Role of the Circadian Clock and Effect of Time-Restricted Feeding in Adenine-Induced Chronic Kidney Disease.

Most physiological functions exhibit circadian rhythmicity that is partly regulated by the molecular circadian clock. Herein, we investigated the relationship between the circadian clock and chronic kidney disease (CKD). The role of the clock gene in adenine-induced CKD and the mechanisms of interaction were investigated in mice in which Bmal1, the master regulator of the clock gene, was knocked out, and Bmal1 knockout (KO) tubule cells. We also determined whether the renoprotective effect of time-restricted feeding (TRF), a dietary strategy to enhance circadian rhythm, is clock gene-dependent. The mice with CKD showed altered expression of the core clock genes with a loss of diurnal variations in renal functions and key tubular transporter gene expression. Bmal1 KO mice developed more severe fibrosis, and transcriptome profiling followed by gene ontology analysis suggested that genes associated with the cell cycle, inflammation, and fatty acid oxidation pathways were significantly affected in the mutant mice. Tubule-specific deletion of BMAL1 in HK-2 cells by CRISPR/Cas9 led to upregulation of p21 and tumor necrosis α and exacerbated epithelial-mesenchymal transition-related gene expression upon transforming growth factor ß stimulation. Finally, TRF in the mice with CKD partially restored the disrupted oscillation of the kidney clock genes, accompanied by improved cell cycle arrest and inflammation, leading to decreased fibrosis. However, the renoprotective effect of TRF was abolished in Bmal1 KO mice, suggesting that TRF is partially dependent on the clock gene. Our data demonstrate that the molecular clock system plays an important role in CKD via cell cycle regulation and inflammation. Understanding the role of the circadian clock in kidney diseases can be a new research field for developing novel therapeutic targets.

Intestinal barrier disruption and dysregulated mucosal immunity contribute to kidney fibrosis in chronic kidney disease.

BACKGROUND: Emerging evidence suggests that intestinal dysbiosis is associated with diverse pathological processes. In this study we demonstrated intestinal barrier disruption and aberrant mucosal immunity in 5/6 nephrectomized mice and the effect of probiotics on chronic kidney disease (CKD). METHODS: CKD was induced in 6-week-old mice by 5/6 nephrectomy. They were fed a lactobacilli mixture for 8 weeks. Serum, urine and stool samples were collected for renal function assessments and gut microbiome analyses. Gut permeability, colon heat shock protein 70 (HSP70) and colon epithelial integrity were evaluated and cytokine levels in colon and kidney were measured. Colon leukocytes were analyzed by flow cytometry and bone marrow-derived cells were cocultured with lactobacilli mixture. RESULTS: In CKD mice, 'leaky gut' was accompanied by decreased colon HSP70 and claudin-1 expression, whereas it increased pore-forming claudin-2 expression and apoptosis. Although the percentage of regulatory T cells did not differ between CKD and control mice, cytokine expression and the ratio of CX3CR1intermediate:CX3CR1high pro-inflammatory/resident macrophages increased in the colon of CKD mice. Orally administered lactobacilli partially mitigated the CKD-induced 'leaky gut'; restored colon epithelial HSP70, claudin-1 and claudin-2 expression and decreased apoptosis. Probiotic treatment also restored the CX3CR1intermediate:CX3CR1high macrophage ratio and increased circular dichroism (CD)103+CD11c+ regulatory dendritic cells in the colon. These changes suppressed systemic inflammation and kidney fibrosis. CONCLUSIONS: Our results suggest that intestinal dysbiosis-associated gut barrier disruption and aberrant mucosal immunity are important for the systemic inflammation and progressive fibrosis of CKD. Targeting the intestine might provide novel therapeutic opportunities for CKD.

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

AIM: 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. METHODS: We performed renal IRI in ADAMTS13 knockout (KO) or wild type (WT) mice. Functional and histological kidney damage, and inflammation were compared and the effect of anti-vWF antibodies in ADAMTS13 KO mice was assessed. RESULTS: Following IRI, the blood and kidney ADAMTS13 levels were significantly decreased. vWF expression was significantly upregulated in both the medulla and cortex of injured kidneys as shown by immunohistochemistry and western blot analyses. There was also an increased level of vWF dimers after IRI. In ADAMTS13 KO mice, kidney vWF levels were further increased and this was associated with greater endothelial and epithelial injury compared to WT mice, suggesting an important role of vWF in renal IRI. In addition, the number of Gr-1+ neutrophils was significantly higher in the kidneys of ADAMTS13 KO mice compared to WT mice, whereas F4/80 macrophage numbers were unchanged. In ADAMTS13 KO mice, administration of anti-vWF antibodies after IRI partially reversed renal injury. CONCLUSION: Our data show that the ADAMTS13-vWF axis is partially involved in the pathophysiology of kidney IRI, suggesting that regulating ADAMTS13- and vWF-dependent mechanisms could have therapeutic potential to limit renal IRI.

Fate of Neutrophils during the Recovery Phase of Ischemia/Reperfusion Induced Acute Kidney Injury.

Effective clearance of inflammatory cells is required for resolution of inflammation. Here, we show in vivo evidence that apoptosis and reverse transendothelial migration (rTEM) are important mechanisms in eliminating neutrophils and facilitating recovery following ischemia/reperfusion injury (IRI) of the kidney. The clearance of neutrophils was delayed in the Bax knockout (KO)(BM) → wild-type (WT) chimera in which bone marrow derived cells are partially resistant to apoptosis, compared to WT(BM) → WT mice. These mice also showed delayed functional, histological recovery, increased tissue cytokines, and accelerated fibrosis. The circulating intercellular adhesion molecule-1 (ICAM-1)⁺ Gr-1⁺ neutrophils displaying rTEM phenotype increased during the recovery phase and blockade of junctional adhesion molecule-C (JAM-C), a negative regulator of rTEM, resulted in an increase in circulating ICAM-1⁺ neutrophils, faster resolution of inflammation and recovery. The presence of Tamm-Horsfall protein (THP) in circulating ICAM-1⁺ neutrophils could suggest that they are derived from injured kidneys. In conclusion, we suggest that apoptosis and rTEM are critically involved in the clearance mechanisms of neutrophils during the recovery phase of 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.

Mechanisms of Piperacillin/Tazobactam Nephrotoxicity: Piperacillin/Tazobactam-Induced Direct Tubular Damage in Mice.

Piperacillin/tazobactam (PT) is one of the most commonly prescribed antibiotics for critically ill patients in intensive care. PT has been reported to cause direct nephrotoxicity; however, the underlying mechanisms remain unknown. We investigated the mechanisms underlying PT nephrotoxicity using a mouse model. The kidneys and sera were collected 24 h after PT injection. Serum blood urea nitrogen (BUN), creatinine, neutrophil gelatinase-associated lipocalin (NGAL), and renal pathologies, including inflammation, oxidative stress, mitochondrial damage, and apoptosis, were examined. Serum BUN, creatinine, and NGAL levels significantly increased in PT-treated mice. We observed increased IGFBP7, KIM-1, and NGAL expression in kidney tubules. Markers of oxidative stress, including 8-OHdG and superoxide dismutase, also showed a significant increase, accompanied by mitochondrial damage and apoptosis. The decrease in the acyl-coA oxidase 2 and Bcl2/Bax ratio also supports that PT induces mitochondrial injury. An in vitro study using HK-2 cells also demonstrated mitochondrial membrane potential loss, indicating that PT induces mitochondrial damage. PT appears to exert direct nephrotoxicity, which is associated with oxidative stress and mitochondrial damage in the kidney tubular cells. Given that PT alone or in combination with vancomycin is the most commonly prescribed antibiotic in patients at high risk of acute kidney injury, caution should be exercised.

Altered gut microbiome plays an important role in AKI to CKD transition in aged mice.

Introduction: This study investigated the role of renal-intestinal crosstalk in the transition from acute kidney injury (AKI) to chronic kidney disease (CKD) in elderly individuals. Methods: Using young and aged mice, we induced bilateral ischemia-reperfusion injury (IRI) and compared intestinal and kidney inflammation over 28 days. To determine the role of the microbiome in gut-kidney crosstalk, we analyzed the microbiome of fecal samples of the young vs. aged mice and examined the effects of probiotic supplementation. Results: In the post-IRI recovery phase, prolonged intestinal and renal inflammation along with dysbiosis were evident in aged vs. younger mice that was associated with severe renal dysfunction and fibrosis progression in aged mice. Probiotic supplementation with Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI alleviated intestinal inflammation but not intestinal leakage, characterized by decreased inflammatory cytokine levels and decreased infiltration of macrophages, neutrophils, and Th17 cells. This was associated with improved M1-dominant renal inflammation and ultimately improved renal function and fibrosis, suggesting that renal-intestinal crosstalk in aged mice contributes to the transition from AKI to CKD. Discussion: Our study findings suggest that exacerbation of chronic inflammation through the gut-kidney axis might be an important mechanism in the transition from AKI to CKD in the elderly.

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.

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.

The role of senescence of bone marrow cells in acute kidney injury.

BACKGROUND: The prevalence of acute kidney injury (AKI) in elderly patients has grown considerably. Age-associated changes in the immune system can be one of the critical factors determining AKI outcomes. This study aimed to investigate the role of senescence of bone marrow (BM)-derived cells in the development of AKI, focusing on the immune response. METHODS: Female 7-week-old C57BL/6 mice were irradiated and treated with BM cells from either 48-week-old or 8-week-old male mice. Ischemia-reperfusion injury (IRI) was induced, and their functional deterioration, histological tubular damage, and inflammatory responses were compared. For the in vitro study, lipopolysaccharide (LPS)-stimulated cytokine production by BM cells from old and young mice were examined. RESULTS: At 24 hours after IRI, there was no significant difference in the number of circulating immune cells between the mice transplanted with old or young BM cells. However, the mice with old BM cells showed less functional deterioration and histological tubular injury than those with young BM cells. Moreover, macrophage infiltration and renal cytokine interleukin (IL)-12 levels were lower in the mice with old BM cells at 24 hours post-IRI. Consistently, the in vitro study showed that LPS-induced production of cytokines interferon-γ, monocyte chemoattractant protein-1, and IL-10 was attenuated in cultured old BM cells, suggesting that age-related functional changes in these cells may lead to reduced inflammation in IRI. CONCLUSION: Immunosenescence could affect the susceptibility and response to renal IRI. Further studies specifically addressing age-related alterations can help in the development of treatment strategies for elderly patients with AKI.

Impact of aging on transition of acute kidney injury to chronic kidney disease.

Acute kidney injury (AKI) increases the risk of end stage renal disease among the elderly, but the precise underlying mechanism is unknown. We investigated the effects of aging on AKI-to-chronic kidney disease (CKD) transition, focusing on renal inflammation. Aged and young C57BL/6 mice were subjected to bilateral ischemia-reperfusion injury (IRI). Baseline proinflammatory cytokine levels of kidneys were elevated in aged mice. After IRI, aged mice also showed persistent M1 dominant inflammation, with increased proinflammatory cytokines during the recovery phase. Persistent M1 inflammation was associated with blunted activation of CSF-1/IRF4 signal for M1/M2 polarization, but in vitro macrophage polarization with cytokine stimulation was not different between young and aged mononuclear cells. The tubular expressions of cell cycle arrest markers increased in aged mice during recovery phase, and in vitro transwell experiments showed that mononuclear cells or M1 macrophages co-cultured with arrested proximal tubular cells at G1 phase significantly impaired M2 polarization, suggesting that prolonged G1 arrest might be involved in persistent M1 inflammation in aged mice. Finally, M1 dominant inflammation in aged mice resulted in fibrosis progression. Our data show that impaired M2 polarization partially driven by senescent tubule cells with cell-cycle arrest may lead to an accelerated progression to CKD in the elderly.

The Impact of Preexisting Chronic Kidney Disease on the Severity and Recovery of Acute Kidney Injury.

BACKGROUND: Recent observational studies have shown that in chronic kidney disease (CKD) patients, a significantly smaller percentage of patients with an episode of acute kidney injury (AKI) have full recovery of renal function compared to those without CKD. However, precise mechanisms involved in the incomplete repair after AKI with preexisting CKD have not been completely ascertained. Here, we assessed the impact of preexisting CKD on the severity and recovery of AKI in a mouse model of 5/6 nephrectomy. METHODS: Male CD-1 mice underwent 5/6 nephrectomy (Nx). Six weeks post surgery, ischemia reperfusion injury (IRI) or a sham operation was performed and functional, histological, and various molecular parameters were compared between them. RESULTS: Serum creatinine level on day 1 after IRI was comparable between control and Nx mice. However, serum creatinine remained significantly higher throughout the recovery phase in Nx mice compared to control mice. mRNA and protein expression of the cell cycle regulatory proteins were persistently elevated in Nx mice and this was associated with significantly increased levels of the G1 cell cycle arrest markers. Treatment with a p53 inhibitor following IRI resulted in not only decreased expression of G1 arrest markers but also decreased fibrosis, suggesting that prolonged epithelial G1 cell cycle arrest might be partially responsible for impaired recovery from superimposed AKI on CKD. CONCLUSION: Taken together, reduced nephron mass have a negative effect on the repair process that is partially mediated by the disruption of the cell cycle regulation.

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 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-ß. 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-ß 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-ß 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.

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.