Endothelin-1 levels and renal function in newborns of various gestational ages

Abstract

BACKGROUND: Renal failure is common in the NICU; Acute Kidney Injury (AKI) occurs in 8–24% of admissions. Although AKI is preventable with early diagnosis, no reliable AKI biomarkers exist. Endothelin-1 (ET-1) has been implicated in renal pathogenesis, and elevated urinary ET-1 (uET-1) levels may correlate with progression of renal dysfunction. The study objectives were to determine whether uET-1 levels correlate with renal function parameters and/or fetal growth restriction, and if uET-1 is a potential neonatal AKI biomarker.

METHODS: Sixty-three neonates were enrolled and divided into gestational age (GA) groups by weeks: 1) (24–30 6/7; n = 24); 2) (31–36 6/7; n = 26); and 3) (37–42; n = 13). Additional preterm subgroups for fetal growth restriction analysis included: 1) Appropriate for GA (AGA; n = 40), and 2) Small for GA (SGA; n = 10). ET-1 levels, measured using enzyme linked immunosorbent assay, were collected at birth (cord blood) and 24 h ( ± 4) of life (blood/urine).

RESULTS: No correlation was found between uET-1 and blood plasma levels at birth (r = 0.15; p > 0.05) or 24 h (r = 0.17; p > 0.05). uET-1 negatively correlated with GA (r = –0.44; p < 0.001) and GFR (r = –0.34; p < 0.01). uET-1 levels did not correlate with creatinine (r = 0.13; p > 0.05), BUN (r = 0.19; p > 0.05), BUN/Cr ratio (r = 0.15; p > 0.05), or urinary output (r = 0.12; p > 0.05). In fetal growth restriction subgroup analyses: uET-1 levels negatively correlated with GFR in the PT–AGA subgroup (r = –0.38; p = 0.017), but not with PT–SGA (r = 0.01; p > 0.05).

CONCLUSION: Plasma and uET-1 levels did not correlate; therefore, renal ET-1 excretion may reflect renal ET-1 production. uET-1 levels correlated negatively with GA and GFR. uET-1 may be a marker of impaired neonatal circulatory regulation and consequent renal injury.

Keywords

Endothelin 1 ET-1 renal function newborn

1 Introduction

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide with a variety of biological effects on different organs. In the neonatal population, although ET-1 has been most widely associated with its involvement in hypoxia-induced pulmonary hypertension and bronchopulmonary dysplasia (BPD) [1 –4], more recent research has also revealed its implication in the pathogenesis of kidney disease. The renal medulla is the major source of ET-1 synthesis and the expression of both ET_A and ET_B receptors [5]. In addition to vasoconstriction activities and maintenance of systemic blood pressure, renal ET-1 promotes diuresis and natriuresis in the collecting duct [6]. Animal studies have demonstrated that the degree of renal damage correlates with ET-1 gene expression and urinary ET-1 (uET-1) excretion [7]. In humans, studies have shown increased uET-1 levels as renal dysfunction progressed in adult patients with obstructive and inflammatory renal injury[8, 9].

Renal failure is frequent in the neonatal period, particularly in preterm infants due to organ immaturity, hypoxic ischemic injuries, and use of nephrotoxic medications. Acute Kidney Injury (AKI) is a common neonatal morbidity with prevalence rates in the NICU ranging between 8 and 24% [10, 11]. AKI is preventable if diagnosed early and treated adequately. Oliguria and elevated serum creatinine are in fact results of glomerular injury rather than markers of the injury itself [11 –13]. The identification of early biomarkers of renal failure in critically ill neonates could improve outcomes and reduce the risk of chronic kidney diseases. uET-1 may be a potential biomarker of renal dysfunction in neonates.

The objective was to evaluate the correlation between plasma and uET-1 levels with parameters of renal function in neonates of various GAs to determine if uET-1 may serve as a biomarker of AKI.

2 Methods

2.1 Study population

This prospective, observational study was designed as a pilot study and conducted between October 2010 and July 2012 in a Level IV NICU. Local Institutional Review Board approval and written parent permission were received prior to the initiation of any study procedures. Inborn infants between 24 0/7 and 42 0/7 weeks GA at birth were eligible for study inclusion. Those with a congenital malformation incompatible with life or other condition at the time of enrollment that, in the opinion of the investigator, would compromise the infant’s safety were excluded from study enrollment. Parents of 136 infants were approached for study participation, and 63 (46.3%) neonates who met all eligibility criteria were enrolled into the study.

Enrolled subjects were divided into three groups based upon GA at the time of birth: 1) Preterm (PT) 1 – 24–30 6/7 weeks (n = 24); 2) PT 2 – 31–36 6/7 weeks (n = 26); and 3) Full Term (FT) – 37–42 weeks (n = 13). Divisions by GA were implemented because neonatal morbidities and mortality differ between infants born extremely and very preterm (PT 1), moderate and late preterm (PT 2), and FT [14]. Also, gestational age-related kidney maturation [15], including pre- and postnatal renal development, may affect urinary ET-1 excretion. All subjects were followed from birth to the first 7 days of life (postnatal age). Data collection included maternal and infant demographic information (Table 1).

To investigate a possible association between ET-1 levels and fetal growth restriction, subjects in the PT 1 and PT 2 groups were divided into two subgroups based upon their birth weight: 1) Appropriate for GA (PT-AGA) (n = 40), and 2) Small for GA (PT-SGA) (n = 10). To qualify for the PT-SGA group, subjects must have had a birth weight and/or length at least two standard deviations (SDs) below the mean for GA [16].

2.2 Specimen collection for ET-1 analyses

A 1.5 ml sample of umbilical cord blood was collected at the time of delivery. At 24 h ( ± 4) of life, a 1 ml blood sample was drawn from an indwelling catheter or heel stick. A 3 ml urine sample was obtained within the first 24 h of life via sterile urine collection bags. Blood and urine samples were immediately placed on ice and/or refrigerated. Within 4 h of collection, all specimens were processed and frozen at –70°C to ensure stability until performance of ET-1 analyses.

2.3 Renal function parameters

Urine output, blood urea nitrogen (BUN), and creatinine (Cr) results performed as part of routine care were obtained for data analysis. GFR was estimated using the modified Bedside Schwartz equation (recommended by the National Institutes of Health and National Institute of Diabetes and Digestive and Kidney Diseases) [17, 18]. To minimize the effect of urinary flow rate, the uET-1 to Cr ratio (uET-1/Cr) was used in analysis of the results[19].

2.4 Estimation of plasma and urine ET-1 levels

Plasma and uET-1 estimations were performed at Midwestern University, Downers Grove, IL, using a commercially available enzyme immunoassay kit (specific to ET-1 with negligible cross reactivity to other ET; Enzo Life Sciences, Farmingdale, NY) [20, 21]. Samples and standards were added to wells coated with a mAb specific for ET-1. The plate was then washed after 1 h of incubation and horseradish peroxidase (HRP) labeled mAb was then added. After 30 minutes of incubation, the plate was washed and a solution of 3,3′,5,5′-tetramethylbenzidine substrate was added which generates a blue color. Hydrochloric acid (1N) was added to stop the substrate reaction and the resulting yellow color was read at 450 nm using DTX 800 Multimode detector. The measured optical density is directly proportional to the concentration of ET-1.

2.5 Statistical analysis

Results are shown as mean ± SEM (mean ± SD for demographic analyses). Pearson’s coefficient of correlation test was performed to examine the relationships between uET-1 levels and renal function parameters. One-way ANOVA test was used to compare means between the independent groups for continuous variables, and the Fisher exact test was applied for categorical variables. Statistical calculations were performed with GraphPad Prism 6.02 (GraphPad, San Diego, CA, USA), and a p value < 0.05 was considered statistically significant.

3 Results

3.1 ET-1 levels and ethnicity, sex and mode of delivery

No significant difference was found in umbilical cord, 24 h plasma and uET-1 levels between female and male neonates (p > 0.05 for all respective analyses). Ethnicity did not impact ET-1 levels in umbilical cord, 24 h plasma, and urine (p > 0.05 for all respective analyses). The mean ET-1 levels in umbilical cord and 24 h plasma as well as uET-1 levels in neonates did not differ with vaginal delivery or cesarean section (p > 0.05 for all respective analyses).

3.2 Plasma ET-1 levels across GA

The mean plasma ET-1 levels in umbilical cord blood did not differ significantly (p > 0.05) among the three GA groups: PT 1 (24–30 6/7 weeks), PT 2 (31–36 6/7 weeks), and FT (37–42 weeks). Also, no significant difference was found (p > 0.05) in the mean plasma ET-1 values in 24 h blood across the GA groups (Table 2).

3.3 Plasma ET-1 levels across preterm AGA/SGA subgroups and FT

No significant difference was found between plasma ET-1 levels of PT-AGA, PT-SGA, and FT neonates in umbilical cord (p > 0.05) or 24 h-blood samples (p > 0.05).

3.4 uET-1 levels and uET-1/Cr ratios across GA

A significant difference was found for both urinary ET-1 levels and ET-1/Cr excretion ratios across the three GA groups. The mean uET-1 levels of both PT groups: PT 1 (2.39 ± 0.30 pg/ml) and PT 2 (1.38 ± 0.41 pg/ml) were significantly higher than in the FT group (0.65 ± 0.20 pg/ml) (p = 0.013) (Figure 1). Similarly, the mean uET-1/Cr ratios of both PT groups: PT 1 (3.36 ± 0.52 pg/ml) and PT 2 (1.75 ± 0.49 pg/ml) were significantly higher than in the FT group (0.93 ± 0.32 pg/ml) (p < 0.01) (Figure 2). uET-1 levels also negatively correlated with GA (r = –0.44; p < 0.001) (Figure 3).

3.5 uET-1 levels and uET-1/Cr ratios across AGA and SGA subgroups

The mean uET-1 levels and uET-1/Cr ratios among the three subgroups: PT–AGA (1.93 ± 0.31 and 2.58 ± 0.41; respectively), PT–SGA (1.53 ± 0.53 and 2.17 ± 0.91; respectively), and FT (0.65 ± 0.20 and 0.93 ± 0.32; respectively) did not differ significantly (p > 0.05 for respective analyses).

3.6 Plasma and uET-1 levels

No significant correlation was found after evaluating the relationship between uET-1 and plasma ET-1 levels in umbilical cord (r = 0.15; p > 0.05) and 24 h-blood samples (r = 0.17; p > 0.05).

3.7 uET-1 and renal function parameters

uET-1 did not correlate with creatinine (r = 0.13; p > 0.05), BUN (r = 0.19; p > 0.05), BUN/Cr ratio (r = 0.15; p > 0.05), or urinary output (r = 0.12; p > 0.05) in subjects across all GAs. However, uET-1 levels negatively correlated with GFR (r = –0.34; p < 0.01) (Figure 4).

No significant correlation was found in either of the PT–AGA or PT–SGA subgroups between uET-1 levels and creatinine (r = 0.26; p > 0.05 and r = –0.21; p > 0.05 respectively), BUN (r = 0.28; p < 0.05and r = 0.001; p > 0.05 respectively), BUN/Cr ratio(r = 0.10; p > 0.05 and r = 0.11; p > 0.05 respectively),or urinary output (r = 0.12; p > 0.05 and r = 0.02;p > 0.05 respectively). uET-1 levels negatively corre-lated with GFR in the PT–AGA subgroup (r = –0.38; p = 0.017), but not with PT–SGA (r = 0.01; p > 0.05).

4 Discussion

From the time of the first published article announcing the discovery of endothelin in 1988 by Yanagisawa et al. [22], a plethora of research has continued to identify its many roles in biological activities and its numerous effects on organ systems, including vasoconstriction, regulation of cellular proliferation, and growth-promoting effects on the developing kidney [23, 24]. The cardiovascular effects of ET-1 are dependent on the activation of ET_A and ET_B receptors located on the vascular endothelial and smooth muscle cells. In physiologic conditions, ET-1 is also involved in the regulation of water and sodium transport mediated through ET_B receptors, and in pathologic states, ET-1 contributes to vasoconstriction and inflammation primarily through activation of ET_A receptors [5]. In neonates, ET-1 is most widely known for its role as a potent pulmonary vasoconstrictor and mediator of vascular remodeling in the pathogenesis of hypoxia-induced pulmonary hypertension and BPD. However, relatively little is known about ET-1’s renal involvement in the neonatal population still today, even though nearly 30 years have passed since its initial discovery.

Research has demonstrated that kidneys are not only an important ET-1 target organ (renal vasoregulation), but a significant source of ET-1 as well. It is believed uET-1 is produced from renal cells because the majority of plasma ET-1 filtered through the kidney is deactivated by endopeptidase [25]. Data from two studies evaluating plasma and uET-1 levels in the neonatal population have demonstrated that uET-1 excretion reflects independent renal production rather than filtrated plasma ET-1 [23, 26]. Our results support this finding because we also found no correlation between plasma and uET-1 levels. Therefore, uET-1 may serve as a useful marker of renal dysfunction in neonates.

Kumar et al. [27] and Kuo et al. [28] found no relationship between plasma ET-1 concentrations and race, sex, mode of delivery, and GA in neonates. Similarly, in our findings, plasma concentrations of ET-1 do not correlate with ethnicity, sex, mode of delivery or GA.

In contrast to plasma, uET-1 levels decrease as GA increases. Results from our study, that present a heterogeneous neonatal population typical of the majority of Level III and IV NICUs (Table 1), clearly demonstrate that the highest uET-1 levels were found in the most preterm group (PT 1 : 24–30 6/7 weeks), and the lowest were those of the FT group. Likewise, we found that uET-1 levels correlated negatively with GA, and uET-1 excretion ratios (ET-1/Cr) were also highest in the PT 1 group and lowest in the FT group. uET-1 levels and excretion ratio may be a reflection of renal maturity. When uET-1 levels are measured in the neonatal population, the GA at the time of testing must be considered.

Taking into consideration ET-1’s vasoconstrictor effects, a subgroup analysis was performed to evaluate whether plasma ET-1 and/or uET-1 levels correlated with fetal growth restriction. A correlation was found between intrauterine growth restriction (IUGR) and maternal plasma ET-1 and leptin in pregnancies complicated with preeclampsia by Nezar et al. [29]. However, data from our study did not demonstrate a similar correlation between urine or blood ET-1 levels among preterm neonates born SGA and AGA. It should also be noted that our study did not include neonates born from pregnancies complicated with preeclampsia. Thus, the difference between our findings and those of the previously-mentioned study suggests that the leading mechanism for IUGR was preeclampsia itself rather than uretroplacental hypoperfusion related to ET-1’s vasoconstrictive properties [30]. Moreover, because GA correlated with uET-1 levels and excretion ratios but SGA did not, our results indicate that developmental maturation plays a more significant role in uET-1invovment than the size of the renal tissueitself.

Although the survival rate of critically ill neonates has improved with the advancement of neonatal medical care, residual morbidity and mortality rates remain high [31]. Neonatal, and especially premature, kidneys are prone to perinatal stress due to immaturity, hypoxic ischemic injuries, and exposure to nephrotoxic medications. This is in addition to their already reduced renal perfusion. Compared with adults whose kidneys receive as much as 20 to 25% of their total cardiac output [32], neonatal kidneys only receive 5 to 18% [33]. Studies have suggested that the high prevalence of AKI in NICU-hospitalized neonates may also pose a significant subsequent risk for the development of chronic kidney disease and hypertension [10].

AKI is preventable if diagnosed early and treated adequately, but a timely diagnosis in neonates is often challenging. Currently, the markers used to identify AKI are elevated serum creatinine levels and oliguria, but these criteria are the sequelae of renal injury rather than early indicators of AKI. In fact, studies have demonstrated that infants with AKI often have non-oliguric renal failure thereby further nullifying oliguria as a useful marker [10]. Moreover, renal function may decrease by as much as 25–50% before elevated serum creatinine levels are observed [12, 34]. The identification of early AKI biomarkers is needed to improve the prevention and treatment of neonatal AKI that will lead to better outcomes for high risk neonates. uET-1 could be indicative of renal hemodynamics or a reflection of impairment in GFR during the early period when urine output and serum creatinine levels remain normal.

Elevated uET-1production has been linked to renal disorders secondary to ischemia-induced kidney injury [26, 35]. In addition, significantly higher uET1 levels have been observed in asphyxiated neonates during their first 3 days of life and uET-1 correlated positively with the severity of asphyxia [23, 26]. Moreover, studies in children with reflux nephropathy, Wilms tumor, urinary tract infections, and hydronephrosis have all demonstrated that uET-1 excretion increases as renal function declines leading the authors to conclude that uET-1 may be a marker for these renal pathologies [19 , 36–38]. Finally, ET_A receptor antagonists have been reported to preserve renal function and decrease the severity of injury in experimental nephrotoxic models as well as in the clinical setting [7 , 40].

We analyzed uET-1 levels against laboratory/clinical parameters of renal function and found no correlation between uET-1 and serum creatinine, BUN, BUN/Cr ratio, or urinary output in both pre- and full term neonates during the first day of life. However, results of our study demonstrated that uET-1 excretion correlated negatively with GFR. According to these results, uET-1 appears to be a useful noninvasive tool to detect impaired glomerular filtration in neonates.

In our study, only three of the enrolled neonates developed Stage 1 AKI (based on the neonatal AKI KDIGO stage classification) [41, 42] between days 5 and 7 of life. As per the study design, plasma and urine samples were obtained at birth and at 24 ( ± 4) hours of life. Therefore, because of the significant time gap between sample collection and the presentation of AKI as well as the very small subgroup of neonates with AKI, statistical analyses on this data were not performed.

Our study is limited by the small sample size of 63 subjects and the variables with which ET-1 was compared in order to assess its feasibility as a prospective biomarker for AKI in neonatal population. Oliguria, serum creatinine, GFR and BUN were chosen for this pilot study because they are the most commonly used indicators of AKI in the neonates. However, in a larger, follow-up study, we plan to compare uET-1 with new generation AKI biomarkers, such as neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 – two markers demonstrated to have greater sensitivity and specificity in neonates with AKI [11 , 15]. Cost effectiveness of these AKI biomarkers will also be evaluated.

With regard to sample size, a larger study with sufficient power is needed to fully evaluate ET-1as a neonatal AKI biomarker. The study design will also be altered in order to evaluate urinary ET-1 levels in neonates with different stages ofAKI.

In conclusion, plasma and urinary ET-1 levels are independent of each other, and renal excretion of ET-1 may be reflective of intrinsic renal ET-1 production. Urinary ET-1 levels correlated negatively with GA and GFR. In the neonatal population, urinary ET-1 may be indicative of impaired renal circulation and consequent renal injury. Further studies are required to establish a more definitive correlation between uET-1 and AKI in the neonatalpopulation.

Disclosure Statements

Financial

This study was funded by a grant from the Advocate Lutheran General Health Partners Endowment.

The authors have no conflicts of interest to disclose.

Human research

The authors affirm that this research involving human subjects was conducted in accordance with the ethical standards of the local institutional review board and the World Medical Association’s Helsinki Declaration.

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