Neal B. Blatt

Signaling Pathways and Effector Mechanisms Pre-Programmed Cell Death

Apoptosis is a complex biochemical process that involves all aspects of the cell from the plasma membrane to the nucleus. Apoptosis stimuli are mediated by many different cellular processes including protein synthesis and degradation, the alteration in protein phosphorylation states, the activation of lipid second messenger systems, and disruption of normal mitochondrial function. Despite this diversity in signal transduction, all apoptotic pathways are believed to converge ultimately with the activation of caspases leading to the characteristic morphological changes of apoptosis. In this review, we discuss what is known about these pathways and its implication for normal cellular function.

Fluid overload and fluid removal in pediatric patients on extracorporeal membrane oxygenation requiring continuous renal replacement therapy.

Objective:In pediatric patients, fluid overload at continuous renal replacement therapy initiation is associated with increased mortality. The aim of this study was to characterize the association between fluid overload at continuous renal replacement therapy initiation, fluid removal during continuous renal replacement therapy, the kinetics of fluid removal and mortality in a large pediatric population receiving continuous renal replacement therapy while on extracorporeal membrane oxygenation. Design:Retrospective chart review. Setting:Tertiary children’s hospital. Patients:Extracorporeal membrane oxygenation patients requiring continuous renal replacement therapy from July 2006 to September 2010. Interventions:None. Measurements and Main Results:Overall intensive care unit survival was 34% for 53 patients that were initiated on continuous renal replacement therapy while on extracorporeal membrane oxygenation during the study period. Median fluid overload at continuous renal replacement therapy initiation was significantly lower in survivors compared to nonsurvivors (24.5% vs. 38%, p = .006). Median fluid overload at continuous renal replacement therapy discontinuation was significantly lower in survivors compared to nonsurvivors (7.1% vs. 17.5%, p = .035). After adjusting for percent fluid overload at continuous renal replacement therapy initiation, age, and severity of illness, the change in fluid overload at continuous renal replacement therapy discontinuation was not significantly associated with mortality (p = .212). Models investigating the rates of fluid removal in different periods, age, severity of illness, and fluid overload at continuous renal replacement therapy initiation found that fluid overload at continuous renal replacement therapy initiation was the most consistent predictor of survival. Conclusions:Our data demonstrate an association between fluid overload at continuous renal replacement therapy initiation and mortality in pediatric patients receiving extracorporeal membrane oxygenation. The degree of fluid overload at continuous renal replacement therapy discontinuation is also associated with mortality, but appears to reflect the effect of fluid overload at initiation. Furthermore, correction of fluid overload to ⩽10% was not associated with improved survival. These results suggest that intervening prior to the development of significant fluid overload may be more clinically effective than attempting fluid removal after significant fluid overload has developed. Our findings suggest a role for earlier initiation of continuous renal replacement therapy in this population, and warrant further clinical studies.

Fluid Overload in Infants Following Congenital Heart Surgery

Objective: To describe postoperative fluid overload patterns and correlate degree of fluid overload with intensive care morbidity and mortality in infants undergoing congenital heart surgery. Design: Prospective, observational study. Fluid overload (%) was calculated by two methods: 1) (Total fluid in – Total fluid out)/(Preoperative weight) × 100; and 2) (Current weight – Preoperative weight)/(Preoperative weight) × 100. Composite poor outcome included: need for renal replacement therapy, upper quartile time to extubation or intensive care length of stay (> 6.5 and 9.9 days, respectively), or death ⩽ 30 days after surgery. Setting: University hospital pediatric cardiac ICU. Patients: Forty-nine infants < 6 months of age undergoing congenital heart surgery with cardiopulmonary bypass during the period of July 2009 to July 2010. Interventions: None. Measurements and Main Results: Patients had a median age of 53 days (21 neonates) and mean weight of 4.5±1.3kg. Forty-two patients (86%) developed acute kidney injury by meeting at least Acute Kidney Injury Network and Kidney Disease Improving Global Outcomes stage 1 criteria (serum creatinine rise of 50% or ≥ 0.3mg/dL). The patients with adverse outcomes (n = 17, 35%) were younger (7 [5 – 10] vs. 98 [33 – 150] days, p = 0.001), had lower preoperative weight (3.7±0.7 vs. 4.9±1.4kg, p = 0.0002), higher postoperative mean peak serum creatinine (SCr) (0.9±0.3 vs. 0.6±0.3mg/dL, p = 0.005), and higher mean maximum fluid overload by both method 1 (12% ± 10% vs. 6% ± 4%, p = 0.03) and method 2 (24% ± 15% vs. 14% ± 8%, p = 0.02). Predictors of a poor outcome from multivariate analyses were cardiopulmonary bypass time, use of circulatory arrest, and increased vasoactive medication requirements postoperatively. Conclusions: Early postoperative fluid overload is associated with suboptimal outcomes in infants following cardiac surgery. Because the majority of patients developed kidney injury without needing renal replacement therapy, fluid overload may be an important risk factor for adverse outcomes with all degrees of acute kidney injury.

Epidemiology and Risk Factors for Thromboembolic Complications of Childhood Nephrotic Syndrome: A Midwest Pediatric Nephrology Consortium (MWPNC) Study

OBJECTIVES To identify clinical variables predictive of the risk of thromboembolism (TE), and to confirm the incidence of TE in primary and secondary childhood nephrotic syndrome (NS). STUDY DESIGN A comprehensive chart review identified 326 children with NS from any cause evaluated between 1999 and 2006. These patients had a total of 1472.8 patient-years of follow-up. Comparison statistics, survival analysis, and logistic regression were used to define TE epidemiology and clinical risk factors. RESULTS We found that 9.2% of our cohort had experienced at least 1 TE. The overall incidence was 20.4 patients with TEs/1000 patient-years. The median time to the first TE was 70.5 days after diagnosis of NS. Deep venous thrombosis was the most common TE (76%) and was frequently associated with the use of a central venous catheter (45%). Significant independent predictors of TE included age > or = 12 years at onset of NS (P < .0001), severity of proteinuria (P < .0001), and history of TE preceding diagnosis of NS (P < .0001). Life- or limb-threatening TEs represented 23.7% of the events. CONCLUSIONS Children with NS should be carefully followed for TE, particularly those who are age 12 years or older, have severe proteinuria, or have a previous history of TE.

Benzodiazepine-induced superoxide signals B cell apoptosis: Mechanistic insight and potential therapeutic utility

The properties of a proapoptotic 1,4-benzodiazepine, Bz-423, identified through combinatorial chemistry and phenotype screening are described. Bz-423 rapidly generated superoxide (O(2)(-)) in transformed Ramos B cells. This O(2)(-) response originated from mitochondria prior to mitochondrial transmembrane gradient collapse and opening of the permeability transition pore. Bz-423-induced O(2)(-) functioned as an upstream signal that initiated an apoptotic program characterized by cytochrome c release, mitochondrial depolarization, and caspase activation. Pretreatment of cells with agents that either block the formation of Bz-423-induced O(2)(-) or scavenge free radicals attenuated the death cascade, which demonstrated that cell killing by Bz-423 depends on O(2)(-). Parallels between Ramos cells and germinal center B cells prompted experiments to determine whether Bz-423 had therapeutic activity in vivo. This possibility was tested using the (NZB x NZW)F(1) murine model of lupus, in which the pathologically enhanced survival and expansion of germinal center B cells mediate disease. Administration of Bz-423 for 12 weeks specifically controlled germinal center hyperplasia and reduced the histological evidence of glomerulonephritis. Collectively, these studies define a new structure-function relationship for benzodiazepines and point to a new target and mechanism that could be of value for developing improved drugs to manage systemic lupus erythematosus and related disorders.

Anti-DNA autoantibodies and systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that affects most of the organs and tissues of the body, causing glomerulonephritis, arthritis, and cerebritis. SLE can be fatal with nephritis, in particular, predicting a poor outcome for patients. In this review, we highlight what has been learned about SLE from the study of mouse models, and pay particular attention to anti-DNA autoantibodies, both as pathological agents of lupus nephritis and as DNA-binding proteins. We summarize the current approaches used to treat SLE and discuss the targeting of anti-DNA autoantibodies as a new treatment for lupus nephritis.

Ligand recognition by murine anti-DNA autoantibodies. II. Genetic analysis and pathogenicity.

Although anti-DNA autoantibodies are an important hallmark of lupus, the relationships among anti-DNA structure, reactivity, and pathogenicity have not been fully elucidated. To further investigate these relationships, we compare the variable genes and primary structure of eight anti-DNA mAbs previously obtained from an MRL/MpJ-lpr/lpr mouse along with the ability of three representative mAbs to induce nephritis in nonautoimmune mice using established adoptive transfer protocols. One monospecific anti-single-stranded (ss) DNA (11F8) induces severe diffuse proliferative glomerulonephritis in nonautoimmune mice whereas another anti-ssDNA with apparently similar in vitro binding properties (9F11) and an anti-double-stranded DNA (4B2) are essentially benign. These results establish a murine model of anti-DNA-induced glomerular injury resembling the severe nephritis seen in lupus patients and provide direct evidence that anti-ssDNA can be more pathogenic than anti-double-stranded DNA. In vitro binding experiments using both protein-DNA complexes and naive kidney tissue indicate that glomerular localization of 11F8 may occur by recognition of a planted antigen in vivo. Binding to this antigen is DNase sensitive which suggests that DNA or a DNA-containing molecule is being recognized.

Structure activity studies of a novel cytotoxic benzodiazepine

Analogues of Bz-423, a pro-apoptotic 1,4-benzodiazepine with potent activity in animal models of systemic lupus erythematosus and rheumatoid arthritis, have been designed, synthesized, and evaluated in cell-culture assays. The results of these experiments have defined the structural elements of this new cytotoxic agent required for activity.

Bz-423 Superoxide Signals Apoptosis via Selective Activation of JNK, Bak, and Bax

Bz-423 is a proapoptotic 1,4-benzodiazepine with potent therapeutic properties in murine models of lupus and psoriasis. Bz-423 modulates the F(1)F(0)-ATPase, inducing the formation of superoxide within the mitochondrial respiratory chain, which then functions as a second messenger initiating apoptosis. Herein, we report the signaling pathway activated by Bz-423 in mouse embryonic fibroblasts containing knockouts of key apoptotic proteins. Bz-423-induced superoxide activates cytosolic ASK1 and its release from thioredoxin. A mitogen-activated protein kinase cascade follows, leading to the specific phosphorylation of JNK. JNK signals activation of Bax and Bak which then induces mitochondrial outer membrane permeabilization to cause the release of cytochrome c and a commitment to apoptosis. The response of these cells to Bz-423 is critically dependent on both superoxide and JNK activation as antioxidants and the JNK inhibitor SP600125 prevents Bax translocation, cytochrome c release, and cell death. These results demonstrate that superoxide generated from the mitochondrial respiratory chain as a consequence of a respiratory transition can signal a sequential and specific apoptotic response. Collectively, these data suggest that the selectivity of Bz-423 observed in vivo results from cell-type specific differences in redox balance and signaling by ASK1 and Bcl-2 proteins.

Fluid Overload Is Associated With Late Poor Outcomes in Neonates Following Cardiac Surgery

Objectives: Acute kidney injury is a severe complication of cardiac surgery associated with increased morbidity and mortality; yet, acute kidney injury classification for neonates remains challenging. We characterized patterns of postoperative fluid overload as a surrogate marker for acute kidney injury and as a risk factor of poor postoperative outcomes in neonates undergoing cardiac surgery. Design: Retrospective cohort study. Setting: Single, congenital heart center destination program. Patients: Four hundred thirty-five neonates undergoing cardiac surgery with cardiopulmonary bypass from January 2006 through December 2010. Interventions: None. Measurements and Main Results: Demographics, diagnosis, and perioperative clinical variables were collected, including daily weights and serum creatinine levels. A composite poor clinical outcome (death, need for renal replacement therapy or extracorporeal life support within 30 postoperative days) was considered the primary outcome measure. Twenty-one neonates (5%) had a composite poor outcome with 7 (2%) requiring renal replacement therapy, 8 (2%) requiring extracorporeal life support, and 14 (3%) dying between 3 and 30 days post surgery. Neonates with a composite poor outcome had significantly higher maximum fluid overload (> 20%) and were slower to diurese. A receiver-operating characteristic curve determined that fluid overload greater than or equal to 16% and serum creatinine greater than or equal to 0.9 on postoperative day 3 were the optimal cutoffs for significant discrimination on the primary outcome (area under the curve = 0.71 and 0.76, respectively). In multivariable analysis, fluid overload greater than or equal to 16% (adjusted odds ratio = 3.7) and serum creatinine adjusted odds ratio 0.9 (adjusted odds ratio = 6.6) on postoperative day 3 remained an independent risk factor for poor outcome. Fluid overload greater than or equal 16% was also significantly associated with cardiac arrest requiring cardiopulmonary resuscitation, prolonged ICU stay, and chest reexploration. Conclusions: This study highlights the importance of monitoring fluid balance in the neonatal cardiac surgical population and suggests that daily fluid overload, a readily available, noninvasive marker of renal function, may be a sensitive and specific predictor of adverse outcomes.