Jeffery V. Bishop

Acute removal of common sepsis mediators does not explain the effects of extracorporeal blood purification in experimental sepsis

The effect of extracorporeal blood purification on clinical outcomes in sepsis is assumed to be related to modulation of plasma cytokine concentrations. To test this hypothesis directly, we treated rats that had a cecal ligation followed by puncture (a standard model of sepsis) with a modest dose of extracorporeal blood purification that did not result in acute changes in a panel of common cytokines associated with inflammation (TNF-α, IL-1β, IL-6, and IL-10). Pre- and immediate post-treatment levels of these cytokines were unchanged compared to the sham therapy of extracorporeal circulation without blood purifying sorbent. The overall survival to 7 days, however, was significantly better in animals that received extracorporeal blood purification compared to those with a sham procedure. This panel of common plasma cytokines along with alanine aminotransferase and creatinine was significantly lower 72 h following extracorporeal blood purification compared to sham-treated rats. Thus, the effects of this procedure on organ function and survival do not appear to be due solely to immediate changes in the usual measured circulating cytokines. These results may have important implications for the design and conduct of future trials in sepsis including defining alternative targets for extracorporeal blood purification and other therapies.

Effects of fluid resuscitation with 0.9% saline versus a balanced electrolyte solution on acute kidney injury in a rat model of sepsis*.

Objective:To compare the acute effects of 0.9% saline versus a balanced electrolyte solution on acute kidney injury in a rat model of sepsis. Design:Controlled laboratory experiment. Setting:University laboratory. Subjects:Sixty adult, male Sprague-Dawley rats. Interventions:We induced sepsis by cecal ligation and puncture and randomized animals to receive fluid resuscitation with either 0.9% saline or Plasma-Lyte solution for 4 hours after 18 hours of cecal ligation and puncture (10 mL/kg in the first hour and 5 mL/kg in the next 3 hr). Blood and urine specimens were obtained from baseline, 18 hours after cecal ligation and puncture, immediately after 4 hours fluid resuscitation, and 24 hours later. We measured blood gas, plasma electrolytes, creatinine, interleukin-6, cystatin C, and neutrophil gelatinase-associated lipocalin concentrations. We also analyzed urine for cystatin C and neutrophil gelatinase-associated lipocalin. We used Risk, Injury, Failure, Loss and End-stage criteria for creatinine to assess severity of acute kidney injury. We observed all animals for survival up to 1 day after resuscitation. Surviving animals were killed for kidney histology. Finally, we carried out an identical study in 12 healthy animals. Measurements and Main Results:Compared with Plasma-Lyte, 0.9% saline resuscitation resulted in significantly greater blood chloride concentrations (p < 0.05) and significantly decreased pH and base excess. Acute kidney injury severity measured by RIFLE criteria was increased with 0.9% saline compared with Plasma-Lyte resuscitation (p < 0.05), and these results were consistent with kidney histology and biomarkers of acute kidney injury. Twenty-four-hour survival favored Plasma-Lyte resuscitation (76.6% vs 53.3%; p = 0.03). Finally, in healthy animals, we found no differences between fluids and no evidence of acute kidney injury. Conclusion:Volume resuscitation with Plasma-Lyte resulted in less acidosis and less kidney injury and improved short-term survival when compared with 0.9% saline in this experimental animal model of sepsis.

Blood Purification in Sepsis: A New Paradigm

Sepsis is one of the main causes of death in critically ill patients. The pathophysiology of sepsis is complex and not completely understood. The proinflammatory and anti-inflammatory response leads to cell and organ dysfunction and, in many cases, death. Thus, the goal of the intervention is to restore the homeostasis of circulating mediators rather than to inhibit selectively the proinflammatory or anti-inflammatory mediators. Blood purification has been reported to remove a wide array of inflammatory mediators. The effects are broad-spectrum and auto-regulating. Blood purification has also been demonstrated to restore immune function through improving antigen-presenting capability, adjusting leukocyte recruitment, oxidative burst and phagocytosis, and improving leukocyte responsiveness. A great deal of work has to be done in order to find and optimize the best extracorporeal blood purification therapy for sepsis. New devices specifically target the pathophysiological mechanisms involved in these conditions. High-volume hemofiltration, hemoadsorption, coupled plasma filtration adsorption, and high cutoff membrane are now being tested in septic patients. Preliminary data indicate the feasibility of these modified techniques in sepsis. Their impact on patient prognosis, however, still needs proof by large randomized clinical trials. Finally, the emerging paradigm of sepsis-induced immune suppression provides additional rationale for the development of extracorporeal blood purification therapy for sepsis.

Differential effects of kidney-lung cross-talk during acute kidney injury and bacterial pneumonia

Acute injuries of the kidney or lung each represent serious, complex clinical problems, and their combination drastically decreases patient survival. However, detailed understanding of interactions between these two organs is scarce. To evaluate this further, we used the folic acid (FA) and myohemoglobinuria models of acute kidney injury (AKI) together with Pseudomonas aeruginosa inhalation to study kidney-lung cross-talk in mice during acute kidney and lung injury. Subgroups of mice received antineutrophil antibody or platelet-depleting serum to assess the role of neutrophil and platelets, respectively. AKI by itself did not cause clinically relevant acute lung injury. Pneumonia was neutrophil dependent, whereas pneumonia-induced AKI was platelet dependent. AKI attenuated pulmonary neutrophil recruitment and worsened pneumonia. Mice with AKI had lower oxygen saturations and greater bacterial load than mice without. Neutrophils isolated from mice with FA-induced AKI also had impaired transmigration and F-actin polymerization in vitro. Thus, during acute kidney and pneumonia-induced lung injury, clinically relevant kidney-lung interactions are both neutrophil and platelet dependent.

Bactericidal Antibiotics temporarily increase inflammation and worsen acute kidney injury in experimental sepsis

Objective: To explore the relationships among bactericidal antimicrobial treatment of sepsis, inflammatory response, severity of acute kidney injury, and outcomes. Design: Controlled laboratory experiment. Setting: University laboratory. Interventions: Sepsis was induced by cecal ligation and puncture in 52 rats and was treated with either bactericidal antibiotics (ampicillin/sulbactam) or placebo (saline). Serial blood specimens were obtained after cecal ligation and puncture for serum creatinine, interleukin-6, and neutrophil gelatinase-associated lipocalin concentrations. RIFLE (Risk, Injury, Failure, Loss, End-stage kidney disease) criteria were used to assess severity of acute kidney injury. All animals were observed for survival up to 1 wk. In a separate experiment, six healthy animals were given antibiotics and renal function was assessed. Another 12 animals were euthanized 2 days after laparotomy for kidney histology. Measurements and Main Results: Survival in the placebo group was 50% compared with 81.8% in the antibiotic group (p < .05). Most animals (93%) without antibiotics developed acute kidney injury, of which 39% exhibited greater than a threefold rise in serum creatinine (RIFLE-F). Furthermore, survival decreased as acute kidney injury severity increased. Surprisingly, all antibiotic-treated animals developed acute kidney injury, of which 68.6% reached RIFLE-F. However, renal dysfunction was less persistent in these animals. Patterns of plasma interleukin-6 were similar to creatinine with higher concentrations seen earlier in antibiotic-treated animals but with faster resolution. Interleukin-6 concentration at 24 hrs was independently associated with the development of RIFLE-F. Histologic findings were consistent with functional parameters showing that antibiotics worsened acute kidney injury. Conclusion: In polymicrobial sepsis, bactericidal antibiotics resulted in more inflammation and more severe acute kidney injury. However, resolution of inflammation and acute kidney injury was faster with antibiotics and correlated best with survival. These results suggest that transient worsening of renal function may be an expected consequence of sepsis therapy. These findings also question the value of peak severity of acute kidney injury as a primary end point and suggest that resolution of acute kidney injury may be more appropriate.

Leukocyte capture and modulation of cell-mediated immunity during human sepsis: an ex vivo study

INTRODUCTION Promising preclinical results have been obtained with blood purification therapies as adjuvant treatment for sepsis. However, the mechanisms by which these therapies exert beneficial effects remain unclear. Some investigators have suggested that removal of activated leukocytes from the circulation might help ameliorate remote organ injury. We designed an extracorporeal hemoadsorption device capable of capturing both cytokines and leukocytes in order to test the hypothesis that leukocyte capture would alter circulating cytokine profiles and influence immunological cell-cell interactions in whole blood taken from patients with sepsis. METHODS We performed a series of ex vivo studies in 21 patients with septic shock and 12 healthy volunteers. Blood circulated for four hours in closed loops with four specially designed miniaturized extracorporeal blood purification devices including two different hemoadsorption devices and a hemofilter in order to characterize leukocyte capture and to assess the effects of leukocyte removal on inflammation and immune function. RESULTS Hemoadsorption was selective for removal of activated neutrophils and monocytes. Capture of these cells led to local release of certain cytokines, especially IL-8, and resulted in complex cell-cell interactions involved in cell-mediated immunity. Inhibition of cell adherence reversed the cytokine release and the effects on lymphocyte function. CONCLUSIONS Monocyte and neutrophil capture using a sorbent polymer results in upregulation of IL-8 and modulation of cell-mediated immunity. Further studies are needed to understand better these cellular interactions in order to help design better blood purification therapies.

N-acetylcysteine is effective for prevention but not for treatment of folic acid-induced acute kidney injury in mice

Objectives:There is controversy regarding the benefits of N-acetylcysteine in acute kidney injury. This study was to compare three commonly used regimens and explore which regimen is best for the protection of acute kidney injury. Design:Prospective experimental study. Setting:University research laboratory. Interventions:Acute kidney injury was induced with folic acid intraperitoneal injection in mice. Mice in pretreatment were treated with a subcutaneous injection of N-acetylcysteine before the folic acid injection. Mice in posttreatment were treated with N-acetylcysteine after folic acid. Mice in pre- + posttreatment were treated with N-acetylcysteine before folic acid and after folic acid. Placebo mice received vehicle only using the pre- + posttreatment protocol. Fourteen healthy animals were given N-acetylcysteine to evaluate for toxicity and the other 24 mice subjected to folic acid were killed for kidney histology and analysis for oxidative injury. The same studies were also carried out in milder acute kidney injury (lower folic acid) model. Measurements and Main Results:Plasma concentrations of creatinine, cystatin C, and reduced glutathione were measured. Survival time was assessed up to 7 days. The survival rates in N-acetylcysteine pretreatment mice were significantly better (73.33% vs. 46.67%, p < .04) and acute kidney injury was significantly less compared with placebo. However, mice with posttreatment exhibited significantly worse survival and more severe acute kidney injury. Histologic findings were consistent with functional parameters. Glutathione levels decreased less in N-acetylcysteine pretreatment but also increased beginning on day 2 compared with placebo (11.5 vs. 8.1 &mgr;g/mL, p < .05). Glutathione levels did not increase in N-acetylcysteine posttreatment. However, three different N-acetylcysteine interventions neither significantly improved nor worsened renal function in the milder acute kidney injury model. Conclusion:N-acetylcysteine pretreatment was effective in reducing the incidence and severity of acute kidney injury as well as in increasing survival. However, N-acetylcysteine posttreatment worsened folic acid toxicity. Only pretreatment was effective in increasing glutathione. These data may help explain the variation from clinical studies of N-acetylcysteine use.

The anti-oxidant effects are not the main mechanism for glutamine's protective effects on acute kidney injury in mice.

Acute kidney injury (AKI) is a common problem characterized by an inflammatory response in the kidney and oxidative stress. However, there are no interventions to prevent AKI. Glutamine is an important precursor of glutathione and has also been shown to induce heat shock proteins (HSP). Thus, glutamine may affect both oxidative stress and inflammation. This study was to explore the effects of glutamine pretreatment on nephrotoxic AKI and to investigate the underlying mechanisms. First, the effects of alternate doses of glutamine were compared in CD-1 mice with AKI induced with folic acid intra-peritoneal injection. Then the effects of glutamine quercetin (an HSP inhibitor), and quercetin+glutamine, were compared in the same AKI model. AKI were assessed with plasma creatinine, urine neutrophil gelatinase-associated lipocalin, and renal histology. Inflammatory response was monitored with renal tumor necrosis factor (TNF-α), chemkines (CXCL1 and CCL2) contents, and neutrophil infiltration. Oxidative injury was detected with reduced glutathione, malondialdehyde, and protein thiol. Glutamine provided dose-dependent renal protection. Pretreatment with quercetin, which was showed to inhibit HSP-70 expression, abolished glutamines renal-protective effects. Quercetin also abrogated glutamines beneficial effects on renal TNF-α, chemokines, and neutrophil infiltration. However, quercetin did not affect glutamines anti-oxidative effects. These results suggest that glutamines renal-protective effects are mainly related to its activation of HSP-70, which mitigates inflammatory response, renal neutrophil infiltration and subsequent AKI. Regulating neutrophil infiltration might be a potential therapeutic target for AKI.