Roles of Human Urotensin II in Volume Resistance Hypertension in Peritoneal Dialysis Patients

Abstract

Human urotensin II (hUII) is a newly discovered substance that can dilate small blood vessels to decrease the blood pressure (BP). Our previous studies showed that some volume-overloaded patients on peritoneal dialysis can maintain normal BP (congestive heart failure excluded), suggesting that these patients have volume resistance capacity. This study is to investigate whether hUII plays an important role in this subgroup of patients on peritoneal dialysis. In this study, 105 patients on continuous ambulatory peritoneal dialysis were enrolled. Volume load was evaluated by the overhydration (OH) value obtained by bioimpedance analysis. OH < 2.0 kg was defined as normal volume (NV), and OH ≥ 2.0 kg as high volume (HV). Systolic blood pressure (SBP) <130 mmHg was defined as normotension (NT) and ≥130 mmHg as hypertension (HT). The patients were thus divided into four subgroups: (1) normotension with normal volume (NT-NV), (2) normotension with high volume (NT-HV), (3) normal volume with hypertension (HT-NV), and (4) high volume with hypertension (HT-HV). hUII was measured using radioimmunoassay method. hUII was significantly higher in normal SBP group than that in high SBP group (p < 0.05). hUII was higher in the NT-HV group compared with that in the HT-HV group (p < 0.05). Our study suggests that hUII may be involved in the pathogenesis of the volume resistance HT in peritoneal dialysis patients.

• human urotensin II
• continuous ambulatory peritoneal dialysis
• volume resistance
• hypertension

INTRODUCTION

Volume overload due to excess salt intake and/or decreased residual renal function is believed to play a pivotal role in the pathogenesis of hypertension (HT) in dialysis patients.¹ In our previous research, although there were presentations of volume overload in many dialysis patients, they could remain normotension (NT; congestive heart failure excluded). Hemodynamic research found that the total peripheral vascular resistance in these patients with high volume (HV) and NT (the so-called volume resistance) was significantly lower than that in other patients.² However, the underlying mechanism for the reduction of the total peripheral vascular resistance in these patients is not fully understood. We suppose that the metabolism of some substances causing the dilation of blood vessels might play a role in this phenomenon.

Urotensin II (UII) is a cyclic peptide of 11 amino acids initially isolated from neurohumoral organ and is expressed in the cardiovascular system as well as the central nervous system, kidney, spleen, small intestine, thymus, prostate, pituitary, and adrenal glands.³ Recent studies have shown that human urotensin II (hUII) has the prominent effect of dilating vessels and decreasing the blood pressure (BP).⁴ So, in this study, we aimed at investigating the effect of hUII on HT and volume resistance in peritoneal dialysis patients.

PATIENTS AND METHODS

Study Population

The patients were enrolled from the Peritoneal Dialysis Center, Division of Nephrology, Peking University Third Hospital. All patients were receiving the standard continuous ambulatory peritoneal dialysis treatment. Exclusion criteria were (1) dialysis vintage <3 months, (2) congestive heart failure (New York Heart Association classes III–IV), (3) severe malnutrition, and (4) peritonitis. Based on these criteria, 105 patients were included in this study. Written informed consent was obtained from all patients. The ethics committee approved this study protocol.

Home Measurement of BP

Home BP was self-measured with a digital sphygmomanometer (Omron HEM 7200; Omron Co., Ltd., Dalian, China).⁵ A cuff with a bladder width of 13 cm was used for subjects with an arm circumference ≤35 cm, and a cuff with a bladder width of 15 cm was used for subjects with an arm circumference >35 cm. Patients received written instructions and individual guidance on how to measure BP correctly. Preparations for self-measured home BP were the same as for clinic BP [finding a quiet place to check patients’ BP; make sure that patients are comfortable and relaxed with a recently emptied bladder (a full bladder may affect their reading); roll up the sleeve on their arm or remove any tight-sleeved clothing and if needed, rest in a chair next to a table for 5–10 min; sit up straight with their back against the chair, legs uncrossed; rest their forearm on the table with the palm of their hand facing up]. Seated BP was measured twice, approximately at a 2-min interval every morning between 6 AM and 9 AM and every evening between 6 PM and 9 PM on 7 consecutive days. Every patient’s home BP was determined as the mean of 14 duplicate measures. The antihypertensive medications prescribed during the previous days were recorded in detail to calculate the defined daily dose.⁶

The patients were divided into two categories: NT group and HT group. HT group was defined as SBP ≥ 130 mmHg regardless of antihypertensive medication. NT group was defined as SBP < 130 mmHg regardless of antihypertensive medication.⁷

Measurement of Volume Status

Hydration status can be measured in terms of extracellular water (ECW) or total body water (TBW) in proportion to body weight and is also dependent on body composition. A recent development has been the method for calculation of normal hydration status, that is, the expected normal values of ECW or TBW that result in healthy kidney function.⁸ As normal ECW (nECW) and TBW can be determined for a given weight and body composition, ΔHS can be defined as the actually measured ECW minus the expected normal ECW. A new device, the Body Composition Monitor (BCM; Fresenius Medical Care, Bad Homburg, Germany), provides a convenient method to obtain ECW and TBW and has been validated previously.⁹ These volumes are determined by the measurement of the whole body impedance at 50 frequencies via electrodes placed on the wrist and ankle. For this study, patients were measured without dialysate in the peritoneum and the result was regarded as valid only if the operative quality score was over 80%. Measurement was performed during a 2-min period, whereby values for ΔHS were recorded. ΔHS > 2.0 L was the reasonable cutoff value for overhydration (OH), and patients with OH ≥ 2.0 kg were considered volume overloaded by our previous studies.¹⁰

Blood Collection

Blood samples were obtained in the early morning after the subjects had fasted for at least 8 h. Whole blood (4 mL) was collected in EDTA tubes and then immediately transported to the analytical laboratory on ice and centrifuged at 3000 rpm (1006 × g) for 15 min at 4°C to separate plasma. The supernatants were frozen at −80°C until the time of assay.

Measurement of hUII

Plasma UII was measured using a radioimmunoassay.¹¹ Plasma samples of 3 mL were deproteinated 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 250 μL of radioimmunoassay buffer.

One hundred microliters of standard UII or assay sample was incubated overnight at 48°C with 100 μL rabbit antiserum. One hundred microliters labeled ¹²⁵I-urotensin II (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 antirabbit antiserum and normal horse serum. Using a gamma counter, the amount of bound ¹²⁵I-urotensin II was measured as picograms per milliliter.

Evaluation of Dialysis Adequacy

Dialysis adequacy was evaluated by small-molecule clearance rate Kt/V.¹² The peritoneal Kt/V (pKt/V) and renal Kt/V (rKt/V) were measured separately, and total Kt/V was the sum of pKt/V and rKt/V. The urea distribution volume was calculated according to the formula of Watson et al.¹³

Measurement of Biochemical Parameters

Biochemical parameters, such as blood urea nitrogen, creatinine, and natrium, were measured using standard methods.

Classification of Subgroups

According to the aim of this study, these patients were classified into four subgroups: normotension with normal volume (NT-NV), normotension with high volume (NT-HV), normal volume with hypertension (HT-NV), and high volume with hypertension (HT-HV).

Statistical Analysis

Statistical analysis was performed using SPSS software, version 13.0 (SPSS Inc., Chicago, IL, USA). Continuous variables are expressed as mean ± SD, while categorical variables are expressed as ratio or percentage. For comparison of categorical variables in the two groups the χ² test was employed. The comparison between independent two groups, such as the clinical conditions of normotension group and HT group and the level of hUII, was analyzed by independent t-test. The comparisons of the subgroup of BP and volume were analyzed using ANOVA. All tests were two-sided. A value of p < 0.05 was taken as statistically significant.

RESULTS

Demographic Data of the Study Population

Of the 105 patients studied, 50 were males and 55 were females (Table 1). The mean age was 61.07 ± 12.76 years. The mean time on dialysis was 30.09 ± 19.52 months. The etiologies of uremia were type 2 diabetes mellitus (30.4%), HT (21.7%), chronic primary glomerulonephritis (10.1%), secondary glomerulonephritis (1.4%), tubulointerstitial nephritis (13.0%), others (8.6%), and unknown (14.5%).

Table 1.  Demographic data of the study population.

Number of patients	105
Male/female	50/55
Mean age (years)	61.07 ± 12.76
Body mass index (kg/m^2)	24.02 ± 4.58
Dialysis vintage (months)	30.09 ± 15.52
Etiology of uremia (%)
Glomerulonephritis	11.5%
Hypertension	21.7%
Diabetes mellitus	30.6%
Tubulointerstitial nephritis	13.0%
Other	13.6%
Unknown	14.5%

Comparison of Demographic Data, Biochemical Parameters, and Dialysis Adequacy among the Subgroups

The patients were classified into two subgroups according to BP: systolic blood pressure (SBP) <130 mmHg for the NT group and SBP ≥130 mmHg for the HT group. There was no significant difference in sex, prevalence of diabetes mellitus, body mass index (BMI), dialysis vintage, residual renal function, dialysis adequacy, defined daily dose of antihypertensive drugs, and diuretics between the NT and HT groups (Table 2). The systolic pressure and diastolic pressure of HT group were significantly higher than those in the NT group, p < 0.05. The OH and nECW of HT group were significantly higher than those of the NT group, p < 0.05. The hUII level of NT group was notably higher than that of HT group, p < 0.05.

Table 2.  Clinical characteristics in the classified subgroups.

	Normotension group (NT)	Hypertension group (HT)
Number of patients	47	58
Male (%)	55.5	44.8
Diabetes mellitus (%)	38.9	39.5
BMI (kg/m^2)	23.03 ± 3.70	24.02 ± 4.58
Dialysis vintage (months)	31.56 ± 24.92	29.00 ± 14.54
Plasma sodium (mmol/L)	138.41 ± 3.14	138.75 ± 2.65
Urea nitrogen (mmol/L)	23.49 ± 4.98	21.53 ± 6.01
Creatinine (μmol/L)	844.81 ± 245.85	818.89 ± 289.56
pKt/V	1.28 ± 0.62	1.44 ± 0.5
rKt/V	0.47 ± 0.64	0.45 ± 0.63
tKt/V	1.75 ± 0.77	1.90 ± 0.55
ECW (L)	14.55 ± 2.89	17.55 ± 3.03
ICW (L)	15.42 ± 3.61	16.49 ± 3.43
TBW (L)	29.97 ± 6.10	34.03 ± 6.02
nECW (L/m)	9.00 ± 1.50	10.54 ± 1.65
OH (L)	2.01 ± 1.20	2.85 ± 1.66
Systolic pressure (mmHg)	110.74 ± 13.36	146.78 ± 14.88
Diastolic pressure (mmHg)	68.49 ± 12.60	83.12 ± 11.57
Diuretics DDD	0.03 ± 0.8	0.04 ± 0.5
Total DDD	2.6 ± 1.6	2.9 ± 1.4
hUII (pg/mL)	13.03 ± 5.74	10.71 ± 4.62

Notes: DDD, defined daily dose; pKt/V, peritoneal dialysis Kt/V; rKt/V, renal Kt/V; tKt/V, total Kt/V; BMI, body mass index; ECW, extracellular water; nECW, normal ECW; TBW, total body water; OH, overhydration; hUII, human urotensin II.

*p < 0.05 versus NT group.

Table 3.  Comparison of human urotensin II (hUII) among subgroups.

	NT-NV	NT-HV	HT-NV	HT-HV
Number of patients	17	30	30	28
Age (years)	62.29 ± 13.99	62.67 ± 11.38	62.43 ± 10.77	57.14 ± 15.03
Male (%)	55.2	55.6	50.0	39.3
Diabetes mellitus (%)	47.6	26.7	40.9	38.1
BMI (kg/m^2)	23.07 ± 3.40	23.01 ± 3.91	24.62 ± 5.04	23.39 ± 4.02
Dialysis vintage (months)	26.56 ± 17.92	34.31 ± 28.09	29.40 ± 15.14	28.53 ± 14.18
Urea nitrogen (mmol/L)	20.75 ± 4.45	21.27 ± 4.54	21.06 ± 5.24	22.04 ± 6.84
Creatinine (μmol/L)	809.40 ± 283.34	868.95 ± 220.43	805.64 ± 248.43	833.72 ± 334.36
Serum sodium (mmol/L)	138.0 ± 2.5	139.0 ± 2.6	138.0 ± 3.2	137.8 ± 3.3
Serum albumin (g/L)	39.87 ± 3.31	39.21 ± 3.16	38.44 ± 4.00	38.69 ± 4.81
pKt/V	1.40 ± 0.58	1.49 ± 0.65	1.55 ± 0.57	1.33 ± 0.39
rKt/V	0.66 ± 0.74	0.44 ± 0.53	0.43 ± 0.52	0.48 ± 0.75
tKt/V	2.06 ± 0.66	1.93 ± 0.78	1.98 ± 0.52	1.81 ± 0.65
hUII (pg/mL)	12.25 ± 5.28	13.47 ± 6.02	11.37 ± 4.49	10.00 ± 4.73
Total DDD	2.1 ± 1.6	3.1 ± 1.7	3.0 ± 1.8	2.8 ± 1.1

Notes: DDD, defined daily dose; pKt/V, peritoneal dialysis Kt/V; rKt/V, renal Kt/V; tKt/V, total Kt/V; BMI, body mass index; hUII, human urotensin II.

*p < 0.05 versus HT-HV group.

hUII of Volume-Tension Subgroups

The comparison of hUII among subgroups is shown in Table 3. There was no significant difference in age, sex, prevalence of diabetes mellitus, BMI, dialysis vintage, serum albumin, creatinine, blood urea nitrogen, pKt/V, rKt/V, and tKt/V among the four subgroups. However, compared with HT-HV group, the hUII of NT-HV group was significantly high (NT-HV group vs. HT-HV group, 13.47 ± 6.02 vs. 10.00 ± 4.73 pg/mL, p < 0.05). No significant difference in the concentration of hUII between other groups was observed.

DISCUSSION

hUII is a cyclic peptide of 11 amino acids, with a molecular weight of 1388 Da, and is expressed in the cardiovascular system as well as the central nervous system, kidney, spleen, small intestine, thymus, prostate, pituitary, and adrenal gland.³ It could stimulate secreting adrenocortical hormone, could influence metabolism of glucose and lipid, and is one of the vaso-excitor material, whose function is 10 times higher than that of endothelin.^14–16 Subsequent researchers found that UII also had noticeable vasodepressor effect. For example, injecting intravenously hUII 10 min elicited decrease of 10 mmHg in mean arterial pressure in SD rats and 20 mmHg in mean arterial pressure in spontaneously hypertensive rats. This vasodepressor effect was obviously stronger than that of bradykinin,¹⁷ which resulted from extension of small arteriolar and was mediated by nitric oxide (NO).⁴ Some studies showed plasma hUII was raised in hypertensive patients compared to normotensive controls and was directly related to SBP, suggesting that hUII may have a compensatory increase in HT.^10,11 However, the role of hUII in HT has not been completely clear. In our study, the hUII of NT group (systolic pressure <130 mmHg) was significantly higher than that of HT group (systolic pressure ≥130 mmHg). And there was statistical difference. This may suggest that hUII plays a role in vasodilation regulation in the human body. Meanwhile, there was no difference in antihypertensive agent, the prevalence of diabetes mellitus, and BMI between NT patients and HT patients. But the OH and nECW of HT group were significantly high. As a result, it was too hard to determine whether systolic pressure rising resulted from the decrease of hUII or volume overload.

For this reason, we evaluated OH of these patients by bioimpedance analysis. The result showed that there was volume overload in 30 normotensive patients, which suggested that these patients had volume resistance. However, in 58 HT patients, there were 30 patients having volume overload. HT-HV suggested that there might be volume sensitiveness in these patients. We found the hUII of NT-HV group was much higher than that of HT-HV group. But there was no difference in the dosage of diuretics and antihypertensive drugs. Moreover, all patients were required to stop their antihypertensive medications when BP and OH were measured. And nearly none of the patients used diuretics due to anuria. This could preclude the influence of drugs to volume and BP and suggest that hUII may play a role in volume resistance. In our previous research, we found that the peripheral resistance of volume resistance patients was significantly lower than others.² And HT patients with volume sensitiveness were usually believed to be associated with increased cardiac output.² The reason why volume resistance patients were characterized by lower total peripheral resistance indices is unclear. In our study, we suspect that hUII might play a role in vasodilatation and reducing BP in BP regulation when volume increases through endothelium-dependent regulation. However, this speculation should be validated by animal studies.

This study has some limitations. This is only a cross-sectional study that may be a confounding factor to affect the BP and volume status.

In conclusion, in normotensive peritoneal dialysis patients, their hUII level was elevated. Furthermore, hUII was higher in the volume resistance group as compared to the volume sensitiveness group. Our study revealed that hUII may be involved in the pathogenesis of the volume resistance in peritoneal dialysis patients.

ACKNOWLEDGMENTS

This study was supported by National Natural Science Foundation (Grant No. 30800522) and Specialized Research Fund for the Doctoral Program of Higher Education (Grant No. 200800011034) to LT Cheng and Peking University Third Hospital Key grant to AH Zhang and National Natural Science Foundation of China (Grant No. 81170706).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.