CCL14 Predicts Oliguria and Dialysis Requirement in Patients with Moderate to Severe Acute Kidney Injury.

INTRODUCTION: AKI is a frequent complication of critical illness and portends poor outcome. CCL14 is a validated predictor of persistent severe AKI in critically ill patients. We examined the association of CCL14 with urine output within 48 h. METHODS: In pooled data from 2 studies of critically ill patients with KDIGO stage 2-3 AKI, CCL14 was measured by NEPHROCLEAR™ CCL14 Test on the Astute 140® Meter (low, intermediate, and high categories [1.3 and 13 ng/mL]). Average hourly urine output over 48 h, stage 3 AKI per urine output criterion on day 2, and composite of dialysis or death within 7 days were examined using multivariable mixed and logistic regression models. RESULTS: Of the 497 subjects with median age of 65 (56-74) years, 49% (242/497) were on diuretics. CCL14 concentration was low in 219 (44%), intermediate in 217 (44%), and high in 61 (12%) patients. In mixed regression analysis, hourly urine output over time was different within each CCL14 risk category based on diuretic use due to significant three-way interaction (p < 0.001). In logistic regression analysis, CCL14 risk category was independently associated with low urine output on day 2 per KDIGO stage 3 (adjusted for diuretic use and baseline clinical variables), and composite of dialysis or death within 7 days (adjusted for urine output within 48 h of CCL14 measurement). CONCLUSIONS: CCL14 measured in patients with moderate to severe AKI is associated with urine output trajectory within 48 h, oliguria on day 2, and dialysis within 7 days.

Clinical Outcomes of Persistent Severe Acute Kidney Injury among Patients with Kidney Disease Improving Global Outcomes Stage 2 or 3 Acute Kidney Injury.

INTRODUCTION: The burden of persistent (≥3 days) severe AKI (PS-AKI) is poorly described among inpatients with stage 2-3 AKI in the ward or ICU. Quantification could motivate targeted interventions to decrease duration of AKI in these high-risk patients. METHODS: This retrospective cohort study included adult patients discharged from January 1, 2017, to December 31, 2019, from US hospitals in the PINC AI Healthcare Database. Patients with KDIGO stage 2 or 3 AKI, length of stay ≥3 days, ≥3 serum creatinine measures, and no history of renal transplant, dialysis, or stage 5 chronic kidney disease were included. Patients were classified as PS-AKI (stage 3 AKI lasting ≥3 days or with death in ≤3 days, or stage 2 or 3 AKI with dialysis in ≤3 days) or not PS-AKI (NPS-AKI) (stage 3 AKI for ≤2 days, or stage 2 AKI without dialysis in ≤3 days). Outcomes during index (initial) hospitalization were PS-AKI incidence, ICU use, and in-hospital mortality, and during 30 days post-discharge were readmissions, in-hospital mortality, dialysis, and "new" dialysis (dialysis among patients without dialysis during index hospitalization). For index outcomes, we used a sensitivity definition, PS-AKISens, that excluded patients who met PS-AKI criteria by dialysis/death in ≤3 days of AKI onset. Multivariable-adjusted logistic regression quantified differences between PS-AKI and NPS-AKI, overall, and separately for ICU and non-ICU patients. RESULTS: Among 126,528 inpatients with stage 2 or 3 AKI, PS-AKI developed in 24.4% (30,916), with 39% of PS-AKI occurring in non-ICU patients. With NPS-AKI as the reference group, adjusted odds ratios (aORs) (95% CI) for PS-AKI and for PS-AKISens were 2.15 (2.09-2.21) and 1.28 (1.24-1.32) for ICU use and 4.58 (4.41-4.75) and 1.79 (1.70-1.89) for in-hospital mortality during index hospitalization. For outcomes during 30 days post-discharge, aORs for PS-AKI versus NPS-AKI were 1.07 (1.02-1.11) for readmissions, 1.33 (1.18-1.49) for in-hospital mortality, 15.66 (13.87-17.67) for dialysis, and 6.80 (5.84-7.93) for new dialysis. Despite higher mortality among ICU patients, aORs for outcomes during index and 30 days post-discharge were similar for ICU and non-ICU patients. CONCLUSION: In and out of the ICU, PS-AKI frequently affected inpatients with stage 2 or 3 AKI and was independently associated with worse clinical outcomes during index hospitalization and during 30 days post-discharge. These results suggest that interventions to prevent persistence of severe AKI may reduce adverse clinical outcomes among patients with stage 2 or 3 AKI in or out of the ICU.

Limiting Acute Kidney Injury Progression In Sepsis: Study Protocol and Trial Simulation.

OBJECTIVES: To describe study design considerations and to simulate a trial of biomarker-guided sepsis management aimed to reduce acute kidney injury (acute kidney injury). Tissue inhibitor of metalloproteinases-2 and insulin-like growth factor-binding protein 7, urinary biomarkers of cell-cycle arrest, and indicators of kidney stress can detect acute kidney injury before clinical manifestations. We sought to determine the event rates for acute kidney injury as a function of serial measurements of urinary (tissue inhibitor of metalloproteinases-2)•(insulin-like growth factor-binding protein 7) in patients at risk of sepsis-associated acute kidney injury, so that an escalating series of kidney-sparing sepsis bundles based on international guidelines could be applied. DESIGN: We described the study protocol of "Limiting acute kidney injury Progression In Sepsis," a phase 4, multicenter, adaptive, randomized controlled trial. We performed simulations to estimate the rates for the trial's primary endpoint using patient-level data from two previous studies (Sapphire and Protocolized Care for Early Septic Shock). SETTING: Academic and community ICUs. PATIENTS: Critically ill patients with sepsis or septic shock, without evidence of stage 2/3 acute kidney injury at enrollment. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Our primary endpoint is progression of two or more stages of acute kidney injury, death, or dialysis within 72 hours after enrollment. In the Sapphire simulation, 45 of 203 patients (22%) with sepsis met the endpoint. In Protocolized Care for Early Septic Shock, 144 of 607 patients (24%) with septic shock met the endpoint. In both simulations, (tissue inhibitor of metalloproteinases-2)•(insulin-like growth factor-binding protein 7) patterns, suggested by Limiting acute kidney injury Progression In Sepsis protocol, stratified the risk for the endpoint from 6% (three negative tests) to 41% (for patients eligible for the highest level of kidney-sparing sepsis bundle) in Sapphire, and 14% (two negative tests) to 46% (for the highest level of kidney-sparing sepsis bundle) in Protocolized Care for Early Septic Shock. CONCLUSIONS: Findings of our Limiting acute kidney injury Progression In Sepsis trial simulation confirmed that (tissue inhibitor of metalloproteinases-2)•(insulin-like growth factor-binding protein 7) could identify patients with different rates of progression to moderate/severe acute kidney injury, death, or dialysis in 72 hours. The Limiting acute kidney injury Progression In Sepsis protocol algorithm is therefore feasible in terms of identifying suitably high-risk individuals for kidney-sparing sepsis bundle.

Use of Biomarkers to Identify Acute Kidney Injury to Help Detect Sepsis in Patients With Infection.

OBJECTIVES: Although early recognition of sepsis is vital to improving outcomes, the diagnosis may be missed or delayed in many patients. Acute kidney injury is one of the most common organ failures in patients with sepsis but may not be apparent on presentation. Novel biomarkers for acute kidney injury might improve organ failure recognition and facilitate earlier sepsis care. DESIGN: Retrospective, international, Sapphire study. SETTING: Academic Medical Center. PATIENTS: Adults admitted to the ICU without evidence of acute kidney injury at time of enrollment. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We stratified patients enrolled in the Sapphire study into three groups-those with a clinical diagnosis of sepsis (n = 216), those with infection without sepsis (n = 120), and those without infection (n = 387) at enrollment. We then examined 30-day mortality stratified by acute kidney injury within each group. Finally, we determined the operating characteristics for kidney stress markers (tissue inhibitor of metalloproteinases-2) × (insulin-like growth factor binding protein 7) for prediction of acute kidney injury as a sepsis-defining organ failure in patients with infection without a clinical diagnosis of sepsis at enrollment. Combining all groups, 30-day mortality was 23% for patients who developed stage 2-3 acute kidney injury within the first 3 days compared with 14% without stage 2-3 acute kidney injury. However, this difference was greatest in the infection without sepsis group (34% vs 11%; odds ratio, 4.09; 95% CI, 1.53-11.12; p = 0.005). Using a (tissue inhibitor of metalloproteinases-2) × (insulin-like growth factor binding protein 7) cutoff of 2.0 units, 14 patients (11.7%), in the infection/no sepsis group, tested positive of which 10 (71.4%) developed stage 2-3 acute kidney injury. The positive test result occurred a median of 19 hours (interquartile range, 0.8-34.0 hr) before acute kidney injury manifested by serum creatinine or urine output. Similar results were obtained using a cutoff of 1.0 for any stage of acute kidney injury. CONCLUSIONS: Use of the urinary (tissue inhibitor of metalloproteinases-2) × (insulin-like growth factor binding protein 7) test could identify acute kidney injury in patients with infection, possibly helping to detect sepsis, nearly a day before acute kidney injury is apparent by clinical criteria.

External validation of urinary C-C motif chemokine ligand 14 (CCL14) for prediction of persistent acute kidney injury.

BACKGROUND: Persistent acute kidney injury (AKI) portends worse clinical outcomes and remains a therapeutic challenge for clinicians. A recent study found that urinary C-C motif chemokine ligand 14 (CCL14) can predict the development of persistent AKI. We aimed to externally validate urinary CCL14 for the prediction of persistent AKI in critically ill patients. METHODS: This was a secondary analysis of the prospective multi-center SAPPHIRE study. We evaluated critically ill patients with cardiac and/or respiratory dysfunction who developed Kidney Disease: Improving Global Outcomes (KDIGO) stage 2-3 AKI within one week of enrollment. The main exposure was the urinary concentration of CCL14 measured at the onset of AKI stage 2-3. The primary endpoint was the development of persistent severe AKI, defined as ≥ 72 h of KDIGO stage 3 AKI or death or renal-replacement therapy (RRT) prior to 72 h. The secondary endpoint was a composite of RRT and/or death by 90 days. We used receiver operating characteristic (ROC) curve analysis to assess discriminative ability of urinary CCL14 for the development of persistent severe AKI and multivariate analysis to compare tertiles of urinary CCL14 and outcomes. RESULTS: We included 195 patients who developed KDIGO stage 2-3 AKI. Of these, 28 (14%) developed persistent severe AKI, of whom 15 had AKI ≥ 72 h, 12 received RRT and 1 died prior to ≥ 72 h of KDIGO stage 3 AKI. Persistent severe AKI was associated with chronic kidney disease, diabetes mellitus, higher non-renal APACHE III score, greater fluid balance, vasopressor use, and greater change in baseline serum creatinine. The AUC for urinary CCL14 to predict persistent severe AKI was 0.81 (95% CI, 0.72-0.89). The risk of persistent severe AKI increased with higher values of urinary CCL14. RRT and/or death at 90 days increased within tertiles of urinary CCL14 concentration. CONCLUSIONS: This secondary analysis externally validates urinary CCL14 to predict persistent severe AKI in critically ill patients.

Exploring the Cost-Utility of a Biomarker Predicting Persistent Severe Acute Kidney Injury: The Case of C-C Motif Chemokine Ligand 14 (CCL14).

Background: Approximately 24% of hospitalized stage 2-3 acute kidney injury (AKI) patients will develop persistent severe AKI (PS-AKI), defined as KDIGO stage 3 AKI lasting ≥3 days or with death in ≤3 days or stage 2 or 3 AKI with dialysis in ≤3 days, leading to worse outcomes and higher costs. There is currently no consensus on an intervention that effectively reverts the course of AKI and prevents PS-AKI in the population with stage 2-3 AKI. This study explores the cost-utility of biomarkers predicting PS-AKI, under the assumption that such intervention exists by comparing C-C motif chemokine ligand 14 (CCL14) to hospital standard of care (SOC) alone. Methods: The analysis combined a 90-day decision tree using CCL14 operating characteristics to predict PS-AKI and clinical outcomes in 66-year-old patients, and a Markov cohort estimating lifetime costs and quality-adjusted life years (QALYs). Cost and QALYs from admission, 30-day readmission, intensive care, dialysis, and death were compared. Clinical and cost inputs were informed by a large retrospective cohort of US hospitals in the PINC AI Healthcare Database. Inputs and assumptions were challenged in deterministic and probabilistic sensitivity analyses. Two-way analyses were used to explore the efficacy and costs of an intervention preventing PS-AKI. Results: Depending on selected costs and early intervention efficacy, CCL14-directed care led to lower costs and more QALYs (dominating) or was cost-effective at the $50,000/QALY threshold. Assuming the intervention would avoid 10% of PS-AKI complications in AKI stage 2-3 patients identified as true positive resulted in 0.066 additional QALYs and $486 reduced costs. Results were robust to substantial parameter variation. Conclusion: The analysis suggests that in the presence of an efficacious intervention preventing PS-AKI, identifying people at risk using CCL14 in addition to SOC is likely to represent a cost-effective use of resources.

CCL14 testing to guide clinical practice in patients with AKI: Results from an international expert panel.

PURPOSE: Urinary C-C motif chemokine ligand 14 (CCL14) is a strong predictor of persistent stage 3 acute kidney injury (AKI). Multiple clinical actions are recommended for AKI but how these are applied in individual patients and how the CCL14 test results may impact their application is unknown. METHODS: We assembled an international panel of 12 experts and conducted a modified Delphi process to evaluate patients at risk for persistent stage 3 AKI (lasting 72 hours or longer). Using a Likert scale, we rated 11 clinical actions based on international guidelines applied to each case before and after CCL14 testing and analyzed the association between the strength and direction of recommendations and CCL14 results. RESULTS: The strength and direction of clinical recommendations were strongly influenced by CCL14 results (P < 0.001 for the interaction). Nine (82%) recommendations for clinical actions were significantly impacted by CCL14 results (P < 0.001 comparing low to highest CCL14 risk category). CONCLUSIONS: Most recommendations for care of patients with stage 2-3 by an international panel of experts were strongly modified by CCL14 test results. This work should set the stage for clinical practice protocols and studies to determine the effects of recommended actions informed by CCL14.

Outcomes, Healthcare Resource Utilization, and Costs of Overall, Community-Acquired, and Hospital-Acquired Acute Kidney Injury in COVID-19 Patients.

Background: In hospitalized patients with COVID-19, acute kidney injury (AKI) is associated with higher mortality, but data are lacking on healthcare resource utilization (HRU) and costs related to AKI, community-acquired AKI (CA-AKI), and hospital-acquired AKI (HA-AKI). Objectives: To quantify the burden of AKI, CA-AKI, and HA-AKI among inpatients with COVID-19. Methods: This retrospective cohort study included inpatients with COVID-19 discharged from US hospitals in the Premier PINC AI™ Healthcare Database April 1-October 31, 2020, categorized as AKI, CA-AKI, HA-AKI, or no AKI by ICD-10-CM diagnosis codes. Outcomes were assessed during index (initial) hospitalization and 30 days postdischarge. Results: Among 208 583 COVID-19 inpatients, 30%, 25%, and 5% had AKI, CA-AKI, and HA-AKI, of whom 10%, 7%, and 23% received dialysis, respectively. Excess mortality, HRU, and costs were greater for HA-AKI than CA-AKI. In adjusted models, for patients with AKI vs no AKI and HA-AKI vs CA-AKI, odds ratios (ORs) (95% CI) were 3.70 (3.61-3.79) and 4.11 (3.92-4.31) for intensive care unit use and 3.52 (3.41-3.63) and 2.64 (2.52-2.78) for in-hospital mortality; mean length of stay (LOS) differences and LOS ratios (95% CI) were 1.8 days and 1.24 (1.23-1.25) and 5.1 days and 1.57 (1.54-1.59); and mean cost differences and cost ratios were $7163 and 1.35 (1.34-1.36) and $19 127 and 1.78 (1.75-1.81) (all P < .001). During the 30 days postdischarge, readmission LOS was ≥6% longer for AKI vs no AKI and HA-AKI vs CA-AKI; outpatient costs were ≥41% higher for HA-AKI vs CA-AKI or no AKI. Only 30-day new dialysis (among patients without index hospitalization dialysis) had similar odds for HA-AKI vs CA-AKI (2.37-2.8 times higher for AKI, HA-AKI, or CA-AKI vs no AKI). Discussion: Among inpatients with COVID-19, HA-AKI had higher excess mortality, HRU, and costs than CA-AKI. Other studies suggest that interventions to prevent HA-AKI could decrease excess morbidity, HRU, and costs among inpatients with COVID-19. Conclusions: In adjusted models among COVID-19 inpatients, AKI, especially HA-AKI, was associated with significantly higher mortality, HRU, and costs during index admission, and higher dialysis and longer readmission LOS during the 30 days postdischarge. These findings support implementation of interventions to prevent HA-AKI in COVID-19 patients.

Serial Urinary C-C Motif Chemokine Ligand 14 and Risk of Persistent Severe Acute Kidney Injury.

To assess the added prognostic value of serial monitoring of urinary C-C motif chemokine ligand 14 (uCCL14) over that of single measurements, which have been shown to be prognostic for development of persistent severe acute kidney injury (AKI) in critically ill patients. DESIGN: Retrospective observational study. SETTING: Data derived from two multinational ICU studies (Ruby and Sapphire). PATIENTS: Critically ill patients with early stage 2-3 AKI. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We analyzed three consecutive uCCL14 measurements at 12-hour intervals after diagnosis of stage 2-3 AKI by Kidney Disease Improving Global Outcomes criteria. Primary outcome was persistent severe AKI, defined as 72 consecutive hours of stage 3 AKI, death, or receipt of dialysis prior to 72 hours. uCCL14 was measured using the NEPHROCLEAR uCCL14 Test on the Astute 140 Meter (Astute Medical, San Diego, CA). Based on predefined, validated cutoffs, we categorized uCCL14 as: low (≤ 1.3 ng/mL), medium (> 1.3 to ≤ 13 ng/mL), or high (> 13 ng/mL). Seventy-five of 417 patients with three consecutive uCCL14 measurements developed persistent severe AKI. Initial uCCL14 category strongly correlated with primary endpoint and, in most cases (66%), uCCL14 category was unchanged over the first 24 hours. Compared with no change and accounting for baseline category, decrease in category was associated with decreased odds of persistent severe AKI (odds ratio [OR], 0.20; 95% CI, 0.08-0.45; p < 0.001) and an increase in category with increased odds (OR, 4.04; 95% CI, 1.75-9.46; p = 0.001). CONCLUSIONS: In one-third of patients with moderate to severe AKI uCCL14 risk category altered over three serial measurements and such changes were associated with altered risk for persistent severe AKI. Serial CCL-14 measurement may detect progression or resolution of underlying kidney pathology and help refine AKI prognosis.

Fluorescence sensor for the quantification of unbound bilirubin concentrations.

BACKGROUND: Hyperbilirubinemia in jaundiced neonates is routinely assessed by use of total serum bilirubin. However, the unbound or free form (B(f)), not total bilirubin, crosses the blood-brain barrier and can be neurotoxic. Although the peroxidase-mediated oxidation of bilirubin can be used to measure plasma concentrations of B(f), this measurement is relatively complex and the assay is not routinely used. We describe a fluorescence sensor for quantifying B(f) in plasma. METHODS: Our method uses a mutated fatty acid binding protein labeled with the fluorescent molecule acrylodan (BL22P1B11), whose fluorescence is quenched upon binding bilirubin. Another configuration (BL22P1B11-Rh) was developed that uses BL22P1B11 together with the fluorophore rhodamine B, which responds by a change in the ratio of its fluorescence. RESULTS: The "B(f) probes" were calibrated with aqueous solutions of bilirubin and yielded similar bilirubin dissociation constants [K(d) = 16 (1.5) nmol/L]. We used the probes to determine B(f) concentrations in equilibrium with human serum albumin (HSA) and in human plasma samples supplemented with bilirubin. We obtained equivalent B(f) values in both systems, and the B(f) probe results were in agreement with the peroxidase assay. B(f) measurements revealed that bilirubin-HSA binding was well described by 2 sites with K(d) values of 15.4 (1) nmol/L and 748 (14) nmol/L. We measured B(f) concentrations in the range expected in jaundiced neonates with a mean CV of approximately 3%. CONCLUSIONS: The BL22P1B11-Rh probe provides accurate plasma sample B(f) concentrations with a single measurement, in 1 min with either a handheld B(f) meter or a laboratory fluorometer.

Performance of a Standardized Clinical Assay for Urinary C-C Motif Chemokine Ligand 14 (CCL14) for Persistent Severe Acute Kidney Injury.

Background: Clinical use of biomarkers requires the development of standardized assays and establishment of cutoffs. Urinary C-C motif chemokine ligand 14 (CCL14) has been validated to predict persistent severe AKI in critically ill patients with established AKI. We now report on the performance of standardized cutoffs using a clinical assay. Methods: A second aim of the multicenter RUBY Study was to establish two cutoffs for the prediction of persistent severe AKI (defined as KDIGO stage 3 AKI for at least 72 consecutive hours). Patients who received renal replacement therapy (RRT) or died before achieving 72 hours in stage 3 AKI were also considered to have reached the end point. Results: A cutoff value for urinary CCL14 of 1.3 ng/ml was determined to achieve high sensitivity (91%; 95% CI, 84% to 96%), and 13 ng/ml achieved high specificity (93%; 95% CI, 89% to 96%). The cutoff of 1.3 ng/ml identifies the majority (91%) of patients who developed persistent severe AKI with a negative predictive value of 92%. The cutoff at 13 ng/ml had a positive predictive value of 72% (with a negative predictive value of 75%). In multivariable adjusted analyses, a CCL14 concentration between 1.3 and 13 ng/ml had an adjusted odds ratio (aOR) of 3.82 (95% CI, 1.73 to 9.12; P=0.001) for the development of persistent severe AKI compared with those with a CCL14 ≤1.3 ng/ml, whereas a CCL14 >13 ng/ml had an aOR of 10.4 (95% CI, 3.89 to 29.9; P<0.001). Conclusions: Using a clinical assay, these standardized cutoffs (1.3 and 13 ng/ml) allow for the identification of patients at high risk for the development of persistent severe AKI. These results have immediate utility in helping to guide AKI patient care and may facilitate future clinical trials.Clinical Trial registry name and registration number: Identification and Validation of Biomarkers of Acute Kidney Injury Recovery, NCT01868724.

Identification and validation of biomarkers of persistent acute kidney injury: the RUBY study.

PURPOSE: The aim of the RUBY study was to evaluate novel candidate biomarkers to enable prediction of persistence of renal dysfunction as well as further understand potential mechanisms of kidney tissue damage and repair in acute kidney injury (AKI). METHODS: The RUBY study was a multi-center international prospective observational study to identify biomarkers of the persistence of stage 3 AKI as defined by the KDIGO criteria. Patients in the intensive care unit (ICU) with moderate or severe AKI (KDIGO stage 2 or 3) were enrolled. Patients were to be enrolled within 36 h of meeting KDIGO stage 2 criteria. The primary study endpoint was the development of persistent severe AKI (KDIGO stage 3) lasting for 72 h or more (NCT01868724). RESULTS: 364 patients were enrolled of whom 331 (91%) were available for the primary analysis. One hundred ten (33%) of the analysis cohort met the primary endpoint of persistent stage 3 AKI. Of the biomarkers tested in this study, urinary C-C motif chemokine ligand 14 (CCL14) was the most predictive of persistent stage 3 AKI with an area under the receiver operating characteristic curve (AUC) (95% CI) of 0.83 (0.78-0.87). This AUC was significantly greater than values for other biomarkers associated with AKI including urinary KIM-1, plasma cystatin C, and urinary NGAL, none of which achieved an AUC > 0.75. CONCLUSION: Elevated urinary CCL14 predicts persistent AKI in a large heterogeneous cohort of critically ill patients with severe AKI. The discovery of CCL14 as a predictor of persistent AKI and thus, renal non-recovery, is novel and could help identify new therapeutic approaches to AKI.

Serial Urinary Tissue Inhibitor of Metalloproteinase-2 and Insulin-Like Growth Factor-Binding Protein 7 and the Prognosis for Acute Kidney Injury over the Course of Critical Illness.

INTRODUCTION: Over the course of critical illness, there is a risk of acute kidney injury (AKI), and when it occurs, it is associated with increased length of stay, morbidity, and mortality. The urinary cell-cycle arrest markers tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor binding protein 7 (IGFBP7) have been utilized to predict the risk of AKI over the next 12 h from the time of sampling. The aim of this analysis was to evaluate the utility of [TIMP-2] × [IGFBP7] measured serially to anticipate the occurrence of AKI over the first 7 days of critical illness. METHODS: This analysis is from a prospective, blinded, observational, international study of patients admitted to intensive care units. We designed the analysis to emulate a clinician-driven serial testing strategy. Urine samples collected every 12 h up to 3 days from 530 patients were considered for analysis. We evaluated [TIMP-2] × [IGFBP7] results for the first 3 measurements (baseline, 12 and 24 h) and continued to evaluate additional results if any of the first 3 were positive >0.3 (ng/mL)2/1,000. Patients were stratified by number of [TIMP-2] × [IGFBP7] results >0.3 (ng/mL)2/1,000 and number of results >2.0 (ng/mL)2/1,000. The primary endpoint was AKI stage 2-3 defined by the Kidney Disease: Improving Global Outcomes (KDIGO) criteria. RESULTS: The median (interquartile range) age was 64 (53-74) years, 61% were men, and 79% were Caucasian. The median APACHE III score was 71 (51-93), and 82% required mechanical ventilation. Baseline serum creatinine was 0.8 mg/dL and 164/530 (31%) developed the primary endpoint by day 7 with a median time from baseline to stage 2/3 AKI of 26 (8-56) h. In patients with negative values for the first 3 tests (≤0.3 (ng/mL)2/1,000), the cumulative incidence of the primary endpoint at 7 days was 13.0%. Conversely, for those with one, two, or three strongly positive values (>2.0 (ng/mL)2/1,000), the cumulative incidence for the primary endpoint at 7 days was 57.7, 75.0, and 94.4%, respectively, p < 0.001 for trend. There were 3.4% with test results between 0.3 and 2.0 (ng/mL)2/1,000 at all measurements; one third of those patients developed the primary endpoint. We observed a graded increase in the primary endpoint in Kaplan-Meier plots for successively positive test results over time. CONCLUSION: Serial urinary [TIMP-2] × [IGFBP7] at baseline, 12 and 24 h, and up through 3 days are prognostic for the occurrence of stage 2/3 AKI over the course of critical illness. Three consecutive negative values (≤0.3 (ng/mL)2/1,000) are associated with very low (13.0%) incidence of stage 2/3 AKI over the course of 7 days. Conversely, emerging or persistent, strongly positive results [>2.0 [ng/mL]2/1,000] predict very high incidence rates (up to 94.4%) of stage 2/3 AKI. There was a low rate of test results between 0.3 and 2.0 (ng/mL)2/1,000, where the primary endpoint was observed in a third of cases.

Transport of 13C-oleate in adipocytes measured using multi imaging mass spectrometry.

The mechanism of long chain free fatty acid (FFA) transport across cell membranes is under active investigation. Here we describe the use of multi imaging mass spectrometry (MIMS) to monitor intracellular concentrations of FFA and provide new insight into FFA transport in cultured adipocytes. Cells were incubated with 13C-oleate:BSA and either dried directly or dried after washing with a medium deprived of 13C-oleate:BSA. Cells were analyzed with MIMS using a scanning primary Cs+ ion beam and 12C-, 13C-, 12C14N-, 13C14N-) (or 12C 15N-) were imaged simultaneously. From these quantitative images the values of the 13C/ 12C ratios were determined in the intracellular lipid droplets, in the cytoplasm and outside the 3T3F442A adipocytes. The results indicate that after incubation with 13C-oleate:BSA the droplet 13C/ 12C ratio was 15 +/- 6%. This value is about 14-fold higher than the 13C/ 12C terrestrial ratio (1.12%). After washing the 13C-oleate:BSA, the droplet 13C/ 12C ratios decreased to 1.6 +/- 0.1%, about 40% greater than the natural abundance. Results for washed cells indicate that relatively little FFA was esterified. The unwashed cell results, together with the value of the lipid water partition coefficient, reveal that intracellular unbound FFA (FFAu) concentrations were on average about 4.5-fold greater than the extracellular FFAu concentrations. These results are consistent with the possibility that FFA may be pumped into adipocytes against their electro-chemical potential. This work demonstrates that MIMS can be used to image and quantitate stable isotope labeled fatty acid in intracellular lipid droplets.

Usefulness of serum unbound free fatty acid levels to predict death early in patients with ST-segment elevation myocardial infarction (from the Thrombolysis In Myocardial Infarction [TIMI] II trial).

Circulating total free fatty acid (FFA) levels are elevated early in myocardial infarction (MI) and have been associated with an increase in mortality. We investigated the association of serum unbound FFA (FFAu) levels with mortality in patients presenting with ST-segment elevation MI in the Thrombolysis In Myocardial Infarction II trial. The Thrombolysis In Myocardial Infarction II trial enrolled patients within 4 hours of chest pain onset. The patients were treated with a recombinant tissue plasminogen activator within 1 hour of enrollment. The FFAu concentration was evaluated in serum samples from 1,834 patients obtained at baseline, before therapy. The FFAu level was an independent risk factor for death as early as at 1 day of hospitalization and continued to be an independent risk factor for the >3.8 years of follow-up. When adjusted for other cardiovascular risk factors, the FFAu levels in the fourth versus the first quartile remained an independent risk factor for death from MI (hazard ratio 5.0, 95% confidence interval 1.9 to 13.0), all cardiac death (hazard ratio 2.4, 95% confidence interval 1.3 to 4.4), and all-cause death (hazard ratio 1.9, 95% confidence interval 1.2 to 3.1). Women were twice as likely to be in the upper 2 FFAu quartiles and had approximately twice the rate of death as men. In conclusion, FFAu elevation is 1 of the earliest molecular biomarkers of mortality in patients with ST-segment elevation MI and was independent of other risk factors known to affect the outcomes after ST-segment elevation MI.

Free fatty acid transport across adipocytes is mediated by an unknown membrane protein pump.

The role of cell membranes in regulating the flux of long chain free fatty acids (FFA) into and out of adipocytes is intensely debated. Four different membrane proteins including, FABPpm, CD36/FAT, caveolin-1, and FATP have been identified as facilitating FFA transport. Moreover, CD36 and caveolin-1 are also reported to mediate transport in conjunction with lipid rafts. The principal evidence for these findings is a correlation of the level of FFA uptake with the expression level of these proteins and with the integrity of lipid rafts. The 3T3-L1 and 3T3-F442A cell lines in their preadipocyte states reveal little or no expression of these proteins and correspondingly low levels of uptake. Here we have microinjected the adipocyte and preadipocyte cell lines with ADIFAB, the fluorescent indicator of FFA. The ADIFAB fluorescence allowed us to monitor the intracellular unbound FFA concentration during FFA influx and efflux. We show that these measurements of transport, in contrast to FFA uptake measurements, correlate neither with expression of these proteins nor with lipid raft integrity in preadipocytes and adipocytes. Transport characteristics, including the generation of an ATP-dependent FFA concentration gradient, are virtually identical in adipocytes and preadipocytes. We suggest that the origin of the discrepancy between uptake and our measurements is that most of the FFA transported into the cells is lost during the uptake but not in the transport protocols. We conclude that long chain fatty acid transport in adipocytes is very likely mediated by an as-yet-unidentified membrane protein pump.

Different mechanisms of free fatty acid flip-flop and dissociation revealed by temperature and molecular species dependence of transport across lipid vesicles.

The mechanism of free fatty acid (FFA) transport across membranes is a subject of intense investigation. We have demonstrated recently that flip-flop is the rate-limiting step for transport of oleic acid across phospholipid vesicles (Cupp, D., Kampf, J. P., and Kleinfeld, A. M. (2004) Biochemistry 43, 4473-4481). To better understand the nature of the flip-flop barrier, we measured the temperature dependence of a series of saturated and monounsaturated FFA. We determined the rate constants for flip-flop and dissociation for small (SUV), large (LUV), and giant (GUV) unilamellar vesicles composed of egg phosphatidylcholine. For all FFA and vesicle types, dissociation was faster than flip-flop, and for all FFA, flip-flop and dissociation were faster in SUV than in LUV or GUV. Rate constants for both flip-flop and dissociation decreased exponentially with increasing FFA size. However, only the flip-flop rate constants increased significantly with temperature; the barrier to flip-flop was virtually entirely due to an enthalpic activation free energy. The barrier to dissociation was primarily entropic. Analysis in terms of a simple free volume (V(f)) model revealed V(f) values for flip-flop that ranged between approximately 12 and 15 Angstroms(3), with larger values for SUV than for LUV or GUV. V(f) values increased with temperature, and this temperature dependence generated the enthalpic barrier to flip-flop. The barrier for dissociation and its size dependence primarily reflect the aqueous solubility of FFA. These are the first results to distinguish the energetics of flipflop and dissociation. This should lead to a better understanding of the mechanisms governing FFA transport across biological membranes.

Fatty acid-specific fluorescent probes and their use in resolving mixtures of unbound free fatty acids in equilibrium with albumin.

We report the first measurements for profiling mixtures of unbound free fatty acids. Measurements utilized fluorescent probes with distinctly different response profiles for different free fatty acids (FFA). These probes were constructed by labeling site-specific mutants of the rat intestinal fatty acid binding protein (rI-FABP) with acrylodan. The probes were produced and screened by high-throughput methods, and from more than 30 000 such probes we selected six that together have sufficient specificity and sensitivity for resolving the profile of unbound FFA (FFAu) in mixtures of different FFAu. We developed analytical methods to determine the FFAu profile from the fluorescence (ratio) response of the different probes and used these methods to determine FFAu profiles for mixtures of arachidonate, linoleate, oleate, palmitate, and stearate in equilibrium with bovine serum albumin (BSA). Measurements were performed using mixtures with a range of total FFAu concentrations, including 0.9 nM, which is similar to normal plasma levels. We also measured single FFA binding isotherms for BSA and found that binding was described well by six to seven sites with the same binding constants (Kd). The Kd values for the FFA (4-38 nM) were inversely related to the aqueous solubility of the FFA. We constructed a model with these parameters to predict the FFAu profile in equilibrium with BSA and found excellent agreement between the profiles measured using the FFA probes and those calculated with this model. These results should lead to a better understanding of albumin's role in buffering FFAu and to profiling FFAu in intra- and extracellular biological fluids.

High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry.

BACKGROUND: Secondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials. RESULTS: The new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as 12C15N- and 13C14N-, it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of 14C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using 13C-oleic acid; to examine nitrogen fixation in bacteria using 15N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using 15N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using 15N-uridine and 81Br of bromodeoxyuridine or 14C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes 12C, 16O, 14N and 31P; and to track a few 15N-labeled donor spleen cells in the lymph nodes of the host mouse. CONCLUSION: MIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments.