Late apoptotic cells are characterized by compromised plasma membrane integrity

to describe their potent antiinflammatory and immunoregulatory actions. Resolvins of the E series are derived from eicosapentaenoic acid, while Resolvins of the D series are biosynthesized from the precursor docosahexaenoic acid. The regulatory role of RvE1 in inflammation has been documented in various mouse models of inflammatory disease; including acute peritonitis, acute Danoprevir site colitis and periodontitis. We have established that administration of RvE1 or RvD1 can significantly reduce fibroblast proliferation and extracellular matrix production in the mouse UUO model. In addition, RvE1 was shown to inhibit platelet-derived growth factor-BB -induced proliferation in rat fibroblast NRK49F cells. RvD1 has also been shown to reduce expression of CCL2 and IL8 in TNF-a activated human aortic endothelial cells. In the current study, we examined whether RvD1 can protect endothelial Endothelial Dysfunction Exacerbates Renal Fibrosis cells from injury as a mechanism underlying its’ anti-proliferative and anti-fibrotic effects in the mouse UUO model. Confocal Microscopy Analysis Cryostat sections of tissues fixed in 4% paraformaldehyde were blocked with 2% bovine serum albumin in PBS and incubated with the following antibodies: rabbit anti-Ki67 antibody followed by goat anti-Alexa Fluor 488 and Cy3-conjugated mouse anti-a-SMA antibody; rabbit anticollagen type I followed by goat anti-rabbit Alexa Fluor 488 and rat anti-CD31 followed by goat anti-rat Alexa Fluor 488. Sections were counterstained with 4,6-diamidino2-phenylindole to visualize nuclei. Sections were analysed with an Olympus Fluoview 1000 confocal microscope, FV10-ASW software and oil UPLFL 660 objective. The number of aSMA-positive/ki67-positive cells was counted directly. 21821671 All scoring was performed on blinded slides. Quantification of peritubular capillaries. In each sample group, 40 randomly selected high power cortical fields were examined under 6600 magnification for assessment of CD31-positive PTC changes. 20571074 PTC changes were expressed per mm2 as previously described. Materials and Methods Experimental Animals Wild-type C57BL6/J mice were purchased from Monash Animal Services, Monash University, Australia. Breeding pairs of NOS3 gene knockout mice were purchased from Jackson Laboratories and maintained at Monash Animal Services. All experiments were performed with the approval of a Monash University Animal Ethics Committee, which adheres to the “Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.”Groups of 5 male wild-type or Nos3-/- C57BL/6J mice were used in each experiment. UUO surgery was performed under isofluorane anaesthesia. The left ureter was visualized following a flank incision and ligated with a vascular clamp. Sham mice underwent the same procedure, except that the ureter was not ligated. Mice were killed at 6 hours, 12 hours, 24 hours, 48 hours, 4 days and 7 days after UUO. Kidney tissues were collected for analysis. In addition, we analysed archival tissue from a previous study in which groups of four C57BL/6J mice that underwent UUO surgery and were given tail vein injections of RvD1 or vehicle, beginning on day 2 after UUO and continuing until they were killed on day 4. Cell Culture Mouse microvascular endothelial cells were purchased from ATCC and cultured in 5% CO2 atmosphere at 37uC in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. MMECs were seeded in 6-well plates at 16106 cells/well, Endothelial Dysfunction Ex