Prion Protein Protects against Renal Ischemia/Reperfusion Injury

The cellular prion protein (PrP^C), a protein most noted for its link to prion diseases, has been found to play a protective role in ischemic brain injury. To investigate the role of PrP^C in the kidney, an organ highly prone to ischemia/reperfusion (IR) injury, we examined wild-type (WT) and PrP^C knockout (KO) mice that were subjected to 30-min of renal ischemia followed by 1, 2, or 3 days of reperfusion. Renal dysfunction and structural damage was more severe in KO than in WT mice. While PrP was undetectable in KO kidneys, Western blotting revealed an increase in PrP in IR-injured WT kidneys compared to sham-treated kidneys. Compared to WT, KO kidneys exhibited increases in oxidative stress markers heme oxygenase-1, nitrotyrosine, and N^ε-(carboxymethyl)lysine, and decreases in mitochondrial complexes I and III. Notably, phosphorylated extracellular signal-regulated kinase (pERK) staining was predominantly observed in tubular cells from KO mice following 2 days of reperfusion, a time at which significant differences in renal dysfunction, histological changes, oxidative stress, and mitochondrial complexes between WT and KO mice were observed. Our study provides the first evidence that PrP^C may play a protective role in renal IR injury, likely through its effects on mitochondria and ERK signaling pathways.     

Dual deficiency of angiotensin‐converting enzyme‐2 and Mas receptor enhances angiotensin II‐induced hypertension and hypertensive nephropathy

Angiotensin‐converting enzyme‐2 (ACE2) and Mas receptor are the major components of the ACE2/Ang 1‐7/Mas axis and have been shown to play a protective role in hypertension and hypertensive nephropathy individually. However, the effects of dual deficiency of ACE2 and Mas (ACE2/Mas) on Ang II‐induced hypertensive nephropathy remain unexplored, which was investigated in this study in a mouse model of hypertension induced in either ACE2 knockout (KO) or Mas KO mice and in double ACE2/Mas KO mice by subcutaneously chronic infusion of Ang II. Compared with wild‐type (WT) animals, mice lacking either ACE2 or Mas significantly increased blood pressure over 7‐28 days following a chronic Ang II infusion (P < .001), which was further exacerbated in double ACE2/Mas KO mice (P < .001). Furthermore, compared to a single ACE2 or Mas KO mice, mice lacking ACE2/Mas developed more severe renal injury including higher levels of serum creatinine and a further reduction in creatinine clearance, and progressive renal inflammation and fibrosis. Mechanistically, worsen hypertensive nephropathy in double ACE2/Mas KO mice was associated with markedly enhanced AT1‐ERK1/2‐Smad3 and NF‐κB signalling, thereby promoting renal fibrosis and renal inflammation in the hypertensive kidney. In conclusion, ACE2 and Mas play an additive protective role in Ang II‐induced hypertension and hypertensive nephropathy. Thus, restoring the ACE2/Ang1‐7/Mas axis may represent a novel therapy for hypertension and hypertensive nephropathy.     

Disruption of Smad7 Promotes ANG II-Mediated Renal Inflammation and Fibrosis via Sp1-TGF-β/Smad3-NF.κB-Dependent Mechanisms in Mice

Smad7 is an inhibitory Smad and plays a protective role in obstructive and diabetic kidney disease. However, the role and mechanisms of Smad7 in hypertensive nephropathy remains unexplored. Thus, the aim of this study was to investigate the role and regulatory mechanisms of Smad7 in ANG II-induced hypertensive nephropathy. Smad7 gene knockout (KO) and wild-type (WT) mice received a subcutaneous infusion of ANG II or control saline for 4 weeks via osmotic mini-pumps. ANG II infusion produced equivalent hypertension in Smad7 KO and WT mice; however, Smad7 KO mice exhibited more severe renal functional injury as shown by increased proteinuria and reduced renal function (both p<0.05) when compared with Smad7 WT mice. Enhanced renal injury in Smad7 KO mice was associated with more progressive renal fibrosis with elevated TGF-β/Smad3 signalling. Smad7 KO mice also showed more profound renal inflammation including increased macrophage infiltration, enhanced IL-1β and TNF-α expression, and a marked activation of NF-κB signaling (all p<0.01). Further studies revealed that enhanced ANG II-mediated renal inflammation and fibrosis in Smad7 KO mice were also associated with up-regulation of Sp1 but downregulation of miR-29b expression. Taken together, the present study revealed that enhanced Sp1-TGF-β1/Smad3-NF-κB signaling and loss of miR-29 may be mechanisms by which deletion of Smad7 promotes ANG II-mediated renal fibrosis and inflammation. Thus, Smad7 may play a protective role in ANG II-induced hypertensive kidney disease.     

Deficiency of Smad7 Enhances Cardiac Remodeling Induced by Angiotensin II Infusion in a Mouse Model of Hypertension

Smad7 has been shown to negatively regulate fibrosis and inflammation, but its role in angiotensin II (Ang II)-induced hypertensive cardiac remodeling remains unknown. Therefore, the present study investigated the role of Smad7 in hypertensive cardiopathy induced by angiotensin II infusion. Hypertensive cardiac disease was induced in Smad7 gene knockout (KO) and wild-type (WT) mice by subcutaneous infusion of Ang II (1.46 mg/kg/day) for 28 days. Although equal levels of high blood pressure were developed in both Smad7 KO and WT mice, Smad7 KO mice developed more severe cardiac injury as demonstrated by impairing cardiac function including a significant increase in left ventricular (LV) mass (P<0.01),reduction of LV ejection fraction(P<0.001) and fractional shortening(P<0.001). Real-time PCR, Western blot and immunohistochemistry detected that deletion of Smad7 significantly enhanced Ang II-induced cardiac fibrosis and inflammation, including upregulation of collagen I, α-SMA, interleukin-1β, TNF-α, and infiltration of CD3⁺ T cells and F4/80⁺ macrophages. Further studies revealed that enhanced activation of the Sp1-TGFβ/Smad3-NF-κB pathways and downregulation of miR-29 were mechanisms though which deletion of Smad7 promoted Ang II-mediated cardiac remodeling. In conclusions, Smad7 plays a protective role in AngII-mediated cardiac remodeling via mechanisms involving the Sp1-TGF-β/Smad3-NF.κB-miR-29 regulatory network.     

Renal ischemia-reperfusion injury and adenosine 2A receptor-mediated tissue protection: the role of CD4+ T cells and IFN-gamma

A(2A) adenosine receptor (A(2A)R)-expressing bone marrow (BM)-derived cells contribute to the renal protective effect of A(2A) agonists in renal ischemia-reperfusion injury (IRI). We performed IRI in mice lacking T and B cells to determine whether A(2A)R expressed in CD4+ cells mediate protection from IRI. Rag-1 knockout (KO) mice were protected in comparison to wild-type (WT) mice when subjected to IRI. ATL146e, a selective A(2A) agonist, did not confer additional protection. IFN-gamma is an important early signal in IRI and is thought to contribute to reperfusion injury. Because IFN-gamma is produced by kidney cells and T cells we performed IRI in BM chimeras in which the BM of WT mice was reconstituted with BM from IFN-gamma KO mice (IFN-gamma KO-->WT chimera). We observed marked reduction in IRI in comparison to WT-->WT chimeras providing additional indirect support for the role of T cells. To confirm the role of CD4+ A(2A)R in mediating protection from IRI, Rag-1 KO mice were subjected to ischemia-reperfusion. The protection observed in Rag-1 KO mice was reversed in Rag-1 KO mice that were adoptively transferred WT CD4+ cells (WT CD4+-->Rag-1 KO) or A(2A) KO CD4+ cells (A(2A) KO CD4+-->Rag-1 KO). ATL146e reduced injury in WT CD4+-->Rag-1 KO mice but not in A(2A) KO CD4+-->Rag-1 KO mice. Rag-1 KO mice reconstituted with CD4+ cells derived from IFN-gamma KO mice (IFN-gamma CD4+-->Rag-1 KO) were protected from IRI; ATL146e conferred no additional protection. These studies demonstrate that CD4+ IFN-gamma contributes to IRI and that A(2A) agonists mediate protection from IRI through action on CD4+ cells.     

β-arrestin2 in Infiltrated Macrophages Inhibits Excessive Inflammation after Myocardial Infarction

Beta-arrestins (β-arrestin1 and β-arrestin2) are known as cytosolic proteins that mediate desensitization and internalization of activated G protein-coupled receptors. In addition to these functions, β-arrestins have been found to work as adaptor proteins for intracellular signaling pathways. β-arrestin1 and β-arrestin2 are expressed in the heart and are reported to participate in normal cardiac function. However, the physiological and pathological roles of β-arrestin1/2 in myocardial infarction (MI) have not been examined. Here, we demonstrate that β-arrestin2 negatively regulates inflammatory responses of macrophages recruited to the infarct area. β-arrestin2 knockout (KO) mice have higher mortality than wild-type (WT) mice after MI. In infarcted hearts, β-arrestin2 was strongly expressed in infiltrated macrophages. The production of inflammatory cytokines was enhanced in β-arrestin2 KO mice. In addition, p65 phosphorylation in the macrophages from the infarcted hearts of β-arrestin2 KO mice was increased in comparison to that of WT mice. These results suggest that the infiltrated macrophages of β-arrestin2 KO mice induce excessive inflammation at the infarct area. Furthermore, the inflammation in WT mice transplanted with bone marrow cells of β-arrestin2 KO mice is enhanced by MI, which is similar to that in β-arrestin2 KO mice. In contrast, the inflammation after MI in β-arrestin2 KO mice transplanted with bone marrow cells of WT mice is comparable to that in WT mice transplanted with bone marrow cells of WT mice. In summary, our present study demonstrates that β-arrestin2 of infiltrated macrophages negatively regulates inflammation in infarcted hearts, thereby enhancing inflammation when the β-arrestin2 gene is knocked out. β-arrestin2 plays a protective role in MI-induced inflammation.     

Interleukin-19 Mediates Tissue Damage in Murine Ischemic Acute Kidney Injury

Inflammation and renal tubular injury are major features of acute kidney injury (AKI). Many cytokines and chemokines are released from injured tubular cells and acts as proinflammatory mediators. However, the role of IL-19 in the pathogenesis of AKI is not defined yet. In bilateral renal ischemia/reperfusion injury (IRI)-induced and HgCl₂-induced AKI animal models, real-time quantitative (RTQ)-PCR showed that the kidneys, livers, and lungs of AKI mice expressed significantly higher IL-19 and its receptors than did sham control mice. Immunohistochemical staining showed that IL-19 and its receptors were strongly stained in the kidney, liver, and lung tissue of AKI mice. In vitro, IL-19 upregulated MCP-1, TGF-β1, and IL-19, and induced mitochondria-dependent apoptosis in murine renal tubular epithelial M-1 cells. IL-19 upregulated TNF-α and IL-10 in cultured HepG2 cells, and it increased IL-1β and TNF-α expression in cultured A549 cells. In vivo, after renal IRI or a nephrotoxic dose of HgCl₂ treatment, IL-20R1-deficient mice (the deficiency blocks IL-19 signaling) showed lower levels of blood urea nitrogen (BUN) in serum and less tubular damage than did wild-type mice. Therefore, we conclude that IL-19 mediates kidney, liver, and lung tissue damage in murine AKI and that blocking IL-19 signaling may provide a potent therapeutic strategy for treating AKI.     

β-Arrestin 2 Promotes Hepatocyte Apoptosis by Inhibiting Akt Protein

Recent studies reveal that multifunctional protein β-arrestin 2 (Arrb2) modulates cell apoptosis. Survival and various aspects of liver injury were investigated in WT and Arrb2 KO mice after bile duct ligation (BDL). We found that deficiency of Arrb2 enhances survival and attenuates hepatic injury and fibrosis. Following BDL, Arrb2-deficient mice as compared with WT controls displayed a significant reduction of hepatocyte apoptosis as demonstrated by the TUNEL assay. Following BDL, the levels of phospho-Akt and phospho-glycogen synthase kinase 3β (GSK3β) in the livers were significantly increased in Arrb2 KO compared with WT mice, although p-p38 increased in WT but not in Arrb2-deficient mice. Inhibition of GSK3β following BDL decreases hepatic apoptosis and decreased p-p38 in WT mice but not in Arrb2 KO mice. Activation of Fas receptor with Jo2 reduces phospho-Akt and increases apoptosis in WT cells and WT mice but not in Arrb2-deficient cells and Arrb2-deficient mice. Consistent with direct interaction of Arrb2 with and regulating Akt phosphorylation, the expression of a full-length or N terminus but not the C terminus of Arrb2 reduces Akt phosphorylation and coimmunoprecipates with Akt. These results reveal that the protective effect of deficiency of Arrb2 is due to loss of negative regulation of Akt due to BDL and decreased downstream GSK3β and p38 MAPK signaling pathways.     

Fractalkine aggravates LPS‐induced macrophage activation and acute kidney injury via Wnt/β‐catenin signalling pathway

Fractalkine (CX3CL1, FKN), a CX3C gene sequence inflammatory chemokine, has been found to have pro‐inflammatory and pro‐adhesion effects. Macrophages are immune cells with a critical role in regulating the inflammatory response. The imbalance of M1/M2 macrophage polarization can lead to aggravated inflammation. This study attempts to investigate the mechanisms through which FKN regulates macrophage activation and the acute kidney injury (AKI) involved in inflammatory response induced by lipopolysaccharide (LPS) by using FKN knockout (FKN‐KO) mice and cultured macrophages. It was found that FKN and Wnt/β‐catenin signalling have a positive interaction in macrophages. FKN overexpression inhibited LPS‐induced macrophage apoptosis. However, it enhanced their cell viability and transformed them into the M2 type. The effects of FKN overexpression were accelerated by activation of Wnt/β‐catenin signalling. In the in vivo experiments, FKN deficiency suppressed macrophage activation and reduced AKI induced by LPS. Inhibition of Wnt/β‐catenin signalling and FKN deficiency further mitigated the pathologic process of AKI. In summary, we provide a novel mechanism underlying activation of macrophages in LPS‐induced AKI. Although LPS‐induced murine AKI was unable to completely recapitulate human AKI, the positive interactions between FKN and Wnt/β‐catenin signalling pathway may be a therapeutic target in the treatment of kidney injury.     

NAD+ ameliorates endotoxin‐induced acute kidney injury in a sirtuin1–dependent manner via GSK‐3β/Nrf2 signalling pathway

Acute kidney injury (AKI) is a substantial worldwide public health concern with no specific and effective therapies in clinic. NAD⁺ is a pivotal determinant of cellular energy metabolism involved in the progression of AKI; however, its mechanism in kidney injury remains poorly understood. Sirtuin 1 (SIRT1) is an NAD⁺‐dependent deacetylase associated with renal protection and acute stress resistance. In this study, we have investigated the role of NAD⁺ in AKI and the potential mechanism(s) involved in its renoprotective effect. NAD⁺ was notably decreased and negatively correlated with kidney dysfunction in AKI, restoring NAD⁺ with NMN significantly ameliorates LPS‐induced oxidative stress and apoptosis and attenuates renal damage. We also found that the protection of NAD⁺ is associated with SIRT1 expressions and performs in a SIRT1‐dependent manner. Inhibition of SIRT1 blunted the protective effect of NAD⁺ and up‐regulated the activity of glycogen synthase kinase‐3β (GSK‐3β) that was concomitant with mitigated Nrf2 nuclear accumulation, thereby exacerbates AKI. These findings suggest that NAD⁺/SIRT1/GSK‐3β/Nrf2 axis is an important mechanism that can protect against AKI which might be a potential therapeutic target for the treatment of AKI.     

PPARα Protein Expression Was Increased by Four Weeks of Intermittent Hypoxic Training via AMPKα2-Dependent Manner in Mouse Skeletal Muscle

Peroxisome proliferator-activated receptor α (PPARα) is critical for muscle endurance due to its role in the regulation of fatty acid oxidation. The 5’-AMP-activated protein kinase (AMPK) is an energy sensor in cells, but its role in PPARα regulation in vivo remains unknown. In this study, we examined PPARα expression in the skeletal muscle of AMPKα2 overexpression (OE), knockout (KO) and wild-type (WT) mice after four weeks of exercise under intermittent hypoxia. WT, OE and KO mice were used at 40 mice/strain and randomly subdivided into four subgroups: control (C), running (R), hypoxia (H), and running plus hypoxia (R+H) at 10 mice/group. The treadmill running was performed at the speed of 12 m/min, 60 min/day with a slope of 0 degree for four weeks. The hypoxia treatment was performed in daytime with normobaric hypoxia (11.20% oxygen, 8 hours/day). In the R+H group, the treadmill running was conducted in the hypoxic condition. AMPKα2, phosphor-AMPKα (p-AMPKα) (Thr172), nuclear PPARα proteins were measured by Western blot and the medium chain acyl coenzyme A dehydrogenase (MCAD) mRNA, the key enzyme for fatty acid oxidation and one of the PPARα target genes, was also measured in skeletal muscles after the interventions. The results showed that nuclear PPARα protein was significantly increased by R+H in WT muscles, the increase was enhanced by 41% (p<0.01) in OE mice, but was reduced by 33% (p<0.01) in KO mice. The MCAD mRNA expression was increased after four weeks of R+H intervention. The change in MCAD mRNA followed a similar trend as that of PPARα protein in OE and KO mice. Our data suggest that the increase in nuclear PPARα protein by four-week exercise training under the intermittent hypoxia was dependent on AMPK activation.     

Increased Cellular Senescence and Vascular Rarefaction Exacerbate the Progression of Kidney Fibrosis in Aged Mice Following Transient Ischemic Injury

Recent findings indicate that elderly patients with acute kidney injury (AKI) have an increased incidence of progression to chronic kidney disease (CKD) due to incomplete recovery from an acute insult. In the current study, a co-morbid model of AKI was developed to better mimic the patient population and to investigate whether age exacerbates the fibrosis and inflammation that develop in the sequelae of progressive kidney disease following acute injury. Young (8–10 weeks) and aged (46–49 weeks) C57BL/6 mice were subjected to 30 min bilateral renal ischemia-reperfusion (I/R) to induce AKI. The aged animals have greater mortality and prolonged elevation of plasma creatinine correlating with less tubular epithelial cell proliferation compared to the young. Six weeks post-reperfusion, interstitial fibrosis is greater in aged kidneys based on picrosirius red staining and immunolocalization of cellular fibronectin, collagen III and collagen IV. Aged kidneys 6 weeks post-reperfusion also express higher levels of p53 and p21 compared to the young, correlating with greater increases in senescence associated (SA) β-galactosidase, a known marker of cellular senescence. A higher influx of F4/80⁺ macrophages and CD4⁺ T lymphocytes is measured and is accompanied by increases in mRNA of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α). Importantly, microvascular density is significantly less, correlating with an increase in nitro-tyrosine, a marker of oxidative stress. Collectively, these data demonstrate that prolonged acute injury in the aged animals results in an accelerated progression of kidney disease in a chronic state.     

Local Immunosuppressive Microenvironment Enhances Migration of Melanoma Cells to Lungs in DJ-1 Knockout Mice

DJ-1 is an oncoprotein that promotes survival of cancer cells through anti-apoptosis. However, DJ-1 also plays a role in regulating IL-1β expression, and whether inflammatory microenvironment built by dysregulated DJ-1 affects cancer progression is still unclear. This study thus aimed to compare the metastatic abilities of melanoma cells in wild-type (WT) and DJ-1 knockout (KO) mice, and to check whether inflammatory microenvironment built in DJ-1 KO mice plays a role in migration of cancer cells to lungs. First, B16F10 melanoma cells (at 6×10⁴) were injected into the femoral vein of mice, and formation of lung nodules, levels of lung IL-1β and serum cytokines, and accumulation of myeloid-derived suppressor cells (MDSCs) were compared between WT and DJ-1 KO mice. Second, the cancer-bearing mice were treated with an interleukin-1 beta (IL-1β) neutralizing antibody to see whether IL-1β is involved in the cancer migration. Finally, cultured RAW 264.7 macrophage and B16F10 melanoma cells were respectively treated with DJ-1 shRNA and recombinant IL-1β to explore underlying molecular mechanisms. Our results showed that IL-1β enhanced survival and colony formation of cultured melanoma cells, and that IL-1β levels were elevated both in DJ-1 KO mice and in cultured macrophage cells with DJ-1 knockdown. The elevated IL-1β correlated with higher accumulation of immunosuppressive MDSCs and formation of melanoma module in the lung of DJ-1 KO mice, and both can be decreased by treating mice with IL-1β neutralizing antibodies. Taken together, these results indicate that immunosuppressive tissue microenvironment built in DJ-1 KO mice can enhance lung migration of cancer, and IL-1β plays an important role in promoting the cancer migration.     

Hypoxia-Inducible Factor Activation Protects the Kidney from Gentamicin-Induced Acute Injury

Gentamicin nephrotoxicity is one of the most common causes of acute kidney injury (AKI). Hypoxia-inducible factor (HIF) is effective in protecting the kidney from ischemic and toxic injury. Increased expression of HIF-1α mRNA has been reported in rats with gentamicin-induced renal injury. We hypothesizd that we could study the role of HIF in gentamicin-induced AKI by modulating HIF activity. In this study, we investigated whether HIF activation had protective effects on gentamicin-induced renal tubule cell injury. Gentamicin-induced AKI was established in male Sprague-Dawley rats. Cobalt was continuously infused into the rats to activate HIF. HK-2 cells were pre-treated with cobalt or dimethyloxalylglycine (DMOG) to activate HIF and were then exposed to gentamicin. Cobalt or DMOG significantly increased HIF-1α expression in rat kidneys and HK-2 cells. In HK-2 cells, HIF inhibited gentamicin-induced reactive oxygen species (ROS) formation. HIF also protected these cells from apoptosis by reducing caspase-3 activity and the amount of cleaved caspase-3, and -9 proteins. Increased expression of HIF-1α reduced the number of gentamicin-induced apoptotic cells in rat kidneys and HK-2 cells. HIF activation improved the creatinine clearance and proteinuria in gentamicin-induced AKI. HIF activation also ameliorated the extent of histologic injury and reduced macrophage infiltration into the tubulointerstitium. In gentamicin-induced AKI, the activation of HIF by cobalt or DMOG attenuated renal dysfunction, proteinuria, and structural damage through a reduction of oxidative stress, inflammation, and apoptosis in renal tubular epithelial cells.     

Gastrointestinal ischemia-reperfusion injury is lectin complement pathway dependent without involving C1q

Complement activation plays an important role in local and remote tissue injury associated with gastrointestinal ischemia-reperfusion (GI/R). The role of the classical and lectin complement pathways in GI/R injury was evaluated using C1q-deficient (C1q KO), MBL-A/C-deficient (MBL-null), complement factor 2- and factor B-deficient (C2/fB KO), and wild-type (WT) mice. Gastrointestinal ischemia (20 min), followed by 3-h reperfusion, induced intestinal and lung injury in C1q KO and WT mice, but not in C2/fB KO mice. Addition of human C2 to C2/fB KO mice significantly restored GI/R injury, demonstrating that GI/R injury is mediated via the lectin and/or classical pathway. Tissue C3 deposition in C1q KO and WT, but not C2/fB KO, mice after GI/R demonstrated that complement was activated in C1q KO mice. GI/R significantly increased serum alanine aminotransferase, gastrointestinal barrier dysfunction, and neutrophil infiltration into the lung and gut in C1q KO and WT, but not C2/fB KO, mice. MBL-null mice displayed little gut injury after GI/R, but lung injury was present. Addition of recombinant human MBL (rhuMBL) to MBL-null mice significantly increased injury compared with MBL-null mice after GI/R and was reversed by anti-MBL mAb treatment. However, MBL-null mice were not protected from secondary lung injury after GI/R. These data demonstrate that C2 and MBL, but not C1q, are necessary for gut injury after GI/R. Lung injury in mice after GI/R is MBL and C1q independent, but C2 dependent, suggesting a potential role for ficolins in this model.     

Deletion of the gabra2 Gene Results in Hypersensitivity to the Acute Effects of Ethanol but Does Not Alter Ethanol Self Administration

Human genetic studies have suggested that polymorphisms of the GABRA2 gene encoding the GABA_A α2-subunit are associated with ethanol dependence. Variations in this gene also convey sensitivity to the subjective effects of ethanol, indicating a role in mediating ethanol-related behaviours. We therefore investigated the consequences of deleting the α2-subunit on the ataxic and rewarding properties of ethanol in mice. Ataxic and sedative effects of ethanol were explored in GABA_A α2-subunit wildtype (WT) and knockout (KO) mice using a Rotarod apparatus, wire hang and the duration of loss of righting reflex. Following training, KO mice showed shorter latencies to fall than WT littermates under ethanol (2 g/kg i.p.) in both Rotarod and wire hang tests. After administration of ethanol (3.5 g/kg i.p.), KO mice took longer to regain the righting reflex than WT mice. To ensure the acute effects are not due to the gabra2 deletion affecting pharmacokinetics, blood ethanol concentrations were measured at 20 minute intervals after acute administration (2 g/kg i.p.), and did not differ between genotypes. To investigate ethanol’s rewarding properties, WT and KO mice were trained to lever press to receive increasing concentrations of ethanol on an FR4 schedule of reinforcement. Both WT and KO mice self-administered ethanol at similar rates, with no differences in the numbers of reinforcers earned. These data indicate a protective role for α2-subunits, against the acute sedative and ataxic effects of ethanol. However, no change was observed in ethanol self administration, suggesting the rewarding effects of ethanol remain unchanged.     

Deletion of the mouse alpha-calcitonin gene-related peptide gene increases the vulnerability of the heart to ischemia-reperfusion injury

Calcitonin gene-related peptide (CGRP), a potent vasodilator released from capsaicin-sensitive C-fiber and Adelta-fiber sensory nerves, has been suggested to play a beneficial role in myocardial ischemia-reperfusion (I/R) injury. Because most previous studies showing a cardioprotective role of CGRP employed pharmacological experiments, the purpose of this study was to utilize a genetic approach by using mice with a targeted deletion of the alpha-CGRP gene to determine whether this neuropeptide had a modulatory function on the severity of I/R injury. To accomplish this goal, isolated, perfused hearts from alpha-CGRP knockout (KO) and wild-type (WT) mice were subjected to 30 min of ischemia followed by 5, 15, and 30 min of reperfusion. Cardiac functional parameters, including coronary flow rates, left ventricular developed pressure, maximum rates of pressure development, and left ventricular end-diastolic pressure, were measured before and after I/R injury, as were levels of creatine kinase, to assess myocardial damage, and malonaldehyde, to assess oxidative stress. Following I/R injury, cardiac performance was significantly reduced in the hearts from the alpha-CGRP KO mice compared with their WT counterparts. The marked reduction in myocardial function in the alpha-CGRP KO hearts compared with WT hearts after I/R injury was associated with a significant elevation in creatine kinase release into the perfusates and malonaldehyde production in the cardiac tissue. Therefore, these data indicate that, in this in vitro setting, deletion of alpha-CGRP makes the heart more vulnerable to I/R injury, possibly due, at least in part, to increased oxidative stress.     

Guanylyl Cyclase-G Modulates Jejunal Apoptosis and Inflammation in Mice with Intestinal Ischemia and Reperfusion

Background

Membrane bound guanylyl cyclase-G (mGC-G), a novel form of GC mediates ischemia and reperfusion (IR)-induced renal injury. We investigated the roles of mGC-G in intestinal IR-induced jejunal damage, inflammation, and apoptosis.

Materials and methods

Male C57BL/6 wild-type (WT) and mGC-G gene knockout (KO) mice were treated with a sham operation or 45 min of superior mesenteric arterial obstruction followed by 3, 6, 12, or 24 h of reperfusion.

Results

Sham-operated KO mice had significantly lower plasma nitrate and nitrite (NOx) levels and jejunal villus height, crypt depth, and protein expression of phosphorylated-nuclear factor-kappa-B (NF-κB), phosphorylated-c-Jun N-terminal kinases (JNK) 2/3, phosphorylated-p38, and B-cell lymphoma-2 (Bcl-2). They had significantly greater jejunal interleukin-6 mRNA, cytochrome c protein, and apoptotic index compared with sham-operated WT mice. Intestinal IR significantly decreased plasma NOx in WT mice and increased plasma NOx in KO mice. The jejunal apoptotic index and caspase 3 activities were significantly increased, and nuclear phosphorylated-NF-κB and phosphorylated-p38 protein were significantly decreased in WT, but not KO mice with intestinal IR. After reperfusion, KO mice had an earlier decrease in jejunal cyclic GMP, and WT mice had an earlier increase in jejunal proliferation and a later increase in cytosol inhibitor of kappa-B-alpha. Intestinal IR induced greater increases in plasma and jejunal interleukin-6 protein in WT mice and a greater increase in jejunal interleukin-6 mRNA in KO mice.

Conclusions

mGC-G is involved in the maintenance of jejunal integrity and intestinal IR-induced inflammation and apoptosis. These results suggest that targeting cGMP pathway might be a potential strategy to alleviate IR-induced jejunal damages.     

AMP Activated Protein Kinase Is Indispensable for Myocardial Adaptation to Caloric Restriction in Mice

Caloric restriction (CR) is a robust dietary intervention known to enhance cardiovascular health. AMP activated protein kinase (AMPK) has been suggested to mediate the cardioprotective effects of CR. However, this hypothesis remains to be tested by using definitive loss-of-function animal models. In the present study, we subjected AMPKα2 knockout (KO) mice and their wild type (WT) littermates to a CR regimen that reduces caloric intake by 20%–40% for 4 weeks. CR decreased body weight, heart weight and serum levels of insulin in both WT and KO mice to the same degree, indicating the effectiveness of the CR protocol. CR activated cardiac AMPK signaling in WT mice, but not in AMPKα2 KO mice. Correspondingly, AMPKα2 KO mice had markedly reduced cardiac function during CR as determined by echocardiography and hemodynamic measurements. The compromised cardiac function was associated with increased markers of oxidative stress, endoplasmic reticulum stress and myocyte apoptosis. Mechanistically, CR down-regulated the expression of ATP5g2, a subunit of mitochondrial ATP synthase, and reduced ATP content in AMPKα2 KO hearts, but not in WT hearts. In addition, CR accelerated cardiac autophagic flux in WT mice, but failed to do so in AMPKα2 KO mice. These results demonstrated that without AMPK, CR triggers adverse effects that can lead to cardiac dysfunction, suggesting that AMPK signaling pathway is indispensible for energy homeostasis and myocardial adaptation to CR, a dietary intervention that normally produces beneficial cardiac effects.