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Item A new perspective on NO pathway in sepsis and ADMA lowering as a potential therapeutic approach(BMC, 2022-08-12) Singh, Jaipal; Lee, Young; Kellum, John A.; Cellular and Integrative Physiology, School of MedicineThe nitric oxide pathway plays a critical role in vascular homeostasis. Increased levels of systemic nitric oxide (NO) are observed in preclinical models of sepsis and endotoxemia. This has led to the postulation that vasodilation by inducible nitric oxide synthase (iNOS) generated NO may be a mechanism of hypotension in sepsis. However, contrary to the expected pharmacological action of a nitric oxide synthase (NOS) inhibitor, clinical studies with L-NAME produced adverse cardiac and pulmonary events, and higher mortality in sepsis patients. Thus, the potential adverse effects of NO in human sepsis and shock have not been fully established. In recent years, the emerging new understanding of the NO pathway has shown that an endogenously produced inhibitor of NOS, asymmetric dimethylarginine (ADMA), a host response to infection, may play an important role in the pathophysiology of sepsis as well as organ damage during ischemia-reperfusion. ADMA induces microvascular dysfunction, proinflammatory and prothrombotic state in endothelium, release of inflammatory cytokines, oxidative stress and mitochondrial dysfunction. High levels of ADMA exist in sepsis patients, which may produce adverse effects like those observed with L-NAME. Several studies have demonstrated the association of plasma ADMA levels with mortality in sepsis patients. Preclinical studies in sepsis and ischemia-reperfusion animal models have shown that lowering of ADMA reduced organ damage and improved survival. The clinical finding with L-NAME and the preclinical research on ADMA "bed to bench" suggest that ADMA lowering could be a potential therapeutic approach to attenuate progressive organ damage and mortality in sepsis. Testing of this approach is now feasible by using the pharmacological molecules that specifically lower ADMA.Item Depression of fast excitatory synaptic transmission in large aspiny neurons of the neostriatum after transient forebrain ischemia(Society for Neuroscience, 2002-12) Pang, Zhi-Ping; Deng, Ping; Ruan, Yi-Wen; Xu, Zao C.; Anatomy and Cell Biology, School of MedicineSpiny neurons in the neostriatum die within 24 hr after transient global ischemia, whereas large aspiny (LA) neurons remain intact. To reveal the mechanisms of such selective cell death after ischemia, excitatory neurotransmission was studied in LA neurons before and after ischemia. The intrastriatally evoked fast EPSCs in LA neurons were depressed < or =24 hr after ischemia. The concentration-response curves generated by application of exogenous glutamate in these neurons were approximately the same before and after ischemia. A train of five stimuli (100 Hz) induced progressively smaller EPSCs, but the proportion of decrease in EPSC amplitude at 4 hr after ischemia was significantly smaller compared with control and at 24 hr after ischemia. Parallel depression of NMDA receptor and AMPA receptor-mediated EPSCs was also observed after ischemia, supporting the involvement of presynaptic mechanisms. The adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked the inhibition of evoked EPSCs at 4 hr after ischemia but not at 24 hr after ischemia. Electron microscopic studies demonstrated that the most presynaptic terminals in the striatum had a normal appearance at 4 hr after ischemia but showed degenerating signs at 24 hr after ischemia. These results indicated that the excitatory neurotransmission in LA neurons was depressed after ischemia via presynaptic mechanisms. The depression of EPSCs shortly after ischemia might be attributable to the enhanced adenosine A1 receptor function on synaptic transmission, and the depression at late time points might result from the degeneration of presynaptic terminals.Item Hydrodynamic delivery for prevention of acute kidney injury(2015) Zhang, Shijun; Atkinson, Simon; Bacallao, Robert; Blazer-Yost, BonnieThe young field of gene therapy offers the promises of significant progress towards the treatment of many different types of human diseases. Gene therapy has been proposed as an innovative way to treat Acute Kidney Injury (AKI). Through proteomic analysis, the upregulation of two enzymes, IDH2 and SULT1C2, within the mitochondrial fraction has been identified following ischemic preconditioning, a treatment by which rat kidneys are protected from ischemia. Using the hydrodynamic fluid gene delivery technique, we were able to upregulate the expression of IDH2 and SULT1C2 in the kidney. We found that the delivery of IDH2 plasmid through hydrodynamic fluid delivery to the kidney resulted in increased mitochondrial oxygen respiration compared with injured kidneys without gene delivery. We also found that renal ischemic preconditioning altered the membrane fluidity of mitochondria. In conclusion, our study supports the idea that upregulated expression of IDH2 in mitochondria can protect the kidney against AKI, while the protective function of upregulated SULT1C2 needs to be further studied.Item Hypoxia Resistance Is an Inherent Phenotype of the Mouse Flexor Digitorum Brevis Skeletal Muscle(Oxford University Press, 2023-03-21) Amorese, Adam J.; Minchew, Everett C.; Tarpey, Michael D.; Readyoff, Andrew T.; Williamson, Nicholas C.; Schmidt, Cameron A.; McMillin, Shawna L.; Goldberg, Emma J.; Terwilliger, Zoe S.; Spangenburg, Quincy A.; Witczak, Carol A.; Brault, Jeffrey J.; Abel, E. Dale; McClung, Joseph M.; Fisher-Wellman, Kelsey H.; Spangenburg, Espen E.; Anatomy, Cell Biology and Physiology, School of MedicineThe various functions of skeletal muscle (movement, respiration, thermogenesis, etc.) require the presence of oxygen (O2). Inadequate O2 bioavailability (ie, hypoxia) is detrimental to muscle function and, in chronic cases, can result in muscle wasting. Current therapeutic interventions have proven largely ineffective to rescue skeletal muscle from hypoxic damage. However, our lab has identified a mammalian skeletal muscle that maintains proper physiological function in an environment depleted of O2. Using mouse models of in vivo hindlimb ischemia and ex vivo anoxia exposure, we observed the preservation of force production in the flexor digitorum brevis (FDB), while in contrast the extensor digitorum longus (EDL) and soleus muscles suffered loss of force output. Unlike other muscles, we found that the FDB phenotype is not dependent on mitochondria, which partially explains the hypoxia resistance. Muscle proteomes were interrogated using a discovery-based approach, which identified significantly greater expression of the transmembrane glucose transporter GLUT1 in the FDB as compared to the EDL and soleus. Through loss-and-gain-of-function approaches, we determined that GLUT1 is necessary for the FDB to survive hypoxia, but overexpression of GLUT1 was insufficient to rescue other skeletal muscles from hypoxic damage. Collectively, the data demonstrate that the FDB is uniquely resistant to hypoxic insults. Defining the mechanisms that explain the phenotype may provide insight towards developing approaches for preventing hypoxia-induced tissue damage.Item Impact of Early Revascularization on Major Adverse Cardiovascular Events in Relation to Automatically Quantified Ischemia(Elsevier, 2021) Azadani, Peyman N.; Miller, Robert J. H.; Sharir, Tali; Diniz, Marcio A.; Hu, Lien-Hsin; Otaki, Yuka; Gransar, Heidi; Liang, Joanna X.; Eisenberg, Evann; Einstein, Andrew J.; Fish, Mathews B.; Ruddy, Terrence D.; Kaufmann, Philipp A.; Sinusas, Albert J.; Miller, Edward J.; Bateman, Timothy M.; Dorbala, Sharmila; Di Carli, Marcelo; Tamarappoo, Balaji K.; Dey, Damini; Berman, Daniel S.; Slomka, Piotr J.; Medicine, School of MedicineObjectives: Using a contemporary, multicenter international single-photon emission computed tomography myocardial perfusion imaging (SPECT-MPI) registry, this study characterized the potential major adverse cardiovascular event(s) (MACE) benefit of early revascularization based on automatic quantification of ischemia. Background: Prior single-center data reported an association between moderate to severe ischemia SPECT-MPI and reduced cardiac death with early revascularization. Methods: Consecutive patients from a multicenter, international registry who underwent 99mTc SPECT-MPI between 2009 and 2014 with solid-state scanners were included. Ischemia was quantified automatically as ischemic total perfusion deficit (TPD). Early revascularization was defined as within 90 days. The primary outcome was MACE (death, myocardial infarction, and unstable angina). A propensity score was developed to adjust for nonrandomization of revascularization; then, multivariable Cox modeling adjusted for propensity score and demographics was used to predict MACE. Results: In total, 19,088 patients were included, with a mean follow-up of 4.7 ± 1.6 years, during which MACE occurred in 1,836 (9.6%) patients. There was a significant interaction between ischemic TPD modeled as a continuous variable and early revascularization (interaction p value: 0.012). In this model, there was a trend toward reduced MACE in patients with >5.4% ischemic TPD and a significant association with reduced MACE in patients with >10.2% ischemic TPD. Conclusions: In this large, international, multicenter study reflecting contemporary cardiology practice, early revascularization of patients with >10.2% ischemia on SPECT-MPI, quantified automatically, was associated with reduced MACE.Item Impaired microvascular circulation in distant organs following renal ischemia(Public Library of Science, 2023-06-02) Dominguez, Jesus H.; Xie, Danhui; Kelly, K. J.; Medicine, School of MedicineMortality in acute kidney injury (AKI) patients remains very high, although very important advances in understanding the pathophysiology and in diagnosis and supportive care have been made. Most commonly, adverse outcomes are related to extra-renal organ dysfunction and failure. We and others have documented inflammation in remote organs as well as microvascular dysfunction in the kidney after renal ischemia. We hypothesized that abnormal microvascular flow in AKI extends to distant organs. To test this hypothesis, we employed intravital multiphoton fluorescence imaging in a well-characterized rat model of renal ischemia/reperfusion. Marked abnormalities in microvascular flow were seen in every organ evaluated, with decreases up to 46% observed 48 hours postischemia (as compared to sham surgery, p = 0.002). Decreased microvascular plasma flow was found in areas of erythrocyte aggregation and leukocyte adherence to endothelia. Intravital microscopy allowed the characterization of the erythrocyte formations as rouleaux that flowed as one-dimensional aggregates. Observed microvascular abnormalities were associated with significantly elevated fibrinogen levels. Plasma flow within capillaries as well as microthrombi, but not adherent leukocytes, were significantly improved by treatment with the platelet aggregation inhibitor dipyridamole. These microvascular defects may, in part, explain known distant organ dysfunction associated with renal ischemia. The results of these studies are relevant to human acute kidney injury.Item Ischemia-Selective Cardioprotection by Malonate for Ischemia/Reperfusion Injury(American Heart Association, 2022) Prag, Hiran A.; Aksentijevic, Dunja; Dannhorn, Andreas; Giles, Abigail V.; Mulvey, John F.; Sauchanka, Olga; Du, Luping; Bates, Georgina; Reinhold, Johannes; Kula-Alwar, Duvaraka; Xu, Zhelong; Pellerin, Luc; Goodwin, Richard J. A.; Murphy, Michael P.; Krieg, Thomas; Surgery, School of MedicineBackground: Inhibiting SDH (succinate dehydrogenase), with the competitive inhibitor malonate, has shown promise in ameliorating ischemia/reperfusion injury. However, key for translation to the clinic is understanding the mechanism of malonate entry into cells to enable inhibition of SDH, its mitochondrial target, as malonate itself poorly permeates cellular membranes. The possibility of malonate selectively entering the at-risk heart tissue on reperfusion, however, remains unexplored. Methods: C57BL/6J mice, C2C12 and H9c2 myoblasts, and HeLa cells were used to elucidate the mechanism of selective malonate uptake into the ischemic heart upon reperfusion. Cells were treated with malonate while varying pH or together with transport inhibitors. Mouse hearts were either perfused ex vivo (Langendorff) or subjected to in vivo left anterior descending coronary artery ligation as models of ischemia/reperfusion injury. Succinate and malonate levels were assessed by liquid chromatography-tandem mass spectrometry LC-MS/MS, in vivo by mass spectrometry imaging, and infarct size by TTC (2,3,5-triphenyl-2H-tetrazolium chloride) staining. Results: Malonate was robustly protective against cardiac ischemia/reperfusion injury, but only if administered at reperfusion and not when infused before ischemia. The extent of malonate uptake into the heart was proportional to the duration of ischemia. Malonate entry into cardiomyocytes in vivo and in vitro was dramatically increased at the low pH (≈6.5) associated with ischemia. This increased uptake of malonate was blocked by selective inhibition of MCT1 (monocarboxylate transporter 1). Reperfusion of the ischemic heart region with malonate led to selective SDH inhibition in the at-risk region. Acid-formulation greatly enhances the cardioprotective potency of malonate. Conclusions: Cardioprotection by malonate is dependent on its entry into cardiomyocytes. This is facilitated by the local decrease in pH that occurs during ischemia, leading to its selective uptake upon reperfusion into the at-risk tissue, via MCT1. Thus, malonate's preferential uptake in reperfused tissue means it is an at-risk tissue-selective drug that protects against cardiac ischemia/reperfusion injury.Item Ischemic preconditioning and hydrodynamic delivery for the prevention of acute kidney injury(2015-07) Lu, Keyin; Atkinson, Simon J.; Bacallao, Robert L.; Blazer-Yost, BonnieAcute Kidney Injury (AKI) is a prevalent and significant problem whose primary treatment is supportive care. Ischemic preconditioning is a strategy used to protect organs from ischemic injury via a prior injury. Ischemic preconditioning in the kidneys has been shown to confer protection onto kidneys from subsequent ischemic insults with attenuated serum creatinine values in treated rats. In the preconditioned kidneys, the enzyme IDH2 was discovered to be upregulated in the mitochondria. Hydrodynamic fluid delivery to the kidney was found to be a viable technique for delivering this gene to the kidney, resulting in artificially upregulated expression of IDH2. Via a two-pronged effort to discern the functional significance of ischemic preconditioning and hydrodynamic IDH2 fluid injections, we performed mitochondrial oxygen respiration assays on both preconditioned and injected kidneys. We found that renal ischemic preconditioning resulted in no significant difference between sham and preconditioned, subsequently injured kidneys, which is similar to the results from the serum creatinine studies. Hydrodynamically IDH2-injected, and subsequently injured kidneys respire significantly better than vehicle injected, and subsequently injured kidneys, which shows that hydrodynamic injections of IDH2 protects kidneys against injury, and partially mimics the effects of preconditioning.Item Major Collateral Vessels Develop from Pre-existing Small Arteries through RAC2/NOX2 Independent Mechanisms(2009-03-18T18:45:14Z) DiStasi, Matthew Robert; Unthank, JosephThere is no consensus on which vascular segment or what size of vessels is most important in the process of collateral growth, the degree to which these vessels can enlarge, or the mechanisms that mediate collateral vessel expansion and its impairment. Chapter I identifies the major collateral vessels that develop in response to femoral arterial occlusion in the pig, rat, and mouse hindlimbs for comparison to humans. Pre-existent small named arteries enlarged ~2-3-fold to become the major collateral vessels in each species, these major collaterals displayed characteristics similar to large arteries experiencing flow-mediated outward remodeling, and important differences in vascular wall thickness were observed between rodents and pigs. Chapter II utilized Rac2-/- and Nox2-/- mice to investigate the hypothesis that Nox2-NAD(P)H oxidase is required for major collateral growth subsequent to femoral arterial occlusion. Previous studies suggest bone marrow cell (BMC)-derived reactive oxygen species (ROS) produced by the Nox2 subunit of NAD(P)H oxidase plays an important role in neovascularization and recovery of hindlimb perfusion subsequent to femoral arterial occlusion; but did not investigate collateral growth. The hematopoietic cell restricted protein Rac2 has been shown to bind to and activate Nox2-NAD(P)H oxidase and Rac2-/- and Nox2-/- leukocytes display impaired ROS related functions. The data demonstrated that Rac2 and Nox2 are not essential for major collateral growth, but both are important for the recovery of hindlimb perfusion and preservation of distal tissue morphology. Chapter III investigated BMC and antioxidant therapy in the age-related impairment of collateral growth. Aging, like all cardiovascular disease risk factors is associated with elevated ROS and impaired collateral growth. Studies also suggest BMCs promote collateral growth by secreting paracrine factors but elevated ROS may affect the efficacy of BMCs. The data revealed that neither BMC injection nor antioxidant therapy via apocynin enhanced the process of major collateral artery growth in aged mice.Item Mechanisms of Cytoskeletal Dysregulation in the Kidney Proximal Tubule During ATP Depletion and Ischemia(2009-08) Zhang, Hao; Atkinson, Simon J.; Harrington, Maureen A.; Marrs, James A.; Quilliam, Lawrence A.Knowledge of the molecular and cellular mechanisms of ischemic injury is necessary for understanding acute kidney injury and devising optimal treatment regimens. The cortical actin cytoskeleton in the proximal tubule epithelial cells of the kidney nephron, playing an important role in both the establishment and maintenance of cell polarity, is drastically disrupted by the onset of ischemia. We found that in LLC-PK cells (a porcine kidney proximal tubule epithelial cell line), cortactin, an important regulator of actin assembly and organization, translocated from the cell cortex to the cytoplasmic regions upon ischemia/ATP-depletion. Meanwhile both the tyrosine phosphorylation level of cortactin and cortactin’s interaction with either F-actin or the actin nucleator Arp2/3 complex were down-regulated upon ischemia/ATP-depletion or inhibition of Src kinase activity. These results suggest that tyrosine phosphorylation plays an important role in regulating cortactin’s cellular function and localization in the scenario of kidney ischemia. The Rho GTPase signaling pathways is also a critical mediator of the effects of ATP depletion and ischemia on the actin cytoskeleton, but the mechanism by which ATP depletion leads to altered RhoA and Rac1 activity is unknown. We propose that ischemia and ATP depletion result in activation of AMP-activated protein kinase (AMPK) and that this affects Rho GTPase activity and cytoskeletal organization (possibly via TSC1/2 complex and/or mTOR complex). We found that AMPK was rapidly activated (≤5 minutes) by ATP depletion in S3 epithelial cells derived from the proximal tubule in mouse kidney, and there was a corresponding decrease in RhoA and Rac1 activity. During graded ATP-depletion, we found intermediate levels of AMPK activity at the intermediate ATP levels, and that the activity of RhoA and Rac1 activity correlated inversely with the activity of AMPK. Activation of AMPK using two different drugs suppressed RhoA activity, and also led to morphological changes of stress fibers. In addition, the inhibition of AMPK activation partially rescued the disruption of stress fibers caused by ATP-depletion. This evidence supports our hypothesis that the activation of AMPK is upstream of the signaling pathways that eventually lead to RhoA inactivation and cytoskeletal dysregulation during ATP-depletion.