Renalase levels and genetic variants are associated with preeclampsia: a hospital based study in Chinese cohort

Pre-eclampsia (PE) is a major contributor of maternal and fetal morbidity and mortality. There are many host related biomolecules that regulate the pathophysiology PE. Critical analysis of these parameters is of crucial importance as some of these may be used as prognostic/diagnostic marker of this serious ailment and can be targeted for developing therapeutic measures against the disease. The aim of the current study is to examine the role of renalse in the context PE in a Chinese cohort. A hospital based case control study was designed to investigate role of renalase in PE. 384 Chinese women consisting of subjects with normotensive pregnancy (n = 105), women with PE (n = 121) and healthy pregnancy (n = 158) were included in the study. Serum renalse level was measured in recruited subjects by ELISA. Renalase gene polymorphisms (rs10887800, rs2576178 and rs2296545) were genotyped by PCR-RFLP. correlation test. Data were as mean ± standard error (SE). Allele and genotype frequency was determined by direct counting. Fisher’s exact test compared the distribution of genotypes and alleles among different P-value, odds ratio, and 95% condence calculated. Case-controls haplotype analysis and linkage were by SNPAlyze The distribution of various genotypes in healthy controls was tested for Hardy-Weinberg equilibrium. For all statistical analyses, a P value was statistically


Abstract Background
Pre-eclampsia (PE) is a major contributor of maternal and fetal morbidity and mortality. There are many host related biomolecules that regulate the pathophysiology PE. Critical analysis of these parameters is of crucial importance as some of these may be used as prognostic/diagnostic marker of this serious ailment and can be targeted for developing therapeutic measures against the disease. The aim of the current study is to examine the role of renalse in the context PE in a Chinese cohort.

Methods
A hospital based case control study was designed to investigate role of renalase in PE. 384 Chinese women consisting of subjects with normotensive pregnancy (n = 105), women with PE (n = 121) and healthy pregnancy (n = 158) were included in the study. Serum renalse level was measured in recruited subjects by ELISA. Renalase gene polymorphisms (rs10887800, rs2576178 and rs2296545) were genotyped by PCR-RFLP.

Results
A higher level of serum renalse in healthy pregnant women compared to controls, whereas, subjects with PE demonstrated a reduced level of this enzyme. Renalse level was negatively correlated with systolic as well as diastolic blood pressure, whereas, a positive association was observed with glomerular ltration rate. Prevalence of homozygous mutant (GG) and minor allele (G) of rs10887800 and rs2576178 polymorphisms were higher in PE patients compared to normotensive pregnant women and healthy controls. Furthermore, association of G-G-C haplotype with susceptibility to PE was observed.

Conclusions
Low level of renalse may be associated with high risk of PE during pregnancy. Renalase gene polymorphisms (rs10887800 and rs2576178) are correlated with with serum renalase and associated with predisposition to development of PE in Chinese cohort.

Background
Pre-eclampsia (PE) is the most common complication during pregnancy that affects about 2-8% of pregnancies and contributes to the leading cause of pregnancy-related mortality in developed countries [1]. The syndrome poses a signi cant risk to both the mother and the fetus. Although the exact cause and pathogenesis of this ailment are not understood with reasonable clarity, it is assumed that the disease is driven by several factors released from trophoblast, induced by placental pressure and promotes an overwhelming maternal in ammatory response [2]. In addition to morbidity and mortality in pregnant women, PE may cause prenatal death, preterm birth, as well as intrauterine growth restriction [3].
The association for American College of Obstetricians and Gynecologists (ACOG) de nes PE as women with hypertension (> 140/90 mmHg), high proteinuria (> 0.3 g/24 hrs) and liver dysfunctions after 20 weeks of gestation [4]. Further, worldwide about 14% of maternal death accounted due to PE and its related hypertensive disorders [5]. There are ample instances of the occurrence of PE in China. In a retrospective study at three different hospitals of China, among 67,746 pregnant women included, the prevalence of mild or severe preeclampsia was 1.42 or 0.49%, respectively [6].
Renalase is a form of monoamine oxidase that directly degrades several catecholamines such as epinephrine, norepinephrine and dopamine. Renalase is synthesized and secreted within the peripheral nervous system, hypothalamus and the pituitary [7]. The major function of renalase enzyme is to reduce blood pressure levels by inhibiting cardiac contractility and maintain optimal heart rate [8,9]. Sicen renalase is directly involved in maintaince of blood pressure, serum renalase is considered as potent biological marker for primary hypertensive disease [10].
The involvement of this enzyme in regulating reproductive health during pregnancy is well known. An augmented level of renalase has been reported in ovaries during gestation [11]. Alteration in serum renalse level has been found to be strongly correlated with pathophysiologic process in pregnant subjects with PE [12]. Additionally, renalase levels are also found to be associated with systolic and diastolic blood pressure levels, glomerular ltration rate and urinary protein excretion in pregnant women with PE [13].
However, pathophysiological role of renalse in Chinese pregnant women with PE is limited and this gap triggered us to examine the possible correlation of this biomolecule with disease pathogenesis during PE.
Renalase gene is located at the long arm of 10th chromosome (q23.33) having 16 exons and spans around 310 kb. Several single nucleotide polymorphism have been reported in the renalase gene, but some polymorphisms with functional relevance have been investigated widely. A signi cant association of GG genotype and minor allele "G" of rs10887800 polymorphism has been associated with susceptibility to development of PE, in Turkiesh PE patients [14]. Similarly, combined homozygous mutnats of rs10887800 and rs rs2576178 polymophisms increased eight fold chances to develop PE clinical phenotype in pregnant women [15].
Based on importance on role of renalase in PE and limited study in Chinese population, we investigated distribution of renalase gene polymorphisms (rs10887800, rs2576178 and rs2296545) and and serum renalse levels between healthy controls, pregnant women with and without PE.

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The current study was undertaken between a period from July 2017 to September 2019 at Department of obstetrics, Daqing peoples's Hospital after approval of study protocol by Institutional Review Board of Daqing people's hospital, Daqing. In total, 384 women aged 25-35 years were recruited in the study.
Essentially, three study populations were considered, namely, subjects with normotensive pregnancy with gestation weeks > 20 weeks (n = 105), women with newly diagnosed PE (n = 121), and healthy nonpregnant women (n = 158). Before the investigation, written informed consent was taken from each study participant.
The major inclusion criteria for patients with PE involved; third-trimester pregnancy complicated with PE, blood pressure > 140/90 with proteinuria > 300 mg in 24 hours (According to ACOG) [4]. In the group with normotensive pregnancy, women with a record of PE in previous pregnancies, hypertension, cardiovascular and renal diseases were excluded. A healthy control group included age-matched normotensive women with no pregnancy and no previous record of PE or hypertension or cardiovascular/renal diseases. Data for clinical characteristics, such as body mass index (BMI), fetal birth weight, blood pressure (systolic as well as diastolic), serum creatinine, glomerular ltration rate, WBC count, serum uric acid and urea were collected from hospital records.
Collection of serum About 2.5 ml of blood (without anticoagulant) was collected intravenously from all participants (pregnant women, non-pregnant controls, and PE patients) at the time of enrollment. 500 ul blood samples were dissolved with anticoagulant, and 2 ml of blood samples were used for isolation of serum. Serum was separated by centrifuging the blood at 2500 rpm for 5 minutes and was stored at -20 °C until further use.

Enzyme-Linked Immunosorbent Assay
Serum level renalase was determined in the subjects from three study categories by ELISA using human renalase ELISA kit (R&D Systems, Inc, USA) following the manufacturer's instructions. All serum samples were measured in duplicate, and the average absorbance value was recorded for a study subject.

Isolation of genomic DNA
From the 500 ul of blood samples collected with anticoagulant, 200 ul of whole blood was used for isolation of genomic DNA by QIAamp DNA Blood Mini kit (Qiagen, Germany). The instructions provided by the manufacturer were followed for isolation of the genomic DNA. The purity of the isolated DNA was accessed by a Nanodrop spectrophotometer.

Statistical analysis
All statistical analysis was performed by using GraphPad Prism version 7.0 (GraphPad Sofware, Inc, La Jolla, CA, USA). For comparison of various parameters among different groups, unpaired Student's t-test or one-way ANOVA were used as appropriate. Further, correlation analysis between the variables was performed by Spearman's correlation test. Data were expressed as mean ± standard error (SE). Allele and genotype frequency was determined by direct counting. Fisher's exact test compared the distribution of genotypes and alleles among different groups. P-value, odds ratio, and 95% con dence interval were calculated. Case-controls haplotype analysis and linkage disequilibrium were accessed by SNPAlyze software. The distribution of various genotypes in healthy controls was tested for Hardy-Weinberg equilibrium. For all statistical analyses, a P value of less than 0.01 was considered statistically signi cant.

Baseline characteristics of participants
The baseline clinical features of the different study groups are demonstrated in Table-1. The results showed a signi cant difference in parameters such as duration of gestation, body mass index (BMI) at the time of blood draw, frequency of standard delivery, percentage of caesarian section, fetal birth weight, systolic/diastolic blood pressure (mmHg), white blood cell count (x10 9 /L), levels of urea and uric acid (mg/dL) and glomerular ltration rate (GFR) among three study populations. However, serum creatinine levels were reasonably similar in three study populations.

Serum renalase levels in study subjects
Serum renalase levels were measured in three clinical categories, and observations are shown in Figure-1.
The mean ± SE renalase levels in PE patients, normotensive pregnant and normotensive non-pregnant women were found to be 170.7 ± 2.38ug/ml, 279.1 ± 3.65ug/ml and 199.6 ± 1.08ug/ml, respectively. The results indicate a signi cant difference in the level of this enzyme among the groups (One way ANOVA, P < 0.0001). Normotensive pregnant subjects had signi cantly higher renalase levels as compared to nonpregnant controls (P < 0.0001). On the other hand, subjects with PE displayed considerably lower levels of renalase when compared normotensive pregnant subjects (P < 0.0001).
Association of serum renalase level with blood pressure and glomerular ltration rate Previously, the role of renalase on the regulation of blood pressure has been well de ned. Thus in the present study, we tested the possible relationship of this enzyme with systolic as well as diastolic blood pressure. A signi cant inverse association was observed between the serum renalase level and systolic blood pressure (r = -0.5949, P < 0.0001) as well as diastolic blood pressure (r = -0.6243, P < 0.0001).
As the renalase productions and circulation in the plasma are regulated by kidney function, we examined the association of these enzyme levels with glomerular ltration rate. Correlation analysis suggested a direct association of renalase with glomerular ltration rate in the study subjects (r = 0.0567, P < 0.0001).
Association of renalase polymorphisms with susceptibility to PE Prevalence of renalase polymorphisms (rs10887800, rs2576178, and rs2296545) was compared among different clinical categories of subjects enrolled in the present investigation by Fisher exact test. As shown in Table- Table-3. Prevalence of G-G-C haplotype (rs10887800-rs2576178-rs2296545) of the renalase gene was signi cantly high in PE patients when compared to NNP (P = 1 × 10 − 3 ) and NP (P = 0) cases. Also, haplotype A-G-G was more frequent in PE patients compared to NP (P = 0), indicating an important role of renalase haplotype on predisposition to the development of PE pathogenesis.
Association of renalase polymorphisms with serum renalase levels, blood pressure, and glomerular ltration rate We observed signi cant differences in serum renalase, blood pressure, and glomerular ltration rate among different groups of enrolled subjects. Based on these observations, we hypothesized that variations in the renalase gene (rs10887800, rs2576178, and rs2296545) would be associated with those parameters. As shown in gure-3A, homozygous mutants (GG) of renalase rs10887800 polymorphism displayed lower serum renalase compared to heterozygous (AG) and wild type (AA). A similar observation was also noticed for rs2576178, AA genotype had higher serum renalase levels than heterozygous (AG) and homozygous mutant (GG) subjects (Figure-3B). However, the other renalase polymorphism (rs2296545) failed to demonstrate a genotype-phenotype relationship (Figure-3C). Also, no signi cant association of renalase polymorphisms with blood pressure and glomerular ltration rate was notice (data not shown).

Discussion
PE is a clinical phenotype in pregnant women associated with unrestricted activation of the in ammatory response. Prior shreds of evidence have suggested a substantial role of many in ammatory mediators (including cytokines) in promoting the pathogenesis of PE. However, other biomolecules in pregnant women with distinct physiological functions during pregnancy should also be simultaneously looked at to understand the pathophysiology of PE in detail. Among other biologically relevant molecules, catecholamine levels have been found to be enhanced in pre-eclamptic women [18]. Catecholamines are degraded by different monoamine oxidases, and renalase is therefore presumed to play a role in regulating the pathogenesis of PE.
Before examining the role of renalase during PE, we analyzed the baseline characteristics in three categories of study populations. Table-1 clearly showed a signi cant reduction in the duration of gestation, the percentage of vaginal delivery, and fetal birth weight in subjects with PE as compared to healthy pregnant women. On the other hand, pre-eclamptic women demonstrated an increased body mass index (BMI), percentage of caesarian section, systolic and diastolic blood pressure, WBC count, serum urea, and uric acid levels as compared to healthy controls. All these data are in accordance with the previous report [19]. Importantly, the glomerular ltration rate (GFR) was found to be signi cantly lower in subjects with PE as compared to healthy pregnant women. Many earlier reports are supporting our results [12], [20]. It is assumed that the decreased GFR in pre-eclamptic subjects may be due to reduced renal blood ow that results due to high renal vascular resistance. An earlier study demonstrated that the decreased GFR in PE subjects is due to a fall in renal blood ow and a reduction in the ultra ltration coe cient in glomerular capillary [21].
A signi cant elevation of serum renalase was noticed in pregnant women with healthy gestations as compared to those without pregnancy. Additionally, the level of the enzyme was signi cantly diminished in subjects with PE. The overall results of the present investigation were in line with previous observations [12,20]. However, our results are in contrary to a report in Pakistani PE patients [22], that demonstrated comparable renalase levels among subjects with PE, healthy pregnant women, and non-pregnant controls. Discrepancies among studies can be attributed to a lower number of subjects enrolled in the reports from the Pakistan population (non-pregnant controls: n = 30, healthy pregnant women: n = 45, and subjects with PE: n = 20). Compared to the earlier report [22], in the present study, we enrolled su ciently large numbers of samples, further strengthing our observations. Renalase is secreted into the peripheral circulation, and its levels are regulated by renal function and catecholamine concentrations [23]. Prior pieces of evidence support the occurrence of systemic and renal hemodynamic alterations during pregnancy [24]. During pregnancy, systemic renal vasodilation is associated with a 30-40% rise in renal blood ow and GFR [24]. Therefore, a higher level of renalase in pregnant subjects in our study may be due to increased GFR. A notable reduction in renalase level in preeclamptic women was observed in the present study when compared to the healthy pregnancies. Previously, it was shown that the manipulation of gonadotropinreleasing hormone (GnRH) might alter the expression of renalase and GnRH antagonists downregulate the renalase secretion [25]. As the placenta has a central role in PE [26], ischemic changes observed during PE possibly accompany low GnRH level and diminish GnRH would facilitate a low level of renalase in pre-eclamptic women.
Prior evidence from both animal and human suggested a regulatory role of renalase on blood pressure [13]. Further, renalase de ciency was found to be associated with hypertension and increased sympathetic activity [27]. Notably, in patients showing resistance to hypertension, the plasma levels of renalase are inversely associated with systolic blood pressure [28]. Our study indicates an inverse association of renalase level with systolic as well as diastolic blood pressure in study subjects. Collectively, all this evidence strongly demonstrated the regulatory importance of renalase with blood pressure.
We found a direct correlation of serum renalase level with GFR. Prior studies have indicated a direct correlation of GFR with renal mass [20]. Additionally, it is also known that the release of renalase into peripheral blood can be in uenced by renal function performance. Since GFR is known as the best index for examining renal function, we speculate the direct relationship of GFR with renalase level. Overall, the results of our investigation and earlier reports highlighted an important pathophysiological relevance of serum renalase in the development of PE.
Genetic association studies on the association of common renalase variants with susceptibility to PE is minimal. To date, two independent studies in Turkish [14] and the Iranian population [15] have demonstrated the importance of renalase mutants on predisposition to PE development. In the present report, we observed a signi cant association of homozygous mutant (GG) and minor allele (G) of rs 10887800 and rs2576178 polymorphisms with risk for development of PE in the Chinese population. These observations are following earlier reports, including Iranian [15] and Turkish patients [14]. However, Bagci et al. failed to demonstrate the association of rs2576178 polymorphism with PE. Furthermore, G-G haplotype (rs10887800-rs2576178) has been associated with susceptibility to PE risk in Iranian patients. Similarly, in the current report rs10887800-G rs2576178-G rs2296545-C haplotype was linked with predisposition to PE when compared to women with or without pregnancy. Our present study is more advantages over the previous report: i) we have included three SNPs of the renalase gene, and ii) normotensive non-pregnant and normotensive pregnant women were enrolled as controls. Collectively results of the present report and earlier studies further strengthn association of renalase polymorphisms with susceptibility to PE.
As we observed signi cant associations of renalase gene polymorphisms with susceptibility to PE development, further, we analyze the functional relevance of those studied SNPs. Elevated serum renalase was observed in homozygous genotype compared to wild type and heterozygous mutant for rs10887800 and rs2576178 polymorphisms. In contrast, an earlier report highlighted the association of elevated renalase levels in the GG genotype of rs10887800 compared to AA genotype and nonexistence of the relationship between rs2576178 polymorphism and plasma renalase levels in patients undergoing hemodialysis [29]. Also, an earlier report showed an association of high SBP and DBP with GG genotype of rs10887800 polymorphism and for GG genotype of rs2576178 only with SBP. However, in the present study, we did not observe any association of renalase polymorphisms with SBP, DBP, and GFR levels in patients and controls.

Conclusions
Renalase levels diminished in PE patients and negatively associated with SBP and DBP. Lower levels of renalase in PE patients are possibly due to a higher prevalence of GG genotype of rs10887800 and rs2576178 polymorphisms in PE patients. Combinely, the present study revealed a signi cant role of renalase with susceptibility and pathophysiology of PE in the Chinese population. However, further studies in other communities are required to validate our ndings.

Declarations
Ethics approval and consent to participate: The study protocol was approved by the institutional Human Ethical Committee and informed consent was obtained from all participants.

Consent for publication: Not Applicable
Availability of data and materials: Data and materials will be available upon requent to corresponding author.
Competing interests: authors declear no con ict of interest.
Funding: No speci c fund has received for this report.    Correlation between serum renalase with blood pressure and glomerular ltration rate. Serum renalase, systolic, and diastolic blood pressure and glomerular ltration rate were measured in all participants and correlation was analyzed by the Spearman rank correlation coe cient. A negative correlation of renalase was observed with diastolic (A), and systolic blood pressure (B) and a positive relationship were noticed with glomerular ltration rate (C). A P-value <0.01was considered as signi cant.