Hyperoxia Increases Kidney Injury During Renal Ischemia and Reperfusion in Mice.

BACKGROUND: Renal ischemia and reperfusion (IR) contribute to perioperative acute kidney injury, and oxygen is a key regulator of this process. We hypothesized that oxygen administration during surgery and renal IR would impact postoperative kidney function and injury in mice. METHODS: Mice were anesthetized, intubated, and mechanically ventilated with a fraction of inspired oxygen (F io2 ) 0.10 (hypoxia), 0.21 (normoxia), 0.60 (moderate hyperoxia), or 1.00 (severe hyperoxia) during 67 minutes of renal IR or sham IR surgery. Additional mice were treated before IR or sham IR surgery with 50 mg/kg tempol, a superoxide scavenger. At 24 hours, mice were sacrificed, and blood and kidney collected. We assessed and compared kidney function and injury across groups by measuring blood urea nitrogen (BUN, primary end point), renal histological injury, renal expression of neutrophil gelatinase-associated lipocalin (NGAL), and renal heme oxygenase 1 ( Ho-1 ), peroxisome proliferator-activated receptor gamma coactivator 1-α ( Pgc1-α ), and glutathione peroxidase 4 ( Gpx-4 ) transcripts, to explore potential mechanisms of any effect of oxygen. RESULTS: Hyperoxia and hypoxia during renal IR surgery decreased renal function and increased kidney injury compared to normoxia. Baseline median (interquartile range) BUN was 22.2 mg/dL (18.4-26.0), and 24 hours after IR surgery, BUN was 17.5 mg/dL (95% confidence interval [CI], 1.3-38.4; P = .034) higher in moderate hyperoxia-treated animals, 51.8 mg/dL (95% CI, 24.9-74.8; P < .001) higher in severe hyperoxia-treated animals, and 64.9 mg/dL (95% CI, 41.2-80.3; P < .001) higher in hypoxia-treated animals compared to animals treated with normoxia ( P < .001, overall effect of hyperoxia). Hyperoxia-induced injury, but not hypoxia-induced injury, was attenuated by pretreatment with tempol. Histological injury scores, renal NGAL staining, and renal transcription of Ho-1 and suppression of Pgc1- α followed the same pattern as BUN, in relation to the effects of oxygen treatment. CONCLUSIONS: In this controlled preclinical study of oxygen treatment during renal IR surgery, hyperoxia and hypoxia impaired renal function, increased renal injury, and impacted expression of genes that affect mitochondrial biogenesis and antioxidant response. These results might have implications for patients during surgery when high concentrations of oxygen are frequently administered, especially in cases involving renal IR.

Endothelial nitric oxide synthase modulates Toll-like receptor 4-mediated IL-6 production and permeability via nitric oxide-independent signaling.

Endothelial dysfunction, characterized by changes in eNOS, is a common finding in chronic inflammatory vascular diseases. These states are associated with increased infectious complications. We hypothesized that alterations in eNOS would enhance the response to LPS-mediated TLR4 inflammation. Human microvascular endothelial cells were treated with sepiapterin or N-nitro-L-arginine methylester (L-NAME) to alter endogenous NO production, and small interfering RNA to knockdown eNOS. Alterations of endogenous NO by sepiapterin, and L-NAME provided no significant changes to LPS inflammation. In contrast, eNOS knockdown greatly enhanced endothelial IL-6 production and permeability in response to LPS. Knockdown of eNOS enhanced LPS-induced p38. Inhibition of p38 with SB203580 prevented IL-6 production, without altering permeability. Knockdown of p38 impaired NF-κB activation. Physical interaction between p38 and eNOS was demonstrated by immunoprecipitation, suggesting a novel, NO-independent mechanism for eNOS regulation of TLR4. In correlation, biopsy samples in patients with systemic lupus erythematous showed reduced eNOS expression with associated elevations in TLR4 and p38, suggesting an in vivo link. Thus, reduced expression of eNOS, as seen in chronic inflammatory disease, was associated with enhanced TLR4 signaling through p38. This may enhance the response to infection in patients with chronic inflammatory conditions.-Stark, R. J., Koch, S. R., Choi, H., Mace, E. H., Dikalov, S. I., Sherwood, E. R., Lamb, F. S. Endothelial nitric oxide synthase modulates Toll-like receptor 4-mediated IL-6 production and permeability via nitric oxide-independent signaling.

Toll-like receptor 3-mediated inflammation by p38 is enhanced by endothelial nitric oxide synthase knockdown.

BACKGROUND: Vascular dysfunction is commonly seen during severe viral infections. Endothelial nitric oxide synthase (eNOS), has been postulated to play an important role in regulating vascular homeostasis as well as propagation of the inflammatory reaction. We hypothesized that the loss of eNOS would negatively impact toll-like receptor 3 (TLR3) signaling and worsen vascular function to viral challenge. METHODS: Human microvascular endothelial cells (HMVECs) were exposed to either control or eNOS siRNA and then treated with Poly I:C, a TLR3 agonist and mimicker of dsRNA viruses. Cells were assessed for protein-protein associations, cytokine and chemokine analysis as well as transendothelial electrical resistance (TEER) as a surrogate of permeability. RESULTS: HMVECs that had reduced eNOS expression had a significantly elevated increase in IL-6, IL-8 and IP-10 production after Poly I:C. In addition, the knockdown of eNOS enhanced the change in TEER after Poly I:C stimulation. Western blot analysis showed enhanced phosphorylation of p38 in sieNOS treated cells with Poly I:C compared to siControl cells. Proximity ligation assays further demonstrated direct eNOS-p38 protein-protein interactions. The addition of the p38 inhibitor, SB203580, in eNOS knockdown cells reduced both cytokine production after Poly I:C, and as well as mitigated the reduction in TEER, suggesting a direct link between eNOS and p38 in TLR3 signaling. CONCLUSIONS: These results suggest that reduction of eNOS increases TLR3-mediated inflammation in human endothelial cells in a p38-dependent manner. This finding has important implications for understanding the pathogenesis of severe viral infections and the associated vascular dysfunction.

Ultrasound Modulates the Splenic Neuroimmune Axis in Attenuating AKI.

We showed previously that prior exposure to a modified ultrasound regimen prevents kidney ischemia-reperfusion injury (IRI) likely via the splenic cholinergic anti-inflammatory pathway (CAP) and α7 nicotinic acetylcholine receptors (α7nAChR). However, it is unclear how ultrasound stimulates the splenic CAP. Further investigating the role of the spleen in ischemic injury, we found that prior splenectomy (-7d) or chemical sympathectomy of the spleen with 6-hydroxydopamine (6OHDA; -14d) exacerbated injury after subthreshold (24-minute ischemia) IRI. 6-OHDA-induced splenic denervation also prevented ultrasound-induced protection of kidneys from moderate (26-minute ischemia) IRI. Ultrasound-induced protection required hematopoietic but not parenchymal α7nAChRs, as shown by experiments in bone marrow chimeras generated with wild-type and α7nAChR(-/-) mice. Ultrasound protection was associated with reduced expression of circulating and kidney-derived cytokines. However, splenocytes isolated from mice 24 hours after ultrasound treatment released more IL-6 ex vivo in response to LPS than splenocytes from sham mice. Adoptive transfer of splenocytes from ultrasound-treated (but not sham) mice to naïve mice was sufficient to protect kidneys of recipient mice from IRI. Ultrasound treatment 24 hours before cecal ligation puncture-induced sepsis was effective in reducing plasma creatinine in this model of AKI. Thus, splenocytes of ultrasound-treated mice are capable of modulating IRI in vivo, supporting our ongoing hypothesis that a modified ultrasound regimen has therapeutic potential for AKI and other inflammatory conditions.

Technical Considerations in Laparoscopic-Assisted Endoscopic Retrograde Cholangiography.

The use of endoscopic retrograde cholangiography (ERCP) for diagnostic and therapeutic interventions on the pancreaticobiliary system has steadily increased, but the standard approach through the oropharynx is prohibited after Roux-en-Y (RYGB) gastric bypass surgery. Laparoscopic access to the gastric remnant allows for the completion of ERCP using the standard side-viewing duodenoscope to facilitate the completion of standard and advanced endoscopic maneuvers. Here, we describe our experience with the technical aspects of safe and effective performance of laparoscopic-assisted ERCP.

Vascular imaging immediately after tourniquet removal does not increase vasospasm risk.

BACKGROUND: Prehospital tourniquet use is now standard in trauma patients with diagnosed or suspected extremity vascular injuries. Tourniquet-related vasospasm is an understudied phenomenon that may confound management by causing erroneous arterial pressure indices (APIs) and abnormalities on computed tomography angiography (CTA) that do not reflect true arterial injuries. We hypothesized that shorter intervals between tourniquet removal and CTA imaging and longer total tourniquet times would be correlated with a higher likelihood of false positive CTA. MATERIALS AND METHODS: We performed a single-institution retrospective cohort study of patients presenting to a busy, urban Level 1 Trauma Center with prehospital tourniquets from 2019 to 2021. Patients who presented with a tourniquet disengaged upon arrival or who died prior to admission to the Trauma Unit were excluded. Tourniquet duration, time between tourniquet removal and CTA imaging (CTA interval), CTA findings, and management of extremity arterial injuries were extracted. The proportion of false positive injuries on CTA was assessed for correlation with increasing time interval from tourniquet removal to CTA imaging and correlation with increasing total tourniquet time using multivariable logistic regression. RESULTS: 251 patients were identified with prehospital tourniquets. 127 underwent CTA of the affected extremity, 96 patients had an abnormal CTA finding, and 57 (45% of total CTA patients) had false positive arterial injuries on imaging. Using multivariable logistic regression, neither the CTA interval nor the tourniquet duration was associated with false positive CTA injuries. Female sex was associated with false positive injuries on CTA (OR 2.91, 95% CI: 1.01 - 8.39). Vasospasm was cited as a possible explanation by radiologists in 40% of false positive CTA reports. CONCLUSIONS: Arterial vasospasm is a frequent finding on CTA after tourniquet use for extremity trauma, but concerns regarding tourniquet-related vasospasm should not alter trauma patient management. Neither the duration of tourniquet application nor the time interval since removal is associated with decreased CTA accuracy, and any delay in imaging does not appear to reduce the likelihood of vasospasm. These findings are important for supporting expedited care of trauma patients with severe extremity injuries.

Targeting Soluble Guanylyl Cyclase during Ischemia and Reperfusion.

Ischemia and reperfusion (IR) damage organs and contribute to many disease states. Few effective treatments exist that attenuate IR injury. The augmentation of nitric oxide (NO) signaling remains a promising therapeutic target for IR injury. NO binds to soluble guanylyl cyclase (sGC) to regulate vasodilation, maintain endothelial barrier integrity, and modulate inflammation through the production of cyclic-GMP in vascular smooth muscle. Pharmacologic sGC stimulators and activators have recently been developed. In preclinical studies, sGC stimulators, which augment the reduced form of sGC, and activators, which activate the oxidized non-NO binding form of sGC, increase vasodilation and decrease cardiac, cerebral, renal, pulmonary, and hepatic injury following IR. These effects may be a result of the improved regulation of perfusion and decreased oxidative injury during IR. sGC stimulators are now used clinically to treat some chronic conditions such as heart failure and pulmonary hypertension. Clinical trials of sGC activators have been terminated secondary to adverse side effects including hypotension. Additional clinical studies to investigate the effects of sGC stimulation and activation during acute conditions, such as IR, are warranted.

SOLUBLE GUANYLYL CYCLASE ACTIVATION RESCUES HYPEROXIA-INDUCED DYSFUNCTION OF VASCULAR RELAXATION.

ABSTRACT: Introduction: Perioperative alterations in perfusion lead to ischemia and reperfusion injury, and supplemental oxygen is administered during surgery to limit hypoxic injury but can lead to hyperoxia. We hypothesized that hyperoxia impairs endothelium-dependent and endothelium-independent vasodilation but not the vasodilatory response to heme-independent soluble guanylyl cyclase activation. Methods: We measured the effect of oxygen on vascular reactivity in mouse aortas. Mice were ventilated with 21% (normoxia), 60% (moderate hyperoxia), or 100% (severe hyperoxia) oxygen during 30 minutes of renal ischemia and 30 minutes of reperfusion. After sacrifice, the thoracic aorta was isolated, and segments mounted on a wire myograph. We measured endothelium-dependent and endothelium-independent vasodilation with escalating concentrations of acetylcholine (ACh) and sodium nitroprusside (SNP), respectively, and we measured the response to heme-independent soluble guanylyl cyclase activation with cinaciguat. Vasodilator responses to each agonist were quantified as the maximal theoretical response ( Emax ) and the effective concentration to elicit 50% relaxation (EC 50 ) using a sigmoid model and nonlinear mixed-effects regression. Aortic superoxide was measured with dihydroethidium probe and high-performance liquid chromatography quantification of the specific superoxide product 2-hydroxyethidium. Results: Hyperoxia impaired endothelium-dependent (ACh) and endothelium-independent (SNP) vasodilation compared with normoxia and had no effect on cinaciguat-induced vasodilation. The median ACh Emax was 76.4% (95% confidence interval = 69.6 to 83.3) in the normoxia group, 53.5% (46.7 to 60.3) in the moderate hyperoxia group, and 53.1% (46.3 to 60.0) in the severe hyperoxia group ( P < 0.001, effect across groups), while the ACh EC 50 was not different among groups. The SNP Emax was 133.1% (122.9 to 143.3) in normoxia, 128.3% (118.1 to 138.6) in moderate hyperoxia, and 114.8% (104.6 to 125.0) in severe hyperoxia ( P < 0.001, effect across groups), and the SNP EC 50 was 0.38 log M greater in moderate hyperoxia than in normoxia (95% confidence interval = 0.18 to 0.58, P < 0.001). Cinaciguat Emax and EC 50 were not different among oxygen treatment groups (median range Emax = 78.0% to 79.4% and EC 50 = -18.0 to -18.2 log M across oxygen groups). Aorta 2-hydroxyethidium was 1419 pmol/mg of protein (25th-75th percentile = 1178-1513) in normoxia, 1993 (1831-2473) in moderate hyperoxia, and 2078 (1936-2922) in severe hyperoxia ( P = 0.008, effect across groups). Conclusions: Hyperoxia, compared with normoxia, impaired endothelium-dependent and endothelium-independent vasodilation but not the response to heme-independent soluble guanylyl cyclase activation, and hyperoxia increased vascular superoxide production. Results from this study could have important implications for patients receiving high concentrations of oxygen and at risk for ischemia reperfusion-mediated organ injury.

Impact of Inhaled Oxygen on Reactive Oxygen Species Production and Oxidative Damage during Spontaneous Ventilation in a Murine Model of Acute Renal Ischemia and Reperfusion.

Introduction: Acute kidney injury (AKI) affects 10% of patients following major surgery and is independently associated with extra-renal organ injury, development of chronic kidney disease, and death. Perioperative renal ischemia and reperfusion (IR) contributes to AKI by, in part, increasing production of reactive oxygen species (ROS) and leading to oxidative damage. Variations in inhaled oxygen may mediate some aspects of IR injury by affecting tissue oxygenation, ROS production, and oxidative damage. We tested the hypothesis that provision of air (normoxia) compared to 100% oxygen (hyperoxia) during murine renal IR affects renal ROS production and oxidative damage. Methods: We administered 100% oxygen or 21% oxygen (air) to 8-9 week-old FVB/N mice and performed dorsal unilateral nephrectomy with contralateral renal ischemia/reperfusion surgery while mice spontaneously ventilated. We subjected mice to 30 minutes of ischemia and 30 minutes of reperfusion prior to sacrifice. We obtained an arterial blood gas (ABG) by performing sternotomy and left cardiac puncture. We stained the kidney with pimonidazole, a marker of tissue hypoxia; 4-HNE, a marker of ROS-production; and we measured F2-isoprostanes in homogenized tissue to quantify oxidative damage. Results: Hyperoxia during IR increased arterial oxygen content compared to normoxia, but both groups of mice were hypoventilating at the time of ABG sampling. Renal tissue hypoxia following reperfusion was similar in both treatment groups. ROS production was similar in the cortex of mice (3.8% area in hyperoxia vs. 3.1% in normoxia, P=0.19) but increased in the medulla of hyperoxia-treated animals (6.3% area in hyperoxia vs. 4.5% in nomoxia, P=0.02). Renal F2-isoprostanes were similar in treatment groups (2.2 pg/mg kidney in hyperoxia vs. 2.1 pg/mg in normoxia, P=0.40). Conclusions: Hyperoxia during spontaneous ventilation in murine renal IR did not appear to affect renal hypoxia following reperfusion, but hyperoxia increased medullary ROS production compared to normoxia.

Upper Extremity Pulse Pressure Predicts Amputation-Free Survival after Lower Extremity Bypass.

Increased pulse pressure reflects pathologic arterial stiffening and predicts cardiovascular events and mortality. The effect of pulse pressure on outcomes in lower extremity bypass patients remains unknown. We thus investigated whether preoperative pulse pressure could predict amputation-free survival in patients undergoing lower extremity bypass for atherosclerotic occlusive disease. An institutional database identified 240 included patients undergoing lower extremity bypass from 2005 to 2014. Preoperative demographics, cardiovascular risk factors, operative factors, and systolic and diastolic blood pressures were recorded, and compared between patients with pulse pressures above and below 80 mm Hg. Factors were analyzed in bi- and multivariable models to assess independent predictors of amputation-free survival. Kaplan-Meier analysis was performed to evaluate the temporal effect of pulse pressure ≥80 mm Hg on amputation-free survival. Patients with a pulse pressure ≥80 mm Hg were older, male, and had higher systolic and lower diastolic pressures. Patients with pulse pressure <80 mm Hg demonstrated a survival advantage on Kaplan-Meier analysis at six months (log-rank P = 0.003) and one year (P = 0.005) postoperatively. In multivariable analysis, independent risk factors for decreased amputation-free survival at six months included nonwhite race, tissue loss, infrapopliteal target, and preoperative pulse pressure ≥80 mm Hg (hazard ratio 2.60; P = 0.02), while only tissue loss and pulse pressure ≥80 mm Hg (hazard ratio 2.30, P = 0.02) remained predictive at one year. Increased pulse pressure is independently associated with decreased amputation-free survival in patients undergoing lower extremity bypass. Further efforts to understand the relationship between increased arterial stiffness and poor outcomes in these patients are needed.