Renal-associated TLR2 mediates ischemia/reperfusion injury in the kidney

TLRs are conserved pattern recognition receptors that detect motifs of pathogens and host material released during injury. For unknown reasons, renal TLR2 mRNA is mainly expressed by tubular cells and is enhanced upon renal ischemia/reperfusion (I/R) injury. We evaluated the role of TLR2 in I/R injury using TLR2-/- and TLR2+/+ mice, TLR2 antisense oligonucleotides, and chimeric mice deficient in leukocyte or renal TLR2. Tubular cells needed TLR2 to produce significant cytokine and chemokine amounts upon ischemia in vitro. TLR2 played a proinflammatory and detrimental role in vivo after I/R injury, as reflected by a reduction in the amount of local cytokines and chemokines, leukocytes, and the level of renal injury and dysfunction in TLR2-/- mice compared with controls. Analysis of chimeric mice suggested that TLR2 expressed on renal parenchyma plays a crucial role in the induction of inflammation and injury. TLR2-antisense treatment protected mice from renal dysfunction, neutrophil influx, and tubular apoptosis after I/R injury compared with nonsense treatment. In summary, we identified renal-associated TLR2 as an important initiator of inflammatory responses leading to renal injury and dysfunction in I/R injury. These data imply that TLR2 blockade could provide a basis for therapeutic strategies to treat or prevent renal ischemic injury.     

C1D is a major autoantibody target in patients with the polymyositis-scleroderma overlap syndrome

OBJECTIVE: To assess whether the recently discovered exosome-associated proteins MPP6, C1D, KIAA0052/hMtr4, hSki2, and hSki8 are targeted by autoantibodies, and to determine whether these autoantibodies are accompanied by antibodies directed to the established exosome-associated autoantigens PM-Scl-75 and PM-Scl-100. METHODS: Complementary DNAs encoding the recently identified human exosome-associated proteins were expressed as His-tagged fusion proteins in Escherichia coli cells. Sera obtained from patients with several different autoimmune diseases were analyzed for the presence of autoantibodies directed to these proteins, in an enzyme-linked immunosorbent assay (ELISA). The ELISA data obtained for C1D were confirmed by Western blot analysis, using recombinant C1D. RESULTS: All exosome-associated proteins were found to be targeted by autoantibodies, although the frequency with which such antibodies occurred in patient sera was relatively low, with the exception of anti-C1D antibodies. Autoantibodies recognizing C1D were detected in 7 of 30 patients (23%) with the polymyositis (PM)-scleroderma overlap syndrome; this frequency was similar to the frequencies for the established autoantigens PM-Scl-75c (27%) and PM-Scl-100 (23%). Importantly, several patients with the PM-scleroderma overlap syndrome had anti-C1D antibodies but no anti-PM-Scl antibodies. Anti-C1D autoantibodies were observed in only 2 of 204 patients with other diseases, including PM, dermatomyositis, and scleroderma. CONCLUSION: Our results demonstrate that the recently identified exosome-associated protein C1D is a major autoantigen in patients with the PM-scleroderma overlap syndrome and suggest that the use of recombinant C1D as an autoantibody target may aid in diagnosis of the PM-scleroderma overlap syndrome.     

Epac-Rap Signaling Reduces Oxidative Stress in the Tubular Epithelium

Activation of Rap1 by exchange protein activated by cAMP (Epac) promotes cell adhesion and actin cytoskeletal polarization. Pharmacologic activation of Epac-Rap signaling by the Epac-selective cAMP analog 8-pCPT-2′-O-Me-cAMP during ischemia-reperfusion (IR) injury reduces renal failure and application of 8-pCPT-2′-O-Me-cAMP promotes renal cell survival during exposure to the nephrotoxicant cisplatin. Here, we found that activation of Epac by 8-pCPT-2′-O-Me-cAMP reduced production of reactive oxygen species during reoxygenation after hypoxia by decreasing mitochondrial superoxide production. Epac activation prevented disruption of tubular morphology during diethyl maleate–induced oxidative stress in an organotypic three-dimensional culture assay. In vivo renal targeting of 8-pCPT-2′-O-Me-cAMP to proximal tubules using a kidney-selective drug carrier approach resulted in prolonged activation of Rap1 compared with nonconjugated 8-pCPT-2′-O-Me-cAMP. Activation of Epac reduced antioxidant signaling during IR injury and prevented tubular epithelial injury, apoptosis, and renal failure. Our data suggest that Epac1 decreases reactive oxygen species production by preventing mitochondrial superoxide formation during IR injury, thus limiting the degree of oxidative stress. These findings indicate a new role for activation of Epac as a therapeutic application in renal injury associated with oxidative stress.Renal ischemia-reperfusion (IR) injury is an important cause of AKI¹ and a significant risk factor for the development of renal dysfunction after kidney transplantation.² During IR injury, morphologic and functional alterations of the proximal tubular epithelium occur that are linked to the development of renal failure and activation of immune cells via release of proinflammatory cytokines.³Exchange protein activated by cAMP (Epac) is a guanine nucleotide exchange factor for the small GTPase Rap1.⁴ Activation of Epac by cAMP or by the Epac-selective cAMP analog 8-pCPT-2′-O-Me-cAMP (also referred to as 007) induces functional activation of Rap1.⁵ Initial studies showed that Epac-Rap signaling enhances cell adhesion by supporting maturation of cell-cell junctions^6,7 and promoting integrin-mediated cell-matrix adhesion.^8,9 In line with these studies, we recently demonstrated that selective activation of Epac reduces proximal tubular epithelial cell (PTEC) detachment during IR injury using in vitro and in vivo models.¹⁰ Activation of Epac-Rap was associated with reduced expression of markers for cellular stress in PTECs. In addition, in vitro cisplatin-induced apoptosis of PTECs could be significantly reduced by activation of Epac and this was also associated with improved adhesion of cells.¹¹ On the basis of these findings, we hypothesized that activation of Epac-Rap signaling may protect against a common cytotoxic event in these injury models.Unbalanced and uncontrolled production of reactive oxygen species (ROS) is an important mediator of cell injury and occurs during cisplatin nephrotoxicity,¹² IR injury,¹³ and renal fibrosis.¹⁴ In renal pathology, intracellular ROS can be produced enzymatically such as by NADPH oxidase (NOX) complexes or derive from dysfunctional mitochondrial activity. Mitochondrial ROS production appears to be the driving force behind hypoxia-reoxygenation cell injury¹⁵ and cisplatin cytotoxicity.¹⁶Here we studied the role of specific proximal tubular activation of Epac and how this protects against renal injury in both in vitro and in vivo models for IR injury. We found that ROS production during reoxygenation after hypoxia was decreased by activation of Epac. Selective proximal tubular activation of Epac by renal targeting of 8-pCPT-2′-O-Me-cAMP conjugated to lysozyme (LZM-007) reduced oxidative stress in an in vivo model for IR injury and significantly decreased IR injury–associated renal failure and tubular damage. Our data show that Epac activation reduces ROS-mediated cellular injury in renal disease and may be a therapeutic strategy for modulation of oxidative stress.     

Left ventricular thrombus

Opinion statement Left ventricular thrombus (LVT) is a frequent complication in patients with acute anterior myocardial infarction (MI) and in those with dilated cardiomyopathy (DCM). The clinical importance of LVT lies in its potential to embolize. The current treatment of patients with acute MI centers on reperfusion, and although controversial, the incidence of LVT complicating acute anterior MI is probably reduced when compared with historical controls. Nevertheless, stroke continues to be a clinically important complication of acute MI and is most common in patients with anterior MI, in part secondary to embolization of LVT. Therapeutic anticoagulation during acute MI reduces the incidence of LVT, and long-term anticoagulation has been associated with a reduction in recurrent infarction and ischemic stroke, but carries hemorrhagic risk. Primary treatment strategies for patients with acute MI center on reperfusion therapy followed by antiplatelet agents and pharmacologic blockade of abnormal neurohumoral mechanisms. Strategies to prevent stroke following infarction include risk stratification for development of LVT and embolism. For patients with anterior MI, particularly those with apical akinesis or dyskinesis, therapeutic anticoagulation reduces the number of LVT and cardioembolic strokes. However, the absolute number of ischemic strokes prevented with this strategy may only be marginal, given the anticoagulation risk, particularly if antiplatelet agents are used concurrently. An attractive alternative strategy is echocardiographic evaluation following anterior infarction with therapeutic anticoagulation reserved for those with demonstrable thrombus. The efficacy of this strategy, however, never has been proven in a clinical study. Primary prevention of cardioembolic stroke through therapeutic anticoagulation is controversial in patients with DCM; the greatest benefit would be expected for those with severe left ventricular dysfunction. If LVT is detected during the course of MI or DCM, therapeutic anticoagulation is usually indicated with the expectation that the majority of thrombi will resolve without clinical evidence of systemic embolism. Additional therapeutic intervention is rarely needed.     

Quantification of oral mucositis due to radiotherapy by determining viability and maturation of epithelial cells

BACKGROUND: An in-vitro assay has been developed for quantitative assessment of chemotherapy induced oral mucositis. In the present study this method was evaluated for assessment of irradiation mucositis at a cellular level. METHODS: Ten patients participated in this consecutive study. All patients were treated with conventional fractionated curative postoperative radiotherapy. Prior to, and weekly during, the irradiation course, oral washings were obtained to determine viability of epithelial cells by trypan blue dye exclusion. Maturation of epithelial cells was assessed from smears (Papanicolaou staining). The viability data were compared with the WHO-score for mucositis. RESULTS: Epithelial cell viability increased during the first three weeks of radiation (P = 0.04), and was seen earlier than the subjective mucosal changes with the WHO-score. Cell maturity shifted from immature and intermediate to mature (P = 0.03). CONCLUSIONS: The cell viability assay can be considered an objective method for following the development of irradiation mucositis, and seems to be more sensitive during the first three weeks of irradiation than the WHO-scoring method.     

Preventive intervention possibilities in radiotherapy- and chemotherapy-induced oral mucositis: results of meta-analyses

The aim of these meta-analyses was to evaluate the effectiveness of interventions for the prevention of oral mucositis in cancer patients treated with head and neck radiotherapy and/or chemotherapy, with a focus on randomized clinical trials. A literature search was performed for reports of randomized controlled clinical studies, published between 1966 and 2004, the aim of which was the prevention of mucositis in cancer patients undergoing head and neck radiation, chemotherapy, or chemoradiation. The control group consisted of a placebo, no intervention, or another intervention group. Mucositis was scored by either the WHO, the National Cancer Institute-Common Toxicity Criteria (NCI-CTC) score, or the absence or presence of ulcerations, or the presence or absence of grades 3 and 4 mucositis. The meta-analyses included 45 studies fulfilling the inclusion criteria, in which 8 different interventions were evaluated: i.e., local application of chlorhexidine; iseganan; PTA (polymyxin E, tobramycine, and amphotericin B); granulocyte macrophage-colony-stimulating factor/granulocyte colony-stimulating factor (GM-CSF/G-CSF); oral cooling; sucralfate and glutamine; and systemic administration of amifostine and GM-CSF/G-CSF. Four interventions showed a significant preventive effect on the development or severity of oral mucositis: PTA with an odds ratio (OR) = 0.61 (95% confidence interval [CI], 0.39-0.96); GM-CSF, OR = 0.53 (CI: 0.33-0.87); oral cooling, OR = 0.3 (CI: 0.16-0.56); and amifostine, OR = 0.37 (CI: 0.15-0.89). To date, no single intervention completely prevents oral mucositis, so combined preventive therapy strategies seem to be required to ensure more successful outcomes.     

Assessment of oral mucositis in clinical trials: impact of training on evaluators in a multi-centre trial

In the assessment of mucositis, the inter-evaluator variability needs to be minimised and would likely to be best accomplished by training. The aim of this study was to evaluate the effect of training on concordance of evaluators in scoring oral mucositis. The evaluators were informed about the pathobiology and clinical appearance of mucositis and were trained in scoring mucositis according the Oral Mucositis Assessment Scale (OMAS). The effect of the training was evaluated by a pre- and post-training test. Each test consisted of 15 slides depicting oral mucositis. The pre- and post-training scores were compared to the reference standard. During 8 months at 6 meetings, 65 evaluators were trained. The mean percentage correctly scored slides according the OMAS increased significantly between the pre- and post-training test (P<0.001). Training evaluators in scoring oral mucositis has a significant improvement on the outcome of mucositis assessment.     

N-Acetylcysteine improves the disturbed thiol redox balance after methionine loading

Methionine loading seems to be accompanied by increased oxidative stress and damage. However, it is not known how this oxidative stress is generated. We performed the present crossover study to further elucidate the effects of methionine loading on oxidative stress in the blood of healthy volunteers, and to examine possible preventative effects of N -acetylcysteine (NAC) administration. A total of 18 healthy subjects were given two oral methionine loads of 100 mg/kg body weight, 4 weeks apart, one without NAC (Met group), and one in combination with supplementation with 2x900 mg doses of NAC (Met+NAC group). Blood samples were collected before and 2, 4, 8 and 24 h after methionine loading for measurements of thiol levels, protein carbonyls, lipid peroxidation, cellular fibronectin and ferric reducing ability of plasma (FRAP; i.e. antioxidant capacity). After methionine loading, whole-blood levels of free and oxidized cysteine and homocysteine were increased in both groups. Furthermore, the total plasma levels of homocysteine were higher, whereas those of cysteine were lower, after methionine loading in both groups. Lower levels of oxidized homocysteine and a higher free/oxidized ratio were found in the Met+NAC group compared with the Met group. Although the antioxidant capacity decreased after methionine loading, no major changes over time were found for protein carbonyls or cellular fibronectin in either group. Our results suggest that methionine loading may initiate the generation of reactive oxygen species by the (auto)-oxidation of homocysteine. In addition, supplementation with NAC seems to be able to partially prevent excessive increases in the levels of homocysteine in plasma and of oxidized homocysteine in whole blood, and might thereby contribute to the prevention of oxidative stress.     

Enhanced mobilization of bone marrow cells does not ameliorate renal fibrosis.

BACKGROUND: The plasticity of bone marrow-derived stem cells, also comprising haematopoietic stem cells, has been shown to extend to renal epithelial lineages. Yet, the low rate of their contribution to the injured kidney has led to questions regarding their significance in tissue repair after acute injury. We describe here the effect of stem cell mobilization therapy on the progression of renal fibrosis in a mouse model of chronic obstructive nephropathy. METHODS: Mice were subjected to unilateral ureter obstruction (UUO) and treated with stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) or saline. Circulating cells were analysed by flow cytometry; labelled bone marrow c-KIT(HIGH) cells were injected into animals subjected to UUO. Granulocytes, macrophages, cellular proliferation or apoptosis and myofibroblasts were detected by immunostaining. Collagen deposition was determined by measuring renal hydroxyproline contents. Cytokine levels were measured by ELISA. RESULTS: SCF/G-CSF treatment of mice induced significant haematopoietic stem and progenitor cell mobilization from the bone marrow. Although these cells are able to migrate to the obstructed kidney, they did not influence renal damage, fibrosis and inflammatory cell influx. CONCLUSIONS: Although SCF/G-CSF treatment significantly enhanced the availability of haematopoietic stem cells to the obstructed kidney, the progression of renal fibrosis could not be delayed or halted. Our results indicate that effective stem cell mobilization does not alter renal fibrosis.