Original Paper Blood Purif 2014;38:188–194 DOI: 10.1159/000368883
Received: April 25, 2014 Accepted: October 3, 2014 Published online: December 16, 2014
Correlation of Lower Concentrations of Hydrogen Sulfide with Atherosclerosis in Chronic Hemodialysis Patients with Diabetic Nephropathy Han Li Su-Juan Feng Gui-Zhi Zhang Shi-Xiang Wang Department of Blood Purification, Beijing Chao-Yang Hospital, Capital Medical University, Nephrology Faculty, Capital Medical University, Beijing, China
Abstract Background/Aims: To explore the relationship between hydrogen sulfide (H2S) and uremic accelerated atherosclerosis (UAAS) in chronic hemodialysis patients with diabetic nephropathy (CHD/DN). Methods: A total of 36 CHD/DN and 32 chronic hemodialyzed non-diabetic patients with chronic glomerulonephritis (CHD/non-DN) were studied. Plasma H2S was measured with a sulfide sensitive electrode. Results: Plasma H2S in CHD/DN was significantly lower than that in CHD/non-DN patients. Plasma H2S was positively correlated with plasma TGF-β1, and negatively correlated with MMP-12 in CHD/DN patients. CHD/DN patients exhibited higher CCAIMT, hsCRP, and lower H2S levels than in CHD/non-DN patients. Moreover, in CHD/DN patients, CCA-IMT was negatively correlated with plasma H2S, and positively correlated with hsCRP and LDL. On multiple regression analysis, H2S levels exhibited independent association with IMT in CHD/DN patients. Conclusions: These findings suggest possible linkage between H2S metabolism and TGF-β/Smad signaling pathway modulation abnormalities that may contribute to the development of UAAS in CHD/DN patients. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 0253–5068/14/0384–0188$39.50/0 E-Mail [email protected] www.karger.com/bpu
Introduction
Atherosclerotic cardiovascular disease (ACVD) is the most common complication and the leading cause of death in chronic hemodialysis patients [1, 2], especially in diabetic patients [3]. The mortality caused by cardiovascular disease in death of end-stage renal disease (ESRD) patients accounted for 45–50% [4]. In this condition, evidence shows that there is an increased incidence and accelerated progress of atherosclerosis compared with that of the conventional ACVD [5]. Hydrogen sulfide (H2S) is a novel gas-signaling molecule, which has the effects of relaxing vascular smooth muscle, inhibiting proliferation of vascular smooth muscle cells and lowering blood pressure [6]. Perna et al. [7] have confirmed that H2S decreased in the plasma of hemodialysis patients and it may have relevance to the pathogenesis of the uremic syndrome manifestations, such as hypertension and atherosclerosis. However, mechanisms of the accelerated formation of atherosclerosis in chronic hemodialysis patients with diabetic nephropathy (CHD/DN) patients are
This work was supported by National Natural Science Foundation of China (81200543), Beijing Natural Science Foundation (7142057) and Beijing Municipal Health Bureau High-level Medical Professionals Promotion Project (2013–3-016).
Shi-Xiang Wang Department of Blood Purification, Beijing Chao-Yang Hospital Capital Medical University, Nephrology Faculty, Capital Medical University Beijing 100020 (China) E-Mail wxy1988 @ 263.net
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Key Words Hemodialysis · Uremic accelerated artherosclerosis · Hydrogen sulfide · TGF-β/Smad signaling pathway
Methods
titia ultrasound interfaces and measured as the distance between the two parallel echogenic lines on the far wall of artery in longitudinal plane image frozen in the screen by electronic calipers [11]. The IMT on the far wall of the bilateral common carotid artery about 10mm proximal to the bifurcation of the carotid artery was measured manually as previously described [12]. The mean IMT was calculated as the average of the three readings of bilateral carotid arteries. Plaque was defined as localized thickening of IMT ≥1.2 mm that did not uniformly involve the whole wall of carotid artery.
Data Sources A total of 36 CHD/DN patients and 32 chronic hemodialyzed non-diabetic patients with chronic glomerulonephritis (CHD/ non-DN) were invited to participate in the study if they were more than 18 years of age, had no residual renal function, and had maintained hemodialysis for more than 3 months. Patients were considered diabetic nephropathy if they were on insulin, oral hypoglycemic agents, diagnosed as diet-controlled diabetes, or had a fasting blood glucose of ≥7 mmol/l, and/or 2 h blood glucose after an oral glucose tolerance test of ≥11.1 mmol/l. Patients were not included in the study if they had heart failure, a recent acute coronary event, cancer, autoimmune disease, and active infection. A standard questionnaire was used for every participant to obtain systematic information regarding conventional cardiovascular risk factors, including hyperlipidemia, hypertension, diabetes, and a family history of cardiovascular disease. As a normal control group, age and gender matched, 30 healthy individuals (15 females and 15 males) were enrolled for measuring plasma H2S in this study. The study was approved by the ethics committee of Beijing Chao-Yang Hospital, Capital Medical University, and written informed consent was obtained from each participant.
Laboratory Investigations Plasma was separated as described above immediately and used freshly for analysis by biochemists, who were blinded to classification of subjects as CHD/DN and CHD/non-DN patients. The concentration of TGF-β1 and MMP-12 was measured in plasma by enzyme linked immunosorbent assay (ELISA). Biochemical and hematological parameters were determined by standard laboratory methods in the clinical laboratory.
Hydrogen Sulfide (H2S) Concentration Measurement The blood of patients was drawn prior to the mid-week dialysis session. Once blood was drawn in plastic Vacutainers using EDTA (1 mg/ml of blood), plasma was immediately obtained through brief 5 min centrifugation at 500 g and rapidly added to the assay mixture. Plasma H2S concentration was measured with a sulfide sensitive electrode as described by Li et al. [8] with modifications. Briefly, 0.5 ml of plasma was added into a test tube containing 0.5 ml of 0.04 g NaOH, 0.035 g EDTA and 0.05 g ascorbic acid. The sulfide sensitive electrode and a reference electrode immersed into the sample together, and record the serum H2S concentration until the reading is stable. H2S concentration was calculated against a calibration curve obtained with known H2S concentrations in a range between 5 and 100 μM, utilizing the H2S donor NaHS [9, 10]. Standard curves were repeated daily with triplicate measurement for each point, and freshly made solutions were utilized at all times.
Subject Characteristics A total number of 68 patients (36 CHD/DN, 32 CHD/ non-DN) with a mean age of 48.1 ± 11.9 (range 20–71 years) and a mean dialysis period of 41.5 ± 18.3 months (range 5–84 months) were included in this study. The CHD/DN group consisted of 20 men and 16 women; the mean age was 48.3 ± 11.5 and the average dialysis period was 39 months (range 5–82 months). The CHD/non-DN group consisted of 17 men and 15 women; the mean age was 47.8 ± 12.5 and the average dialysis period was 45 months (range 8–84 months). There was no significant difference between the two groups in terms of age, sex ratio, dialysis duration, smoking, BMI, Kt/V, Hb, serum creatinine, BUN, TG, Tch, etc.
Common Carotid Artery Ultrasonography Carotid artery ultrasonography was performed by an experienced specialist physician who was specifically trained for the vascular ultrasonography. Carotid B-mode ultrasound measurements were performed using HDI 5000 equipped with a 5–12 MHz linear array transducer (Philips, USA). Subjects were examined in a supine position with the head turned 45° contralateral to the side of scanning. Intima-media thickness (IMT) was defined as the distance between the lumen-intima and the media-adven-
H2S and CCA-IMT in CHD/DN and CHD/Non-DN Patients Plasma H2S level in CHD patients was significantly lower than that in the control group, and the plasma H2S level in the CHD/DN group was significantly lower than that in the CHD/non-DN group. Furthermore, in CHD/ DN patients, the plasma H2S level was positively corre-
H2S and UAAS
Statistical Analysis The SPSS version 13.0 statistics package was employed for the statistical analysis. Measurement data was presented as mean value ± standard deviation (±SD). Comparisons were performed using one-way ANOVA with post hoc analysis (LSD), independentsamples t test or chi-square test. In addition, bivariate correlation analysis and multiple regression analysis were performed. A p < 0.05 was regarded as statistically significant.
Results
Blood Purif 2014;38:188–194 DOI: 10.1159/000368883
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not yet clear. The role of H2S remains unclear in this uremic accelerated atherosclerosis (UAAS) process. Therefore, we evaluated the relationship between H2S and atherosclerosis in chronic hemodialysis patients with diabetes mellitus nephropathy.
Table 1. Characteristics of both study groups
Items
CHD/DN group (n = 36)
CHD/non-DN group (n = 32)
t/χ2 value
p value
Age, years Gender, male/female Dialysis duration, months BMI, kg/m2 Smoking, n (%) Hypertension, n (%) SBP, mm Hg DBP, mm Hg Kt/V Hb, g/l Alb, g/l Creatinine, μmol/l BUN, mmol/l ALT, U/l AST, U/l TG, mmol/l Tch, mmol/l LDL-C, mmol/l RASI, n (%) CCB, n (%) β-blocker, n (%) Mononitrite, n (%)
48.3±11.5 20/16 38.9±18.5 23.6±2.3 10 (27.8) 30 (83.3) 139.8±7.3 80.2±7.1 2.3±0.3 115.9±7.9 34.1±2.7 882.5±102.3 23.7±6.0 18.1±6.4 19.2±7.9 1.45±0.65 4.06±1.07 2.23±0.63 30 (83.3) 28 (77.8) 10 (27.8) 7 (19.4)
47.8±12.5 17/15 44.5±17.8 23.1±1.4 8 (25.0) 26 (81.3) 142.4±10.9 82.6±5.9 2.4±0.3 118.7±8.8 34.8±3.4 902.4±100.3 23.8±4.3 16.8±9.1 16.2±7.3 1.28±0.70 3.92±0.79 2.33±0.56 26 (81.3) 25 (78.1) 8 (25.0) 7 (21.9)
0.180 0.040 1.263 1.184 0.067 0.051 1.179 1.547 0.385 1.398 0.934 0.809 0.077 0.653 1.641 1.058 0.596 0.080 0.051 0.001 0.067 0.061
0.858 0.841 0.211 0.241 0.796 0.822 0.243 0.127 0.702 0.167 0.354 0.421 0.939 0.516 0.106 0.294 0.553 0.937 0.822 0.973 0.796 0.805
Values are means ± SD, unless specified otherwise. BMI = Body mass index; SBP = systolic blood pressure; DBP = diastolic blood pressure; Hb = hemoglobin; Alb = albumin; BUN = blood urea nitrogen; ALT = alaninetransarninase; AST = aspartate aminotransferase; TG = triglyceride; Tch = total cholesterol; LDL-C = low density lipoprotein-cholesterol; CCB = calcium channel blocker; RASI = renin abguitensin system inhibitor.
Correlations Between CCA-IMT and Risk Factors for Atherosclerosis and H2S in CHD/DN Patients In CHD/DN patients, bivariate correlation analysis showed that CCA-IMT was negatively correlated with plasma H2S (r = –0.923, p = 0.000), and positively correlated with hsCRP (r = 0.449, p = 0.006) and serum LDL (r = 0.363, p = 0.029). No correlation with age, gender, dialysis duration, serum TG, Tch, smoking and hypertension (table 3). On multiple regression analysis, the hydrogen sulfide level exhibited independent association with IMT in CHD/DN patients (table 4). 190
Blood Purif 2014;38:188–194 DOI: 10.1159/000368883
Table 2. Plasma level of H2S and CCA-IMT in CHD/DN and CHD/non-DN patients
Items
CHD/DN group (n = 36)
CHD/non-DN group (n = 32)
Control group (n = 30)
CCA-IMT, mm hsCRP, mmol/l H2S, μmol/l
1.16±0.10a 2.24±0.92a 22.9±8.3a, b
0.91±0.10 1.63±1.12 29.1±7.2b
56.6±6.9
a p < 0.05, compared with CHD/non-DN group; b p < 0.05, compared with the control group.
Discussion
In this study, we found that plasma H2S in CHD/ DN was significantly lower than that in CHD/non-DN patients, and it was positively correlated with plasma Li/Feng/Zhang/Wang
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lated with plasma TGF-β1 (r = 0.887, p = 0.000), and negatively correlated with MMP-12 (r = –0.892, p = 0.000). On the other hand, table 2 also showed that CHD/ DN patients exhibited higher CCA-IMT, hsCRP (highsensitivity C-reactive protein), and lower plasma H2S levels than in CHD/non-DN patients (table 2).
Variables
R
p value
H2S hsCRP Age Gender Dialysis durations Smoking Hypertension TG Tch LDL-C
–0.923 0.449 0.259 0.140 0.144 0.105 0.286 0.097 0.235 0.364
0.000 0.006 0.128 0.416 0.402 0.542 0.091 0.573 0.168 0.029
Table 4. Variables predicting IMT in multiple regression analysis in CHD/DN patients
Variables
Standardized coefficient β
Standard error
t value
p value
Constant H2S hsCRP LDL
1.355 –0.859 0.093 0.140
0.041 0.001 0.009 0.013
32.998 11.241 1.146 1.836
0.000 0.000 0.260 0.075
TGF-β1, and negatively correlated with MMP-12 in CHD/DN patients. CHD/DN patients also exhibited higher CCA-IMT, hsCRP, and lower H2S levels than in CHD/non-DN patients. Moreover, in CHD/DN patients, CCA-IMT was negatively correlated with plasma H2S, and positively correlated with hsCRP and LDL. Finally, multiple regression analysis indicated that H2S level was independent association with IMT in CHD/DN patients. To our knowledge, cardiovascular diseases remain the leading cause of morbidity and mortality in patients with chronic kidney disease (CKD), especially with diabetes mellitus [13, 14]. The prevalence of cardiovascular death in CKD patients, especially in those on dialysis therapy, is 10–20 times greater than the age-, gender-, and racematched general population [15, 16]. Some studies have demonstrated that atherosclerosis could induce the increase of the arterial IMT and arterial stiffening, and eventually lead to luminal obstruction with consequent ischemic events, such as myocardial infarction and stroke [17, 18]. In the 1970s, Lindner et al. proposed that atherosclerosis was the main cause of cardiovascular disease in patients with CKD and that its progression was accelerH2S and UAAS
ated by long-term dialysis [19]. Subsequent investigations elucidated abnormal atherosclerosic pathology in patients with CKD may be classified as atherosclerosis, arteriosclerosis, and vascular calcification [20–23]. H2S can be considered the third endogenous gas with modulating actions after nitric oxide and carbon monoxide, which is generated from L-cysteine catalyzed by cystathionine-β-synthase in the brain and cystathionineγ-lyase in the cardiovascular system [24]. Numerous present studies in animals and humans have shown H2S possess important physiological and pathophysiologic functions, such as the regulation of blood pressure [25], inflammation [26], renal damage [27], neurodegenerative diseases [28, 29], and metabolic disorders, including obesity and diabetes [30]. Liu et al. found that H2S can improve wound healing by restoring endothelial progenitor cell functions and activation of Ang-1 in type 2 diabetic mice [31]. Ahmad et al. demonstrated that administration of exogenous H2S donor sodium hydrosulfide (NaHS) can lower the blood pressure and decreases the progression of nephropathy in spontaneously hypertensive rats [32]. Meanwhile, H2S was also confirmed to protect neurons from oxidative stress by increasing the production of the antioxidant glutathione, enhancing cystine/cysteine transporters and suppressing the excessive increase in the intracellular Ca2+ [33, 34]. In the cardiovascular system, H2S protects cardiomyocytes from ischemia-reperfusion injury by preserving mitochondrial function [35] and inducing a hypometabolic state in mice, which is associated with anti-apoptotic, anti-inflammatory, and antioxidant properties [36]. A similar protective effect was also observed in the kidney [37], intestinal [38], pulmonary [39], and hepatic [40] I/R injury. H2S deficiency is implicated in the pathogenesis of hypertension and atherosclerosis [6, 41]. Zhao et al. showed that intravenous bolus injection of H2S could reduce blood pressure in rats and induce vasodilation in isolated blood vessels [42]. A survey from the patients with coronary heart disease has also manifested that plasma H2S is lower compared to normal subjects [43]. By using apolipoprotein E knock-out mice, Wang et al. also found the involvement of the CSE/H2S pathway and its regulatory role in the development and progression of atherosclerosis [44]. It is worth noting that Perna et al. found the low blood levels of H2S in hemodialysis patients for the first time [7]; this declining trend may correlate to the prevalence of hypertension and atherosclerosis, which are important factors influencing the high cardiovascular mortality present in CKD patients. Similarly, our current study further confirmed that the decrease of plasma H2S Blood Purif 2014;38:188–194 DOI: 10.1159/000368883
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Table 3. Correlation coefficients for CCA-IMT and other variables in CHD/DN patients
CHD/DN
H2Sଯ
TGF-DŽ/Smad signaling pathway
TGF-DŽଯ
003ଭ
CCA-IMTଭ
Fig. 1. Interactions of H2S and TGF-β/Smad signaling pathways.
In chronic hemodialysis patients with diabetic nephropathy, the plasma H2S level decreased; this can induce the activation of TGF-β/Smad signaling pathways and cause the downregulation of TGF-β1 and upregulation of MMP-12, eventually resulted in CCA-IMT increase.
Given that high-frequency B-mode ultrasonography is a noninvasive and effective method of detecting carotid artery wall and provides measurement of IMT and presence of plaques [48] , and the increased IMT as well as formation of carotid artery plaque can predict future events of coronary heart disease and systemic subclinical atherosclerosis [49, 50], we evaluated the CCA-IMT in CHD patients. According to the results obtained in this study, the CHD patients with dialysis were further grouped into CHD/DN and CHD/non-DN group, and the content of plasma H2S in these two groups were compared. We found that CHD/DN patients exhibited higher CCA-IMT and lower H2S levels than in CHD/non-DN patients. In CHD/DN patients, CCA-IMT was negatively correlated with plasma H2S and positively correlated with hsCRP and serum LDL. But on multiple regression analysis, H2S level exhibited independent association with IMT in CHD/DN patients. In conclusion, our findings suggested possible linkage between H2S metabolism and TGF-β/Smad signaling pathway modulation abnormalities that may contribute to the development of UAAS in CHD/DN patients.
Acknowledgments
192
Blood Purif 2014;38:188–194 DOI: 10.1159/000368883
This work was supported by National Natural Science Foundation of China (81200543), Beijing Natural Science Foundation (7142057) and Beijing Municipal Health Bureau High-level Medical Professionals Promotion Project (2013–3-016).
Disclosure Statement The authors have no potential conflicts of interest to be disclosed in this article.
References
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Received: April 25, 2014 Accepted: October 3, 2014 Published online: December 16, 2014
Correlation of Lower Concentrations of Hydrogen Sulfide with Atherosclerosis in Chronic Hemodialysis Patients with Diabetic Nephropathy Han Li Su-Juan Feng Gui-Zhi Zhang Shi-Xiang Wang Department of Blood Purification, Beijing Chao-Yang Hospital, Capital Medical University, Nephrology Faculty, Capital Medical University, Beijing, China
Abstract Background/Aims: To explore the relationship between hydrogen sulfide (H2S) and uremic accelerated atherosclerosis (UAAS) in chronic hemodialysis patients with diabetic nephropathy (CHD/DN). Methods: A total of 36 CHD/DN and 32 chronic hemodialyzed non-diabetic patients with chronic glomerulonephritis (CHD/non-DN) were studied. Plasma H2S was measured with a sulfide sensitive electrode. Results: Plasma H2S in CHD/DN was significantly lower than that in CHD/non-DN patients. Plasma H2S was positively correlated with plasma TGF-β1, and negatively correlated with MMP-12 in CHD/DN patients. CHD/DN patients exhibited higher CCAIMT, hsCRP, and lower H2S levels than in CHD/non-DN patients. Moreover, in CHD/DN patients, CCA-IMT was negatively correlated with plasma H2S, and positively correlated with hsCRP and LDL. On multiple regression analysis, H2S levels exhibited independent association with IMT in CHD/DN patients. Conclusions: These findings suggest possible linkage between H2S metabolism and TGF-β/Smad signaling pathway modulation abnormalities that may contribute to the development of UAAS in CHD/DN patients. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 0253–5068/14/0384–0188$39.50/0 E-Mail [email protected] www.karger.com/bpu
Introduction
Atherosclerotic cardiovascular disease (ACVD) is the most common complication and the leading cause of death in chronic hemodialysis patients [1, 2], especially in diabetic patients [3]. The mortality caused by cardiovascular disease in death of end-stage renal disease (ESRD) patients accounted for 45–50% [4]. In this condition, evidence shows that there is an increased incidence and accelerated progress of atherosclerosis compared with that of the conventional ACVD [5]. Hydrogen sulfide (H2S) is a novel gas-signaling molecule, which has the effects of relaxing vascular smooth muscle, inhibiting proliferation of vascular smooth muscle cells and lowering blood pressure [6]. Perna et al. [7] have confirmed that H2S decreased in the plasma of hemodialysis patients and it may have relevance to the pathogenesis of the uremic syndrome manifestations, such as hypertension and atherosclerosis. However, mechanisms of the accelerated formation of atherosclerosis in chronic hemodialysis patients with diabetic nephropathy (CHD/DN) patients are
This work was supported by National Natural Science Foundation of China (81200543), Beijing Natural Science Foundation (7142057) and Beijing Municipal Health Bureau High-level Medical Professionals Promotion Project (2013–3-016).
Shi-Xiang Wang Department of Blood Purification, Beijing Chao-Yang Hospital Capital Medical University, Nephrology Faculty, Capital Medical University Beijing 100020 (China) E-Mail wxy1988 @ 263.net
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Key Words Hemodialysis · Uremic accelerated artherosclerosis · Hydrogen sulfide · TGF-β/Smad signaling pathway
Methods
titia ultrasound interfaces and measured as the distance between the two parallel echogenic lines on the far wall of artery in longitudinal plane image frozen in the screen by electronic calipers [11]. The IMT on the far wall of the bilateral common carotid artery about 10mm proximal to the bifurcation of the carotid artery was measured manually as previously described [12]. The mean IMT was calculated as the average of the three readings of bilateral carotid arteries. Plaque was defined as localized thickening of IMT ≥1.2 mm that did not uniformly involve the whole wall of carotid artery.
Data Sources A total of 36 CHD/DN patients and 32 chronic hemodialyzed non-diabetic patients with chronic glomerulonephritis (CHD/ non-DN) were invited to participate in the study if they were more than 18 years of age, had no residual renal function, and had maintained hemodialysis for more than 3 months. Patients were considered diabetic nephropathy if they were on insulin, oral hypoglycemic agents, diagnosed as diet-controlled diabetes, or had a fasting blood glucose of ≥7 mmol/l, and/or 2 h blood glucose after an oral glucose tolerance test of ≥11.1 mmol/l. Patients were not included in the study if they had heart failure, a recent acute coronary event, cancer, autoimmune disease, and active infection. A standard questionnaire was used for every participant to obtain systematic information regarding conventional cardiovascular risk factors, including hyperlipidemia, hypertension, diabetes, and a family history of cardiovascular disease. As a normal control group, age and gender matched, 30 healthy individuals (15 females and 15 males) were enrolled for measuring plasma H2S in this study. The study was approved by the ethics committee of Beijing Chao-Yang Hospital, Capital Medical University, and written informed consent was obtained from each participant.
Laboratory Investigations Plasma was separated as described above immediately and used freshly for analysis by biochemists, who were blinded to classification of subjects as CHD/DN and CHD/non-DN patients. The concentration of TGF-β1 and MMP-12 was measured in plasma by enzyme linked immunosorbent assay (ELISA). Biochemical and hematological parameters were determined by standard laboratory methods in the clinical laboratory.
Hydrogen Sulfide (H2S) Concentration Measurement The blood of patients was drawn prior to the mid-week dialysis session. Once blood was drawn in plastic Vacutainers using EDTA (1 mg/ml of blood), plasma was immediately obtained through brief 5 min centrifugation at 500 g and rapidly added to the assay mixture. Plasma H2S concentration was measured with a sulfide sensitive electrode as described by Li et al. [8] with modifications. Briefly, 0.5 ml of plasma was added into a test tube containing 0.5 ml of 0.04 g NaOH, 0.035 g EDTA and 0.05 g ascorbic acid. The sulfide sensitive electrode and a reference electrode immersed into the sample together, and record the serum H2S concentration until the reading is stable. H2S concentration was calculated against a calibration curve obtained with known H2S concentrations in a range between 5 and 100 μM, utilizing the H2S donor NaHS [9, 10]. Standard curves were repeated daily with triplicate measurement for each point, and freshly made solutions were utilized at all times.
Subject Characteristics A total number of 68 patients (36 CHD/DN, 32 CHD/ non-DN) with a mean age of 48.1 ± 11.9 (range 20–71 years) and a mean dialysis period of 41.5 ± 18.3 months (range 5–84 months) were included in this study. The CHD/DN group consisted of 20 men and 16 women; the mean age was 48.3 ± 11.5 and the average dialysis period was 39 months (range 5–82 months). The CHD/non-DN group consisted of 17 men and 15 women; the mean age was 47.8 ± 12.5 and the average dialysis period was 45 months (range 8–84 months). There was no significant difference between the two groups in terms of age, sex ratio, dialysis duration, smoking, BMI, Kt/V, Hb, serum creatinine, BUN, TG, Tch, etc.
Common Carotid Artery Ultrasonography Carotid artery ultrasonography was performed by an experienced specialist physician who was specifically trained for the vascular ultrasonography. Carotid B-mode ultrasound measurements were performed using HDI 5000 equipped with a 5–12 MHz linear array transducer (Philips, USA). Subjects were examined in a supine position with the head turned 45° contralateral to the side of scanning. Intima-media thickness (IMT) was defined as the distance between the lumen-intima and the media-adven-
H2S and CCA-IMT in CHD/DN and CHD/Non-DN Patients Plasma H2S level in CHD patients was significantly lower than that in the control group, and the plasma H2S level in the CHD/DN group was significantly lower than that in the CHD/non-DN group. Furthermore, in CHD/ DN patients, the plasma H2S level was positively corre-
H2S and UAAS
Statistical Analysis The SPSS version 13.0 statistics package was employed for the statistical analysis. Measurement data was presented as mean value ± standard deviation (±SD). Comparisons were performed using one-way ANOVA with post hoc analysis (LSD), independentsamples t test or chi-square test. In addition, bivariate correlation analysis and multiple regression analysis were performed. A p < 0.05 was regarded as statistically significant.
Results
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not yet clear. The role of H2S remains unclear in this uremic accelerated atherosclerosis (UAAS) process. Therefore, we evaluated the relationship between H2S and atherosclerosis in chronic hemodialysis patients with diabetes mellitus nephropathy.
Table 1. Characteristics of both study groups
Items
CHD/DN group (n = 36)
CHD/non-DN group (n = 32)
t/χ2 value
p value
Age, years Gender, male/female Dialysis duration, months BMI, kg/m2 Smoking, n (%) Hypertension, n (%) SBP, mm Hg DBP, mm Hg Kt/V Hb, g/l Alb, g/l Creatinine, μmol/l BUN, mmol/l ALT, U/l AST, U/l TG, mmol/l Tch, mmol/l LDL-C, mmol/l RASI, n (%) CCB, n (%) β-blocker, n (%) Mononitrite, n (%)
48.3±11.5 20/16 38.9±18.5 23.6±2.3 10 (27.8) 30 (83.3) 139.8±7.3 80.2±7.1 2.3±0.3 115.9±7.9 34.1±2.7 882.5±102.3 23.7±6.0 18.1±6.4 19.2±7.9 1.45±0.65 4.06±1.07 2.23±0.63 30 (83.3) 28 (77.8) 10 (27.8) 7 (19.4)
47.8±12.5 17/15 44.5±17.8 23.1±1.4 8 (25.0) 26 (81.3) 142.4±10.9 82.6±5.9 2.4±0.3 118.7±8.8 34.8±3.4 902.4±100.3 23.8±4.3 16.8±9.1 16.2±7.3 1.28±0.70 3.92±0.79 2.33±0.56 26 (81.3) 25 (78.1) 8 (25.0) 7 (21.9)
0.180 0.040 1.263 1.184 0.067 0.051 1.179 1.547 0.385 1.398 0.934 0.809 0.077 0.653 1.641 1.058 0.596 0.080 0.051 0.001 0.067 0.061
0.858 0.841 0.211 0.241 0.796 0.822 0.243 0.127 0.702 0.167 0.354 0.421 0.939 0.516 0.106 0.294 0.553 0.937 0.822 0.973 0.796 0.805
Values are means ± SD, unless specified otherwise. BMI = Body mass index; SBP = systolic blood pressure; DBP = diastolic blood pressure; Hb = hemoglobin; Alb = albumin; BUN = blood urea nitrogen; ALT = alaninetransarninase; AST = aspartate aminotransferase; TG = triglyceride; Tch = total cholesterol; LDL-C = low density lipoprotein-cholesterol; CCB = calcium channel blocker; RASI = renin abguitensin system inhibitor.
Correlations Between CCA-IMT and Risk Factors for Atherosclerosis and H2S in CHD/DN Patients In CHD/DN patients, bivariate correlation analysis showed that CCA-IMT was negatively correlated with plasma H2S (r = –0.923, p = 0.000), and positively correlated with hsCRP (r = 0.449, p = 0.006) and serum LDL (r = 0.363, p = 0.029). No correlation with age, gender, dialysis duration, serum TG, Tch, smoking and hypertension (table 3). On multiple regression analysis, the hydrogen sulfide level exhibited independent association with IMT in CHD/DN patients (table 4). 190
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Table 2. Plasma level of H2S and CCA-IMT in CHD/DN and CHD/non-DN patients
Items
CHD/DN group (n = 36)
CHD/non-DN group (n = 32)
Control group (n = 30)
CCA-IMT, mm hsCRP, mmol/l H2S, μmol/l
1.16±0.10a 2.24±0.92a 22.9±8.3a, b
0.91±0.10 1.63±1.12 29.1±7.2b
56.6±6.9
a p < 0.05, compared with CHD/non-DN group; b p < 0.05, compared with the control group.
Discussion
In this study, we found that plasma H2S in CHD/ DN was significantly lower than that in CHD/non-DN patients, and it was positively correlated with plasma Li/Feng/Zhang/Wang
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lated with plasma TGF-β1 (r = 0.887, p = 0.000), and negatively correlated with MMP-12 (r = –0.892, p = 0.000). On the other hand, table 2 also showed that CHD/ DN patients exhibited higher CCA-IMT, hsCRP (highsensitivity C-reactive protein), and lower plasma H2S levels than in CHD/non-DN patients (table 2).
Variables
R
p value
H2S hsCRP Age Gender Dialysis durations Smoking Hypertension TG Tch LDL-C
–0.923 0.449 0.259 0.140 0.144 0.105 0.286 0.097 0.235 0.364
0.000 0.006 0.128 0.416 0.402 0.542 0.091 0.573 0.168 0.029
Table 4. Variables predicting IMT in multiple regression analysis in CHD/DN patients
Variables
Standardized coefficient β
Standard error
t value
p value
Constant H2S hsCRP LDL
1.355 –0.859 0.093 0.140
0.041 0.001 0.009 0.013
32.998 11.241 1.146 1.836
0.000 0.000 0.260 0.075
TGF-β1, and negatively correlated with MMP-12 in CHD/DN patients. CHD/DN patients also exhibited higher CCA-IMT, hsCRP, and lower H2S levels than in CHD/non-DN patients. Moreover, in CHD/DN patients, CCA-IMT was negatively correlated with plasma H2S, and positively correlated with hsCRP and LDL. Finally, multiple regression analysis indicated that H2S level was independent association with IMT in CHD/DN patients. To our knowledge, cardiovascular diseases remain the leading cause of morbidity and mortality in patients with chronic kidney disease (CKD), especially with diabetes mellitus [13, 14]. The prevalence of cardiovascular death in CKD patients, especially in those on dialysis therapy, is 10–20 times greater than the age-, gender-, and racematched general population [15, 16]. Some studies have demonstrated that atherosclerosis could induce the increase of the arterial IMT and arterial stiffening, and eventually lead to luminal obstruction with consequent ischemic events, such as myocardial infarction and stroke [17, 18]. In the 1970s, Lindner et al. proposed that atherosclerosis was the main cause of cardiovascular disease in patients with CKD and that its progression was accelerH2S and UAAS
ated by long-term dialysis [19]. Subsequent investigations elucidated abnormal atherosclerosic pathology in patients with CKD may be classified as atherosclerosis, arteriosclerosis, and vascular calcification [20–23]. H2S can be considered the third endogenous gas with modulating actions after nitric oxide and carbon monoxide, which is generated from L-cysteine catalyzed by cystathionine-β-synthase in the brain and cystathionineγ-lyase in the cardiovascular system [24]. Numerous present studies in animals and humans have shown H2S possess important physiological and pathophysiologic functions, such as the regulation of blood pressure [25], inflammation [26], renal damage [27], neurodegenerative diseases [28, 29], and metabolic disorders, including obesity and diabetes [30]. Liu et al. found that H2S can improve wound healing by restoring endothelial progenitor cell functions and activation of Ang-1 in type 2 diabetic mice [31]. Ahmad et al. demonstrated that administration of exogenous H2S donor sodium hydrosulfide (NaHS) can lower the blood pressure and decreases the progression of nephropathy in spontaneously hypertensive rats [32]. Meanwhile, H2S was also confirmed to protect neurons from oxidative stress by increasing the production of the antioxidant glutathione, enhancing cystine/cysteine transporters and suppressing the excessive increase in the intracellular Ca2+ [33, 34]. In the cardiovascular system, H2S protects cardiomyocytes from ischemia-reperfusion injury by preserving mitochondrial function [35] and inducing a hypometabolic state in mice, which is associated with anti-apoptotic, anti-inflammatory, and antioxidant properties [36]. A similar protective effect was also observed in the kidney [37], intestinal [38], pulmonary [39], and hepatic [40] I/R injury. H2S deficiency is implicated in the pathogenesis of hypertension and atherosclerosis [6, 41]. Zhao et al. showed that intravenous bolus injection of H2S could reduce blood pressure in rats and induce vasodilation in isolated blood vessels [42]. A survey from the patients with coronary heart disease has also manifested that plasma H2S is lower compared to normal subjects [43]. By using apolipoprotein E knock-out mice, Wang et al. also found the involvement of the CSE/H2S pathway and its regulatory role in the development and progression of atherosclerosis [44]. It is worth noting that Perna et al. found the low blood levels of H2S in hemodialysis patients for the first time [7]; this declining trend may correlate to the prevalence of hypertension and atherosclerosis, which are important factors influencing the high cardiovascular mortality present in CKD patients. Similarly, our current study further confirmed that the decrease of plasma H2S Blood Purif 2014;38:188–194 DOI: 10.1159/000368883
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Table 3. Correlation coefficients for CCA-IMT and other variables in CHD/DN patients
CHD/DN
H2Sଯ
TGF-DŽ/Smad signaling pathway
TGF-DŽଯ
003ଭ
CCA-IMTଭ
Fig. 1. Interactions of H2S and TGF-β/Smad signaling pathways.
In chronic hemodialysis patients with diabetic nephropathy, the plasma H2S level decreased; this can induce the activation of TGF-β/Smad signaling pathways and cause the downregulation of TGF-β1 and upregulation of MMP-12, eventually resulted in CCA-IMT increase.
Given that high-frequency B-mode ultrasonography is a noninvasive and effective method of detecting carotid artery wall and provides measurement of IMT and presence of plaques [48] , and the increased IMT as well as formation of carotid artery plaque can predict future events of coronary heart disease and systemic subclinical atherosclerosis [49, 50], we evaluated the CCA-IMT in CHD patients. According to the results obtained in this study, the CHD patients with dialysis were further grouped into CHD/DN and CHD/non-DN group, and the content of plasma H2S in these two groups were compared. We found that CHD/DN patients exhibited higher CCA-IMT and lower H2S levels than in CHD/non-DN patients. In CHD/DN patients, CCA-IMT was negatively correlated with plasma H2S and positively correlated with hsCRP and serum LDL. But on multiple regression analysis, H2S level exhibited independent association with IMT in CHD/DN patients. In conclusion, our findings suggested possible linkage between H2S metabolism and TGF-β/Smad signaling pathway modulation abnormalities that may contribute to the development of UAAS in CHD/DN patients.
Acknowledgments
192
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This work was supported by National Natural Science Foundation of China (81200543), Beijing Natural Science Foundation (7142057) and Beijing Municipal Health Bureau High-level Medical Professionals Promotion Project (2013–3-016).
Disclosure Statement The authors have no potential conflicts of interest to be disclosed in this article.
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