Ischemic preconditioning is a powerful technique to protect organs, including the kidney, from ischemia-reperfusion injury. In the current study we have demonstrated that delayed kidney IPC specifically inhibits the innate inflammation (kidney neutrophil and macrophage accumulation) associated with kidney IRI. We also report the novel finding that mild kidney IRI induces a delayed increase in the number of FoxP3+ and IL-10+ Treg cells in the kidney, such that at the time of the subsequent more severe renal IRI, preconditioned mice have 3-fold more FoxP3+ and 8-fold more IL-10+ Treg cells in the kidney than non-preconditioned mice. In support of a protective role for Tregs in IPC, partial Treg depletion reversed the anti-inflammatory effects of delayed IPC and significantly inhibited the functional and histological protection. Finally, adoptive transfer of Treg cells, prior to IRI in naïve mice mimicked ischemic preconditioning in terms of blocking inflammation and preventing renal injury.
There are two methods to induce ischemic preconditioning in the kidney. One type is induced by repeated short ischemic times followed by periods of reperfusion (i.e. 4 cycles of 5 min ischemia and 5 min reperfusion). This type of preconditioning partially protects the kidney from 30 min of ischemia immediately after the last preconditioning cycle and at 24 hr after the last cycle, but not at 6 hours after preconditioning (29
). Delayed kidney IPC involves a longer single initial ischemic time (i.e. >20 min) followed by a recovery period of 6 to 8 days (11
). After this procedure, mice are completely protected from subsequent ischemic injury for greater than one week and partially protected for up to twelve weeks (14
). We used a model of delayed ischemic preconditioning that resulted in complete functional protection of the kidney from severe ischemic injury 7 days after the initial preconditioning surgery. This model of IPC markedly decreased the infiltration of neutrophils and macrophages in the post-ischemic kidney, which are both known to cause renal injury (5
A report from Ascon et al.
) demonstrated that adoptive transfer of lymphocytes from kidneys of preconditioned mice conferred protection against renal IRI in naïve T cell-deficient mice, suggesting that renal IRI causes kidney accumulation of lymphocytes with a protective (potentially anti-inflammatory) phenotype. Characterization of the specific lymphocyte (T or B cell) subset(s) responsible for the protection was not reported. We hypothesized that the highly immunosuppressive CD4+
Treg cells (expressing FoxP3+
and/or IL-10) could be important mediators of kidney ischemic preconditioning for several reasons. First, we have recently shown that CD4+
Treg cells are an intrinsic protective mechanism against kidney IRI in naïve mice, which directly and potently block the innate immune response to IRI (17
). Second, WT, but not IL-10 KO, Treg cells could suppress IRI in lymphocyte-deficient RAG-1 KO mice (17
). Furthermore, the trafficking and accumulation of Treg cells in areas of ongoing inflammation has been documented in other models (23
). We found that the initial ischemic insult in our preconditioning model resulted in a kidney-specific increase in the number of kidney CD4+
Treg cells and the number and percentage of CD4+
Treg cells expressing IL-10 in the kidney. Our findings are in agreement with a recent report from Gandolfo et al.
) demonstrating renal accumulation of Treg cells after more severe IRI. The preconditioned mice, which were completely protected from ischemic injury, possessed significantly more kidney Treg cells than non-preconditioned mice that were susceptible to IRI. In addition, the localization of the Treg cells in the preconditioned kidneys was distinct, in that Tregs were consistently observed in the outer medulla interstitium (which is also the area of the most extensive inflammation and tubular necrosis following kidney IRI). In summary, the preconditioned kidney has a larger population of anti-inflammatory Treg cells and they are located in the most ischemia-susceptible region of the kidney, the outer medulla.
To determine the role of Treg cells in kidney IPC, the Treg cell-depleting antibody, PC61 was administered to preconditioned mice, resulting in a ~50% reduction in Treg cell numbers, compared to control IgG-treated preconditioned mice, at the time of the second ischemic insult. Decreasing Treg cell numbers, in preconditioned mice, caused inhibition of the anti-inflammatory effect of IPC and partially blocked the functional and histological protection. These results reveal an important role for Treg cells in kidney IPC. The finding that mice are not completely protected from IRI-induced dysfunction at 3 days after the initial preconditioning surgery (prior to kidney Treg cell accumulation) adds additional support for the contribution of Treg cells to the protection of IPC.
Although Treg cell-deficient mice, FoxP3-deficient Scurfy mice, are available, they develop extensive autoimmune disease and early death (31
) precluding their use in our studies. Furthermore, our goal was to test the effectiveness of IPC in kidneys that contained the basal number of Treg cells found in non-preconditioned mice. We were able to create these conditions in two ways: 1) use of preconditioned mice 3 days after the initial surgery and prior to the increase in kidney Treg cell numbers, and 2) by treating preconditioned mice with PC61 to reduce the number of kidney Treg cells at 7 days after the initial surgery. Both methods resulted in partial reversal of the functional protection of IPC.
Other immune cells may participate in kidney preconditioning based upon studies that have demonstrated their role in kidney IRI or in actual preconditioning. B cells do accumulate in the post ischemic kidney (9
) and may have a pathogenic role in kidney IRI (3
). However no studies have examined their role in preconditioning. Although macrophage infiltration may contribute to the recovery following kidney injury they do not contribute to ischemic preconditioning in kidneys (32
). To our knowledge there are no studies demonstrating a role for neutrophils in ischemic preconditioning in kidneys. Further studies that focus on immune cells may reveal additional mechanisms of ischemic preconditioning.
While our results demonstrate that Treg cells are important mediators of the anti-inflammatory action of IPC, other mechanisms protect the preconditioned kidney, even in the presence of extensive inflammatory leukocyte accumulation. In delayed kidney IPC, several non-immune mechanisms have previously been described including upregulation of the renal pro-survival signals HSP 27 expression (14
) and Akt phosphorylation (13
), and decreased phosphorylation of cell death promoting p38 and Jun kinase (14
). These changes presumably occur in the epithelial and endothelial cells of the preconditioned kidney and make them more resistant to ischemia and inflammatory mediator (reactive oxygen species, proteases, etc.)-induced toxicity, leading to the substantial residual protection observed in preconditioned mice treated with PC61.
Our goal in studying IPC is to discover novel and less invasive strategies to protect the kidney from IRI. As a first step in this direction we assessed the effectiveness of adoptive transfer of Treg cells to a naïve WT recipient prior to kidney IRI. We found that, in contrast to an equivalent number of non-Treg CD4+
lymphocytes, Treg cells could completely inhibit inflammation and renal dysfunction induced by severe bilateral renal ischemia. These results complement our previous finding that Treg cells, but not non-Treg lymphocytes, can inhibit renal neutrophil accumulation and IRI in lymphocyte-deficient RAG-1 KO mice (17
). Therefore, in the presence or absence of other leukocytes, Treg cells suppress the innate inflammation and subsequent loss of function induced by kidney ischemia-reperfusion. Our findings suggest that adoptive transfer or pharmacologic induction of Treg cells may represent less invasive methods to protect organs from ischemic insult.
The source of the renal Treg cells, accumulating after IRI, is not currently known. At least 2 different explanations are possible, which are not mutually exclusive. First, existing Treg cells from the circulation could be recruited to the post-ischemic kidney by chemokines. The CCR2 chemokine receptor directs Tregs towards arthritic joints (33
). Monocyte chemoattractant protein-1 (MCP-1/CCL-2) is the major ligand for CCR2 and the expression of this chemokine is highly upregulated in the kidney after IRI (34
). A second potential source of the Treg cells could be kidney non-Treg lymphocytes, activated by T cell receptor stimulation in the presence of TGF-β, which has been shown to induce FoxP3 expression and a suppressive phenotype (36
). While TGF-β is produced in the kidney after ischemia (37
), so is IL-6 (38
), and the combination of these 2 cytokines promotes the induction of Th17 cells rather than Treg cells (39
). However, the time course and the intra-kidney differences in concentrations of these cytokines may still permit the induction of Treg cells in the post-ischemic kidney. The origin of Treg cells accumulating after IRI is under investigation and may lead to more specific ways to mimic IPC in different organs.
In summary, IPC markedly inhibits inflammation, acute tubular necrosis and loss of function induced by ischemia-reperfusion. We demonstrate that the initial ischemic insult (preconditioning) causes a kidney-specific increase in Treg cells, such that the preconditioned kidney has several-fold more Treg cells than non-preconditioned kidneys. Partial depletion of Treg cells in preconditioned mice restores the accumulation of neutrophils and macrophages, induced by IRI, and partially reverses functional and histological protection. Finally, increasing Treg cell numbers in WT mice, by adoptive transfer, mimics the anti-inflammatory and renal protective effects of IPC. Taken together these results suggest that strategies designed to increase regulatory T cells may represent a novel therapy for ischemia-reperfusion injury.