Given the observed up-regulation

Given the observed up-regulation Regorafenib mechanism of Gpnmb in M in the post-IRI kidneys and motivated by recent studies of the role of inflammatory Ms during the repair of liver, heart, and skeletal muscle (4, 6, 7), we analyzed M content in IRI kidneys by flow cytometry. Whereas the number of monocyte/Ms (CD11b+, NK1.1?, Ly6G?) in the kidneys increased little over 24 h following injury; by 72 h following injury, we observed a marked increase in Ms within the tissue (Fig. 3A). This increase persisted through 7 and 10 d post-IRI. Thus, the kinetics of M recruitment coincided with repair, not injury, and these data were further supported by immunostaining and scoring tissue sections from parallel samples for CD11b+ cells (detecting neutrophils and monocytes/Ms) or F4/80+ (Ms only) cells (Fig. 3B).

In contrast to M populations, neutrophil recruitment occurred earlier following injury and did not persist and was thus more consistent with an injury response (not shown). Therefore, to study the function of Ms during repair of the kidney, conditional M ablation in vivo was performed using the Cd11b-DTR transgenic mouse model we developed previously (5). In this in vivo model, administration of minute amounts of diphtheria toxin (DT) results in rapid specific ablation of monocytes and kidney Ms (3,�C5, 19, 32). Cohorts of mice with identical IRI were randomized at 1 d post-IRI to receive DT from 2�C6 d or vehicle (Fig. 3C�CF). Ablation was successful when F480+ kidney Ms were quantified (Fig. 3C, D). In those mice that retained the normal complement of Ms there was recovery of injury as assessed by plasma creatinine level and tubular injury score.

However, in mice that had M ablation, plasma creatinine levels did not recover. Coincident with this, tubules showed severe persistent injury, and the tubule injury score remained also markedly elevated compared with mice that had the normal M complement (Fig. 3C, F). Tubules remained severely injured with flattened morphology, and there was a persistence of debris within the tubules. These observations provide strong evidence that Ms are functioning in a reparative capacity in this model. Figure 3. M ablation in Cd11b-DTR mice prevents normal kidney repair following IRI. A) Percentage of kidney cells that are CD11b+, NK1.1?, Ly6G? Ms, as assessed by flow cytometry. B) CD11b+ and F4/80+ cells in the kidney following …

Gpnmb localizes to LC3-containing vesicle compartments To further dissect the cellular mechanism by which Gpnmb promotes repair, we determined its subcellular localization. Untagged Gpnmb or Gpnmb fused to either GFP or RFP (Gpnmb-GFP or Gpnmb-RFP) was expressed in the porcine kidney epithelial cell line LLC-PK1. Both the untagged (detected by antibody; not shown) and fluorescently tagged Gpnmb proteins localized to intracellular vesicles (Fig. 4A). The protein was primarily Cilengitide detected associated with the membranes of these vesicles.

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