1. Volume 81, Issue 9, Pages 844-855 (May 2012)
Endogenous Tim-1 (Kim-1) promotes T-cell responses and cell-mediated injury in experimental crescentic glomerulonephritis 
Yuji Nozaki, David J. Nikolic-Paterson, Sarah L. Snelgrove, Hisaya Akiba, Hideo Yagita, Stephen R. Holdsworth, A Richard Kitching 
Kidney International 
Volume 81, Issue 9, Pages (May 2012)
DOI: /ki
Copyright © 2012 International Society of Nephrology Terms and Conditions

2. Figure 1
Anti-T-cell immunoglobulin mucin 1 (Tim-1) antibodies reduce the severity of experimental crescentic glomerulonephritis. Representative photomicrographs of glomerular cross-sections at high power, × 400, in mice with glomerulonephritis given (a) rIgG or (b) anti-Tim-1 antibodies. Nonimmune rat IgG (rIgG)–injected mice developed proliferative and crescentic glomerulonephritis, which was diminished in anti-Tim-1 antibody–treated mice (c), with decreased interstitial disease (d). Compared with that in rIgG-treated mice, proteinuria (e) in anti-Tim-1 antibody–treated mice was not different in the heterologous phase (day 1) but was decreased at day 14. There was a trend toward a lesser rise in serum creatinine (f) in anti-Tim-1 antibody–treated mice. Dotted lines represent mean values from normal C57BL/6 mice without nephritis. *P<0.05, **P<0.005.
Kidney International  , DOI: ( /ki )
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3. Figure 2
Anti-T-cell immunoglobulin mucin 1 (Tim-1) treatment limits glomerular and interstitial leukocyte accumulation. On day 14, anti-Tim-1 antibody–treated mice had less glomerular leukocyte infiltrates, including fewer CD4+ cells (a), macrophages (b), and neutrophils (c). Findings for interstitial CD4+ cells (d) and macrophages (e) were similarly reduced in anti-Tim-1 antibody–treated mice, but interstitial neutrophils were not significantly reduced (f). Interstitial foxp3+ cells, shown as black dots at high power, × 400, were detected in both rat IgG (rIgG) (g)- and anti-Tim-1 antibody–treated mice (h), but anti-Tim-1-treated mice had more foxp3+ positive cells in the interstitium (i). Foxp3+ cells were not found in glomeruli. Intrarenal foxp3 mRNA expression in anti-Tim-1 antibody–treated mice was increased compared with rIgG-treated mice (j). Dotted lines represent mean values from normal C57BL/6 mice without nephritis. c/gcs, cells per glomerular cross-section; c/hpf, cells per high-power field. *P<0.05, **P<0.005.
Kidney International  , DOI: ( /ki )
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4. Figure 3
Anti-T-cell immunoglobulin mucin 1 (Tim-1) antibody treatment reduces intrarenal proliferation and apoptosis. There were more intraglomerular Ki-67+ cells, shown as black dots at high power, × 400, in rat IgG (rIgG)-treated mice than in anti-Tim-1 antibody–treated mice both in the glomerulus (a–c) and in the interstitium (d–f). In comparison with rIgG-treated mice, cleaved caspase-3+ cells in the tubulointerstitium were reduced in anti-Tim-1 antibody–treated mice (g–i). Cleaved caspase-3+ cells were not found in glomeruli. Photomicrograhs were taken at high power, × 400. *P<0.05.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

5. Figure 4
Effects of anti-T-cell immunoglobulin mucin 1 (Tim-1) antibodies on CD4+ T-cell responses. Splenic CD4+ cell apoptosis, proliferation, and cytokine production on day 14 were assessed by flow cytometric analysis of annexin-V binding, bromodeoxyuridine (BrdU) uptake, and intracellular interferon-gamma (IFN-γ) and interleukin (IL)-17A. Results are expressed as the percentage of CD4+ cells that were also BrdU+ (a), annexin-V+ (b), IFN-γ+ (c), and IL-17A+ (d). In comparison with rat IgG (rIgG)-treated mice, anti-Tim-1 antibody–treated mice developed less CD4+ cell proliferation (a) and apoptosis (b), with fewer CD4+ cells positive for BrdU or annexin-V. The proportion of CD4+ cells expressing IFN-γ was significantly reduced (c), but reductions in CD4+ cell IL-17A expression (d) did not reach significance. PI, propidium iodide. *P<0.05.
Kidney International  , DOI: ( /ki )
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6. Figure 5
Systemic immune responses in control IgG- and anti-T-cell immunoglobulin mucin 1 (Tim-1) antibody–treated mice with glomerulonephritis. Antigen-stimulated splenocyte production of interferon-gamma (IFN-γ) (a) and interleukin (IL)-17A (b) was reduced in anti-Tim-1 antibody–treated mice. However, IL-4 (c) and IL-10 (d) production were unaffected. Total antigen (sheep globulin)–specific IgG (e) and the Th2-associated subclass IgG1 (f) were increased in anti-Tim-1–treated mice, but antigen-specific IgG3 titers were unaffected (g). OD, optical density. *P<0.05.
Kidney International  , DOI: ( /ki )
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7. Figure 6
Expression of T-cell immunoglobulin mucin 1 (Tim-1) on intrarenal effector CD4+ cells. Kidneys were digested from normal mice and mice with glomerulonephritis (not injected with rat IgG (rIgG) or anti-Tim-1 antibodies). There was a progressive increase in CD4+ cells from day 6 to 14 (a), and although Tim-1+ CD4+ cells were scarce in normal mice, an increasing proportion (b) and number (c) of effector T cells (CD4+CD44+) expressed Tim-1 from days 6 to 14 of disease. *P<0.05, **P<0.005.
Kidney International  , DOI: ( /ki )
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8. Figure 7
Anti-T-cell immunoglobulin mucin 1 (Tim-1) antibodies reduce intrarenal proinflammatory cytokine gene expression in glomerulonephritis. Cytokine gene expression was measured 14 days after injection of anti-GBM antibodies in mice treated with rat IgG (rIgG) or anti-Tim-1 antibodies. IFN-γ, IL-17A, and IL-1β expression were significantly reduced, reductions in IL-12p40 and tumor necrosis factor (TNF) did not reach significance, and IL-4 and IL-10 mRNAs were increased after anti-Tim-1 antibody treatment. Expression levels were normalized to the expression of 18S rRNA and expressed relative to saline-treated normal mice without nephritis. *P<0.05, **P<0.005.
Kidney International  , DOI: ( /ki )
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9. Figure 8
Renal expression of intercellular adhesion molecule 1 (ICAM-1) in control IgG- and anti-T-cell immunoglobulin mucin 1 (Tim-1) antibody–treated mice with glomerulonephritis. Representative photomicrographs of ICAM-1 in glomeruli (a, b, e) and tubules (c, d, f) of mice with glomerulonephritis on day 14, showing reduced ICAM-1 in both glomeruli and tubules of anti-Tim-1 antibody–treated mice compared with that in rat IgG (rIgG)-treated mice. Anti-Tim-1 antibodies also reduced intrarenal ICAM-1 mRNA expression (g). Photomicrographs were taken at high power, × 400. *P<0.05, **P<0.005.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

10. Figure 9
Renal kidney injury molecule-1 (Kim-1) in glomerulonephritis. (a) No Kim-1 is present in normal kidneys. (b) On day 14 of nephritis, in rat IgG (rIgG)-treated mice, Kim-1+ proximal tubules were clearly present with immunostaining in apical regions and cytoplasm (arrowheads). (c) In anti-T-cell immunoglobulin mucin 1 (Tim-1) antibody–treated mice, there were fewer Kim-1+ proximal tubules. (d) Intrarenal Kim-1 mRNA expression was suppressed at day 14 in anti-Tim-1 antibody–treated mice compared with rIgG-treated mice. (e) Urinary Kim-1 excretion from days 13–14 was markedly increased compared with excretion from days 1–2 in both groups. However, compared with rIgG-treated mice, urinary Kim-1 excretion from days 13–14 was decreased in anti-Tim-1 antibody–treated mice. In normal mice, urinary Kim-1 excretion was below the level of detection of the assay. (f, g) Western blot analysis and densitometric analysis showing reduced renal Kim-1 expression on day 14. Photomicrographs were taken at high power, × 400. †P<0.05 vs. day 1, *P<0.05 vs. rIgG-treated mice.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions

11. Figure 10
Anti-T-cell immunoglobulin mucin 1 (Tim-1) antibodies have no effect on the early autologous phase of disease. On day 6, proteinuria, serum creatinine, the proportion of abnormal glomeruli, and the modest tubulointerstitial injury seen were similar in both groups (a). Dotted lines represent mean values from normal C57BL/6 mice without nephritis. Measuring splenocyte cytokine production at day 6 (b), there were trends toward decreased antigen-stimulated splenocyte production of interferon-gamma (IFN-γ) and interleukin (IL)-17A in anti-Tim-1 antibody–treated mice, but there was no difference in IL-4 production. During the early autologous phase, kim-1–positive proximal tubules (high power, × 400) were present to a similar degree in rat IgG (rIgG)- and anti-Tim-1–treated mice (c). Similarly, on day 6, intrarenal kidney injury molecule-1 (Kim-1) mRNA expression was similar in the two groups.
Kidney International  , DOI: ( /ki )
Copyright © 2012 International Society of Nephrology Terms and Conditions