The primer sequences are shown in Table 1

The primer sequences are shown in Table 1. Table 1. Sequences of primers used in the study for 15min at 4C, the supernatant was carefully removed and its protein concentration was measured. production and macrophage infiltration in the NTN kidney. mice (17) or NZB NZW mice (18). The murine nephrotoxic serum (NTS) nephritis (NTN) model resembles human Goodpastures disease (19) by virtue of its dependence on the targeting effect of anti-glomerular basement membrane (GBM) antibodies to direct Biapenem immune complex deposition to the GBM and initiate an inflammatory response (20). It is also a leptin-dependent disease model (21), and IL-17-producing CD4+ T cells (Th17 cells), as well as T cells have been shown to play important roles in the development of NTN that had been thought to be Th1-dominant previously (22C24). IL-17 drives germinal center formation and class switch recombination (25); therefore, IL-17 is critical in the generation of a humoral immune response (25) and triggers various inflammatory diseases, including the NTN model (26). IL-23 is a potent inducer of IL-17 in murine T cells (27C29), and is also reported to be essential in the development of NTN (23), even though the producer of IL-23 in the nephritis model is still to be elucidated. Furthermore, the effect of leptin on macrophages infiltrating the nephritic kidney and playing a pivotal role as direct effector cells (20) is still unknown. In the current study, the effect of leptin on regulation of the IL-23/IL-17 axis and macrophage activation was investigated in the NTN mouse model. Methods Mice Male C57BL/6J mice, male C57BL/6J-mice, male BKS Cg-+mice were under food restriction (2.5g of food per day) from 5 to 12 weeks of age. Eight hundred micrograms of pentobarbital was injected intraperitoneally for anesthesia before sacrifice. All animal experiments were performed following protocols approved by the Institutional Animal Care and Ethics Committee at Niigata University. Induction of NTN Sheep NTS was prepared as described previously (20). NTS was heat inactivated, and then absorbed with Biapenem an excess of murine red blood cells. Twelve-week-old mice were preimmunized intraperitoneally with 200 g of sheep IgG (AbD Serotec, Oxford, UK) in a 50:50 mix with complete Freunds adjuvant (Sigma-Aldrich, St. Louis, MO, USA), followed by intravenous injection of sheep NTS (2.0 l of serum per gram of mouse) 4 days later. Mice were sacrificed 7 days after NTS administration to collect blood, kidney, and spleen samples under anesthesia. Blood urea nitrogen level in sera was measured using a DetectX Urea Nitrogen Colorimetric Detection Kit (Arbor Assays, Ann Arbor, MI, USA). Serum creatinine and cystatin C concentration was measured using Creatinine Colorimetric Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) and Mouse/Rat Cystatin C Immunoassay (R & D Systems, Minneapolis, MN, USA), respectively. The serum leptin level was quantified using Mouse/Rat Leptin Quantikine ELISA Kit (R & D Systems). Biapenem Histological analysis Kidneys were removed from mice, fixed in 10% buffered formalin, and embedded in paraffin. Four-micrometer paraffin sections were stained with periodic acid-Schiff (PAS) and assessed in 20 glomeruli per mouse by light microscopy in a blind manner. The clinical scores of glomerular injury were graded into five grades; grade 0 (no PAS-positive material), grade 1 (0 to 25% of glomerular cross-section PAS-positive), grade 2 (25 Plxna1 to 50%), grade 3 (50 to 75%) and grade 4 (75 to 100%) (20). Immunohistochemical and immunofluorescence staining For immunohistochemical analysis, 4-m paraffin sections were subjected to heat-mediated antigen retrieval and stained for F4/80 (AbD Serotec), CD3 (HistoBioTec, Miami Beach, FL, USA), IL-17 (Abcam, Cambridge, UK), leptin receptor (Abcam) and IL-23 (Abnova, Taipei, Taiwan). These specific antibodies were incubated either HRP-conjugated goat anti-rabbit IgG or alkaline phosphatase (ALP)-conjugated goat anti-rat IgG (Sigma-Aldrich). Detection of immune complexes was performed using 3,3-diaminobenzidine for HRP (Nichirei Biosciences, Tokyo, Japan) or Vector Red for ALP (Vector laboratories, Burlingame, CA, USA). The number of F4/80 positive cells was assessed in a minimum of 20 randomly selected high-power fields per animal. For immunofluorescence staining, 4-m frozen sections were fixed in acetone and stained with FITC-conjugated anti-sheep IgG and tetramethylrhodamine-conjugated anti-mouse IgG (Sigma-Aldrich), respectively. For quantification of immunofluorescence, blinded sections were examined at 100-fold.