Early urinary angiotensinogen excretion in critically ill neonates
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Early urinary angiotensinogen excretion in critically ill neonates
Introduction: Urinary angiotensinogen is considered a reliable biomarker for intrarenal renin-angiotensin system activity. The aims of this study were to assess the urinary angiotensinogen level during the first day of life and to evaluate its correlation with renal function in critically ill neonates. Methods: Urinary angiotensinogen concentration during the first 24 hours of life was measured in 98 critically ill neonates. Neonatal renal function was assessed by urinary levels of cystatin-C, albumin and α1-microglobulin and urinary electrolyte excretion. Results: Urinary angiotensinogen level decreased with increasing gestational age and body weight in critically ill neonates (P<0.001). After adjustment for gestational age, urinary angiotensinogen level correlated with urinary fractional excretion of sodium and urinary levels of cystatin-C and α1-microglobulin. Multivariate linear regression identified a significant impact of urinary cystatin-C on urinary angiotensinogen level (P<0.001). Furthermore, urinary angiotensinogen was significantly increased in neonates with a urinary cystatin-C-to-creatinine ratio ⩾2500 ng/mg, which was the optimal cut-off value to predict acute kidney injury in our previous study. Conclusions: The urinary angiotensinogen level correlates with the overall maturity of renal function during the early postnatal period in critically ill neonates and an increased urinary angiotensinogen level might reflect renal injury in immature neonates.
eol>Acute kidney injury; critically ill neonates; renal function; renin-angiotensin system; urinary angiotensinogen; urinary cystatin-C; urinary fractional excretion of electrolytes
The renin–angiotensin system (RAS) is known to play an
important role in the regulation of blood pressure and fluid
and electrolyte homeostasis. There is an increased
emphasis on the local RAS, which plays a significant role in
specific tissues.1 The intrarenal RAS is an independent system,
and inappropriate activation of this system is an important
contributor to renal injury.2
Angiotensinogen, as a hormone precursor, is the
principal substrate for renin, which is the rate-limiting enzyme
of the RAS. It is well documented that urinary
angiotensinogen (uAGT) can serve as a reliable marker to reflect
intrarenal RAS activity accurately,3–7 although one study
showed that uAGT was not an effective marker of renal
RAS activity.8 Previous studies indicate that uAGT is
locally produced and secreted into the tubular lumens
directly by proximal tubular cells,2,6,9,10 and no correlation
exists between the plasma angiotensinogen concentration
and uAGT excretion.11
1Department of Neonatology, Children’s Hospital affiliated to Soochow
2Department of Nephrology, Children’s Hospital affiliated to Soochow
3Institute of Pediatric Research, Children’s Hospital affiliated to
Soochow University, China
The first two authors share first authorship.
In adult patients with chronic kidney disease (CKD),
uAGT was inversely correlated to the estimated glomerular
filtration rate (GFR). There was also a significant positive
correlation between the uAGT concentration and the
urinary albumin–creatinine ratio, fractional excretion of
sodium and serum creatinine level.12 Thus, uAGT is
considered to be a potential biomarker of the severity of
CKD.12–14 In addition, the uAGT level was elevated in adult
patients with cardiac injury who developed more severe
acute kidney injury (AKI) after sample collection.13
During the neonatal or infantile period, the RAS is
more activated compared to later in childhood,15 and this
activity is functionally independent of the maternal RAS
system and important for normal kidney development.16
However, the effect of the intrarenal RAS on immature
renal function during the neonatal period remains unclear,
and little is known about the uAGT level during neonatal
life. The aims of this study were to assess the uAGT level
during the first 24 hours of life and to evaluate its
correlation with renal glomerular and tubular function in critically
The early postnatal period is characterised by the
maturation of renal function. Neonates, especially critically ill
premature neonates, are affected by multiple factors that
influence kidney function and that may predispose to the
development of AKI.17,18 Creatinine clearance, as an
assessment of GFR, is unreliable in neonates, unless
bladder catheterisation is used to assure an accurately timed
urine collection. Moreover, the serum creatinine level of
neonates reflects the maternal levels during the first few
days of life,17 and serum creatinine and estimated GFR
based on the Schwartz formula therefore cannot be used to
assess renal function at this time.
Cystatin-C (CysC) is normally filtered, completely
reabsorbed and catabolised within the proximal tubule.
The urinary cystatin-C (uCysC) concentration can be used
as a marker of renal function.19,20 The ratio of uCysC to
urinary creatinine is considered to be a reliable screening
tool for detecting decreased GFR in children,20 and we and
other authors have demonstrated that increased uCysC is
an independent predictor of AKI during the first days of
life in neonates.21,22 Therefore, the urinary levels of CysC,
albumin, and α1-microglobulin as well as urinary
electrolyte excretion were measured to evaluate neonatal renal
glomerular and tubular function in this study.
Materials and methods
Neonates admitted to the neonatal intensive care unit
(NICU) from August 2011 to August 2012 were eligible
for this study. The exclusion criteria were as follows:
neonates admitted after 24 hours of life; severe congenital
anomalies; antenatal exposure to angiotensin-converting
enzyme inhibitors (ACEIs) or angiotensin receptor
blockers; receiving treatment with diuretics or nephrotoxic
antibiotics; failure to collect a urine sample during the first 24
hours of life; and refusal to participate by parents. The
study was performed in accordance with the Declaration of
Helsinki, with approval of the Institutional Review Board
of the Children’s Hospital of Soochow University. Written
informed consent was obtained from the parents.
Clinical and laboratory data collection
Maternal data including gestational diabetes mellitus,
hypertension, pre-eclampsia, premature rupture of the
membranes >24 hours, antenatal steroids and mode of
delivery, as well as neonatal data including gestational age,
birth weight, gender and the Apgar scores were collected.
The score for neonatal acute physiology (SNAP) was
calculated based on 28 items collected during the first 24
hours of admission as described previously,22,23 which is a
valid measure of illness severity and an important
predictor of neonatal mortality and clinical outcomes.24,25
Clinical status, medication and therapeutic interventions
of the neonates were recorded daily during the
hospitalisation. The diagnostic criteria for respiratory distress
syndrome (RDS), patent ductus arteriosus, early-onset sepsis,
hypoxic-ischaemic encephalopathy and intraventricular
haemorrhage, as well as the indications for mechanical
ventilation during the first day of life were in accordance
with our previous study.25
Clinical laboratory results from the first 24 hours of life
such as serum electrolytes, urea nitrogen, creatinine,
osmolality and bicarbonate were recorded for each
neonate as part of routine care.
Neonatal renal function tests
The urinary concentrations of albumin, α1-microglobulin
and CysC, as well as the urinary fractional excretion of
sodium (FENa), potassium (EFK) and chloride (FECl),
were measured to evaluate renal glomerular and tubular
Urine sample collection and measurement
A urine sample after admission to the NICU during the
first 24 hours of life was collected using a plastic bag and
stored at −80°C. The concentrations of angiotensinogen
from single-voided urine samples were measured using a
human total angiotensinogen enzyme-linked
immunosorbent assay (ELISA) kit (code no. 27412; Immuno-Biological
Laboratories Co. Ltd., Takasaki-Shi, Japan) that
specifically detects human angiotensinogen. The cross-reactivity
of the kit for angiotensin is ⩽0.1%. The assay was
performed according to the manufacturer’s protocol. Briefly,
angiotensinogen standards or diluted urine samples were
applied onto the precoated plates and incubated for 60
minutes at 37°C. After washing with wash buffer,
streptavidin-horseradish peroxidase conjugated anti-human
angiotensinogen antibody was incubated in the plates at 37°C
for 30 minutes. After washing, tetramethylbenzidine
substrate solution was added for 30 minutes at room
temperature in the dark before adding stop solution. Finally, the
angiotensinogen concentration was measured at 450 nm
wavelength in each plate. Linearity was observed in the
range of 0.31–20 ng/ml, with a correlation coefficient (R2)
>0.99. The detection limit for angiotensinogen was 0.03
ng/ml. The coefficient of variation of inter-assay and
intraassay reproducibility for angiotensinogen concentration
ranges from 4% to 7%, corresponding to that reported by
the kit manufacturer. The measurements were repeated on
eight ‘standard’ samples that were run with each set of
patient samples to confirm the reliability of the results.26
The final angiotensinogen concentration was expressed in
micrograms per gram of urinary creatinine (µg/g uCr).
The urinary concentrations of albumin, α1-microglobulin
and CysC were measured as described previously.22,23
Urinary concentrations of sodium, potassium and chloride
from the same samples were measured in the clinical
laboratory in the First Hospital of Soochow University. FENa,
EFK and FECl were calculated as 100 × Px × Ucr /Pcr ×
Ux, when Ux and Px are the urinary and plasma
concentrations of sodium, potassium or chloride, respectively, and
Ucr and Pcr are the urinary and plasma concentrations of
Plasma sample collection
To determine whether the uAGT level is related to the
plasma angiotensinogen concentration, a blood sample
was collected during the first 24 hours of life. The sample
was collected directly into a tube containing anticoagulant
and the plasma was stored at −80°C. The plasma
angiotensinogen concentration was measured as above and
expressed in microgram per milliliter (µg/ml).
Statistical analyses were performed using SPSS Statistics
13.0. Continuous variables were expressed as medians
(min–max range) and categorical variables as proportions.
Because the continuous variables violated the assumptions
of normality and homogeneity of variance, Spearman’s
analysis was used to examine correlations. The Mann–
Whitney U-test was used to evaluate differences between
the two groups.
To meet the assumptions of regression analysis and the
analysis of covariance (ANCOVA), the data for continuous
variables were log-transformed. A univariate linear
regression was performed to examine the contribution of clinical
and laboratory factors to the level of uAGT. Variables with
P<0.2 in the univariate analysis were entered into a
stepwise multivariate regression model. The assumptions of
regression analysis were verified by residual plots.
ANCOVA or general linear model (GLM) univariate
analysis was used to adjust for confounders. Differences with P
values <0.05 were considered to be statistically significant.
All probability values are two-sided.
The study enrolled 98 neonates. Written informed parental
consent was obtained from 112 neonates admitted to the
NICU on the first day of life during the study period. The
following 14 neonates were excluded: one infant
diagnosed with severe congenital anomalies; two exposed to
antenatal ACEIs and 11 due to failure to obtain a urine
sample during the first 24 hours of life. The patient
characteristics are shown in Table 1. None of the neonates
received diuretics, vancomycin, ibuprofen or vaccines.
The present study evaluated a heterogeneous population of
critically ill neonates with various gestational ages, and
uAGT levels were detectable within a wide range in 96
samples. The median uAGT level during the first 24 hours
of life was 96.90 ng/ml (range 0–241.56) and the median
uAGT/creatinine ratio was 722.1 µg/g (range 0–16178.1).
In addition, uCysC was detectable in 86 samples. For those
samples with undetectable levels, the values of uAGT and
uCysC were arbitrarily recorded as 0.03 ng/ml and 10 ng/
ml, respectively, corresponding to the detection limit of the
assay as indicated by the manufacturer. The results of
uAGT and uCysC with adjustment for urinary creatinine
were analysed in the study.
The correlation analysis of the uAGT level and various
patient characteristics is shown in Table 1. The negative
correlation of the log-transformed level of uAGT with
gestational age and birth weight is shown in Figure 1.
The contribution of potential factors to the uAGT level
Univariate and multivariate linear regression analysis was
used to examine the contribution of clinical and laboratory
factors to the level of uAGT, as shown in Tables 2 and 3.
The following factors were significantly associated with
the uAGT level in univariate analyses: gestational age,
birth weight, SNAP, mechanical ventilation, RDS,
surfactant, FENa and the urinary levels of albumin,
α1microglobulin and CysC. In the univariate analyses, there
were no significant associations between the uAGT level
and gender, Apgar score, EFK, FECl or any of the
aData were collected on the first day of life.
Values are median (min–max range).
rs: Spearman’s rank correlation coefficient; SNAP: the score for neonatal acute physiology.
following serum values, which were measured on the same
day: creatinine, urea nitrogen, sodium, potassium,
chloride, bicarbonate and osmolality (Table 2).
Variables with P<0.2 in the univariate analysis were
entered into a stepwise multivariate linear regression
analysis. The final model confirmed the impact of uCysC
(unstandardised coefficient B=0.572, P<0.001) on the
level of AGT in the urine (R2=0.287) (Table 3).
Correlation between the uAGT level and urinary electrolyte excretion
Spearman’s rank correlation demonstrated that the uAGT
level was significantly positively correlated with FENa
(R2=0.161, P<0.001), FEK (R2=0.089, P=0.004) and
FECl (R2=0.069, P=0.012). There was also a significant
positive correlation between the uAGT level and urinary
sodium/potassium ratio (R2=0.067, P0.010). However,
only FENa remained significantly correlated with the
uAGT level after adjustment for gestational age (P=0.043,
GLM univariate analysis, the data were log-transformed).
Correlation analyses of log-transformed data are shown
in Figure 2.
Correlation between uAGT and the urinary
levels of CysC, albumin and α1-microglobulin
The uAGT level was significantly positively correlated with
the urinary levels of CysC (R2=0.482, P<0.001), albumin
(R2=0.123, P<0.001) and α1-microglobulin (R2=0.218,
P<0.001) in Spearman’s rank analysis. The correlation
between uAGT and uCysC levels remained significant after
adjustment for gestational age, birth weight, SNAP, FENa,
FEK, FECl, urinary sodium/potassium ratio, urinary albumin
and urinary α1-microglobulin (P<0.001). However, the
correlation of the uAGT level with the urinary level of albumin
and α1-microglobulin did not remain significant after
adjustment for the level of uCysC (P>0.05, GLM univariate
analysis, the data were log-transformed). The positive correlation
between the uAGT level and the urinary level of CysC,
albumin or α1-microglobulin performed with log-transformed
data is shown in Figure 3.
Comparison of the uAGT level in neonates with
urinary CysC-to-creatinine ratio ⩾2500 ng/mg
to those with a ratio <2500 ng/mg
The concentration of uAGT was significantly higher in
critically ill neonates with a urinary CysC-to-creatinine
ratio ⩾2500 ng/mg (2985.8 (652.3–9094.3) vs. 634.5
(0.19–16178.1) µg/g uCr; P=0.009) as compared to those
with a ratio <2500 ng/mg, which was shown to be the
optimal cut-off value to predict AKI development in critically
ill neonates in our previous study.22 Moreover, this
difference remained significant after adjustment for gestational
aTo meet the assumptions of univariate linear regression analysis, the data for continuous variables were log-transformed. For samples with
undetectable levels, the values of urinary angiotensinogen and cystatin-C were arbitrarily recorded as 0.03 ng/ml and 10 ng/ml, respectively,
corresponding to the detection limit.
bAssociation remains significant after controlling for gestational age by using general linear model univariate analysis.
cAssociation does not remain significant after controlling for gestational age.
SNAP: the score for neonatal acute physiology.
Total R2=0.287, adjusted R2=0.278.
aVariables with P<0.2 in the univariate analysis (Table 2) were entered into a stepwise multivariate linear regression analysis. To meet the
assumptions of multivariate linear regression analysis, the data for continuous variables were log-transformed. For samples with undetectable levels,
the values of urinary angiotensinogen and cystatin-C were arbitrarily recorded as 0.03 ng/ml and 10 ng/ml, respectively, corresponding to the
age, birth weight and SNAP by ANCOVA (P<0.001). The
comparison of log-transformed uAGT levels between
these two groups is shown in Figure 4.
Correlation between uAGT and the plasma level of angiotensinogen
To determine whether the uAGT level is related to the
plasma angiotensinogen concentration, we analysed the
correlation between plasma and urinary levels of
angiotensinogen. Because of the difficulty in collecting blood
samples during the first 24 hours of life, venous blood
samples were obtained in only 48 patients. Plasma
angiotensinogen levels were detectable in all the samples (86.78
(25.06–239.50) µg/ml, n=48). There was no significant
correlation between the uAGT level (104.31 (0.03–241.27)
ng/ml, n=48) and the level of angiotensinogen in plasma
(R2=0.015, P=0.411, n=48).
In addition, there was no significant difference between
the uAGT level in 48 patients and in all 98 patients (104.31
(0.03–241.27) ng/ml vs. 96.90 (0.03–241.56) ng/ml,
This study provides initial data on the uAGT level during
the first 24 hours of life in critically ill neonates. We
evaluated the impact of multiple covariates on the uAGT level
and determined the correlation of uAGT with renal
function. Although previous studies suggest that uAGT
originates from angiotensinogen in the kidney, rather than from
angiotensinogen in the plasma,2,27 the data are not entirely
consistent. By measuring uAGT in patients with type 2
diabetes, Terami et al.28 suggested that plasma
angiotensinogen is filtered through glomerular capillaries and
that uAGT is mainly derived from the plasma. In the
present study, there was no relationship between plasma and
urinary levels of angiotensinogen. Thus, it is unlikely that
the increased uAGT is accounted for by leakage across the
glomerular filtration barrier in immature neonates. Our
observations support the concept that uAGT originates
from angiotensinogen in the kidney.
Because multiple factors that affect renal function and
limit postnatal renal functional adaptation to endogenous
and exogenous stress are present in critically ill neonates,29
this study examined the impact of multiple covariates,
including gestational age, birth weight, gender, Apgar
score and the severity of illness as assessed by SNAP, on
the level of uAGT.
We found that the uAGT level in the first day of life
decreased with increasing gestational age and birth weight,
suggesting that the uAGT level might decrease with
increasing maturity of the neonates. Our results are in
accordance with previous studies. It is well known that the
RAS is functional during fetal life.16,30 The renin
concentration in umbilical venous blood and the activity of the
angiotensin-l-converting enzyme in fetal blood samples
exceed the corresponding maternal values, which suggests
that the activity of the fetal RAS is independent from the
maternal RAS system.31–33 In addition, RAS activity is
higher during the fetal period compared to postnatal
life.15,32 Accordingly, angiotensin-l-converting enzyme
activity measured in the peripheral blood during the first
24 hours of life was significantly higher in premature
infants compared to full-term infants.34
The urinary fractional excretion of electrolytes is high in
neonates and even higher in premature neonates, indicating
immature renal tubular function.35,36 The correlation
between uAGT and FENa suggests that the increased
uAGT level in neonates might be partially due to renal
immaturity. The uAGT level was positively correlated with
the urinary sodium/potassium ratio in the present study, and
the increased urinary sodium loss accompanied by limited
renal potassium excretion seems to suggest an important
role of the RAS system.37 Previous studies have suggested
that the urinary sodium/potassium ratio is closely
associated with aldosterone activity, reflecting both the
aldosterone blood level and responsiveness of kidney
tubules to aldosterone action. The increased urinary
sodium/potassium ratio also indicates low aldosterone
activity in neonates.26,38 However, the weak correlation
between the uAGT level and the urinary sodium/potassium
ratio did not remain statistically significant after adjusting
for gestational age. Thus, this correlation does not
necessarily indicate a relationship between uAGT and
aldosterone activity, but instead more likely reflects the immaturity
in renal tubular handling of sodium and potassium.
The major finding of this study was the positive
relationship between uAGT and uCysC levels in critically ill
neonates, even after adjustment for gestational age, birth weight,
the severity of illness and the urinary levels of albumin and
α1-microglobulin. Our results are in line with previously
published findings, suggesting that uAGT is correlated with
urinary albumin and α1-microglobulin.12,28 In the present
study, however, the correlation between uAGT and the
urinary levels of albumin and α1-microglobulin did not remain
significant after controlling for uCysC.
The increased concentration of CysC in the urine
reflects renal tubular injury and impairment, independent
of the GFR.39 Urinary CysC is an independent predictor of
AKI in neonates.21,22 Therefore, the independent
correlation between uAGT and uCysC levels in the present study
suggests that higher uAGT levels might result from greater
renal injury in immature neonates. This result is consistent
with a recently published finding in adult patients with
cardiac surgery, showing that elevated urinary
angiotensinogen is associated with worsening of AKI.13 Our data
indicate that the presence of uAGT is not a non-specific
result of proteinuria. In particular, the increased urinary
excretion of angiotensinogen in neonates reflects renal
injury, which may promote intrarenal RAS activation and
the production of tubular angiotensinogen. Our findings
imply that the RAS may be critical for normal renal
maturation, but also may be mechanistically involved in renal
injury during renal maturation. This is supported by
previous studies suggesting the association between
pharmacological inhibition of the RAS and the risk of developing
AKI, although there are inconsistent reports.40,41 It has
been demonstrated that uAGT reflects renal angiotensin II
production, the most important component of the RAS.
Positive correlation between uAGT and intrarenal
angiotensin II has been shown in clinical and experimental
The concentration of uAGT during the first 24 hours of
life was increased in neonates with RDS, even after
adjustment for gestational age. It is possible that the high levels of
uAGT in neonates with RDS are due to kidney immaturity
and potential kidney injury, as RDS occurs almost
exclusively in premature infants, and critically ill neonates with
RDS are predisposed to the development of kidney injury.29
However, although our findings suggest a large amount of
uAGT is released by the kidneys, the possibility that the
increased uAGT level in neonates with RDS may have
been caused by activation of the circulating RAS must be
considered. These results are consistent with a previous
study in which serum angiotensin-l-converting enzyme
activity was significantly elevated in newborn infants with
idiopathic RDS, reflecting activation of the
renin–angiotensin–aldosterone system in response to stress.44
To our knowledge, this is the first clinical study
reporting urinary levels of AGT during early life in critically ill
neonates. Our data suggest the potential of uAGT as a
novel tool for assessing intrarenal RAS activity in
neonates. However, our study has certain limitations. First, the
physiological mechanism underlying the association
between uAGT and renal injury in neonates is uncertain.
Although activation of the RAS is clearly established to be
linked to chronic diseases such as hypertension and
CKD,12,43 its link to acute injury remains
controversial.13,40,41 Further studies are needed to elucidate the
underlying mechanism of the association between the
RAS and renal injury in immature neonates. Second, we
demonstrated significantly higher concentrations of uAGT
in critically ill neonates with a urinary CysC-to-creatinine
ratio ⩾2500 ng/mg, which was the optimal cut-off value
shown to predict AKI in our previous study.22 Howerer, the
current study was unable to address whether uAGT, as a
biomarker, is associated with the risk of AKI in critically
ill neonates. Third, the urine samples used for the
measurement of angiotensinogen were collected only during the
first 24 hours of life. Thus, time-dependent changes in this
parameter during postnatal maturation were not evaluated.
In particular, it remains to be evaluated whether changes in
the uAGT concentration over time could reflect the
clinical responses of critically ill neonates to treatment. Fourth,
among all the 98 patients, blood samples were obtained in
only 48 patients for analysing the correlation between
plasma and urinary levels of angiotensinogen. Nevertheless,
there was no difference between the uAGT level in 48
patients and in all the 98 patients. Fifth, food intake was
not recorded, although the total amount of food consumed
during the first day of life is quite small. Whether dietary
sodium intake, which is considered to be an important
modifier of the intrarenal RAS,45 could impact the
production of uAGT remains to be elucidated.
In conclusion, the current study demonstrated that the
uAGT level was negatively associated with gestational
age, but independently positively associated with the
uCysC level in critically ill neonates. Thus, our data
suggest that the uAGT level correlates with the overall
maturity of renal function during the early postnatal period and
that increased uAGT levels may reflect renal injury in
immature neonates. Further studies are necessary to clarify
whether elevated uAGT represents a specific indicator of
AKI in neonates.
Conflict of interest
The authors declare that there is no conflict of interest.
This work was supported by grants from the National Natural
Science Foundation of China (81370773), the Natural Science
Foundation of Jiangsu Province (BK2012604), and the Natural
Science Foundation for Research Projects in the Colleges and
Universities of Jiangsu Province (12KJB320006).
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