Multiparameter Fluorescence-Activated Cell Sorting of Human Lymphatic Endothelial Cells.

Multiparameter fluorescence-activated cell sorting (FACS) procedure separates target cells from a total population of cells by using specific signatures that the target cell expresses on their cell surface. For human lymphatic endothelial cells (LECs) this relates to cell surface expression of the CD34LowCD31HighVEGFR-3HighPodoplaninHigh profile that permits their separation from blood vascular endothelial cells and other cells likely to be present in the digested tissue sample. In addition, FACS allows the evaluation of LEC size, volume, granularity, and purity at the time of sorting.

Improving Compliance with a Rounding Checklist through Low- and High-technology Interventions: A Quality Improvement Initiative.

INTRODUCTION: Checklists aid in ensuring consistency and completeness in medical care delivery. However, using an improvement and safety checklist during rounds was variable in our neonatology intensive care unit (NICU), and completion was not tracked sustainably. This quality improvement (QI) initiative's primary aim was to increase compliance with checklist completion from 31% to >75% within 1 year. METHODS: A multidisciplinary QI team identified barriers to checklist completion and implemented a human factors-focused low-technology intervention (redesign of a hard-copy checklist) and later a high-technology clinical decision support tool within the electronic health record. The primary outcome measure was percent compliance with the use of the checklist. Process metrics included the duration of checklist completion. Balancing measures included staff perceptions of work burden and question relevance. RESULTS: Major barriers to checklist utilization were inability to remember, rounding interruptions, and perceived lack of question relevance to patients. Average biweekly checklist compliance improved from 31% before interventions to 80% after interventions. Average checklist completion time decreased from 46 to 11 seconds. Follow-up surveys demonstrated more respondents found questions "completely relevant" (34% pre versus 43% post) but perceived increased work burden (26% pre versus 31% post). CONCLUSIONS: Using QI methodology, human factors-based interventions, and a novel clinical decision support tool, we significantly improved efficiency and checklist compliance and created an automated, sustainable method for monitoring completion and responses. This foundational project provides an infrastructure broadly applicable to QI work in other healthcare settings.

A platform for discovery of functional cell-penetrating peptides for efficient multi-cargo intracellular delivery.

Cell penetrating peptides (CPPs) offer great potential to deliver therapeutic molecules to previously inaccessible intracellular targets. However, many CPPs are inefficient and often leave their attached cargo stranded in the cell's endosome. We report a versatile platform for the isolation of peptides delivering a wide range of cargos into the cytoplasm of cells. We used this screening platform to identify multiple "Phylomer" CPPs, derived from bacterial and viral genomes. These peptides are amenable to conventional sequence optimization and engineering approaches for cell targeting and half-life extension. We demonstrate potent, functional delivery of protein, peptide, and nucleic acid analog cargos into cells using Phylomer CPPs. We validate in vivo activity in the cytoplasm, through successful transport of an oligonucleotide therapeutic fused to a Phylomer CPP in a disease model for Duchenne's muscular dystrophy. This report thus establishes a discovery platform for identifying novel, functional CPPs to expand the delivery landscape of druggable intracellular targets for biological therapeutics.

Colonoscopy vs. Fecal Immunochemical Test in Reducing Mortality From Colorectal Cancer (CONFIRM): Rationale for Study Design.

RATIONALE: Colorectal cancer (CRC) is preventable through screening, with colonoscopy and fecal occult blood testing comprising the two most commonly used screening tests. Given the differences in complexity, risk, and cost, it is important to understand these tests' comparative effectiveness. STUDY DESIGN: The CONFIRM Study is a large, pragmatic, multicenter, randomized, parallel group trial to compare screening with colonoscopy vs. the annual fecal immunochemical test (FIT) in 50,000 average risk individuals. CONFIRM examines whether screening colonoscopy will be superior to a FIT-based screening program in the prevention of CRC mortality measured over 10 years. Eligible individuals 50-75 years of age and due for CRC screening are recruited from 46 Veterans Affairs (VA) medical centers. Participants are randomized to either colonoscopy or annual FIT. Results of colonoscopy are managed as per usual care and study participants are assessed for complications. Participants testing FIT positive are referred for colonoscopy. Participants are surveyed annually to determine if they have undergone colonoscopy or been diagnosed with CRC. The primary endpoint is CRC mortality. The secondary endpoints are (1) CRC incidence (2) complications of screening colonoscopy, and (3) the association between colonoscopists' characteristics and neoplasia detection, complications and post-colonoscopy CRC. CONFIRM leverages several key characteristics of the VA's integrated healthcare system, including a shared medical record with national databases, electronic CRC screening reminders, and a robust national research infrastructure with experience in conducting large-scale clinical trials. When completed, CONFIRM will be the largest intervention trial conducted within the VA (ClinicalTrials.gov identifier: NCT01239082).

GFP-complementation assay to detect functional CPP and protein delivery into living cells.

Efficient cargo uptake is essential for cell-penetrating peptide (CPP) therapeutics, which deliver widely diverse cargoes by exploiting natural cell processes to penetrate the cell's membranes. Yet most current CPP activity assays are hampered by limitations in assessing uptake, including confounding effects of conjugated fluorophores or ligands, indirect read-outs requiring secondary processing, and difficulty in discriminating internalization from endosomally trapped cargo. Split-complementation Endosomal Escape (SEE) provides the first direct assay visualizing true cytoplasmic-delivery of proteins at biologically relevant concentrations. The SEE assay has minimal background, is amenable to high-throughput processes, and adaptable to different transient and stable cell lines. This split-GFP-based platform can be useful to study transduction mechanisms, cellular imaging, and characterizing novel CPPs as pharmaceutical delivery agents in the treatment of disease.

The efficacy of tumor debulking surgery is improved by adjuvant immunotherapy using imiquimod and anti-CD40.

BACKGROUND: Tumor debulking surgery followed by adjuvant chemotherapy or radiotherapy is a standard treatment for many solid malignancies. Although this approach can be effective, it often has limited success against recurrent or metastatic cancers and new multimodality approaches are needed. Adjuvant immunotherapy is another potentially effective approach. We therefore tested the efficacy of the TLR7 agonist imiquimod (IMQ) combined with agonistic anti-CD40 in an incomplete debulking model of malignant mesothelioma. METHODS: Established subcutaneous murine ABA-HA mesothelioma tumors in BALB/c mice were surgically debulked by 75% and treated with either: i) saline; ii) intratumoral IMQ; iii) systemic anti-CD40 antibody, or using a combination of IMQ and anti-CD40. Tumour growth and survival were monitored, and the role of anti-tumor CD4 and CD8 T cells in therapeutic responses was determined. RESULTS: The combination therapy of partial debulking surgery, IMQ and anti-CD40 significantly delayed tumor growth in a CD8 T cell dependent manner, and promoted tumor regression in 25% of animals with establishment of immunological memory. This response was associated with an increase in ICOS+ CD8 T cells and tumor-specific CTL activity in tumor draining lymph nodes along with an increase in ICOS+ CD8 T cells in responding tumours. CONCLUSIONS: We show that the post-surgical environment can be significantly altered by the co-administration of adjuvant IMQ and anti-CD40, resulting in strong, systemic anti-tumor activity. Both adjuvants are available for clinical use/trial, hence this treatment regimen has clear translational potential.

Neoadjuvant anti-tumor vaccination prior to surgery enhances survival.

BACKGROUND: This study was conducted to determine if anti-tumor vaccination administered prior to partial debulking surgery could improve survival using a murine solid tumour model. METHODS: Tumor incidence and survival rates were compared in mice bearing subcutaneous AB1-HA mesothelioma tumors that received either sham surgery, debulking surgery or vaccination prior to debulking surgery. Additionally, mice were depleted of CD4 and/or CD8 T lymphocytes during vaccination to assess their involvement in vaccine induced anti-tumor immunity. Flow cytometry was performed to characterise changes in the proportion and activation status of immune cells associated with anti-tumor immunity. RESULTS: Neoadjuvant vaccination combined with debulking surgery resulted in decreased tumor burden, increased survival and generation of tumor-specific immunity compared to surgery alone. Depletion of CD8 T cells completely abrogated any vaccine induced anti-tumor immune response. Conversely, CD4 depletion enhanced CD8 T cell activation resulting in complete tumor regression in 70% of mice treated with combined surgery and vaccination therapy. Tumor free survival was associated with established immunological memory as defined by the induction of effector memory T cells and resistance to rechallenge with parental AB1 mesothelioma cells. CONCLUSIONS: Neoadjuvant anti-cancer vaccination combined with partial debulking surgery induced CD8-dependent anti-tumor immunity that significantly delayed tumor outgrowth relative to surgery alone. Complete tumor eradication was observed when vaccination and surgery were performed in CD4 T cell depleted animals. This demonstrates that adjuvant immunotherapy can improve post-surgical survival following cancer debulking surgery and provides a scientific rational for clinical trials of such an approach.

Long-term exposure of mesothelial cells to SV40 and asbestos leads to malignant transformation and chemotherapy resistance.

Simian virus 40 (SV40) has been implicated in the development of several cancers including malignant mesothelioma. A definitive role for the virus in human mesothelioma has not been unequivocally demonstrated but has been rigorously debated. The virus clearly has oncogenic potential: the TAg is one of the most potent transforming proteins known and acts synergistically with crocidolite asbestos to transform mesothelial cells. In this study, we show that SV40 oncogenes alone can cause malignant transformation and that asbestos-induced DNA damage and apoptosis occurs principally in cycling cells. After long-term exposure (up to 100 days) to both SV40 and asbestos, cells become resistant to stress-induced senescence. Significantly, these cells demonstrate resistance to chemotherapy-induced apoptosis. This finding has implications for the development of effective treatment options for patients with mesothelioma.

Combined angiotensin inhibition for the treatment of diabetic nephropathy.

BACKGROUND: Combination therapy with angiotensin-converting-enzyme (ACE) inhibitors and angiotensin-receptor blockers (ARBs) decreases proteinuria; however, its safety and effect on the progression of kidney disease are uncertain. Methods We provided losartan (at a dose of 100 mg per day) to patients with type 2 diabetes, a urinary albumin-to-creatinine ratio (with albumin measured in milligrams and creatinine measured in grams) of at least 300, and an estimated glomerular filtration rate (GFR) of 30.0 to 89.9 ml per minute per 1.73 m(2) of body-surface area and then randomly assigned them to receive lisinopril (at a dose of 10 to 40 mg per day) or placebo. The primary end point was the first occurrence of a change in the estimated GFR (a decline of ≥ 30 ml per minute per 1.73 m(2) if the initial estimated GFR was ≥ 60 ml per minute per 1.73 m(2) or a decline of ≥ 50% if the initial estimated GFR was <60 ml per minute per 1.73 m(2)), end-stage renal disease (ESRD), or death. The secondary renal end point was the first occurrence of a decline in the estimated GFR or ESRD. Safety outcomes included mortality, hyperkalemia, and acute kidney injury. Results The study was stopped early owing to safety concerns. Among 1448 randomly assigned patients with a median follow-up of 2.2 years, there were 152 primary end-point events in the monotherapy group and 132 in the combination-therapy group (hazard ratio with combination therapy, 0.88; 95% confidence interval [CI], 0.70 to 1.12; P=0.30). A trend toward a benefit from combination therapy with respect to the secondary end point (hazard ratio, 0.78; 95% CI, 0.58 to 1.05; P=0.10) decreased with time (P=0.02 for nonproportionality). There was no benefit with respect to mortality (hazard ratio for death, 1.04; 95% CI, 0.73 to 1.49; P=0.75) or cardiovascular events. Combination therapy increased the risk of hyperkalemia (6.3 events per 100 person-years, vs. 2.6 events per 100 person-years with monotherapy; P<0.001) and acute kidney injury (12.2 vs. 6.7 events per 100 person-years, P<0.001). Conclusions Combination therapy with an ACE inhibitor and an ARB was associated with an increased risk of adverse events among patients with diabetic nephropathy. (Funded by the Cooperative Studies Program of the Department of Veterans Affairs Office of Research and Development; VA NEPHRON-D ClinicalTrials.gov number, NCT00555217.).

Piecewise analysis of patient survival after onset of AKI.

BACKGROUND AND OBJECTIVES: AKI affects approximately 2%-7% of hospitalized patients and >35% of critically ill patients. Survival after AKI may be described as having an acute phase (including an initial hyperacute component) followed by a convalescent phase, which may itself have early and late components. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Data from the Veterans Affairs/National Institutes of Health Acute Renal Failure Trial Network (ATN) study was used to model mortality risk among patients with dialysis-requiring AKI. This study assumed that the mortality hazard can be described by a piecewise log-linear function with change points. Using an average likelihood method, the authors tested for the number of change points in a piecewise log-linear hazard model. The maximum likelihood approach to locate the change point(s) was then adopted, and associated parameters and standard errors were estimated. RESULTS: There were 1124 ATN participants with follow-up to 1 year. The mortality hazard of AKI decreased over time with inflections in the rate of decrease at days 4, 42, and 148, with the sharpest change at day 42. The daily rate of decline in the log of the hazard for death was 0.220 over the first 4 days, 0.046 between day 4 and day 42, 0.017 between day 42 and day 148, and 0.003 between day 148 and day 365. CONCLUSIONS: There appear to be two major phases of mortality risk after AKI: an early phase extending over the first 6 weeks and a late phase from 6 weeks to 1 year. Within the first 42 days, this can be further divided into hyperacute (days 1-4) and acute (days 4-42) phases. After 42 days, there appear to be early (days 42-148) and late (after day 148) convalescent phases. These findings may help to inform the design of AKI clinical trials and assist critical care physicians in prognostic stratification.

Characterization of the novel broad-spectrum kinase inhibitor CTx-0294885 as an affinity reagent for mass spectrometry-based kinome profiling.

Kinase enrichment utilizing broad-spectrum kinase inhibitors enables the identification of large proportions of the expressed kinome by mass spectrometry. However, the existing inhibitors are still inadequate in covering the entire kinome. Here, we identified a novel bisanilino pyrimidine, CTx-0294885, exhibiting inhibitory activity against a broad range of kinases in vitro, and further developed it into a Sepharose-supported kinase capture reagent. Use of a quantitative proteomics approach confirmed the selectivity of CTx-0294885-bound beads for kinase enrichment. Large-scale CTx-0294885-based affinity purification followed by LC-MS/MS led to the identification of 235 protein kinases from MDA-MB-231 cells, including all members of the AKT family that had not been previously detected by other broad-spectrum kinase inhibitors. Addition of CTx-0294885 to a mixture of three kinase inhibitors commonly used for kinase-enrichment increased the number of kinase identifications to 261, representing the largest kinome coverage from a single cell line reported to date. Coupling phosphopeptide enrichment with affinity purification using the four inhibitors enabled the identification of 799 high-confidence phosphosites on 183 kinases, ∼10% of which were localized to the activation loop, and included previously unreported phosphosites on BMP2K, MELK, HIPK2, and PRKDC. Therefore, CTx-0294885 represents a powerful new reagent for analysis of kinome signaling networks that may facilitate development of targeted therapeutic strategies. Proteomics data have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with the data set identifier PXD000239.

Synthesis of conformationally constrained benzoylureas as BH3-mimetics.

The design of small molecules that mimic the BH3 domain and bind to Bcl-2 proteins has emerged as a promising approach to discovering novel anti-cancer therapeutics. We reveal the design and synthesis of conformationally constrained benzoylurea scaffolds as conformational probes. Central to helix mimicry, the intramolecular hydrogen bond in the benzoylurea plays a key role in the pre-organisation of the acyclic substrates for cyclisation via ring closing metathesis, providing efficient access to the constrained mimetics.

Health-related quality of life as a predictor of mortality among survivors of AKI.

BACKGROUND AND OBJECTIVES: This study examined the relationship between health-related quality of life and subsequent mortality among AKI survivors treated with renal replacement therapy. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Multivariable Cox regression models were used to assess the associations between Health Utilities Index Mark 3 (HUI3) and ambulation, emotion, cognition, and pain scores at 60 days and all-cause mortality at 1 year in 60-day AKI survivors (n=439 with evaluable HUI3 assessments) from a randomized multicenter study comparing less- with more-intensive renal replacement therapies. RESULTS: The median 60-day HUI3 index score was 0.32. Patients with evaluable HUI3 data who died between 60 days and 1 year (n=99) were more likely to have lower 60-day median HUI3 scores, higher comorbidity scores, and longer initial hospital stays, and they were more likely to be dialysis-dependent. A 0.1 higher HUI3 index score was associated with a 17% decrease (hazard ratio, 0.83; 95% confidence interval 0.77-0.89) in all-cause mortality after controlling for clinical risk factors. Similar associations were observed for HUI3 ambulation, emotion, cognition, and pain attribute scores. CONCLUSIONS: Health-related quality of life measured by HUI3 is an independent predictor of mortality among survivors of AKI after adjusting for clinical risk variables. Poor ambulation and other health-related quality of life attributes are also associated with increased risk of death. Health-related quality of life may provide clinicians with additional information to help identify patients at high risk of mortality after AKI that required renal replacement therapy.

An efficient high-throughput screening method for MYST family acetyltransferases, a new class of epigenetic drug targets.

Epigenetic aberrations are increasingly regarded as key factors in cancer progression. Recently, deregulation of histone acetyltransferases (HATs) has been linked to several types of cancer. Monocytic leukemia zinc finger protein (MOZ) is a member of the MYST family of HATs, which regulate gene expression in cell proliferation and differentiation. Deregulation of these processes through constitutively active MOZ fusion proteins gives rise to the formation of leukemic stem cells, rendering MOZ an excellent target for treating myeloid leukemia. The authors implemented a hit discovery campaign to identify small-molecule inhibitors of MOZ-HAT activity. They developed a robust, homogeneous assay measuring the acetylation of synthetic histone peptides. In a primary screening campaign testing 243 000 lead-like compounds, they identified inhibitors from several chemical classes. Secondary assays were used to eliminate assay-interfering compounds and prioritize confirmed hits. This study establishes a new high-throughput assay for HAT activity and could provide the foundation for the development of a new class of drugs for the treatment of leukemias.

Model to predict mortality in critically ill adults with acute kidney injury.

BACKGROUND AND OBJECTIVES: Acute kidney injury (AKI) requiring dialysis is associated with high mortality. Most prognostic tools used to describe case complexity and to project patient outcome lack predictive accuracy when applied in patients with AKI. In this study, we developed an AKI-specific predictive model for 60-day mortality and compared the model to the performance of two generic (Sequential Organ Failure Assessment [SOFA] and Acute Physiology and Chronic Health Evaluation II [APACHE II]) scores, and a disease specific (Cleveland Clinic [CCF]) score. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Data from 1122 subjects enrolled in the Veterans Affairs/National Institutes of Health Acute Renal Failure Trial Network study; a multicenter randomized trial of intensive versus less intensive renal support in critically ill patients with AKI conducted between November 2003 and July 2007 at 27 VA- and university-affiliated centers. RESULTS: The 60-day mortality was 53%. Twenty-one independent predictors of 60-day mortality were identified. The logistic regression model exhibited good discrimination, with an area under the receiver operating characteristic (ROC) curve of 0.85 (0.83 to 0.88), and a derived integer risk score yielded a value of 0.80 (0.77 to 0.83). Existing scoring systems, including APACHE II, SOFA, and CCF, when applied to our cohort, showed relatively poor discrimination, reflected by areas under the ROC curve of 0.68 (0.64 to 0.71), 0.69 (0.66 to 0.73), and 0.65 (0.62 to 0.69), respectively. CONCLUSIONS: Our new risk model outperformed existing generic and disease-specific scoring systems in predicting 60-day mortality in critically ill patients with AKI. The current model requires external validation before it can be applied to other patient populations.

Methodological issues in comparative effectiveness research: clinical trials.

The US Department of Veterans Affairs (VA) Cooperative Studies Program has been conducting comparative effectiveness clinical trials for nearly 4 decades in many disease areas, including cardiovascular disease/surgery, diabetes mellitus, mental health, neurologic disorders, cancer, infectious diseases, and rheumatoid arthritis. The features that have made this program advantageous for conducting comparative effectiveness clinical trials are described along with methodological considerations for future trials based on lessons learned from its experience conducting these types of studies. Some of the lessons learned involve managing risk factors, clinical equipoise, patient preferences, evolving technology, the use of usual care as a comparator and pharmaceutical issues related to study drug blinding. These issues are not unique to the VA but can play an important role in enabling valid comparisons between treatments that may have differences in delivery or mechanisms of action and could affect the execution and feasibility of conducting a clinical trial with a comparative effectiveness aim. We also outline some future directions for comparative effectiveness clinical trials.

Prevalence of antibodies to four human coronaviruses is lower in nasal secretions than in serum.

Little is known about the prevalence of mucosal antibodies induced by infection with human coronaviruses (HCoV), including HCoV-229E and -OC43 and recently described strains (HCoV-NL63 and -HKU1). By enzyme-linked immunosorbent assay, we measured anti-HCoV IgG antibodies in serum and IgA antibodies in nasal wash specimens collected at seven U.S. sites from 105 adults aged 50 years and older (mean age, 67 ± 9 years) with chronic obstructive pulmonary disease. Most patients (95 [90%]) had at least one more chronic disease. More patients had serum antibody to each HCoV strain (104 [99%] had antibody to HCoV-229E, 105 [100%] had antibody to HCoV-OC43, 103 [98%] had antibody to HCoV-NL63, and 96 [91%] had antibody to HCoV-HKU1) than had antibody to each HCoV strain in nasal wash specimens (12 [11%] had antibody to HCoV-229E, 22 [22%] had antibody to HCoV-OC43, 8 [8%] had antibody to HCoV-NL63, and 31 [31%] had antibody to HCoV-HKU1), respectively (P < 0.0001). The proportions of subjects with IgA antibodies in nasal wash specimens and the geometric mean IgA antibody titers were statistically higher for HCoV-OC43 and -HKU1 than for HCoV-229E and -NL63. A higher proportion of patients with heart disease than not had IgA antibodies to HCoV-NL63 (6 [16%] versus 2 [3%]; P = 0.014). Correlations were highest for serum antibody titers between group I strains (HCoV-229E and -NL63 [r = 0.443; P < 0.0001]) and between group II strains (HCoV-OC43 and -HKU1 [r = 0.603; P < 0.0001]) and not statistically significant between HCoV-NL63 and -OC43 and between HCoV-NL63 and -HKU1. Patients likely had experienced infections with more than one HCoV strain, and IgG antibodies to these HCoV strains in serum were more likely to be detected than IgA antibodies to these HCoV strains in nasal wash specimens.

Predictors of health utility among 60-day survivors of acute kidney injury in the Veterans Affairs/National Institutes of Health Acute Renal Failure Trial Network Study.

BACKGROUND AND OBJECTIVES: Health-related quality of life (HRQOL) after acute kidney injury (AKI) is an area of great importance to patients. It was hypothesized that HRQOL after AKI would relate to intensity of dialysis during AKI and dialysis dependence at follow-up. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: The Veterans Affairs/National Institutes of Health Acute Renal Failure Trial Network Study was a multicenter, prospective, randomized trial of intensive versus less intensive renal replacement therapy in critically ill patients with AKI. Of 1124 participants, 415 survived at least 60 days and completed the Health Utilities Index (HUI), which measures 8 health attributes and calculates an overall HRQOL score, also called a utility score. How strongly pre-intensive care unit (ICU) health, severity of illness, hospital course, intensity of dialysis, and outcome were associated with 60-day HUI scores was assessed, after adjustment for demographics. RESULTS: The overall HUI score was 0.40 +/- 0.37, indicating severely compromised health utility and was associated with only admission from home and hospital and ICU length of stay (LOS). Ambulation was better among those with a shorter hospital and ICU LOS. Better cognition was associated with dialysis independence and with fewer comorbid chronic illnesses. Emotion was associated with only hospital LOS. Pain was associated with ICU LOS. CONCLUSIONS: Health utility was low in this cohort of patients after AKI, and intensity of dialysis did not affect subsequent health utility. The effects of a lengthy hospitalization generally outweighed the effects of delayed recovery of kidney function on HRQOL after AKI.

Intensity of renal replacement therapy in acute kidney injury: perspective from within the Acute Renal Failure Trial Network Study.

Determination of the optimal dose of renal replacement therapy in critically ill patients with acute kidney injury has been controversial. Questions have recently been raised regarding the design and execution of the US Department of Veterans Affairs/National Institutes of Health Acute Renal Failure Trial Network (ATN) Study, which demonstrated no improvement in 60-day all-cause mortality with more intensive management of renal replacement therapy. In the present article we present our rationale for these aspects of the design and conduct of the study, including our use of both intermittent and continuous modalities of renal support, our approach to initiation of study therapy and the volume management during study therapy. In addition, the article presents data on hypotension during therapy and recovery of kidney function in the perspective of other studies of renal support in acute kidney injury. Finally, we address the implications of the ATN Study results for clinical practice from the perspective of the study investigators.