Associations of Prematurity and Low Birth Weight With Blood Pressure and Kidney Function in Middle-aged Participants of the Brazilian Longitudinal Study of Adult Health – ELSA-Brasil

Based on the Developmental Origins of Health and Disease, adverse intrauterine environment – reected by low birth weight (LBW) and prematurity – may induce fetal programming that favors kidney dysfunction in adulthood. We examined the association of LBW and prematurity with blood pressure (BP) and kidney function markers in non-diabetic middle-aged adults without kidney disease from the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil). A cross-sectional analysis of 768 subjects aged 35-54 years was conducted. Comparisons were performed according to birth weight: LBW (<2.5 kg) or normal birth weight (NBW) (2.5-4.0 kg). Associations of LBW and prematurity with BP levels and kidney function markers (glomerular ltration rate - eGFR, albumin-creatinine ratio - ACR and serum cystatin C) were tested by multiple linear regression using adjustments based on Directed Acyclic Graphs. Propensity score matching was applied to control imbalances. BP levels, and of LBW with markers of kidney function, in adulthood support that early life events predict risk for hypertension and kidney dysfunction in adulthood. The study design precluded the inferring of causality, and prospective studies are needed to further investigate the hypothesis raised.


Introduction
Based on the Developmental Origins of Health and Disease (DOHaD) theory, early life events can increase the risk of noncommunicable diseases in adulthood 1 . Nutrition-related and other stressors during pregnancy may result in low birth weight (LBW), considered a proxy of an adverse intrauterine environment 2 and commonly the result of intrauterine growth restriction or prematurity 3 . Given the shortand long-term adverse outcomes in LBW neonates, this condition remains a major public health concern.
The World Health Organization (WHO) estimates that LBW occurs in 15-20% of all births, with rates varying widely according to country income level 4 . Compelling evidence has indicated that LBW may predict cardiometabolic and renal disorders 5,6 .
Perinatal distress has been associated with blood pressure level elevation in adulthood 7 . Underlying mechanisms for the association of early life events with blood pressure elevation have been proposed in animal and human models 8,9 . Changes in RAS gene expression, activity of the angiotensin-converting enzyme and sympathetic nervous system, may contribute to development of hypertension in adult life [10][11][12] . Sustained increased blood pressure levels with gradual glomerular damage may precipitate chronic renal failure.
Nephrogenesis begins at early stages of embryo life with an increase in glomeruli number in the last weeks of gestation 13 . Insults during intrauterine life (undernutrition, toxic exposures and/or placental abnormalities) 14 can negatively impact renal functional reserve. These adverse events can induce fetal programming with resultant reduction in nephron formation, hypertrophy of the remaining nephrons, leading to adaptive hyper ltration and increased risk of kidney dysfunction later in life 15,16 . An early sign of hypertension-dependent glomerular damage is urinary protein loss 17 .
Issues regarding whether LBW adults born at full-term or prematurely are prone to exhibit different longterm consequences, and whether these changes can be detected early in the natural history of hypertension and chronic kidney disease, remain understudied.
The Brazilian Longitudinal Study of Adult Health (ELSA-Brasil) is a large cohort that provides an opportunity to investigate the relationship of exposures with several outcomes in adulthood 18,19 . In particular, this allows the testing of associations of early life events with blood pressure levels and renal function markers in adults without overt kidney diseases. We examined the association of LBW and prematurity with blood pressure levels and kidney function markers in middle-aged non-diabetic participants of ELSA-Brasil without kidney disease.

Study design and population
A cross-sectional analysis was carried out of the baseline cohort from the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil), a multicenter cohort study aimed at investigating biological, behavioral, environmental, occupational, psychological, and social factors related to incidence and progression of diabetes and cardiovascular disease 19 . Methodological details have been reported elsewhere 18,19 . Brie y, from August 2008 to December 2010, ELSA-Brasil included employees aged 35-74 years working in universities and research institutions located in the Northeast, Southeast and South regions of Brazil. The current study involved a random sample of 998 non-diabetic individuals aged 35-54 years, drawn from 5061 participants at the São Paulo center of the ELSA-Brasil for whom information on biomarkers of in ammation and serum Cystatin C (sCys C) was available 20 . The study was approved by the local Ethics Committee and informed consent was obtained from all participants.

Eligibility criteria
Of the initial 998 participants, 17 with newly diagnosed diabetes and 16 with low estimated glomerular ltration rate (eGFR < 60 mL/min/1.73m 2 ) were excluded, as were 8 underweight individuals and 50 participants born with macrosomia. Considering thyroid and liver dysfunctions can affect plasma sCys C levels, participants with TSH < 0.1 or ≥ 10.0 µUI/mL (n = 18) and with ALT/AST levels three-fold the normal range (n = 2) were excluded. Participants with missing data regarding exposure (birth weight) or outcomes (sCys C, eGFR and ACR) were also excluded (n = 119), as shown in Fig. 1. A nal total of 768 participants were thus included in the present study.

Clinical and laboratory data
Interview and anthropometric data were collected by trained personnel using standardized questionnaires 21 . Prematurity (yes or no) and birth weight were self-reported. Participants were asked to recall their weight at birth and the variable was classi ed into "< 2.5 kg", "2.5-4.0 kg", "> 4.0 kg" or "unknown". All participants were asked to provide their speci c birth weight and to recall their body weight at 20 years of age.
Sociodemographic and health factors of interest for this study were: age (years), sex (male or female), self-reported skin color (black, white, brown, yellow and indigenous), parental history of diabetes and hypertension (yes or no) and educational levels of the participant and their mother. For the purpose of the present study, skin colors were grouped into white and non-white categories.
Blood pressure was measured using an Omron HEM 705CPINT device (Omron Co, Kyoto, Japan) after a 5-minute rest in a sitting position. Three measurements were taken at 1-min intervals and mean values calculated. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. After overnight fasting, blood and urine samples were obtained and participants then underwent a 2-hour 75 g oral glucose tolerance test. Aliquots of biological samples were frozen at -80 o C for further analyses 22,23 .
Fasting and 2-hour post-load plasma glucose were measured using the hexokinase method (ADVIA Chemistry; Siemens, Deer eld, Illinois, USA) and glycated hemoglobin by high-pressure liquid chromatography (HPLC) (Bio-Rad Laboratories, Hercules, California, USA), according to the National Glycohemoglobin Standardization Program (NGSP) certi ed method. Glomerular ltration rate was estimated using the equations proposed by the Chronic Kidney Disease Epidemiology Collaboration (eGFR CKD-EPI) 24 . Albuminuria was determined in a 12-h overnight sample by nephelometry and was expressed as albumin-to-creatinine ratio (ACR). Serum cystatin C was measured using a human cystatin C ELISA kit (Elabscience Biotechnology, Houston, Texas, USA). Intra-assay and inter-assay coe cient of variability ranges were 5.05-5.38% and 3.29-6.48%, respectively.

De nitions
Prematurity was de ned by an a rmative answer to the question: "Were you a premature baby, that is, were you born earlier than expected?". Birth weight was classi ed into 3 categories: low birth weight (< 2.5 kg), normal birth weight (2.5-4.0 kg) and macrosomia (> 4.0 kg). Outcomes were blood pressure (BP) levels, eGFR, ACR and sCys C and were analyzed as continuous variables.
Hypertension was diagnosed when systolic or diastolic blood pressure levels were ≥ 140 or 90 mmHg, respectively, or when individual was in use of antihypertensive drugs. Kidney function was considered altered for eGFR < 60 mL/min/1.73m 2 or urinary albumin excretion > 1,000 mg/g creatinine, according to KDIGO 2012 25 . Normal ranges of sCys C were 0.64-0.84 mg/L for men and 0.57-0.74 mg/L for women 26 .

Statistical analysis
Continuous variables with normal distribution were expressed as mean ± standard deviation (SD) and compared using Student´s t-test. Non-normal distributed parametric variables were expressed as median and interquartile range and compared using the Wilcoxon rank test. Categorical variables were expressed as absolute and relative frequencies and compared using the chi-squared test.
Associations of LBW (exposure) with outcomes (blood pressure levels and kidney function parameters) were initially analyzed by simple linear regression. Direct Acyclic Graphs (DAG) were used to build theoretical models to analyze independent associations of exposure with outcomes in multiple linear regression analyses. The DAG is a causal diagram which allows the input of scienti c evidence regarding the relationships among variables in graphics software to reach the ideal set of covariables (minimum su cient adjustment) for the model to prevent bias and overadjustments 27,28 . The gures were created using DAGitty software, version 3.0 (www.dagitty.net), included in the Supplementary material ( Figure S1 -Panels A and B).
Based on DAGs, linear regression models for the association of LBW with blood pressure levels were adjusted for race/skin color, sex, parental history of hypertension, and prematurity. Linear regression models for the associations of LBW with kidney function parameters (eGFR, ACR and sCys C) were adjusted for skin color and prematurity.
Considering the contrasting sample sizes of the groups with normal and LBW, as well as possible selection bias due to the observational nature of the study, propensity score matching was employed to create more comparable groups 29,30 . The nearest neighbor-matching algorithm, within a caliper of 0.1 SD of logit function of propensity score was used. Firstly, to apply the propensity score matching, a multiple logistic regression model was used, adjusted for DAG-based covariates (blood pressure was adjusted for skin color, sex, parental history of hypertension and prematurity; while kidney function markers were adjusted for skin color and prematurity), and the probability of each participant being LBW versus normal birth weight was estimated. Balance between the groups was assessed by comparing each covariate.
When standardized mean difference lay in the − 0.1 to 0.1 range, groups were considered balanced. This matching was able to reduce all covariate imbalance in the sample. Using the matched sample, multiple linear regression analyses were then performed to test associations of LBW with blood pressure and kidney function markers (outcomes) adjusted for the same DAG-based covariates. Tests were performed using "MatchIt", "rbounds", "Matching", "twang" and "survey" packages in the R statistical environment.
Additionally, sensitivity analyses were performed by selecting the participants with self-reported preterm birth (prematurity). Also, when blood pressure levels were outcomes, sensitivity analyses were carried out excluding participants who reported use of antihypertensive agents (Supplementary Table S1). Since similar results were obtained in both analyses, preterm and hypertensive participants were retained for subsequent analysis.
All analyses were performed using R Project for Statistical Computing (R version 3.5.2) and statistical signi cance was set at a p-value of 0.05.

Results
In the sample of 768 participants, mean age was 45.5 ± 4.6 years, 56.8% were female, and 60% reported white skin color. A total of 64 (8.3%) participants reported being LBW and 39 (5.0%) born preterm. The LBW group comprised participants that were predominantly female (54.6%), white skinned (56.0%) and had mothers with low educational level (71.9%). These characteristics showed a similar pattern in the preterm subgroup (67.0% female and 61.0% white skin color).
Only 133 participants ful lled the diagnostic criteria for hypertension. Among the hypertensive participants, 8.1 % and 14 % were receiving antihypertensive treatment in the normal and LBW groups, respectively. Hypertension rates differed between normal and LBW groups (16.2% versus 29.7%, p = 0.024).
Low birth weight participants were, on average, older than normal birth weight subjects (47. Participants with LBW had higher mean blood pressure and ACR, and lower eGFR, than individuals with normal birth weight, but there were no group differences for sCys C ( Table 1).
The preterm subgroup also had signi cantly higher mean systolic and diastolic blood pressure levels, but no difference in kidney function markers was detected (Supplementary Table S2).
On multiple linear regression analysis, LBW was associated with blood pressure levels but this association did not persist after adjustment for prematurity, which remained associated with systolic and diastolic blood pressure ( Table 2). Associations of LBW with eGFR and ACR in the fully adjusted models had borderline signi cance (p = 0.05) ( Table 3). In a separate analysis of only preterm participants, prematurity was independently associated with both systolic and diastolic blood pressure (Supplementary Table S3).    Table S4).
Associations of LBW with blood pressure and kidney function markers after propensity score matching The multiple linear regression model, adjusted for skin color and prematurity, showed that being born with low weight was directly associated with ACR values (ß 1.34; 95%CI 0.47 − 2.20; p = 0.003). Even when reaching adequate balance, LBW was not associated with eGFR or with systolic and diastolic blood pressure levels (Table 4).

Discussion
We hypothesized that being born full-term or preterm, with LBW, is associated with altered blood pressure levels and kidney function markers in adulthood, indicating an insult during intrauterine life. The ndings showing both LBW and preterm participants had higher mean blood pressure levels, and that the LBW group also exhibited lower eGFR and higher ACR, than individuals with normal birth weight corroborated this hypothesis. Enhancing our analysis by propensity score matching and DAG-based adjustments on multiple linear regression boosted con dence in the associations found between early-life events and outcomes in adulthood. We suggest that a subset of individuals with LBW may be at increased risk of renal abnormalities even when routine parameters are within normal values.
Hypertension is a major cause of chronic kidney disease (CKD) and both are prevalent disorders globally 31,32 . Additionally, these conditions increase the risk for cardiovascular events and deaths 33 . Therefore, studies have sought to identify predictors for prevention of these diseases. There is evidence that the pathophysiological process starts in early life, congruent with the DOHaD theory 34,35 . Our main results are in line with the DOHaD, which holds that insults in fetal life induce programming and that LBW is a surrogate of this condition. An interesting nding of the present study was that prematurity was associated with blood pressure levels, yet LBW was not. Previous studies have reported an association of prematurity with elevated blood pressure levels 36,37 . Proposed mechanisms linking prematurity to higher blood pressure levels in adult life involve pre-and postnatal factors. Premature birth has been associated with increased vascular resistance 38,39 , endothelial dysfunction 40 , immature autonomic blood pressure regulation 41 and high sympathetic nervous system activity 12 . The extent of these abnormalities are dependent on the fetal adaptations and cardiovascular system programming in response to an adverse intrauterine environment during a critical period of development 42 . It is known that the full number of nephrons is achieved at between 32 and 36 weeks of gestation and that glomerular ltration starts during intrauterine life 13,43 . Therefore, prematurity may cause morphological and functional renal changes that lead to hemodynamic disturbances and hypertension 44 . Most preterm infants have accelerated postnatal growth during the rst years of life 45 , where this compensatory catch-up growth is commonly associated with rapid body weight gain and obesity in childhood 46 . We speculate that the overweight participants in the present study have an increased risk of hypertension later in life.
Several nutritional, toxic or emotional determinants of LBW -regardless of gestational age at deliverycan impact adult kidney function [47][48][49] , assessed by a number of parameters such as eGFR, serum creatinine, sCys C and albuminuria in clinical practice. Elevated ACR is a recognized biomarker of kidney dysfunction, even when eGFR is within normal ranges 50 . In the present study, mean values of eGFR and ACR were signi cantly worse in the LBW group compared to the other groups.
Furthermore, having employed the propensity score matching, a strong association of LBW was found with albuminuria, but not with eGFR or sCys C. We speculate that ACR might be a useful marker for early detection of kidney dysfunction in adults born with LBW. The higher albuminuria seen in LBW individuals might have been due, in part, to fetal glomeruli morphological alterations and prenatal programming.
This notion is supported by evidence demonstrating that LBW individuals have fewer nephrons 51 , lower glomerular volume at birth 52 , abnormal glomeruli structure with enlarged Bowman's capsule and altered glomerular tufts 53,54 , factors that, in the long-term, could result in increased glomerular permeability and albuminuria. The present sample of LBW individuals without overt nephropathy may have had glomerular hypertrophy of remaining nephrons and hyper ltration. The current mean eGFR of this group was within the normal range, as was albuminuria, but remained signi cantly higher than the group with normal birth weight. Whether ACR, periodically measured, serves as a suitable marker of early kidney dysfunction in LBW individuals requires investigation by studies with the appropriate design. This is relevant considering the synergistic impact of the nephron mass on the development of hypertension favoring CKD in later life.
We elected to measure sCys C based on previous reports suggesting its greater accuracy than creatinine for detecting decreased renal function [55][56][57] . Serum Cystatin C determination, combined with creatinine, can be used in GFR estimation (KDIGO 2012) 25 . However, in the present sample with normal eGFR, sCys C failed to detect kidney dysfunction in LBW individuals.
This study has limitations in relation to recall bias, given that data about early life events were collected retrospectively. However, our sample was middle-aged and previous studies have shown that perinatalrelated events can be accurately reported during adult life [58][59][60] . We also used the exposure variable (LBW or prematurity) as a categorical variable, where this may have reduced the statistical power of the analysis. This variable was chosen so as to minimize information bias from participants unable to accurately remember their birth weight. The frequency of self-reported prematurity in our sample was comparable to the rate reported in the Brazilian population at large 61 . A strength of the present study was its methodological approach. DAG was employed to avoid overadjustments 27,28 when performing the regression analysis. However, although several covariates were incorporated into our theoretical model, including body weight at age 20, other exposures throughout the participants' life course were unavailable. Size difference of the groups with normal birthweight and LBW was also a concern; therefore, propensity score matching was applied to reduce potential selection confounders from observational studies 29,30 and su ciently balanced groups was achieved.

Conclusion
In conclusion, prematurity was found to be associated with higher blood pressure levels and LBW with albuminuria in adulthood. Considering the magnitude of the ELSA-Brasil and potentialities of its cohort 19,23 , our ndings are important to raise awareness of early life events and to be alert to a subset of heathy adults born with LBW at increased risk for hypertension and kidney dysfunction. Long birth cohort studies could help con rm the hypothesis raised in this study. Informed consent was obtained from all participants in the written format during an interview with the assistance of trained personnel.

CONSENT FOR PUBLICATION
Informed consent was obtained from all participants for data publication.

AVAILABILITY OF DATA
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.  Figure 1