ABSTRACT
We describe a human lung disease caused by autosomal recessive, complete deficiency of the monocyte chemokine receptor C-C motif chemokine receptor 2 (CCR2). Nine children from five independent kindreds have pulmonary alveolar proteinosis (PAP), progressive polycystic lung disease, and recurrent infections, including bacillus Calmette Guérin (BCG) disease. The CCR2 variants are homozygous in six patients and compound heterozygous in three, and all are loss-of-expression and loss-of-function. They abolish CCR2-agonist chemokine C-C motif ligand 2 (CCL-2)-stimulated Ca2+ signaling in and migration of monocytic cells. All patients have high blood CCL-2 levels, providing a diagnostic test for screening children with unexplained lung or mycobacterial disease. Blood myeloid and lymphoid subsets and interferon (IFN)-γ- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated immunity are unaffected. CCR2-deficient monocytes and alveolar macrophage-like cells have normal gene expression profiles and functions. By contrast, alveolar macrophage counts are about half. Human complete CCR2 deficiency is a genetic etiology of PAP, polycystic lung disease, and recurrent infections caused by impaired CCL2-dependent monocyte migration to the lungs and infected tissues.
Subject(s)
Pulmonary Alveolar Proteinosis , Receptors, CCR2 , Child , Humans , Lung/metabolism , Macrophages, Alveolar/metabolism , Pulmonary Alveolar Proteinosis/genetics , Pulmonary Alveolar Proteinosis/diagnosis , Receptors, CCR2/deficiency , Receptors, CCR2/genetics , Receptors, CCR2/metabolism , Reinfection/metabolismABSTRACT
Acute kidney injury (AKI) frequently complicates major surgery and can be associated with hypertension and progress to chronic kidney disease, but reports on blood pressure normalization in AKI are conflicting. In the present study, we investigated the effects of an angiotensin-converting enzyme inhibitor, enalapril, and a soluble epoxide hydrolase inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea (TPPU), on renal inflammation, fibrosis, and glomerulosclerosis in a mouse model of ischemia-reperfusion injury (IRI)-induced AKI. Male CD1 mice underwent unilateral IRI for 35 min. Blood pressure was measured by tail cuff, and mesangial matrix expansion was quantified on methenamine silver-stained sections. Renal perfusion was assessed by functional MRI in vehicle- and TPPU-treated mice. Immunohistochemistry was performed to study the severity of AKI and inflammation. Leukocyte subsets were analyzed by flow cytometry, and proinflammatory cytokines were analyzed by quantitative PCR. Plasma and tissue levels of TPPU and lipid mediators were analyzed by liquid chromatography mass spectrometry. IRI resulted in a blood pressure increase of 20 mmHg in the vehicle-treated group. TPPU and enalapril normalized blood pressure and reduced mesangial matrix expansion. However, inflammation and progressive renal fibrosis were severe in all groups. TPPU further reduced renal perfusion on days 1 and 14. In conclusion, early antihypertensive treatment worsened renal outcome after AKI by further reducing renal perfusion despite reduced glomerulosclerosis.
Subject(s)
Acute Kidney Injury/drug therapy , Antihypertensive Agents/pharmacology , Blood Pressure/drug effects , Enzyme Inhibitors/pharmacology , Glomerulonephritis/prevention & control , Hypertension/drug therapy , Phenylurea Compounds/pharmacology , Piperidines/pharmacology , Reperfusion Injury/drug therapy , Acute Kidney Injury/etiology , Acute Kidney Injury/pathology , Acute Kidney Injury/physiopathology , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Animals , Antihypertensive Agents/toxicity , Disease Models, Animal , Disease Progression , Enalapril/pharmacology , Enzyme Inhibitors/toxicity , Epoxide Hydrolases/antagonists & inhibitors , Fibrosis , Glomerular Mesangium/drug effects , Glomerular Mesangium/pathology , Glomerular Mesangium/physiopathology , Glomerulonephritis/etiology , Glomerulonephritis/pathology , Glomerulonephritis/physiopathology , Hypertension/etiology , Hypertension/physiopathology , Male , Mice , Phenylurea Compounds/toxicity , Piperidines/toxicity , Reperfusion Injury/complications , Reperfusion Injury/physiopathologyABSTRACT
Background: Doxycycline is currently the most frequently used treatment in patients with scrub typhus. However, doxycycline-resistant strains have been found, necessitating the development of a new treatment. Rifampin is known to be effective even for such strains. Our aim in this study was to compare the effects of rifampin and doxycycline treatment in patients with scrub typhus in areas in which resistance to doxycycline has not been reported. Methods: Patients admitted to Chosun University Hospital and regional network hospitals between 2007 and 2009 with a body temperature ≥37.5°C and suspected to have scrub typhus were randomly assigned to 1 of 2 treatment groups: a group administered doxycycline 100 mg twice daily for 5 days and a group administered rifampin 600 mg once daily for 5 days. For treatment outcomes, fever, headache, muscle ache, and rash clearance times were compared between the groups. Results: The rifampin and doxycycline groups showed equivalence in all treatment outcomes evaluated. The proportions of patients with fever clearance within 48 hours were similar between groups. Furthermore, there was no significant difference in the occurrence of side effects following drug administration between groups. Conclusions: On the basis of the finding that equivalent treatment effects and safety were found in patient groups that received 600 mg of rifampin and 200 mg of doxycycline, respectively, for 5 days to treat scrub typhus, rifampin may be considered an alternative treatment to doxycycline. Clinical Trials Registration: NCT00568711.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Doxycycline/therapeutic use , Rifampin/therapeutic use , Scrub Typhus/drug therapy , Aged , Exanthema , Female , Fever , Humans , Male , Middle Aged , Treatment OutcomeABSTRACT
Renal ischemia-reperfusion injury (IRI) is a severe complication of major surgery and a risk factor for increased morbidity and mortality. Here, we investigated mechanisms that might contribute to IRI-induced progression to chronic kidney disease (CKD). Acute kidney injury (AKI) was induced by unilateral IRI for 35 min in CD1 and C57BL/6 (B6) mice. Unilateral IRI was used to overcome early mortality. Renal morphology, NGAL upregulation, and neutrophil infiltration as well as peritubular capillary density were studied by immunohistochemistry. The composition of leukocyte infiltrates in the kidney after IRI was investigated by flow cytometry. Systemic blood pressure was measured with a tail cuff, and renal perfusion was quantified by functional magnetic resonance imaging (fMRI). Mesangial matrix expansion was assessed by silver staining. Following IRI, CD1 and B6 mice developed similar morphological signs of AKI and increases in NGAL expression, but neutrophil infiltration was greater in CD1 than B6 mice. IRI induced an increase in systemic blood pressure of 20 mmHg in CD1, but not in B6 mice; and CD1 mice also had a greater loss of renal perfusion and kidney volume than B6 mice ( P < 0.05). CD1 mice developed substantial interstitial fibrosis and decreased peritubular capillary (PTC) density by day 14 while B6 mice showed only mild renal scarring and almost normal PTC. Our results show that after IRI, CD1 mice develop more inflammation, hypertension, and later mesangial matrix expansion than B6 mice do. Subsequently, CD1 animals suffer from CKD due to impaired renal perfusion and pronounced permanent loss of peritubular capillaries.
Subject(s)
Acute Kidney Injury/complications , Hypertension/etiology , Kidney/blood supply , Renal Circulation , Renal Insufficiency, Chronic/etiology , Reperfusion Injury/complications , Acute Kidney Injury/metabolism , Acute Kidney Injury/pathology , Acute Kidney Injury/physiopathology , Animals , Blood Flow Velocity , Blood Pressure , Cell Proliferation , Disease Models, Animal , Disease Progression , Fibrosis , Glomerular Mesangium/pathology , Hypertension/metabolism , Hypertension/pathology , Hypertension/physiopathology , Immunohistochemistry , Kidney/metabolism , Kidney/pathology , Lipocalin-2/metabolism , Magnetic Resonance Imaging , Male , Mice, Inbred C57BL , Neutrophil Infiltration , Renal Insufficiency, Chronic/metabolism , Renal Insufficiency, Chronic/pathology , Renal Insufficiency, Chronic/physiopathology , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Reperfusion Injury/physiopathology , Time FactorsABSTRACT
OBJECTIVES: To evaluate T1 mapping as a non-invasive, functional MRI biomarker in patients shortly after solid organ transplantation to detect acute postsurgical kidney damage and to correlate T1 times with renal function. METHODS: 101 patients within 2 weeks after solid organ transplantation (49 kidney transplantation, 52 lung transplantation) and 14 healthy volunteers were examined by MRI between July 2012 and April 2015 using the modified Look-Locker inversion recovery (MOLLI) sequence. T1 times in renal cortex and medulla and the corticomedullary difference were compared between groups using one-way ANOVA adjusted for multiple comparison with the Tukey test, and T1 times were correlated with renal function using Pearson's correlation. RESULTS: Compared to healthy volunteers T1 times were significantly increased after solid organ transplantation in the renal cortex (healthy volunteers 987 ± 102 ms; kidney transplantation 1299 ± 101 ms, p < 0.001; lung transplantation 1058 ± 96 ms, p < 0.05) and to a lesser extent in the renal medulla. Accordingly, the corticomedullary difference was diminished shortly after solid organ transplantation. T1 changes were more pronounced following kidney compared to lung transplantation, were associated with the stage of renal impairment and significantly correlated with renal function. CONCLUSIONS: T1 mapping may be helpful for early non-invasive assessment of acute kidney injury and renal pathology following major surgery such as solid organ transplantation. KEY POINTS: ⢠Renal cortical T1 relaxation times are prolonged after solid organ transplantation. ⢠Cortical T1 values increase with higher stages of renal function impairment. ⢠Corticomedullary difference decreases with higher stages of renal function impairment. ⢠Renal cortical T1 relaxation time and corticomedullary difference correlate with renal function. ⢠T1 mapping may be helpful for non-invasive assessment of post-operative renal pathology.
Subject(s)
Acute Kidney Injury/diagnosis , Kidney Transplantation/adverse effects , Kidney/pathology , Lung Transplantation/adverse effects , Magnetic Resonance Imaging/methods , Postoperative Complications/diagnosis , Adult , Female , Humans , Male , Middle AgedABSTRACT
Chronic exposure to commercial glucose-based peritoneal dialysis fluids during peritoneal dialysis induces peritoneal membrane damage leading to ultrafiltration failure. In this study the role of protein kinase C (PKC) α in peritoneal membrane damage was investigated in a mouse model of peritoneal dialysis. We used 2 different approaches: blockade of biological activity of PKCα by intraperitoneal application of the conventional PKC inhibitor Go6976 in C57BL/6 wild-type mice and PKCα-deficient mice on a 129/Sv genetic background. Daily administration of peritoneal dialysis fluid for 5 weeks induced peritoneal upregulation and activation of PKCα accompanied by epithelial-to-mesenchymal transition of peritoneal mesothelial cells, peritoneal membrane fibrosis, neoangiogenesis, and macrophage and T cell infiltration, paralleled by reduced ultrafiltration capacity. All pathological changes were prevented by PKCα blockade or deficiency. Moreover, treatment with Go6976 and PKCα deficiency resulted in strong reduction of proinflammatory, profibrotic, and proangiogenic mediators. In cell culture experiments, both treatment with Go6976 and PKCα deficiency prevented peritoneal dialysis fluid-induced release of MCP-1 from mouse peritoneal mesothelial cells and ameliorated transforming growth factor-ß1-induced epithelial-to-mesenchymal transition and peritoneal dialysis fluid-induced MCP-1 release in human peritoneal mesothelial cells. Thus, PKCα plays a crucial role in the pathophysiology of peritoneal membrane dysfunction induced by peritoneal dialysis fluids, and we suggest that its therapeutic inhibition might be a valuable treatment option for peritoneal dialysis patients.
Subject(s)
Carbazoles/therapeutic use , Dialysis Solutions/adverse effects , Enzyme Inhibitors/therapeutic use , Glucose/adverse effects , Peritoneal Dialysis/adverse effects , Peritoneal Fibrosis/prevention & control , Protein Kinase C-alpha/antagonists & inhibitors , Animals , Cell Line , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Epithelial Cells/physiology , Epithelial-Mesenchymal Transition , Female , Flow Cytometry , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Peritoneum/cytology , Peritoneum/pathology , Primary Cell Culture , Protein Kinase C-alpha/genetics , Protein Kinase C-alpha/metabolism , Transforming Growth Factor beta1/metabolism , Up-RegulationABSTRACT
Delayed graft function (DGF) after kidney transplantation is not uncommon, and it is associated with long-term allograft impairment. Our aim was to compare renal perfusion changes measured with noninvasive functional MRI in patients early after kidney transplantation to renal function and allograft histology in biopsy samples. Forty-six patients underwent MRI 4-11 days after transplantation. Contrast-free MRI renal perfusion images were acquired using an arterial spin labeling technique. Renal function was assessed by estimated glomerular filtration rate (eGFR), and renal biopsies were performed when indicated within 5 days of MRI. Twenty-six of 46 patients had DGF. Of these, nine patients had acute rejection (including borderline), and eight had other changes (e.g., tubular injury or glomerulosclerosis). Renal perfusion was significantly lower in the DGF group compared with the group with good allograft function (231 ± 15 vs. 331 ± 15 ml·min(-1)·100 g(-1), P < 0.001). Living donor allografts exhibited significantly higher perfusion values compared with deceased donor allografts (P < 0.001). Renal perfusion significantly correlated with eGFR (r = 0.64, P < 0.001), resistance index (r = -0.57, P < 0.001), and cold ischemia time (r = -0.48, P < 0.01). Furthermore, renal perfusion impairment early after transplantation predicted inferior renal outcome and graft loss. In conclusion, noninvasive functional MRI detects renal perfusion impairment early after kidney transplantation in patients with DGF.
Subject(s)
Delayed Graft Function/pathology , Delayed Graft Function/physiopathology , Graft Survival/physiology , Kidney Transplantation , Kidney/physiopathology , Magnetic Resonance Imaging , Adult , Aged , Female , Glomerular Filtration Rate/physiology , Graft Rejection/pathology , Humans , Kidney Transplantation/adverse effects , Kidney Transplantation/methods , Magnetic Resonance Imaging/methods , Male , Middle Aged , Perfusion/methods , Transplantation, Homologous/adverse effects , Transplantation, Homologous/methodsABSTRACT
BACKGROUND: Rice prolamin has been reported to possess antioxidative, anti-inflammatory and immune-promoting properties. This study is aimed to examine the protective effects of dietary rice prolamin extract (RPE) against dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD)-like skin lesions in mice. METHODS: BALB/c mice were fed diet supplemented with 0-0.1 % RPE for 6 weeks. For the last 2 weeks, 1 % or 0.2 % DNCB was applied repeatedly to the back skin of mice to induce AD-like lesions. Following AD induction, the severity of skin lesions was examined macroscopically and histologically. In addition, the serum levels of IgE, IgG1 and IgG2a were determined by ELISA, and the mRNA expression of IL-4 and IFN-γ in the skin was determined by real-time PCR. RESULTS: Dietary RPE suppressed the clinical symptoms of DNCB-induced dermatitis as well as its associated histopathological changes such as epidermal hyperplasia and infiltration of mast cells and eosinophils in the dermis. RPE treatment also suppressed the DNCB-induced increase in transepidermal water loss. Dietary RPE inhibited the DNCB-induced enhancement of serum IgE and IgG1 levels, whereas it increased the serum IgG2a level in DNCB-treated mice. In addition, dietary RPE upregulated the IFN-γ mRNA expression and downregulated the IL-4 mRNA expression in the skin of DNCB-treated mice. CONCLUSIONS: The above results suggest that dietary RPE exerts a protective effect against DNCB-induced AD in mice via upregulation of Th1 immunity and that RPE may be useful for the treatment of AD.
Subject(s)
Dermatitis, Atopic/drug therapy , Dermatitis, Atopic/immunology , Oryza , Phytotherapy , Prolamins/therapeutic use , Skin/drug effects , Animals , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Dermatitis, Atopic/blood , Dermatitis, Atopic/pathology , Disease Models, Animal , Female , Mice , Mice, Inbred BALB C , Plant Extracts/pharmacology , Plant Extracts/therapeutic use , Prolamins/pharmacologyABSTRACT
BACKGROUND: There is a significant demand for intermediate-scale bioreactors in academic and industrial institutions to produce cells for various applications in drug screening and/or cell therapy. However, the application of these bioreactors in cultivating hiPSC-derived immune cells and other blood cells is noticeably lacking. To address this gap, we have developed a xeno-free and chemically defined intermediate-scale bioreactor platform, which allows for the generation of standardized human iPSC-derived hematopoietic organoids and subsequent continuous production of macrophages (iPSC-Mac). METHODS: We describe a novel method for intermediate-scale immune cell manufacturing, specifically the continuous production of functionally and phenotypically relevant macrophages that are harvested on weekly basis for multiple weeks. RESULTS: The continuous production of standardized human iPSC-derived macrophages (iPSC-Mac) from 3D hematopoietic organoids also termed hemanoids, is demonstrated. The hemanoids exhibit successive stage-specific embryonic development, recapitulating embryonic hematopoiesis. iPSC-Mac were efficiently and continuously produced from three different iPSC lines and exhibited a consistent and reproducible phenotype, as well as classical functionality and the ability to adapt towards pro- and anti-inflammatory activation stages. Single-cell transcriptomic analysis revealed high macrophage purity. Additionally, we show the ability to use the produced iPSC-Mac as a model for testing immunomodulatory drugs, exemplified by dexamethasone. CONCLUSIONS: The novel method demonstrates an easy-to-use intermediate-scale bioreactor platform that produces prime macrophages from human iPSCs. These macrophages are functionally active and require no downstream maturation steps, rendering them highly desirable for both therapeutic and non-therapeutic applications.
Subject(s)
Bioreactors , Induced Pluripotent Stem Cells , Macrophages , Organoids , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Macrophages/cytology , Macrophages/metabolism , Organoids/cytology , Organoids/metabolism , Cell Differentiation , Cell Culture Techniques/methods , Cell Culture Techniques/instrumentation , HematopoiesisABSTRACT
BACKGROUND: Macrophages have recently become attractive therapeutics in cancer immunotherapy. The potential of macrophages to infiltrate and influence solid malignancies makes them promising targets for the chimeric antigen receptor (CAR) technology to redirect their stage of polarization, thus enhancing their anticancer capacities. Given the emerging interest for CAR-macrophages, generation of such cells so far mainly depends on peripheral blood monocytes, which are isolated from the respective donor prior to genetic manipulation. This procedure is time-intensive and cost-intensive, while, in some cases, insufficient monocyte amounts can be recovered from the donor, thus hampering the broad applicability of this technology. Hence, we demonstrate the generation and effectiveness of CAR-macrophages from various stem cell sources using also modern upscaling technologies for next generation immune cell farming. METHODS: Primary human hematopoietic stem and progenitor cells and induced pluripotent stem cells were used to derive anti-CD19 CAR-macrophages. Anticancer activity of the cells was demonstrated in co-culture systems, including primary material from patients with leukemia. Generation of CAR-macrophages was facilitated by bioreactor technologies and single-cell RNA (scRNA) sequencing was used to characterize in-depth response and behavior of CAR-macrophages. RESULTS: Irrespective of the stem-cell source, CAR-macrophages exhibited enhanced and antigen-dependent phagocytosis of CD19+ target cancer cells with increased pro-inflammatory responses. Phagocytic capacity of CAR-macrophages was dependent on target cell CD19 expression levels with superior function of CAR-macrophages against CD19+ cancer cell lines and patient-derived acute lymphocytic leukemia cancer cells. scRNA sequencing revealed CAR-macrophages to be distinct from eGFP control cells after co-culture with target cells, which includes the activation of pro-inflammatory pathways and upregulation of chemokines and cytokines associated with adaptive immune cell recruitment, favoring the repolarization of CAR-macrophages to a pro-inflammatory state. Taken together, the data highlight the unique features of CAR-macrophages in combination with the successful upscaling of the production pipeline using a three-dimensional differentiation protocol and intermediate scale bioreactors. CONCLUSION: In summary, our work provides insights into the seminal use and behavior of CAR-macrophages which are derived from various sources of stem cells, while introducing a unique technology for CAR-macrophage manufacturing, all dedicated to the clinical translation of CAR-macrophages within the field of anticancer immunotherapies.
Subject(s)
Induced Pluripotent Stem Cells , Leukemia , Neoplasms , Receptors, Chimeric Antigen , Humans , Receptors, Chimeric Antigen/genetics , Receptors, Antigen, T-Cell , Induced Pluripotent Stem Cells/metabolism , T-Lymphocytes , Leukemia/therapy , Macrophages/metabolismABSTRACT
Nephrotoxic drugs can cause acute kidney injury (AKI) and analgesic nephropathy. Diclofenac is potentially nephrotoxic and frequently prescribed for pain control. In this study, we investigated the effects of single and repetitive oral doses of diclofenac in the setting of pre-existing subclinical AKI on the further course of AKI and on long-term renal consequences. Unilateral renal ischemia-reperfusion injury (IRI) for 15 min was performed in male CD1 mice to induce subclinical AKI. Immediately after surgery, single oral doses (100 mg or 200 mg) of diclofenac were administered. In a separate experimental series, repetitive treatment with 100 mg diclofenac over three days was performed after IRI and sham surgery. Renal morphology and pro-fibrotic markers were investigated 24 h and two weeks after the single dose and three days after the repetitive dose of diclofenac treatment using histology, immunofluorescence, and qPCR. Renal function was studied in a bilateral renal IRI model. A single oral dose of 200 mg, but not 100 mg, of diclofenac after IRI aggravated acute tubular injury after 24 h and caused interstitial fibrosis and tubular atrophy two weeks later. Repetitive treatment with 100 mg diclofenac over three days aggravated renal injury and caused upregulation of the pro-fibrotic marker fibronectin in the setting of subclinical AKI, but not in sham control kidneys. In conclusion, diclofenac aggravated renal injury in pre-existing subclinical AKI in a dose and time-dependent manner and already a single dose can cause progression to chronic kidney disease (CKD) in this model.
ABSTRACT
BACKGROUND: The clinical significance of the Enterobacter nimipressuralis as human pathogens remains unclear. CASE PRESENTATIONS: The microbiologic culture monitoring system of sterile body fluids revealed on an episode of Enterobacter cloacae and Enterobacter amnigenus in blood culture results on the same day; the antibiotic sensitivity and MIC were nearly the same for both species. First patient was a healthy woman with postmenopausal syndrome, while second patient with herpes zoster. Both patients had febrile sensations without signs of bacteremia. E. amnigenus was also cultured from the unused package of salined cotton in the container through epidemiologic investigation. The cultured Enterobacter species were all identified as E. nimipressuralis through hsp60 gene sequencing and infrequent-restriction-site PCR (IRS-PCR). CONCLUSION: When an unusual microorganisms such as E. nimipressuralis is isolated from blood of a patient with no clinical signs of sepsis, a pseudobacteremia should be suspected. When the antibiogram and MIC test results of bacterial cultures from two or more patients are nearly the same, although the species involved may appear different, it may be necessary to prove that they are the same species through molecular methods. The microbiologic cultures monitoring system will probably help to detect pseudobacteremia and other pseudo infections through reliable and fast identification.
Subject(s)
Bacteremia/diagnosis , Bacteremia/microbiology , Enterobacter/classification , Enterobacter/isolation & purification , Enterobacteriaceae Infections/diagnosis , Adult , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Blood/microbiology , Chaperonin 60/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Enterobacter/drug effects , Enterobacteriaceae Infections/microbiology , Female , Herpes Zoster/complications , Humans , Microbial Sensitivity Tests , Middle Aged , Postmenopause , Sequence Analysis, DNAABSTRACT
To assess whether MR diffusion imaging may be applied for non-invasive detection of renal changes correlating with clinical diagnosis of acute kidney injury (AKI) in patients after lung transplantation (lutx).Fifty-four patients (mean age 49.6, range 26-64 years) after lutx were enrolled in a prospective clinical study and underwent functional MR imaging of the kidneys in the early postoperative period. Baseline s-creatinine ranged from 39 to 112âµmol/L. For comparison, 14 healthy volunteers (mean age 42.1, range 24-59 years) underwent magnetic resonance imaging (MRI) using the same protocol. Renal tissue injury was evaluated using quantification of diffusion and diffusion anisotropy with diffusion-weighted (DWI) and diffusion-tensor imaging (DTI). Renal function was monitored and AKI was defined according to Acute-Kidney-Injury-Network criteria. Statistical analysis comprised one-way ANOVA and Pearson correlation.67% of lutx patients (36/54) developed AKI, 47% (17/36) had AKI stage 1, 42% (15/36) AKI stage 2, and 8% (3/36) severe AKI stage 3. Renal apparent diffusion coefficients (ADCs) were reduced in patients with AKI, but preserved in transplant patients without AKI and healthy volunteers (2.07â±â0.02 vs 2.18â±â0.05 vs 2.21â±â0.03â×â10âmm/s, Pâ<â.05). Diffusion anisotropy was reduced in all lutx recipients compared with healthy volunteers (AKI: 0.27â±â0.01 vs no AKI: 0.28â±â0.01 vs healthy: 0.33â±â0.02; Pâ<â.01). Reduction of renal ADC correlated significantly with acute loss of renal function after lutx (decrease of renal function in the postoperative period and glomerular filtration rate on the day of MRI).MR diffusion imaging enables non-invasive assessment of renal changes correlating with AKI early after lutx. Reduction of diffusion anisotropy was present in all patients after lutx, whereas marked reduction of renal ADC was observed only in the group of lutx recipients with AKI and correlated with renal function impairment.
Subject(s)
Acute Kidney Injury/diagnostic imaging , Acute Kidney Injury/etiology , Diffusion Magnetic Resonance Imaging/methods , Lung Transplantation/adverse effects , Acute Kidney Injury/pathology , Adult , Anisotropy , Creatinine/blood , Female , Glomerular Filtration Rate , Humans , Male , Middle Aged , Prospective Studies , Severity of Illness IndexABSTRACT
Ischemia reperfusion injury (IRI) is linked with inflammation in kidney transplantation (ktx). The chemokine CXCL13, also known as B lymphocyte chemoattractant, mediates recruitment of B cells within follicles of lymphoid tissues and has recently been identified as a biomarker for acute kidney allograft rejection. The goal of this study was to explore whether IRI contributes to the up-regulation of CXCL13 levels in ktx. It is demonstrated that systemic levels of CXCL13 were increased in mouse models of uni- and bilateral renal IRI, which correlated with the duration of IRI. Moreover, in unilateral renal IRI CXCL13 expression in ischemic kidneys was up-regulated. Immunohistochemical studies revealed infiltration of CD22+ B-cells and, single-cell RNA sequencing analysis a higher number of cells expressing the CXCL13 receptor CXCR5, in ischemic kidneys 7 days post IRI, respectively. The potential relevance of these findings was also evaluated in a mouse model of ktx. Increased levels of serum CXCL13 correlated with the lengths of cold ischemia times and were further enhanced in allogenic compared to isogenic kidney transplants. Taken together, these findings indicate that IRI is associated with increased systemic levels of CXCL13 in renal IRI and ktx.
Subject(s)
B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Chemokine CXCL13/metabolism , Chemotaxis, Leukocyte/immunology , Kidney Transplantation , Reperfusion Injury/etiology , Reperfusion Injury/metabolism , Animals , Animals, Genetically Modified , Biomarkers , Chemokine CXCL13/blood , Chemokine CXCL13/genetics , Cytokines , Disease Models, Animal , Gene Expression , Kidney/immunology , Kidney/metabolism , Kidney/pathology , Kidney Transplantation/adverse effects , Leukocytes/immunology , Leukocytes/metabolism , Leukocytes/pathology , Male , Mice , Reperfusion Injury/pathologyABSTRACT
Background: Ischemia reperfusion injury (IRI) plays a major role in solid organ transplantation. The length of warm ischemia time is critical for the extent of tissue damage in renal IRI. In this experimental study we hypothesized that local release of labile heme in renal tissue is triggered by the duration of warm ischemia (15 vs. 45 min IRI) and mediates complement activation, cytokine release, and inflammation. Methods: To induce IRI, renal pedicle clamping was performed in male C57BL/6 mice for short (15 min) or prolonged (45 min) time periods. Two and 24 h after experimental ischemia tissue injury labile heme levels in the kidney were determined with an apo-horseradish peroxidase assay. Moreover, renal injury, cytokines, and C5a and C3a receptor (C5aR, C3aR) expression were determined by histology, immunohistochemistry and qPCR, respectively. In addition, in vitro studies stimulating bone marrow-derived macrophages with LPS and the combination of LPS and heme were performed and cytokine expression was measured. Results: Inflammation and local tissue injury correlated with the duration of warm ischemia time. Labile heme concentrations in renal tissue were significantly higher after prolonged (45 min) as compared to short (15 min) IRI. Notably, expression of the inducible heme-degrading enzyme heme oxygenase-1 (HO-1) was up-regulated in kidneys after prolonged, but not after short IRI. C5aR, the pro-inflammatory cytokines IL-6 and TNF-α as well as pERK were up-regulated after prolonged, but not after short ischemia times. Consecutively, neutrophil infiltration and up-regulation of pro-fibrotic cytokines such as CTGF and PAI were more pronounced in prolonged IRI in comparison to short IRI. In vitro stimulation of macrophages with LPS revealed that IL-6 expression was enhanced in the presence of heme. Finally, administration of the heme scavenger human serum albumin (HSA) reduced the expression of pro-inflammatory cytokines, C3a receptor and improved tubular function indicated by enhanced alpha 1 microglobulin (A1M) absorption after IRI. Conclusions: Our data show that prolonged duration of warm ischemia time increased labile heme levels in the kidney, which correlates with IRI-dependent inflammation and up-regulation of anaphylatoxin receptor expression.
Subject(s)
Complement Activation , Heme/immunology , Kidney Diseases/immunology , Kidney/immunology , Reperfusion Injury/immunology , Animals , Cytokines/immunology , Inflammation/immunology , Inflammation/pathology , Kidney/pathology , Kidney Diseases/pathology , Male , Mice , Receptor, Anaphylatoxin C5a/immunology , Receptors, G-Protein-Coupled/immunology , Reperfusion Injury/pathologyABSTRACT
OBJECTIVES: Heart transplantation (HTX) in mice is used to characterize gene-deficient mice and to test new treatment strategies. The purpose was to establish noninvasive magnetic resonance imaging techniques in mice to monitor pathophysiological changes of the allograft during rejection. MATERIALS AND METHODS: Magnetic resonance imaging was performed at baseline and days 1 and 6 after isogenic (n = 10, C57BL/6) and allogenic (n = 12, C57BL/6 to BALB/c) heterotopic HTX on a 7 T small animal scanner. Respiratory- and electrocardiogram-gated multislice multi-echo spin echo sequences were acquired, and parameter maps of T2 relaxation time were generated. T2 times in septal, anterior, lateral, and posterior myocardial segments as well as global T2 times were calculated and compared between groups. At day 7 animals were sacrificed and graft pathology was assessed by semiquantitative regional analysis and correlated with magnetic resonance imaging results. RESULTS: Myocardial T2 relaxation time was significantly increased in allogenic (33.4 ± 0.1 ms) and isogenic cardiac grafts (31.8 ± 1.8 ms) on day 1 after HTX compared with healthy donor hearts at baseline (23.1 ± 0.3 ms, P < 0.001). Until day 6 after HTX, myocardial T2 further increased markedly in allografts but not in isografts (43.4 ± 1.9 vs 31.2 ± 1.1 ms, P < 0.001). Mean segmental T2 values as well as mean global T2 values in allogenic compared with isogenic cardiac grafts on day 6 were significantly higher (P < 0.01). Histologically, isogenic grafts were almost normal besides small focal leukocyte infiltrates and signs of interstitial edema, most likely due to ischemia reperfusion injury (histological sum score, 0.9 ± 0.4). In allogenic HTX, histology revealed severe inflammation and tissue edema representing allograft rejection with increased histological scores (5.3 ± 0.7, P < 0.001). Higher histological scores of rejection were significantly associated with increased T2 times on a segmental and a global level. CONCLUSIONS: We could show that T2 mapping is a suitable noninvasive imaging method to monitor global and regional HTX pathologies in experimental heart transplantation in mice. Progressive prolongation of T2 time was significantly associated with pathological signs of rejection.
Subject(s)
Allografts , Edema/diagnostic imaging , Graft Rejection/diagnostic imaging , Heart Transplantation , Magnetic Resonance Imaging/methods , Postoperative Complications/diagnostic imaging , Acute Disease , Animals , Disease Models, Animal , Edema/pathology , Graft Rejection/pathology , Heart/diagnostic imaging , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Postoperative Complications/pathologyABSTRACT
OBJECTIVES: IL-17A contributes to acute kidney injury and fibrosis. Therefore, we asked whether IL-17A deficiency or treatment with a IL-17A blocking antibody impacts severe renal ischaemia reperfusion injury (IRI) and the progression to chronic kidney disease (CKD). METHODS: IL-17A-deficient and wild-type (WT) mice underwent transient unilateral renal pedicle clamping for 45 min to induce IRI and subsequent renal fibrosis. Furthermore, a neutralizing anti-IL-17A antibody (mAb) was injected into WT mice before induction of renal IRI intravenously. On days 1, 7 and 21, inflammation, fibrosis, leukocyte infiltration and pro-inflammatory and pro-fibrotic cytokine expression were assessed in kidneys using histology, qPCR and flow cytometry. KEY FINDINGS: IL-17A was significantly increased after renal IRI in WT kidneys. Levels of pro-inflammatory (MCP-1) cytokine and pro-fibrotic (collagen 1α1, fibronectin) transcripts were similar in the experimental groups studied. IL-17A deficiency had no effect on renal T-cell influx or the number, inflammatory phenotype, or spatial distribution of macrophages. Similarly, administration of an IL-17A blocking antibody did not attenuate inflammation. CONCLUSIONS: Despite the effects of IL-17 in other inflammation models, neither genetic IL-17A deficiency nor treatment with an IL-17A blocking antibody attenuated IRI and progression to CKD. We conclude that in severe renal IRI IL-17A is not crucially involved in disease progression.
Subject(s)
Acute Kidney Injury/physiopathology , Interleukin-17/genetics , Renal Insufficiency, Chronic/prevention & control , Reperfusion Injury/physiopathology , Acute Kidney Injury/immunology , Animals , Antibodies, Monoclonal/pharmacology , Cytokines/metabolism , Disease Models, Animal , Disease Progression , Fibrosis , Flow Cytometry , Inflammation/immunology , Inflammation/physiopathology , Interleukin-17/antagonists & inhibitors , Interleukin-17/immunology , Macrophages/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Renal Insufficiency, Chronic/immunology , Reperfusion Injury/immunologyABSTRACT
PURPOSE: The purpose was to characterize acute kidney injury (AKI) in C57BL/6 (B6)- and 129/Sv (Sv)-mice by noninvasive measurement of renal perfusion and tissue edema using functional MRI. METHODS: Different severities of AKI were induced in B6- and Sv-mice by renal ischemia reperfusion injury (IRI). Unilateral clamping of the renal pedicle for 35 min (moderate AKI) or 45 min (severe AKI) was done. MRI (7-Tesla) was performed 1, 7 and 28 days after surgery using a flow alternating inversion recovery (FAIR) arterial spin labeling (ASL) sequence. Maps of perfusion and T1-relaxation time were calculated. Relative MRI-parameters of the IRI kidney compared to the contralateral not-clipped kidney were compared between AKI severities and between mouse strains using unpaired t-tests. In addition, fibrosis was assessed by Masson Trichrome and collagen IV staining. RESULTS: After moderate AKI relative perfusion impairment was significantly higher in B6- than in Sv-mice at d7 (55±7% vs. 82±8%, p<0.05) and d28 (76±7% vs. 102±3%, p<0.01). T1-values increased in the early phase after AKI in both mouse strains. T1-increase was more severe after prolonged ischemia times of 45 min compared to 35 min in both mouse strains, measured in the renal cortex and outer stripe of outer medulla. Kidney volume loss (compared to the contralateral kidney) occurred already after 7 days but proceeded markedly towards 4 weeks in severe AKI. Early renal perfusion impairment was predictive for later kidney volume loss. The progression to chronic kidney disease (CKD) in the severe AKI model was similar in both mouse strains as revealed by histology. CONCLUSION: Quantification of renal perfusion and tissue edema by functional MRI allows characterization of strain differences upon AKI. Renal perfusion impairment was stronger in B6- compared to Sv-animals following moderate AKI. Prolonged ischemia times were associated with more severe perfusion impairment and edema formation in the early phase and progression to CKD within 4 weeks of observation.
Subject(s)
Acute Kidney Injury/diagnostic imaging , Disease Models, Animal , Kidney/diagnostic imaging , Magnetic Resonance Imaging , Mice, 129 Strain , Mice, Inbred C57BL , Acute Kidney Injury/pathology , Acute Kidney Injury/physiopathology , Animals , Disease Progression , Edema/diagnostic imaging , Edema/pathology , Edema/physiopathology , Fibrosis/diagnostic imaging , Fibrosis/pathology , Fibrosis/physiopathology , Immunohistochemistry , Kidney/pathology , Kidney/physiopathology , Macrophages/pathology , Macrophages/physiology , Male , Organ Size , Reperfusion Injury , Severity of Illness Index , Species Specificity , Time FactorsABSTRACT
OBJECTIVES: The aims of this experimental study were to investigate renal allograft pathophysiology by multiparametric functional magnetic resonance imaging (MRI) and to directly correlate MRI parameters with renal histopathology in mouse models of allogenic and isogenic kidney transplantation (ktx). MATERIALS AND METHODS: Allograft rejection was induced by transplantation of C57BL/6 (B6) donor kidneys into BALB/c recipients (allogenic ktx). B6 mice that received B6 kidneys served as controls (isogenic ktx). Three weeks after ktx, MRI was performed using a 7-T small-animal scanner. Flow sensitive alternating inversion recovery echoplanar imaging arterial spin labeling, multiecho turbo spin echo, and diffusion-weighted imaging sequences were acquired. Maps of renal perfusion, T2 and T1 relaxation times, and apparent diffusion coefficients were calculated. Histological changes in the kidney were evaluated according to Banff criteria. Renal cell infiltrates and fibrosis were quantified by immunohistochemistry. Differences between groups were assessed using the Mann-Whitney U test, and the correlation of MRI parameters with renal histopathology was determined by Spearman correlation analysis. RESULTS: After allogenic, but not isogenic, ktx, animals developed acute allograft rejection. Allogenic grafts were infiltrated by macrophages and T-lymphocytes and exhibited marked renal fibrosis. Magnetic resonance imaging revealed stronger impairment of renal perfusion (56 ± 7 vs 293 ± 44 mL/[min × 100 g]; P < 0.01) and more pronounced increases in T2 (60.1 ± 2.0 vs 45.7 ± 1.2 milliseconds, P < 0.01) and T1 relaxation times (1938 ± 53 vs 1350 ± 27 milliseconds, P < 0.01) in allogenic than in isogenic kidneys. Apparent diffusion coefficient was reduced to 1.39 ± 0.14 × 10(-3) mm2/s in kidneys with an acute rejection and was 1.83 ± 0.05 × 10(-3) mm2/s in isogenic kidneys without rejection (P < 0.05). Magnetic resonance imaging parameters significantly correlated with the amount of cellular infiltration and renal fibrosis observed histologically. CONCLUSIONS: Functional MRI allows detection of acute renal allograft rejection after allogenic ktx in mice. Functional MRI parameters correlate with cell infiltrates and fibrosis. Thus, MRI may be used noninvasively and longitudinally to investigate mechanisms of renal allograft rejection and evaluate novel therapeutic strategies in experimental studies.