Abstract
Background and objectives: Mineral metabolism abnormalities and inflammation are concerns in chronic kidney disease (CKD). Interrelationships among these parameters have not been analyzed.
Design, setting, participants, & measurements: The study included 133 patients with CKD not on dialysis and not receiving calcium (Ca) supplements, phosphate binders, or vitamin D. Estimated GFR (eGFR) was 34.1 ± 6.8 ml/min/1.73 m2; 107 participants had stage 3 CKD, and 26 had stage 4.
Results: Patients were classified by tertiles of Ca, phosphorus (P), Ca-P product (Ca x P), and parathyroid hormone (PTH). After adjustment for age, gender, and eGFR, the levels of C-reactive protein (CRP) and IL-6 (IL-6) of the third tertile of P, Ca x P, and PTH were significantly higher than those of the first and second tertiles. Serum P and Ca x P directly correlated with CRP and IL-6, whereas HDL-cholesterol and eGFR inversely correlated with the levels of the inflammatory parameters. After partial correlation analysis, the previous associations between CRP and eGFR, and serum P, as well as the relationship between IL-6 and eGFR, and serum P, remained significant. Multiple regression analysis demonstrated that eGFR and serum P were independently associated with CRP and IL-6. Finally, logistic regression analysis using the presence/absence of an inflammatory state as the dependent variable showed that eGFR was a protective factor, whereas serum P was an independent risk factor for the presence of an inflammatory state.
Conclusions: Elevated serum P might play a role in the development of inflammation in CKD.
Alteration of mineral metabolism is a prevalent condition in chronic kidney disease (CKD). Large epidemiologic studies have shown a strong relationship between elevated levels of calcium (Ca), phosphorus (P), Ca-P product (Ca x P), and parathyroid hormone (PTH) with cardiovascular morbidity and mortality (1–3). Among abnormalities of mineral metabolism, one of the most prominent and relevant is hyperphosphatemia. Elevated serum P has been related to cardiovascular morbidity and mortality in both hemodialysis and predialysis patients. Vascular and coronary artery calcification have been suggested as the link between abnormal mineral metabolism in general, and hyperphosphatemia in particular, and cardiovascular events in this population (4–6). Hyperphosphatemia has been pointed out as the primary culprit in the process of cardiovascular calcification (4,8–10), an event already present in the early phases of renal failure (7,8). More interesting, a significant association between the progression of coronary artery calcification and serum P concentration was observed in CKD patients, despite serum P being in the normal range. Faster progression was found in patients with a high-normal serum P, which was accompanied by more frequent cardiovascular events (11). However, despite these previous findings, the mechanisms by which serum P contributes to cardiovascular disease are not completely known.
Vascular and coronary artery calcification are markers of atherosclerotic disease. In the last decades, intensive investigations have led to a paradigm shift in the interpretation of atherosclerosis, from a purely metabolic process (i.e., mainly driven by hypercholesterolemia) to a disease where inflammation is the dominant pathophysiological and biochemical alteration (12). Cardiovascular disease is up to 20-fold more frequent in ESRD patients and accounts for up to 50% of all deaths, with accelerated atherosclerosis being consistently implicated in this process (13). Traditional cardiovascular risk factors cannot completely explain the prevalence of atherosclerosis, the elevated cardiovascular risk, and the disproportional predisposition for adverse cardiovascular outcomes in this population. Therefore, novel emergent cardiovascular risk factors are suggested to contribute to atherogenesis and have been associated with cardiovascular risk. Among these, it is now known that inflammation is a highly prevalent condition in CKD patients, with diverse studies having documented increased concentrations of inflammatory mediators in dialysis patients as well as subjects with advanced renal failure (14,15). More importantly, it has been demonstrated that several inflammatory parameters, mainly C-reactive protein (CRP) and IL-6 (IL-6), are strong and independent predictors of all-cause and cardiovascular mortality (16–19).
In spite of the important prevalence and relevance of alterations of mineral metabolism and inflammation in CKD patients, the interrelationships among these factors have been scarcely analyzed, especially in predialysis patients. Most previous studies have been performed in hemodialysis, with a limited number of subjects, and with the potentially confounding effect of concomitant treatment with Ca supplements, phosphate binders, or vitamin D derivates (20,21). The aim of this study was to examine the interrelationships among the main parameters of mineral metabolism (Ca, P, Ca x P, and PTH) and inflammation (CRP and IL-6) in patients with advanced CKD.
Materials and Methods
Participants
A cohort of 313 outpatients with CKD not yet on dialysis was evaluated. Finally, based on the inclusion/exclusion criteria, this cross-sectional study involved 133 patients (Figure 1), with 41 of them (30.8%) suffering from diabetes. Entry criteria included age older than 18 yr; ability to provide informed consent for participation; CKD stages 3 to 4 according to the National Kidney Foundation criteria (22) (with an estimated GFR [eGFR] lower than 45 ml/min/1.73m2, calculated using the four-variable Modification of Diet in Renal Disease Study equation) (23); and no treatment with Ca supplements, phosphate binders, or vitamin D derivates. Exclusion criteria included current smoking habit (defined as patients smoking at least one cigarette per day during the previous 12 mo), history of any clinical manifestations of cardiovascular disease (coronary artery disease, cerebral vascular disease, and/or peripheral vascular disease) or chronic inflammatory disease, an acute inflammatory or infectious disease, institutionalized subjects, and patients on immunotherapy or immunosuppressive treatment. Before the definitive inclusion, the possible existence of immunologic diseases, malignancy, and infections was investigated. White blood cell count was lower than 10,000/mm3 in all cases. Determination of tumoral markers including carcino-embryonic antigen, α-fetoprotein, cancer antigen 125, and prostate-specific antigen were negative. Serologic tests for antinuclear antibodies, antineutrophil cytoplasmic antibodies, rheumatoid factor, inmunoglobulins, and complement were negative or within the normal range. Urine cultures and serology to hepatitis B, hepatitis C, and HIV were also negative. Weight and height data were collected from each individual to calculate body mass index (BMI) as body weight divided by height squared. The protocol was in accordance with the Declaration of Helsinki and was approved by the local committee, and all subjects gave their informed consent.
Flow diagram for enrolment in the study.
Laboratory Analyses
Blood samples were drawn from each patient before breakfast in the morning (between 8 and 11 a.m.), after an 8-hr to 12-h overnight fast. Samples were collected in sterile tubes, centrifuged at 3000 g for 10 min at 4°C, and then stored at −80°C until assayed. Serum concentrations of Ca, P, intact PTH, triglycerides, total cholesterol, and HDL- and LDL-cholesterol were measured. The concentration of high-sensitive C-reactive protein (CRP) was determined by ultrasensitive competitive immunoassay (Calbiochem, La Jolla, CA), with an interassay coefficient of variation of 8.4%. Mean concentration in healthy individuals is 0.63 mg/L. Serum concentrations of IL-6 were measured by a solid-phase, chemiluminescent immunometric assay. The analytical sensitivity was 2 pg/ml, and the intra- and interassay coefficients of variability were 6.2% and 7.5%, respectively. Mean concentration in healthy individuals is 2.1 pg/ml. Urinary albumin excretion (UAE) was determined as albumin-to-creatinine ratio in the first morning urine sample.
Statistics
Results are expressed as means and SD (SD), except for CRP and IL-6, which are presented as geometric means and ranges. The Shapiro-Wilk W test was used in testing for normality. Due to non-normal distribution, CRP and IL-6 were logarithmically transformed for analyses and then back-transformed to their natural units for presentation in tables and figures. Mineral metabolism parameters (Ca, P, and PTH) were analyzed by dividing the distribution into tertiles. For comparisons among the Ca, P, and PTH tertiles, the ANOVA post hoc Scheffé test and the chi-square test were used, as appropriate. Correlation analysis was performed to evaluate the associations between the inflammatory parameters and the other variables. Partial correlation analysis was performed to determine the extent to which a relationship was altered after adjusting for the other variables. A forward stepwise multiple regression analysis was performed to determine the independent association between diverse independent variables (age; gender; presence or absence of diabetes; BMI; serum total and LDL- and HDL-cholesterol; albumin; eGFR; UAE; and serum concentrations of Ca, P, and PTH) and CRP and IL-6 as the dependent variables. Polynomial regression analysis was used to test for potential nonlinearity. Finally, multiple logistic regression analysis was performed to determine risk factors for the presence of an inflammatory state in the patients. The study sample was divided into two groups based on level of CRP (high CRP ≥3 mg/L versus low CRP <3 mg/L) as defined in a recent guideline from the Centers for Disease Control and Prevention, and the American Heart Association (24). A p-value <0.05 was considered to be statistically significant. SPSS version 15.0 for Windows (SPSS Inc., Chicago, IL) was used to analyze the data.
Results
Three hundred thirteen patients initially were evaluated for study participation, and 133 were finally included in the study. The main characteristics of the 133 participants are shown in Table 1. One hundred seven participants (80.4%) had stage 3 CKD, and 26 (19.5%) had stage 4. For the purposes of this study, the presence of an inflammatory state in a patient was defined when the serum concentration of CRP was ≥3 mg/L. Thus, 26 patients (19.5%) presented an inflammatory state.
Clinical characteristics of 133 study participants
Characteristics of participants by tertiles of Ca, P, Ca x P, and PTH are showed in Tables 2, 3, 4, and 5, respectively. The only difference among patients classified by Ca tertile was the higher value of Ca x P observed in patients in the highest tertile. No differences were observed in the levels of CRP and IL-6. Regarding P, patients in the highest tertile exhibited a significantly lower eGFR, with a higher proportion of patients in CKD stage 4. Serum PTH and Ca x P, as well as inflammatory parameters, were also higher in patients in the highest tertile. Similar results were found when patients were classified according to Ca x P tertiles. Finally, patients in the highest tertile of PTH showed higher concentrations of P, Ca x P, CRP, and IL-6. In this group, the proportion of patients with CKD stage 4 was higher, and the mean eGFR lower, than was observed in patients in the first and second tertiles. After adjustement for age, gender, and eGFR, the levels of CRP and IL-6 of the third tertile of P and PTH were significantly higher than CRP and IL-6 of the first and second tertiles.
Clinical characteristics of participants stratified by tertile of calcium
Clinical characteristics of participants stratified by tertile of phosphorus
Clinical characteristics of participants stratified by tertile of calcium x phosphorus product
Clinical characteristics of participants stratified by tertile of parathyroid hormone
Table 6 lists findings from the bivariate correlation analysis examining CPR, IL-6, and potential correlates. Serum P and Ca x P most strongly directly correlated with CRP and IL-6, followed by PTH. On the contrary, HDL-cholesterol and eGFR inversely correlated with the levels of the inflammatory parameters. However, after adjusting for the effect of other variables by partial correlation analysis, only the previous associations between CRP and eGFR (r = −0.43, P < 0.0001), and serum P (r = 0.35, P < 0.01) remained significant. Likewise, the relationship between IL-6 and eGFR (r = −0.70, P < 0.0001), and serum P (r = 0.38, P < 0.0001) remained significant.
Correlation coefficients (r) between inflammatory parameters and clinical characteristics
To test the hypothesis of an independent association between variables of mineral metabolism and inflammatory parameters, forward stepwise multiple regression analysis was performed with CRP and IL-6 as the dependent variables. Age; gender; the presence or absence of diabetes; BMI; serum total and LDL- and HDL-cholesterol; albumin; eGFR; UAE; and serum concentrations of Ca, P, and PTH were considered as possible predictors of inflammatory parameters. The result showed that from the different independent variables, eGFR and serum P were independently associated with CRP (adjusted R2 = 0.41, P < 0.0001) and IL-6 (adjusted R2 = 0.66, P < 0.001) (Table 7). A marginal association was present between CRP and serum LDL-cholesterol (P = 0.14), and between IL-6 and age (P = 0.18), serum total and HDL-cholesterol (P = 0.25 and P = 0.19, respectively), and BMI (P = 0.30). No significant associations were observed for serum levels of Ca or PTH. There was NS collinearity between the independent predictors. Finally, analysis for potential nonlinearity of the associations was performed, which did not show any significant relationship between inflammatory and mineral metabolism parameters.
Multiple stepwise regression analysis for C-reactive protein and IL-6 as dependent variablesa
For logistic regression analysis, patients with a serum concentration of CRP ≥3 mg/L were classified as having an inflammatory state. Therefore, the presence/absence of this inflammatory state was used as the dependent variable. The logistic regression analysis showed that higher eGFR (0.80 [0.70 to 0.91], P = 0.001) was a protective factor, whereas higher serum P (1.86 [1.08 to 3.19], P < 0.05) was an independent risk factor for the presence of a significant inflammatory state (Table 8).
Multivariable logistic regression analysis for the presence of an inflammatory state
Discussion
In this cross-sectional study, we explored the associations of markers of mineral metabolism (Ca, P, and PTH) and parameters of inflammation (CRP and IL-6). Numerous studies have documented the importance of abnormal mineral metabolism (1–3) and inflammation (16–19) as pivotal factors for the increased cardiovascular risk in CKD patients. However, the inter-relationship between both factors has been scarcely analyzed. Two previous studies in hemodialysis, with a limited number of patients, showed that a high Ca x P was associated with high CRP concentrations (20,21). Consequently, there is a potential basis to speculate that alterations of mineral metabolism may be associated with inflammation in CKD.
The main finding of the present study was that, in this sample of patients with CKD stages 3 to 4, without a history or symptoms of cardiovascular disease, and with no previous treatment with Ca supplements, phosphate binders, or vitamin D derivates, the level of the inflammatory parameters CRP and IL-6 was significantly higher in the highest serum P tertile even after adjustment for age, gender, and eGFR. Moreover, serum P concentration was positive, significant, and independently associated with CRP and IL-6, and it was an independent risk factor for the presence of an inflammatory state. In addition, our results show that eGFR is a protective factor for the presence of this inflammatory condition.
Inflammation is recognized in up to 50% of CKD patients, being a common feature of advanced renal disease (25,26). The most extensively studied biomarkers of inflammation in cardiovascular disease are CRP and IL-6. The Cardiovascular Health Study reported that levels of CRP and IL-6 were significantly higher in patients with renal insufficiency compared with patients with normal kidney function (27), whereas several studies have associated changes in GFR with biomarkers of inflammation, particularly in patients with advanced disease (28,29). Diverse factors, including genetic background, volume overload, co-morbidity, advanced glycation end products, reactive oxygen species, autonomic dysfunction, peripheral polymorphonuclear leukocyte priming, and reduced clearance of pro-inflammatory cytokines as the GFR declines, have been involved as causes of the highly prevalent state of inflammation in CKD (30–32). The results of the present study indicate that elevated serum P is an independent predictor of increased levels of inflammatory parameters in these patients, suggesting that hyperphosphatemia may promote and/or facilitate the development of inflammation in CKD patients. In addition, and interestingly, numerous studies in the last years have demonstrated that P and inflammation run in parallel with similar points regarding deleterious consequences in CKD. Therefore, both elevated serum P levels and increased serum inflammatory biomarkers are considered important factors in the progression of CKD (33–35), abnormalities of cardiac structure and function (36–38), vascular calcification (39–42), as well as cardiovascular events and mortality (1,43–45). On the other hand, interesting data derive from studies reporting potential anti-inflammatory properties of phosphate binders, specifically sevelamer. In a randomized prospective study of 108 chronic hemodialysis patients, treatment with sevelamer resulted in significant reductions in serum CRP levels, as well as in serum P and total and LDL-cholesterol (46,47). In those studies, no data are available about a potential correlation between reduction of P and decrease of CRP. However, a more recent study by Yamada et al. (48) in hemodialysis subjects showed that CRP experienced a significant decrease during sevelamer therapy, and that the reduction rate of CRP was significantly correlated with the change of P. In this study, a titration protocol was used, and it is possible that patients with better P control and higher P reduction received a higher sevelamer dosage. Therefore, the effects on CRP could be related to more of the pleiotropic instead of the P lowering effects of sevelamer.
The mechanisms potentially involved in the stimulation of inflammation by P are unknown. However, since important steps of inflammatory pathways are regulated by phosphorylation events, it is possible to speculate that increased P concentration may trigger phosphorylation-driven signaling inflammatory cascades (49–52). In addition, interesting data regarding basic Ca phosphate (BCP) crystals are known from the study of degenerative arthritis. In this setting, BCP crystals have been shown to activate synovial fibroblasts, inducing the activation of intracellular signaling pathways, including protein kinase C, MAP kinase, and NFκB (53). Furthermore, in vitro studies have shown the ability of macrophages to interact with BCP crystals, resulting in cytokine production (54). Finally, Nadra et al. (55) elegantly demonstrated that BCP crystals are able to interact with and activate human macrophages in a pro-inflammatory manner. In this study, the interaction of human macrophages with BCP crystals results in the secretion of biologically significant quantities of pro-inflammatory cytokines, which were capable of stimulating the activation of endothelial cells and recruitment of mononuclear cells. Therefore, increase of serum P may induce or favor the formation of BCP crystals, which may act similarly to antigens that activate the main cell types participating in the chronic inflammatory response— namely monocytes and macrophages.
A relevant strength in the present study is the lack of interference of potential confounders such as smoking habit, presence of clinical cardiovascular disease, and, importantly, the effect of treatments affecting mineral metabolism parameters (Ca supplements, phosphate binders, or vitamin D analogues). On the other hand, several limitations need also to be taken into account: the relatively small sample size, which may have prevented some of the detected associations from being statistically significant at the P < 0.05 level; and the cross-sectional design and the potential influence of unmeasured confounders. Therefore, our study does not indicate certainty about a direct causality relationship between elevated serum P and the development of inflammation, but we believe, conceptually, that it is hypothesis-generating. Despite these limitations, to the best of our knowledge, this is the first study that investigates the potential association between mineral metabolism and inflammation in patients with advanced CKD who were not undergoing dialysis, showing that serum P is a risk factor for the presence of an inflammatory state in these subjects. Our observation on the association between serum P and pro-inflammatory cytokines requires further exploration and confirmation by longitudinal prospective studies.
Disclosures
None.
Acknowledgments
This study was supported by ACINEF.
Footnotes
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Published online ahead of print. Publication date available at www.cjasn.org.
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Access to UpToDate on-line is available for additional clinical information at http://www.cjasn.org/
- Received April 10, 2009.
- Accepted July 12, 2009.
- Copyright © 2009 by the American Society of Nephrology
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