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Isolation of WAT stromal vascular cells and adipocyte differentiation To isolate the stromal vascular fraction, minced iWAT (combined depots from 45 mice per genotype) from 46-week-old mice was digested in 1 mg ml 1 collagenase D (Roche) with 1.5% BSA in sterile Hanks Balanced Salt Solution and incubated in a 37 C shaking water bath for 1 h

Figure 4 Epithelial cells Similar to their effects on mesangial cells, AGE also induce the production of RAGE, ROS, TGF-1 and type I collagen in tubular epithelial cells in vitro (Figure 2) and also activates AMP-activated protein kinase (AMPK)- ( Although the mechanisms involved are not fully elucidated, AMPK- regulation of RAGE could involve increased tubular epithelial cell production of the antioxidant thioredoxin ( AGE-induced type I collagen production in epithelial cells is regulated by both mitogen-activated protein kinase (MAPK)-induced/TGF-1-independent and TGF-1-dependent Smad cell signals ( Moreover, both AGE-induced MAPK activity and epithelial mesenchymal transition (EMT) in rat NRK-52E cells are blocked by treatment with AT1R blockers (e.g., olemesartan, irbesartan, losartan) ( In summary, RAGE activation in epithelial cells induces cell signals (DIAPH1, MAPK, SMAD2/3, NF-B) and promotes the production of molecules (ROS, TGF-1, type I collagen, -SMA, HMGB1) commonly associated with fibrogenesis, EMT, and inflammation

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