Therapeutic α-1-microglobulin ameliorates kidney ischemia-reperfusion injury.

α-1-Microglobulin (A1M) is a circulating glycoprotein with antioxidant, heme-binding, and mitochondrial protection properties. The investigational drug RMC-035, a modified therapeutic A1M protein, was assessed for biodistribution and pharmacological activity in a broad set of in vitro and in vivo experiments, supporting its clinical development. Efficacy and treatment posology were assessed in various models of kidney ischemia and reperfusion injury (IRI). Real-time glomerular filtration rate (GFR), functional renal biomarkers, tubular injury biomarkers (NGAL and KIM-1), and histopathology were evaluated. Fluorescently labeled RMC-035 was used to assess biodistribution. RMC-035 demonstrated consistent and reproducible kidney protection in rat IRI models as well as in a model of IRI imposed on renal impairment and in a mouse IRI model, where it reduced mortality. Its pharmacological activity was most pronounced with combined dosing pre- and post-ischemia and weaker with either pre- or post-ischemia dosing alone. RMC-035 rapidly distributed to the kidneys via glomerular filtration and selective luminal uptake by proximal tubular cells. IRI-induced expression of kidney heme oxygenase-1 was inhibited by RMC-035, consistent with its antioxidative properties. RMC-035 also dampened IRI-associated inflammation and improved mitochondrial function, as shown by tubular autofluorescence. Taken together, the efficacy of RMC-035 is congruent with its targeted mechanism(s) and biodistribution profile, supporting its further clinical evaluation as a novel kidney-protective therapy.NEW & NOTEWORTHY A therapeutic A1M protein variant (RMC-035) is currently in phase 2 clinical development for renal protection in patients undergoing open-chest cardiac surgery. It targets several key pathways underlying kidney injury in this patient group, including oxidative stress, heme toxicity, and mitochondrial dysfunction. RMC-035 is rapidly eliminated from plasma, distributing to kidney proximal tubules, and demonstrates dose-dependent efficacy in numerous models of ischemia-reperfusion injury, particularly when administered before ischemia. These results support its continued clinical evaluation.

Alpha1-antitrypsin improves survival in murine abdominal sepsis model by decreasing inflammation and sequestration of free heme.

Background: Excessive inflammation, hemolysis, and accumulation of labile heme play an essential role in the pathophysiology of multi-organ dysfunction syndrome (MODS) in sepsis. Alpha1-antitrypsin (AAT), an acute phase protein with heme binding capacity, is one of the essential modulators of host responses to inflammation. In this study, we evaluate the putative protective effect of AAT against MODS and mortality in a mouse model of polymicrobial abdominal sepsis. Methods: Polymicrobial abdominal sepsis was induced in C57BL/6N mice by cecal ligation and puncture (CLP). Immediately after CLP surgery, mice were treated intraperitoneally with three different forms of human AAT-plasma-derived native (nAAT), oxidized nAAT (oxAAT), or recombinant AAT (recAAT)-or were injected with vehicle. Sham-operated mice served as controls. Mouse survival, bacterial load, kidney and liver function, immune cell profiles, cytokines/chemokines, and free (labile) heme levels were assessed. In parallel, in vitro experiments were carried out with resident peritoneal macrophages (MPMΦ) and mouse peritoneal mesothelial cells (MPMC). Results: All AAT preparations used reduced mortality in septic mice. Treatment with AAT significantly reduced plasma lactate dehydrogenase and s-creatinine levels, vascular leakage, and systemic inflammation. Specifically, AAT reduced intraperitoneal accumulation of free heme, production of cytokines/chemokines, and neutrophil infiltration into the peritoneal cavity compared to septic mice not treated with AAT. In vitro experiments performed using MPMC and primary MPMΦ confirmed that AAT not only significantly decreases lipopolysaccharide (LPS)-induced pro-inflammatory cell activation but also prevents the enhancement of cellular responses to LPS by free heme. In addition, AAT inhibits cell death caused by free heme in vitro. Conclusion: Data from the septic CLP mouse model suggest that intraperitoneal AAT treatment alone is sufficient to improve sepsis-associated organ dysfunctions, preserve endothelial barrier function, and reduce mortality, likely by preventing hyper-inflammatory responses and by neutralizing free heme.

Validation of CSF-1 receptor (CD115) staining for analysis of murine monocytes by flow cytometry.

CD115, the receptor for colony stimulating factor 1, is essential for survival and differentiation of monocytes and macrophages and is therefore frequently used to define monocyte subsets and their progenitors in immunological assays. However, CD115 surface expression and detection by flow cytometry is greatly influenced by cell isolation and processing methods, organ source, and disease context. In a systematic analysis of murine monocytes, we define experimental conditions that preserve or limit CD115 surface expression and staining by flow cytometry. We also find that, independent of conditions, CD115 surface levels are consistently lower in Ly6Clo monocytes than in Ly6Chi monocytes, with the exception of Ly6Clo monocytes in the bone marrow. Furthermore, in contrast to IL-34, the presence of colony stimulating factor 1 impairs CD115 antibody staining in a dose-dependent manner, which, in a model of ischemic kidney injury with elevated levels of colony stimulating factor 1, influenced quantification of kidney monocytes. Thus, staining and experimental conditions affect quantitative and qualitative analysis of monocytes and may influence experimental conclusions.

Free heme and hemopexin in acute kidney injury after cardiopulmonary bypass and transient renal ischemia.

Free heme is released from hemoproteins during hemolysis or ischemia reperfusion injury and can be pro-inflammatory. Most studies on nephrotoxicity of hemolysis-derived proteins focus on free hemoglobin (fHb) with heme as a prosthetic group. Measurement of heme in its free, non-protein bound, form is challenging and not commonly used in clinical routine diagnostics. In contrast to fHb, the role of free heme in acute kidney injury (AKI) after cardiopulmonary bypass (CPB) surgery is unknown. Using an apo-horseradish peroxidase-based assay, we identified free heme during CPB surgery as predictor of AKI in patients undergoing cardiac valve replacement (n = 37). Free heme levels during CPB surgery correlated with depletion of hemopexin (Hx), a heme scavenger-protein. In mice, the impact of high levels of circulating free heme on the development of AKI following transient renal ischemia and the therapeutic potential of Hx were investigated. C57BL/6 mice were subjected to bilateral renal ischemia/reperfusion injury for 15 min which did not cause AKI. However, additional administration of free heme in this model promoted overt AKI with reduced renal function, increased renal inflammation, and reduced renal perfusion on functional magnetic resonance imaging. Hx treatment attenuated AKI. Free heme administration to sham operated control mice did not cause AKI. In conclusion, free heme is a predictor of AKI in CPB surgery patients and promotes AKI in transient renal ischemia. Depletion of Hx in CPB surgery patients and attenuation of AKI by Hx in the in vivo model encourage further research on Hx therapy in patients with increased free heme levels during CPB surgery.

A Modified Surgical Technique for Kidney Transplantation in Mice.

Kidney transplantation in mice is a complicated and challenging surgery procedure. There are very few publications demonstrating the key steps of this operation. Therefore, this article introduces the technique and points out the surgical caveats associated with this operation. In addition, important modifications in comparison to the conventional procedure are demonstrated. Firstly, a patch of the abdominal aorta is cut and prepared so that the proximal bifurcations of the renal artery, including the ureteral artery are transected together with the donor kidney en bloc. This reduces the risk of a ureter necrosis and avoids the development of a urinary tract occlusion. Secondly, a new method of the vascular anastomosis is demonstrated that allows the operator to flexibly increase or decrease the size of the anastomosis after renal transplant reperfusion has already been initiated. This avoids the development of vessel strictures and intraabdominal bleeding. Thirdly, a technique that enables the anastomosis of the delicate donor ureter and the recipient bladder that does not cause a trauma is shown. Adopting this protocol can shorten the operation time and reduces the damage to the recipient's bladder, thereby significantly increasing the operation success rate for the recipient mice.

A Single Oral Dose of Diclofenac Causes Transition of Experimental Subclinical Acute Kidney Injury to Chronic Kidney Disease.

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.

SLAMF8 Participates in Acute Renal Transplant Rejection via TLR4 Pathway on Pro-Inflammatory Macrophages.

Background: Acute rejection (AR) in kidney transplantation is an established risk factor that reduces the survival rate of allografts. Despite standard immunosuppression, molecules with regulatory control in the immune pathway of AR can be used as important targets for therapeutic operations to prevent rejection. Methods: We downloaded the microarray data of 15 AR patients and 37 non-acute rejection (NAR) patients from Gene Expression Omnibus (GEO). Gene network was constructed, and genes were classified into different modules using weighted gene co-expression network analysis (WGCNA). Kyoto Encyclopedia of Genes and Genomes (KEGG) and Cytoscape were applied for the hub genes in the most related module to AR. Different cell types were explored by xCell online database and single-cell RNA sequencing. We also validated the SLAMF8 and TLR4 levels in Raw264.7 and human kidney tissues of TCMR. Results: A total of 1,561 differentially expressed genes were filtered. WGCNA was constructed, and genes were classified into 12 modules. Among them, the green module was most closely associated with AR. These genes were significantly enriched in 20 pathway terms, such as cytokine-cytokine receptor interaction, chemokine signaling pathway, and other important regulatory processes. Intersection with GS > 0.4, MM > 0.9, the top 10 MCC values and DEGs in the green module, and six hub genes (DOCK2, NCKAP1L, IL2RG, SLAMF8, CD180, and PTPRE) were identified. Their expression levels were all confirmed to be significantly elevated in AR patients in GEO, Nephroseq, and quantitative real-time PCR (qRT-PCR). Single-cell RNA sequencing showed that AR patient had a higher percentage of native T, CD1C+_B DC, NKT, NK, and monocytes in peripheral blood mononuclear cells (PBMCs). Xcell enrichment scores of 20 cell types were significantly different (p<0.01), mostly immune cells, such as B cells, CD4+ Tem, CD8+ T cells, CD8+ Tcm, macrophages, M1, and monocytes. GSEA suggests that highly expressed six hub genes are correlated with allograft rejection, interferon γ response, interferon α response, and inflammatory response. In addition, SLAMF8 is highly expressed in human kidney tissues of TCMR and in M1 phenotype macrophages of Raw264.7 cell line WGCNA accompanied by high expression of TLR4. Conclusion: This study demonstrates six hub genes and functionally enriched pathways related to AR. SLAMF8 is involved in the M1 macrophages via TLR4, which contributed to AR process.

Peritoneal dialysate-range hypertonic glucose promotes T-cell IL-17 production that induces mesothelial inflammation.

Peritoneal dialysis (PD) employs hypertonic glucose to remove excess water and uremic waste. Peritoneal membrane failure limits its long-term use. T-cell cytokines promote this decline. T-cell differentiation is critically determined by the microenvironment. We here study how PD-range hypertonic glucose regulates T-cell polarization and IL-17 production. In the human peritoneal cavity, CD3+ cell numbers increased in PD. Single cell RNA sequencing detected expression of T helper (Th) 17 signature genes RORC and IL23R. In vitro, PD-range glucose stimulated spontaneous and amplified cytokine-induced Th17 polarization. Osmotic controls l-glucose and d-mannose demonstrate that induction of IL-17A is a substance-independent, tonicity dose-dependent process. PD-range glucose upregulated glycolysis and increased the proportion of dysfunctional mitochondria. Blockade of reactive-oxygen species (ROS) prevented IL-17A induction in response to PD-range glucose. Peritoneal mesothelium cultured with IL-17A or IL17F produced pro-inflammatory cytokines IL-6, CCL2, and CX3CL1. In PD patients, peritoneal IL-17A positively correlated with CX3CL1 concentrations. PD-range glucose-stimulated, but neither identically treated Il17a-/- Il17f-/- nor T cells cultured with the ROS scavenger N-acetylcysteine enhanced mesothelial CX3CL1 expression. Our data delineate PD-range hypertonic glucose as a novel inducer of Th17 polarization in a mitochondrial-ROS-dependent manner. Modulation of tonicity-mediated effects of PD solutions may improve membrane survival.

TLR4 Response to LPS Is Reinforced by Urokinase Receptor.

GPI-anchored uPAR is the receptor for the extracellular serine protease urokinase-type plasminogen activator (uPA). Though uPAR role in inflammatory processes is documented, underlying mechanisms are not fully understood. In this study we demonstrate that uPAR is a part of Toll-like receptor 4 (TLR4) interactome. Downregulation of uPAR expression resulted in diminished LPS-induced TLR4 signaling, less activation of NFκB, and decreased secretion of inflammatory mediators in myeloid and non-myeloid cells in vitro. In vivo uPAR-/- mice demonstrated better survival, strongly diminished inflammatory response and better organ functions in cecal ligation and puncture mouse polymicrobial sepsis model. Mechanistically, GPI-uPAR and soluble uPAR colocalized with TLR4 on the cell membrane and interacted with scavenger receptor CD36. Our data show that uPAR can interfere with innate immunity response via TLR4 and this mechanism represents a potentially important target in inflammation and sepsis therapy.

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.

Calcium dobesilate reduces VEGF signaling by interfering with heparan sulfate binding site and protects from vascular complications in diabetic mice.

Inhibiting vascular endothelial growth factor (VEGF) is a therapeutic option in diabetic microangiopathy. However, VEGF is needed at physiological concentrations to maintain glomerular integrity; complete VEGF blockade has deleterious effects on glomerular structure and function. Anti-VEGF therapy in diabetes raises the challenge of reducing VEGF-induced pathology without accelerating endothelial cell injury. Heparan sulfate (HS) act as a co-receptor for VEGF. Calcium dobesilate (CaD) is a small molecule with vasoprotective properties that has been used for the treatment of diabetic microangiopathy. Preliminary evidence suggests that CaD interferes with HS binding sites of fibroblast growth factor. We therefore tested the hypotheses that (1) CaD inhibits VEGF signaling in endothelial cells, (2) that this effect is mediated via interference between CaD and HS, and (3) that CaD ameliorates diabetic nephropathy in a streptozotocin-induced diabetic mouse model by VEGF inhibition. We found that CaD significantly inhibited VEGF165-induced endothelial cell migration, proliferation, and permeability. CaD significantly inhibited VEGF165-induced phosphorylation of VEGFR-2 and suppressed the activity of VEGFR-2 mediated signaling cascades. The effects of CaD in vitro were abrogated by heparin, suggesting the involvement of heparin-like domain in the interaction with CaD. In addition, VEGF121, an isoform which does not bind to heparin, was not inhibited by CaD. Using the proximity ligation approach, we detected inhibition of interaction in situ between HS and VEGF and between VEGF and VEGFR-2. Moreover, CaD reduced VEGF signaling in mice diabetic kidneys and ameliorated diabetic nephropathy and neuropathy, suggesting CaD as a VEGF inhibitor without the negative effects of complete VEGF blockade and therefore could be useful as a strategy in treating diabetic nephropathy.

CX3CL1-CX3CR1 interaction mediates macrophage-mesothelial cross talk and promotes peritoneal fibrosis.

Peritoneal dialysis (PD) is limited by chronic fibrotic remodeling of the peritoneal wall, a transforming growth factor-ß (TGF-ß)-mediated process. The fractalkine (CX3CL1) receptor CX3CR1 is expressed on macrophages and monocytes, where it is a marker of TGFß expression. Detection of its ligand CX3CL1 on the peritoneal mesothelium led us to hypothesize a pathophysiologic role of CX3CL1-CX3CR1 interaction in peritoneal fibrosis. We found that CX3CL1 was expressed on peritoneal mesothelial cells from PD patients and in a murine PD model. CX3CR1, mostly expressed on macrophages in the peritoneal wall, promoted fibrosis induced by chronic dialysate exposure in the mouse model. Our data suggest a positive feedback loop whereby direct interaction with CX3CR1-expressing macrophages promotes mesothelial expression of CX3CL1 and TGFß expression. In turn, TGFß upregulates CX3CR1 in murine and human monocytic cells. Upstream, macrophage cytokines including interleukin-1ß (IL-1ß) promote mesothelial CX3CR1 and TGFß expression, providing a starting point for CX3CL1-CX3CR1 interaction. IL-1ß expression was enhanced by exposure to dialysate both in vitro and in the mouse models. Our data suggest that macrophage-mesothelial cell crosstalk through CX3CR1-CX3CL1 interaction enhances mesothelial TGFß production, promoting peritoneal fibrosis in response to dialysate exposure. This interaction could be a novel therapeutic target in PD-associated chronic peritoneal fibrosis.

Protein kinase C beta deficiency increases glucose-mediated peritoneal damage via M1 macrophage polarization and up-regulation of mesothelial protein kinase C alpha.

BACKGROUND: Peritoneal membrane (PM) damage during peritoneal dialysis (PD) is mediated largely by high glucose (HG)-induced pro-inflammatory and neo-angiogenic processes, resulting in PM fibrosis and ultrafiltration failure. We recently demonstrated a crucial role for protein kinase C (PKC) isoform α in mesothelial cells. METHODS: In this study we investigate the role of PKCß in PM damage in vitro using primary mouse peritoneal macrophages (MPMΦ), human macrophages (HMΦ) and immortalized mouse peritoneal mesothelial cells (MPMCs), as well as in vivo using a chronic PD mouse model. RESULTS: We demonstrate that PKCß is the predominant classical PKC isoform expressed in primary MPMΦ and its expression is up-regulated in vitro under HG conditions. After in vitro lipopolysaccharides stimulation PKCß-/- MPMΦ demonstrates increased levels of interleukin 6 (IL-6), tumour necrosis factor α, and monocyte chemoattractant protein-1 and drastically decrease IL-10 release compared with wild-type (WT) cells. In vivo, catheter-delivered treatment with HG PD fluid for 5 weeks induces PKCß up-regulation in omentum of WT mice and results in inflammatory response and PM damage characterized by fibrosis and neo-angiogenesis. In comparison to WT mice, all pathological changes are strongly aggravated in PKCß-/- animals. Underlying molecular mechanisms involve a pro-inflammatory M1 polarization shift of MPMΦ and up-regulation of PKCα in MPMCs of PKCß-/- mice. Finally, we demonstrate PKCß involvement in HG-induced polarization processes in HMΦ. CONCLUSIONS: PKCß as the dominant PKC isoform in MPMΦ is up-regulated by HG PD fluid and exerts anti-inflammatory effects during PD through regulation of MPMΦ M1/M2 polarization and control of the dominant mesothelial PKC isoform α.

Antagonism of profibrotic microRNA-21 improves outcome of murine chronic renal allograft dysfunction.

Chronic renal allograft dysfunction (CAD) is a major limiting factor of long-term graft survival. It is characterized by interstitial fibrosis and tubular atrophy. The underlying pathomechanisms are incompletely understood. MicroRNAs are powerful regulators of gene expression and may have an impact on various diseases by direct mRNA decay or translational inhibition. A murine model of allogenic kidney transplantation was used resulting in CAD at 6 weeks after kidney transplantation. We identified fibrosis-associated miR-21a-5p by whole miRNAome expression analysis to be among the most highly upregulated miRNAs. In vitro in renal fibroblasts, miR-21a-5p was transcriptionally activated by interleukin 6-induced signal transducer and activator of transcription 3. Co-culture of LPS-activated macrophages with renal fibroblasts increased expression levels of miR-21a-5p and markers of fibrosis and inflammation. In addition, mature miR-21a-5p was secreted by macrophages in small vesicles, which were internalized by renal fibroblasts, thereby promoting profibrotic and proinflammatory effects. Notch2 receptor was identified as a potential target of miR-21a-5p and validated by luciferase gene reporter assays. Therapeutic silencing of miR-21a-5p in mice after allogenic kidney transplantation resulted in an amelioration of CAD, as indicated by a reduction in fibrosis development, inflammatory cell influx, tissue injury and BANFF lesion scoring. In a life-supporting model, miR-21a-5p antagonism had beneficial effects on kidney function. miR-21a-5p silencing may therefore be a viable therapeutic option in the treatment of patients following kidney transplantation to halt the development of CAD.

Protein kinase C α inhibition prevents peritoneal damage in a mouse model of chronic peritoneal exposure to high-glucose dialysate.

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.

Characterization of changes in plasma and tissue oxylipin levels in LPS and CLP induced murine sepsis.

OBJECTIVE: The present study aimed to comprehensively investigate the changes in oxylipins during murine sepsis induced by lipopolysaccharide (LPS) or cecal ligation and puncture (CLP). METHODS: Twenty-four hours after induction of sepsis in male C57BL/6 mice by LPS or CLP, plasma and liver, lung, kidney and heart tissues were sampled. Oxylipin levels in plasma and tissue were quantified by means of LC-MS. Moreover, clinical chemistry parameters were determined in plasma and interleukin levels (MCP-1 and IL-6) were determined in kidney and liver. RESULTS: Elevation of liver function plasma parameters at 24 h revealed that both models were successful in the induction of sepsis. LPS induced sepsis resulted in a dramatic increase of plasma PGE2 (2,100% change in comparison to control) and other cyclooxygenase metabolites, whereas this effect was less pronounced in CLP induced sepsis (97% increase of PGE2). Plasma epoxy-fatty acids (FAs) and hydroxy-FAs and most of the dihydroxy-FAs were elevated in both models of sepsis. Changes of tissue oxylipin concentrations were organ dependent. Only few changes were detected in the lung and liver tissue, epoxy-FAs were elevated in the kidney. In the heart tissue a trend towards lower levels of hydroxy-FAs and epoxy-FAs was observed. CONCLUSION: Both murine models of sepsis are characterized by changes of oxylipins formed in all branches of the arachidonic acid (AA) cascade. The more pronounced effects in the LPS model make this model more suitable for the investigation of the AA cascade and its pharmacological modulation in sepsis.

Food Polyphenols Fail to Cause a Biologically Relevant Reduction of COX-2 Activity.

Epidemiologic studies show a correlation between the dietary intake of food polyphenols and beneficial health effects. Several in vitro studies indicate that the anti-inflammatory potential of polyphenols is, at least in part, mediated by a modulation of the enzymes of the arachidonic acid cascade, such as the prostaglandin forming cyclooxygenases (COXs). Evidence that this mode of action can be transferred to the situation in vivo is scarce. This study characterized effects of a subset of polyphenols on COX-2 expression and activity in vitro and compared the potency with known drugs. Next, the in vivo relevance of the observed in vitro effects was tested. Enzyme assays and incubations of polyphenols with the cancer cell line HCA-7 and lipopolysaccharide (LPS) stimulated primary monocytes support the hypothesis that polyphenols can effect COX-2 expression and activity in vitro. The effects were most pronounced in the monocyte assay for wogonin, apigenin, resveratrol and genistein with IC50 values of 1.5 µM, 2.6 µM, 2.8 µM and 7.4 µM. However, these values are 100- to 1000-fold higher in comparison to those of the known pharmaceuticals celecoxib, indomethacin and dexamethasone. In an animal model of LPS induced sepsis, pretreatment with polyphenols (i. p. 100 mg/kg bw) did not result in decreased plasma or tissue prostaglandin levels, whereas the positive control celecoxib effectively attenuated LPS induced prostaglandin formation. These data suggest that despite the moderate potency in vitro, an effect of polyphenols on COX-2 during acute inflammation is unlikely, even if a high dose of polyphenols is ingested.

Renal PKC-ε deficiency attenuates acute kidney injury and ischemic allograft injury via TNF-α-dependent inhibition of apoptosis and inflammation.

Acute kidney injury (AKI) increases the risk of morbidity and mortality after major surgery and transplantation. We investigated the effect of PKC-ε deficiency on AKI and ischemic allograft damage after kidney transplantation. PKC-ε-deficient and wild type (WT) control mice were subjected to 35 min of renal pedicle clamping to induce AKI. PKC-ε deficiency was associated with a marked improvement in survival and an attenuated loss of kidney function. Furthermore, functional MRI experiments revealed better renal perfusion in PKC-ε-deficient mice than in WT mice one day after IRI. Acute tubular necrosis and neutrophil infiltration were markedly reduced in PKC-ε-deficient mice. To determine whether this resistance to ischemia-reperfusion injury resulted from changes in local renal cells or infiltrating leukocytes, we studied a life-supporting renal transplant model of ischemic graft injury. We transplanted kidneys from H(2b) PKC-ε-deficient mice (129/SV) and their corresponding WT littermates into major histocompatibility complex-incompatible H(2d) recipients (BALB/c) and induced ischemic graft injury by prolonged cold ischemia time. Recipients of WT allografts developed severe renal failure and died within 10 days of transplantation. Recipients of PKC-ε-deficient allografts had better renal function and survival; they had less generation of ROS and upregulation of proinflammatory proteins (i.e., ICAM-1, inducible nitric oxide synthase, and TNF-α) and showed less tubular epithelial cell apoptosis and inflammation in their allografts. These data suggest that local renal PKC-ε expression mediates proapoptotic and proinflammatory signaling and that an inhibitor of PKC-ε signaling could be used to prevent hypoxia-induced AKI.

Autoimmunity in CD73/Ecto-5'-nucleotidase deficient mice induces renal injury.

Extracellular adenosine formed by 5'-ectonucleotidase (CD73) is involved in tubulo-glomerular feedback in the kidney but is also known to be an important immune modulator. Since CD73(-/-)mutant mice exhibit a vascular proinflammatory phenotype, we asked whether long term lack of CD73 causes inflammation related kidney pathologies. CD73(-/-)mice (13 weeks old) showed significantly increased low molecule proteinuria compared to C57BL6 wild type controls (4.8 ≥ 0.52 vs. 2.9 ± 0.54 mg/24 h, p<0.03). Total proteinuria increased to 5.97 ± 0.78 vs. 2.55 ± 0.35 mg/24 h at 30 weeks (p<0.01) whereas creatinine clearance decreased (0.161 ± 0.02 vs. 0.224 ± 0.02 ml/min). We observed autoimmune inflammation in CD73(-/-)mice with glomerulitis and peritubular capillaritis, showing glomerular deposition of IgG and C3 and enhanced presence of CD11b, CD8, CD25 as well as GR-1-positive cells in the interstitium. Vascular inflammation was associated with enhanced serum levels of the cytokines IL-18 and TNF-α as well as VEGF and the chemokine MIP-2 (CXCL-2) in CD73(-/-)mice, whereas chemokines and cytokines in the kidney tissue were unaltered or reduced. In CD73(-/-)mice glomeruli, we found a reduced number of podocytes and endothelial fenestrations, increased capillaries per glomeruli, endotheliosis and enhanced tubular fibrosis. Our results show that adult CD73(-/-)mice exhibit spontaneous proteinuria and renal functional deterioration even without exogenous stress factors. We have identified an autoimmune inflammatory phenotype comprising the glomerular endothelium, leading to glomeruli inflammation and injury and to a cellular infiltrate of the renal interstitium. Thus, long term lack of CD73 reduced renal function and is associated with autoimmune inflammation.

Protein kinase C-θ is required for murine neutrophil recruitment and adhesion strengthening under flow.

Protein kinase C (PKC)-θ is involved in T cell activation via regulating the avidity of the ß(2) integrin LFA-1 in the immunological synapse. LFA-1 also mediates leukocyte adhesion. To investigate the role of PKC-θ in neutrophil adhesion, we performed intravital microscopy in cremaster venules of mice reconstituted with bone marrow from LysM-GFP(+) (wild-type [WT]) and PKC-θ gene-deficient (Prkcq(-/-)) mice. Following stimulation with CXCL1, both WT and Prkcq(-/-) cells became adherent. Although most WT neutrophils remained adherent for at least 180 s, 50% of Prkcq(-/-) neutrophils were detached after 105 s and most by 180 s. Upon CXCL1 injection, rolling of all WT neutrophils stopped for 90 s, but rolling of Prkcq(-/-) neutrophils started 30 s after CXCL1 stimulation. A similar neutrophil adhesion defect was seen in vitro, and spreading of Prkcq(-/-) neutrophils was delayed. Prkcq(-/-) neutrophil recruitment was impaired in fMLP-induced transmigration into the cremaster muscle, thioglycollate-induced peritonitis, and LPS-induced lung injury. We conclude that PKC-θ mediates integrin-dependent neutrophil functions and is required to sustain neutrophil adhesion in postcapillary venules in vivo. These findings suggest that the role of PKC-θ in outside-in signaling following engagement of neutrophil integrins is relevant for inflammation in vivo.