Peritoneal Dialysis International, Vol. 35, pp. 43–51 doi: 10.3747/pdi.2013.00150
0896-8608/15 $3.00 + .00 Copyright © 2015 International Society for Peritoneal Dialysis
DECREASED CIRCULATING KLOTHO LEVELS IN PATIENTS UNDERGOING DIALYSIS AND RELATIONSHIP TO OXIDATIVE STRESS AND INFLAMMATION
Hyung Jung Oh,1,* Bo Young Nam,2,* Mi Jung Lee,1 Chan Ho Kim,1 Hyang Mo Koo,1 Fa Mee Doh,1 Jae Hyun Han,1 Eun Jin Kim,1 Ji Suk Han,1 Jung Tak Park,1 Tae-Hyun Yoo,1 Shin-Wook Kang,1,2 Dae-Suk Han,1 and Seung Hyeok Han1 Division of Nephrology,1 Department of Internal Medicine, College of Medicine, Brain Korea 21,2 Severance Biomedical Science Institute, Yonsei University, Seoul, Korea
Perit Dial Int 2015; 35(1):43–51 www.PDIConnect.com epub ahead of print: 04 Feb 2014 doi:10.3747/pdi.2013.00150
KEY WORDS: ESRD; inflammation; klotho; oxidative stress. * Oh HJ and Nam BY equally contributed to this work.
C
ardiovascular disease is the leading cause of death in patients with end-stage renal disease (ESRD) (1,2). ESRD patients have both traditional and nontraditional risk factors for cardiovascular disease including anemia, inflammation, oxidative stress and abnormal calcium phosphorus metabolism (3–5). Oxidative stress and inflammation are proposed to play a pivotal role in the pathophysiology of uremia and development of cardiovascular complications (6). These two features increase atherosclerotic burden, amplifying cardiovascular risk (3,4), and are clearly evident in patients receiving dialysis (3,6–8). Klotho, an anti-aging gene, is predominantly expressed in the distal convoluted tubules of the kidney, but is also expressed in the brain choroid plexus, pituitary gland, pancreas, and reproductive organs (9). Previous studies showed that klotho is downregulated after kidney injury. Expression of klotho mRNA and protein in the kidney is reduced in experimental models of angiotensin II-induced hypertensive renal damage (10), diabetic nephropathy (11–13), and ischemia-reperfusion acute kidney injury (14). Although findings in humans are lacking, Koh et al. showed that klotho mRNA and protein expression in the kidneys of patients with ESRD and advanced stages of chronic kidney disease (CKD) were significantly lower than in healthy controls (15). Furthermore, we recently showed that circulating klotho levels decrease as CKD stage advances and that levels are inversely correlated with estimated glomerular filtration rate (eGFR) (16). Klotho has two forms. The membrane form is a cofactor in fibroblast growth factor 23 (FGF23) signaling in the kidney. This complex modulates phosphate homeostasis Correspondence to: Seung Hyeok Han, Department of Internal Medicine, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-Gu, Seoul, Korea, 120-752. [email protected] Received 4 June 2013; accepted 18 August 2013. 43
Downloaded from http://www.pdiconnect.com/ at SEVEN OAKS GEN HOSP on June 15, 2015
♦ Introduction: It has been reported that klotho deficiency is associated with oxidative stress and inflammation in experimental kidney disease models. Patients with endstage renal disease (ESRD) are particularly characterized by increased oxidative stress and inflammation. However, little is known about the relationship between these features and klotho in patients with ESRD. ♦ Methods: We conducted a single-center, cross-sectional study of 78 patients receiving peritoneal dialysis (PD). Serum concentrations of klotho, high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), and 8-isoprostane were measured by enzyme-linked immunosorbent assay. To define factors independently associated with klotho, we determined Spearman’s correlation coefficients for between co-variates and conducted multiple linear regression analyses. ♦ Results: Patients were classified by median concentration of klotho. In patients with klotho levels > 329.6 pg/ mL, serum 8-isoprostane and IL-6 levels were significantly higher than in those with klotho levels < 329.6 pg/mL. In correlation analyses, log 8-isoprostane (γ = –0.310, p = 0.006) and log IL-6 (γ = –0.343, p = 0.002) were inversely correlated with log klotho. After adjustment for age, gender, mean arterial pressure, log intact parathyroid hormone, and log IL-6, log 8-isoprostane was independently associated with log klotho (β = –0.158, p = 0.040). However, the significant relationship between klotho and IL-6 was not seen in an adjusted model. ♦ Conclusions: This study showed that circulating klotho levels were significantly associated with 8-isoprostane levels in patients undergoing PD, suggesting a potential link between klotho deficiency and enhanced oxidative stress in ESRD patients.
OH et al.
and vitamin D metabolism (17,18). The soluble form of klotho acts like a hormone, with anti-oxidative stress (19,20) and anti-inflammatory effects (21). These findings and the distinct features of ESRD patients led us to hypothesize that klotho deficiency might contribute to increased oxidative stress and inflammation in ESRD patients. However, no human studies have addressed this issue. Therefore, this study aimed to identify the relationship between klotho, oxidative stress, and inflammation in patients receiving peritoneal dialysis (PD). MATERIALS AND METHODS
JANUARY 2015 - VOL. 35, NO. 1
PDI
centrifuged at 1500 x g, 4°C for 10 min, and supernatants were stored in aliquots at –80°C. Serum soluble klotho was measured by enzyme-linked immunosorbent assay (ELISA) kit (Immuno Biological Laboratories Co., Ltd, Tokyo, Japan) as previously reported (22). Means ± standard deviation (SD) were 6.5 ± 3.3% for interassay and 3.6 ± 1.5% for intra-assay coefficients of variability. Interleukin-6 (IL-6) serum concentration was determined by ELISA (R&D Systems Europe, Abingdon, Oxon, UK) and 8-isoprostane, an oxidative stress marker, was measured in duplicate using a specific enzyme immunoassay kit (Cayman Chemicals, Ann Arbor, MI, USA).
PATIENTS DIALYSIS ADEQUACY, RESIDUAL RENAL FUNCTION AND GLUCOSE EXPOSURE
Urea kinetic studies were conducted using a 24-hour collection of dialysate and urine at study enrollment. Dialysis adequacy was determined by measuring total weekly urea clearance (Kt/Vurea) using the Watson equation (23), and residual renal function (RRF) was determined as the average of urea and creatinine clearance from a 24-hour urine collection. Estimated average glucose exposure was calculated by multiplying the instilled dialysate volume by the respective glu cose concentration. STATISTICAL ANALYSIS
Statistical analysis used SPSS for Windows, version 18.0 (SPSS Inc., Chicago, IL, USA). All data were
DATA COLLECTION
Demographic and clinical data were recorded at study entry. After an overnight fast, venous samples were drawn to assess the following: hemoglobin, blood urea nitrogen, creatinine, albumin, calcium, phosphorus, intact parathyroid hormone (iPTH), total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride. The preceding overnight dwell was regulated to 1.5% dextrose dialysate to standardize glucose load. Body weight and blood pressure were measured in the morning on the same day when the first dialysate of the day was drained out. SERUM SOLUBLE KLOTHO AND INFLAMMATORY AND OXIDATIVE STRESS MARKERS
For measurement of klotho and inflammatory and oxidative stress markers, blood samples were immediately 44
Figure 1 — Flow diagram of patient recruitment and exclusion. ESRD = end-stage renal disease; PD = peritoneal dialysis.
Downloaded from http://www.pdiconnect.com/ at SEVEN OAKS GEN HOSP on June 15, 2015
This single-center, cross-sectional study examined 135 ESRD patients receiving PD at Yonsei University Severance Hospital, Seoul, Korea. Patients were eligible if they were 20 to 75 years old and had maintained PD > 3 months. Exclusion criteria were history of overt infection during the 3 months prior to the study, history of malignancy or other chronic inflammatory disease (e.g., rheumatoid arthritis or systemic lupus erythematosus), and prior history of kidney transplantation. After exclusions, 78 patients were included (Figure 1). We included healthy controls (n = 30) who underwent a routine health examination, had no history of medical disease and were not taking regular medication. This study protocol was approved by the Institutional Review Boards at our institution and all patients provided their written informed consent to participate in the study.
PDI
JANUARY 2015 - VOL. 35, NO. 1
KLOTHO DEFICIENCY AND OXIDATIVE STRESS IN PD
RESULTS BASELINE DEMOGRAPHIC, CLINICAL AND LABORATORY FINDINGS
Circulating klotho levels were significantly lower in patients receiving PD than in healthy controls (329.6 (215.8–464.8) vs 717.8 (490.3–1080.2) pg/mL, p < 0.001), whereas serum concentrations of inflammatory and oxidative stress markers were significantly higher in PD patients compared with in healthy controls (Table 1). Baseline characteristics of study patients are presented in Table 2. Mean age was 48.9 ± 11.0 years, 38 patients (48.7%) were male, and mean PD duration was 76.7 ± 50.6 months. A total of 93.6% and 17.9% of patients
had hypertension and diabetes mellitus, respectively. In addition, 16.7% of patients had cardiovascular disease defined as a history of coronary artery disease, peripheral artery occlusive disease, or cerebrovascular disease. Average body mass index (BMI) was 22.8 ± 3.0 kg/m2, mean arterial pressure (MAP) was 101.3 ± 10.6 mmHg and over 70% of the patients were using renin-angiotensin system blockades. In addition, 23 of 78 patients (29.5%) used one daily exchange of a long-dwell 2.0 L icodextrin PD solution (Extraneal, Baxter Health Corporation, Singapore). Median values were 329.6 (215.8–464.8) pg/mL for klotho, 308.9 (197.5–487.7) ng/mL for 8-isoprostane, and 7.3 (4.8–8.5) pg/mL for IL-6. DEMOGRAPHIC, CLINICAL AND LABORATORY PARAMETERS BY MEDIAN KLOTHO LEVEL
Patients were stratif ied into two groups using median klotho level (329.6 pg/mL) and demographic, clinical, and laboratory parameters were compared between the two groups (Table 1). Compared to patients with klotho ≤ 329.6 pg/mL (L group), patients with klotho > 329.6 pg/mL (H group) had significantly lower MAP (98.9 ± 9.4 vs 103.7 ± 11.4 mmHg, p = 0.042), log 8-isoprostane(2.4 ± 0.4 vs 2.6 ± 0.3, p = 0.023), log IL-6 (0.7 ± 0.1 vs 0.9 ± 0.2, p = 0.044), and serum uric acid levels (6.3 ± 1.0 vs 6.9 ± 1.4 mg/dL, p = 0.019). However, iPTH was significantly higher in the H group than the L group (253.1 vs 189.1 pg/mL, p = 0.037). No significant differences were observed between the two groups in age, gender, BMI, dialysis duration, medication use, comorbid conditions, peritonitis episodes, dialysis adequacy and RRF, or other serum biomarkers.
TABLE 1 Serum Concentrations of Klotho, IL-6, and 8-isoprostane in Healthy Controls and Patients with PD
Variables
PD (n=78)
Control (n=30)
p-value
Age (years) 48.9±11.0 48.4±12.5 0.751 Male gender, n (%) 38 (48.7%) 20 (66.7%) 0.244 BMI (kg/m2) 22.8±3.0 22.6±4.0 0.553 MAP (mmHg) 101.3±10.6 87.1±10.9
0896-8608/15 $3.00 + .00 Copyright © 2015 International Society for Peritoneal Dialysis
DECREASED CIRCULATING KLOTHO LEVELS IN PATIENTS UNDERGOING DIALYSIS AND RELATIONSHIP TO OXIDATIVE STRESS AND INFLAMMATION
Hyung Jung Oh,1,* Bo Young Nam,2,* Mi Jung Lee,1 Chan Ho Kim,1 Hyang Mo Koo,1 Fa Mee Doh,1 Jae Hyun Han,1 Eun Jin Kim,1 Ji Suk Han,1 Jung Tak Park,1 Tae-Hyun Yoo,1 Shin-Wook Kang,1,2 Dae-Suk Han,1 and Seung Hyeok Han1 Division of Nephrology,1 Department of Internal Medicine, College of Medicine, Brain Korea 21,2 Severance Biomedical Science Institute, Yonsei University, Seoul, Korea
Perit Dial Int 2015; 35(1):43–51 www.PDIConnect.com epub ahead of print: 04 Feb 2014 doi:10.3747/pdi.2013.00150
KEY WORDS: ESRD; inflammation; klotho; oxidative stress. * Oh HJ and Nam BY equally contributed to this work.
C
ardiovascular disease is the leading cause of death in patients with end-stage renal disease (ESRD) (1,2). ESRD patients have both traditional and nontraditional risk factors for cardiovascular disease including anemia, inflammation, oxidative stress and abnormal calcium phosphorus metabolism (3–5). Oxidative stress and inflammation are proposed to play a pivotal role in the pathophysiology of uremia and development of cardiovascular complications (6). These two features increase atherosclerotic burden, amplifying cardiovascular risk (3,4), and are clearly evident in patients receiving dialysis (3,6–8). Klotho, an anti-aging gene, is predominantly expressed in the distal convoluted tubules of the kidney, but is also expressed in the brain choroid plexus, pituitary gland, pancreas, and reproductive organs (9). Previous studies showed that klotho is downregulated after kidney injury. Expression of klotho mRNA and protein in the kidney is reduced in experimental models of angiotensin II-induced hypertensive renal damage (10), diabetic nephropathy (11–13), and ischemia-reperfusion acute kidney injury (14). Although findings in humans are lacking, Koh et al. showed that klotho mRNA and protein expression in the kidneys of patients with ESRD and advanced stages of chronic kidney disease (CKD) were significantly lower than in healthy controls (15). Furthermore, we recently showed that circulating klotho levels decrease as CKD stage advances and that levels are inversely correlated with estimated glomerular filtration rate (eGFR) (16). Klotho has two forms. The membrane form is a cofactor in fibroblast growth factor 23 (FGF23) signaling in the kidney. This complex modulates phosphate homeostasis Correspondence to: Seung Hyeok Han, Department of Internal Medicine, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-Gu, Seoul, Korea, 120-752. [email protected] Received 4 June 2013; accepted 18 August 2013. 43
Downloaded from http://www.pdiconnect.com/ at SEVEN OAKS GEN HOSP on June 15, 2015
♦ Introduction: It has been reported that klotho deficiency is associated with oxidative stress and inflammation in experimental kidney disease models. Patients with endstage renal disease (ESRD) are particularly characterized by increased oxidative stress and inflammation. However, little is known about the relationship between these features and klotho in patients with ESRD. ♦ Methods: We conducted a single-center, cross-sectional study of 78 patients receiving peritoneal dialysis (PD). Serum concentrations of klotho, high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), and 8-isoprostane were measured by enzyme-linked immunosorbent assay. To define factors independently associated with klotho, we determined Spearman’s correlation coefficients for between co-variates and conducted multiple linear regression analyses. ♦ Results: Patients were classified by median concentration of klotho. In patients with klotho levels > 329.6 pg/ mL, serum 8-isoprostane and IL-6 levels were significantly higher than in those with klotho levels < 329.6 pg/mL. In correlation analyses, log 8-isoprostane (γ = –0.310, p = 0.006) and log IL-6 (γ = –0.343, p = 0.002) were inversely correlated with log klotho. After adjustment for age, gender, mean arterial pressure, log intact parathyroid hormone, and log IL-6, log 8-isoprostane was independently associated with log klotho (β = –0.158, p = 0.040). However, the significant relationship between klotho and IL-6 was not seen in an adjusted model. ♦ Conclusions: This study showed that circulating klotho levels were significantly associated with 8-isoprostane levels in patients undergoing PD, suggesting a potential link between klotho deficiency and enhanced oxidative stress in ESRD patients.
OH et al.
and vitamin D metabolism (17,18). The soluble form of klotho acts like a hormone, with anti-oxidative stress (19,20) and anti-inflammatory effects (21). These findings and the distinct features of ESRD patients led us to hypothesize that klotho deficiency might contribute to increased oxidative stress and inflammation in ESRD patients. However, no human studies have addressed this issue. Therefore, this study aimed to identify the relationship between klotho, oxidative stress, and inflammation in patients receiving peritoneal dialysis (PD). MATERIALS AND METHODS
JANUARY 2015 - VOL. 35, NO. 1
PDI
centrifuged at 1500 x g, 4°C for 10 min, and supernatants were stored in aliquots at –80°C. Serum soluble klotho was measured by enzyme-linked immunosorbent assay (ELISA) kit (Immuno Biological Laboratories Co., Ltd, Tokyo, Japan) as previously reported (22). Means ± standard deviation (SD) were 6.5 ± 3.3% for interassay and 3.6 ± 1.5% for intra-assay coefficients of variability. Interleukin-6 (IL-6) serum concentration was determined by ELISA (R&D Systems Europe, Abingdon, Oxon, UK) and 8-isoprostane, an oxidative stress marker, was measured in duplicate using a specific enzyme immunoassay kit (Cayman Chemicals, Ann Arbor, MI, USA).
PATIENTS DIALYSIS ADEQUACY, RESIDUAL RENAL FUNCTION AND GLUCOSE EXPOSURE
Urea kinetic studies were conducted using a 24-hour collection of dialysate and urine at study enrollment. Dialysis adequacy was determined by measuring total weekly urea clearance (Kt/Vurea) using the Watson equation (23), and residual renal function (RRF) was determined as the average of urea and creatinine clearance from a 24-hour urine collection. Estimated average glucose exposure was calculated by multiplying the instilled dialysate volume by the respective glu cose concentration. STATISTICAL ANALYSIS
Statistical analysis used SPSS for Windows, version 18.0 (SPSS Inc., Chicago, IL, USA). All data were
DATA COLLECTION
Demographic and clinical data were recorded at study entry. After an overnight fast, venous samples were drawn to assess the following: hemoglobin, blood urea nitrogen, creatinine, albumin, calcium, phosphorus, intact parathyroid hormone (iPTH), total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride. The preceding overnight dwell was regulated to 1.5% dextrose dialysate to standardize glucose load. Body weight and blood pressure were measured in the morning on the same day when the first dialysate of the day was drained out. SERUM SOLUBLE KLOTHO AND INFLAMMATORY AND OXIDATIVE STRESS MARKERS
For measurement of klotho and inflammatory and oxidative stress markers, blood samples were immediately 44
Figure 1 — Flow diagram of patient recruitment and exclusion. ESRD = end-stage renal disease; PD = peritoneal dialysis.
Downloaded from http://www.pdiconnect.com/ at SEVEN OAKS GEN HOSP on June 15, 2015
This single-center, cross-sectional study examined 135 ESRD patients receiving PD at Yonsei University Severance Hospital, Seoul, Korea. Patients were eligible if they were 20 to 75 years old and had maintained PD > 3 months. Exclusion criteria were history of overt infection during the 3 months prior to the study, history of malignancy or other chronic inflammatory disease (e.g., rheumatoid arthritis or systemic lupus erythematosus), and prior history of kidney transplantation. After exclusions, 78 patients were included (Figure 1). We included healthy controls (n = 30) who underwent a routine health examination, had no history of medical disease and were not taking regular medication. This study protocol was approved by the Institutional Review Boards at our institution and all patients provided their written informed consent to participate in the study.
PDI
JANUARY 2015 - VOL. 35, NO. 1
KLOTHO DEFICIENCY AND OXIDATIVE STRESS IN PD
RESULTS BASELINE DEMOGRAPHIC, CLINICAL AND LABORATORY FINDINGS
Circulating klotho levels were significantly lower in patients receiving PD than in healthy controls (329.6 (215.8–464.8) vs 717.8 (490.3–1080.2) pg/mL, p < 0.001), whereas serum concentrations of inflammatory and oxidative stress markers were significantly higher in PD patients compared with in healthy controls (Table 1). Baseline characteristics of study patients are presented in Table 2. Mean age was 48.9 ± 11.0 years, 38 patients (48.7%) were male, and mean PD duration was 76.7 ± 50.6 months. A total of 93.6% and 17.9% of patients
had hypertension and diabetes mellitus, respectively. In addition, 16.7% of patients had cardiovascular disease defined as a history of coronary artery disease, peripheral artery occlusive disease, or cerebrovascular disease. Average body mass index (BMI) was 22.8 ± 3.0 kg/m2, mean arterial pressure (MAP) was 101.3 ± 10.6 mmHg and over 70% of the patients were using renin-angiotensin system blockades. In addition, 23 of 78 patients (29.5%) used one daily exchange of a long-dwell 2.0 L icodextrin PD solution (Extraneal, Baxter Health Corporation, Singapore). Median values were 329.6 (215.8–464.8) pg/mL for klotho, 308.9 (197.5–487.7) ng/mL for 8-isoprostane, and 7.3 (4.8–8.5) pg/mL for IL-6. DEMOGRAPHIC, CLINICAL AND LABORATORY PARAMETERS BY MEDIAN KLOTHO LEVEL
Patients were stratif ied into two groups using median klotho level (329.6 pg/mL) and demographic, clinical, and laboratory parameters were compared between the two groups (Table 1). Compared to patients with klotho ≤ 329.6 pg/mL (L group), patients with klotho > 329.6 pg/mL (H group) had significantly lower MAP (98.9 ± 9.4 vs 103.7 ± 11.4 mmHg, p = 0.042), log 8-isoprostane(2.4 ± 0.4 vs 2.6 ± 0.3, p = 0.023), log IL-6 (0.7 ± 0.1 vs 0.9 ± 0.2, p = 0.044), and serum uric acid levels (6.3 ± 1.0 vs 6.9 ± 1.4 mg/dL, p = 0.019). However, iPTH was significantly higher in the H group than the L group (253.1 vs 189.1 pg/mL, p = 0.037). No significant differences were observed between the two groups in age, gender, BMI, dialysis duration, medication use, comorbid conditions, peritonitis episodes, dialysis adequacy and RRF, or other serum biomarkers.
TABLE 1 Serum Concentrations of Klotho, IL-6, and 8-isoprostane in Healthy Controls and Patients with PD
Variables
PD (n=78)
Control (n=30)
p-value
Age (years) 48.9±11.0 48.4±12.5 0.751 Male gender, n (%) 38 (48.7%) 20 (66.7%) 0.244 BMI (kg/m2) 22.8±3.0 22.6±4.0 0.553 MAP (mmHg) 101.3±10.6 87.1±10.9
