Atherosclerosis 233 (2014) 415e418
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Association of plasma soluble a-klotho with pro-endothelin-1 in patients with type 2 diabetes Jian-Jun Liu a, Sylvia Liu a, Nils G. Morgenthaler b, Melvin D.S. Wong a, Subramaniam Tavintharan c, Chee Fang Sum c, Su Chi Lim c, * a b c
Clinical Research Unit, Khoo Teck Puat Hospital, Republic of Singapore Institut für Experimentelle Endokrinologie, Charité Universitätsmedizin Berlin, Germany Diabetes Centre, Khoo Teck Puat Hospital, 90 Yishun Central, Singapore 768828, Republic of Singapore
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 October 2013 Received in revised form 9 January 2014 Accepted 10 January 2014 Available online 22 January 2014
Objectives: To study the relationship between plasma soluble klotho (sKlotho) and pro-endothelin-1 (proET-1) in patients with type 2 diabetes (T2DM). Subjects and methods: In this cross-sectional study, we recruited 175 T2DM subjects and 56 non-diabetic controls. Plasma sKlotho, proET-1 and extracellular superoxide dismutase (SOD) were measured by ELISA and ILMA, respectively. Results: Plasma sKlotho level in patients with T2DM was lower compared to that in non-diabetic controls (416.8 148.1 vs. 494.6 134.3 pg/ml, p ¼ 0.001) and showed significant interaction with diabetes status in its association with proET-1. Plasma sKlotho was inversely correlated with proET-1 in T2DM (Rho ¼ 0.410, p < 0.0001) but not in non-diabetic controls (Rho ¼ 0.091, p ¼ 0.505). Multivariable linear regression models revealed that sKlotho was independently associated with proET-1 after adjustment for renal filtration function, albuminuria, diabetes duration, HbA1c, systolic and diastolic blood pressure. Conclusions: Plasma sKlotho was associated with proET-1 independent of renal function in patients with T2DM. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Soluble klotho Endothelin-1 Oxidative stress Vascular complication Type 2 diabetes
1. Introduction Vascular complications are the main cause of morbidity and mortality in patients with type 2 diabetes (T2DM) [1]. Although the mechanisms of vasculopathy in patients with diabetes are incompletely understood, chronic inflammation, excessive oxidative stress and endothelial dysfunction may underlie the pathogenesis of atherosclerosis and increased cardiovascular risk in these patients [2]. The aging suppressor a-klotho (often refers to as klotho) is first discovered as membrane protein [3]. Later, it is found that Klotho can also be released into circulation by ectodomain shedding and becomes soluble klotho (sKlotho) [4,5]. Kidney has been thought as the major contributor to plasma sKlotho although it is still being debated [6]. In addition to maintenance of mineral homeostasis and renal protection, klotho plays an important role in vasoprotection [6e8]. At the cellular level, sKlotho exerts its vasoprotective property by reducing oxidative stress and improving endothelial function [4,9].
* Corresponding author. Tel.: þ65 6602 2353; fax: þ65 6602 3772. E-mail address: [email protected] (S.C. Lim). 0021-9150/$ e see front matter Ó 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2014.01.024
Expression of klotho in kidney of db/db diabetic mouse is markedly reduced after only 12 weeks of hyperglycemia, presumably attributes to accumulation of advanced glycation end products [10]. Consistently, klotho expression in kidney in patients with diabetes and preserved renal function is also significantly lower compared with that in non-diabetic controls [11]. These results suggest that reduction in klotho expression may be an early event in diabetic nephropathy, one of the most common microvascular complications in patients with diabetes. Plasma endothelin-1 levels are elevated in patients with T2DM and higher endothelin-1 is associated with poor glycemic control, renal insufficiency and impaired cerebral and cardiovascular circulation [12,13]. It is believed that endothelin-1 is an important mediator of vascular complications in diabetes [12]. Interestingly, recent studies in animal models showed that silencing klotho expression by siRNA led to increased expression of endothelin-1. These data suggest that deficiency in klotho may contribute to pathogenesis of vasculopathy by up-regulating vasoconstrictor endothelin-1 although the relevance of these findings in human is still unknown [14,15]. In this work, we aim to study whether the level of plasma sKlotho is associated with endothelin-1 in patients with T2DM.
416
J.-J. Liu et al. / Atherosclerosis 233 (2014) 415e418
2. Subjects and methods 2.1. Subjects We recruited 175 Chinese subjects with T2DM in a secondary hospital in Singapore. The exclusion criteria include pregnancy, active inflammation, active cancer, fasting plasma glucose above 15 mM or HbA1c above 12%. Subjects with late stage chronic kidney disease (MDRD eGFR < 15 ml/min/1.73 m2) were also excluded from the study. Non-diabetic controls were recruited from a health screening program among healthcare workers from the same hospital. Selection of non-diabetic controls was based on age (45 years old and above) and exclusion criteria. Subjects with active cancer, acute or chronic infection, laboratory evidence of kidney or liver diseases, chronic vascular diseases (stroke, ischemia heart disease and peripheral arterial diseases) and those on immunosuppressive therapy and anti-hyperglycemia medications were excluded from the control group. The study has been approved by Singapore National Health Group Domain Specific Ethical Committee. Written informed consent has been obtained from each participant. 2.2. Biochemical measurements HbA1c was measured using immunoturbidimetric method. Urinary albumin was measured by competitive chemiluminescent enzymatic immunoassay. Estimated glomerular filtration rate (eGFR) was calculated based on Modified Diet in Renal Disease (MDRD) formula. Plasma pro-endothelin-1 (proET-1) and copper/ zinc superoxide dismutase (Cu/Zn-SOD) assay kits were from B.R.A.H.M.S GmbH (Thermo Fisher Scientific, Hennigsdorf, Germany) and these immunoluminometric assays (ILMA) have been described elsewhere [16,17]. Plasma sKlotho was assayed by ELISA (Immuno-Biological Laboratories, Fujioka-shi, Gunma, Japan). 2.3. Statistical analysis Statistical analysis was performed using SPSS for Windows version 21. Normally distributed continuous data were expressed as mean SD. Skewed variables (urinary ACR and proET-1) were expressed as median (inter-quartile) and log-transformed before data analysis. Bivariate correlation was determined by Spearman rank correlation analysis. Multivariable linear regression models were used to study if sKlotho was independently associated with proET-1. Log-transformed proET-1 was entered as a dependent variable, sKlotho was entered as an independent variable, age and gender were entered as covariates (model 1). The model was additionally adjusted for renal function and diabetes disease burden (model 2), blood pressure (model 3), lipids profile (model 4) and Cu/Zn-SOD (model 5). A 2-tailed p value of
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Association of plasma soluble a-klotho with pro-endothelin-1 in patients with type 2 diabetes Jian-Jun Liu a, Sylvia Liu a, Nils G. Morgenthaler b, Melvin D.S. Wong a, Subramaniam Tavintharan c, Chee Fang Sum c, Su Chi Lim c, * a b c
Clinical Research Unit, Khoo Teck Puat Hospital, Republic of Singapore Institut für Experimentelle Endokrinologie, Charité Universitätsmedizin Berlin, Germany Diabetes Centre, Khoo Teck Puat Hospital, 90 Yishun Central, Singapore 768828, Republic of Singapore
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 October 2013 Received in revised form 9 January 2014 Accepted 10 January 2014 Available online 22 January 2014
Objectives: To study the relationship between plasma soluble klotho (sKlotho) and pro-endothelin-1 (proET-1) in patients with type 2 diabetes (T2DM). Subjects and methods: In this cross-sectional study, we recruited 175 T2DM subjects and 56 non-diabetic controls. Plasma sKlotho, proET-1 and extracellular superoxide dismutase (SOD) were measured by ELISA and ILMA, respectively. Results: Plasma sKlotho level in patients with T2DM was lower compared to that in non-diabetic controls (416.8 148.1 vs. 494.6 134.3 pg/ml, p ¼ 0.001) and showed significant interaction with diabetes status in its association with proET-1. Plasma sKlotho was inversely correlated with proET-1 in T2DM (Rho ¼ 0.410, p < 0.0001) but not in non-diabetic controls (Rho ¼ 0.091, p ¼ 0.505). Multivariable linear regression models revealed that sKlotho was independently associated with proET-1 after adjustment for renal filtration function, albuminuria, diabetes duration, HbA1c, systolic and diastolic blood pressure. Conclusions: Plasma sKlotho was associated with proET-1 independent of renal function in patients with T2DM. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Soluble klotho Endothelin-1 Oxidative stress Vascular complication Type 2 diabetes
1. Introduction Vascular complications are the main cause of morbidity and mortality in patients with type 2 diabetes (T2DM) [1]. Although the mechanisms of vasculopathy in patients with diabetes are incompletely understood, chronic inflammation, excessive oxidative stress and endothelial dysfunction may underlie the pathogenesis of atherosclerosis and increased cardiovascular risk in these patients [2]. The aging suppressor a-klotho (often refers to as klotho) is first discovered as membrane protein [3]. Later, it is found that Klotho can also be released into circulation by ectodomain shedding and becomes soluble klotho (sKlotho) [4,5]. Kidney has been thought as the major contributor to plasma sKlotho although it is still being debated [6]. In addition to maintenance of mineral homeostasis and renal protection, klotho plays an important role in vasoprotection [6e8]. At the cellular level, sKlotho exerts its vasoprotective property by reducing oxidative stress and improving endothelial function [4,9].
* Corresponding author. Tel.: þ65 6602 2353; fax: þ65 6602 3772. E-mail address: [email protected] (S.C. Lim). 0021-9150/$ e see front matter Ó 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2014.01.024
Expression of klotho in kidney of db/db diabetic mouse is markedly reduced after only 12 weeks of hyperglycemia, presumably attributes to accumulation of advanced glycation end products [10]. Consistently, klotho expression in kidney in patients with diabetes and preserved renal function is also significantly lower compared with that in non-diabetic controls [11]. These results suggest that reduction in klotho expression may be an early event in diabetic nephropathy, one of the most common microvascular complications in patients with diabetes. Plasma endothelin-1 levels are elevated in patients with T2DM and higher endothelin-1 is associated with poor glycemic control, renal insufficiency and impaired cerebral and cardiovascular circulation [12,13]. It is believed that endothelin-1 is an important mediator of vascular complications in diabetes [12]. Interestingly, recent studies in animal models showed that silencing klotho expression by siRNA led to increased expression of endothelin-1. These data suggest that deficiency in klotho may contribute to pathogenesis of vasculopathy by up-regulating vasoconstrictor endothelin-1 although the relevance of these findings in human is still unknown [14,15]. In this work, we aim to study whether the level of plasma sKlotho is associated with endothelin-1 in patients with T2DM.
416
J.-J. Liu et al. / Atherosclerosis 233 (2014) 415e418
2. Subjects and methods 2.1. Subjects We recruited 175 Chinese subjects with T2DM in a secondary hospital in Singapore. The exclusion criteria include pregnancy, active inflammation, active cancer, fasting plasma glucose above 15 mM or HbA1c above 12%. Subjects with late stage chronic kidney disease (MDRD eGFR < 15 ml/min/1.73 m2) were also excluded from the study. Non-diabetic controls were recruited from a health screening program among healthcare workers from the same hospital. Selection of non-diabetic controls was based on age (45 years old and above) and exclusion criteria. Subjects with active cancer, acute or chronic infection, laboratory evidence of kidney or liver diseases, chronic vascular diseases (stroke, ischemia heart disease and peripheral arterial diseases) and those on immunosuppressive therapy and anti-hyperglycemia medications were excluded from the control group. The study has been approved by Singapore National Health Group Domain Specific Ethical Committee. Written informed consent has been obtained from each participant. 2.2. Biochemical measurements HbA1c was measured using immunoturbidimetric method. Urinary albumin was measured by competitive chemiluminescent enzymatic immunoassay. Estimated glomerular filtration rate (eGFR) was calculated based on Modified Diet in Renal Disease (MDRD) formula. Plasma pro-endothelin-1 (proET-1) and copper/ zinc superoxide dismutase (Cu/Zn-SOD) assay kits were from B.R.A.H.M.S GmbH (Thermo Fisher Scientific, Hennigsdorf, Germany) and these immunoluminometric assays (ILMA) have been described elsewhere [16,17]. Plasma sKlotho was assayed by ELISA (Immuno-Biological Laboratories, Fujioka-shi, Gunma, Japan). 2.3. Statistical analysis Statistical analysis was performed using SPSS for Windows version 21. Normally distributed continuous data were expressed as mean SD. Skewed variables (urinary ACR and proET-1) were expressed as median (inter-quartile) and log-transformed before data analysis. Bivariate correlation was determined by Spearman rank correlation analysis. Multivariable linear regression models were used to study if sKlotho was independently associated with proET-1. Log-transformed proET-1 was entered as a dependent variable, sKlotho was entered as an independent variable, age and gender were entered as covariates (model 1). The model was additionally adjusted for renal function and diabetes disease burden (model 2), blood pressure (model 3), lipids profile (model 4) and Cu/Zn-SOD (model 5). A 2-tailed p value of
