Regulatory T cells in acute and chronic kidney diseases.

Foxp3-expressing CD4+ regulatory T cells (Tregs) make up one subset of the helper T cells (Th) and are one of the major mechanisms of peripheral tolerance. Tregs prevent abnormal activation of the immune system throughout the lifespan, thus protecting from autoimmune and inflammatory diseases. Recent studies have elucidated the role of Tregs beyond autoimmunity. Tregs play important functions in controlling not only innate and adaptive immune cell activation, but also regulate nonimmune cell function during insults and injury. Inflammation contributes to a multitude of acute and chronic diseases affecting the kidneys. This review examines the role of Tregs in pathogenesis of renal inflammatory diseases and explores the approaches for enhancing Tregs for prevention and therapy of renal inflammation.

IL233, A Novel IL-2 and IL-33 Hybrid Cytokine, Ameliorates Renal Injury.

CD4+Foxp3+ regulatory T cells (Tregs) protect the kidney during AKI. We previously found that IL-2, which is critical for Treg homeostasis, upregulates the IL-33 receptor (ST2) on CD4+ T cells, thus we hypothesized that IL-2 and IL-33 cooperate to enhance Treg function. We found that a major subset of Tregs in mice express ST2, and coinjection of IL-2 and IL-33 increased the number of Tregs in lymphoid organs and protected mice from ischemia-reperfusion injury (IRI) more efficiently than either cytokine alone. Accordingly, we generated a novel hybrid cytokine (IL233) bearing the activities of IL-2 and IL-33 for efficient targeting to Tregs. IL233 treatment increased the number of Tregs in blood and spleen and prevented IRI more efficiently than a mixture of IL-2 and IL-33. Injection of IL233 also increased the numbers of Tregs in renal compartments. Moreover, IL233-treated mice had fewer splenic Tregs and more Tregs in kidneys after IRI. In vitro, splenic Tregs from IL233-treated mice suppressed CD4+ T cell proliferation better than Tregs from saline-treated controls. IL233 treatment also improved the ability of isolated Tregs to inhibit IRI in adoptive transfer experiments and protected mice from cisplatin- and doxorubicin-induced nephrotoxic injury. Finally, treatment with IL233 increased the proportion of ST2-bearing innate lymphoid cells (ILC2) in blood and kidneys, and adoptive transfer of ILC2 also protected mice from IRI. Thus, the novel IL233 hybrid cytokine, which utilizes the cooperation of IL-2 and IL-33 to enhance Treg- and ILC2-mediated protection from AKI, bears strong therapeutic potential.

The spleen as a bidirectional signal transducer in acute kidney injury.

In acute kidney injury models, the lung is damaged through an interleukin-6-dependent inflammatory response. Clinically, development of lung injury requiring mechanical ventilation markedly increases in-hospital acute kidney injury mortality. Andres-Hernando et al. demonstrate that the spleen coordinates interleukin-6-dependent interleukin-10 production, which lessens lung injury during experimental acute kidney injury. This study highlights the beneficial influence of the spleen on the lung, and dovetails with other recent publications, to demonstrate the integral role of the spleen in acute kidney injury.

The Influence of Hemodialysis on T Regulatory Cells: A Meta-Analysis and Systematic Review.

AIMS: The study aimed to determine whether the available literature supports a positive or negative influence of dialysis on regulatory T-cells (Tregs). METHODS: We performed a systematic search and a meta-analysis. Mean differences in Tregs number of chronic kidney disease stages G5 on dialysis patients (CKD G5D) and healthy controls (HCs) were compared. Random effects model was applied. The software used was general package for meta-analysis (version 4.3-0, depends R (≥2.9.1)). RESULTS: Five studies were included in the meta-analysis. The mean difference in percentage of Tregs on CD4+ T-cells between CKD G5D and HCs was not statistically different. Moreover, CKD GFR stages G5 not on dialysis (CKD G5) versus HC (p = 0.002; mean difference in Treg percentage was -2.47% in CKD G5 vs. HC) and CKD G5 versus CKD G5D (not significant). CONCLUSION: This meta-analysis demonstrates an association between the uremic state and lower Tregs, and supports the hypothesis that hemodialysis alter Tregs. Our findings highlight the need for new clinical studies. Video Journal Club 'Cappuccino with Claudio Ronco' at http://www.karger.com/?doi=449242.

Natural IgM Switches the Function of Lipopolysaccharide-Activated Murine Bone Marrow-Derived Dendritic Cells to a Regulatory Dendritic Cell That Suppresses Innate Inflammation.

We have previously shown that polyclonal natural IgM protects mice from renal ischemia/reperfusion injury (IRI) by inhibiting the reperfusion inflammatory response. We hypothesized that a potential mechanism involved IgM modulation of dendritic cells (DC), as we observed high IgM binding to splenic DC. To test this hypothesis, we pretreated bone marrow-derived DC (BMDC) with polyclonal murine or human IgM prior to LPS activation and demonstrated that 0.5 × 10(6) IgM/LPS-pretreated BMDC, when injected into wild-type C57BL/6 mice 24 h before renal ischemia, protect mice from developing renal IRI. We show that this switching of LPS-activated BMDC to a regulatory phenotype requires modulation of BMDC function that is mediated by IgM binding to nonapoptotic BMDC receptors. Regulatory BMDC require IL-10 and programmed death 1 as well as downregulation of CD40 and p65 NF-κB phosphorylation to protect in renal IRI. Blocking the programmed death ligand 1 binding site just before i.v. injection of IgM/LPS-pretreated BMDC or using IL-10 knockout BMDC fails to induce protection. Similarly, IgM/LPS-pretreated BMDC are rendered nonprotective by increasing CD40 expression and phosphorylation of p65 NF-κB. How IgM/LPS regulatory BMDC suppress in vivo ischemia-induced innate inflammation remains to be determined. However, we show that suppression is dependent on other in vivo regulatory mechanisms in the host, that is, CD25(+) T cells, B cells, IL-10, and circulating IgM. There was no increase in Foxp3(+) regulatory T cells in the spleen either before or after renal IRI. Collectively, these findings show that natural IgM anti-leukocyte Abs can switch BMDC to a regulatory phenotype despite the presence of LPS that ordinarily induces BMDC maturation.

Both PD-1 ligands protect the kidney from ischemia reperfusion injury.

Acute kidney injury (AKI) is a common problem in hospitalized patients that enhances morbidity and mortality and promotes the development of chronic and end-stage renal disease. Ischemia reperfusion injury (IRI) is one of the major causes of AKI and is characterized by uncontrolled renal inflammation and tubular epithelial cell death. Our recent studies demonstrated that regulatory T cells (Tregs) protect the kidney from ischemia reperfusion-induced inflammation and injury. Blockade of programmed death-1 (PD-1) on the surface of Tregs, prior to adoptive transfer, negates their ability to protect against ischemic kidney injury. The present study was designed to investigate the role of the known PD-1 ligands, PD-L1 and PD-L2, in kidney IRI. Administration of PD-L1 or PD-L2 blocking Abs prior to mild or moderate kidney IRI significantly exacerbated the loss of renal function, renal inflammation, and acute tubular necrosis compared with mice receiving isotype control Abs. Interestingly, blockade of both PD-1 ligands resulted in worse injury, dysfunction, and inflammation than did blocking either ligand alone. Genetic deficiency of either PD-1 ligand also exacerbated kidney dysfunction and acute tubular necrosis after subthreshold ischemia. Bone marrow chimeric studies revealed that PD-L1 expressed on non-bone marrow-derived cells is critical for this resistance to IRI. Finally, blockade of either PD-1 ligand negated the protective ability of adoptively transferred Tregs in IRI. These findings suggest that PD-L1 and PD-L2 are nonredundant aspects of the natural protective response to ischemic injury and may be novel therapeutic targets for AKI.

Sphingosine 1-phosphate receptor-1 enhances mitochondrial function and reduces cisplatin-induced tubule injury.

Sphingosine 1-phosphate (S1P), the natural sphingolipid ligand for a family of five G protein- coupled receptors (S1P1-S1P5Rs), regulates cell survival and lymphocyte circulation. We have shown that the pan-S1PR agonist, FTY720, attenuates kidney ischemia-reperfusion injury by directly activating S1P1 on proximal tubule (PT) cells, independent of the canonical lymphopenic effects of S1P1 activation on B and T cells. FTY720 also reduces cisplatin-induced AKI. Therefore, in this study, we used conditional PT-S1P1-null (PepckCreS1pr1(fl/fl)) and control (PepckCreS1pr1(w/wt)) mice to determine whether the protective effect of FTY720 in AKI is mediated by PT-S1P1. Cisplatin induced more renal injury in PT-S1P1-null mice than in controls. Although FTY720 produced lymphopenia in both control and PT-S1P1-null mice, it reduced injury only in control mice. Furthermore, the increase in proinflammatory cytokine (CXCL1, MCP-1, TNF-α, and IL-6) expression and infiltration of neutrophils and macrophages induced by cisplatin treatment was attenuated by FTY720 in control mice but not in PT-S1P1-null mice. Similarly, S1P1 deletion rendered cultured PT cells more susceptible to cisplatin-induced injury, whereas S1P1 overexpression protected PT cells from injury and preserved mitochondrial function. We conclude that S1P1 may have an important role in stabilizing mitochondrial function and that FTY720 administration represents a novel strategy in the prevention of cisplatin-induced AKI.

Expanding role of T cells in acute kidney injury.

PURPOSE OF REVIEW: Recent advances in T cell biology have shed light on the role of T cell subsets in the pathogenesis of acute kidney injury (AKI). The purpose of this review is to harness our understanding of recent advances in T cell biology in tissue injury and repair and provide a mechanistic insight into the role of T cells in the inflammation of AKI. RECENT FINDINGS: New specific reagents and genetic animal models have led to advances in our understanding of the role of T cell subsets involved in renal injury. Whereas some T cells promote innate renal inflammation and injury, other T cells promote protection and repair. Recent studies illuminated the pathogenic mechanisms of invariant natural killer T (NKT) cells and T helper1-type responses, and the beneficial functions of regulatory T cells and NKT cells are just beginning to be explored. Pharmacologic and cell-based therapies that influence T cell responses to experimental AKI suggest that this is a promising approach to preserve renal function. SUMMARY: The recent insights gained into how T cells modulate renal injury suggest that strategies targeting specific types of T cells, to either inhibit or enhance their activity, may ameliorate renal injury in patients.

Regulatory T cells in AKI.

Human AKI is manifested by inflammation, and an early feature in the pathogenesis is the accumulation of immune cells in the kidney. To understand the pathophysiology of AKI, results from animal models have shown a causal relation between the leukocyte activation and infiltration to the kidney after kidney ischemia-reperfusion. Blocking the activation or trafficking of proinflammatory leukocytes into the kidney preserves renal function and histologic integrity. In contrast, the anti-inflammatory lymphocytes called regulatory T cells have an intrinsic renal-protective function and may represent a novel therapeutic approach and/or target for pharmacological manipulation to ameliorate AKI. This review will highlight the recent insight gained into the role and mechanisms of regulatory T cells in AKI.

Dendritic cells tolerized with adenosine A2AR agonist attenuate acute kidney injury.

DC-mediated NKT cell activation is critical in initiating the immune response following kidney ischemia/reperfusion injury (IRI), which mimics human acute kidney injury (AKI). Adenosine is an important antiinflammatory molecule in tissue inflammation, and adenosine 2A receptor (A2AR) agonists protect kidneys from IRI through their actions on leukocytes. In this study, we showed that mice with A2AR-deficient DCs are more susceptible to kidney IRI and are not protected from injury by A2AR agonists. In addition, administration of DCs treated ex vivo with an A2AR agonist protected the kidneys of WT mice from IRI by suppressing NKT production of IFN-γ and by regulating DC costimulatory molecules that are important for NKT cell activation. A2AR agonists had no effect on DC antigen presentation or on Tregs. We conclude that ex vivo A2AR-induced tolerized DCs suppress NKT cell activation in vivo and provide a unique and potent cell-based strategy to attenuate organ IRI.

Autocrine adenosine signaling promotes regulatory T cell-mediated renal protection.

Regulatory T cells (Tregs) suppress the innate inflammation associated with kidney ischemia-reperfusion injury (IRI), but the mechanism is not well understood. Tregs express CD73, the final enzyme involved in the production of extracellular adenosine, and activation of the adenosine 2A receptor (A(2A)R) on immune cells suppresses inflammation and preserves kidney function after IRI. We hypothesized that Treg-generated adenosine is required to block innate immune responses in kidney IRI and that the Treg-generated adenosine would signal through A(2A)Rs on inflammatory cells and, in an autocrine manner, on Tregs themselves. We found that adoptively transferred wild-type Tregs protected wild-type mice from kidney IRI, but the absence of adenosine generation (CD73-deficient Tregs) or adenosine responsiveness (A(2A)R-deficient Tregs) led to inhibition of Treg function. Pharmacologic stimulation of A(2A)R before adoptive transfer augmented the ability of wild-type and CD73-deficient Tregs to suppress kidney IRI. Microarray analysis and flow cytometry revealed that A(2A)R activation enhanced surface PD-1 expression on Tregs in the absence of any other activation signal. Treatment of Tregs with a PD-1 blocking antibody before adoptive transfer reversed their protective effects, even if pretreated with an A(2A)R agonist. Taken together, these results demonstrate that the simultaneous ability to generate and respond to adenosine is required for Tregs to suppress innate immune responses in IRI through a PD-1-dependent mechanism.

Monocyte/macrophage chemokine receptor CCR2 mediates diabetic renal injury.

Monocyte/macrophage recruitment correlates strongly with the progression of renal impairment in diabetic nephropathy (DN). C-C chemokine receptor (CCR)2 regulates monocyte/macrophage migration into injured tissues. However, the direct role of CCR2-mediated monocyte/macrophage recruitment in diabetic kidney disease remains unclear. We report that pharmacological blockade or genetic deficiency of CCR2 confers kidney protection in Ins2(Akita) and streptozotocin (STZ)-induced diabetic kidney disease. Blocking CCR2 using the selective CCR2 antagonist RS504393 for 12 wk in Ins2(Akita) mice significantly attenuated albuminuria, the increase in blood urea nitrogen and plasma creatinine, histological changes, and glomerular macrophage recruitment compared with vehicle. Furthermore, mice lacking CCR2 (CCR2(-/-)) mimicked CCR2 blockade by reducing albuminuria and displaying less fibronectin mRNA expression and inflammatory cytokine production compared with CCR2(+/+) mice, despite comparable blood glucose levels. Bone marrow-derived monocytes from CCR2(+/+) or CCR2(-/-) mice adoptively transferred into CCR2(-/-) mice reversed the renal tissue-protective effect in diabetic CCR2(-/-) mice as evaluated by increased urinary albumin excretion and kidney macrophage recruitment, indicating that CCR2 is not required for monocyte migration from the circulation into diabetic kidneys. These findings provide evidence that CCR2 is necessary for monocyte/macrophage-induced diabetic renal injury and suggest that blocking CCR2 could be a novel therapeutic approach in the treatment of DN.

Pathogenesis of acute kidney injury: foundation for clinical practice.

The pathogenesis of acute kidney injury (AKI) is complex, involving such factors as vasoconstriction, leukostasis, vascular congestion, cell death, and abnormal immune modulators and growth factors. Many targeted clinical therapies have failed, are inconclusive, or have yet to be tested. Given the complexity of the pathogenesis of AKI, it may be naive to expect that one therapeutic intervention would have success. Some examples of detrimental processes that can be blocked in preclinical models to improve kidney function and survival are apoptotic cell death in tubular epithelial cells, complement-mediated immune system activation, and impairment of cellular homeostasis and metabolism. Modalities with the potential to decrease morbidity and mortality in patients with AKI include vasodilators, growth factors, anti-inflammatory agents, and cell-based therapies. Pharmacologic agents that target these diverse pathways are being used clinically for other indications. Using combinatorial approaches in future clinical trials may improve our ability to prevent and treat AKI.

Profiling of fatty acids released during calcium-induced mitochondrial permeability transition in isolated rabbit kidney cortex mitochondria.

Increases in intracellular Ca(2+) during cellular stress often lead to the mitochondrial permeability transition (MPT). We examined changes in fatty acids (FAs) released from isolated renal cortical mitochondria subjected to Ca(2+)-induced MPT. Exposing mitochondria to Ca(2+) stimulated mitochondrial swelling and release of FAs such as arachidonic (20:4) and docosahexenoic acids which increased 71% and 32%, respectively, and linoleic (18:2) which decreased 23% compared to controls. Stearic (18:0), oleic (18:1), and linoleic (18:3) acids were unchanged. To elucidate a mechanism for FA release, mitochondria were pre-treated with bromoenolactone (BEL) to inhibit Ca(2+)-independent phospholipase A(2) gamma activity (iPLA(2)γ). BEL blocked Ca(2+)-induced release of arachidonic and behenic (22:0) acids. Finally, four FAs were released in the absence of Ca(2+) in a BEL-sensitive manner, including arachidonic and docosatrienoic acids. Thus, extensive FA release occurs during Ca(2+)-induced MPT, and that mitochondrial iPLA(2)γ maintains mitochondrial arachidonic acid homeostasis under both basal and Ca(2+)-induced stress conditions.

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

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.

Immune mechanisms and novel pharmacological therapies of acute kidney injury.

Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) and both innate and adaptive immunity contribute to the pathogenesis. Kidney resident cells promote inflammation after IRI by increasing endothelial cell adhesion molecule expression and vascular permeability. Kidney epithelial cells bind complement and express toll-like receptors and resident and infiltrating cells produce cytokines/chemokines. Early activation of kidney dendritic cells (DCs) initiates a cascade of events leading to accumulation of interferon-gamma-producing neutrophils, infiltrating macrophages, CD4(+) T cells, B cells and invariant natural killer T (NKT) cells. Recent studies from our laboratory now implicate the IL23/IL17 pathway in kidney IRI. Following the initial early phase of inflammation, the late phase involves infiltration of anti-inflammatory cells including regulatory T cells, alternatively activated macrophages and stem cells leading to attenuation of inflammation and initiation of repair. Based upon these immune mechanisms of injury, recent studies hold promise for novel drug therapies. These pharmacological agents have been shown to reduce inflammation or cytotoxicity in rodent models of AKI and some show early promise in clinical trials. This review summarizes recent advances to further our understanding of the immune mechanisms of AKI and potential pharmacological therapies.

Regulatory T cells suppress innate immunity in kidney ischemia-reperfusion injury.

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.