Volume 61, Issue 4, Pages 1339-1350 (April 2002) FK506 ameliorates proteinuria and glomerular lesions induced by anti-Thy 1.1 monoclonal antibody 1-22-3  Yohei Ikezumi, Katsue Kanno, Hiroko Koike, Masayuki Tomita, Makoto Uchiyama, Fujio Shimizu, Hiroshi Kawachi  Kidney International  Volume 61, Issue 4, Pages 1339-1350 (April 2002) DOI: 10.1046/j.1523-1755.2002.00259.x Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 1 Effect of FK506 treatment on kinetics of proteinuria after injection of monoclonal antibody 1-22-3 (mAb 1-22-3). Significant reduction of proteinuria was observed in the 1.0 mg/kg body weight (BW) or 0.3 mg/kg BW of the FK506-treated group. Symbols are: (○) placebo-treated control group; (×) 1.0 mg/kg BW of the FK506-treated group; (▵) 0.3 mg/kg BW of the FK506-treated group; (♦) 0.1 mg/kg BW of the FK506-treated group. Results were expressed as mean ± SD (N = 5). aP < 0.01, bP < 0.05 compared with the placebo-treated control group at the same time point. Kidney International 2002 61, 1339-1350DOI: (10.1046/j.1523-1755.2002.00259.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 2 Effect of FK506 treatment on rat glomerular changes. Glomerular changes evaluated by total number of cells per glomerular cross-section (A), matrix expansion (B), crescent formation rate (C), α-smooth muscle actin (αSMA) staining score (D) and collagen type I staining score (E) were compared between the FK506-treated groups and the control group on day 14. The FK506 treatment ameliorated the glomerular morphological alterations in a dose-dependent manner. Symbols are: (□) placebo control; (▪) FK506 1.0 mg/kg; () FK506 0.3 mg/kg; () FK506 0.1 mg/kg. Each value is expressed as mean ± SD (N = 5). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the placebo-treated control group. #No crescents were observed in rats treated with 1.0 mg/kg BW of FK506. Kidney International 2002 61, 1339-1350DOI: (10.1046/j.1523-1755.2002.00259.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 3 Photomicrographs of periodic acid-Schiff (PAS) staining (A, B) and immunofluorescence (IF) staining for α-smooth muscle actin (α-SMA) (C, D) and collagen type I (E, F) of kidney sections from rats treated with 1.0 mg/kg BW of FK506 (B, D, F), and from a placebo-treated control group (A, C, E) 14 days after injection of mAb 1-22-3. (Original magnification ×200) Kidney International 2002 61, 1339-1350DOI: (10.1046/j.1523-1755.2002.00259.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 4 Representative immunofluorescence findings of bound mAb 1-22-3 and C9 in glomeruli (original magnification ×200). Representative IF findings stained with FITC-conjugated anti-mouse Igs (A, B) and stained with anti-rat C9/FITC-conjugated anti-rabbit IgG (C, D). No difference of staining intensity of bound mAb 1-22-3, or rat C9 in glomeruli was observed between the rats pretreated with 1.0 mg/kg BW of FK506 (B, D) and with placebo (A, C). Kidney International 2002 61, 1339-1350DOI: (10.1046/j.1523-1755.2002.00259.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 5 Effect of FK506 treatment on proteinuria from one day after the induction of glomerulonephritis (GN). The treatment with 0.3 mg/kg BW of FK506 daily from day 1 also reduced the proteinuria on 14th day. Symbols are: (○) placebo-treated control group; (•) 0.3 mg/kg BW of the FK506-treated group. Data are expressed as mean ± SD (N = 5). *P < 0.05 compared with the placebo-treated control group. Kidney International 2002 61, 1339-1350DOI: (10.1046/j.1523-1755.2002.00259.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 6 Effect of treatment with 0.3 mg/kg BW of FK506 daily from 1 day after the induction of GN on glomerular changes on day 14. Symbols are: (□) placebo control; () FK506 0.3 mg/kg. The treatment with 0.3 mg/kg BW of FK506 from day 1 also ameliorated the glomerular morphological alterations. Data are expressed as mean ± SD (N = 5). *P < 0.05, **P < 0.01 compared with the placebo-treated control group. Kidney International 2002 61, 1339-1350DOI: (10.1046/j.1523-1755.2002.00259.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 7 Effect of 1.0 mg/kg BW(A) and 0.3 mg/kg BW(B) of FK506 treatment from one day after the induction of GN on glomerular filtration of leukocytes on day 5. Symbols are: (□) placebo control; (▪) FK506 1.0 mg/kg; () FK506 0.3 mg/kg. Numbers of OX19+ cells (T lymphocytes), OX38+ cells (CD4 T lymphocytes), ED1+ cells (monocytes/macrophages) and ED3+ cells (activated macrophages) per glomerular cross section (gcs) were counted in 50 randomly selected full-sized glomeruli. The FK506 treatment did not affect the glomerular accumulation of OX19+ cells, OX38+ cells nor ED1+ cells, whereas it reduced the glomerular accumulation of ED3+ cells. Data are expressed as mean ± SD (N = 5). *P < 0.05 compared with the placebo-treated control group. NS is not significant. Kidney International 2002 61, 1339-1350DOI: (10.1046/j.1523-1755.2002.00259.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 8 Immunofluorescence findings of glomerular staining for ED3+ cells (activated macrophages) in rats treated with placebo(A) or 1.0 mg/kg BW of FK506(B) on day 5 after induction of GN (original magnification ×200). Kidney International 2002 61, 1339-1350DOI: (10.1046/j.1523-1755.2002.00259.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 9 Effects of FK506 treatment on glomerular mRNA expression for Th1 cytokine and PDGF on day 5.(A) Representative semiquantitative measurements made by reverse transcription-polymerase chain reaction (RT-PCR) amplification of mRNA of five rats from the 1.0 mg/kg BW of FK506-treated group (F1-F5) and the placebo-treated control group (#1-#5). The representative agarose gel electrophoretic patterns are shown in the left panel. The ratios of the densitometric signals of cytokines to that of the internal control (GAPDH) were computed (right panel), and the results were compared between the FK506-treated group (▪) and the placebo-treated control group (□). The optimal cycle numbers were 20 cycles (for GAPDH), 30 cycles (for IFN-γ), 35 cycles (for IL-2) and 25 cycles (for PDGF-BB). The FK506 treatment reduced the mRNA expression of IFN-γ and IL-2, whereas it did not affect PDGF expression. Data are expressed as mean ± SD (N = 5). (B) RT-PCR findings of 0.3 mg/kg BW of FK506-treated group and the placebo-treated control group. The representative agarose gel electrophoretic patterns are shown in the upper panel. The ratios to the placebo control were shown in the bottom panel. (C) Real-time RT-PCR analysis of IL-2 and IFN-γ in rats with 0.3 mg/kg BW of FK506. On the x-axis the number of PCR cycles is shown, on the y-axis the difference between measured fluorescence and the baseline. Decreased expression of IL-2 and IFN-γ in rats treated with FK506 was confirmed. (a) Placebo control GAPDH; (b) FK506 0.3 mg/kg; (c, left panel) placebo control IL-2; (c, right panel) placebo control IFN-γ; (d, left panel) FK506 0.3 mg/kg IL-2; (d right panel) FK506 0.3 mg/kg IFN-γ. Kidney International 2002 61, 1339-1350DOI: (10.1046/j.1523-1755.2002.00259.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Kidney International 2002 61, 1339-1350DOI: (10. 1046/j. 1523-1755 Kidney International 2002 61, 1339-1350DOI: (10.1046/j.1523-1755.2002.00259.x) Copyright © 2002 International Society of Nephrology Terms and Conditions