Expressions of irisin and urotensin II and their relationships with blood pressure in patients with preeclampsia

ABSTRACT

The aims of this study are to observe irisin and urotensin II (UII) levels in serum and placenta in normal pregnant and preeclamptic women and investigate the relationship between expressions irisin and UII, and their association with blood pressure. A total of 67 pregnant subjects were recruited, including 31 healthy and 36 preeclamptic pregnant women. Serum irisin and UII concentrations were measured. Expressions of fibronectin type III domain-containing protein 5 (FNDC5) (irisin precursor) and UII in placenta specimens were performed. There was no significant difference of serum irisin levels between severe preeclamptic (SPE)) patients, mild preeclamptic (MPE) patients and normal controls, while serum UII was significantly higher in preeclamptic women than normal pregnancy. There was no relationship between serum UII and irisin levels. In patients with preeclampsia, serum irisin was negatively associated with systolic and diastolic blood pressure(r = −0.350, P = 0.004, r = −0.307, P = 0.011), while serum UII was positively associated with systolic blood pressure (r = 0.291, P = 0.031). Serum irisin, UII, urinary protein level, BMI and serum creatinine were the independent determinants of blood pressure in preeclampsia by multiple regression analysis. Protein expression of FNDC5 and UII was upregulated in placenta of patients with SPE and positively correlated with systolic blood pressure and urinary protein level. We firstly verify that serum irisin and placental irisin precursor expressions have differently correlated with blood pressure. Expressions of irisin and urotensin II have relationships with blood pressure in patients with preeclampsia

KEYWORDS:

• Blood pressure
• irisin
• placenta
• preeclampsia
• pregnancy
• urotensin II

Introduction

Irisin is a novel myokine, encoded by the fibronectin type III domain-containing protein 5 (FNDC5) precursor genes, and secreted from skeletal muscle after exercise. Irisin mediates exercise-related energy expenditure by turning white adipose tissue (WAT) into brown adipose tissue (BAT) (1). This conversion of white adipocytes to brown adipocytes and the resultant increase in thermogenesis promotes improved insulin sensitivity, reductions in body weight, and glucose tolerance in mice (1, 2). Previous clinical studies have also proved that irisin was correlated with insulin resistance and BMI (3–5). Yuksel et al. reported that maternal serum irisin levels of patients with gestational diabetes mellitus are significantly lower compared with non-gestational diabetes mellitus controls (6).

UII is a vasoactive, somatostatin-like cyclic peptide of 11 amino acids initially isolated from the urophysis of the goby fish. UII is generally agreed to be the most potent endogenous vasoconstrictor discovered to date (7). It has been demonstrated that UII may participate in the pathophysiology of hypertensive disorders in pregnancy (8). Our previous study also found that circulating UII levels increased in preeclamptic women, and UII protein in placenta was associated with endoplasmic reticulum stress in patients with severe preeclampsia (9). Studies showed that UII may have a role in the pathophysiology of insulin resistance (10–12).

Contemporary scientific data show that there is a link between insulin resistance and preeclampsia (13–15). According to previous studies, UII may aggravate insulin resistant (11, 16), while irisin may improve insulin resistant (1, 17). On the other hand, we found that UII had positive correlated with blood pressure in patients with preeclampsia in our previous study (10), and recently, it was reported that central irisin (irisin injected by brain ventricle) can increase blood pressure and peripheral irisin (irisin injection by intravenous) could decrease blood pressure in rats (18), and we presume that UII, irisin, preeclampsia and hypertension have relationships through link with insulin resistant. The aims of this study are to test irisin levels and UII levels in serum and placenta in normal pregnant and preeclamptic women and investigate the relationships between expressions of irisin and urotensin II and blood pressure in patients with preeclampsia.

Methods

Study Participants

From December 30 2014 to March 30 2015, a total of 67 pregnant subjects were recruited, including 31 healthy pregnancies and 36 preeclamptic patients (6 patients with mild preeclampsia and 30 patients with severe preeclampsia). All subjects are nulliparous. Histological studies and western blot were performed on placental tissues of thirty-six patients with preeclampsia (Six patients with mild preeclampsia and thirty patients with severe preeclampsia) and 31 healthy pregnancies. Consent forms were signed by all the participants. Peking University Third Hospital ethical committee approved this study.

Definition of mild preeclampsia (19): Systolic blood pressure 140 mm Hg or higher or diastolic blood pressure 90mm Hg or higher occurring after 20 weeks of gestation in a woman whose blood pressure was previously normal. Proteinuria, with excretion of 0.3 g or more in a 24-hour period；definition of severe preeclampsia (one or more of following): SBP ≥ 160 mm Hg or DBP ≥ 110 mm Hg on two occasions 6 or more hours apart in a pregnant woman on bed rest, proteinuria with excretion > 2g in 24-hour urine, oliguria, with less than 500ml over 24 hours, pulmonary edema or cyanosis, impairment of liver function, visual or cerebral disturbances, pain in epigastric area or right upper quadrant, thrombocytopenia, intrauterine growth restriction (19).

The inclusive criteria of preeclamptic patients were that patients must have pregnant-induced hypertension and proteinuria, with a living fetus. The exclusive criteria in preeclamptic group were pregnancies with infection, twin pregnancies, cancer, and diabetes mellitus, chronic liver disease, chronic kidney disease and chronic heart disease. The control group was chosen at the same time admission for delivery in our hospital and obtained consent form for their blood sample or placenta. Inclusive criteria for women in the control group were the participants delivery with single living fetus after 37 weeks and were obtained consent form for their blood sample or placenta tissues; exclusive criteria for normal control were patients with hypertension, diabetes mellitus, primary liver disease, chronic kidney disease or other diseases. The way of delivery in normal control group was natural labor, and only five participants have done caesarean birth for scarreduterus; all the preeclpamtic patients have done caesarean birth.

Demographic data including age, body weight, height, etc. were recorded. Body mass indices (BMI) of all patients were determined as weight before gestation divided by squared height. Blood pressures of all subjects were measured with sphygmomanometer at 8:00 am by qualified physicians.

Laboratory Assays

Venous blood samples were collected from 31 healthy pregnancies and 36 patients with preeclampsia in the fasting state before delivery. None of the patients were in labor at the time of sampling. Blood samples were collected and were immediately centrifuged after clotting. Aliquots of serum were stored at −80°C and were not thawed until analyzed. Complete blood counts, routine urinary and biochemical analysis were measured with standard laboratory methods in our clinical laboratory. Twenty-four hours urine was collected, and urine protein quantitation was tested.

Measurement of Serum Irisin and Insulin

Serum irisin concentration was measured in duplicate by using the enzyme-linked immunosorbent assay (ELISA) kits (Phoenix Pharmaceuticals, Burlingame, CA, USA) in accordance with the manufacturer’s instructions. The sensitivity of the assay was 0.1 ng/ml, and the linear range of the standard was 0.1–1000 ng/ml. The intra- and interassay coefficients of variation (CV) were 4.5% and 8%, respectively. Serum insulin concentration was measured by using the enzyme-linked immunosorbent assay (ELISA) kits (DRG, Germany) in accordance with the manufacturer’s instructions. The linear range of the standard was 1.76–100uIU/ml. The intra- and interassay coefficients of variation (CV) were 2.6% and 2.9%, respectively. Homeostasis model assessment of estimated insulin resistance (HOMA-IR) was determined using the formula: HOMA-IR = [fasting glucose (mmol/l) × fasting insulin (uU/ml)]/22.5 (20).

Radioimmunoassay of UII

UII concentrations were measured by radioimmunoassay according to our previous publications and other literatures (21, 22), and samples were redeproteinized with 0.75 ml 2 mol/l hydrochloric acid. After centrifugation for 20 min at 6000 g, the supernatant was loaded onto cartridges that had been activated with 3 ml 100% methanol and 3 ml double-distilled deionized water. The cartridges were then washed twice with 3 ml 0.1% trifluoroacetic acid (TFA) and eluted with 3 ml 60% acetonitrile in 0.1% TFA. The eluants were freeze-dried overnight and resuspended in 250μL of radioimmunoassay buffer. Then, 100 μL of standard UII or assay sample was incubated overnight at 4°C with 100 μl rabbit antiserum. A total of 100 μL of labeled ¹²⁵I-UII (Phoenix Pharmaceuticals, Inc., Belmont, CA, USA) was added to each tube and incubated for a further 24 h. Antibody-bound UII was precipitated using a goat anti-rabbit antiserum and normal horse serum. Using a gamma counter, the amount of bound ¹²⁵I-UII was measured as pg/ml. Urinary UII was concentration calibrated by urine creatinine and expressed as the ratio of urinary UII concentrations to grams of urinary creatinine (ng/g).

Immunohistochemical Analyses

Histological studies were performed on placental tissues of patients with preeclampsia (6 MPE patients and 30 SPE patients) and 31 healthy pregnancies. All participants signed their written informed consents. The placental tissues were embedded in optimal cutting temperature compound. The tissues were sectioned at a thickness of 10μm. For immunohistochemical analysis, 5% hydrogen peroxide was used to deplete endogenous peroxidase activity. Following pre-incubation with 5% bovine serum albumin for 30 min to prevent nonspecific staining, the sections were incubated with rabbit anti-human UII (1:1500) and FNDC5(1:1000) at 4°C overnight (all antibodies were bought from Phoenix Pharmaceuticals, Inc. and all other chemicals were of analytical grade from commercial suppliers). The sections were then incubated with horseradish peroxidase-coupled goat anti-rabbit IgG antibody for 20 min, followed by incubation with strep-avidin-biotin-peroxidase complex (SABC) for 20 min at 37°C. The peroxidase was visualized by incubation with 3, 3’-diaminobenzidine (DAB) in the dark for 50s for UII and FNDC5 antibody. The sections were counterstained with hematoxylin, dehydrated, and observed under a light microscope. Negative controls were established using PBS as a substitute for the primary antibody. Positive controls for UII were established using renal tissues (UII) expressions in kidney are mainly located in renal tubular epithelium. Positive controls for FNDC5 were chosen using skeletal muscle tissues. Positive staining was indicated by brown deposits. For semi-quantitative analysis, 10 high-power microscope fields were randomly selected, and the pathological image analysis system was used to calculate the integral optical density (IOD) of positive staining for irisin precursor FNDC5 and UII in placenta in normal control, MPE patients and SPE patients.

Western Blot Analysis for Placentas

Proteins were extracted from the placentas of both patients with preeclampsia and healthy pregnancies which were frozen in liquid nitrogen immediately after obtained by naturally labor or caesarean birth. Immunoprecipitation and western blot analysis of UII, FNDC5 were performed. Briefly, the protein samples were denatured at 95°C for 5 min separated on a 10% SDS-PAGE gel prior to being transferred to NC membranes (Applygen Technologies Inc. Beijing, China). Membranes were subsequently incubated with primary rabbit polyclonal anti-UII, anti-FNDC5 antibodies (1:1000; ab21685, Abcam) overnight at 4℃, followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies (1:500, Zhongshan Gold Bridge Biotechnology Co., Ltd Beijing, China). Semi-quantitative grayscale intensity was generated with Odssey Software v1.2.

Statistical Analysis

Data are presented as means ± standard deviation (SD), whereas data with non-normal distribution were reported as median and interquartile range. Independent-samples t-test or one way ANOVA test was applied in statistical analysis. LSD was performed for post hoc analysis after we used ANOVA. Mann–Whitney U-test was used to test the difference for non-normal distribution. Non-normal distributed data were normalized to its natural logarithm. Then, multiple linear regressions were carried out. Pearson correlation coefficients were calculated to evaluate the relationship between irisin, UII, blood pressure and other parameters, and then, multiple regressions was carried out. To evaluate the relationship between blood pressure and irisin and other parameters multiple linear regression analyses were performed. All data were analyzed by using the statistical package spss 17.0 (SPSS, Inc., Chicago, IL, USA). A two sided P＜0.05 was considered statistically significant.

Results

Clinical Characteristics of Participants

The clinical characteristics and biochemical data of the control subjects and preeclamptic patients are summarized in Table 1 . Notably, preeclamptic patients had higher levels of body mass index (BMI), systolic blood pressure, diastolic blood pressure, aspartate aminotransferase (ALT), blood urea nitrogen (BUN), blood creatinine, urea, creatinine kinase MB (CKMB), thrombin time (TT) and circulating UII levels than those of normal controls (P < 0.05), whereas gestational age, total serum protein, serum albumin and International standard rate (INR) were significantly lower (P < 0.05) in preeclamptic patients. Moreover, there were significantly higher urinary protein quantitative level and CK-MB, significantly lower GA, TP and Alb levels in SPE patients compared to MPE patients, (P<0.05) There were no significant differences in serum irisin, Ln HOMA-IR and urinary UII between preeclamptic patients and normal pregnancy.

Table 1. Comparison of clinical parameters between preeclamptic patients and normal control.

Variables control	Normal (n = 31)	MPE patients (n = 6)	SPE patients（n = 30）
Age(years)	30.7 ± 3.8	32.0 ± 4.6	31.3 ± 5.7
BMI(kg/m^2)	27.2 ± 3.1	30.6 ± 4.6*	29.8.0 ± 4.8*
GA(week)	38.2 ± 2.6	36.4 ± 2.5	34.1 ± 2.1*#
SBP(mmHg)	116.8 ± 9.5	139.1 ± 19.9*	144.3 ± 17.7*
DBP(mmHg)	72.8 ± 8.7	90.7 ± 12.9*	89.1 ± 13.3*
Urinary protein quantitative（g/24hr）		0.997* (0.317,1.987)	4.449*# (2.053, 19.841)
WBC(×10^9)	8.66 ± 1.83	9.17 ± 1.98	11.29 ± 2.49
Hb(g/L)	114.1 ± 14.7	112.6 ± 18.1	116.7 ± 19.7
PLT(×10^9)	197.8 ± 40.6	210.6 ± 70.8	165.7 ± 78.8*
ALT(U/l)	11.9 ± 6.0	15.2 ± 17.7	18.9 ± 16.7
AST(U/l)	17.9 ± 3.7	23.0 ± 10.0	28.1 ± 17.0*
TP(g/l)	62.9 ± 7.2	57.0 ± 4.4*	51.0 ± 3.4*#
Alb(g/l)	36.5 ± 3.2	31.4 ± 2.8*	28.8 ± 3.1*#
BUN(mmol/l)	2.9 ± 0.7	3.9 ± 1.9*	4.8 ± 1.7*
Cr(μmol/l)	47.0 ± 9.3	59.2 ± 12.7*	64.5 ± 12.9*
UA(mmol/l)	234.0 ± 76.5	354.0 ± 95.8*	427.0 ± 93.2*
Glu(mmol/l)	4.5 ± 0.4	4.0 ± 0.5	4.1 ± 0.9
HbA1c(％)	5.4 ± 0.6	5.0 ± 0.5	4.6 ± 1.5
CK(IU/l)	43 ± 16.6	44 ± 31	98 ± 129
CK-MB（IU/l)	9.6 ± 6.2	9.78 ± 12.1	17.8 ± 12.4*#
TC(mmol/l)	6.23 ± 2.37	6.85 ± 1.31	6.29 ± 1.84
TG(mmol/l)	1.75 ± 0.56	3.9 ± 0.45	4.0 ± 1.5
LDL(mmol/l)	2.60 ± 0.84	4.02 ± 1.31*	3.44 ± 1.44*
HDL(mmol/l)	1.50 ± 0.48	1.63 ± 0.24	1.86 ± 1.03
PT(s)	9.6 ± 0.4	9.4 ± 0.5	9.3 ± 0.7
TT(s)	12.0 ± 2.2	12.2 ± 1.2	14.3 ± 1.4*
INR	0.91 ± 0.44	0.88 ± 0.44	0.87 ± 0.41
Serum insulin(uIU/ml)	16.51 ± 8.03	14.79 ± 7.81	12.13 ± 6.87
Ln(HOMA-IR)	1.09 ± 0.51	0.81 ± 0.50	0.93 ± 0.47
Serum irisin (ng/ml)	160.87 ± 15.92	154.47 ± 21.96	159.18 ± 22.86
Serum UII（pg/ml）	23.44 ± 13.20	31.59 ± 15.02	42.59 ± 22.27*
Urinary UII (ng/g)	10.35(7.23,19.69)	13.89(8.13,21.67)	17.14(9.16,23.21)a

P< 0.05 have been accepted to be significant. Normal distribution data are presented as means ± SD. Abnormal distribution data are presented as median, 25 percentile and 75 percentile. MPE: mild preeclampsia, SPE: Severe preeclampsia, BMI：body mass index, GA：gestational age, SBP： systolic blood pressure, DBP：diastolic blood pressure, WBC：white blood cell, Hb：hemoglobin, PLT：platelet, ALT：alanine aminotransferase, AST：aspartate aminotransferase, Alb：albumin，TP：total protein，BUN：blood urea nitrogen Cr：creatinine，UA: uric acid， Glu：glucose, HbA1C：Hemoglobin A1c, CK：creatine kinase, CK-MB：creatine kinase MB, TC：total cholesterol, TG：triglyceride, HDL：high density lipoprotein; LDL：low density lipoprotein, PT：prothrombin time, TT：thrombin time, INR：international normalized ratio, Ln: the natural logarithm transform, HOMA-IR ：Homeostasis model assessment of estimated insulin resistance. *P < 0.05 compared with normal control; # P<0.05 compared with MPE patients.

Comparison of Expressions of UII and FNDC5 in Placenta between Preeclamptic Patients and Normal Pregnancy by IHC and Western Blot

UII expressions were upregulated in placenta of patients with SPE in comparison with normal control by immunohistochemical methods, and they were mainly located in the cytoplasm of placental trophoblastic cells and syncytiotrophoblast cells (brown deposits) (Figure 1C, D, E). There was significantly higher integral optical density (IOD) of UII expression in placenta in SPE in comparison with normal control by semi-quantitative analysis (Figure 1F).

Figure 1. Expressions of UII in placental tissue by Immunochemistry. Expressions of UII and in SPE group were highly increased and were mainly located in the cytoplasm of placental trophoblastic cells and syncytiotrophoblast cells (brown deposits), A for PBS solution replace antibody as a negative control; B for positive control (human kidney tissue);C for normal pregnancy; D for MPE group and E for SPE group. There was significantly higher integral optical density (IOD) of UII expression in placenta in SPE in comparison with normal control by semi-quantitative analysis，**P<0.01 Vs. Normal control ( F ).

FNDC5 expressions (irisin precursor) were upregulated in placenta of patients with SPE in comparison with normal control by immunohistochemical methods, and they were mainly located in the cytoplasm of placental trophoblastic cells and syncytiotrophoblast cells (brown deposits) (Figure 2C, D, E), There was higher integral optical density (IOD) of FNDC5 expression in placenta in SPE in comparison with normal control by semi-quantitative analysis (Figure 2F).

Figure 2. Expressions of irisin precursor (FNDC5) in placental tissue by Immunochemistry. Expressions of FNDC5 in SPE group were highly increased and were mainly located in the cytoplasm of placental trophoblastic cells and syncytiotrophoblast cells (brown deposits), A for PBS solution replace antibody as a negative control; B for a positive control (skeletal muscle tissues); C for normal pregnancy; D for MPE group and E for SPE group. There was higher integral optical density (IOD) of FNDC5 expression in placenta in SPE in comparison with normal control by semi-quantitative analysis, **P<0.01 Vs. Normal control （F）.

Western blot analysis showed that expressions of UII and FNDC5 were significantly higher in SPE group in comparison with normal control (Figure 3A and B Figure 3 first mention.).

Figure 3. Protein expressions of UII and FNDC5 in placentas with preeclampsia and normal control (NC). There were significantly higher protein expressions of UII and FNDC5 in placentas with SPE by western blot compared with NC (**P<0.01 compared with NC, *P<0.05 compared with NC). NC: Normal control.

Moreover, expression of FNDC5 in placenta was positively correlated with systolic blood pressure (r = 0.889, P = 0.021) and urinary protein level. (r = 0.906, P = 0.016), expression of UII in placenta was also positively with urinary protein levels(r = 0.514, P = 0.023) and systolic blood pressure (r = −0.489, P = 0.015).

Relationships between Circulating Irisin, UII and Other parameters with Blood Pressure in Patients with Preeclampsia

Bivariate Pearson correlation analysis, we did not observe a significant correlation between circulating irisin with circulating UII (r = −0.183, P = 0.285, Figure 4 first mention.). We found that circulating irisin was negatively correlated with systolic blood pressure (r = 0.291, P = 0.031). and diastolic blood pressure (r = −0.307, P = 0.011),and circulating UII was positively also correlated with systolic blood pressure (r = 0.286, P = 0.033) (Table 2).

Table 2. Relationship between Circulating Irisin, UII and Other parameters with systolic blood pressure in patients with Preeclampsia.

Variables	Systolic blood pressure
irisin	r = −0.350, P = 0.004
Serum UII	r = 0.291, P = 0.031
GA	r = −0.569, P = 0.000
BMI	r = 0.431, P = 0.000
TP	r = −0.398, P = 0.001
Alb	r = −0.413, P = 0.000
BUN	r = 0.476, P = 0.000
Cr	r = 0.498, P = 0.000
UA	r = 0.524, P = 0.000
Urinary protein level*	r = 0.637, P = 0.000
Urinary UII	r = 0.135, P = 0.368

*Urinary protein level: Defined urinary protein grade according to urine tipstick

Pro (++) or 24 hour urinary protein excretion≥ 2.0g = 2, Pro (+) or 24 hour urinary protein excretion >0.3g but <2.0g = 1, Pro (-) = 0

BMI：body mass index, GA：gestational age, SBP：systolic blood pressure, DBP：diastolic blood pressure, Alb：albumin，TP：total protein，BUN：blood urea nitrogen Cr：creatinine.

Figure 4. Correlation analysis of circulating irisin and circulating UII in patients with preeclampsia. There was no significantly relationship between circulating irisin and circulating UII in patients with preeclampsia by Pearson correlation analysis (r = −0.185, P = 0.285).

Besides these, we found that systolic blood pressure and diastolic blood pressure had negatively correlated with GA, TP and Alb, while systolic blood pressure and diastolic blood pressure had positively correlated with BMI, BUN, serum creatinine and urinary protein level in patients with preeclampsia (Table 2 and Table 3).

Table 3. Relationship between Circulating Irisin, UII and Other parameters with diastolic blood pressure in Patients with Preeclampsia

Variables	Diastolic blood pressure
irisin	r = −0.307, P = 0.011
Serum UⅡ	r = 0.253, P = 0.058
GA	r = −0.419, P = 0.000
BMI	r = 0.351, P = 0.006
TP	r = −0.339, P = 0.005
Alb	r = −0.353, P = 0.004
BUN	r = 0.417, P = 0.000
Cr	r = 0.369, P = 0.002
Urinary protein level*	r = 0.547, P = 0.000
Urinary UII	r = 0.148, P = 0.367

*Urinary protein level: Defined urinary protein grade according to urine tipstick

Pro (++) or 24 hour urinary protein excretion≥ 2.0g = 2, Pro (+) or 24-hour urinary protein excretion >0.3g but <2.0g = 1, Pro (-) = 0

BMI：body mass index, GA：gestational age, SBP：systolic blood pressure, DBP：diastolic blood pressure, Alb：albumin，TP：total protein，BUN：blood urea nitrogen Cr：creatinine.

Moreover, our results showed that irisin had weak negative correlated with pregnant age in severe preeclamptic patients (r = −0.397, P = 0.045)

We did not observe there were correlationships between circulating irisin and HOMA-IR or circulating UII and HOMA-IR.

Multiple Regression Analysis of Blood Pressure with Other Parameters

A multivariate linear regression model was used to study which kinds of clinical and biochemical factors were independently associated with systolic blood pressure. Our model showed that circulating irisin levels, urotensin II levels, urinary protein level, BMI and serum creatinine are independent determinant factors of blood pressure (Table 4).

Table 4. Multiple regression analysis for systolic blood pressure.

Variables	B	Beta	P value	95% CI for B
Constant	129.02		0.000	85.90～173.14
Urinary protein level*	13.036	0.519	0.000	7.604～17.479
irisin	−0.317	−0.291	0.001	−0.483～−0.135
Cr	0.297	0.208	0.027	0.035～0.569
BMI	0.818	0.178	0.043	0.022～1.625
Serum UII	0.249	0.277	0.041	0.008～0.452
Urinary UII	0.127	0.099	0.481	−0.215～0.469

*Urinary protein level: Defined urinary protein grade according to urine tip stick Pro (++) or 24-hour urinary protein excretion≥ 2.0g = 2, Pro (+) or 24-hour urinary protein excretion >0.3g but <2.0g = 1, Pro (-) = 0

BMI = body mass index, Cr = serum creatinine.

Discussion

Zhang, et al. (18) reported that central administration of irisin increased blood pressure and cardiac contractibility. In contrast, peripheral administration of irisin reduced blood pressure in both control and spontaneously hypertensive rats. Both endothelial cells and smooth muscle cells are affected by irisin. Irisin was able to dilate the vessel rings without endothelium, but dilation effect was greater in vessel rings with intact endothelium. ATP sensitive potassium channel (KATP) was associated with peripheral irisin-lowered blood pressure. Pretreatment vessel rings with glibenclamide, a K ATP channel blocker, dramatically abolished irisin-induced vessel dilation (18).

In our current study, we first demonstrated that circulating irisin had a negative correlation with blood pressure in preeclamptic patients. We provide that the evidence for irisin may regulate blood pressure level in preeclapmtic patents. The results are consistent with previous studies of Zhang’s animal study (18).

Garcés (23) reported that serum irisin levels did not change throughout gestation in preeclamptic women; however, there were lower irisin levels during the third trimester when compared with the normal pregnant group. But in our current study, there was no change in serum irisin throughout gestation in preeclampsia. Yuksel et al. (6) previous verified that maternal serum irisin levels of patients with gestational diabetes mellitus are significantly lower compared with non-gestational diabetes mellitus controls. However, they also found that there was no significant difference between cord blood irisin levels of patients with gestational diabetes mellitus and healthy pregnant women. In our current study, we did not found serum irisin significantly decreased in patients with preeclampsia.

Our results showed that irisin had weak negative correlated with pregnant age in severe preeclamptic patients, but pregnant age had no correlated with blood pressure in preeclamptic patients. There was no significant difference in pregnant age between normal control and preeclamptic patients, so we believe that older pregnancy might not affect blood pressure via through irisin pathway in preeclapmtic patients.

It was reported that UII could inhibit glucose transport in skeletal muscle in diabetic mice and aggravate insulin resistance (16), while irisin could improve insulin resistance (17). But our results showed that there was no relationship between irisin and UII levels, although circulating irisin and UII were both correlated with blood pressure, and all both the independent determinants in patients with preeclampsia.

We found that there were no differences in insulin resistance (HOMA-IR) between patients with preeclampsia and healthy pregnancies. This was consistent with the study of Valério E G, et al. (24) and Kun A (25).

We have shown that there was UII expression in human placenta and higher expression in preeclampsia than that in normal pregnancy in our previous study (9). We have confirmed that again in this study by IHC and western blot analysis, which is consistent with the circulating levels.

It was reported that irisin was secreted and synthesized by skeletal muscle, partly by adipose tissue in previous studies. Barjia-Fernandez S et al. (26) just have found that the body fat mass increases after intervention with high-fat diet accompanying with a decrease in the secretion of FNDC5 from the stomach and a diminution in the FNDC5 circulating levels, and they verified that FNDC5 could be produced by stomach and associated with body composition.

Garcés MF et al. (23) found that FNDC5 expression in decidal, cytotrophoblast and syncytotrophoblst in placenta of normal pregnancy. We first verified that FNDC5 protein expression (irisin precusor) in placenta was upregulated in severe preeclamptic pregnancy in comparison with that of normal pregnancy by IHC and western blot analysis in our current study, and they both positively correlated with systolic blood pressure and urinary protein level in these patients. This means circulating irisin and placental FNDC5 (irisin precursor) may play different roles on preeclampsia patients; on the other hand, placental FNDC5 may not be the mainly source of circulating. This need further study.

There were several limitations of our current study. The sample size of this cross-sectional study was relatively small. In addition, the correlation between circulating irisin and blood pressure in the present clinical study provides correlated evidence, but does not address the cause–effect relationship in pregnant women.

In summary, we firstly verify that serum irisin and UII, urinary protein level, BMI and serum creatinine are independent determinants of blood pressure in preeclampsia by multiple regression analysis. Protein expression of FNDC5 and UII is upregulated in placenta of patients with SPE and positively correlated with systolic blood pressure and urinary protein level. Expressions of irisin and urotensin II have relationships with blood pressure in patients with preeclampsia.

Conflict Of Interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of this paper.

Acknowledgments

This study was supported by National Natural Science Foundation (Grant No. 81170706, Grant No 81341022, and Grant No 81570663) to Ai-Hua Zhang. Major diseases of funding of Beijing Municipal Science & technology commission (No.SCW 2009-8) to Ai-Hua Zhang.