Irisin is Associated with Urotensin II and Protein Energy Wasting in Hemodialysis Patients

Hemodialysis treatment and some concurrent clinical conditions during the course of the treatment will cause persistent inflammation, which can lead to muscle depletion in chronic kidney disease patients. These patients usually have insulin resistance and metabolic syndrome which elevate their risk for the development of cardiovascular disease [1,2]. Insulin resistance is also an important cause of muscle atrophy [3].

Boströmʼs lab first identified irisin in 2012, a novel myokine released to blood after cleavage and shedding of the membrane fraction of fibronectin type III domain containing 5 (FNDC5). Irisin acts on white adipose cells to stimulate brown fat-like development which will increase energy expenditure [4,5]. Recent research found that irisin levels were decreased in patients with chronic kidney disease (CKD) and also in HD patients when compared to that of the healthy subjects [6,7].

Urotensin II (UII) was originally isolated from the urophysis of teleost fish. It is a somatostatin-like cyclic undecapeptide, identified as the most potent mammalian vasoconstrictor to date [8,9]. Previous studies showed that UII levels are increased in diabetes. Furthermore, it was reported that UII could inhibit glucose transport in skeletal muscle in diabetic mice and aggravate insulin resistance [10] while irisin also might be associated with insulin resistance [11]. Building upon the findings of the previous studies, we speculate that UII not only can induce insulin resistance but also can be associated with irisin and skeletal muscle protein wasting. In CKD patients, skeletal muscle protein wasting and atrophy is also one of the main criteria of protein energy wasting (PEW) [2]. Since PEW is prevalent among hemodialysis patients, this also led us to investigate if irisin levels may be associated with hemodialysis patients with PEW. In the present study, we combined these thoughts and studied the association among irisin, UII, and PEW in HD patients.

From Aug 2014 to Oct 2014, 139 maintenance hemodialysis patients were recruited initially, but only 128 patients finished all of the lab tests. 40 healthy subjects were selected as control subjects. Inclusive criteria: Patients had to have received hemodialysis for at least 3 months; patients who received dialysis treatment for less than 3 months previously or had intermittent dialysis treatment were not eligible for the study. Consent forms were signed by all the subjects. Peking University Third Hospital ethical committee approved this study.

Blood samples were obtained from participants after a 12-h fast. The patients' blood and anthropometric data were collected in a subsequent session of the week. Aliquots of serum and plasma were stored at −80°C and were not thawed until the analysis. Serum irisin concentrations were measured in duplicates 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 0.1ng/ml and the linear range of the standard was 0.1-1000ng/ml. The intra- and inter-assay coefficients of variation (CV) were 4.5% and 8%, respectively. Serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglyceride, and high-density lipoprotein cholesterol (HDL-C) were measured enzymatically. Serum creatinine was measured by picrate method. Blood glucose levels were measured as fasting blood glucose (FBG) and hemoglobin A1C (HbA1C). HbA1c value was measured by high-performance chromatography.

The blood samples avoided repeated freeze/thaw cycles and were redeproteinated with 0.75ml 2mol/L hydrochloric acid. After centrifugation for 20 min at 6000g, 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. One hundred microliter of standard UII or assay sample was incubated overnight at 4°C with 100µL rabbit antiserum. One hundred microliters labeled 125I-UII (Phoenix Pharmaceuticals, Inc., Belmont, CA, USA) were added to each tube and incubated for an additional 24h. 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 picograms per milliliter.

Every subjectʼs body composition was analyzed by Bioelectrical Impedance after his or her HD treatment session (BIA, Tsinghua Science and Technology Co., Ltd, Beijing, China). The collected data included body fat (% and kg), lean body mass (kg), muscle mass (kg), and visceral fat rating (1-59 grade). Grade 1-12 is considered healthy while grade more than or equal to 13 corresponds to excessive abdominal fat and indicates central obesity. The measurements were performed by skilled staff members who were experienced at collecting these measurements.

Our patients and the normal control subjects did not exercise intentionally and were not sedentary. We assessed the subjects' physical activity according to the international physical activity questionnaire long forms [12]. In the international physical activity long form, physical activity types consist of work-related activity (walking, moderate physical activity, and vigorous physical activity), transportation-related activity (walking and bicycling) domestic and garden-related activity (moderate physical activity and vigorous physical activity), and leisure related activity (walking, moderate physical activity, and vigorous physical activity). Walking MET=3.4METs*walking time, moderate physical activity=4METs*activity time, vigorous physical activity=8METs*activity times. We calculated the subject's total physical activity MET-minutes/week=sum of total work+total transport+total domestic and garden+total leisure time MET-minutes/week scores.

According to the cirteria for the diagnosis of PEW [13], PEW is diagnosed if three characteristics are present: low serum levels of albumin (<38g/l or total cholesterol<100mg/dl), reduced body mass (weight loss 5% within 3 months or reduced skeletal muscle mass), and reduced intake of dietary protein (<0.8g/kg/d). Dietary assessment was performed by a dedicated dietitian based on patients' dietary records for 3 days. Prior to the assessment, all patients received intensive instruction on how to record dietary food correctly with the help of portion-sized food model of dietary protein intake. Dietary protein intake was calculated and normalized by each patient's ideal body weight (kg), which is equal to height (cm) minus 105.

Data are presented as means ± standard deviation (SD) or median (25%-75% quartile). Independent Student's t-test was used to test the differences of numerical variables for normal distribution between two groups. Non-parameter test was used to test difference for non-normal distribution. Chi-square test was used to compare categorical variables and nominal variables. Non-normal distributed data, such as circulation irisin, were changed to Ln (irisin) (the natural logarithm of irisin) for Pearson correlated analysis. The data were analyzed by using the statistical package SPSS 19.0 (SPSS, Inc., Chicago, IL, USA).

The clinical characteristics and biochemical data of the control subjects and hemodialysis patients were summarized in Table 1. There were 68 males and 60 females in the HD patients group. The primary renal diseases in the HD patients were diabetes (30 patients), chronic glomerulonephritis (40 patients), hypertensive glomerulosclerosis (35 patients), other causes (18 patients), and unknown causes (5 patients). Mean dialysis treatment vintage was 60±33 months.

Notably, hemodialysis patients had higher level of systolic blood pressure (pre-hemodialysis), UA, creatinine, potassuim, phosphorous (pre-hemodialysis), triglyceride, and PTH than that of the normal controls. In contrast, hemoglobin, irisin, Ln (irisin) (P< 0.01), UII, albumin, CO2CP, and body fat proportion were significantly lower (P < 0.05) in HD patients when compared to that of the control subjects. Moreover, the international physical activity long form showed that normal control subjects had higher physical activity score than those of hemodialysis patients (1508±305minutes/week vs. 697±324 minutes /week, P=0.001).

Bivariate correlation analysis revealed that circulating Ln (irisin) (the natural logarithm of irisin) was negatively correlated with circulating UII (r=-0.315, P=0.006) but was positively correlated with ALB (r=-0.299, P=0.016), muscle mass (r=0.265, P=0.030), and lean body mass (r=0.274, P=0.025). Furthermore, circulating UII were also negatively related with skeletal muscle mass (r=-0.337, P=0.026) and lean body mass (r=-0.312, P=0.034) in hemodialysis patients. We did not observe a significant correlation between Ln (irisin), age, hemoglobin, KT/V, BMI, UA, body fat, and visceral fat rate (Table 2). Additionally, irisin levels were not correlated with international physical activity scores in our current study. There were no difference in irisin levels between higher physical activity scores and lower physical acvity scores in normal control subjects or hemodialysis patients.

Our results showed lower irisin levels in hemodialysis patients with PEW when compared to that of the hemodialysis patients without PEW. Hemodialysis patients with PEW also have lower albumin level, lower hemoglobin, lower body fat, and lower lean body mass in comparison to those of hemodialysis patients without PEW (Table 3).

A multivariate linear regression model (the variables entered by univariate analysis P≤0.1) was used to study which kinds of clinical and biochemical factors were independently associated with circulating irisin levels. In our model, body fat proportion, skeletal muscle mass, and age were excluded from the linear model. Our model showed that circulating UII, PEW, and lean body mass were independently associated with circulating irisin in HD patients (Table 4).

PEW is common in patients with CKD and is associated with adverse clinical outcomes, especially in individuals receiving maintenance dialysis therapy [2]. During the development of PEW, persistent muscle protein catabolism in CKD results in striking losses of muscle proteins as well as skeletal muscle wasting and atrophy. Mechanisms of muscle wasting in CKD are multifactorial. Some of the notable causes of muscle wasting in CKD include metabolic acidosis, insulin resistance, inflammation, increased angiotensin II level, and abnormal appetite regulation [3,14].

Irisin, a hormone secreted by myocytes, can act as a muscle-derived energy-expenditure signal [4,5]. This particular myokine may also play an important role in adjusting skeletal muscle metabolism. Recent studies showed that plasma irisin levels in HD patients were lower than that of the healthy subjects [7]. In our current study, we found that circulating irisin concentrations were decreased in HD patients. The mechanisms underlying a significant reduction in circulating irisin in late-stage chronic kidney disease (CKD) could be multifactorial. An important reason for the reduction could be that uremic toxins, such as indoxyl sulfate, might decrease FNDC5 expression. Additionally, CKD patients might have lower muscle volume, and irisin, which is produced within muscle, can be affected by total muscle volume [6].

UII level is significantly increased in many kinds of diseases, such as hypertension, atherosclerosis, and diabetes [9,15]. Conflicting results have been reported by researchers measuring blood values of UII in patients with kidney disease. Some studies observed a lower UII concentration among subjects with CKD and ESRD whereas other studies reported higher UII concentration in patients with kidney disease [16,17]. We found that UII is significantly lower in our current study, and this is consistent with Mosenkis, et al's results [18] but differs from that of Tostune, et al. [8,9]. The reduction in UII levels in hemodialysis patients can suggest reduced production, greater clearance, or both [18]. On the other hand, some authors believed that the difference of UII levels can be a result of using different antibodies for the UII BIA assessment (such as anti-UII polyclonal antibody or anti-UII monoclonal antibody) [18].

Our results are the first to verify that UII level is negatively correlated with skeletal muscle mass and lean body mass in our HD patients. Moreover, we are the first to demonstrate that circulating irisin is negatively correlated with circulating UII in HD patients. Recently, Wang, et al. [10] reported that UII could inhibit glucose transport in skeletal muscle in diabetic mouse and aggravate insulin resistance while irisin could alleviate insulin resistance in an animal model [17]. Insulin resistance is an important cause of muscle atrophy [3]. Building upon the results of the past studies, it is reasonable to speculate that the lower level of circulating irisin in HD subjects observed in our study may be associated with the UII that are induced by skeletal muscle atrophy. In addition to inherent skeletal atrophy, uremic toxins and other causes can also contribute to muscle volume reduction. Our results showed that there was lower skeletal muscle mass in HD patient. In these patients, circulating irisin was positively associated with muscle mass while circulating UII was negatively associated with muscle mass. The result of our study supported our hypothesis. At the same time, there were lower body fat and visceral fat rate in our HD patients. Some authors reported that myocytes cells or adipose tissues also secreted irisin in addition to skeletal muscle cells [19]. This which may also be another reason for the lower irisin level in HD patients. It is important to note that the independent determinants of irisin level in our study are UII level and lean body mass in our HD patients by multiple linear regression analysis. Our results show that UII level might be the important adjusting factor for irisin level in HD patients. However, the HD patients' circulating UII level was not increased in our current study when the circulating irisin level had already decreased in these HD patients. This may hint that there are other factors affecting circulating irisin level besides UII. Some authors of past literatures believed that low UII may reflect malnutrition and wasting (UII is positively correlated with albumin levels) [20]. Malnutrition and Wasting is consequently associated with low skeletal muscle mass and low irisin level.

In order to verify this hypothesis on a molecular level, further study needs to be conducted. It will be important to investigate whether skeletal muscle cells can release irisin after culturing it with UII. It will also be interesting to see if UII and UII receptor expressions are negatively associated with the expression of FNDC5 in the skeletal muscle of 5/6 nephrectomized animal model. Additionally, experiments studying the level of irisin and the expression of FNDC5 using urotension II receptor knockout mice should also be conducted.

Irisin may be stimulated by exercise [5]. Our patients and the normal control were not instructed to excercise intentionally but are not sedentary. We assessed the subjects' physical activity according to the international physical activity questionnaire long form. Through the assessment, it was found that the normal controls had higher scores of physical activity than that of the hemodialysis patients, but irisin levels were not correlated with the international physical activity scores in our current study. There were no differences in irisin levels between higher physical activity scores and lower physical activity scores in normal controls and hemodialysis patients. It could be that there is still no conclusive results that demonstrate that irisin can be stimulated by exercise [7,21]; moreover, the subjects in our study were not put under intentional exercise regimen.

Skeletal muscle protein wasting and atrophy is the predominant characteristic of PEW [13]. Irisin is a hormone that is secreted by myocytes [4]. We speculate that irisin may be associated with PEW. It is very interesting that hemodialysis patients with PEW had lower irisin level, lower skeletal muscle, and body fat than those of patients without PEW. Lower irisin levels were correlated with PEW in our current study. Reduced skeletal muscle mass and body fat might be the cause of lower irisin levels in hemodialysis patients with PEW. However, irisin is also a myokine factor, and it regulates skeletal metabolism, however, irisin itself may also induce skeletal muscle atrophy in hemodialysis with PEW. The cause and effect of low irisin, UII, and PEW is worthy of further study.

At present, there are no reliable methods to prevent CKD-induced muscle wasting, but mechanisms that control cellular protein turnover have been identified. This suggests that therapeutic strategies can be developed to suppress or block protein loss. From the results of our study, UII inhibition and irisin might help develop new therapeutic directions for blocking muscle protein wasting in CKD. There are several limitations in our current study. The sample size of this cross-sectional study is relatively small; hemodialysis patients also have many confounding factors affecting our results.

In summary, our study is the first to provide the clinical evidence of the association among irisin, UII, and protein energy wasting. Our results suggest that UII and protein energy wasting might inhibit the release or synthesis of irisin from skeletal muscles in HD patients.

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

This study was supported by National Natural Science Foundation (Grant No. 81170706, Grant No 81341022, 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.

Wan-Yu He and Fei Wu contributed equally to this work and therefore share first authorship.