NS Mice Research Articles

Nicotinamide protects against diabetic kidney disease through regulation of Sirt1

PurposeTo investigate the effect of nicotinamide (Nam) on diabetic kidney disease (DKD) in mice and explore its mechanism.MethodsThirty DBA/2 J mice were randomly assigned to three groups. After 8 weeks of hyperglycemia induced by streptozocin (STZ), Nam and saline were administrated to STZ + Nam and STZ + NS mice, respectively, for 8 weeks. Non-diabetic mice (NDM) were used as control group. Twenty In2−/− Akita mice were randomly divided into two groups. After 8 weeks of hyperglycemia, Nam and saline were administered to Akita + Nam and Akita + NS mice, respectively, for 6 weeks. Wild-type littermates were used as control group. Markers of renal injury were analyzed, and the molecular mechanisms were explored in human proximal tubular HK2 cells.ResultsUrinary albumin-to-creatinine ratio (UACR) and kidney injury molecule 1 (KIM-1) decreased in the STZ + Nam and Akita + Nam groups. Pathological analysis showed that Nam improved the structure of glomerular basement membrane, ameliorated glomerular sclerosis, and decreased the accumulation of extracellular matrix and collagen. Compared to the diabetic control group, renal fibrosis, inflammation, and oxidative stress were reduced in the Nam-treated mice. The expression of sirtuin 1 (Sirt1) in human proximal tubular HK2 cells was inhibited by high glucose and Nam treatment enhanced its expression. However, in HK2 cells with Sirt1 knockdown, the protective effect of Nam was abolished, indicating that the beneficial effect of Nam was partially dependent on Sirt1.ConclusionsNam has a renoprotective effect against renal injury caused by hyperglycemia and may be a potential target for the treatment of DKD.

Circadian misalignment by environmental light/dark shifting causes circadian disruption in colon.

BackgroundPhysiological circadian rhythms (CRs) are complex processes with 24-hour oscillations that regulate diverse biological functions. Chronic weekly light/dark (LD) shifting (CR disruption; CRD) in mice results in colonic hyperpermeability. However, the mechanisms behind this phenomenon are incompletely understood. One potential innovative in vitro method to study colonic CRs are colon organoids. The goals of this study were to utilize circadian clock gene Per2 luciferase reporter (Per2::Luc) mice to measure the effects of chronic LD shifting on colonic tissue circadian rhythmicity ex vivo and to determine if organoids made from shifted mice colons recapitulate the in vivo phenotype.MethodsNon-shifted (NS) and shifted (S) BL6 Per2::Luc mice were compared after a 22-week experiment. NS mice had a standard 12h light/12h dark LD cycle throughout. S mice alternated 12h LD patterns weekly, with light from 6am-6pm one week followed by shifting light to 6pm-6am the next week for 22 weeks. Mice were tested for intestinal permeability while colon tissue and organoids were examined for CRs of bioluminescence and proteins of barrier function and cell fate.ResultsThere was no absolute difference in NS vs. S 24h circadian period or phase. However, chronic LD shifting caused Per2::Luc S mice colon tissue to exhibit significantly greater variability in both the period and phase of Per2::Luc rhythms than NS mice colon tissue and organoids. Chronic LD shifting also resulted in increased colonic permeability of the Per2::Luc mice as well as decreased protein markers of intestinal permeability in colonic tissue and organoids from shifted Per2:Luc mice.ConclusionsOur studies support a model in which chronic central circadian disruption by LD shifting alters the circadian phenotype of the colon tissue and results in colon leakiness and loss of colonic barrier function. These CRD-related changes are stably expressed in colon stem cell derived organoids from CRD mice.

A Novel Natural Autoantibody Targeted Complement Inhibitor Protects against Lung Transplant Ischemia Reperfusion Injury

Purpose Chronic obstructive pulmonary disease is characterized by chronic inflammation and severe lung parenchyma damage that is in part driven by autoimmunity. Lung specific autoreactive antibodies (AAb) are indicative of disease progression and severity. The purpose of this study was to elucidate how pre-existing AAb contribute to ischemia reperfusion injury (IRI) and graft rejection following lung transplant (LTx). We hypothesize that pre-existing AAb bind cryptic antigens exposed during IRI which exacerbates lung graft injury in a complement (C)-dependent pathway. Methods We generated a clinically relevant cigarette smoke (CS)-induced mouse model that exhibits increased serum AAb compared to non-smoke exposed age-matched (NS) controls. We analyzed the impact of a full pre-existing AAb repertoire by creating an orthotopic syngeneic left lung LTx model in which we transplanted the left lungs from age-matched donor mice into NS and CS mice. Furthermore, we developed a novel bifunctional inhibitor that blocks IgM binding and inhibits complement. Results CS exposure significantly increased graft injury and immune infiltration 48 hours after LTx. Syngeneic LTx into Rag−/- mice reconstituted with serum from NS and CS mice demonstrated that AAb present in CS are responsible for the observed graft injury. Immunofluorescent staining showed that IgM/IgG binding was increased in CS recipients and colocalized with C3d deposition. These data suggested that AAb-mediated graft injury may occur via the C pathway. To elucidate the role of C in AAb-mediated graft injury, we treated CS LTx recipients with a novel bifunctional C inhibitor, C2scFv-Crry, that blocks IgM binding and C activation. C2scFv-Crry treatment following LTx dramatically reduced acute graft injury and immune infiltration, and significantly decreased de novo AAb production compared to CS control LTx mice. Conclusion Ultimately, these data reveal that CS induces pre-existing AAb production that exacerbate IRI following LTx in a C-dependent manner. Furthermore, we demonstrate the efficacy of a novel C inhibitor in reducing IRI and acute graft injury.

Motor co-ordinative roles of Nigella sativa oil in mice models of phenol-induced essential tremor

Background: Essential tremor, regarded as the world's most common movement disorder, is a neuronal disorder characterized by uncontrollable shaking (tremor) of different parts of the body. Nigella sativa is a medicinal herb with pharmacologically proven therapeutic potentials in various ailments including neurological disorders. Objective: To evaluate the therapeutic roles of Nigella sativa on cerebellar phenotypes in phenol-induced mice models of essential tremor. Methods: Tremor response, body weight, temperature, motor coordination (using the parallel bars and static rods tests), relative brain weights, cerebellar glutamate and Glutathione peroxidase (GPX) and histoarchitecture were assayed in 75 adult male BALB/cm ice weighing 21-30g. The animals, equally divided into five groups were respectively administered feed and water only (CTRL); 100mg/kg phenol and 1ml/kg Nigella sativa oil concurrently (PNSC); 100mg/kg phenol (P); 1ml/kg Nigella sativa oil followed by 100mg/kg phenol (NSP); and 1ml/kg Nigella sativa oil (NS). Results: The PNSC, NSP and NS mice displayed significant weight reduction. Histoachitectural defects, stagnancy in weight, high Glutathione peroxidase (GPX) and high glutamate levels and poor motor coordination were exhibited by the P group. The CTRL and NS animals demonstrated good motor coordination while the PNSC and NSP groups showed better coordination than the untreated P group. The CTRL group showed no histoarchitectural defects while the NS and PNSC animals showed histoarchitectural regeneration. Conclusion: This research affirmed the weight-reducing, neuroprotective, neuroregenerative and motor coordinating effects of Nigella sativa in the modelled tremor condition.

Emphysema-associated Autoreactive Antibodies Exacerbate Post-Lung Transplant Ischemia-Reperfusion Injury.

Chronic obstructive pulmonary disease-associated chronic inflammation has been shown to lead to an autoimmune phenotype characterized in part by the presence of lung autoreactive antibodies. We hypothesized that ischemia-reperfusion injury (IRI) liberates epitopes that would facilitate preexisting autoantibody binding, thereby exacerbating lung injury after transplant. We induced emphysema in C57BL/6 mice through 6 months of cigarette smoke (CS) exposure. Mice with CS exposure had significantly elevated serum autoantibodies compared with non-smoke-exposed age-matched (NS) mice. To determine the impact of a full preexisting autoantibody repertoire on IRI, we transplanted BALB/c donor lungs into NS or CS recipients and analyzed grafts 48 hours after transplant. CS recipients had significantly increased lung injury and immune cell infiltration after transplant. Immunofluorescence staining revealed increased IgM, IgG, and C3d deposition in CS recipients. To exclude confounding alloreactivity and confirm the role of preexisting autoantibodies in IRI, syngeneic Rag1-/- (recombination-activating protein 1-knockout) transplants were performed in which recipients were reconstituted with pooled serum from CS or NS mice. Serum from CS-exposed mice significantly increased IRI compared with control mice, with trends in antibody and C3d deposition similar to those seen in allografts. These data demonstrate that pretransplant CS exposure is associated with increased IgM/IgG autoantibodies, which, upon transplant, bind to the donor lung, activate complement, and exacerbate post-transplant IRI.

Prenatal stress leads to chromatin and synaptic remodeling and excessive alcohol intake comorbid with anxiety-like behaviors in adult offspring

Epidemiologic evidence suggests that individuals during their prenatal development may be especially vulnerable to the effects of environmental factors such as stress that predisposes them to psychiatric disorders including alcohol use disorder (AUD) later in life. Currently, the epigenetic mechanisms of anxiety comorbid with AUD induced by prenatal stress (PRS) remain to be elucidated. Here, we examined anxiety-like and alcohol drinking behaviors in adult offspring of prenatally stressed dam (PRS-mice) using elevated plus maze, light/dark box and two-bottle free-choice paradigm. It was found that PRS-mice exhibit heightened anxiety-like behaviors and increased alcohol intake in adulthood and these behavioral deficits were associated with a significant decrease in dendritic spine density (DSD) in medial prefrontal cortex (mPFC) relative to non-stressed mice (NS mice). To determine the mechanisms by which PRS reduces DSD, we examined the expressions of key genes associated with synaptic plasticity, including activity regulated cytoskeleton associated protein (Arc), spinophilin (Spn), postsynaptic density 95 (Psd95), tropomyosin receptor kinase B (TrkB), protein kinase B (Akt), mammalian target of rapamycin (mTOR) and period 2 (Per2) in mPFC of PRS and NS mice. The mRNA levels of these genes were significantly decreased in PRS mice. Methylated DNA and chromatin immunoprecipitation studies revealed hyper DNA methylation or reduced histone H3K14 acetylation on promoters of above genes suggesting that epigenetic dysregulation may be responsible for the deficits in their expression. Findings from this study suggest that prenatal stress induced abnormal epigenetic mechanisms and synaptic plasticity-related events may be associated with anxiety-like and alcohol drinking behaviors in adulthood.

Polymorphisms in FcγRII (CD32) mediate abnormal antibody half-life in NOD SCID mice and reduce antibody-mediated tumour cell deletion.

Abstract NOD SCID (NS) mice are commonly used for the assessment of monoclonal antibody (mAb) therapeutics and form the genetic background of NSG mice widely used in human patient derived xenograft models. However, relatively little is known regarding how the NS background effects monoclonal antibody (mAb) pharmacokinetics and how this might influence therapeutic response. To address this we examined mAb half-life and clearance of hCD20 transgenic EμTCL-1 tumours in NS mice following administration of anti-hCD20 hIgG1 mAb and showed mAb half-life to be significantly shorter in NS versus SCID mice, correlating with reduced therapy. Reduced half-life was also seen with mIgG2a but not mIgG1 or hIgG2 isotypes. Additionally, NS mice lacking CD32 or the FcR γ-chain demonstrated this rapid loss was dependent on the expression of CD32 and not activatory FcγRs. Sequencing confirmed the NOD strains carry the rare ly17.1 variant of CD32. Recombinant ly17.1 displayed increased affinity for hIgG1 compared to the more common ly17.2 variant, providing a potential mechanism for its more rapid removal from serum. Importantly, the reduced half-life was dependent on the presence of both SCID and NOD mutations. SCID mice lack endogenous IgG and so we reasoned this might accentuate the effects of the NOD CD32 polymorphic variant. Accordingly, reconstitution of NS mice with physiological levels of mIgG restored normal half-life. These findings highlight the importance of examining mAb pharmacokinetics in complex mouse strains and demonstrate that therapeutic studies in mice on the NS background should be performed in the presence of exogenous IgG.

Persistent p55TNFR expression impairs T cell responses during chronic tuberculosis and promotes reactivation.

The pleiotropic activities of TNF are mediated by two structurally related but functionally distinct type I transmembrane receptors, p55TNFR and p75TNFR expressed in most cell types, that can be cleaved and act as TNF scavengers. Here, we investigated the effect of persistent p55TNFR cell surface expression during aerosol inhalation challenge with virulent M. tuberculosis H37Rv. We demonstrated that persistency of p55TNFR in macrophage cultures increased the synthesis of soluble TNF, p75TNFR and NO, however, had no effects on bacteria killing ability. Furthermore, it did not facilitate enhanced protection to primary acute M. tuberculosis infection in p55∆NS mice. Without exacerbated lung inflammation, we found a compensatory increase in p75TNFR shedding and decrease in bioactive TNF in BAL of p55∆NS mice after M. tuberculosis challenge. Defective expressions of CD44 and INFγ attributed to an impaired T cell response during persistent p55TNFR expression that caused marginal transient susceptibility during chronic infection. Moreover, persistent p55TNFR expression induced early reactivation during latent tuberculosis infection. These data indicate a prominent role of p55TNFR shedding in Th1 mediated protection against chronic and latent tuberculosis infection.

Stress perception alters cellular and molecular immunity in the immune response in the brain to a neurotropic viral pathogen (VIR1P.1154)

Abstract Vesicular stomatitis virus (VSV) applied intranasally produces a well-characterized encephalitis. However, mice that experienced controllable stress via an escapable foot-shock (ES) demonstrated an enhanced immune response relative to mice that experienced uncontrollable stress (inescapable foot shock, IS) or non-stressed control mice (NS). These differences began as early as 1 day post-infection, where IS mice show a more robust cytokine response than ES or NS mice. By 3 days post-infection, however, ES mice showed enhanced inflammatory cytokine responses in the olfactory bulb. We used the Ingenuity Pathways Analysis (IPA) platform to identify regulatory genes responsible for the differences. IPA identified miRNA146a as an upstream regulator of the antiviral response and qPCR found elevated levels (~5-fold) of miRNA146a in the olfactory bulb from ES versus IS mice. miRNA levels in the non-stressed group were below detectable limits. These results suggested increased inflammation in the brains of ES mice, which were supported by more aggressive weight loss in ES mice compared to IS or NS mice as the encephalitis progressed. By 28 days post-infection, surviving mice subjected to ES continue to show increased immune activation as evidenced by increased leukocyte infiltration and enhanced production or infiltration of CD103+ (memory phenotype) T cells relative to IS or NS mice.

Fatty acid oxidation changes and the correlation with oxidative stress in different preeclampsia-like mouse models.

BackgroundLong-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) expression is decreased in placenta of some cases of preeclampsia (PE) which may result in free fatty acid (FFA) increased. High FFA level will induce oxidative stress, so abnormal long-chain fatty acid-oxidation may participate in the pathogenesis of PE through oxidative stress pathway.MethodsPE-like groups were ApoC3 transgenic mice with abnormal fatty acid metabolism, classical PE-like models with injection of Nw-nitro-L-arginine-methyl ester (L-NA) or lipopolysaccharide (LPS) and the antiphospholipid syndrome (APS) mouse model with β2GPI injection (ApoC3+NS, ApoC3+L-NA, L-NA, LPS and β2GPI groups). The control group was wild-type mice with normal saline injection. Except for β2GPI mice, the other mice were subdivided into pre-implantation (Pre) and mid-pregnancy (Mid) subgroups by injection time.ResultsAll PE-like groups showed hypertension and proteinuria except ApoC3+NS mice only showed hypertension. Serum FFA levels increased significantly except in LPS group compared to controls (P<0.05). LCHAD mRNA and protein expression in the liver and placenta was significantly higher for ApoC3+NS, ApoC3+L-NA and β2GPI mice and lower for L-NA mice than controls (P<0.05) but did not differ between LPS mice and controls. P47phox mRNA and protein expression in the liver significantly increased in all PE-like groups except LPS group, while P47phox expression in the placenta only significantly increased in L-NA and β2GPI groups.ConclusionsAbnormal long-chain fatty acid-oxidation may play a different role in different PE-like models and in some cases participate in the pathogenesis of PE through oxidative stress pathway.

Contribution of arginase activation to vascular dysfunction in cigarette smoking

BackgroundCigarette smoke increases the risk of several cardiovascular diseases and has synergistic detrimental effects when present with other risks that contribute to its pathogenesis. Oxidative injury to the endothelium via reactive oxygen species (ROS) and nitric oxide (NO) dysregulation is a common denominator of smoking-induced alterations in vascular function. However, the mechanisms underlying ROS and NO dysregulation due to smoking remain unclear. We tested if arginase (Arg) activation/upregulation contributes to this phenomenon by constraining nitric oxide synthase (NOS) activity. MethodsArg2 knockout (Arg2−/−) and control C57BL/6J mice were either exposed to cigarette smoke, 6 h/day/2 weeks (Second Hand Smoking; SHS) or housed in normal environment (Non Smoking; NS). Arg activity, NO and ROS levels were determined by measuring urea production, fluorescent dye (DAF), and dihydroethedium (DHE) respectively in isolated mouse aorta. ResultsArg activity and ROS levels were higher NO lower in SHS compared to NS mice. SHS failed to lower NO levels in Arg2−/− mice. Endothelial dependent vasodilation (EDV) was attenuated in SHS mice as compared to controls (78.80% ± 8 vs 46.58% ± 5). This impaired EDV was abolished in Arg2−/− mice (67.48% ± 7 in SHS vs. 78.80% ± 8 in NS). Vascular stiffness was increased in SHS mice as compared to NS controls but remained unchanged in Arg2−/− mice. ConclusionEndothelial NOS is uncoupled by smoking exposure, leading to endothelial dysfunction and vascular stiffness, a process that is prevented by Arg2 deletion. Hence, we identify Arg2 upregulation as a critical pathogenic factor and target for therapy in oxidative injury following smoking exposure through reciprocal regulation of endothelial NOS.

Deficiency in Serine Protease Inhibitor Neuroserpin Exacerbates Ischemic Brain Injury by Increased Postischemic Inflammation

The only approved pharmacological treatment for ischemic stroke is intravenous administration of plasminogen activator (tPA) to re-canalize the occluded cerebral vessel. Not only reperfusion but also tPA itself can induce an inflammatory response. Microglia are the innate immune cells of the central nervous system and the first immune cells to become activated in stroke. Neuroserpin, an endogenous inhibitor of tPA, is up-regulated following cerebral ischemia. To examine neuroserpin-dependent mechanisms of neuroprotection in stroke, we studied neuroserpin deficient (Ns−/−) mice in an animal model of temporal focal ischemic stroke. Infarct size and neurological outcome were worse in neuroserpin deficient mice even though the fibrinolytic activity in the ischemic brain was increased. The increased infarct size was paralleled by a selective increase in proinflammatory microglia activation in Ns−/− mice. Our results show excessive microglial activation in Ns−/− mice mediated by an increased activity of tPA. This activation results in a worse outcome further underscoring the potential detrimental proinflammatory effects of tPA.

Distinct Human Cell Populations Produce Rapidly Detectable Levels of Neutrophils and Platelets in NOD/SCID-IL-2Receptor Gamma Chain Null Mice Transgenically Engineered to Produce Human Myeloid Growth Factors

Abstract 1892Cord blood (CB) is becoming an increasingly utilized source of cells for cancer patients who are eligible for therapies that require a transplant to rescue them from toxic side effects on their own hematopoietic cells but lack a suitable HLA-matched donor. This strategy is now routinely used in children, but delayed neutrophil and platelet recovery remain unsolved problems and these problems are exacerbated in adults. To address this issue, we first surveyed the variability in 8 individual CB harvests of parameters routinely used to predict the utility of CB units as transplants (i.e., CD34+ and in vitro myeloid clonogenic progenitor cell frequencies). In addition, we compared their 3-week outputs of CD33/15/66+ cells (neutrophils and monocytes) in the marrow and CD41a+ platelets in the blood of sublethally irradiated NSG mice after the IV transplantation of ∼104 CD34+ cells. These latter assessments were based on ongoing experiments in our lab demonstrating that, at this transplant dose, the outputs measured are linearly related to the number of CD34+ cells injected and detect transplantable progenitor cell types that are biologically distinct from cells with longer term repopulating activity. The results showed variation between CBs in all parameters, a marked lack of correlation between %CD34+ cells or % total CFCs in initial cells and %CD41a+ cells regenerated at 3 weeks/104 CD34+ cells transplanted (R=-0.28 and 0.35, respectively), and a weak correlation between the %CD33/15/66+ cells regenerated at 3 weeks/104 CD34+ cells transplanted and %CD34+ cells or % total CFCs in the initial CB cells (R values of 0.46–0.64). However, although engraftment of primitive human cells in NSG mice appears highly efficient, terminal differentiation of the myeloid lineages in these mice is poor. One possible explanation for this deficiency in mature cell output is that several of the murine growth factors responsible for regulating the production and release of these cells into the circulation in mice are not cross-reactive on human cells. We therefore hypothesized that engineering NSG mice to produce the human counterparts might significantly improve the detection of short term repopulating human cells whose maximum clone size might be limiting in NSG mice. Three potential relevant factors are IL-3, GM-CSF and Steel factor. We therefore backcrossed a line of transgenic NS mice we had created to express human IL-3, GM-CSF and Steel factor onto the NSG strain to produce homozygous NSG mice expressing all 3 of these human factors (NSG-3GS mice). We then compared these NSG-3GS mice with NSG mice in terms of their ability to stimulate the production within 3 weeks of human neutrophil-monocytes and platelets from intravenously transplanted CD34+ cells isolated from pooled CB harvests. The results showed that the levels of neutrophils and monocytes generated in the marrow of the NSG-3GS mice were elevated to levels of >50% of the marrow in 90% of the mice, even at the lowest number of CD34+ cells transplanted. Human neutrophils and monocytes were also elevated in the blood of the NSG-3GS mice where, despite the observed “saturation” of the marrow, there was a linear dose-response in the number of human neutrophils and monocytes present in the blood with increasing CD34+ cells infused. These findings are consistent with the reported activities of these molecules in vitro and in patients suggesting their physiological relevance in this murine xenograft model. We next utilized this assay to characterize the cells responsible for the neutrophil/monocyte and platelet repopulating activities detected in NSG-3GS mice. Preliminary assessment of the CD34+CD45RA- population on the basis of CD123 (IL-3 receptor alpha chain) expression indicates that the CD123+ fraction is enriched for short term (3-week) neutrophil/monocyte repopulating activity, while the CD123- fraction is enriched for short term (3-week) platelet repopulating activity. In summary, NSG-3GS mice significantly enhance the output of human cells with short term human myeloid repopulating ability thereby enabling neutrophil/monocyte outputs as well as platelet outputs to be assessed by analysis of peripheral blood samples. We have also used this tool to obtain evidence that these two outputs are derived from distinct cell types. Direct quantification of these may add to future predictions of graft quality. Disclosures:No relevant conflicts of interest to declare.

Intervention effects of enriched environment on brain damage of MPTP-induced senescence-accelerated prone mice

Objective To observe the effects of enriched environmen(EE) on brain-derived neurotrophic factor(BDNF) in substantia nigra(SN) of senescence-accelerated prone mice,so as to explore the possible mechanism of EE in alleviating MPTP induced damage and in protecting dopaminergic neurons in the SN.Methods Totally 80 3-month old female SAMP8 mice were averagely assigned to EE and standard environment(SE) groups at random.After three months,the mice in each group were further divided into 2 subgroups at random:MPTP group and NS group(n = 20).The MPTP groups received subcutaneous injection of MPTP,and the NS mice were treated with an equal volume of NS.At the seventh day,the mice were sacrificed for RT-PCR and immunohistochemistry staining.RT-PCR was used to examine BDNF mRNA expression and immunohistochemistry staining was used to examine BDNF-ir expression.Results Compared with SE + NS mice,EE + NS mice had significantly increased BDNF mRNA expression(P 0.01),and the number of BDNF-ir cell and COD values in SN were also significantly increased(P 0.01).Compared with SE + NS mice,SE + MPTP mice showed significantly decreased BDNF mRNA expression(P 0.001),and the number of BDNF-ir cells and the COD values in SN of mice were significantly decreased(P 0.01).Compared with SE + MPTP mice,EE + MPTP mice showed increased BDNF mRNA expression(P 0.01),and the number of BDNF-ir cells and the COD values in SN were significantly increased(P 0.01).Conclusion EE can increase BDNF mRNA expression and BDNF-ir cell number in SN of SAMP8 mice,alleviating MPTP-induced SN damage.

Decreased Arteriolar Tetrahydrobiopterin is Linked to Superoxide Generation from Nitric Oxide Synthase in Mice Fed High Salt

Impaired endothelium-dependent arteriolar dilation in mice fed high salt (HS) is due to local oxidation of nitric oxide (NO) by superoxide anion (O(2) (-)). We explored the possibility that "uncoupled" endothelial nitric oxide synthase (eNOS) is the source of this O(2) (-). Levels of L-arginine (L-Arg), tetrahydrobiopterin (BH(4)), and O(2) (-) (hydroethidine oxidation) were measured in spinotrapezius muscle arterioles of mice fed normal salt (0.45%, NS) or (4%, HS) diets for 4 weeks, with or without dietary L-Arg supplementation. The contribution of NO to endothelium-dependent dilation was determined from the effect of N(omega)-nitro-L-arginine methyl ester (L-NAME) on responses to acetylcholine (ACh). Arterioles in HS mice had lower [BH(4)] and higher O(2) (-) levels than those in NS mice. ACh further increased arteriolar O(2) (-) in HS mice only. L-Arg supplementation prevented the reduction in [BH(4)] in arterioles of HS mice, and O(2) (-) was not elevated in these vessels. Compared to NS mice, arteriolar ACh responses were diminished and insensitive to L-NAME in HS mice, but not in HS mice supplemented with L-Arg. These findings suggest that eNOS uncoupling due to low [BH(4)] is responsible for O(2) (-) generation and reduced NO-dependent dilation in arterioles of mice fed a HS diet.

Superoxide from uncoupled nitric oxide synthase reduces endothelium‐dependent arteriolar dilation in mice fed high salt

We have previously reported that the impaired endothelium‐dependent dilation of arterioles in rodents on a high salt diet is due to oxidation of nitric oxide (NO) by superoxide anion (O2−) in the arteriolar wall. The aim of this study was to test the hypothesis that "uncoupled" nitric oxide synthase (NOS) is the predominant source of this O2−. Arterial pressure in C57BL/6J mice fed a high salt (4%, HS) diet for 4 weeks was not significantly different from that in mice fed a normal salt (0.45%, NS) diet. However, compared to NS mice, spinotrapezius muscle arterioles in HS mice had elevated arteriolar wall oxidant activity (assessed by hydroethidine oxidation) and a large (75‐80%) reduction in dilator responses to acetylcholine. In HS mice, arteriolar oxidant stress was abolished by acute application of the NOS inhibitor NG monomethyl L‐arginine and by long‐term supplementation of L‐arginine intake (1.51% in drinking water for 4 weeks), which has been shown to increase vascular tetrahydrobiopterin (BH4) levels. L‐arginine supplementation also prevented the reduction in arteriolar ACh responses in HS mice but had no effect on these responses in NS mice. These findings suggest that NOS uncoupling due to reduced BH4 availability is primarily responsible for O2− accumulation and the resultant impairment of NO‐dependent arteriolar dilation in mice fed a high salt diet. (Support AHA Grant‐in‐Aid 0755264B and NIH ES‐015022)

High-salt diet enhances mouse aortic relaxation through adenosine A2Areceptor via CYP epoxygenases

We hypothesize that A(2A) adenosine receptors (A(2A) AR) promote aortic relaxation in mice through cytochrome P450 (CYP)-epoxygenases and help to avoid salt sensitivity. Aortas from male mice maintained on a high-salt (HS; 7% NaCl) or normal-salt (NS; 0.45% NaCl) diet for 4-5 wks were used. Concentration-response curves (10(-11)-10(-5) M) for 5'-N-ethylcarboxamidoadenosine (NECA; a nonselective adenosine analog) and CGS 21680 (A(2A) AR agonist) were obtained with different antagonists including ZM 241385 (A(2A) AR antagonist; 10(-6) M), SCH 58261 (A(2A) AR antagonist; 10(-6) M), N(omega)-nitro-l-arginine methyl ester (l-NAME; endothelial nitric oxide synthase inhibitor; 10(-4) M) and inhibitors including methylsulfonyl-propargyloxyphenylhexanamide (MS-PPOH; CYP epoxygenases inhibitor; 10(-5)M), 14,15-epoxyeicosa-5(z)-enoic acid (14,15-EEZE; EET antagonist; 10(-5)M), dibromo-dodecenyl-methylsulfimide (DDMS; CYP4A inhibitor; 10(-5)M), and HET0016 (20-HETE inhibitor; 10(-5)M). At 10(-7) M of NECA, significant relaxation in HS (+22.58 +/- 3.12%) was observed compared with contraction in NS (-10.62 +/- 6.27%, P < 0.05). ZM 241385 changed the NECA response to contraction (P < 0.05) in HS. At 10(-7) M of CGS 21680, significant relaxation in HS (+32.04 +/- 3.08%) was observed compared with NS (+10.45 +/- 1.34%, P < 0.05). SCH 58261, l-NAME, MS-PPOH, and 14,15-EEZE changed the CGS 21680-induced relaxation to contraction (P < 0.05) in HS. Interestingly, DDMS and HET0016 changed CGS 21680 response to relaxation (P < 0.05) in NS; however, there was no significant difference found between DDMS, HET0016-treated HS and NS vs. nontreated HS group (P > 0.05). CYP2C29 protein was 55% and 74% upregulated in HS vs. NS (P < 0.05) mice aorta and kidney, respectively. CYP4A protein was 30.30% and 35.70% upregulated in NS vs. HS (P < 0.05) mice aorta and kidneys, respectively. A(1) AR was downregulated, whereas A(2A) AR was upregulated in HS compared with NS. These data suggest that HS may activate CYP2C29 via A(2A) AR, causing relaxation, whereas NS may contribute to the upregulation of CYP4A causing contraction.

Human acute leukemia cells injected in NOD/LtSz‐scid/IL‐2Rγ null mice generate a faster and more efficient disease compared to other NOD/scid‐related strains

Transplantation of human acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) primary cells and cell lines in different strains of immunodeficient mice has led to preclinical models extensively used to investigate acute leukemia stem cells, biology and drug sensitivity. We studied the engraftment kinetics of AML and ALL cell lines and primary cells in 3 strains of NOD.CB17-Prkdc(scid) (NOD/scid, NS)-related mice (NOD.Cg-Prkdc(scid)B2m(tm1Unc)/J, abbreviated NOD/scid/beta2 null, NSB; and NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll)/SzJ, abbreviated NOD/scid/IL-2Rgamma null, NSG). The engraftment of human malignant cells was investigated by means of clinicopathological criteria, flow cytometry, PCR and immunohistochemistry. In NSG mice, we observed a significantly faster development of leukemia-related symptoms and a higher percentage of leukemia cells in the blood, in the marrow and in the spleen. The leukemia-related angiogenic switch (measured as the number of circulating endothelial cells and progenitors) was faster in NSG compared to NS and NSB mice. These models will be instrumental to studies on leukemia-initiating stem cells, leukemia biology, preclinical treatment studies, and to obtain patient-specific preclinical models to design and investigate patient-tailored therapies.

Interleukin‐1 in chronic angiotensin II‐high salt diet induced hypertension

IL‐6 and TNF‐α are implicated in hypertension induced by angiotensin II infusion plus high salt diet (AngII‐HS). Since IL‐1β can stimulate production of both cytokines and is implicated in some renal injury models, we investigated the role of IL‐1β in AngII‐HS hypertension. In rats, 14‐day AngII‐HS (AngII at 65 ng/min s.c. + 8% NaCl diet) increased 24‐h average mean arterial pressure (191±3 vs 108±3 mmHg in normal NaCl control rats [NS], P&lt;0.001) and renal cortical immunoreactive IL‐1β content (44±6 vs 21±3 pg/ml, P&lt;0.01). AngII‐HS rats also displayed significantly increased urinary protein (431±33 mg/d) and albumin excretion (2.5±0.3 mg/d) compared to NS rats (22±6 &amp; 0.8±0.3 mg/d, P&lt;0.001). To test whether IL‐1β contributes to renal injury in AngII‐HS hypertension, the effects of 14‐day AngII‐HS (90 ng/min s.c. + 4% NaCl diet) were compared in wild‐type and IL‐1 receptor −/− mice. After a 24‐h baseline urine collection, mice were randomized to AngII‐HS or NS treatment groups. Baseline urine flow and Na+, protein and albumin excretion rates did not significantly differ between the four groups. At day 14, protein excretion was significantly increased by AngII‐HS in wild‐type and IL‐1 receptor −/− mice (3.7±0.9 &amp; 5.2±1.5 mg/d) compared to NS wild‐type and IL‐1 receptor −/− mice (1.3±0.3 &amp; 0.9±0.1 mg/d; P&lt;0.05), however this effect was not significantly different between genotypes. Similarly, urine flow, Na+ and albumin excretion were significantly increased in AngII‐HS mice compared to NS mice (P&lt;0.05), but there were no differences between genotypes. Preliminary data also suggests that the degree of hypertension induced by AngII‐HS is similar in wild‐type and IL‐1 receptor −/− mice. Our results indicate that although IL‐1β production is increased in the renal cortex in AngII‐HS hypertension, IL‐1 receptor signalling does not appear to contribute to the development of proteinuria in this model.

High salt intake reduces endothelium-dependent dilation of mouse arterioles via superoxide anion generated from nitric oxide synthase

In skeletal muscle arterioles of normotensive rats fed a high salt diet, the bioavailability of endothelium-derived nitric oxide (NO) is reduced by superoxide anion. Because the impact of dietary salt on resistance vessels in other species is largely unknown, we investigated endothelium-dependent dilation and oxidant activity in spinotrapezius muscle arterioles of C57BL/6J mice fed normal (0.45%, NS) or high salt (7%, HS) diets for 4 wk. Mean arterial pressure in HS mice was not different from that in NS mice, but the magnitude of arteriolar dilation in response to different levels of ACh was 42-57% smaller in HS mice than in NS mice. Inhibition of nitric oxide synthase (NOS) with N(G) monomethyl L-arginine (L-NMMA) significantly reduced resting diameters and reduced responses to ACh (by 45-63%) in NS mice but not in HS mice. Arteriolar wall oxidant activity, as assessed by tetranitroblue tetrazolium reduction or hydroethidine oxidation, was greater in HS mice than in NS mice. Exposure to the superoxide scavenger 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) + catalase reduced this oxidant activity to normal and restored normal arteriolar responsiveness to ACh in HS mice but had no effect in NS mice. L-NMMA also restored arteriolar oxidant activity to normal in HS mice. ACh further increased arteriolar oxidant activity in HS mice but not in NS mice, and this effect was prevented with L-NMMA. These data suggest that a high salt diet promotes increased generation of superoxide anion from NOS in the murine skeletal muscle microcirculation, thus impairing endothelium-dependent dilation through reduced NO bioavailability.