement, and albuminuria. The significant 29% reduction in glomerular filtration rate observed in OVE26 mice was completely prevented by deletion of RAGE. Increased transcription of the genes for plasminogen activator inhibitor 1, Tgf-beta1, Tgf-beta-induced, and alpha1-(IV) collagen observed in OVE26 renal cortex was significantly reduced in OVE26 RKO kidney cortex. ROCK1 activity was significantly lower in OVE26 RKO compared with OVE26 kidney cortex. CONCLUSIONS: These data provide compelling evidence for critical roles for RAGE in the pathogenesis of diabetic nephropathy and suggest that strategies targeting RAGE in long-term diabetes may prevent loss of renal function.

The receptor for advanced glycation endproducts (RAGE) binds diverse ligands linked to chronic inflammation and disease. NMR spectroscopy and x-ray crystallization studies of the extracellular domains of RAGE indicate that RAGE ligands bind by distinct charge- and hydrophobicity-dependent mechanisms. The cytoplasmic tail (ct) of RAGE is essential for RAGE ligand-mediated signal transduction and consequent modulation of gene expression and cellular properties. RAGE signaling requires interaction of ctRAGE with the intracellular effector, mammalian diaphanous 1 or DIAPH1. We screened a library of 58,000 small molecules and identified 13 small molecule competitive inhibitors of ctRAGE interaction with DIAPH1. These compounds, which exhibit in vitro and in vivo inhibition of RAGE-dependent molecular processes, present attractive molecular scaffolds for the development of therapeutics against RAGE-mediated diseases, such as those linked to diabetic complications, Alzheimer's disease, and chronic inflammation, and provide support for the feasibility of inhibition of protein-protein interaction (PPI).

BACKGROUND: Advanced glycation end-products (AGEs) have been implicated in diverse pathological settings including diabetes, inflammation and acute ischemia/reperfusion injury in the heart. AGEs interact with the receptor for AGEs (RAGE) and transduce signals through activation of MAPKs and proapoptotic pathways. In the current study, adult cardiomyocytes were studied in an in vitro ischemia/reperfusion (I/R) injury model to delineate the molecular mechanisms underlying RAGE-mediated injury due to hypoxia/reoxygenation (H/R). METHODOLOGY/PRINCIPAL FINDINGS: Cardiomyocytes isolated from adult wild-type (WT), homozygous RAGE-null (RKO), and WT mice treated with soluble RAGE (sRAGE) were subjected to hypoxia for 30 minutes alone or followed by reoxygenation for 1 hour. In specific experiments, RAGE ligand carboxymethyllysine (CML)-AGE (termed "CML" in this manuscript) was evaluated in vitro. LDH, a marker of cellular injury, was assayed in the supernatant in the presence or absence of signaling inhibitor-treated cardiomyocytes. Cardiomyocyte levels of heterogeneous AGEs were measured using ELISA. A pronounced increase in RAGE expression along with AGEs was observed in H/R vs. normoxia in WT cardiomyocytes. WT cardiomyocytes after H/R displayed increased LDH release compared to RKO or sRAGE-treated cardiomyocytes. Our results revealed significant increases in phospho-JNK in WT cardiomyocytes after H/R. In contrast, neither RKO nor sRAGE-treated cardiomyocytes exhibited increased phosphorylation of JNK after H/R stress. The impact of RAGE deletion on GSK-3beta phosphorylation in the cardiomyocytes subjected to H/R revealed significantly higher levels of phospho-GSK-3beta/total GSK-3beta in RKO, as well as in sRAGE-treated cardiomyocytes versus WT cardiomyocytes after H/R. Further investigation established a key role for Akt, which functions upstream of GSK-3beta, in modulating H/R injury in adult cardiomyocytes. CONCLUSIONS/SIGNIFICANCE: These data illustrate key roles for RAGE-ligand interaction in the pathogenesis of cardiomyocyte injury induced by hypoxia/reoxygenation and indicate that the effects of RAGE are mediated by JNK activation and dephosphorylation of GSK-3beta. The outcome in this study lends further support to the potential use of RAGE blockade as an adjunctive therapy for protection of the ischemic heart.

The macro- and microvascular complications of type 1 and 2 diabetes lead to increased disease severity and mortality. The receptor for advanced glycation end products (RAGE) can bind AGEs and multiple proinflammatory ligands that accumulate in diabetic tissues. Preclinical studies indicate that RAGE antagonists have beneficial effects on numerous complications of diabetes. However, these antagonists target the extracellular domains of RAGE, which bind distinct RAGE ligands at diverse sites in the immunoglobulin-like variable domain and two constant domains. The cytoplasmic tail of RAGE (ctRAGE) binds to the formin, Diaphanous-1 (DIAPH1), and this interaction is important for RAGE signaling. To comprehensively capture the breadth of RAGE signaling, we developed small-molecule antagonists of ctRAGE-DIAPH1 interaction, termed RAGE229. We demonstrated that RAGE229 is effective in suppressing RAGE-DIAPH1 binding, Förster resonance energy transfer, and biological activities in cellular assays. Using solution nuclear magnetic resonance spectroscopy, we defined the molecular underpinnings of the interaction of RAGE229 with RAGE. Through in vivo experimentation, we showed that RAGE229 assuaged short- and long-term complications of diabetes in both male and female mice, without lowering blood glucose concentrations. Last, the treatment with RAGE229 reduced plasma concentrations of TNF-α, IL-6, and CCL2/JE-MCP1 in diabetic mice, in parallel with reduced pathological and functional indices of diabetes-like kidney disease. Targeting ctRAGE-DIAPH1 interaction with RAGE229 mitigated diabetic complications in rodents by attenuating inflammatory signaling.