Here, we have carried out preliminary analysis of M1 and M2 macrophages in glomeruli of STZ + HFD mice by studying gene expression levels of CD11c (or Itgax) and CD206 (or Mrc1) as markers of M1 and M2 subtypes, respectively [77, 78] (Fig. 6). In wild-type mice, treatment with STZ alone does not affect glomerular expression of CD11c and CD206 genes, and addition of HFD to STZ causes a 100 % increase in CD11c and a 30 % increase in CD206, suggesting relative predominance of M1 subtype in diabetic-hyperlipidemic conditions. Furthermore, in Tlr4 KO mice, the stimulatory effects of HFD upon STZ treatment are canceled

both for CD11c and CD206 genes, and simple STZ treatment increases CD11c expression by two-fold and

increases CD206 expression by three-fold, suggesting the presence of M2 predominant status. These results imply that TLR4-mediated signal SC79 is check details partially suppressing M2 subtype in STZ-normal diet mice and enhancing M1 subtype in STZ-HFD mice. These findings are in good agreement with previous reports indicating that treatment of macrophages with MRP8 induces M1 subtype (through TLR4 as lipopolysaccharide does) [61, 72, 76] and MRP8-expressing macrophages exhibits M1 characteristics by secretion of TNF-α and interleukin-6 [74, 79]. Formally, M1/M2 subtype analysis had to be carried out by analyzing isolated macrophages extracted from tissues. Fig. 6 Glomerular gene expression of M1 (a) and M2 (b) macrophage markers in STZ-HFD mice Selleck Temsirolimus determined by TaqMan real-time PCR. Data are mean ± SEM. n = 4–11. *p < 0.05, **p < 0.01. # p < 0.05, ## p < 0.01 for similarly treated Tlr4 KO versus wild-type Furthermore, in STZ + HFD animals, the levels of macrophage infiltration and extracellular matrix accumulation are proportional and progressive, suggesting that M1–M2 switching does not occur spontaneously Palbociclib mouse in this model of DN. In glomeruli of STZ + HFD mice, >80 % of MRP8 signals co-localize

with macrophage marker Mac2 (or Lgals3) [5], whereas collecting duct epithelial cells are the main source of MRP8 expression in unilateral ureteral obstruction [76]. In conclusion, a number of epidemiological and experimental studies have revealed that glucotoxicity and lipotoxicity cause synergistic effects upon the development and progression of DN. Macrophages have emerged as a potential contributor for mediating glucolipotoxicity through activation of MRP8/TLR4 signaling in diabetic glomeruli in our experiments. Although further studies are needed to understand regulation and potential role of MRP8/TLR4 signaling, targeting key molecules involved in this pathway may lead to novel therapeutic strategy to combat DN.