Daniel Escobar

Augmenting Autophagy to Treat Acute Kidney Injury during Endotoxemia in Mice

Objective To determine that 1) an age-dependent loss of inducible autophagy underlies the failure to recover from AKI in older, adult animals during endotoxemia, and 2) pharmacologic induction of autophagy, even after established endotoxemia, is of therapeutic utility in facilitating renal recovery in aged mice. Design Murine model of endotoxemia and cecal ligation and puncture (CLP) induced acute kidney injury (AKI). Setting Academic research laboratory. Subjects C57Bl/6 mice of 8 (young) and 45 (adult) weeks of age. Intervention Lipopolysaccharide (1.5 mg/kg), Temsirolimus (5 mg/kg), AICAR (100 mg/kg). Measurements and Main Results: Herein we report that diminished autophagy underlies the failure to recover renal function in older adult mice utilizing a murine model of LPS-induced AKI. The administration of the mTOR inhibitor temsirolimus, even after established endotoxemia, induced autophagy and protected against the development of AKI. Conclusions These novel results demonstrate a role for autophagy in the context of LPS-induced AKI and support further investigation into like interventions that have potential to alter the natural history of disease.

Adenosine monophosphate-activated protein kinase activation protects against sepsis-induced organ injury and inflammation

BACKGROUND Mortality in sepsis is most often attributed to the development of multiple organ failure. In sepsis, inflammation-mediated endothelial activation, defined as a proinflammatory and procoagulant state of the endothelial cells, has been associated with severity of disease. Thus, the objective of this study was to test the hypothesis that adenosine monophosphate-activated protein kinase (AMPK) activation limits inflammation and endothelium activation to protect against organ injury in sepsis. 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), which is an adenosine monophosphate analog, has been used to upregulate activity of AMPK. Compound C is a cell-permeable pyrrazolopyrimidine compound that inhibits AMPK activity. METHODS Wild-type mice underwent cecal ligation and puncture (CLP) or sham surgery. Mice were randomized to vehicle, AICAR, or compound C. Mouse kidney endothelial cells were used for in vitro experiments. Renal and liver function were determined by serum cystatin C, blood urea nitrogen (BUN), creatinine, and alanine aminotransferase. Serum cytokines were measured by enzyme-linked immunosorbent assay. Microvascular injury was determined using Evans blue dye and electron microscopy. Immunohistochemistry was used to measure protein levels of phospho-AMPK (p-AMPK), microtubule-associated protein 1A/1B-light chain 3 (LC3), and intracellular adhesion molecule. LC3 levels were used as a measure of autophagosome formation. RESULTS AICAR decreased liver and kidney injury induced by CLP and minimized cytokine elevation in vivo and in vitro. CLP increased renal and hepatic phosphorylation of AMPK and autophagic signaling as determined by LC3. Inhibition of AMPK with compound C prevented CLP-induced autophagy and exacerbated tissue injury. Additionally, CLP led to endothelial injury as determined by electron microscopy and Evans blue dye extravasation, and AICAR limited this injury. Furthermore, AICAR limited CLP and lipopolysaccharide (LPS)-induced upregulation of intracellular adhesion molecule in vivo and in vitro and decreased LPS-induced neutrophil adhesion in vitro. CONCLUSIONS In this model, activation of AMPK was protective, and AICAR minimized organ injury by decreasing inflammatory cytokines and endothelial activation. These data suggest that AMPK signaling influences sepsis or LPS-induced endothelial activation and organ injury.

Carbon Monoxide Protects Against Hemorrhagic Shock and Resuscitation-Induced Microcirculatory Injury and Tissue Injury

ABSTRACT Traumatic injury is a significant cause of morbidity and mortality worldwide. Microcirculatory activation and injury from hemorrhage contribute to organ injury. Many adaptive responses occur within the microcirculatory beds to limit injury including upregulation of heme oxygenase (HO) enzymes, the rate-limiting enzymes in the breakdown of heme to carbon monoxide (CO), iron, and biliverdin. Here we tested the hypothesis that CO abrogates trauma-induced injury and inflammation protecting the microcirculatory beds. Methods: C57Bl/6 mice underwent sham operation or hemorrhagic shock to a mean arterial pressure of 25 mmHg for 120 minutes. Mice were resuscitated with lactated Ringer’s at 2× the volume of maximal shed blood. Mice were randomized to receive CO-releasing molecule or inactive CO-releasing molecule at resuscitation. A cohort of mice was pretreated with tin protoporphyrin-IX to inhibit endogenous CO generation by HOs. Primary mouse liver sinusoidal endothelial cells were cultured for in vitro experiments. Results: Carbon monoxide–releasing molecule protected against hemorrhagic shock/resuscitation organ injury and systemic inflammation and reduced hepatic sinusoidal endothelial injury. Inhibition of HO activity with tin protoporphyrin-IX exacerbated liver hepatic sinusoidal injury. Hemorrhagic shock/resuscitation in vivo or cytokine stimulation in vitro resulted in increased endothelial expression of adhesion molecules that was associated with decreased leukocyte adhesion in vivo and in vitro. Conclusions: Hemorrhagic shock/resuscitation is associated with endothelial injury. Heme oxygenase enzymes and CO are involved in part in diminishing this injury and may prove useful as a therapeutic adjunct that can be harnessed to protect against endothelial activation and damage.

Polymicrobial sepsis is associated with decreased hepatic oxidative phosphorylation and an altered metabolic profile

BACKGROUND Organ failure in sepsis accounts for significant mortality worldwide. Mitochondrial and metabolic responses are central to the overall response of the cell, and thus of the organ and organism. Adaptive responses in metabolism are critical to the recovery at the cellular level. The purpose of these investigations was to test the hypothesis that sepsis is associated with decreased aerobic respiration and significant metabolic changes in the liver. METHODS C57BL/6 mice underwent cecal ligation and puncture (CLP) with a 21 gauge needle or an operation without CLP. Mice were euthanized from 0-24 h after the procedure and liver tissue was harvested. Tissue oxygen consumption and mitochondrial complex activity were measured. Global biochemical profiles of 311 metabolites were performed at the 8-h time point (n = 8/group) and analyzed by gas chromatography-mass spectrometry and liquid chromatography tandem mass spectrometry platforms by Metabolon (Durham, North Carolina). The influence of lipopolysaccharide (LPS) on aerobic and anaerobic respiration in primary mouse hepatocytes was also investigated. RESULTS CLP in vivo or LPS in vitro resulted in a significant decrease in hepatic oxygen consumption. There was a significant decrease in oxidative phosphorylation measured at 12 h. LPS also resulted in a significant increase in anaerobic respiration in hepatocytes. Interestingly, the metabolomic analysis resulted in a metabolic shift in the liver from carbohydrate-based energy to utilization of fatty acids and amino acids. This included an increase in every tricarboxylic acid cycle intermediate and derivative, suggesting an increased flux into the cycle from fatty acid beta-oxidation and anaplerotic contributions from amino acids. CONCLUSIONS Sepsis results in a metabolic response and profile consistent with increased anaerobic respiration, which occurs prior to significant changes in hemodynamics. The metabolic responses of cells and organs may be important adaptive responses to prevent organ failure and death.

Inhaled Carbon Monoxide Protects against the Development of Shock and Mitochondrial Injury following Hemorrhage and Resuscitation

Aims Currently, there is no effective resuscitative adjunct to fluid and blood products to limit tissue injury for traumatic hemorrhagic shock. The objective of this study was to investigate the role of inhaled carbon monoxide (CO) to limit inflammation and tissue injury, and specifically mitochondrial damage, in experimental models of hemorrhage and resuscitation. Results Inhaled CO (250 ppm for 30 minutes) protected against mortality in severe murine hemorrhagic shock and resuscitation (HS/R) (20% vs. 80%; P<0.01). Additionally, CO limited the development of shock as determined by arterial blood pH (7.25±0.06 vs. 7.05±0.05; P<0.05), lactate levels (7.2±5.1 vs 13.3±6.0; P<0.05), and base deficit (13±3.0 vs 24±3.1; P<0.05). A dose response of CO (25–500 ppm) demonstrated protection against HS/R lung and liver injury as determined by MPO activity and serum ALT, respectively. CO limited HS/R-induced increases in serum tumor necrosis factor-α and interleukin-6 levels as determined by ELISA (P<0.05 for doses of 100–500ppm). Furthermore, inhaled CO limited HS/R induced oxidative stress as determined by hepatic oxidized glutathione:reduced glutathione levels and lipid peroxidation. In porcine HS/R, CO did not influence hemodynamics. However, CO limited HS/R-induced skeletal muscle and platelet mitochondrial injury as determined by respiratory control ratio (muscle) and ATP-linked respiration and mitochondrial reserve capacity (platelets). Conclusion These preclinical studies suggest that inhaled CO can be a protective therapy in HS/R; however, further clinical studies are warranted.

Factors associated with hysterectomy among older women from Latin America and the Caribbean.

ABSTRACT To identify factors associated with hysterectomy, data collected from 1999–2000 were assessed from seven cities of the Health, Well-Being and Aging in Latin America and the Caribbean Study on 6,549 women, aged 60 years and older. Hysterectomy prevalence ranged from 12.8% in Buenos Aires (Argentina) to 30.4% in Bridgetown (Barbados). The median age for having had a hysterectomy ranged from 45 to 50 years across the cities and was 47 years in the pooled sample. Ethnic differences in hysterectomy rates were partially explained by differences across cities. Factors significantly associated with lower odds for hysterectomy included older age, household crowding conditions, and having public/military or no health insurance, compared to having private health insurance. Women who had three or more children were less likely to have had a hysterectomy, a finding that differs from most previous studies. Socioeconomic position related to rates of hysterectomy in late life rather than hysterectomies earlier in life. However, the nature of these differences varied across birth cohorts. The findings suggested that adverse socioeconomic factors were most likely related to hysterectomy risk by affecting access to health care, whereas parity was most likely acting through an effect on decision-making processes.

Sepsis-Induced Autophagy is a Protective Mechanism against Cell Death

In this chapter we discuss sepsis-induced inflammation and organ dysfunction, as well as the role of autophagy as a protective cell signaling mechanism. Sepsis is a leading cause of death worldwide, and is the most common precipitant of organ dysfunction. It has been noted that a minimal amount of cell death is present in patients who succumb to sepsis-induced organ failure and that, after the septic insult has passed, organs have the potential to regain function, suggesting a hibernating state of the cell in order to protect it from apoptosis and death. The cellular and molecular processes involved in sepsis are complex, and continue to be elucidated. Autophagy has been extensively studied in recent decades and an exponential increase in knowledge regarding this physiological pathway used by cells has been found. It is known to be activated under stress conditions in order to protect the cell from dying. In the setting of sepsis it has been demonstrated to play an important role for recycling of unhealthy mitochondria and damaged organelles, thus decreasing injury by ROS and providing nutrients and amino acids that the cell can use in this stressful environment. The purpose of this chapter is to review what is known about autophagy in sepsis and describe the contributing mechanisms by which autophagy can protect against organ injury.