|Home | About | Journals | Submit | Contact Us | Français|
Acute kidney injury (AKI) is common in premature infants and is associated with poor outcomes. Novel biomarkers can detect AKI promptly. Because premature infants are born with underdeveloped kidneys, baseline biomarker values may differ. We describe baseline values of urinary neutrophil gelatinase associated lipocalin, (NGAL), Interleukin - 18 (IL-18), Kidney Injury Molecule -1 (KIM -1), Osteopontin (OPN), beta-2 microglobulin (B2mG) and Cystatin-C (Cys-C). Next, we test the hypothesis that these biomarkers are inversely related to gestational age (GA). Candidate markers were compared according to GA categories in 123 infants. Mixed linear regression models were performed to determine the independent association between demographics/interventions and baseline biomarker values. We found that urine NGAL, KIM-1, Cys-C and B2mG decreased with increasing GA. With correction for urine creatinine (cr), these markers and OPN/cr decreased with increasing GA. IL-18 (with or without correction for urine creatinine) did not differ across GA categories. Controlling for other potential clinical and demographic confounders with regression analysis shows that, NGAL/cr, OPN/cr and B2mG/cr are independently associated with GA. We conclude that urine values of candidate AKI biomarkers are higher in the most premature infants. These findings should be considered when designing neonatal AKI validation studies.
Although outcomes in very-low birth weight (VLBW) premature infants have improved over the last few decades, morbidity and mortality continue to be high(1). Acute kidney injury (AKI) is common and may be independently associated with mortality (2–4) in VLBW infants. Our ability to improve outcomes in those with AKI is hampered by the inability to detect AKI early in the disease process. Novel urine biomarkers of AKI have been discovered and promise to reliably detect AKI prior to a rise in serum creatinine (SCr) in different critically ill populations (5–14). Many of the biomarkers available have been tested in newborns undergoing cardiopulmonary bypass (15, 16) but must be evaluated in VLBW infants who are born with underdeveloped glomeruli and tubules.
Our current methods to diagnose AKI using SCr - based definitions are problematic for the following reasons (17–19): a) changes in SCr represent a functional abnormality which occurs as a consequence of injury, not a marker of injury; b) SCr concentrations may not change until 25–50% of the kidney function has already been lost and thus it may take days before a significant rise in SCr is seen; c) SCr varies by muscle mass, hydration status, age and gender; d) SCr reflects maternal levels at birth and normally decreases to represent the infant’s kidney function in the first few weeks of life (depending on the level of prematurity); and lastly e), blood analysis is not without consequences in premature infants as their total blood volume can be quite low (estimated blood volume of 500g infant is 40 ml).
Recent advances in the field of clinical proteomics have greatly accelerated the discovery of novel urinary proteins which promptly rise in response to renal tubular injury. Some of the most promising urine AKI biomarkers include neutrophil gelatinase associated lipocalin, (NGAL)(20), Interleukin – 18 (IL-18) (16), Kidney Injury Molecule -1 (KIM -1) (21, 22), Osteopontin (OPN), beta-2 microglobulin (B2mG) and Cystatin-C (Cys-C) (23). These and other biomarkers are being tested extensively in different critically ill populations, including children (3, 6, 14, 18, 24, 25) and hold the promise to change our approach to AKI as they can detect AKI hours after an insult as opposed to SCr which may take days to rise after an injury. Development of these biomarkers has advanced such that point-of-care biomarker assessment kits are now becoming available for serum and urine NGAL (7, 19) and KIM-1 (26).
Because glomerular and tubular development continues until 34 weeks post gestational age, baseline levels of candidate biomarkers may be different depending on the degree of prematurity. Baseline evaluation of urine NGAL in premature infants has been performed and shows that urine biomarkers are inversely related to both gestational age and birthweight(27). Confirmation of these findings and exploration of the effect that patient demographics and clinical interventions have on other candidate biomarkers are needed. In order to determine the baseline levels of candidate AKI biomarkers in premature infants, we collected urine from premature infants during the first 7 postnatal days to test the hypothesis that urine levels of NGAL, KIM-1, IL-18, OPN, B2mG and Cys-C are dependent on the degree of prematurity.
VLBW infants with birth weight between 500–1500 g admitted to the regional quaternary care neonatal intensive care unit at the University of Alabama at Birmingham (UAB) between February 2008 and July 2009 were enrolled in the study. Parental consent was obtained and the study was approved by the Institutional Review Board at UAB. Infants were excluded if they did not survive to 48 hrs of life, or if they had known congenital abnormality of the kidney.
Infant demographic data obtained consisted of gestational age, birth weight, race, small for gestational age, and sex. Clinical data included administration of aminoglycoside antibiotics and/or indomethacin, ventilator support, and Apgar scores at 1 and 5 minutes. Maternal demographic data collected included: age, diabetes, pregnancy induced hypertension and preeclampsia. Maternal clinical data included administration of corticosteroids, aminoglycosides, vancomycin, and indomethacin.
Definition of AKI was made if an infant met criteria for Stage 1 definition (a rise in SCr by >0.3mg/dl in 48 hours or a rise of >50% from a previous value) of the AKIN (Acute Kidney Injury Network) classification, within the first 7 postnatal days. (28). SCr was analyzed from laboratory results obtained as part of routine care and if available, from remnant samples which would have otherwise been discarded.
Urine was collected during the first 7 postnatal days with cotton balls placed near the perineum. Urine was extracted and centrifuged for 10 minutes to remove any cotton fibers or cellular elements, and then frozen at −70°C until sample evaluation. Urine biomarker analysis was performed by Core A of the NIH P30 O’Brien Core Center for AKI research (www.obrienaki.org) using the Meso Scale Discovery (MSD); Gaithersburg, MD).
NGAL, Cys-C, OPN and B2mG were measured in urine with a prototype four value multiplex (4-plex) assay. Interleukin-18 and Kidney injury molecule-1 (IL-18/KIM-1) were measured with a prototype duplex (2-plex) assay developed for this study.
Capture antibodies to the four antigens (NGAL, Cys-C, OPN & B2mG) were arrayed by the manufacturer onto separate spots on the carbon electrodes of 4-spot 96-well MSD MULTI-SPOT® plates. Capture antibodies against IL-18 and KIM-1 were arrayed by the manufacturer onto separate spots on the carbon electrodes of 4-spot 96-well MSD MULTI-SPOT® plates, the other two spots were blocked with bovine serum albumin. MSD supplied the calibrators for the 4-plex assay. Calibrators and Biotinylated detection antibodies to IL-18 and KIM-1 were purchased from R&D Systems (Minneapolis, MN). Urine samples for 4-plex assay were prediluted 1 to 200 with sample dilution buffer (MSD Diluent 2). No dilution of samples was required for the 2-plex assay. Wells were blocked with MSD diluent 2 for 30 min and 25 μL samples and calibrators were added to predestined wells. Plates were incubated for 2 hours at room temperature on a shaker set to 600 rpm. Plates were then washed 5 times with Phosphate buffered Saline containing 0.05% Tween-20 (PBS-T) using SkanWasher-300 (Molecular Devices, Sunnyvale, CA).
For the 4-plex assay (NGAL/Cys-C/OPN/B2mG) detection antibodies coupled to a Ruthenium(II)-tris-bipyridine ([Ru(bpy)32+], SULFO-TAG™) were blended together and added to wells. For the 2-plex (IL-18/KIM-1) assay biotinylated detection antibodies were blended with Streptavidin coupled SULFO-TAG and added to wells. Plates were incubated at room temperature for 2 hours on a shaker (600 rpm). Plates were washed 5 times with PBS-T on SkanWasher-300 followed by addition of read buffer (buffered Tripropylamine).
Detection is based on electrochemical oxidation of Ruthenium(II)-tris-bipyridine ([Ru(bpy)32+] in the presence of Tripropylamine (TPA), an electrochemiluminescence co-reactant, leading efficient generation of electrochemiluminescence glow via the high energy electron transfer reaction between Ru(bpy)33+ and TPA radical ). The electrochemical signal emitted as light was detected in a Sector Imager 2400 with a charge-coupled device and the signal analyzed using MSD Workbench and Data Analysis Toolbox v3.0 software. Sample concentrations were back fitted from standard curves generated with a 4-parameter logistic curve fit model with 1/y2 weighting. The intra and inter assay precisions were <3% and <5% respectively for both 4-plex and 2-plex assays. Standard back-calculated recoveries were 90–110%. Calibrators and sample duplicate correlation variability ranged from 0.12% – 7.9%.
Descriptive statistics were performed to determine differences between infant and maternal characteristics among four gestational age groups of VLBW infants (Table 1). Shapiro–Wilk test and normal probability plot were used to test for normality of data. Because biomarker values were not normally distributed, Kruskal-Wallis test was used to describe variation in biomarker values based on gestational age categories. Categorical variables were analyzed using Chi-square or Fisher’s exact test as appropriate. For all descriptive statistics, a p-value < 0.05 was considered statistically significant.
For the regression analysis, biomarkers were converted to natural log in order to gain normal distribution. For each urine sample obtained, the log of the urine biomarker/creatinine (cr) was calculated. A mixed model analysis, with one random intercept included per child, was performed to explore the impact of the measured variables on the biomarker/creatinine values. Gestational age and AKI were forced into all models. All other maternal and infant demographic variables were explored and removed in a stepwise backward selection model keeping variables with p value <0.2. The formula (exp(beta)-1)*100% from the regression coefficients was used to express the % change. SAS 9.2 (SAS Institute Inc., Cary, NC, USA) was used for the all statistical analysis.
The demographic variability for 123 premature infants categorized by gestational age (Table 1), shows differences in infant characteristics as expected (birthweight, exposure to indomethacin and aminoglycosides, respiratory support, Apgar scores at 1 and 5 minutes, acute kidney injury, and small for gestational age (all p<0.01). Maternal pre-eclampsia (p<0.02) and hypertension (p<0.001) were different among groups (p<0.01).
As gestational age increased, urine NGAL, KIM-1, Cys-C, and B2mG values progressively decreased, but no significant differences were seen for other biomarkers (Table 2). When corrected for urine cr, all biomarker/cr ratios progressively declined with gestational age except for IL-18/cr (Table 3).
After controlling for other potential confounders, gestational age independently impacted NGAL/cr, OPN/cr, B2MG/cr, but not KIM-1/cr, IL-18/cr, nor Cys-C/cr. Birthweight was independently associated with Cys-C/cr. The use of blood pressure support medication and indomethacin were independently associated with Kim-1/cr levels. Female gender was independently associated with higher NGAL/cr, IL-18/cr and lower Kim-1/cr. Vancomycin was independently associated with OPN/cr values. Race was independently associated with OPN/cr and B2mG/cr values. (Table 4).
Baseline values of urine NGAL, KIM-1, Cys-C and B2mG decrease with increasing gestational age. With correction to urine cr, these markers and OPN decreased with increasing gestational age. IL-18 (with or without correction for urine creatinine) did not differ acrossgestational age categories. Controlling for other potential clinical and demographic confounders with regression analysis shows that NGAL/cr, OPN/cr and B2mG/cr are independently associated with gestational age.
Even though this study was not designed to determine if these biomarkers predict AKI, we forced AKI into the models to determine if variables such as gestational age were simply a common variable to explain high biomarker values and AKI. We found that gestational age continued to be associated with higher biomarker values, even when controlling for AKI, which suggests that these biomarkers are affected by gestational age and not simply that infants with higher gestational age have more AKI.
Our conclusions and values for the differences in urine NGAL values based on birthweight (data not shown) were similar to those documented by Lavery et al(27). Besides confirming previous results on baseline values of urine NGAL in VLBW infants, our study provides baseline values and evidence that these biomarkers are affected by gestational age in other urine biomarkers (KIM-1, Cys-C, B2-MG, and OPN), but not IL-18. This normative data will help investigators design and analyze AKI biomarker studies.
In 2008, Huynh et al(29) described urine NGAL levels of infants (birthweight >750 grams) and showed differences between males and females, but did not stratify them according to gestational age or birthweight categories. Interestingly, the baseline urine NGAL values in our cohort were similar to values found by Huynh (29) for those whose birthweight is >750 grams, but the overall values in our cohort were higher likely because our population included many infants with birthweight < 750 grams. Also, our sample showed higher variability among NGAL levels likely due to differences in our sample population (Huynh excluded those with clinical risk factors for AKI, whereas we included a more heterogeneous premature infant population). Our data show an independent association between urine biomarker values and female gender which confirms the findings by Huynh et al which showed consistently higher urine NGAL values in premature females.
Compared to other critically ill pediatric patients who were on ventilator support and required ionotropic medications (14, 30), baseline NGAL values are significantly higher in our population while urine IL-18 levels were similar, likely because their cohort was made up of children with multi-organ failure. Similarly baseline values of urine NGAL (but not IL-18, B2MG or OPN) were higher in a cohort of children evaluated in a pediatric emergency department (31).
The strength of this study includes the heterogeneous group of VLBW infants and the evaluation of 6 candidate biomarkers. However, we acknowledge several limitations. Even though we report the number of infants with known AKI in each category, we acknowledge that some infants may have sustained AKI but were “missed” if SCr levels around the time of insult were not performed; thus, from our study, the impact of AKI on these biomarkers cannot be ascertained. Although we controlled for AKI in our regression model, it is possible that the reason higher levels were seen in those with lower gestational age is due to higher incidence of AKI. Second, we acknowledge that there could be other variables which we did not account for that could explain variations in biomarker levels. Therefore, inferences from this analysis should be taken in context of the above limitations. Future studies to determine the ability of these and other biomarkers to detect AKI and mortality are greatly needed.
In conclusion, the normative urine values of NGAL, KIM-1, OPN, Cys-C and B2mG (but not IL-18) are higher in the most premature. One must acknowledge these differences when designing validation studies to find candidate biomarkers of AKI. Whether clinicians and researchers will need to adjust for gestational age, birth weight and gender differences when using these biomarkers is yet to be determined.
We thank Amy Logue, RN, BSN, Faisal Schwab, Shamima Akhter, Amandi Liwo, and Sonja Jordan for their assistance in data collection and management of the study. We thank all of the nurses in the neonatal intensive care unit for their diligence in collecting samples.
This study was made possible by a pilot and feasibility grant from the NIH O’Brien Center for Acute Kidney Injury, the National Kidney Foundation Young Investigator’s Award, and the Kaul Pediatric Research Institute grant.