Introduction
Hemodialysis (HD) has dominated the renal replacement therapy for decades among more than 2,000,000 patients afflicted with end-stage renal disease (ESRD) [
1,
2]. Despite tremendous progress in HD techniques, the mortality and morbidity of complications (cardiovascular, cerebrovascular and infection diseases especially) remain extremely high [
3]. Chronic kidney disease (CKD) is proved as an independent risk factor for all-cause mortality as well as cardiovascular and cerebrovascular diseases (CCDs) [
4,
5]. Although maintenance HD contributes to extending the patients’ life against kidney failure, it also poses vascular injury on the already compromised cardio-cerebrovascular system [
6,
7].
Recent decades have witnessed a series of studies about risk factors for mortality and complications in HD patients [
8]. Apart from traditional risk factors (such as aging, comorbidities, obesity and dyslipidemia), more emerging risk factors (such as oxidative stress, endothelial dysfunction and chronic inflammation) [
9] are identified to be substantially significant. Since the first report about the influence of HD on complement system [
10], complement activation during HD has been thoroughly investigated [
11‐
14]. Previously, our cross-sectional study also observed complement activation among 108 HD patients, representing a decreased level of plasma C3c and complement factor B (CFB), and an elevated level of plasma mannose-binding lectin (MBL), C3a and C5a, compared with normal controls [
15]. Further, diverse complement proteins, including MBL [
16‐
18], C3 [
19], C1q-adiponectin [
2], membrane attack complex (MAC) [
19], complement factor H (CFH) [
20] and complement receptor 1 (CR1) [
21] were confirmed as significant predictors for the incidence of cardiovascular events or death for HD patients. Besides, a body of large studies targeting normal population also revealed plasma C3, C4 [
22] and MBL [
23,
24] level were risk factors for cardiovascular diseases.
In the present study, we took advantage of a prospective cohort to identify the pivotal components of complement system associated with the outcomes of HD patients. Other risk factors were also taken into consideration to control confounding variables. Determination of the optimal cut-off points for the identified factors is of considerable reference value for the prediction of the adverse events.
Discussion
The present study showed an association between the baseline plasma C4 level and the adverse outcomes, including all-cause mortality and CCEs, among patients receiving maintenance HD. Both in the unadjusted and adjusted models, plasma C4 level substantially showed a predictive value. Patients whose baseline plasma C4 > 0.47 g/L or 0.44 g/L in our cohort exhibited higher all-cause mortality or incidence of CCEs. Meanwhile, the level of plasma C4 manifested a positive linear trend with HR for death, CCEs and either of them. In our cohort, baseline plasma C4 levels had correlations with blood lipids, which were widely acknowledged as risk factors for the development of cardiovascular diseases. These findings suggested that C4 may participate in the pathological processes in patients with maintenance HD and excess plasma C4 predicted a worse prognosis for HD patients.
A body of evidence indicated that multiple complement components related to outcomes of HD patients, covering MBL [
16‐
18], C3 [
19], C1q-adiponectin [
2], MAC [
19], CFH [
20] and CR1 [
21]. A higher level of plasma C3 before an HD session, was reported to be associated with a higher probability of cardiovascular events [
19]. Baseline sC5b-9 levels was predicted to be correlated with cardiovascular events and mortality. A lower level of serum C1q-adiponectin/C1q ratios were also identified as a prognostic marker of cardiovascular diseases [
2]. Thus, a possible explanation would be that both an elevated complement activation and an intensified complement activity have been the risk factors for cardiovascular diseases. Additionally, Satomura et al. revealed that a lower MBL level could independently predict all-cause mortality in HD patients [
16], which was also proposed to be linked with the morbidity of cardiovascular diseases in HD patients [
17] and linked to accelerating arterial stiffness in HD patients [
33].
Significantly, the diversity of the above conclusions mainly resulted from the heterogeneity of the patients. The patients’ characteristics varied among different HD centers, particularly such as, age, ethnicity, HD duration, the primary cause of ESRD and comorbidities. These differences profoundly influenced the distribution of the plasma complement levels among patients. Specifically, patients in Satomura’s study (9.054 ± 5.115 μg/ml) had a higher level of MBL than ours [4.346(1.415, 8.979)μg/ml] likely because of heterogeneity. Although a lower level of MBL wasn’t regarded as a significant risk factor in the current study, we indeed found a slight tendency likewise in our cohort. There was a considerable amount of the death whose MBL level was lower than 2.5 μg/ml [as 7 of 17 patients (41.2%)] (Figure S
3). According to the above, conclusions in this field should be restricted to the certain population for higher accuracy. Furthermore, HD patients with any suspicious risk values should be carefully monitored and cared to decrease mortality and the incidence of CCEs.
Our previous cross-sectional study in the same cohort, found that the complement system was activated in patients on hemodialysis and a higher plasma C3a level prior a dialysis session was associated with severe abdominal aortic calcification [
15]. Thus, we included all measured plasma complement factors to identify the critical components associated with the outcomes. As a consequence, an elevated level of plasma C4 was proposed to be the risk factor that significantly increased all-cause mortality and incidence of CCEs, independent of other risk factors reported previously [
8]. Although plasma C4 level among hemodialysis patients (0.312 g/L (0.25 g/L,0.38 g/L)) wasn’t prominently higher (
P = 0.10) than the normal (0.285 g/L (0.22 g/L,0.39 g/L)) [
15], an increased level of plasma C4 could discriminate patients with adverse outcomes.
In the complement cascades, C4 contributes to the formation of C3 convertase in the classical and lectin pathway. Circulating C4 and C3 mainly derive from hepatocytes [
34] and are also related to adipose tissue variables [
35] and involved in the development of visceral adiposity [
36]. In healthy individuals, the polymorphism of C4 genes, including the variation of the gene copy number, the gene size and the C4 isotypes (C4A and C4B), largely determines the plasma levels and functions of C4 [
37]. An elevated level of plasma C4, as well as C3, are reported as strong inflammatory indicators of metabolic syndrome [
38,
39], cardiovascular diseases [
22], thrombotic diseases [
40] and allergic diseases [
41]. During the pathological process, C4 and C4a may play pivotal roles in chronic inflammation and tissue injury, rather than defending against pathogens and cleaning immune complex and cells [
42]. The elevated systemic C4 and C3 levels were probably correlated with metabolic syndrome [
38,
39,
42], which is proved to raise the risk of cardiovascular disease, diabetes and all-cause mortality among general population [
43]. Although our study confirmed the critical impact of plasma C4, plasma C3 level wasn’t measured in the analyses. As we mentioned above, in those studies reported the correlation between C3 and prognosis, the plasma C4 levels were not measured. Further investigations are also needed to answer the relationship between C3 and C4, and their predictive value for prognosis in patients with maintenance HD.
Apart from the well-known conjunction in complement pathways, C4 may have distinct effects on metabolism and chronic inflammation [
38]. Studies of human populations have shown that C3 and C4 are associated with the incidence of myocardial infarction and stroke [
22], as well as with their risk factors, such as obesity, hypertension, hyperlipemia and diabetes [
28‐
31]. Analogous associations were sighted in our HD cohort between plasma C4 and the incidence of CCEs and hyperlipemia. Cytokines stimulating the hepatic production of C4 may also induce hyperlipemia and undermine insulin sensitivity. C4 binding protein (C4BP) inhibits the classical and lectin pathway by binding to C4b and is reported as a protective factor for desired blood pressure, fasting blood glucose and cell function [
44,
45]. Furthermore, C4a, the product of C4 activation, may participate in cardiac remodeling and inflammation [
46] by binding to protease-activated receptor I (PAR1) [
47]. Other components participating in the C4 activation, for example, platelets and endothelial cells [
38], are receiving increasing attention due to their crosstalk in inflammation and vascular injury [
48,
49]. In our study, plasma C4 showed strong correlations with CFH, CFB, C1q and C3c. Thus, the impact of C4 on the prognosis in HD patients is likely to exert through the classic and alternative pathways.
Other baseline variables in our study, including demographic characteristics and laboratory measurements, were also included in the analyses as confounding factors. According to the univariate Cox regression, age, albumin, blood pressure, mCCI and comorbidity conditions were associated with prognosis, in consistent with previous studies [
8,
25,
50‐
55]. Besides, a low level of blood platelet count was identified as a risk factor in our study. However, previous studies revealed that those with a high platelet count (> 300 × 10^9/L) exhibited higher cardiovascular mortality [
56]. Given the fact above, therapies targeting or affecting platelet need to be individualized and refined among HD patients. The dialyzer is considered to exert remarkable impacts on the count, morphology and function of platelet [
57,
58], worsening the already undesired platelet dysfunction (thrombosis and bleeding diathesis) in patients with ESRD [
56]. Considerable activation of platelet can occur during HD session, owing to the exposure to dialysis membrane [
57]. Whether the platelet activation by dialyzer contributes to the elevated all-cause mortality and incidence of CCEs in HD patients remains inconclusive.
Nevertheless, there are some limitations in our study. The case volume of the prospective analyses was relatively low, so limited endpoint events were observed. These defects might weaken the power of tests, especially the multivariate Cox regression. Owing to the potential possibility of overfitting, the multivariate Cox regression models could only reveal the significance of plasma C4 levels rather than be applied as the predict tools for HD patients. Patients enrolled in our study were used as the training population to determine the hazard thresholds of plasma C4 level, thus a validation population is needed to further confirm the optimal value. C4 is cleaved by C1s [
59] and mannan-binding lectin-associated serine protease 2(MASP2) [
60] to release C4a and C4b to produce C3 convertase subsequently. Thus, whether the downstream fragments of C4 activation, such as C4a and C4d, have the correlations with the prognosis in HD patients is further to be excavated.
In conclusion, a high level of baseline plasma C4 was confirmed to be associated with all-cause mortality and the incidence of CCEs. Consequently, plasma C4 level is recommended as an innovative clinical predictor for HD patients, together with other risk-related variables, such as age, blood pressure, albumin, blood platelet count, etc. Further studies are required to thoroughly elucidate the significance and mechanisms of plasma C4 in HD patients.
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