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Both innate and adaptive mechanisms participate in the pathogenesis of kidney ischemia-reperfusion injury (IRI), but the role of regulatory immune mechanisms is unknown. We hypothesized that the anti-inflammatory effects of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) protect against renal IRI. Partial depletion of Tregs with an anti-CD25 mAb potentiated kidney damage induced by IRI. Reducing the number of Tregs resulted in more neutrophils, macrophages, and innate cytokine transcription in the kidney after IRI but did not affect CD4(+) T cells or B cells. We performed adoptive transfer of lymph node cells from wild-type mice or FoxP3-deficient Scurfy mice into T cell- and B cell-deficient RAG-1 knockout mice to generate mice with and without FoxP3(+) Tregs, respectively. FoxP3(+) Treg-deficient mice accumulated a greater number of inflammatory leukocytes after renal IRI than mice containing Tregs. To confirm that a lack of Tregs potentiated renal injury, we co-transferred isolated Tregs and Scurfy lymph node cells; Treg repletion significantly attenuated IRI-induced renal injury and leukocyte accumulation. Furthermore, although adoptive transfer of wild-type Tregs into RAG-1 knockout mice was sufficient to prevent kidney IRI, transfer of IL-10-deficient Tregs was not. Taken together, these results demonstrate that Tregs modulate injury after kidney IRI through IL-10-mediated suppression of the innate immune system.

During renal ischemia-reperfusion, local and distant tissue injury is caused by an influx of neutrophils into the affected tissues. Here we measured the kinetics of margination and transmigration of neutrophils in vivo in the kidney and lungs following renal ischemia-reperfusion. After bilateral renal injury, kidney neutrophil content increased threefold at 24 h. The neutrophils were found primarily in the interstitium and to a lesser degree marginated to the vascular endothelium. These interstitial neutrophils had significantly lower levels of intracellular IFN-gamma, IL-4, IL-6, and IL-10 a tendency for decreased amounts of IL-4 and TNF-alpha compared to the marginated neutrophils. Localization of the neutrophils to the kidney interstitium was confirmed by high resolution microscopy and these sites of transmigration were directly associated with areas of increased vascular permeability. Activation of the adenosine 2A receptor significantly decreased both kidney neutrophil transmigration by about half and vascular permeability by about a third. After unilateral renal ischemia-reperfusion, the unclipped kidney and lungs did not accumulate interstitial neutrophils or have increased vascular permeability despite a marked increase of neutrophil margination in the lungs. Our findings suggest there is a sequential recruitment and transmigration of neutrophils from the vasculature into the kidney interstitium at the site of tissue injury following renal ischemia-reperfusion.

Sphingosine-1-phosphate (S1P), produced by sphingosine kinase 1 (SphK1) or kinase 2 (SphK2), mediates biological effects through intracellular and/or extracellular mechanisms. Here we determined a role for these kinases in kidney injury of wild-type mice following ischemia-reperfusion. SphK1 but not SphK2 mRNA expression and activity increased in the kidney following injury relative to sham-operated animals. Although SphK1(-/-) mice had no alteration in renal function following injury, mice with a disrupted SphK2 gene (SphK2(tr/tr)) had histological damage and impaired function. The immune-modulating pro-drug, FTY720, an S1P agonist failed to provide protection in SphK2(tr/tr) mice. Injured kidneys of these mice showed increased neutrophil infiltration and neutrophil chemokine expression along with a 3- to 5-fold increase in expression of the G-protein-coupled receptor S1P(3) compared to heterozygous SphK2(+/tr) mice. Kidney function and reduced vascular permeability were preserved in S1P(3)(-/-) compared to S1P(3)(+/-) mice after ischemia-reperfusion injury, suggesting increased S1P(3) mRNA may play a role in the injury of SphK2(tr/tr) mice. Our study suggests that constitutive expression of SphK2 may contribute to reduced ischemia-reperfusion injury of the kidney, and its absence may enhance injury due to increased neutrophil infiltration and S1P(3) activation. We also confirm that SphK2 is necessary to mediate the protective effects of FTY720.

Chemokines and their receptors such as CCR2 and CX3CR1 mediate leukocyte adhesion and migration into injured tissue. To further define mechanisms of monocyte trafficking during kidney injury we identified two groups of F4/80-positive cells (F4/80(low) and F4/80(high)) in the normal mouse kidney that phenotypically correspond to macrophages and dendritic cells, respectively. Following ischemia and 3 h of reperfusion, there was a large influx of F4/80(low) inflamed monocytes, but not dendritic cells, into the kidney. These monocytes produced TNF-alpha, IL-6, IL-1alpha and IL-12. Ischemic injury induced in CCR2(-/-) mice or in CCR2(+/+) mice, made chimeric with CCR2(-/-) bone marrow, resulted in lower plasma creatinine levels and their kidneys had fewer infiltrated F4/80(low) macrophages compared to control mice. CX3CR1 expression contributed to monocyte recruitment into inflamed kidneys, as ischemic injury in CX3CR1(-/-) mice was reduced, with fewer F4/80(low) macrophages than controls. Monocytes transferred from CCR2(+/+) or CX3CR1(+/-) mice migrated into reperfused kidneys better than monocytes from either CCR2(-/-) or CX3CR1(-/-) mice. Adoptive transfer of monocytes from CCR2(+/+) mice, but not CCR2(-/-) mice, reversed the protective effect in CCR2(-/-) mice following ischemia-reperfusion. Egress of CD11b(+)Ly6C(high) monocytes from blood into inflamed kidneys was CCR2- and CX3CR1-dependent. Our study shows that inflamed monocyte migration, through CCR2- and CX3CR1-dependent mechanisms, plays a critical role in kidney injury following ischemia reperfusion.

The mechanisms involved in renal ischemia-reperfusion injury (IRI) are complex and appear to involve the early participation of bone marrow-derived cells. T lymphocytes participate in the pathogenesis of IRI. Sphingosine 1-phosphate (S1P) induces peripheral T cell depletion. Therefore, we hypothesized that S1P1 receptor activation protects kidney from IRI. FTY-720, a non-receptor-selective sphingosine analog, was given intraperitoneally to C57BL/6 mice, and animals were subjected to ischemia for 32 min followed by reperfusion for 24 h. Plasma creatinine, blood count, myeloperoxidase (MPO) activity, and renal histology were determined. IRI led to a marked increase in plasma creatinine, MPO activity, leukocyte infiltration, and vascular permeability. FTY-720 significantly decreased plasma creatinine in a dose-response manner with a maximal reduction of ∼73 and ∼69% with doses of 240 and 48 μg/kg, respectively. MPO, leukocyte infiltration, vascular permeability, and peripheral blood lymphocyte counts were markedly decreased with FTY-720 treatment. The protective effect of FTY-720 was reversed with VPC-44116, a selective S1P1 receptor antagonist. Furthermore, SEW-2871, a selective S1P1 agonist, significantly decreased plasma creatinine in a dose-response manner with a maximal reduction of ∼70% with a dose of 10 mg/kg. Analysis of kidneys by light microscopy revealed minimal histological signs of ischemic injury with FTY-720 or SEW-2871 treatment compared with the vehicle group. Using RT-PCR, we found a time-dependent increase in the S1P1 mRNA expression following IRI that begins after 2 h with the maximum expression at ∼4 h. We conclude that the protective effect of FTY-720 is due primarily to activation of S1P1 receptors. The mechanism of protection is not known but may be related to peripheral lymphocyte depletion or direct effects on kidney cells expressing S1P1 receptor. [ABSTRACT FROM AUTHOR]

T cells are activated by antigen-independent as well as antigen-dependent mechanisms during ischemia-reperfusion injury to the kidney. In the current issue, a study by Savransky et al. suggests that antigen-dependent activation of T-cell receptors further contributes to the pathogenesis of ischemia-reperfusion injury.Kidney International (2006) 69, 208–210. doi:10.1038/sj.ki.5000128 [ABSTRACT FROM AUTHOR]

Sphingosine-1-phosphate (S1P), produced by sphingosine kinase 1 (SphK1) or kinase 2 (SphK2), mediates biological effects through intracellular and/or extracellular mechanisms. Here we determined a role for these kinases in kidney injury of wild-type mice following ischemia–reperfusion. SphK1 but not SphK2 mRNA expression and activity increased in the kidney following injury relative to sham-operated animals. Although SphK1−/− mice had no alteration in renal function following injury, mice with a disrupted SphK2 gene (SphK2tr/tr) had histological damage and impaired function. The immune-modulating pro-drug, FTY720, an S1P agonist failed to provide protection in SphK2tr/tr mice. Injured kidneys of these mice showed increased neutrophil infiltration and neutrophil chemokine expression along with a 3- to 5-fold increase in expression of the G-protein-coupled receptor S1P3 compared to heterozygous SphK2+/tr mice. Kidney function and reduced vascular permeability were preserved in S1P3−/− compared to S1P3+/− mice after ischemia–reperfusion injury, suggesting increased S1P3 mRNA may play a role in the injury of SphK2tr/tr mice. Our study suggests that constitutive expression of SphK2 may contribute to reduced ischemia–reperfusion injury of the kidney, and its absence may enhance injury due to increased neutrophil infiltration and S1P3 activation. We also confirm that SphK2 is necessary to mediate the protective effects of FTY720.Kidney International (2009) 75, 167–175; doi:10.1038/ki.2008.400; published online 29 October 2008 [ABSTRACT FROM AUTHOR]