Despite technological advances and optimization

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1 Peritoneal Dialysis International, Vol. 23, pp Printed in Canada. All rights reserved /03 $ Copyright 2003 International Society for Peritoneal Dialysis C-REACTIVE PROTEIN AND ANTI-CHLAMYDIA PNEUMONIAE ANTIBODIES AS RISK FACTORS OF CARDIOVASCULAR DEATH IN INCIDENT PATIENTS ON PERITONEAL DIALYSIS Ramón Paniagua, 1 Yolanda Frías, 1 Maria de Jesús Ventura, 1 Ernesto Rodríguez, 1 María Elena Hurtado, 2 Guadalupe Alcántara, 2 Roberto Vázquez, 3 Ruth Ortiz, 3 Mario Salcedo, 4 Maria Elena Rios, 4 Julio Kaji, 5 and Dante Amato 1 Unidad de Investigación Médica en Enfermedades Nefrológicas, Hospital de Especialidades CMN S-XXI IMSS 1 ; Hospital General Regional 25 IMSS 2 ; Hospital General de Zona IMSS, 3 Juárez, Chihuahua; Hospital General de Zona IMSS, 4 Durango, Durango; Hospital Regional 1 de Octubre, 5 ISSSTE, Mexico City, México Objective: Recently it has been pointed out that inflammation and infections caused by germs such as Chlamydia pneumoniae are independent cardiovascular risk factors for the general population, but information about these relationships in dialysis patients is scarce. This work was done to analyze the association of C-reactive protein (CRP) and IgG anti-chlamydia pneumoniae antibodies (anti-chlp-igg) as independent cardiovascular risk factors in incident patients on continuous ambulatory peritoneal dialysis (CAPD). Design: Single-cohort, prospective observational study. Setting: Three CAPD centers from the Instituto Mexicano del Seguro Social, and one from the Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado, Mexico. Patients: A cohort of 75 adult incident patients on CAPD, without clinical signs of congestive heart failure, coronary heart disease, or peripheral arterial insufficiency. No restrictions for age, gender, or cause of renal failure were applied. Primary Outcome: Mortality. Methods: Demographic variables, body composition by electrical bioimpedance, serum glucose, urea, creatinine, lipids, homocysteine, nutritional markers (albumin, prealbumin, and transferrin), CRP, and anti-chlp-igg were measured and registered at the time of the first admission. When a patient died, the cause of death was determined by review of the clinical chart. Results: Mean follow-up time was patientmonths. There were 14 cardiovascular deaths. CRPwas positive (> 10 mg/l) in 64% of the patients, and anti-chlp- IgG in 64%; 29% of the patients were positive for both markers. The relative risk for cardiovascular mortality was 6.23 for patients positive for either CRP or anti-chlp-igg, and increased to 9.52 when both markers were positive. Correspondence to: R. Paniagua, Coordinación de Investigación en Salud, 4 Piso, Bloque B, Unidad de Congresos, Centro Médico Nacional S-XXI, IMSS, Av. Cuauhtémoc 330,Col. Doctores, México. jpaniaguas@cis.gob.mx Received 29 July 2002; accepted 24 October Multivariate analysis revealed that CRP and anti-chlp-igg were stronger cardiovascular death predictors than age, diabetes, and nutritional status. Conclusion: These data suggest that inflammation and the presence of Chlamydia pneumoniae infections are important predictors of cardiovascular death in patients on CAPD. Perit Dial Int 2003; 23: KEY WORDS: C-reactive protein; Chlamydia pneumoniae; cardiovascular death risk; end-stage renal disease; mortality. Despite technological advances and optimization of dialysis programs, mortality rates remain high in patients with end-stage renal disease (ESRD). The leading causes of mortality are cardiovascular disorders, accounting for 50% of deaths. The risk for cardiovascular death is 30-fold higher in patients with ESRD than in the general population (1,2). The most frequent causes of death are ischemic heart disease with lesions of the coronary arteries or lesser vessels, and congestive heart failure. This wide predominance of cardiovascular mortality among ESRD patients has prompted an exhaustive search for the underlying pathophysiological mechanisms and modifiable risk factors. Age, arterial hypertension, diabetes, obesity, and tobacco smoking have been recognized as the main risk factors among ESRD patients, as in the general population (1,2). Other risk factors, such as hyperlipidemia, remain controversial. For instance, there is a U-shaped relationship between cholesterol and cardiovascular disease; the risk of dying is higher in both extremes of the distribution and lower at the middle part, that is, the normal range of cholesterol concentrations (3). Even the roles of substances strongly associated with cardiovascular risk have been questioned, such as

2 PDI MARCH 2003 VOL. 23, NO. 2 CRP, CHLAMYDIA PNEUMONIAE, AND MORTALITY IN CAPD lipoprotein(a) since it is considered an acute-phase reaction marker (4,5). Homocysteine (Hcy) level has also been considered a cardiovascular risk marker (6), but there are reports in which the association between high Hcy levels and increased cardiovascular risk could not be demonstrated(7). In recent years, evidence has been accumulating on the importance of nontraditional cardiovascular risk factors. Hypoalbuminemia, initially regarded only as a nutritional marker, is now also considered a negative, acute-phase reaction marker (8,9). Therefore, hypoalbuminemia in peritoneal dialysis (PD) may signify protein malnutrition, chronic inflammation, and/ or extracellular volume excess. The concept that hypoalbuminemia may be a chronic inflammation marker is further supported by the inverse relationship between serum albumin concentration and C-reactive protein (CRP), widely accepted as an inflammation marker, and by the realization that CRP is a strong and independent cardiovascular risk marker (9 11). The acceptance of inflammation as a pathophysiologic mechanism involved in cardiovascular mortality calls for the identification of the source of the inflammatory process. In ESRD patients, the uremia itself may be the source. For both non-kidney and kidney patients, it has been suggested that infections caused by bacterial agents, such as Chlamydia pneumoniae or Helicobacter pylori, or viral agents, such as cytomegalovirus, may be directly related to the development of atheromatous lesions, or at least may be the source of the inflammatory process (12 17). The objective of the present study was to assess the relationship of cardiovascular death and nontraditional risk factors, such as CRP, anti-chlamydia pneumoniae antibodies, and serum Hcy levels, in patients beginning treatment with continuous ambulatory peritoneal dialysis (CAPD). MATERIAL AND METHODS DESIGN This was a study of a cohort of incident CAPD patients from three centers of the Instituto Mexicano del Seguro Social, and one from the Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado, Mexico. The study protocol was approved by the Institutional Review Boards of all the participant dialysis centers. Individual informed consent was obtained from every patient. PATIENTS Adult patients (age 18 years) with less than 30da ys from the beginning of dialysis treatment were considered for inclusion. No further selection by age, gender, or cause of ESRD was done. Patients with clinical data of coronary artery disease, congestive heart failure, or peripheral artery disease at the beginning of the study were not considered for inclusion. Patients receiving immunosuppressive therapy, that were seropositive for hepatitisb or HIV, or that had malignancies were not included. Patients lost to follow-up and those that moved to another town or refused to remain in the study were excluded. All the participants received a standardized dialysis dose of four 2-L exchanges per 24 hours. PROCEDURES Demographic data, cause of ESRD, and cardiovascular comorbidity data were gathered and registered. Body composition was assessed by electrical bioimpedance (Biodynamics, Seattle, Washington, USA). A venous blood sample was drawn from an antecubital vein and the serum was separated and kept frozen at 70 C until assayed. MEASUREMENTS Glucose, urea, creatinine, total cholesterol, highdensity lipoprotein (HDL) cholesterol, and triglycerides were measured in the serum sample using conventional methods (Synchron CX-5; Beckman Instruments, Brea, California, USA). Serum Hcy was measured by HPLC with a fluorescence detector (Waters474; Waters Corp., Milford, Massachusetts, USA), using mercaptopropionylglycine as internal standard (18). Albumin, prealbumin, transferrin, apolipoproteina-1 (apoa-1), apolipoproteinb (apob), and CRP were measured by nephelometry using specific antibodies (Array; Beckman Instruments), considered high sensitivity methods. IgG type anti-chlamydia antibody (anti-chlp-igg) titers were measured by solid-phase enzyme immunoassay (Lab Systems OY, Helsinki, Finland). CRP was considered positive when levels were 10mg/L, and anti-chlp- IgG when titers were 35U. ASSESSMENTS The cause of death was determined by review of the clinical charts by a team experienced in such tasks (RP, DA, MJV). There were no records missing. A composite index for nutritional status, including albumin, prealbumin, transferrin, and lean body mass, was used for the regression analysis. POWER CALCULATION Because of the high odds ratio (OR) and relative risk (RR) values reported for association between 133

3 PANIAGUA et al. MARCH 2003 VOL. 23, NO. 2 PDI mortality and anti-chlamydia antibodies or CRP [ranging from 6 to 20 (12,13,15)], the sample size of 75patients is powered at 90% to detect a significant association, with a 95% confidence level. STATISTICS Results are presented as mean ± standard deviation for continuous variables, and as proportions for nominal variables. Univariate analysis was done by one-way ANOVA for continuous variables and chisquared for proportions. Pearson s correlation coefficient was calculated for albumin and CRP. Multivariate analysis was done by logistic regression. Only the variables that were significantly associated with mortality in the univariate analyses were introduced into the logistic regression model: age, gender, diabetes mellitus, cholesterol, HDL cholesterol, apoa/ apob ratio, CRP, anti-chlp-igg, and the composite nutritional index, including albumin, prealbumin, transferrin, and lean body mass (as measured by electrical bioimpedance). Only anti-chlp-igg and CRP were included as dichotomous values; the rest of the quantitative variables were considered continuous scales. All the statistical analyses were done with the software SPSS v8.0 for Windows (SPSS, Chicago, Illinois, USA). RESULTS The studied cohort consisted of 75patients followedup for patient-months (range months). Fifteen patients underwent renal transplantation (they were censored at transplantation time) and 2more were exc luded from the analysis because they were lost to follow-up. There were 18deaths, 14 from cardiovascular causes. The cardiovascular death rate was 21.87deaths/100 patient-years. The specific causes of death are shown in Table1. Forty-eight patients (64%) were positive for CRP and 48 (64%) were positive for anti-chlp-igg; 29patients (39%) were positive for both markers. When the patients were classified according to CRP or anti- Chlp-IgG, subgroups positive for CRP and positive for anti-chlp-igg showed lower serum albumin (1.74 ± 0.67g/dL vs 2.41 ± 0.77g/dL, p< 0.01) and triglyceride (146 ± 77mg/dL vs 189 ± 102mg/dL, p< 0.05) levels than the other subgroups. There were no differences for age, gender, or diabetic status (Table2). The RR for cardiovascular mortality was 6.23 [95% confidence interval (CI) , p< 0.01] for the positive CRP subgroup, and the RR for cardiovascular mortality was also 6.23 (95% CI , p< 0.01) for the positive anti-chlp-igg group. When both markers were positive, the RR was higher, 9.52 (95% CI , p< 0.001). 134 TABLE 1 Specific Causes of Death in Cardiovascular (CVD) andnoncardiovascular Death Subgroups Non-CVD CVD Cause of death death death Total Pneumonia 2 2 Electrolytic disorders 1 1 Sepsis 1 1 Acute myocardial infarction 3 3 Congestive heart failure 3 3 Arrhythmia 2 2 Stroke 2 2 Hypertensive crisis 1 1 Hypotension 1 1 Pericarditis 1 1 Pulmonary edema 1 1 Total There was a negative correlation between serum albumin and CRP (r= 0.41, p< 0.01), determining a high colinearity of these two variables in the multivariate models. Therefore, a composite index of the nutritional status, including albumin, prealbumin, transferrin, and lean body mass, was introduced in the regression analysis. Table3 shows the results of the multivariate analysis. The only statistically significant independent variables were positive CRP and positive anti-chlp-igg. DISCUSSION Our data suggest that the presence of an inflammatory process, probably of infectious origin, plays a relevant role in the development of cardiovascular mortality in incident patients on CAPD. The significance of inflammation as a risk factor for cardiovascular death may be higher than that of other traditional risk factors, such as lipid disorders, age, and malnutrition. However, the inflammatory component may be a covariant rather than a causal factor. The study has some limitations. Well-known lifestyle-related risk factors such as smoking and exercising were not considered. The CRP and anti-chlp- IgG levels were measured only at the beginning of dialysis therapy and there are no follow-up data regarding these variables. Additionally, some causes of death classified as being from cardiovascular origin actually may not be related to atherosclerosis; for instance, hypertensive crisis, pulmonary edema, and congestive heart failure may depend more on bad fluid control than on inflammatory or vascular diseases, and pericarditis could have had a viral origin. On the other hand, with respect to external validity, it should be remembered that Mexican PD patients have substantially lower serum albumin values, their diets con-

4 PDI MARCH 2003 VOL. 23, NO. 2 CRP, CHLAMYDIA PNEUMONIAE, AND MORTALITY IN CAPD TABLE 2 Demographic, Biochemical, and Mortality Data in Patients Grouped by Positivity for C-Reactive Protein (CRP) and Anti-Chlamydia Pneumoniae Antibodies (Anti-Chlp-IgG) CRP(+) CRP(+) CRP( ) CRP( ) Variable Anti-Chlp-IgG(+) Anti-Chlp-IgG( ) Anti-Chlp-IgG(+) Anti-Chlp-IgG( ) N Gender (M/F) 19/10 12/7 9/10 3/5 Diabetes (+) Age (years) 54.10± ± ± ±14.61 Systolic blood pressure (mmhg) 138±15 143±26 147±20 151±24 Diastolic blood pressure (mmhg) 89±10 84±16 86±15 90±12 Serum creatinine (mg/dl) 11.3± ± ± ±2.4 BUN (mg/dl) 65.5± ± ± ±29.8 Total cholesterol (mg/dl) 190±84 188±74 194±51 195±38 Triglycerides (mg/dl) 146±77 a 192± ± ±49 Apolipoprotein A-1 (mg/dl) 115±28 123±30 124±26 123±25 Apolipoprotein B (mg/dl) 101±52 98±36 105±32 102±22 HDL (mg/dl) 39.2± ± ± ±9.3 Hemoglobin (g/dl) 8.5± ± ± ±1.8 Homocysteine (mg/dl) 42.0± ± ± ±19.2 Albumin (g/dl) 1.74±0.67 b 2.16± ± ±0.79 Prealbumin (mg/dl) 20.7± ± ± ±2.5 Transferrin (mg/dl) 210±43 224±38 224±36 231±43 Weight (% of ideal weight) 91.0± ± ± ±17.3 Body fat (% of body weight) 18.8± ± ± ±5.6 Deaths 15 b BUN = blood urea nitrogen; HDL = high-density lipoprotein. a p < 0.05 versus other groups. b p < 0.01 versus other groups. TABLE 3 Results of the Multivariate Analysis with Cardiovascular Death as the Dependent Variable Factor Reference group (OR=1.00) OR 95% CI p Value Age Per 1-year increment Gender Male Diabetes mellitus Without diabetes Cholesterol Per 1-mg increment HDL cholesterol Per 1-mg increment ApoA/apoB Per 1-unit increment CRP Negative (<1.0 mg/dl) Anti-Chlp-IgG Negative (<35 units) Nutritional index a Per 1-unit increment b OR= odds ratio; CI = confidence interval; HDL = high-density lipoprotein; Apo= apolipoprotein; CRP= C-reactive protein; Anti-Chlp-IgG= anti- Chlamydia pneumoniae antibodies. a Composed of albumin, prealbumin, transferrin, and lean body mass (measured by electrical bioimpedance). b Arbitrary units. tain less protein, and their height and weight tend to be lower than those of patients from industrialized countries. Therefore, generalization of these findings to different populations should be done with caution. For a long time, it has been recognized that patients with ESRD are at a higher risk for cardiovascular mortality than is the general population (1,2). The pathophysiological substrate is an accelerated atherosclerotic process. Among the many atherosclerosis-related metabolic factors, we decided to analyze different types of lipids. As a group, our whole population had high total cholesterol, triglycerides, apoa-1, and apob levels, as well as low HDL cholesterol levels. However, we did 135

5 PANIAGUA et al. MARCH 2003 VOL. 23, NO. 2 PDI not find any relationship between lipid levels and cardiovascular mortality. This lack of correlation may be due to malnutrition. As shown in Table2, nutritional markers and body composition are compatible with significant malnutrition in the whole population, and more strikingly in the subgroup of patients positive for CRP and anti-chlp-igg. Individually, some patients that died from cardiovascular causes had low cholesterol and triglyceride levels, which may be indicative of malnutrition. There are reports that the association between cholesterol levels and mortality is Ushaped. Therefore, mortality is higher in patients with high or low cholesterol concentration compared with patients with normal cholesterol concentration (3). This nonlinear relationship of mortality and risk of death has been attributed to the confounding effect of malnutrition. Our population, as a whole, showed Hcy levelshigher than 15mg/dL, a figure considered the upper normal limit. However, there was no correlation between Hcy and CRP or anti-chlp-igg. Moreover, Hcy levels were not different between the subgroup of patients that died from cardiovascular causes and the rest of the subjects. The univariate analysis did not disclose Hcy level as a risk factor for cardiovascular mortality. This result is not surprising. Hyperhomocysteinemia has been associated with atherosclerosis-related clinical outcomes, in both the general population and ESRD patients (6), but there are also reports that a low Hcy level is an independent predictor of death (7). This situation may be analogous to the relationship between cardiovascular death and cholesterol levels previously commented on. In that report (7), an inverse correlation between Hcy and serum albumin was found, suggesting a role for malnutrition as a confounding factor for the interpretation of the Hcy level effect. In the present study and in others, CRP was strongly associated with cardiovascular mortality and it was also correlated with hypoalbuminemia. In previous reports, it has been pointed out that CRP is frequently increased in ESRD patients, both before and after the beginning of substitutive therapy (11). The mechanism of why ESRD patients have high CRP remains unknown, but it has been attributed to the underlying renal disease itself, and to the bioincompatibility of hemodialysis membranes and PD solutions. Our results support the concept that endothelial damage and atherosclerosis have an inflammatory background. C-reactive protein is a nonspecific inflammatory marker. It is synthesized by the liver under the stimulation of cytokines generated by tissue damage, such as interleukin-6, interleukin-1, and tumor necrosis factor (19). Several evidence lines have related inflammation, and more specifically CRP, with endothelial damage. C-reactive protein can bind to 136 the damaged cells and fix complement. It can also bind to low- and very-low density lipoproteins and promote the synthesis of several inflammation mediators (11). Moreover, CRP levels can rise in response to different infections. Recently, the association of cardiovascular risk with inflammation-infection has been reviewed and a probable infectious cause for atherosclerosis has been proposed. Among the infections probably associated with atherosclerosis, the most consistent has been that caused by Chlamydia pneumoniae, but Helicobacter pylori and cytomegalovirus have also been mentioned as possibly related (12 17). The suspicion of the participation of C. pneumoniae in atherogenesis was raised because it is a bacterium frequently involved in upper respiratory track infections in the general population, and the germ itself, or its DNA, has been identified within arterial lesions and in circulating monocytes; also, antibodies against C.pneumoniae have been found in patients with arterial lesions (15). There are also reports that roxithromycin, a macrolide antibiotic active against Chlamydia, reduces the frequency of ischemic events in patients with acute myocardial infarction (20). The pathophysiologic relation of C.pneumoniae and atherosclerosis is complex. The bacterium can cause the lesion, but it also may cause an immunological reaction against heat shock proteins of the micro-organism. Produced antibodies show crossreactivity with human heat shock proteins, since they have high interspecies homology. This may be the reason why initially infectious damage may progress to autoimmune damage (12). Patients with ESRD frequently have anti-chlp-igg or chlamydial DNA in peripheral blood mononuclear cells (16,17). There are also associations between inflammation markers, C.pneumoniae infection, and vascular access atherosclerosis or thrombosis in hemodialysis patients (16,21 23). The frequency of patients with anti-chlp-igg in the present study does not differ from previous reports. On the other hand, there are reports of carotid artery atherosclerosis and anti-chlp-igg present in ESRD patients (21,23), but there is only one report on anti-chlp-igg and mortality (22). That report shows that patients positive for IgA antibodies against C.pneumoniae had a lower survival rate than seronegative patients. Recent studies show that, in pediatric and adult patients with carotid atherosclerosis on hemodialysis, C.pneumoniae-related inflammatory processes may be associated with the arteriopathy (21,24,25). The present data lend strong support to the association of infectioninflammation and mortality in CAPD patients because this was a prospective study including only incident patients, assessing clinically relevant outcomes such as mortality. Our data also suggest that

6 PDI MARCH 2003 VOL. 23, NO. 2 CRP, CHLAMYDIA PNEUMONIAE, AND MORTALITY IN CAPD the relationship of CRP and anti-chlp-igg with mortality in this type of patient is stronger than the relationship of mortality with traditionally accepted markers. In conclusion, our results suggest that inflammation and the presence of Chlamydia pneumoniae infection are important predictors of cardiovascular death in patients on CAPD. ACKNOWLEDGMENT This work received grant support from Baxter México S.A. and the Instituto Mexicano del Seguro Social, Mexico. REFERENCES 1. Lindner A, Charra B, Sherrard DJ, Scribner BH. Accelerated atherosclerosis in prolonged maintenance hemodialysis. NEngl J Med 1994; 290: USRDS. Excerpt from the United States Renal Data System 1999 Annual Data Report. Patient mortality and survival in ESRD. Am J Kidney Dis1999; 34(Suppl 1):S Degoulet P, Legrain M, Reach I, Aime F, Devries C, Rojas P, et al. Mortality risk factors in patients treated by chronic hemodialysis. 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