Hee Young Lee

Depletion of kidney CD11c+ F4/80+ cells impairs the recovery process in ischaemia/reperfusion-induced acute kidney injury

BACKGROUND Recent studies provided evidence of the potential role of CD11c(+) F4/80(+) dendritic subset in mediating injury and repair. The purpose of this study was to examine the role of kidney CD11c(+) F4/80(+) dendritic subset in the recovery phase of ischaemia/reperfusion injury (IRI). METHODS Following ischaemia/reperfusion (I/R), liposome clodronate or phosphate buffered saline (PBS) was administered, and on day 7 biochemical and histologic kidney damage was assessed. Activation and depletion of CD11c(+) F4/80(+) dendritic subset were confirmed by flow cytometry. Isolation of kidney CD11c(+) cells on days 1 and 7 with in vitro culture for measuring cytokines was performed to define functional characteristics of these cells, and adoptive transfer of CD11c(+) cells was also done. RESULTS Following kidney IRI, the percentage of CD11c(+) F4/80(+) kidney dendritic cell subset that co-expresses maturation marker increased. Liposome clodronate injection after I/R resulted in preferential depletion of CD11c(+) F4/80(+) kidney dendritic subset, and depletion of these cells was associated with persistent kidney injury, more apoptosis, inflammation and impaired tubular cell proliferation. CD11c(+) F4/80(+) cell depletion was also associated with higher tissue levels of pro-inflammatory cytokines and lower level of IL-10, indicating the persistence of inflammatory milieu. Isolated kidney CD11c(+) cells on day 7 showed different phenotype with increased production of IL-10 compared with those on day 1. Adoptive transfer of CD11c(+) cells partially reversed impaired tissue recovery. CONCLUSION Our results suggest that kidney CD11c(+) F4/80(+) dendritic subset might contribute to the recovery process by dynamic phenotypic change from pro-inflammatory to anti-inflammatory with modulation of immune response.

Distinct pathophysiologic mechanisms of septic acute kidney injury: role of immune suppression and renal tubular cell apoptosis in murine model of septic acute kidney injury.

Objective:Sepsis is the most common cause of acute kidney injury in critically ill patients; however, the mechanisms leading to acute kidney injury in sepsis remain elusive. Although sepsis has been considered an excessive systemic inflammatory response, clinical trials that inhibit inflammation have been shown to have no effect. The purpose of this study was to examine the pathophysiology of septic acute kidney injury focusing on immune responses and renal tubular cell apoptosis by providing an on-site quantitative comparison between septic- and ischemia/reperfusion-induced acute kidney injury. Design:Twenty-four hours after cecal ligation and puncture or ischemia/reperfusion injury, biochemical, histologic, and cytokine changes were compared in C57BL/6 mice. Apoptosis was assessed, and the effect of caspase 3 inhibition on renal function was also examined. The percentage of regulatory T cells and the effect of depletion were determined and compared with ischemia/reperfusion-induced acute kidney injury. The effect of interleukin-10 blocking was also compared. Measurements and Main Results:Despite comparable renal dysfunction, acute tubular necrosis or inflammation was minimal in septic kidneys. However, tubular cell apoptosis was prominent, and caspase 3 activity was positively correlated with renal dysfunction. A decrease in apoptosis by caspase 3 inhibitor resulted in attenuation of renal dysfunction. In assessment of systemic immunity, septic acute kidney injury was associated with an increase in interleukin-10, and also showed massive immune cell apoptosis with increased regulatory T cells. In contrast to ischemia/reperfusion injury in which depletion of regulatory T cells aggravated renal injury, depletion of regulatory T cells before cecal ligation and puncture resulted in renoprotection. In addition, blocking interleukin-10 rescued septic mice from the development of acute kidney injury, whereas it had no effect in ischemia/reperfusion injury. Conclusions:Pathogenesis of septic acute kidney injury is thought to be different from that of ischemia/reperfusion-induced acute kidney injury. Our data showed a link between apoptosis, immune suppression, and the development of acute kidney injury during sepsis and suggest that strategies targeting apoptosis or enhancing immunity might be a potential therapeutic strategy for septic acute kidney injury.

CD4 + CD25 + regulatory T cells partially mediate the beneficial effects of FTY720, a sphingosine-1-phosphate analogue, during ischaemia/reperfusion-induced acute kidney injury

BACKGROUND The synthetic sphingosine-1-phosphate (S1P) analogue, FTY720, attenuates ischaemia/reperfusion (I/R) injury by inducing peripheral lymphopaenia. Recent studies suggest that FTY720 may also exert protective effects by modulating dendritic cell (DC) function or directly affecting regulatory T cells (Tregs). The purpose of the present study was to examine whether the beneficial effect of FTY720 in I/R-induced acute kidney injury (AKI) involves modulation of DCs or Tregs. METHODS Mice underwent bilateral ischaemia, and FTY720 or vehicle was then administered. Biochemical values, histological kidney damage and tissue inflammation were assessed. Phenotype and function of DCs in blood/spleen or kidney were also examined by flow cytometry or mixed lymphocyte reaction (MLR) assay. Percent Tregs or FoxP3 mRNA expression was examined in kidney and spleen, and depletion and adoptive transfer of Tregs were also performed. RESULTS Treatment with FTY720 attenuated I/R kidney injury and reduced inflammation. The beneficial effect of FTY720 was associated with expansion of peripheral CD11b( +) CD11c( +) DC and with maturation of spleen CD11c( +) DC, which showed impaired allostimulatory capacity. FTY720-treated animals also showed a higher frequency of CD4( +) CD25( +) Tregs and an upregulation of FoxP3 mRNA expression in spleen and kidney. In vitro experiments showed that FTY720 induced expansion of Tregs, possibly via conversion from non-Tregs to Tregs. Depletion and adoptive transfer of Tregs were associated with loss and recovery of the beneficial effects of FTY720. CONCLUSION These results suggest that the beneficial effects of FTY720 in I/R injury may be partially mediated by DC modulation or by increasing Treg activity. Further studies that identify tolerance induction mechanisms will be useful for developing strategies for the prevention or treatment of AKI.

The heat-shock protein-70–induced renoprotective effect is partially mediated by CD4+CD25+Foxp3+ regulatory T cells in ischemia/reperfusion-induced acute kidney injury

Recent reports suggest the presence of heat-shock protein (HSP)-reactive T cells with a regulatory phenotype in various inflammatory diseases. To test whether HSP exerts renoprotective effects through regulatory T cells (Tregs), ischemia/reperfusion injury was done with or without heat preconditioning in mice. Splenocytes from heat-preconditioned mice had Treg expansion and a reduced proliferative response upon mitogenic stimulus. T cells from heat-preconditioned mice failed to reconstitute postischemic injury when adoptively transferred to T cell-deficient nu/nu mice in contrast to those from control mice. Tregs were also increased in heat-preconditioned ischemic kidneys. Depleting Tregs before heat preconditioning abolished the renoprotective effect, while adoptive transfer of these cells back into Treg-depleted mice partially restored the beneficial effect of heat preconditioning. Inhibition of HSP70 by quercetin suppressed Treg expansion, as well as renoprotective effects. Transferring Tregs in quercetin-treated heat-preconditioned mice partially restored the beneficial effect of heat preconditioning. The specificity of immune cell HSP70 in renoprotection was confirmed by partial restoration of kidney injury when T cells from HSP70-deficient heat preconditioned mice were adoptively transferred to nu/nu mice. Thus, the renoprotective effect of HSP70 may be partially mediated by a direct immunomodulatory effect through Tregs. Better understanding of immunomodulatory mechanisms of various stress proteins might facilitate discovery of new preventive strategies in acute kidney injury.

The Role of M2 Macrophages in the Progression of Chronic Kidney Disease following Acute Kidney Injury

Introduction Acute kidney injury (AKI) is a major risk factor in the development of chronic kidney disease (CKD). However, the mechanisms linking AKI to CKD remain unclear. We examined the alteration of macrophage phenotypes during an extended recovery period following ischemia/reperfusion injury (IRI) and determine their roles in the development of fibrosis. Methods The left renal pedicle of mice was clamped for 40 min. To deplete monocyte/macrophage, liposome clodronate was injected or CD11b-DTR and CD11c-DTR transgenic mice were used. Results Throughout the phase of IRI recovery, M2-phenotype macrophages made up the predominant macrophage subset. On day 28, renal fibrosis was clearly shown with increased type IV collagen and TGF-β. The depletion of macrophages induced by the liposome clodronate injection improved renal fibrosis with a reduction of kidney IL-6, type IV collagen, and TGF-β levels. Additionally, the adoptive transfer of the M2c macrophages partially reversed the beneficial effect of macrophage depletion, whereas the adoptive transfer of the M1 macrophages did not. M2 macrophages isolated from the kidneys during the recovery phase expressed 2.5 fold higher levels of TGF-β than the M1 macrophages. The injection of the diphtheria toxin into CD11b or CD11c-DTR transgenic mice resulted in lesser depletion or no change in M2 macrophages and had little impact on renal fibrosis. Conclusion Although M2 macrophages are known to be indispensible for short-term recovery, they are thought to be main culprit in the development of renal fibrosis following IRI.

Renoprotective effect of paricalcitol via a modulation of the TLR4-NF-κB pathway in ischemia/reperfusion-induced acute kidney injury.

BACKGROUND The pathophysiology of ischemic acute kidney injury (AKI) is thought to include a complex interplay between vascular endothelial cell dysfunction, inflammation, and tubular cell damage. Several lines of evidence suggest a potential anti-inflammatory effect of vitamin D in various kidney injury models. In this study, we investigated the effect of paricalcitol, a synthetic vitamin D analog, on renal inflammation in a mouse model of ischemia/reperfusion (I/R) induced acute kidney injury (AKI). METHODS Paricalcitol was administered via intraperitoneal (IP) injection at 24h before ischemia, and then I/R was performed through bilateral clamping of the renal pedicles. Twenty-four hours after I/R, mice were sacrificed for the evaluation of injury and inflammation. Additionally, an in vitro experiment using HK-2 cells was also performed to examine the direct effect of paricalcitol on tubular cells. RESULTS Pre-treatment with paricalcitol attenuated functional deterioration and histological damage in I/R induced AKI, and significantly decreased tissue neutrophil and macrophage infiltration and the levels of chemokines, the pro-inflammatory cytokine interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). It also decreased IR-induced upregulation of Toll-like receptor 4 (TLR4), and nuclear translocation of p65 subunit of NF-κB. Results from the in vitro study showed pre-treatment with paricalcitol suppressed the TNF-α-induced depletion of cytosolic IκB in HK-2 cells. CONCLUSION These results demonstrate that pre-treatment with paricalcitol has a renoprotective effect in ischemic AKI, possibly by suppressing TLR4-NF-κB mediated inflammation.

Role of inflammation in the pathogenesis of cardiorenal syndrome in a rat myocardial infarction model

BACKGROUND Cardiorenal syndrome is now frequently recognized, and the combined dysfunction of heart and kidney increases morbidity and mortality. This study aimed to investigate possible mechanisms that underlie renal damage following heart dysfunction using a rat myocardial infarction model, focusing on the inflammatory pathway. METHODS Rats were randomized into four groups: normal, volume depletion, sham operation and myocardial infarction (MI). MI was induced by the ligation of the left coronary artery and a volume depletion model was produced by low-salt diet and furosemide injection. Biochemical, histological and flow cytometric analyses were performed at 3 days and 4 and 8 weeks after MI. RESULTS On Day 3 following MI, the development of subclinical acute kidney injury was identified through significantly increased serum and urine neutrophil gelatinase-associated lipocalin level. We detected the increase of activated monocytes (CC chemokine receptor 2(+) ED-1(+)) in peripheral blood, along with the infiltration of ED-1(+) macrophages and the increment of nuclear p65 in the kidney of MI rats, suggesting the contribution of nuclear factor-kappa B-mediated inflammation in the development of Type 1 cardiorenal syndrome (CRS). The inflammatory cytokines, interleukin-6 and tumour necrosis factor-α (TNF-α) mRNA expression, as well as microvascular endothelial permeability and tubular cell apoptosis, significantly increased in the kidneys of MI rats. At 4 and 8 weeks after MI, tubular cell apoptosis, ED-1(+) macrophage infiltration and interstitial fibrosis increased in MI rats, and these chronic changes were significantly mitigated by systemic monocyte/macrophage depletion using liposome clodronate. CONCLUSION This study identifies the possible important role of inflammatory response as a mediator of heart-kidney crosstalk in CRS.

CD11c + Cells Partially Mediate the Renoprotective Effect Induced by Bone Marrow-Derived Mesenchymal Stem Cells

Previous studies have shown that induction of immune tolerance by mesenchymal stem cells (MSCs) is partially mediated via monocytes or dendritic cells (DCs). The purpose of this study was to determine the role of CD11c+ cells in MSC-induced effects on ischemia/reperfusion injury (IRI). IRI was induced in wildtype (WT) mice and CD11c+-depleted mice following pretreatment with or without MSCs. In the in-vitro experiments, the MSC-treated CD11c+ cells acquired regulatory phenotype with increased intracellular IL-10 production. Although splenocytes cocultured with MSCs showed reduced T cell proliferation and expansion of CD4+FoxP3+ regulatory T cells (Tregs), depletion of CD11c+ cells was associated with partial loss of MSCs effect on T cells. In in-vivo experiment, MSCs’ renoprotective effect was also associated with induction of more immature CD11c+ cells and increased FoxP3 expression in I/R kidneys. However all these effects induced by the MSCs were partially abrogated when CD11c+ cells were depleted in the CD11c+-DTR transgenic mice. In addition, the observation that adoptive transfer of WT CD11c+ cells partially restored the beneficial effect of the MSCs, while transferring IL-10 deficient CD11c+ cells did not, strongly suggest the important contribution of IL-10 producing CD11c+ cells in attenuating kidney injury by MSCs. Our results suggest that the CD11c+ cell-Tregs play critical role in mediating renoprotective effect of MSCs.

Blocking junctional adhesion molecule C promotes the recovery of cisplatin-induced acute kidney injury

Background/Aims Recent findings have demonstrated the occurrence of neutrophil transendothelial migration in the reverse direction (reverse TEM) and that endothelial junctional adhesion molecule C (JAM-C) is a negative regulator of reverse TEM. In this study, we tested the effects of a JAM-C blocking antibody on the resolution of kidney injuries and inflammation in a mouse model of cisplatin-induced acute kidney injury (AKI). Methods Cisplatin was administered via intraperitoneal injection. A JAM-C blocking antibody or a control immunoglobulin G was administered intraperitoneal at 1.5 mg/kg, with the injection being delayed until day 4 following cisplatin administration to restrict the effect of antibodies on recovery. Results After cisplatin injection, serum creatinine and histologic injuries peaked on day 4. Treatment with a JAM-C blocking antibody on days 4 and 5 promoted the functional and histologic recovery of cisplatin-induced AKI on days 5 and 6. Facilitating recovery with a JAM-C blocking antibody correlated with significantly increased circulating intercellular adhesion molecule 1+ Tamm-Horsfall protein+ neutrophils and significantly decreased renal neutrophil infiltration, indicating that facilitating reverse the TEM of neutrophils from the kidney to the peripheral circulation partially mediated the resolution of inflammation and recovery. Conclusions These results demonstrated that reverse TEM is involved in the resolution of neutrophilic inflammation in cisplatin-induced AKI and that JAM-C is an important regulator of this process.

The ADAMTS13-von Willebrand factor axis is involved in the pathophysiology of kidney ischemia-reperfusion injury.

The ADAMTS13‐von Willebrand factor (vWF) axis has been suggested to play a critical role in the pathophysiology of ischaemia‐reperfusion injury (IRI) in the heart or brain. Therefore, we aimed to investigate whether this axis was involved in the pathophysiology of IRI‐induced acute kidney injury.