Regulatory T cells contribute to the protective effect of ischemic preconditioning in the kidney - PubMed (original) (raw)

Regulatory T cells contribute to the protective effect of ischemic preconditioning in the kidney

Gilbert R Kinsey et al. Kidney Int. 2010 May.

Abstract

Reperfusion following ischemia is associated with acute kidney injury and inflammation. Using a mouse model, we exposed the kidney to a nonlethal period of ischemia, rendering it refractory to future ischemia-induced dysfunction. This ischemic preconditioning is partially mediated by Treg lymphocytes that suppress immune responses. We found that this maneuver significantly inhibited the accumulation of neutrophils and macrophages, tubular necrosis, and loss of kidney function caused by a subsequent ischemia/reperfusion injury 1 week later. The initial ischemia/reperfusion caused a significant increase in CD4(+)CD25(+)FoxP3(+) and CD4(+)CD25(+)IL-10(+) Treg cells within the kidney at 7 days of reperfusion. Treatment of preconditioned mice with a Treg cell-depleting antibody (PC61) reversed the effect of preconditioning on kidney neutrophil accumulation and partially inhibited the functional and histological protection of preconditioning. Adoptive transfer of Treg cells in naive mice, before ischemia/reperfusion, mimicked the protective and anti-inflammatory effects of ischemic preconditioning on the kidney. These studies highlight the role of Treg cells in ischemic preconditioning.

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Figures

Figure 1

Figure 1. Ischemic preconditioning prevents ischemia-reperfusion-induced kidney dysfunction and acute tubular necrosis

C57Bl/6 mice underwent sham surgery or bilateral renal pedicle clamping (IRI) for 24 min on day 0 and were allowed to recover for 7 days. On day 7 mice underwent 28 min IRI. Plasma creatinine was measured periodically to assess renal function (A). Twenty four hrs after the second surgery, kidney sections were fixed and stained with H&E to assess outer medulla acute tubular necrosis (ATN) in the different treatment groups (B), pictures are representative of at least 8 mice per group (C,D,E). Data are presented as the mean ± SEM; * - P<0.01 compared to Sham/Sham control group; # - P<0.01 compared to Sham/IRI, non-preconditioned group.

Figure 2

Figure 2. Ischemic preconditioning inhibits innate leukocyte accumulation after IRI

Twenty four hours after the 2nd surgery, kidneys were harvested, digested and stained with neutrophil or macrophage markers and analyzed by flow cytometry as described in the methods section. The absolute number of neutrophils (A) and macrophages (B) per gram kidney in each group is represented graphically. Frozen kidney sections were stained with an antibody that recognizes neutrophils and recently emigrated monocytes (7/4) to assess renal accumulation in the different treatment groups, pictures are representative of at least 3 mice per group. Data are presented as the mean + SEM; * - P< 0.01 vs. Sham/Sham, control mice; # - P<0.05 compared to Sham/IRI, non-preconditioned group.

Figure 3

Figure 3. Kidney ischemia reperfusion injury causes kidney regulatory T cell accumulation

At 3, 7 and 14 days after 24 min IRI (preconditioned) or sham (non-preconditioned) surgery, mouse kidneys and spleen were harvested, digested and stained for Treg cell markers as described in the Methods section. The absolute number of Treg cells per gram in preconditioned mice is represented as a percentage of their respective sham control (A). Data are presented as the mean + SEM, * - P< 0.05, compared to non-preconditioned mice, n = 4–8 per group. Frozen sections from sham-operated (B) or 24 min IRI (C,D) kidneys were stained with anti-mouse CD4 (red) and anti-mouse FoxP3 (green) antibodies. Images are from the outer medulla and representative of at least 3 separate animals in each group.

Figure 4

Figure 4. Kidney ischemia reperfusion injury causes increased IL-10 production by Treg cells

At 7 days after 24 min IRI (preconditioned) or sham (non-preconditioned) surgery, mouse kidneys harvested, digested and stained for Treg cell surface markers CD4 and CD25, and stained intracellularly for the cytokine IL-10 as described in the Methods section. The percentage (A) and absolute number (B) of IL-10+ Treg cells per kidney in preconditioned and non-preconditioned mice is represented as the mean + SEM, * - P< 0.05, compared to non-preconditioned mice, n = 6 per group.

Figure 5

Figure 5. PC61 administration significantly reduces kidney Treg cells in preconditioned mice

Preconditioned mice were injected with the α-CD25 monoclonal antibody, PC61, or control IgG on day 2 of the IPC protocol (A). On day 7 of reperfusion, kidney Treg cell numbers were determined by flow cytometry (B). Data are presented as the mean + SEM; * - P< 0.01 vs. IRI/IRI-IgG.

Figure 6

Figure 6. Treg cell depletion is associated with inhibition of the anti-inflammatory effects of ischemic preconditioning

Preconditioned mice were injected with the α-CD25 monoclonal antibody, PC61, or control IgG on day 2 of the IPC protocol. On day 8 (24 hr after the second surgery), kidney neutrophil (CD45+7-AAD−CD11b+GR-1high; A) and macrophage (CD45+7-AAD−F4/80intCD11b+; B) accumulation was assessed by flow cytometry. Data are presented as the mean + SEM; n.s. – not significant.

Figure 7

Figure 7. Treg cell depletion inhibits the kidney functional and histological protection of ischemic preconditioning

Renal function was assessed by measuring plasma creatinine at 24 hr after the first surgery (Day 1), at the time of the second surgery (Day 7) and 24 hr after the second surgery (Day 8) in non-preconditioned mice treated with IgG or preconditioned mice treated with IgG or PC61 (A). Twenty four hrs after the second surgery, kidney sections were fixed and stained with H&E to assess outer medulla tubular necrosis in preconditioned mice treated with IgG (B) or PC61 (C), pictures are representative of at least 4 mice per group. Data are presented as the mean + SEM; * - P<0.05 compared to Sham/IRI-IgG; # - P<0.01 compared to IRI/IRI-IgG; n.s. – not significant.

Figure 8

Figure 8. Adoptive transfer of regulatory T cells protects the kidney from subsequent ischemia-reperfusion injury

Eighteen hr prior to 28 min bilateral renal ischemia, naïve C57Bl/6 mice were administered either 1 × 106 CD4+CD25− non-Treg lymphocytes (CD25−), 1 × 105 or 1 × 106 CD4+CD25+ Treg cells by tail vein injection. Twenty four hr after sham surgery or IRI, renal function was assessed by measuring plasma creatinine levels (A). Twenty four hr after IRI kidney sections were fixed and stained with H&E to assess outer medulla tubular necrosis in the different treatment groups (B,C,D,E). Data are presented as the mean + SEM; * - P<0.05 compared to Sham; # - P<0.01 compared to CD25− - IRI; ** - P<0.001 compared to CD25− - IRI; n.d. – not done.

Figure 9

Figure 9. Adoptive transfer of regulatory T cells prevents ischemia-reperfusion-induced inflammation

Eighteen hr prior to 28 min bilateral renal ischemia, naïve C57Bl/6 mice were administered either 1 × 106 CD4+CD25− non-Treg lymphocytes (CD25−), 1 × 105 or 1 × 106 CD4+CD25+ Treg cells by tail vein injection. Twenty four hr after sham surgery or IRI, kidney neutrophil (A) and macrophage (B) accumulation was measured by flow cytometry. Frozen kidney sections were stained with an antibody that recognizes neutrophils and recently emigrated monocytes (7/4) to assess renal accumulation in the different treatment groups (C,D,E). Data are presented as the mean + SEM, means with different superscripts are significantly different from each other, P<0.01; n.d. – not done.

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