Different Acute Kidney Injury Patterns after Renal Ischemia Reperfusion Injury and Extracorporeal Membrane Oxygenation in Mice.

The use of extracorporeal membrane oxygenation (ECMO) is associated with acute kidney injury (AKI) in thoracic organ transplantation. However, multiple other factors contribute to AKI development after these procedures such as renal ischemia-reperfusion injury (IRI) due to hypo-perfusion of the kidney during surgery. In this study, we aimed to explore the kidney injury patterns in mouse models of ECMO and renal IRI. Kidneys of C57BL/6 mice were examined after moderate (35 min) and severe (45 min) unilateral transient renal pedicle clamping and 2 h of veno-venous ECMO. Renal injury markers, neutrophil infiltration, tubular transport function, pro-inflammatory cytokines, and renal heme oxygenase-1 (HO-1) expression were determined by immunofluorescence and qPCR. Both procedures caused AKI, but with different injury patterns. Severe neutrophil infiltration of the kidney was evident after renal IRI, but not following ECMO. Tubular transport function was severely impaired after renal IRI, but preserved in the ECMO group. Both procedures caused upregulation of pro-inflammatory cytokines in the renal tissue, but with different time kinetics. After ECMO, but not IRI, HO-1 was strongly induced in tubular cells indicating contact with hemolysis-derived proteins. After IRI, HO-1 was expressed on infiltrating myeloid cells in the tubulo-interstitial space. In conclusion, renal IRI and ECMO both caused AKI, but kidney damage after renal IRI was more pronounced including severe neutrophil infiltration and tubular transport impairment. Enhanced HO-1 expression in tubular cells after ECMO encourages limitation of hemolysis as a therapeutic approach to reduce ECMO-associated AKI.

Cell-Free Hemoglobin in Acute Kidney Injury after Lung Transplantation and Experimental Renal Ischemia/Reperfusion.

Cell-free hemoglobin (CFH), a pro-oxidant and cytotoxic compound that is released in hemolysis, has been associated with nephrotoxicity. Lung transplantation (LuTx) is a clinical condition with a high incidence of acute kidney injury (AKI). In this study, we investigated the plasma levels of CFH and haptoglobin, a CFH-binding serum protein, in prospectively enrolled LuTx patients (n = 20) with and without AKI. LuTx patients with postoperative AKI had higher CFH plasma levels at the end of surgery compared with no-AKI patients, and CFH correlated with serum creatinine at 48 h. Moreover, CFH levels inversely correlated with haptoglobin levels, which were significantly reduced at the end of surgery in LuTx patients with AKI. Because multiple other factors can contribute to AKI development in the complex clinical setting of LuTx, we next investigated the role of exogenous CFH administration in a mouse model of mild bilateral renal ischemia reperfusion injury (IRI). Exogenous administration of CFH after reperfusion caused overt AKI with creatinine increase, tubular injury, and enhanced markers of renal inflammation compared with vehicle-treated animals. In conclusion, CFH is a possible factor contributing to postoperative AKI after LuTx and promotes AKI in an experimental model of mild transient renal ischemia. Targeting CFH might be a therapeutic option to prevent AKI after LuTx.

Tissue-resident macrophages mediate neutrophil recruitment and kidney injury in shiga toxin-induced hemolytic uremic syndrome.

Enterohaemorrhagic E. coli cause major epidemics worldwide with significant organ damage and very high percentages of death. Due to the ability of enterohaemorrhagic E. coli to produce shiga toxin these bacteria damage the kidney leading to the hemolytic uremic syndrome. A therapy against this serious kidney disease has not been developed yet and the impact and mechanism of leukocyte activation and recruitment are unclear. Tissue-resident macrophages represent the main leukocyte population in the healthy kidney, but the role of this important cell population in shiga toxin-producing E. coli-hemolytic uremic syndrome is incompletely understood. Using state of the art microscopy and mass spectrometry imaging, our preclinical study demonstrated a phenotypic and functional switch of tissue-resident macrophages after disease induction in mice. Kidney macrophages produced the inflammatory molecule TNFα and depletion of tissue-resident macrophages via the CSF1 receptor abolished TNFα levels in the kidney and significantly diminished disease severity. Furthermore, macrophage depletion did not only attenuate endothelial damage and thrombocytopenia, but also activation of thrombocytes and neutrophils. Moreover, we observed that neutrophils infiltrated the kidney cortex and depletion of macrophages significantly reduced the recruitment of neutrophils and expression of the neutrophil-attracting chemokines CXCL1 and CXCL2. Intravital microscopy revealed that inhibition of CXCR2, the receptor for CXCL1 and CXCL2, significantly reduced the infiltration of neutrophils and reduced kidney injury. Thus, our study shows activation of tissue-resident macrophages during shiga toxin-producing E. coli-hemolytic uremic syndrome leading to the production of disease-promoting TNFα and CXCR2-dependent recruitment of neutrophils.

Pre-ischemic renal lavage protects against renal ischemia-reperfusion injury by attenuation of local and systemic inflammatory responses.

Postischemic acute kidney injury (AKI) is a common clinical complication and often fatal, with no effective treatment available. Little is known about the role of leukocytes trapped in renal vessels during ischemia-reperfusion injury (IRI) in the postischemic AKI. We designed a new animal model in rats with preforming renal artery lavage prior to IRI to investigate the effect of diminishing the residual circulating leukocytes on kidney damage and inflammation. Moreover, the functional changes of macrophages in hypoxia reoxygenation condition were also analyzed. We found pre-ischemic renal lavage significantly decreased the serum creatinine and blood urea nitrogen levels, and downregulated the mRNA and protein expressions in kidneys and urinary secretion of kidney injury molecule-1 of rats after IRI. The renal pathological damage caused by IRI was also ameliorated by pre-ischemic renal lavage, as evidenced by fewer cast formation, diminished morphological signs of AKI in the tissue at 24 hours after IRI. Pre-ischemic renal lavage reduced the numbers of infiltrating CD68+ macrophages and MPO+ neutrophils. The mRNA expression of pro-inflammatory mediator in IRI kidneys and the levels of pro-inflammatory cytokines in circulatory system and urine were also reduced due to pre-ischemic lavage. Compared with nontreated rats with IRI, pre-ischemic renal lavage significantly reduced the phosphorylation levels of ERK and p65 subunit of NF-κB in the kidney after IRI. In addition, we found hypoxia/reoxygenation could promote the expression of pro-inflammatory mediators and inhibit the expression of anti-inflammatory factors by regulating ERK/NF-κB signaling pathway. Thus, pre-ischemic renal lavage could clearly reduce the renal damage after IRI by attenuating inflammation, and macrophages trapped in renal vessels during IRI could be important pathogenic factors driving tissue injury.

Targeting muscle-enriched long non-coding RNA H19 reverses pathological cardiac hypertrophy.

AIMS: Pathological cardiac remodelling and subsequent heart failure represents an unmet clinical need. Long non-coding RNAs (lncRNAs) are emerging as crucial molecular orchestrators of disease processes, including that of heart diseases. Here, we report on the powerful therapeutic potential of the conserved lncRNA H19 in the treatment of pathological cardiac hypertrophy. METHOD AND RESULTS: Pressure overload-induced left ventricular cardiac remodelling revealed an up-regulation of H19 in the early phase but strong sustained repression upon reaching the decompensated phase of heart failure. The translational potential of H19 is highlighted by its repression in a large animal (pig) model of left ventricular hypertrophy, in diseased human heart samples, in human stem cell-derived cardiomyocytes and in human engineered heart tissue in response to afterload enhancement. Pressure overload-induced cardiac hypertrophy in H19 knock-out mice was aggravated compared to wild-type mice. In contrast, vector-based, cardiomyocyte-directed gene therapy using murine and human H19 strongly attenuated heart failure even when cardiac hypertrophy was already established. Mechanistically, using microarray, gene set enrichment analyses and Chromatin ImmunoPrecipitation DNA-Sequencing, we identified a link between H19 and pro-hypertrophic nuclear factor of activated T cells (NFAT) signalling. H19 physically interacts with the polycomb repressive complex 2 to suppress H3K27 tri-methylation of the anti-hypertrophic Tescalcin locus which in turn leads to reduced NFAT expression and activity. CONCLUSION: H19 is highly conserved and down-regulated in failing hearts from mice, pigs and humans. H19 gene therapy prevents and reverses experimental pressure-overload-induced heart failure. H19 acts as an anti-hypertrophic lncRNA and represents a promising therapeutic target to combat pathological cardiac remodelling.

Early antihypertensive treatment and ischemia-induced acute kidney injury.

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.

Lymphangiogenesis in a mouse model of renal transplant rejection extends life span of the recipients.

Renal allograft rejection can be prevented by immunological tolerance, which may be associated with de novo formed lymphatic vessels in the donor kidney after transplantation in man. A suitable mouse model of renal allograft rejection in which lymphangiogenesis can be deliberately induced in the graft is critical for elucidating the mechanisms responsible for the association between attenuated transplant rejection and abundance of lymphatic vessels. Here we describe the development of a novel mouse model of rapid renal transplant rejection in which transgenic induction of lymphangiogenesis in the immune-incompatible graft greatly extends its survival time. Thus, our novel approach may facilitate exploitation of lymphangiogenesis in the grafted organ.

Acetaminophen-induced liver injury is mediated by the ion channel TRPV4.

Overdosing of the analgesic acetaminophen (APAP) is one of the most common causes for acute liver failure in modern countries. Although the exact molecular mechanisms mediating hepatocellular necrosis are still elusive, it is preceded by oxidative stress triggered by excessive levels of the metabolite N-acetyl-para-benzoquinone imine (NAPQI). Here, we describe the role of the redox-sensitive transient receptor potential (TRP) ion channel TRP vanilloid 4 (TRPV4) for APAP-induced hepatoxicity. Both pharmacological inhibition and genetic deletion of TRPV4 ameliorate APAP-induced necrosis in mouse and human hepatocytes in vitro. Liver injury caused by a systemic overdose of APAP is reduced in TRPV4-deficient mice and in wild-type mice treated with a TRPV4 inhibitor. The reduction of hepatotoxicity accomplished by systemic TRPV4 inhibition is comparable to the protective effects of the antioxidant N-acetyl-cysteine. Although TRPV4 does not modulate intrahepatic levels of glutathione, both its inhibition and genetic deletion attenuate APAP-induced oxidative and nitrosative stress as well as mitochondrial membrane depolarization. NAPQI evokes a calcium influx by activating heterologously expressed TRPV4 channels and endogenous TRPV4 channels in hepatoma cells but not in primary mouse hepatocytes. Taken together, our data suggest that TRPV4 mediates APAP-induced hepatotoxicity and thus may be a suitable target for treatment of this critical side effect.-Echtermeyer, F., Eberhardt, M., Risser, L., Herzog, C., Gueler, F., Khalil, M., Engel, M., Vondran, F., Leffler, A. Acetaminophen-induced liver injury is mediated by the ion channel TRPV4.

Synthetic retinoid AM80 inhibits IL-17 production of gamma delta T cells and ameliorates biliary atresia in mice.

BACKGROUND & AIMS: Recent evidence suggests that Interleukin (IL)-17-producing gamma delta ( γδ ) T cells are the dominant pathogenic cellular component in designated autoimmune or inflammatory diseases, including biliary atresia (BA). We have previously demonstrated that retinoids effectively suppress T-helper cell (Th) 17 differentiation. METHODS: Here, we established an in vitro system, enabling investigations of the effect of AM80 on the IL-17 production of γδ T cells. Additionally, we tested the therapeutic effect of AM80 in the Rotavirus-induced mouse model of BA. Co-incubation of γδ T cells with IL-23 and anti-CD28 mAb proved most effective in inducing an IL-17 response in vitro. The effect of AM80 on human CCR6+CD26+ V δ 2 cells was assessed by flow cytometry. RESULTS: AM80 efficiently reduced IL-17 production by murine γδ T cells and the expression of the master transcription factor Retinoid-Orphan-Receptor- γ t (ROR γτ ) in a dose-dependent manner. The fraction of human CCR6+CD26+ V δ 2 cells was significantly reduced by co-incubation with AM80. Moreover, AM80 also inhibited IL-17 production by liver-infiltrating γδ T cells isolated from animals suffering from BA. Intraperitoneal treatment with AM80 ameliorated BA-associated inflammation. However, AM80 treatment was not sufficient to control disease progression in the murine model, despite reduced inflammatory activity in the animals. CONCLUSIONS: Retinoids are very efficient in down-regulating IL-17 production by γδ T cells in vitro and, to a lesser extent, in the BA mouse model. However, retinoids do not suffice for the control of disease progression. Thus, our data suggest that IL-17 is not the only factor contributing to the pathogenesis of BA. LAY SUMMARY: Biliary atresia (BA) is a rare disease which affects infants, causing progressive liver failure in most children, and is the most common indication for paediatric liver transplantation. We have previously demonstrated that IL-17, produced by γδ T cells, contributes to hepatic inflammation in the murine model of BA and is increased in the livers of infants suffering from the disease. In the study at hand, we demonstrate that treatment with AM80, a synthetic retinoid with superior pharmacological properties, effectively inhibits the IL-17 production of gamma delta T cells without generating systemic immunosuppression. Although all-trans retinoic acid (ATRA) has been demonstrated to suppress differentiation of IL-17-producing conventional T-helper cells (Th17) in vitro, the therapeutic application of ATRA in vivo is limited by the compound's potential side effects caused by its instability and lack of receptor specificity. Our study is the first to show that AM80 suppresses the IL-17 production of γδ T cells in a very efficient manner and that hepatic inflammation is ameliorated in mice suffering from BA. However, AM80 treatment does not suffice to block the disease progression. We conclude that factors other than IL-17 drive the progressive inflammation in BA. The addition of retinoids to the treatment regime of children suffering from BA might decrease the disease burden; however, further research is needed to clarify the pathomechanism and possible therapeutic interventions in humans.

Dietary omega-3 PUFA improved tubular function after ischemia induced acute kidney injury in mice but did not attenuate impairment of renal function.

BACKGROUND: Acute kidney injury (AKI) is an important complication after major surgery and solid organ transplantation. Here, we present a dietary omega-3 polyunsaturated fatty acid (n3-PUFA) supplementation study to investigate whether pre-treatment can reduce ischemia induced AKI in mice. METHODS: Male 12-14 week old C57BL/6 J mice received a linoleic acid rich sunflower oil based standard diet containing 10 % fat (STD) or the same diet enriched with n3-PUFA (containing 1 % EPA and 1 % DHA) (STD + n3). After 14 days of feeding bilateral 30 min renal ischemia reperfusion injury (IRI) was conducted to induce AKI and mice were sacrificed at 24 h. Serum creatinine and blood urea nitrogen (BUN) as well as liver enzyme elevation were measured. Kidney damage was analyzed by histology and immunohistochemistry. Furthermore, pro-inflammatory cytokines (IL-6, MCP-1) were determined by qPCR. FA and oxylipin pattern were quantified in blood and kidneys by GC-FID and LC-MS/MS, respectively. RESULTS: n3-PUFA supplementation prior to renal IRI increased systemic and renal levels of n3-PUFA. Consistently, eicosanoids and other oxylipins derived from n3-PUFA including precursors of specialized pro-resolving mediators were elevated while n6-PUFA derived mediators such as pro-inflammatory prostaglandins were decreased. Feeding of n3-PUFA did not attenuate renal function impairment, morphological renal damage and inflammation characterized by IL-6 and MCP-1 elevation or neutrophil infiltration. However, the tubular transport marker alpha-1 microglobulin (A1M) was significantly higher expressed in proximal tubular epithelial cells of STD + n3 compared to STD fed mice. This indicates a better integrity of proximal tubular epithelial cells and thus significant protection of tubular function. In addition, heme oxygenase-1 (HO-1) which protects tubular function was also up-regulated in the treatment group receiving n3-PUFA supplemented chow. DISCUSSION: We showed that n3-PUFA pre-treatment did not affect overall renal function or renal inflammation in a mouse model of moderate ischemia induced AKI, but tubular transport was improved. In conclusion, dietary n3-PUFA supplementation altered the oxylipin levels significantly but did not protect from renal function deterioration or attenuate ischemia induced renal inflammation.

Clinical blood sampling for oxylipin analysis - effect of storage and pneumatic tube transport of blood on free and total oxylipin profile in human plasma and serum.

Quantitative analysis of oxylipins in blood samples is of increasing interest in clinical studies. However, storage after sampling and transport of blood might induce artificial changes in the apparent oxylipin profile due to ex vivo formation/degradation by autoxidation or enzymatic activity. In the present study we investigated the stability of free (i.e. non-esterified) and total oxylipins in EDTA-plasma and serum generated under clinical conditions assessing delays in sample processing and automated transportation: Free cytochrome P450 monooxygenase and 5-lipoxygenase (LOX) formed oxylipins as well as autoxidation products were marginally affected by storage of whole blood up to 4 h at 4 °C, while total (i.e. the sum of free and esterified) levels of these oxylipins were stable up to 24 h and following transport. Cyclooxygenase (COX) products (TxB2, 12-HHT) and 12-LOX derived hydroxy-fatty acids were prone to storage and transport induced changes due to platelet activation. Total oxylipin patterns were generally more stable than the concentration of free oxylipins. In serum, coagulation induced higher levels of COX and 12-LOX products showing a high inter-individual variability. Overall, our results indicate that total EDTA-plasma oxylipins are the most stable blood oxylipin marker for clinical samples. Here, storage of blood before further processing is acceptable for a period up to 24 hours at 4 °C. However, levels of platelet derived oxylipins should be interpreted with caution regarding potential ex vivo formation.

Interplay of Heme with Macrophages in Homeostasis and Inflammation.

Macrophages are an integral part of the mononuclear phagocyte system that is critical for maintaining immune homeostasis. They play a key role for initiation and modulation of immunological responses in inflammation and infection. Moreover, macrophages exhibit a wide spectrum of tissue-specific phenotypes in steady-state and pathophysiological conditions. Recent clinical and experimental evidence indicates that the ubiquitous compound heme is a crucial regulator of these cells, e.g., in the differentiation of monocytes to tissue-resident macrophages and/ or in activation by inflammatory stimuli. Notably, heme, an iron containing tetrapyrrole, is essential as a prosthetic group of hemoproteins (e.g., hemoglobin and cytochromes), whereas non-protein bound free or labile heme can be harmful via pro-oxidant, pro-inflammatory, and cytotoxic effects. In this review, it will be discussed how the complex interplay of heme with macrophages regulates homeostasis and inflammation via modulating macrophage inflammatory characteristics and/ or hematopoiesis. A particular focus will be the distinct roles of intra- and extracellular labile heme and the regulation of its availability by heme-binding proteins. Finally, it will be addressed how heme modulates macrophage functions via specific transcriptional factors, in particular the nuclear repressor BTB and CNC homologue (BACH)1 and Spi-C.

CCR2-dependent Gr1high monocytes promote kidney injury in shiga toxin-induced hemolytic uremic syndrome in mice.

The hemolytic uremic syndrome (HUS) is a life-threatening disease of the kidney that is induced by shiga toxin-producing E.coli. Major changes in the monocytic compartment and in CCR2-binding chemokines have been observed. However, the specific contribution of CCR2-dependent Gr1high monocytes is unknown. To investigate the impact of these monocytes during HUS, we injected a combination of LPS and shiga toxin into mice. We observed an impaired kidney function and elevated levels of the CCR2-binding chemokine CCL2 after shiga toxin/LPS- injection, thus suggesting Gr1high monocyte infiltration into the kidney. Indeed, the number of Gr1high monocytes was strongly increased one day after HUS induction. Moreover, these cells expressed high levels of CD11b suggesting activation after tissue entry. Non-invasive PET-MR imaging revealed kidney injury mainly in the kidney cortex and this damage coincided with the detection of Gr1high monocytes. Lack of Gr1high monocytes in Ccr2-deficient animals reduced neutrophil gelatinase-associated lipocalin and blood urea nitrogen levels. Moreover, the survival of Ccr2-deficient animals was significantly improved. Conclusively, this study demonstrates that CCR2-dependent Gr1high monocytes contribute to the kidney injury during HUS and targeting these cells is beneficial during this disease.

Formation of trans-epoxy fatty acids correlates with formation of isoprostanes and could serve as biomarker of oxidative stress.

In mammals, epoxy-polyunsaturated fatty acids (epoxy-PUFA) are enzymatically formed from naturally occurring all-cis PUFA by cytochrome P450 monooxygenases leading to the generation of cis-epoxy-PUFA (mixture of R,S- and S,R-enantiomers). In addition, also non-enzymatic chemical peroxidation gives rise to epoxy-PUFA leading to both, cis- and trans-epoxy-PUFA (mixture of R,R- and S,S-enantiomers). Here, we investigated for the first time trans-epoxy-PUFA and the trans/cis-epoxy-PUFA ratio as potential new biomarker of lipid peroxidation. Their formation was analyzed in correlation with the formation of isoprostanes (IsoP), which are commonly used as biomarkers of oxidative stress. Five oxidative stress models were investigated including incubations of three human cell lines as well as the in vivo model Caenorhabditis elegans with tert-butyl hydroperoxide (t-BOOH) and analysis of murine kidney tissue after renal ischemia reperfusion injury (IRI). A comprehensive set of IsoP and epoxy-PUFA derived from biologically relevant PUFA (ARA, EPA and DHA) was simultaneously quantified by LC-ESI(-)-MS/MS. Following renal IRI only a moderate increase in the kidney levels of IsoP and no relevant change in the trans/cis-epoxy-PUFA ratio was observed. In all investigated cell lines (HCT-116, HepG2 and Caki-2) as well as C. elegans a dose dependent increase of both, IsoP and the trans/cis-epoxy-PUFA ratio in response to the applied t-BOOH was observed. The different cell lines showed a distinct time dependent pattern consistent for both classes of autoxidatively formed oxylipins. Clear and highly significant correlations of the trans/cis-epoxy-PUFA ratios with the IsoP levels were found in all investigated cell lines and C. elegans. Based on this, we suggest the trans/cis-epoxy-PUFA ratio as potential new biomarker of oxidative stress, which warrants further investigation.

Chemokine CXCL13 as a New Systemic Biomarker for B-Cell Involvement in Acute T Cell-Mediated Kidney Allograft Rejection.

The presence of B-cell clusters in allogenic T cell-mediated rejection (TCMR) of kidney allografts is linked to more severe disease entities. In this study we characterized B-cell infiltrates in patients with TCMR and examined the role of serum CXCL-13 in these patients and experimentally. CXCL-13 serum levels were analyzed in 73 kidney allograft recipients at the time of allograft biopsy. In addition, four patients were evaluated for CXCL13 levels during the first week after transplantation. ELISA was done to measure CXCL-13 serum levels. For further mechanistic understanding, a translational allogenic kidney transplant (ktx) mouse model for TCMR was studied in BalbC recipients of fully mismatched transplants with C57BL/6 donor kidneys. CXCL-13 serum levels were measured longitudinally, CD20 and CD3 composition and CXCL13 mRNA in tissue were examined by flow cytometry and kidneys were examined by histology and immunohistochemistry. We found significantly higher serum levels of the B-cell chemoattractant CXCL13 in patients with TCMR compared to controls and patients with borderline TCMR. Moreover, in patients with acute rejection within the first week after ktx, a >5-fold CXCL13 increase was measured and correlated with B-cell infiltrates in the biopsies. In line with the clinical findings, TCMR in mice correlated with increased systemic serum-CXCL13 levels. Moreover, renal allografts had significantly higher CXCL13 mRNA expression than isogenic controls and showed interstitial CD20+ B-cell clusters and CD3+ cell infiltrates accumulating in the vicinity of renal vessels. CXCL13 blood levels correlate with B-cell involvement in TCMR and might help to identify patients at risk of a more severe clinical course of rejection.

Renal ischemia-reperfusion injury causes hypertension and renal perfusion impairment in the CD1 mice which promotes progressive renal fibrosis.

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.

Enhanced activation of interleukin-10, heme oxygenase-1, and AKT in C5aR2-deficient mice is associated with protection from ischemia reperfusion injury-induced inflammation and fibrosis.

Severe ischemia reperfusion injury (IRI) results in rapid complement activation, acute kidney injury and progressive renal fibrosis. Little is known about the roles of the C5aR1 and C5aR2 complement receptors in IRI. In this study C5aR1-/- and C5aR2-/- mice were compared to the wild type in a renal IRI model leading to renal fibrosis. C5a receptor expression, kidney morphology, inflammation, and fibrosis were measured in different mouse strains one, seven and 21 days after IRI. Renal perfusion was evaluated by functional magnetic resonance imaging. Protein abundance and phosphorylation were assessed with high content antibody microarrays and Western blotting. C5aR1 and C5aR2 were increased in damaged tubuli and even more in infiltrating leukocytes after IRI in kidneys of wild-type mice. C5aR1-/- and C5aR2-/- animals developed less IRI-induced inflammation and showed better renal perfusion than wild-type mice following IRI. C5aR2-/- mice, in particular, had enhanced tubular and capillary regeneration with less renal fibrosis. Anti-inflammatory IL-10 and the survival/growth kinase AKT levels were especially high in kidneys of C5aR2-/- mice following IRI. LPS caused bone marrow-derived macrophages from C5aR2-/- mice to release IL-10 and to express the stress response enzyme heme oxygenase-1. Thus, C5aR1 and C5aR2 have overlapping actions in which the kidneys of C5aR2-/- mice regenerate better than those in C5aR1-/- mice following IRI. This is mediated, at least in part, by differential production of IL-10, heme oxygenase-1 and AKT.

Assessment of liver ischemia reperfusion injury in mice using hepatic T2 mapping: Comparison with histopathology.

BACKGROUND: Liver ischemia reperfusion injury (IRI) occurs during liver surgery or transplantation resulting in an inflammatory response, tissue damage, and functional impairment of the organ. PURPOSE: To assess the feasibility of T2 mapping for noninvasive quantification of liver edema after partial liver IRI in mice. STUDY TYPE: Prospective, experimental study. ANIMAL MODEL: Partial liver IRI was induced in C57BL/6-mice by transient clamping of the left lateral and median liver lobes for 35 (n = 8), 45 (n = 6), 60 (n = 17), or 90 minutes (n = 5). For comparison, healthy C57BL/6-mice were examined as controls (n = 9). FIELD STRENGTH/SEQUENCE: Functional liver MRI was performed on a 7T scanner using a respiratory-triggered multiecho spin-echo sequence. ASSESSMENT: Healthy control mice and mice with partial liver IRI on day 1 after surgery, and additionally on day 7 in a subgroup with 60 minutes IRI (n = 8) were examined. Maps of T2 relaxation time of liver tissue were used to assess distribution, severity of tissue edema (mean T2 time), and the percentage of edematous liver tissue. STATISTICAL TEST: One-way analysis of variance (ANOVA) with Tukey's honest significant difference (HSD), paired t-tests, Pearson's test for correlation of MRI parameters with levels of liver enzymes, and histopathology, receiver operating characteristic (ROC) analysis. RESULTS: Significant tissue edema induced by liver IRI as compared to the control group was detected by increased mean T2 times in groups with 60 minutes (P < 0.001) and 90 minutes IRI (P < 0.001). The percentage of edematous liver tissue significantly increased with longer ischemia times (controls 3.4 ± 0.4%, 35 minutes 5.3 ± 0.6%, 45 minutes 23.3 ± 7.6%, 60 minutes 39.7 ± 3.6%, 90 minutes 51.3 ± 4.5%). Mean T2 times and the percentage of edematous liver tissue significantly correlated with elevation of liver enzymes (P < 0.001), histological evidence of liver injury (r = 0.80 and r = 0.82, P < 0.001), and neutrophil infiltration (r = 0.70 and r = 0.74, P < 0.001). In the subgroup with follow-up, the severity (P < 0.01) and extent of liver edema decreased significantly over time (P < 0.01). DATA CONCLUSION: T2 mapping allows quantification and follow-up of liver injury in mice. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2018;48:1586-1594.

Assessment of acute kidney injury with T1 mapping MRI following solid organ transplantation.

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.

Gd-EOB-DTPA-enhanced MRI for quantitative assessment of liver organ damage after partial hepatic ischaemia reperfusion injury: correlation with histology and serum biomarkers of liver cell injury.

OBJECTIVE: To evaluate Gd-EOB-DTPA-enhanced MRI for quantitative assessment of liver organ damage after hepatic ischaemia reperfusion injury (IRI) in mice. METHODS: Partial hepatic IRI was induced in C57Bl/6 mice (n = 31) for 35, 45, 60 and 90 min. Gd-EOB-DTPA-enhanced MRI was performed 1 day after surgery using a 3D-FLASH sequence. A subgroup of n = 9 animals with 60 min IRI underwent follow-up with MRI and histology 7 days after IRI. The total liver volume was determined by manual segmentation of the entire liver. The volume of functional, contrast-enhanced liver parenchyma was quantified by a region growing algorithm (visual threshold) and an automated segmentation (Otsu's method). The percentages of functional, contrast-enhanced and damaged non-enhanced parenchyma were calculated according to these volumes. MRI data was correlated with serum liver enzyme concentrations and histologically quantified organ damage using periodic acid-Schiff (PAS) staining. RESULTS: The percentage of functional (contrasted) liver parenchyma decreased significantly with increasing ischaemia times (control, 94.4 ± 3.3%; 35 min IRI, 89.3 ± 4.1%; 45 min IRI, 87.9 ± 3.3%; 60 min IRI, 68 ± 10.5%, p < 0.001 vs. control; 90 min IRI, 55.9 ± 11.5%, p < 0.001 vs. control). The percentage of non-contrasted liver parenchyma correlated with histologically quantified liver organ damage (r = 0.637, p < 0.01) and serum liver enzyme elevations (AST r = 0.577, p < 0.01; ALT r = 0.536, p < 0.05). Follow-up MRI visualized recovery of functional liver parenchyma (71.5 ± 8.7% vs. 84 ± 2.1%, p < 0.05), consistent with less histological organ damage on day 7. CONCLUSION: We demonstrated the feasibility of Gd-EOB-DTPA-enhanced MRI for non-invasive quantification of damaged liver parenchyma following IRI in mice. This novel methodology may refine the characterization of liver disease and could have application in future studies targeting liver organ damage. KEY POINTS: • Prolonged ischaemia times in partial liver IRI increase liver organ damage. • Gd-EOB-DTPA-enhanced MRI at hepatobiliary phase identifies damaged liver volume after hepatic IRI. • Damaged liver parenchyma quantified with MRI correlates with histological liver damage. • Hepatobiliary phase Gd-EOB-DTPA-enhanced MRI enables non-invasive assessment of recovery from liver injury.