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Gastrointestinal-related Uremic Toxins in Peritoneal Dialysis: A Pilot Study with a 5-year Follow-up
Archives of Medical Research 44 (2013) 535e541
ORIGINAL ARTICLE
Gastrointestinal-related Uremic Toxins in Peritoneal Dialysis: A Pilot Study with a 5-year Follow-up Cheng-jui Lin,a,b,c Chi-feng Pan,a,b Chih-kuang Chuang,c,d,e Hsuan-liang Liu,c Fang-ju Sun,b,f Tuen-jen Wang,g Han-hsiang Chen,a,b and Chih-jen Wua,b,h a
Division of Nephrology, Department of Internal Medicine, Mackay Memorial Hospital, Taipei, Taiwan b Mackay Medicine, Nursing and Management College, Taipei, Taiwan c Institute of Biotechnology, National Taipei University of Technology, Taipei, Taiwan d Division of Genetics and Metabolism, Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan e College of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan f Department of Medical Research, gDepartment of Department of Laboratory Medicine, Mackay Memorial Hospital, Taipei, Taiwan h Graduate Institute of Medical Science, Taipei Medical University, Taipei, Taiwan Received for publication March 2, 2013; accepted September 5, 2013 (ARCMED-D-13-00125).
Background and Aims. P-cresyl sulfate (PCS) and indoxyl sulfate (IS) were not only novel but essential factors associated with cardiovascular disease and mortality in patients with chronic kidney disease and hemodialysis. However, little evidence exams the effect in peritoneal dialysis (PD) patients. Methods. This pilot study recruited 46 stable PD patients in a single medical center. Serum levels of IS, PCS and biochemistry were measured concurrently. Clinical outcomes including cardiovascular, all-cause mortality and PD failure event were recorded during a 5-year follow-up. Results. Serum levels of free and total PCS were lower in patients with residual renal function (11.67 6.92, p 5 0.014, 0.77 0.48, p 5 0.046, respectively). Multivariate Cox regression analysis showed age (HR: 1.07, p 5 0.01), serum CO2 (HR: 0.67, p 5 0.02) and total PCS (HR: 1.05, p !0.01) were independently associated with cardiovascular events; only free PCS (HR: 1.42, p !0.01) reached significant correlation with allcause mortality. Total IS (HR: 1.27, p 5 0.03) significantly correlated with PD failure event after adjusting other confounding factors. KaplaneMeier analysis revealed that patients with higher total and free PCS levels had higher cardiovascular events (log rank p !0.01, log rank p 5 0.05, respectively) and mortality event (log rank p 5 0.02, log rank p 5 0.03, respectively) than those with lower levels. In addition, total IS (log rank p 5 0.04), total PCS (log rank p 5 0.01) and free PCS (log rank p !0.01) could independently predict PD failure event during the study period. Conclusions. Our findings suggest PCS and IS may be a valuable surrogate in predicting poor clinical outcomes in PD patients. Ó 2013 IMSS. Published by Elsevier Inc. Key Words: Protein-bound uremic toxin, p-Cresyl sulfate, Indoxyl sulfate, Peritoneal dialysis.
Introduction
Address reprint requests to: Chih-jen Wu, MD, PhD, Division of Nephrology, Department of Internal Medicine, Mackay Memorial Hospital, 92 Chung San North Road, Section 2, Taipei 104, Taiwan; Phone: (þ886) (2) 25433535; FAX: (þ886) (2) 25433642; E-mail: lincj@ms1. mmh.org.tw
Evidence has shown that cardiovascular disease (CVD) (1,2) is the main cause of death in patients with chronic kidney disease (CKD)—especially in those with end-stage renal disease (ESRD) (3). Atherosclerosis may be preceded by endothelial dysfunction and result in arterial stiffness in dialysis patients. Associated cardiovascular risk factors
0188-4409/$ - see front matter. Copyright Ó 2013 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2013.09.007
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include traditional risk factors such as smoking, diabetes, hyperlipidemia and hypertension (4,5) as well as nontraditional risk factors such as hyperhomocysteinemia, calcium phosphate abnormality, intact parathyroid hormone excess and protein-bound uremic toxins (6e9). Some investigators have recognized that indoxyl sulfate (IS) and P-cresyl sulfate (PCS), two protein-bound uremic toxins, had deleterious effects on endothelial function (10,11) and induced the production of reactive oxygen species in in vitro studies (12,13). IS and PCS were also regarded as a novel surrogate marker in predicting infection event, cardiovascular and all-cause mortality not only in CKD but also in hemodialysis (HD) patients (14e18). Moreover, our recent research showed serum levels of PCS and IS were associated with peripheral artery disease (PAD) and total PCS could be an important determinant of access viability other than traditional or nontraditional risk factors in HD patients (19). These results indicated that IS and PCS played a critical role on clinical outcomes in patients with uremia. However, current studies exploring the effect of IS and PCS on clinical events were focused on a CKD or HD population. Whether this effect can be demonstrated in PD patients remains unclear. In this pilot study we aimed to elucidate the effect of IS and PCS on clinical outcomes in patients undergoing PD during a 5-year follow up. Patients and Methods
determination of albumin. Residual renal function was calculated and expressed as renal Kt/V (rKt/V). Total Kt/ V is equal to peritoneal Kt/V plus renal Kt/V. Patients with daily urine !100 mL were regarded as nonresidual renal function. The normalized protein catabolic rate (nPCR) (g/kg/day) was calculated as a measure of daily protein intake. Serum PCS, IS and hs-CRP were measured twice to obtain an average value. Other biochemical values were measured at the first sampling during after enrollment. Serum IS and PCS were analyzed with LC-MS/MS (4000 QTRAP, Vernon Hills, IL). Briefly, serum samples were prepared and deproteinized by heat denaturation. HPLC was performed at room temperature using a dC18 column (3.0 50 mm, Atlantis, Waters, Milford, MA). The buffers used were (A) 0.1% formic acid and (B) 1 mmol NH4OAc þ 0.1% formic acid in 100% acetonitrile. The flow rate was 0.6 mL/min with a 3.5 min gradient cycling from 90% A/10% Be10% A/90% B. Under these conditions, both PCS and IS were eluted at 2.73 and 2.48 min, respectively. Standard curves for PCS and IS were set at 1, 5, 10, 50, 250, 500 and 1000 mg/L; both were processed in the same manner as the serum samples and correlated with the serum samples with average r2 values of 0.996 0.003. These samples were diluted if the IS or PCS concentration exceeded standard curve. Quantitative results were obtained and calculated in terms of their concentrations (mg/L). The sensitivity of this assay was 1 mg/L for PCS and 1 mg/L for IS.
Study Patients
Endpoint Evaluation
Our study recruited 46 stable ESRD patients on PD from JuneeJuly 2007 in a single medical center. Patients with acute infection and cardiovascular events in the past 3 months, those with malignancy, or those !18 years were excluded from this study. The cause of ESRD included cGN, type 2 diabetic nephropathy, polycystic kidney disease and lupus nephritis. Patient characteristics and biochemical parameters were examined concurrently. Our study was performed in accordance with the principles of the Declaration of Helsinki and approved by the Ethics Committee of the Mackay Memorial Hospital. Informed consent was obtained from all patients.
Patients were followed-up until July 31, 2012. During the study period, clinical events including PD failure event, cardiovascular events and all-cause mortality were reviewed by one independent physician. In order to control the data accuracy, the medical charts of study patients were reviewed for all dialysis and for surgeries. Only patients who experienced PD failure and were switched to HD were recorded as having PD failure events in this study. Cardiovascular event was defined as patients with any one of the following cardiovascular events including death from cardiac causes, myocardial ischemia, nonfatal myocardial infarction, ischemic stroke, or new onset of peripheral vascular disease, whichever developed first. Only one event of cardiovascular event per subject was included in the analysis. Deaths were accurately recorded and the cause of death was categorized as cardiovascular, infectious or other.
Laboratory Assessment Serum samples were obtained twice during the same week after signed permit for all patients. The following tests were performed: blood urea nitrogen (BUN, md/dL), creatinine (Cr, mg/dL), hemoglobin (Hb, g/dL), hematocrit (Hct, %), albumin (g/dL), bicarbonate (mmol/L), calcium (Ca, mg/ dL), phosphate (P, mg/dL), intact-PTH (i-PTH, pg/mL), high-sensitive C-reactive protein (hs-CRP, mg/dL), IS (mg/L) and PCS (mg/L). Serum levels of hs-CRP were measured using a Behring Nephelometer II (Dade Behring, Tokyo, Japan). The bromocresol green method was used for
Statistical Analysis Demographic data were expressed as mean standard deviation (SD). Paired and unpaired t tests were applied for comparison between groups of PD patients (residual vs. nonresidual function or with vs. without event). Cox regression model was used to analyze the relationship between
P-Cresyl and Indoxyl Sulfate in Peritoneal Dialysis
independent variables and clinical outcomes including cardiovascular event, PD failure event and all-cause mortality. All variables with a statistically significant p value in the univariate analysis were included in the multivariate analysis. The KaplaneMeier method was used to estimate cumulative event free rate of time to first cardiovascular event, PD failure event, and overall mortality in PD patients with PCS or IS level above and below the median value. Differences of serum levels of PCS or IS in patients who had or did not have clinical events were examined by paired and unpaired t tests; p !0.05 was considered statistically significant. All statistical analyses were conducted using the SPSS v.17.0 software program (SPSS, Chicago, IL). Results Study Population Demographic and clinical characteristic of study patients are shown in Table 1. Forty six stable patients undergoing PD were recruited into the study and consisted of 21 males (45.6%) and 25 females (54.4%) with a mean age of 47.4 21.8 years. Six patients had diabetes mellitus (13%); 38
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Table 2. Effect of residual renal function on serum levels of uremic toxins No (n 5 25)
Residual renal function Total IS (mg/L) Total PCS (mg/L) Free IS (mg/L) Free PCS (mg/L)
41.96 22.30 4.65 1.35
14.24 13.89 2.56 0.88
Yes (n 5 21)
p
0.345 0.014 0.191 0.046
39.79 11.67 3.85 0.77
9.12 6.92 1.22 0.48
See abbreviations in Table 1.
patients had chronic glomerulosclerosis (82.6%); one patient had polycystic kidney disease (2.2%) and one patient had lupus nephritis (2.2%). The mode of PD included continuous ambulatory peritoneal dialysis (CAPD, n 5 28) and automated peritoneal dialysis (APD, n 5 18) in this study. The average serum total and free PCS was 17.4 14.6, 1.1 1.0 mg/L, respectively; total and free IS was 40.9 15.7, 4.3 2.6 mg/L, respectively. Table 2 showed serum total and free PCS levels ( p 5 0.014, 0.046, respectively) were lower in patients with residual renal function than those without. Serum total and free IS levels did not show significant differences between them.
Table 1. Baseline characteristics of the patients
Event Analysis Patient demographics
PD patients (n 5 46)
Median age (years) Males Time of HD (months) Diabetes cGN PKD SLE CAPD/APD CAD SBP (mmHg) DBP (mmHg) Kt/V rK/V nPCR (g/kg/day) Hct (%) BUN (mg/dL) Creatinine (mg/dL) CO2 (mmol/L) Albumin (g/dL) Calcium (mg/dL) Phosphate (mg/dL) i-PTH (pg/mL) hs-CRP (mg/dL) Total PCS (mg/L) Free PCS (mg/L) Total IS (mg/L) Free IS (mg/L)
47.4 12.8 21 (45.6%) 37.4 27.1 6 (13%) 38 (82.6%) 1 (2.2%) 1 (2.2%) 28/18 3 (6.5%) 137.2 22.2 82.1 15.6 2.1 0.3 0.23 0.3 1.1 0.2 30.9 5.5 61.1 17.3 13.3 3.4 26.2 2.2 3.9 0.3 9.5 0.7 5.7 1.0 382.9 279.4 0.7 0.6 17.4 14.6 1.1 1.0 40.9 15.7 4.3 2.6
PKD, Polycystic kidney disease; SLE, systemic lupus erythematosus; HD, hemodialysis, PD, peritoneal dialysis; CAPD, continuous ambulatory peritoneal dialysis; APD, automated peritoneal dialysis; hs-CRP, high-sensitive C-reactive protein; PCS, p-cresyl sulfate; IS, indoxyl sulfate; i-PTH, intact parathyroid hormone; SBP, systolic blood pressure; DBP, diastolic blood pressure; Hct, hematocrit; BUN, blood urea nitrate. Note: Values expressed as mean SD or percent.
At the end of study, 11 patients were switched to HD due to PD failure with recurrent peritonitis (9) or inadequate dialysis (2). Seventeen of 46 patients were recorded as experiencing a new cardiovascular event. Moreover, seven patients died (three from cardiovascular causes and three from infectious disease) in the final study. Table 3 showed the association between independent parameters and clinical outcomes including cardiovascular event, all-cause mortality and PD failure event. For cardiovascular event analysis, age, Cr, bicarbonate, total PCS and free PCS had significant difference in the univariate Cox regression analysis. Only age (HR: 1.07, p 5 0.01), bicarbonate (HR: 0.67, p 5 0.02) and total PCS (HR: 1.05, p !0.01) were markedly associated with this event in the multivariate analysis. However, it showed that total PCS and total IS were related to PD failure event first, and only total IS (HR: 1.27, p 5 0.03) reached significance after adjusting for confounding factors. As for all-cause mortality, there was no association between independent variables and this event in multivariate analysis, although BUN and free PCS had a significant correlation initially. Figure 1 showed KaplaneMeier curves of time to the first cardiovascular event. Patients were divided into two groups based on the medium value of total (14.42 mg/L) and free PCS (0.81 mg/L). Our results indicated that patients with higher total and free PCS levels were strongly associated with higher rate of cardiovascular event during a 5-year follow up (log rank p !0.01, p 5 0.05, respectively) (Figure 1A and B). There was a significant correlation for total and free PCS levels in regard to all-cause
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Table 3. Uni- and multivariate logistic regression model for evaluating the relationship between independent variables and clinical outcomes in HD patients Cardiovascular event Univariate Cox Regression Analysis Variables
HR
95% CI
p
Gender (F/M) Age (years) DM/Non-DM Dialysis period (months) HTN rKt/V Kt/V nPCR BUN (mg/dL) Creatinine (mg/dL) Hct (%) CO2 (mmol/L) Albumin (g/dL) Ca (mg/dL) P (mg/dL) i-PTH (pg/mL) hs-CRP (mg/dL) Total PCS (mg/L) Free PCS (mg/L) Total IS (mg/L) Free IS (mg/L)
0.53 1.05 2.74 0.99
0.18e1.53 1.01e1.10 0.61e12.41 0.99e1.01
NS 0.03 NS NS
0.47 2.77 0.50 1.22 1.00 0.78 0.99 0.73 1.26 0.76 0.75 0.99 1.02 1.04 1.59 1.01 1.11
0.16e1.35 0.81e9.39 0.08e3.08 0.08e19.08 0.97e1.03 0.63e0.98 0.91e1.09 0.55e0.96 0.27e5.76 0.36e1.57 0.44e1.27 0.99e1.00 0.51e2.06 1.01e1.06 1.13e2.23 0.98e1.05 0.93e1.33
All-cause mortality
Multivariate Cox Regression Analysis HR
95% CI
1.07 1.01e1.39
p
Univariate Cox Regression Analysis HR
0.76 0.01 0.99 1.96 0.99
PD failure event Univariate Cox Regression Analysis
95% CI
p
HR
95% CI
p
0.06e1.98 0.92e1.06 0.22e16.79 0.97e1.01
NS NS NS NS
0.68 1.00 0.42 0.99
0.19e2.37 0.95e1.05 0.00e38.24 0.97e1.01
NS NS NS NS
NS 0.81 0.14e4.40 NS 1.63 0.34e7.68 NS 0.21 0.01e7.28 NS 0.88 0.14e5.61 NS 1.00 0.01e3.18 NS 0.54 0.06e4.87 NS 5.19 0.95e28.01 NS 3.16 0.15e6.55 NS 1.04 1.00e1.08 0.05 1.02 0.98e1.05 0.03 1.14 0.94e1.39 NS 1.01 0.83e1.23 NS 1.06 0.94e1.20 NS 1.04 0.92e1.18 0.02 0.67 0.48e0.95 0.02 1.02 0.71e1.47 NS 1.10 0.82e1.46 NS 0.24 0.18e3.28 NS 0.31 0.04e2.27 NS 1.14 0.39e3.37 NS 0.72 0.28e1.81 NS 1.42 0.70e2.88 NS 1.01 0.55e1.82 NS 0.99 0.99e1.00 NS 0.99 0.99e1.00 NS 1.41 0.62e3.20 NS 0.88 0.33e2.37 !0.01 1.05 1.02e1.08 !0.01 1.02 0.98e1.07 NS 1.03 1.00e1.08 !0.01 1.42 1.10e1.84 !0.01 1.02 0.99e1.06 NS 1.04 0.99e1.11 NS 1.28 1.02e1.60 NS 1.35 0.75e2.43 NS 1.42 0.99e2.16
Multivariate Cox Regression Analysis HR
95% CI
p
NS NS NS NS NS NS NS NS NS NS NS NS NS 0.04 NS 0.02 1.27 1.03e1.61 0.03 NS
NS, not significant; CI, confidence interval.
mortality (log rank p 5 0.02, p 5 0.03, respectively) (Figure 2A and B). Figure 3 revealed KaplaneMeier curves of time to the PD failure event. Serum total PCS (log rank p 5 0.01), free PCS (log rank p !0.01) and total IS (log rank p 5 0.04) were significantly associated with this event. Moreover, average PCS and IS levels in patients with different clinical event were compared. Our results showed that serum total PCS levels were higher in patients with mortality than those without (23.99 9.85 vs. 16.05 13.28 mg/L, p !0.05). Patients with cardiovascular event had higher total and free PCS levels than those without (27.43 15.91 vs. 8.57 9.02 mg/L, 1.74 1.17 vs. 0.31 0.54 mg/L, p !0.01, respectively). In addition, serum levels of total PCS (24.37 13.68 vs. 14.63 14.01 mg/L), free PCS (1.59 0.71 vs. 0.88 1.03 mg/L) and total IS (48.67 11.92 vs. 37.88 16.06 mg/L) were markedly elevated in patients experiencing PD failure event as compared to those without ( p !0.05). Discussion This is the first study to explore the effect of PCS and IS on clinical events with a 5-year follow-up in PD patients. Our study suggested the total, free PCS and total IS levels were independently associated with cardiovascular event and PD
failure event. It indicated PCS and IS may be an alternative marker in predicting clinical outcomes in patient with PD. Cardiovascular mortality in patients on PD or HD therapy is about 10- to 30- fold higher than in the general population (20). In addition to well-known traditional risk factors, there are some specific nontraditional risk factors for dialysis patients (6,7). Accumulation of protein-bound uremic toxins such as PCS or IS was also regarded as a potential risk factor in the development of cardiovascular disease (8,9). Both toxins derived from gut and shared some pathological similarity. Thus, investigators were prompted to conduct studies to elucidate the deleterious effect of the two toxins from in vitro to in vivo. Previous studies have shown that the two toxins will induce the production of reactive oxygen species and result in poor wound healing and endothelial dysfunction, one main reason contributing to CVD in CKD (10e13). Moreover, both toxins have also been regarded as a surrogate marker of infection event, cardiovascular and all-cause mortality in CKD including pre-dialysis and HD patients (9,14e18). Our recent study also suggests that PCS and IS levels were associated with peripheral artery disease and total PCS measurements could possibly be used to predict vascular access failure in HD subjects (19). However, whether these effects can be seen in patients undergoing PD remained unclear until now.
P-Cresyl and Indoxyl Sulfate in Peritoneal Dialysis
539
Figure 1. Kaplan-Meier curves of time to first cardiovascular event. (A) Patients with high (O14.42 mg/L) total PCS concentrations are compared to low (#14.42 mg/L) total PCS concentrations (log rank p !0.01). (B) Patients with high (O0.81 mg/L) free PCS concentrations are compared to low (#0.81 mg/ L) free PCS concentrations (log rank p 5 0.05).
This is the first study to explore the effect of PCS and IS on clinical outcomes including PD failure event, cardiovascular event and all-cause mortality in PD patients during a 5-year follow-up. Our results showed that PCS could predict cardiovascular and mortality events in PD patients after
adjusting for confounding factors, which was consistent with previous results in HD subjects (9,15). It indicated that patients with higher serum PCS levels had a higher rate of CVD and future mortality. In addition, IS and PCS were also found to correlate with PD failure event. Peritonitis
Figure 2. Kaplan-Meier curves of mortality event. (A) Patients with high (O14.42 mg/L) total PCS concentrations are compared to low (#14.42 mg/L) total PCS concentrations (log rank p 5 0.02). (B) Patients with high (O0.81 mg/L) free PCS concentrations are compared to low (#0.81 mg/L) free PCS concentrations (log rank p 5 0.03).
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Figure 3. Kaplan-Meier curves of PD failure event. (A) Patients with high (O14.42 mg/L) total PCS concentrations are compared to low (#14.42 mg/L) total PCS concentrations (log rank p 5 0.01). (B) Patients with high (O0.81 mg/L) free PCS concentrations are compared to low (#0.81 mg/L) free PCS concentrations (log rank p !0.01). (C) Patients with high (O39.33 mg/L) total IS concentrations are compared to low (#39.33 mg/L) total IS concentrations (log rank p 5 0.04).
or tunnel infections were the most common causes for technique failure leading to PD failure (21,22). At the end of study, 11 patients were recorded to have PD failure event including nine patients with recurrent peritonitis and two patients with inadequate dialysis. This inferred that infection was the most frequent cause resulting in PD failure for our patients. Evidence has shown that uremic toxins are involved in the impaired immune response in CKD patients (23,24), which is correlated to an increased risk of infection. Previous in vitro study also revealed that p-cresol will decrease the response of activated
polymorphonuclear leucocytes (25) and inhibits the release of platelet-activating factor by macrophages (26). Thus, it was reasonable to link PCS to PD failure event resulting from infection. Our finding was in line with one previous result that PCS could predict infection-related hospitalization event in an HD cohort (15). On the other hand, IS was first reported to be associated with PD failure event in this study. Based on published data, IS was linked to renal function progression, cardiovascular disease and mortality in pre-dialysis patients (17,18). The precise mechanism regarding the role of IS predicting
P-Cresyl and Indoxyl Sulfate in Peritoneal Dialysis
PD failure event is unclear. From our study we extended the scope of evidence of vascular toxicity for both toxins from patients with pre-dialysis, HD to PD. However, there are some limitations to our study. First, the small patient size was the main limitation and all subjects were enrolled from one regional hospital. Residual renal function, a critical factor related to survival, was not correlated with mortality in this study. Small sample size and low mortality rate of our patients were possible reasons. Second, the effect of peritoneum on the clearance of IS and PCS has not been clarified. Third, how IS is involved in PD failure event is uncertain. Fourth, whether clinical outcomes were affected by AST120 (27,28), one kind of oral charcoal absorbent reported to have significant effect on lowering serum uremic toxins remain to be determined. In conclusion, this pilot study infers that PCS and IS are not consistent; total PCS was related with all-cause mortality and technique failure but not with cardiovascular mortality, supposedly the most susceptible factor for endothelial damage induced by GI toxins. However, further larger, multicenter designed prospective studies are required to verify these issues. Conflict of Interest The authors declare no conflict of interest.
Acknowledgments The authors thank all patients who were involved in this study. This study was supported in part by a grant from the Taiwan National Science Council (NSC 102-2314-B-195-004) and Mackay Memorial Hospital (MMH-103-31).
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ORIGINAL ARTICLE
Gastrointestinal-related Uremic Toxins in Peritoneal Dialysis: A Pilot Study with a 5-year Follow-up Cheng-jui Lin,a,b,c Chi-feng Pan,a,b Chih-kuang Chuang,c,d,e Hsuan-liang Liu,c Fang-ju Sun,b,f Tuen-jen Wang,g Han-hsiang Chen,a,b and Chih-jen Wua,b,h a
Division of Nephrology, Department of Internal Medicine, Mackay Memorial Hospital, Taipei, Taiwan b Mackay Medicine, Nursing and Management College, Taipei, Taiwan c Institute of Biotechnology, National Taipei University of Technology, Taipei, Taiwan d Division of Genetics and Metabolism, Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan e College of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan f Department of Medical Research, gDepartment of Department of Laboratory Medicine, Mackay Memorial Hospital, Taipei, Taiwan h Graduate Institute of Medical Science, Taipei Medical University, Taipei, Taiwan Received for publication March 2, 2013; accepted September 5, 2013 (ARCMED-D-13-00125).
Background and Aims. P-cresyl sulfate (PCS) and indoxyl sulfate (IS) were not only novel but essential factors associated with cardiovascular disease and mortality in patients with chronic kidney disease and hemodialysis. However, little evidence exams the effect in peritoneal dialysis (PD) patients. Methods. This pilot study recruited 46 stable PD patients in a single medical center. Serum levels of IS, PCS and biochemistry were measured concurrently. Clinical outcomes including cardiovascular, all-cause mortality and PD failure event were recorded during a 5-year follow-up. Results. Serum levels of free and total PCS were lower in patients with residual renal function (11.67 6.92, p 5 0.014, 0.77 0.48, p 5 0.046, respectively). Multivariate Cox regression analysis showed age (HR: 1.07, p 5 0.01), serum CO2 (HR: 0.67, p 5 0.02) and total PCS (HR: 1.05, p !0.01) were independently associated with cardiovascular events; only free PCS (HR: 1.42, p !0.01) reached significant correlation with allcause mortality. Total IS (HR: 1.27, p 5 0.03) significantly correlated with PD failure event after adjusting other confounding factors. KaplaneMeier analysis revealed that patients with higher total and free PCS levels had higher cardiovascular events (log rank p !0.01, log rank p 5 0.05, respectively) and mortality event (log rank p 5 0.02, log rank p 5 0.03, respectively) than those with lower levels. In addition, total IS (log rank p 5 0.04), total PCS (log rank p 5 0.01) and free PCS (log rank p !0.01) could independently predict PD failure event during the study period. Conclusions. Our findings suggest PCS and IS may be a valuable surrogate in predicting poor clinical outcomes in PD patients. Ó 2013 IMSS. Published by Elsevier Inc. Key Words: Protein-bound uremic toxin, p-Cresyl sulfate, Indoxyl sulfate, Peritoneal dialysis.
Introduction
Address reprint requests to: Chih-jen Wu, MD, PhD, Division of Nephrology, Department of Internal Medicine, Mackay Memorial Hospital, 92 Chung San North Road, Section 2, Taipei 104, Taiwan; Phone: (þ886) (2) 25433535; FAX: (þ886) (2) 25433642; E-mail: lincj@ms1. mmh.org.tw
Evidence has shown that cardiovascular disease (CVD) (1,2) is the main cause of death in patients with chronic kidney disease (CKD)—especially in those with end-stage renal disease (ESRD) (3). Atherosclerosis may be preceded by endothelial dysfunction and result in arterial stiffness in dialysis patients. Associated cardiovascular risk factors
0188-4409/$ - see front matter. Copyright Ó 2013 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2013.09.007
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include traditional risk factors such as smoking, diabetes, hyperlipidemia and hypertension (4,5) as well as nontraditional risk factors such as hyperhomocysteinemia, calcium phosphate abnormality, intact parathyroid hormone excess and protein-bound uremic toxins (6e9). Some investigators have recognized that indoxyl sulfate (IS) and P-cresyl sulfate (PCS), two protein-bound uremic toxins, had deleterious effects on endothelial function (10,11) and induced the production of reactive oxygen species in in vitro studies (12,13). IS and PCS were also regarded as a novel surrogate marker in predicting infection event, cardiovascular and all-cause mortality not only in CKD but also in hemodialysis (HD) patients (14e18). Moreover, our recent research showed serum levels of PCS and IS were associated with peripheral artery disease (PAD) and total PCS could be an important determinant of access viability other than traditional or nontraditional risk factors in HD patients (19). These results indicated that IS and PCS played a critical role on clinical outcomes in patients with uremia. However, current studies exploring the effect of IS and PCS on clinical events were focused on a CKD or HD population. Whether this effect can be demonstrated in PD patients remains unclear. In this pilot study we aimed to elucidate the effect of IS and PCS on clinical outcomes in patients undergoing PD during a 5-year follow up. Patients and Methods
determination of albumin. Residual renal function was calculated and expressed as renal Kt/V (rKt/V). Total Kt/ V is equal to peritoneal Kt/V plus renal Kt/V. Patients with daily urine !100 mL were regarded as nonresidual renal function. The normalized protein catabolic rate (nPCR) (g/kg/day) was calculated as a measure of daily protein intake. Serum PCS, IS and hs-CRP were measured twice to obtain an average value. Other biochemical values were measured at the first sampling during after enrollment. Serum IS and PCS were analyzed with LC-MS/MS (4000 QTRAP, Vernon Hills, IL). Briefly, serum samples were prepared and deproteinized by heat denaturation. HPLC was performed at room temperature using a dC18 column (3.0 50 mm, Atlantis, Waters, Milford, MA). The buffers used were (A) 0.1% formic acid and (B) 1 mmol NH4OAc þ 0.1% formic acid in 100% acetonitrile. The flow rate was 0.6 mL/min with a 3.5 min gradient cycling from 90% A/10% Be10% A/90% B. Under these conditions, both PCS and IS were eluted at 2.73 and 2.48 min, respectively. Standard curves for PCS and IS were set at 1, 5, 10, 50, 250, 500 and 1000 mg/L; both were processed in the same manner as the serum samples and correlated with the serum samples with average r2 values of 0.996 0.003. These samples were diluted if the IS or PCS concentration exceeded standard curve. Quantitative results were obtained and calculated in terms of their concentrations (mg/L). The sensitivity of this assay was 1 mg/L for PCS and 1 mg/L for IS.
Study Patients
Endpoint Evaluation
Our study recruited 46 stable ESRD patients on PD from JuneeJuly 2007 in a single medical center. Patients with acute infection and cardiovascular events in the past 3 months, those with malignancy, or those !18 years were excluded from this study. The cause of ESRD included cGN, type 2 diabetic nephropathy, polycystic kidney disease and lupus nephritis. Patient characteristics and biochemical parameters were examined concurrently. Our study was performed in accordance with the principles of the Declaration of Helsinki and approved by the Ethics Committee of the Mackay Memorial Hospital. Informed consent was obtained from all patients.
Patients were followed-up until July 31, 2012. During the study period, clinical events including PD failure event, cardiovascular events and all-cause mortality were reviewed by one independent physician. In order to control the data accuracy, the medical charts of study patients were reviewed for all dialysis and for surgeries. Only patients who experienced PD failure and were switched to HD were recorded as having PD failure events in this study. Cardiovascular event was defined as patients with any one of the following cardiovascular events including death from cardiac causes, myocardial ischemia, nonfatal myocardial infarction, ischemic stroke, or new onset of peripheral vascular disease, whichever developed first. Only one event of cardiovascular event per subject was included in the analysis. Deaths were accurately recorded and the cause of death was categorized as cardiovascular, infectious or other.
Laboratory Assessment Serum samples were obtained twice during the same week after signed permit for all patients. The following tests were performed: blood urea nitrogen (BUN, md/dL), creatinine (Cr, mg/dL), hemoglobin (Hb, g/dL), hematocrit (Hct, %), albumin (g/dL), bicarbonate (mmol/L), calcium (Ca, mg/ dL), phosphate (P, mg/dL), intact-PTH (i-PTH, pg/mL), high-sensitive C-reactive protein (hs-CRP, mg/dL), IS (mg/L) and PCS (mg/L). Serum levels of hs-CRP were measured using a Behring Nephelometer II (Dade Behring, Tokyo, Japan). The bromocresol green method was used for
Statistical Analysis Demographic data were expressed as mean standard deviation (SD). Paired and unpaired t tests were applied for comparison between groups of PD patients (residual vs. nonresidual function or with vs. without event). Cox regression model was used to analyze the relationship between
P-Cresyl and Indoxyl Sulfate in Peritoneal Dialysis
independent variables and clinical outcomes including cardiovascular event, PD failure event and all-cause mortality. All variables with a statistically significant p value in the univariate analysis were included in the multivariate analysis. The KaplaneMeier method was used to estimate cumulative event free rate of time to first cardiovascular event, PD failure event, and overall mortality in PD patients with PCS or IS level above and below the median value. Differences of serum levels of PCS or IS in patients who had or did not have clinical events were examined by paired and unpaired t tests; p !0.05 was considered statistically significant. All statistical analyses were conducted using the SPSS v.17.0 software program (SPSS, Chicago, IL). Results Study Population Demographic and clinical characteristic of study patients are shown in Table 1. Forty six stable patients undergoing PD were recruited into the study and consisted of 21 males (45.6%) and 25 females (54.4%) with a mean age of 47.4 21.8 years. Six patients had diabetes mellitus (13%); 38
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Table 2. Effect of residual renal function on serum levels of uremic toxins No (n 5 25)
Residual renal function Total IS (mg/L) Total PCS (mg/L) Free IS (mg/L) Free PCS (mg/L)
41.96 22.30 4.65 1.35
14.24 13.89 2.56 0.88
Yes (n 5 21)
p
0.345 0.014 0.191 0.046
39.79 11.67 3.85 0.77
9.12 6.92 1.22 0.48
See abbreviations in Table 1.
patients had chronic glomerulosclerosis (82.6%); one patient had polycystic kidney disease (2.2%) and one patient had lupus nephritis (2.2%). The mode of PD included continuous ambulatory peritoneal dialysis (CAPD, n 5 28) and automated peritoneal dialysis (APD, n 5 18) in this study. The average serum total and free PCS was 17.4 14.6, 1.1 1.0 mg/L, respectively; total and free IS was 40.9 15.7, 4.3 2.6 mg/L, respectively. Table 2 showed serum total and free PCS levels ( p 5 0.014, 0.046, respectively) were lower in patients with residual renal function than those without. Serum total and free IS levels did not show significant differences between them.
Table 1. Baseline characteristics of the patients
Event Analysis Patient demographics
PD patients (n 5 46)
Median age (years) Males Time of HD (months) Diabetes cGN PKD SLE CAPD/APD CAD SBP (mmHg) DBP (mmHg) Kt/V rK/V nPCR (g/kg/day) Hct (%) BUN (mg/dL) Creatinine (mg/dL) CO2 (mmol/L) Albumin (g/dL) Calcium (mg/dL) Phosphate (mg/dL) i-PTH (pg/mL) hs-CRP (mg/dL) Total PCS (mg/L) Free PCS (mg/L) Total IS (mg/L) Free IS (mg/L)
47.4 12.8 21 (45.6%) 37.4 27.1 6 (13%) 38 (82.6%) 1 (2.2%) 1 (2.2%) 28/18 3 (6.5%) 137.2 22.2 82.1 15.6 2.1 0.3 0.23 0.3 1.1 0.2 30.9 5.5 61.1 17.3 13.3 3.4 26.2 2.2 3.9 0.3 9.5 0.7 5.7 1.0 382.9 279.4 0.7 0.6 17.4 14.6 1.1 1.0 40.9 15.7 4.3 2.6
PKD, Polycystic kidney disease; SLE, systemic lupus erythematosus; HD, hemodialysis, PD, peritoneal dialysis; CAPD, continuous ambulatory peritoneal dialysis; APD, automated peritoneal dialysis; hs-CRP, high-sensitive C-reactive protein; PCS, p-cresyl sulfate; IS, indoxyl sulfate; i-PTH, intact parathyroid hormone; SBP, systolic blood pressure; DBP, diastolic blood pressure; Hct, hematocrit; BUN, blood urea nitrate. Note: Values expressed as mean SD or percent.
At the end of study, 11 patients were switched to HD due to PD failure with recurrent peritonitis (9) or inadequate dialysis (2). Seventeen of 46 patients were recorded as experiencing a new cardiovascular event. Moreover, seven patients died (three from cardiovascular causes and three from infectious disease) in the final study. Table 3 showed the association between independent parameters and clinical outcomes including cardiovascular event, all-cause mortality and PD failure event. For cardiovascular event analysis, age, Cr, bicarbonate, total PCS and free PCS had significant difference in the univariate Cox regression analysis. Only age (HR: 1.07, p 5 0.01), bicarbonate (HR: 0.67, p 5 0.02) and total PCS (HR: 1.05, p !0.01) were markedly associated with this event in the multivariate analysis. However, it showed that total PCS and total IS were related to PD failure event first, and only total IS (HR: 1.27, p 5 0.03) reached significance after adjusting for confounding factors. As for all-cause mortality, there was no association between independent variables and this event in multivariate analysis, although BUN and free PCS had a significant correlation initially. Figure 1 showed KaplaneMeier curves of time to the first cardiovascular event. Patients were divided into two groups based on the medium value of total (14.42 mg/L) and free PCS (0.81 mg/L). Our results indicated that patients with higher total and free PCS levels were strongly associated with higher rate of cardiovascular event during a 5-year follow up (log rank p !0.01, p 5 0.05, respectively) (Figure 1A and B). There was a significant correlation for total and free PCS levels in regard to all-cause
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Table 3. Uni- and multivariate logistic regression model for evaluating the relationship between independent variables and clinical outcomes in HD patients Cardiovascular event Univariate Cox Regression Analysis Variables
HR
95% CI
p
Gender (F/M) Age (years) DM/Non-DM Dialysis period (months) HTN rKt/V Kt/V nPCR BUN (mg/dL) Creatinine (mg/dL) Hct (%) CO2 (mmol/L) Albumin (g/dL) Ca (mg/dL) P (mg/dL) i-PTH (pg/mL) hs-CRP (mg/dL) Total PCS (mg/L) Free PCS (mg/L) Total IS (mg/L) Free IS (mg/L)
0.53 1.05 2.74 0.99
0.18e1.53 1.01e1.10 0.61e12.41 0.99e1.01
NS 0.03 NS NS
0.47 2.77 0.50 1.22 1.00 0.78 0.99 0.73 1.26 0.76 0.75 0.99 1.02 1.04 1.59 1.01 1.11
0.16e1.35 0.81e9.39 0.08e3.08 0.08e19.08 0.97e1.03 0.63e0.98 0.91e1.09 0.55e0.96 0.27e5.76 0.36e1.57 0.44e1.27 0.99e1.00 0.51e2.06 1.01e1.06 1.13e2.23 0.98e1.05 0.93e1.33
All-cause mortality
Multivariate Cox Regression Analysis HR
95% CI
1.07 1.01e1.39
p
Univariate Cox Regression Analysis HR
0.76 0.01 0.99 1.96 0.99
PD failure event Univariate Cox Regression Analysis
95% CI
p
HR
95% CI
p
0.06e1.98 0.92e1.06 0.22e16.79 0.97e1.01
NS NS NS NS
0.68 1.00 0.42 0.99
0.19e2.37 0.95e1.05 0.00e38.24 0.97e1.01
NS NS NS NS
NS 0.81 0.14e4.40 NS 1.63 0.34e7.68 NS 0.21 0.01e7.28 NS 0.88 0.14e5.61 NS 1.00 0.01e3.18 NS 0.54 0.06e4.87 NS 5.19 0.95e28.01 NS 3.16 0.15e6.55 NS 1.04 1.00e1.08 0.05 1.02 0.98e1.05 0.03 1.14 0.94e1.39 NS 1.01 0.83e1.23 NS 1.06 0.94e1.20 NS 1.04 0.92e1.18 0.02 0.67 0.48e0.95 0.02 1.02 0.71e1.47 NS 1.10 0.82e1.46 NS 0.24 0.18e3.28 NS 0.31 0.04e2.27 NS 1.14 0.39e3.37 NS 0.72 0.28e1.81 NS 1.42 0.70e2.88 NS 1.01 0.55e1.82 NS 0.99 0.99e1.00 NS 0.99 0.99e1.00 NS 1.41 0.62e3.20 NS 0.88 0.33e2.37 !0.01 1.05 1.02e1.08 !0.01 1.02 0.98e1.07 NS 1.03 1.00e1.08 !0.01 1.42 1.10e1.84 !0.01 1.02 0.99e1.06 NS 1.04 0.99e1.11 NS 1.28 1.02e1.60 NS 1.35 0.75e2.43 NS 1.42 0.99e2.16
Multivariate Cox Regression Analysis HR
95% CI
p
NS NS NS NS NS NS NS NS NS NS NS NS NS 0.04 NS 0.02 1.27 1.03e1.61 0.03 NS
NS, not significant; CI, confidence interval.
mortality (log rank p 5 0.02, p 5 0.03, respectively) (Figure 2A and B). Figure 3 revealed KaplaneMeier curves of time to the PD failure event. Serum total PCS (log rank p 5 0.01), free PCS (log rank p !0.01) and total IS (log rank p 5 0.04) were significantly associated with this event. Moreover, average PCS and IS levels in patients with different clinical event were compared. Our results showed that serum total PCS levels were higher in patients with mortality than those without (23.99 9.85 vs. 16.05 13.28 mg/L, p !0.05). Patients with cardiovascular event had higher total and free PCS levels than those without (27.43 15.91 vs. 8.57 9.02 mg/L, 1.74 1.17 vs. 0.31 0.54 mg/L, p !0.01, respectively). In addition, serum levels of total PCS (24.37 13.68 vs. 14.63 14.01 mg/L), free PCS (1.59 0.71 vs. 0.88 1.03 mg/L) and total IS (48.67 11.92 vs. 37.88 16.06 mg/L) were markedly elevated in patients experiencing PD failure event as compared to those without ( p !0.05). Discussion This is the first study to explore the effect of PCS and IS on clinical events with a 5-year follow-up in PD patients. Our study suggested the total, free PCS and total IS levels were independently associated with cardiovascular event and PD
failure event. It indicated PCS and IS may be an alternative marker in predicting clinical outcomes in patient with PD. Cardiovascular mortality in patients on PD or HD therapy is about 10- to 30- fold higher than in the general population (20). In addition to well-known traditional risk factors, there are some specific nontraditional risk factors for dialysis patients (6,7). Accumulation of protein-bound uremic toxins such as PCS or IS was also regarded as a potential risk factor in the development of cardiovascular disease (8,9). Both toxins derived from gut and shared some pathological similarity. Thus, investigators were prompted to conduct studies to elucidate the deleterious effect of the two toxins from in vitro to in vivo. Previous studies have shown that the two toxins will induce the production of reactive oxygen species and result in poor wound healing and endothelial dysfunction, one main reason contributing to CVD in CKD (10e13). Moreover, both toxins have also been regarded as a surrogate marker of infection event, cardiovascular and all-cause mortality in CKD including pre-dialysis and HD patients (9,14e18). Our recent study also suggests that PCS and IS levels were associated with peripheral artery disease and total PCS measurements could possibly be used to predict vascular access failure in HD subjects (19). However, whether these effects can be seen in patients undergoing PD remained unclear until now.
P-Cresyl and Indoxyl Sulfate in Peritoneal Dialysis
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Figure 1. Kaplan-Meier curves of time to first cardiovascular event. (A) Patients with high (O14.42 mg/L) total PCS concentrations are compared to low (#14.42 mg/L) total PCS concentrations (log rank p !0.01). (B) Patients with high (O0.81 mg/L) free PCS concentrations are compared to low (#0.81 mg/ L) free PCS concentrations (log rank p 5 0.05).
This is the first study to explore the effect of PCS and IS on clinical outcomes including PD failure event, cardiovascular event and all-cause mortality in PD patients during a 5-year follow-up. Our results showed that PCS could predict cardiovascular and mortality events in PD patients after
adjusting for confounding factors, which was consistent with previous results in HD subjects (9,15). It indicated that patients with higher serum PCS levels had a higher rate of CVD and future mortality. In addition, IS and PCS were also found to correlate with PD failure event. Peritonitis
Figure 2. Kaplan-Meier curves of mortality event. (A) Patients with high (O14.42 mg/L) total PCS concentrations are compared to low (#14.42 mg/L) total PCS concentrations (log rank p 5 0.02). (B) Patients with high (O0.81 mg/L) free PCS concentrations are compared to low (#0.81 mg/L) free PCS concentrations (log rank p 5 0.03).
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Figure 3. Kaplan-Meier curves of PD failure event. (A) Patients with high (O14.42 mg/L) total PCS concentrations are compared to low (#14.42 mg/L) total PCS concentrations (log rank p 5 0.01). (B) Patients with high (O0.81 mg/L) free PCS concentrations are compared to low (#0.81 mg/L) free PCS concentrations (log rank p !0.01). (C) Patients with high (O39.33 mg/L) total IS concentrations are compared to low (#39.33 mg/L) total IS concentrations (log rank p 5 0.04).
or tunnel infections were the most common causes for technique failure leading to PD failure (21,22). At the end of study, 11 patients were recorded to have PD failure event including nine patients with recurrent peritonitis and two patients with inadequate dialysis. This inferred that infection was the most frequent cause resulting in PD failure for our patients. Evidence has shown that uremic toxins are involved in the impaired immune response in CKD patients (23,24), which is correlated to an increased risk of infection. Previous in vitro study also revealed that p-cresol will decrease the response of activated
polymorphonuclear leucocytes (25) and inhibits the release of platelet-activating factor by macrophages (26). Thus, it was reasonable to link PCS to PD failure event resulting from infection. Our finding was in line with one previous result that PCS could predict infection-related hospitalization event in an HD cohort (15). On the other hand, IS was first reported to be associated with PD failure event in this study. Based on published data, IS was linked to renal function progression, cardiovascular disease and mortality in pre-dialysis patients (17,18). The precise mechanism regarding the role of IS predicting
P-Cresyl and Indoxyl Sulfate in Peritoneal Dialysis
PD failure event is unclear. From our study we extended the scope of evidence of vascular toxicity for both toxins from patients with pre-dialysis, HD to PD. However, there are some limitations to our study. First, the small patient size was the main limitation and all subjects were enrolled from one regional hospital. Residual renal function, a critical factor related to survival, was not correlated with mortality in this study. Small sample size and low mortality rate of our patients were possible reasons. Second, the effect of peritoneum on the clearance of IS and PCS has not been clarified. Third, how IS is involved in PD failure event is uncertain. Fourth, whether clinical outcomes were affected by AST120 (27,28), one kind of oral charcoal absorbent reported to have significant effect on lowering serum uremic toxins remain to be determined. In conclusion, this pilot study infers that PCS and IS are not consistent; total PCS was related with all-cause mortality and technique failure but not with cardiovascular mortality, supposedly the most susceptible factor for endothelial damage induced by GI toxins. However, further larger, multicenter designed prospective studies are required to verify these issues. Conflict of Interest The authors declare no conflict of interest.
Acknowledgments The authors thank all patients who were involved in this study. This study was supported in part by a grant from the Taiwan National Science Council (NSC 102-2314-B-195-004) and Mackay Memorial Hospital (MMH-103-31).
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