Neutral ceramidase deficiency protects against cisplatin-induced acute kidney injury

Cisplatin is a commonly used chemotherapeutic for the treatment of many solid organ cancers; however, its effectiveness is limited by the development of acute kidney injury (AKI) in 30% of patients. AKI is driven by proximal tubule cell death, leading to rapid decline in renal function. It has previously been shown that sphingolipid metabolism plays a role in regulating many of the biological processes involved in cisplatin-induced AKI. For example, neutral ceramidase (nCDase) is an enzyme responsible for converting ceramide into sphingosine, which is then phosphorylated to become sphingosine-1-phosphate, and our lab previously demonstrated that nCDase knockout (nCDase?/?) in mouse embryonic fibroblasts led to resistance to nutrient and energy deprivation–induced cell death via upregulation of autophagic flux. In this study, we further characterized the role of nCDase in AKI by demonstrating that nCDase?/? mice are resistant to cisplatin-induced AKI. nCDase?/? mice display improved kidney function, reduced injury and structural damage, lower rates of apoptosis, and less ER stress compared to wild-type mice following cisplatin treatment. Although the mechanism of protection is still unknown, we propose that it could be mediated by increased autophagy, as chloroquine treatment resensitized nCDase?/? mice to AKI development. Taken together, we conclude that nCDase may represent a novel target to prevent cisplatin-induced nephrotoxicity.     

Genetic deficiency of neuronal RAGE protects against AGE-induced synaptic injury

Synaptic dysfunction and degeneration is an early pathological feature of aging and age-related diseases, including Alzheimer''s disease (AD). Aging is associated with increased generation and deposition of advanced glycation endproducts (AGEs), resulting from nonenzymatic glycation (or oxidation) proteins and lipids. AGE formation is accelerated in diabetes and AD-affected brain, contributing to cellular perturbation. The extent of AGEs'' involvement, if at all, in alterations in synaptic structure and function is currently unknown. Here we analyze the contribution of neuronal receptor of AGEs (RAGE) signaling to AGE-mediated synaptic injury using novel transgenic neuronal RAGE knockout mice specifically targeted to the forebrain and transgenic mice expressing neuronal dominant-negative RAGE (DN-RAGE). Addition of AGEs to brain slices impaired hippocampal long-term potentiation (LTP). Similarly, treatment of hippocampal neurons with AGEs significantly decreases synaptic density. Such detrimental effects are largely reversed by genetic RAGE depletion. Notably, brain slices from mice with neuronal RAGE deficiency or DN-RAGE are resistant to AGE-induced LTP deficit. Further, RAGE deficiency or DN-RAGE blocks AGE-induced activation of p38 signaling. Taken together, these data show that neuronal RAGE functions as a signal transducer for AGE-induced synaptic dysfunction, thereby providing new insights into a mechanism by which the AGEs–RAGE-dependent signaling cascade contributes to synaptic injury via the p38 MAP kinase signal transduction pathway. Thus, RAGE blockade may be a target for development of interventions aimed at preventing the progression of cognitive decline in aging and age-related neurodegenerative diseases.Advanced glycation endproducts (AGEs) are members of a heterogeneous class of molecules, which modify cellular function by distinct mechanisms, including ligation and activation of signal transduction receptors. The products of non-enzymatic glycation (or oxidation) of proteins and lipids, AGEs contribute to the normal aging process and when accelerated have a causative role in the vasculature complications of diabetes mellitus and several neurodegenerative diseases, including Alzheimer''s (AD), Parkinson''s, and Huntington''s diseases.^{1, 2, 3, 4, 5} In diabetic patients, the concentration of circulating AGEs (serum AGE level) has been reported at 7.2–22 mU/ml (equivalent to 30–88 μg/ml AGE-BSA), which is significantly higher than that of non-diabetic patients (3 mU/ml, equivalent to 12 μg/ml AGE-BSA).^{6, 7, 8} The brain AGE level was also increased to 5-6 μM (equivalent to 325–390 μg/ml AGE-BSA) in the diabetic animal model.⁹ Excess AGE accumulation is detrimental to neurons and is believed to be a key to the pathogenesis of cognitive decline in normal aging and specific chronic diseases of aging. For example, in a recent clinical study, peripheral AGE levels were associated with cognitive decline in older adults with and without diabetes.¹⁰ Diabetes complications affect the brain, increasing risk for depression, dementia, and AD. In fact, patients with type 2 diabetes are at twofold to threefold increased relative risk for AD^{11, 12, 13, 14, 15, 16, 17, 18} and accelerated cognitive dysfunction.Long-lived proteins such as β-amyloid peptide (Aβ) and hyperphosphorylated tau protein that accumulate in AD brain are highly susceptible to AGE modification.^{19, 20, 21, 22} AGE-modified Aβ or tau protein results in increased oxidative stress and chronic inflammation, accelerating AD pathology and neuronal perturbation.^{19, 20, 22, 23, 24, 25} Moreover, Aβ or tau glycation results in increased aggregation and subsequent formation of senile plaques or neurofibrillary tangles, the major pathological feature of AD,^{19, 22} suggesting that AGE modification is an important risk factor for neurodegenerative diseases.²⁶ Although increased accumulation of AGEs in brain, as seen in aging, diabetes, or neurodegenerative diseases, speeds up oxidative damage to neurons contributing to synaptic dysfunction and cognitive decline, its underlying mechanisms are not well understood.Receptor for advanced glycation endproduct (RAGE) was first identified as a cell surface receptor of the immunoglobulin superfamily for AGEs.^{27, 28} Increased expression of RAGE occurs in neuronal and non-neuronal cells in the peripheral and central nervous system in aging, diabetes, and AD-affected individuals, where RAGE ligands are upregulated.^{29, 30} Although it has been shown that AGEs–RAGE interaction contributes to cellular perturbation relevant to the pathogenesis of the cardiovascular disease and the diabetes vascular complications,^{31, 32, 33} little is known about the role of AGEs and its interaction with RAGE on synaptic dysfunction. To understand the mechanisms involved in AGE-mediated synaptic damage, the following questions need to be addressed: (1) ‘Do AGEs alter synaptic structure and function? If so, are these changes dependent on RAGE signaling?'' (2) ‘Does RAGE blockage by genetic depletion protect from AGE-induced synaptic dysfunction and loss?'' and (3) ‘What is the impact of neuronal RAGE in AGE-induced aberrant synaptic function?''. Thus it is important to evaluate the impact of AGEs–RAGE interaction on synaptic dysfunction and to explore the mechanism underlying AGE–RAGE-dependent signal transduction and its contribution to synaptic damage.Here we investigate neuronal RAGE signaling in AGE-induced synaptic injury using our novel conditional RAGE knockout mice targeted to cortical neurons as well as transgenic mice that overexpress signal transduction-deficient mutants of RAGE in neurons. Given that neuronal and non-neuronal cells in the brain may contribute to AGE-induced sustained neuronal and synaptic stress and dysfunction, we assessed the impact of global RAGE deletion in this setting and further delineated the mechanism by which RAGE-dependent activation of p38 MAP kinase potentiates AGE-insulted synaptic injury.     

Genetic inactivation of RIP1 kinase activity in rats protects against ischemic brain injury

RIP1 kinase-mediated inflammatory and cell death pathways have been implicated in the pathology of acute and chronic disorders of the nervous system. Here, we describe a novel animal model of RIP1 kinase deficiency, generated by knock-in of the kinase-inactivating RIP1(D138N) mutation in rats. Homozygous RIP1 kinase-dead (KD) rats had normal development, reproduction and did not show any gross phenotypes at baseline. However, cells derived from RIP1 KD rats displayed resistance to necroptotic cell death. In addition, RIP1 KD rats were resistant to TNF-induced systemic shock. We studied the utility of RIP1 KD rats for neurological disorders by testing the efficacy of the genetic inactivation in the transient middle cerebral artery occlusion/reperfusion model of brain injury. RIP1 KD rats were protected in this model in a battery of behavioral, imaging, and histopathological endpoints. In addition, RIP1 KD rats had reduced inflammation and accumulation of neuronal injury biomarkers. Unbiased proteomics in the plasma identified additional changes that were ameliorated by RIP1 genetic inactivation. Together these data highlight the utility of the RIP1 KD rats for target validation and biomarker studies for neurological disorders.Subject terms: Cell death in the nervous system, Diseases of the nervous system     

Leptin protects hippocampal CA1 neurons against ischemic injury

Leptin is an adipose hormone with well characterized roles in regulating food intake and energy balance. A novel neuroprotective role for leptin has recently been discovered; however, the underlying mechanisms are not clearly defined. The purpose of this study was to determine whether leptin protects against delayed neuronal cell death in hippocampal CA1 following transient global cerebral ischemia in rats and to study the signaling mechanism responsible for the neuroprotective effects of leptin. Leptin receptor antagonist, protein kinase inhibitors and western blots were used to assess the molecular signaling events that were altered by leptin after ischemia. The results revealed that intracerebral ventricle infusion of leptin markedly increased the numbers of survival CA1 neurons in a dose-dependent manner. Infusion of a specific leptin antagonist 10 min prior to transient global ischemia abolished the pro-survival effects of leptin, indicating the essential role of leptin receptors in mediating this neuroprotection. Both the Akt and extracellular signal-related kinase 1/2 (ERK1/2) signaling pathways appear to play a critical role in leptin neuroprotection, as leptin infusion increased the phosphorylation of Akt and ERK1/2 in CA1. Furthermore, pharmacological inhibition of either pathway compromised the neuroprotective effects of leptin. Taken together, the results suggest that leptin protects against delayed ischemic neuronal death in the hippocampal CA1 by maintaining the pro-survival states of Akt and ERK1/2 MAPK signaling pathways.     

Estradiol protects against ischemic brain injury in middle-aged rats

Several clinical studies suggest that estradiol acts as a potent growth and protective factor in the adult brain. Postmenopausal women experience permanent hypoestrogenicity and suffer from increased risk of brain injury associated with neurodegenerative diseases such as stroke and Alzheimer's disease. Estrogen replacement therapy appears to decrease the risk and severity of these neurodegenerative conditions. Studies using animal models have shown that estradiol exerts similar effects in rodents and can enhance cell survival and induce synaptic plasticity. Therefore, we undertook studies to assess whether estradiol treatment can decrease brain injury and cell death induced by an experimental model of ischemia and whether aging animals remain responsive to the protective effects of estradiol. We will review results from recent studies that demonstrate that 1) in young animals, estrogens exert profound protective effects against ischemic brain injury induced by cerebral artery occlusion and 2) the response of aging animals has been tested with varying results. We will discuss and compare our experimental findings that utilize a permanent cerebral artery occlusion model and physiological levels of estradiol replacement therapy in young and middle-aged rats with those of previous studies. These observations provide important insights into the potential protective actions of estrogen replacement therapy on age- and disease-related processes in the brain.     

Transduced Tat-SOD fusion protein protects against ischemic brain injury

Reactive oxygen species (ROS) are implicated in reperfusion injury after transient focal cerebral ischemia. The antioxidant enzyme, Cu,Zn-superoxide dismutase (SOD), is one of the major means by which cells counteract the deleterious effects of ROS after ischemia. Recently, we reported that when Tat-SOD fusion protein is transduced into pancreatic beta cells it protects the beta cells from destruction by relieving oxidative stress in ROS-implicated diabetes (Eum et al., 2004). In the present study, we investigated the protective effects of Tat-SOD fusion protein against neuronal cell death and ischemic insults. When Tat-SOD was added to the culture medium of neuronal cells, it rapidly entered the cells and protected them against paraquat-induced cell death. Immunohistochemical analysis revealed that Tat-SOD injected intraperitoneally (i.p.) into mice has access to various tissues including brain neurons. When i.p. injected into gerbils, Tat-SOD prevented neuronal cell death in the hippocampus in response to transient fore-brain ischemia. These results suggest that Tat-SOD provides a strategy for therapeutic delivery in various hu-man diseases, including stroke, related to this anti-oxidant enzyme or to ROS.     

SCD1 deficiency protects mice against ethanol-induced liver injury

Stearoyl-CoA desaturase 1 (SCD1) is a delta-9 fatty acid desaturase that catalyzes the synthesis of mono-unsaturated fatty acids (MUFA). SCD1 is a critical control point regulating hepatic lipid synthesis and β-oxidation. Scd1 KO mice are resistant to the development of diet-induced non-alcoholic fatty liver disease (NAFLD). Using a chronic-binge protocol of ethanol-mediated liver injury, we aimed to determine if these KO mice are also resistant to the development of alcoholic fatty liver disease (AFLD).Mice fed a low-fat diet (especially low in MUFA) containing 5% ethanol for 10 days, followed by a single ethanol (5 g/kg) gavage, developed severe liver injury manifesting as hepatic steatosis. This was associated with an increase in de novo lipogenesis and inflammation. Using this model, we show that Scd1 KO mice are resistant to the development of AFLD. Scd1 KO mice do not show accumulation of hepatic triglycerides, activation of de novo lipogenesis nor elevation of cytokines or other pro-inflammatory markers. Incubating HepG2 cells with a SCD1 inhibitor induced a similar resistance to the effect of ethanol, confirming a role for SCD1 activity in mediating ethanol-induced hepatic injury.Taken together, our study shows that SCD1 is a key player in the development of AFLD and associated deleterious effects, and suggests SCD1 inhibition as a therapeutic option for the treatment of this hepatic disease.     

Smad3 deficiency in mast cells provides efficient host protection against acute septic peritonitis

Mast cells play an important role in innate immunity as well as in allergic reaction. However, regulatory mechanisms underlying mast cell-mediated innate immune responses remain largely unknown. Here we determined whether Smad3, a major signal transducer of TGF-beta, regulates innate immune response by mast cells against Gram-negative bacteria. Bone marrow-derived mast cells (BMMC) obtained from Smad3 null mutant mice showed augmented capacity to produce proinflammatory cytokines upon stimulation with a Gram-negative bacteria-associated product, LPS. In acute septic peritonitis model induced by cecal ligation and puncture, mast cell-deficient W/W(v) mice reconstituted with Smad3 null BMMC had significantly higher survival rate than W/W(v) mice reconstituted with wild-type BMMC, which was associated with higher production of proinflammatory cytokines in the peritoneal cavity. These in vitro and in vivo results suggest that Smad3 in mast cells functions as inhibitory for mast cell-mediated innate immune response against Gram-negative bacteria. Suppression of Smad3 expression in mast cells may thus have therapeutic potential for Gram-negative bacterial infection such as acute septic peritonitis by augmenting innate immune responses of mast cells.     

MiR-150-5p protects against septic acute kidney injury via repressing the MEKK3/JNK pathway

BackgroundSeptic acute kidney injury (AKI) is associated with increased morbidity and mortality in critically ill patients. MicroRNA is reportedly involved in sepsis-induced organ dysfunction, while the role of miR-150 in septic AKI remains ambiguous.MethodsQuantitative real-time PCR (qRT-PCR) was carried out to examine miR-150-5p expression in both septic AKI patients and volunteers without septic AKI. Lipopolysaccharide (LPS) was used to treat renal tubular epithelial cell line HK-2 and C57/BL6 mice to establish in vitro and in vivo sepsis-induced AKI models. Cell apoptosis was determined using TdT-mediated dUTP nick end labeling (TUNEL) staining and flow cytometry. Cell viability was tested using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Renal pathological changes were examined via Hematoxylin-Eosin (H&E) staining, and renal function was measured via blood urea nitrogen (BUN) and creatinine (Cre) measurements. The MEKK3/JNK profile and oxidative stress markers (including COX2 and iNOS) were examined by immunoblot analysis, and the expression levels of inflammatory cytokines (TNF-α, IL-6, and IL-1β) and oxidative stress markers (MDA, SOD, and CAT) were evaluated by ELISA.ResultsMiR-150-5p was down-regulated in the serum of patients with septic AKI (compared to healthy volunteers). Moreover, miR-150-5p levels were lower in LPS-treated HK-2 cell lines and in the septic AKI mouse model. Additionally, Stat-3 activation mediated the decrease of miR-150-5p. Functionally, miR-150-5p agomir attenuated LPS-induced apoptosis in HK-2 cells, in addition to renal inflammatory responses and oxidative stress. In contrast, inhibition of miR-150-5p aggravated LPS-induced apoptosis, inflammatory reactions and oxidative stress. Furthermore, miR-150-5p agomir decreased BUN and Scr levels in the septic AKI mice model repressed TNF-α, IL-6 and IL-1β, and up-regulated SOD and CAT down-regulated MDA in the kidney tissues. Moreover, miR-150-5p was identified as a target gene for Stat3, and the overexpression of Stat3 partially promoted the effect of down-regulating miR-150-5p on LPS-induced HK2 cell injury. Mechanistically, the MEKK3/JNK pathway was identified as a functional target of miR-150-5p, and the knockdown of MEKK3 showed protective effects against LPS mediated HK-2 cell apoptosis.ConclusionStat3-mediated miR-150-5p exerted protective effects in sepsis-induced acute kidney injury by regulating the MEKK3/JNK pathway.     

Genetic ablation of Pannexin1 protects retinal neurons from ischemic injury

Pannexin1 (Panx1) forms large nonselective membrane channel that is implicated in paracrine and inflammatory signaling. In vitro experiments suggested that Panx1 could play a key role in ischemic death of hippocampal neurons. Since retinal ganglion cells (RGCs) express high levels of Panx1 and are susceptible to ischemic induced injury, we hypothesized that Panx1 contributes to rapid and selective loss of these neurons in ischemia. To test this hypothesis, we induced experimental retinal ischemia followed by reperfusion in live animals with the Panx1 channel genetically ablated either in the entire mouse (Panx1 KO), or only in neurons using the conditional knockout (Panx1 CKO) technology. Here we report that two distinct neurotoxic processes are induced in RGCs by ischemia in the wild type mice but are inactivated in Panx1KO and Panx1 CKO animals. First, the post-ischemic permeation of RGC plasma membranes is suppressed, as assessed by dye transfer and calcium imaging assays ex vivo and in vitro. Second, the inflammasome-mediated activation of caspase-1 and the production of interleukin-1β in the Panx1 KO retinas are inhibited. Our findings indicate that post-ischemic neurotoxicity in the retina is mediated by previously uncharacterized pathways, which involve neuronal Panx1 and are intrinsic to RGCs. Thus, our work presents the in vivo evidence for neurotoxicity elicited by neuronal Panx1, and identifies this channel as a new therapeutic target in ischemic pathologies.     

Matrix metalloproteinase-10 protects against acute kidney injury by augmenting epidermal growth factor receptor signaling

Matrix metalloproteinase-10 (MMP-10) is a zinc-dependent endopeptidase involved in regulating a wide range of biologic processes, such as apoptosis, cell proliferation, and tissue remodeling. However, the role of MMP-10 in the pathogenesis of acute kidney injury (AKI) is unknown. In this study, we show that MMP-10 was upregulated in the kidneys and predominantly localized in the tubular epithelium in various models of AKI induced by ischemia/reperfusion (IR) or cisplatin. Overexpression of exogenous MMP-10 ameliorated AKI, manifested by decreased serum creatinine, blood urea nitrogen, tubular injury and apoptosis, and increased tubular regeneration. Conversely, knockdown of endogenous MMP-10 expression aggravated kidney injury. Interestingly, alleviation of AKI by MMP-10 in vivo was associated with the activation of epidermal growth factor receptor (EGFR) and its downstream AKT and extracellular signal-regulated kinase-1 and 2 (ERK1/2) signaling. Blockade of EGFR signaling by erlotinib abolished the MMP-10-mediated renal protection after AKI. In vitro, MMP-10 potentiated EGFR activation and protected kidney tubular cells against apoptosis induced by hypoxia/reoxygenation or cisplatin. MMP-10 was colocalized with heparin-binding EGF-like growth factor (HB-EGF) in vivo and activated it by a process of proteolytical cleavage in vitro. These studies identify HB-EGF as a previously unrecognized substrate of MMP-10. Our findings also underscore that MMP-10 can protect against AKI by augmenting EGFR signaling, leading to promotion of tubular cell survival and proliferation after injury.Subject terms: Apoptosis, Cell growth     

Schizandrin protects H9c2 cells against lipopolysaccharide‐induced injury by downregulating Smad3

Schizandrin is a major bioactive constituent of Schisandra chinensis (Turcz.) Baill with antioxidant and anti‐inflammatory properties. The objective of this study was to explore the potential effects of schizandrin on a cell model of myocarditis. The H9c2 cells were treated with schizandrin alone or in combination with lipopolysaccharide (LPS), after which, cell survival, migration, and the release of inflammatory cytokines were assessed. Moreover, downstream effectors and signaling pathways were studied to reveal the possible underlying mechanism. As a result, LPS stimulation induced significant cell damage as cell viability was repressed and the apoptosis was induced. In the meantime, LPS promoted the release of proinflammatory cytokines including interleukin 1β (IL‐1β), IL‐8, IL‐6, and tumor necrosis factor (TNF‐α) while repressing the release of the anti‐inflammatory cytokine IL‐10. Schizandrin could promote H9c2 cell migration and long‐term treatment (7 days) enhanced cell viability. More interestingly, pretreatment with schizandrin attenuated LPS‐induced cell loss and inflammatory response. Besides this, Smad3 was a downstream effector of schizandrin. The beneficial effects of schizandrin on the H9c2 cells were attenuated when Smad3 was overexpressed. Moreover, the silencing of Smad3 deactivated c‐Jun N‐terminal kinase (JNK) and nuclear factor κB (NF‐κB) pathways. This study preliminarily demonstrated that schizandrin prevented LPS‐induced injury in the H9c2 cells and promoted the recovery of myocardial tissues by enhancing cell viability and migration. Schizandrin conferred its beneficial effects possibly by downregulating Smad3 and inhibiting the activation of JNK and NF‐κB pathways.     

Genetic deficiency of Smad3 protects the kidneys from atrophy and interstitial fibrosis in 2K1C hypertension

Although the two-kidney, one-clip (2K1C) model is widely used as a model of human renovascular hypertension, mechanisms leading to the development of fibrosis and atrophy in the cuffed kidney and compensatory hyperplasia in the contralateral kidney have not been defined. Based on the well-established role of the transforming growth factor (TGF)-β signaling pathway in renal fibrosis, we tested the hypothesis that abrogation of TGF-β/Smad3 signaling would prevent fibrosis in the cuffed kidney. Renal artery stenosis (RAS) was established in mice with a targeted disruption of exon 2 of the Smad3 gene (Smad3 KO) and wild-type (WT) controls by placement of a polytetrafluoroethylene cuff on the right renal artery. Serial pulse-wave Doppler ultrasound assessments verified that blood flow through the cuffed renal artery was decreased to a similar extent in Smad3 KO and WT mice. Two weeks after surgery, systolic blood pressure and plasma renin activity were significantly elevated in both the Smad3 KO and WT mice. The cuffed kidney of WT mice developed renal atrophy (50% reduction in weight after 6 wk, P < 0.0001), which was associated with the development of interstitial fibrosis, tubular atrophy, and interstitial inflammation. Remarkably, despite a similar reduction of renal blood flow, the cuffed kidney of the Smad3 KO mice showed minimal atrophy (9% reduction in weight, P = not significant), with no significant histopathological alterations (interstitial fibrosis, tubular atrophy, and interstitial inflammation). We conclude that abrogation of TGF-β/Smad3 signaling confers protection against the development of fibrosis and atrophy in RAS.     

BNIP3L/NIX-mediated mitophagy protects against ischemic brain injury independent of PARK2

Cerebral ischemia induces massive mitochondrial damage. These damaged mitochondria are cleared, thus attenuating brain injury, by mitophagy. Here, we identified the involvement of BNIP3L/NIX in cerebral ischemia-reperfusion (I-R)-induced mitophagy. Bnip3l knockout (bnip3l^?/?) impaired mitophagy and aggravated cerebral I-R injury in mice, which can be rescued by BNIP3L overexpression. The rescuing effects of BNIP3L overexpression can be observed in park2^?/? mice, which showed mitophagy deficiency after I-R. Interestingly, bnip3l and park2 double-knockout mice showed a synergistic mitophagy deficiency with I-R treatment, which further highlighted the roles of BNIP3L-mediated mitophagy as being independent from PARK2. Further experiments indicated that phosphorylation of BNIP3L serine 81 is critical for BNIP3L-mediated mitophagy. Nonphosphorylatable mutant BNIP3L^S81A failed to counteract both mitophagy impairment and neuroprotective effects in bnip3l^?/? mice. Our findings offer insights into mitochondrial quality control in ischemic stroke and bring forth the concept that BNIP3L could be a potential therapeutic target for ischemic stroke, beyond its accepted role in reticulocyte maturation.     

Partial netrin-1 deficiency aggravates acute kidney injury

The netrin family of secreted proteins provides migrational cues in the developing central nervous system. Recently, netrins have also been shown to regulate diverse processes beyond their functions in the brain, incluing the ochrestration of inflammatory events. Particularly netrin-1 has been implicated in dampening hypoxia-induced inflammation. Here, we hypothesized an anti-inflammatory role of endogenous netrin-1 in acute kidney injury (AKI). As homozygous deletion of netrin-1 is lethal, we studied mice with partial netrin-1 deletion (Ntn-1(+/-) mice) as a genetic model. In fact, Ntn-1(+/-) mice showed attenuated Ntn-1 levels at baseline and following ischemic AKI. Functional studies of AKI induced by 30 min of renal ischemia and reperfusion revealed enhanced kidney dysfunction in Ntn-1(+/-) mice as assessed by measurements of glomerular filtration, urine flow rate, urine electrolytes, serum creatinine and creatinine clearance. Consistent with these findings, histological studies indicated a more severe degree kidney injury. Similarly, elevations of renal and systemic inflammatory markers were enhanced in mice with partial netrin-1 deficiency. Finally, treatment of Ntn-1(+/-) mice with exogenous netrin-1 restored a normal phenotype during AKI. Taking together, these studies implicate endogenous netrin-1 in attenuating renal inflammation during AKI.     

Urocortin protects against ischemic and reperfusion injury via a MAPK-dependent pathway

Urocortin (UCN) is a peptide related to hypothalamic corticotrophin-releasing hormone and binds with high affinity to corticotrophin-releasing hormone receptor-2beta, which is expressed in the heart. In this study, we report that UCN prevented cell death when administered to primary cardiac myocyte cultures both prior to simulated hypoxia/ischemia and at the point of reoxygenation after simulated hypoxia/ischemia. UCN-mediated cell survival was measured by trypan blue exclusion, 3'-OH end labeling of DNA (TUNEL), annexin V, and fluorescence-activated cell sorting. To explore the mechanisms that could be responsible for this effect, we investigated the involvement of MAPK-dependent pathways. UCN caused rapid phosphorylation of ERK1/2-p42/44, and PD98059, which blocks the MEK1-ERK1/2-p42/44 cascade, also inhibited the survival-promoting effect of UCN. Most important, UCN reduced damage in isolated rat hearts ex vivo subjected to regional ischemia/reperfusion, with the protective effect being observed when UCN was given either prior to ischemia or at the time of reperfusion after ischemia. This suggests a novel function of UCN as a cardioprotective agent that could act when given after ischemia, at reperfusion.     

Ischemic preconditioning protects cardiomyocytes against ischemic injury by inducing GRP78

Ischemic preconditioning (IP) conferred by brief ischemia-reperfusion induces resistance to cell injury due to the following lethal ischemia. This study aimed to elucidate whether 78-kDa glucose-regulated protein (GRP78), a main ER molecular chaperone, contributes to IP-mediated protection against ischemic myocardial injury. In a rat coronary artery occlusion model, the GRP78 protein level increased to 210% of the sham level by early IP with three cycles of 4-min ischemia and 4-min reperfusion. The IP reduced infarct size in subsequent lethal ischemia. In primary cardiomyocytes, the simulated IP procedure, incubation in oxygen-glucose deprivation (OGD) medium, also increased the GRP78 expression and suppressed the cell death caused by lethal ischemia. Transfection of grp78 antisense oligonucleotide attenuated the IP-mediated resistance to ischemia. This study showed for the first time that early IP up-regulates myocardial GRP78. It was suggested that GRP78 induced by early IP contributes to protect cardiomyocytes against ischemic injury.     

Treatment with outgrowth endothelial cells protects cerebral barrier against ischemic injury

Background and aimsWe have previously reported that outgrowth endothelial cells (OECs) restore cerebral endothelial cell integrity through effective homing to the injury site. This study further investigates whether treatment with OECs can restore blood–brain barrier (BBB) function in settings of ischemia-reperfusion injury both in vitro and in vivo.MethodsAn in vitro model of human BBB was established by co-culture of astrocytes, pericytes, and human brain microvascular endothelial cells (HBMECs) before exposure to oxygen-glucose deprivation alone or followed by reperfusion (OGD±R) in the absence or presence of exogenous OECs. Using a rodent model of middle cerebral artery occlusion (MCAO), we further assessed the therapeutic potential of OECs in vivo.ResultsOwing to their prominent antioxidant, proliferative, and migratory properties, alongside their inherent capacity to incorporate into brain vasculature, treatments with OECs attenuated the extent of OGD±R injury on BBB integrity and function, as ascertained by increases in transendothelial electrical resistance and decreases in paracellular flux across the barrier. Similarly, intravenous delivery of OECs also led to better barrier protection in MCAO rats as evidenced by significant decreases in ipsilateral brain edema volumes on day 3 after treatment. Mechanistic studies subsequently showed that treatment with OECs substantially reduced oxidative stress and apoptosis in HBMECs subjected to ischemic damages.ConclusionThis experimental study shows that OEC-based cell therapy restores BBB integrity in an effective manner by integrating into resident cerebral microvascular network, suppressing oxidative stress and cellular apoptosis.     

Splenectomy exacerbates lung injury after ischemic acute kidney injury in mice

Patients with acute kidney injury (AKI) have increased serum proinflammatory cytokines and an increased occurrence of respiratory complications. The aim of the present study was to examine the effect of renal and extrarenal cytokine production on AKI-mediated lung injury in mice. C57Bl/6 mice underwent sham surgery, splenectomy, ischemic AKI, or ischemic AKI with splenectomy and kidney, spleen, and liver cytokine mRNA, serum cytokines, and lung injury were examined. The proinflammatory cytokines IL-6, CXCL1, IL-1β, and TNF-α were increased in the kidney, spleen, and liver within 6 h of ischemic AKI. Since splenic proinflammatory cytokines were increased, we hypothesized that splenectomy would protect against AKI-mediated lung injury. On the contrary, splenectomy with AKI resulted in increased serum IL-6 and worse lung injury as judged by increased lung capillary leak, higher lung myeloperoxidase activity, and higher lung CXCL1 vs. AKI alone. Splenectomy itself was not associated with increased serum IL-6 or lung injury vs. sham. To investigate the mechanism of the increased proinflammatory response, splenic production of the anti-inflammatory cytokine IL-10 was determined and was markedly upregulated. To confirm that splenic IL-10 downregulates the proinflammatory response of AKI, IL-10 was administered to splenectomized mice with AKI, which reduced serum IL-6 and improved lung injury. Our data demonstrate that AKI in the absence of a counter anti-inflammatory response by splenic IL-10 production results in an exuberant proinflammatory response and lung injury.     

Cyclophilin D deficiency protects against acetaminophen-induced oxidant stress and liver injury

Acetaminophen (APAP) hepatotoxicity is the main cause of acute liver failure in humans. Although mitochondrial oxidant stress and induction of the mitochondrial permeability transition (MPT) have been implicated in APAP-induced hepatotoxicity, the link between these events is unclear. To investigate this, this study evaluated APAP hepatotoxicity in mice deficient of cyclophilin D, a protein component of the MPT. Treatment of wild type mice with APAP resulted in focal centrilobular necrosis, nuclear DNA fragmentation and formation of reactive oxygen (elevated glutathione disulphide levels) and peroxynitrite (nitrotyrosine immunostaining) in the liver. CypD-deficient (Ppif(-/-)) mice were completely protected against APAP-induced liver injury and DNA fragmentation. Oxidant stress and peroxynitrite formation were blunted but not eliminated in CypD-deficient mice. Thus, mitochondrial oxidative stress and induction of the MPT are critical events in APAP hepatotoxicity in vivo and at least part of the APAP-induced oxidant stress and peroxynitrite formation occurs downstream of the MPT.