In this study, we examined the effect of CD on the progression of the AoACS (AoACS progression as defined by ΔAoACS > 0%) and blood CPP levels and showed that the use of CD attenuated blood CPP and suppressed the progression of vascular calcification in patients on maintenance haemodialysis.
The physiological role of primary CPP is to transport calcium-phosphorus, a raw material for teeth and bone, to these tissues, without it precipitating in soft tissue. 9, 10 The uremic environment may cause transformation of primary CPP to secondary CPP to be phagocytosed by the macrophages in the vessel wall, which may contribute to atherosclerosis13 and vascular calcification in the tunica media.14 In fact, it has been reported that CPP levels measured by the gel-filtration method were positively correlated with coronary plaque assessed by intravascular ultrasound, even after adjusting for a number of confounding factors.15 Due to this positive association between blood CPP and arterial calcification in CKD, the Ca-chelating effect of citric acid in CD may have inhibited the formation of blood secondary CPP, which in turn inhibited the progression of vascular calcification. In the present study, ΔCPP and ΔAoACS were negatively correlated in the CD group (Fig. 2b), suggesting that citric acid may have inhibited the formation of primary to secondary CPP and promoted the incorporation of primary CPP into bone. There is a lack of consensus on whether FGF23 in blood is associated with vascular calcification in CKD patients. 16, 17 In our study, we did not find any significant association between blood FGF23 and AoACS (Fig. 2c, 2d). From the viewpoint that FGF23 is a phosphaturic hormone, in the early stage of CKD, when renal function is preserved, FGF23 acts on the kidney to produce Pi-diuretic effect and inhibition of vitamin D activation, and thus FGF23 is thought to have a protective effect against vascular calcification. However, in CKD patients undergoing maintenance hemodialysis, renal function is abolished, so the effects of Pi-diuresis and vitamin D inhibition in the kidney are not obtained and are not associated with vascular calcification. In the present study, we did find a weak but significant positive correlation between ΔCPP and ΔFGF23 in blood (Fig. 2e, 2f). FGF23 in blood is probably produced by osteoblasts and osteocytes upon primary CPP stimulation, while secondary CPP acts on vascular calcification because it cannot pass through bone sinuses due to its large size. 12, 18 As compared to AD, CD has the potential to improve prognosis in patients on maintenance haemodialysis.19 This may be due to the inhibition of secondary CPP production by citrate.12 CPP is thought to induce FGF23 secretion,12, 18 and FGF23 is associated with mortality and cardiovascular disease in patients on maintenance haemodialysis.20 An in vivo rat study has reported that recombinant FGF23 directly causes cardiac hypertrophy and that there was a significant positive correlation between blood FGF23 and cardiac hypertrophy assessed by echocardiography in patients with CKD.21 Similar to our findings, the use of CD has previously been reported to inhibit CPP ripening, as indicated by T50 (T50 is 1/2 of the time it takes for the turbidity of primary CPP in the blood to rapidly increase to secondary CPP).22, 23 Based on previous reports, primary CPP excess, in other words, excess of both Pi and Ca in the blood is thought to be associated with worse prognosis via FGF23 secretion, and secondary CPP is thought to be associated with worse prognosis via vascular calcification.
Another reason for the greater efficacy of CD than AD in suppressing arterial calcification may be related to the following: An ex vivo model showed that citric acid, at concentrations of 733 µmol/L or higher, inhibited rat aortic ring calcification.24 The post-dialysis blood citrate concentration in haemodialysis patients using CD has been reported to be 771.6 ± 184.3 µmol/L, which was approximately five times higher than that of AD.24 Moreover, bicarbonate promoted rat aortic ring calcification in a concentration-dependent manner.24 The use of AD in haemodialysis patients was reported to result in a higher post-dialysis blood bicarbonate concentrations and lower citric acid concentrations than those obtained with CD.24 Moreover, it was reported that no excess bicarbonate correction was observed after dialysis with CD.25 Switching from AD to CD was reported to significantly reduce malondialdehyde-modified LDL(MDA-LDL) (an indicator of lipid oxidation) and pentosidine (an indicator of glycoxidation) level in the blood.26 Therefore, it is likely that CD inhibits lipid oxidation, which is associated with coronary artery calcification,27 as well as glycation, which causes atherosclerosis28 in patients on maintenance haemodialysis, thereby exerting anti-atherosclerotic effects. In subgroup analysis, CPP dissolution from bone was enhanced in patients with secondary hyperparathyroidism,29 suggesting a significant benefit of CD use in patients with controlled PTH (iPTH༜132 pg/mL) (P = 0.036) (Fig. 3). If Pi is controlled (Pi ≦ 6 mg/dL), PTH will also be suppressed, and the anti-calcification effect of CD use will be more effective (P = 0.008). Typically, patients on maintenance haemodialysis have chronic inflammation due to the uremic environment, low nutrition, the dialysis equipment used, and the use of AD.30 In vitro analysis showed that CD significantly inhibited the induction of oxidative stress in the blood of these patients, as compared to AD.31 Thus, CD is less inflammatory than AD and may inhibit the transition to a vicious cycle of atherosclerosis (i.e. malnutrition-inflammation-atherosclerosis syndrome). Therefore, if there is persistent inflammation in hemodialysis patients, AD may further induce inflammation, and CD may reduce inflammation. Subgroup analysis showed that CD was significantly more favorable than AD in inhibiting the progression of vascular calcification when CRP levels were ≧ 0.1 mg/dL (P = 0.041). Under the conditions of low Ca levels and no CaCO3 use, the chelating effect of citric acid on Ca ions was more effective, and CD was thought to have a more anti-calcification effect than AD (P = 0.044 under cCa < 9 mg/dL, P = 0.036 without CaCO3 use). CD had a significant anti-calcification effect compared to AD when the AoACS was < 19 (%). The reason for this is that it has been reported that when calcification is already highly advanced, treatment is unlikely to have an inhibitory effect on progression.32
Warfarin induces vascular calcification by inhibiting matrix Gla protein, an inhibitor of calcification in arteries, in both models of CKD33 and in clinical trials of patients on haemodialysis.34 Therefore, patients on warfarin were excluded from our study. Patients using bisphosphonates were also excluded as bisphosphonate use reportedly reduced the coronary artery calcification scores in patients on maintenance haemodialysis35 and inhibited rat aortic calcification by reducing bone-derived CPP.29
There are several limitations in the present study. First, we did not measure blood Mg levels. Administration of magnesium oxide (MgO) significantly inhibited the progression of coronary artery calcification in patients with CKD,36 and basic experiments in vascular smooth muscle cells showed that Mg dose-dependently inhibited calcification,37 Mg citrate treatment reduced the carotid intima-media thickening in haemodialysis patients,38 and elevation of Mg concentration in the dialysate prolonged the T50.39 Thus, blood Mg levels and oral medications containing Mg are associated with vascular calcification and atherosclerosis in dialysis patients. Nevertheless, none of the patients in this study were receiving oral medications containing Mg. Second, blood bicarbonate and citrate levels may be associated with vascular calcification, but these were not measured in this study. Third, observational study is not of high quality as a research method, and the number of patients included in this study was small.
In conclusion, for patients on maintenance haemodialysis, CD is more effective than AD in inhibiting the progression of vascular calcification as assessed by ΔAoACS. Larger randomised controlled trials are warranted to validate the anti-atherosclerotic results obtained with the use of CD, and to elucidate the mechanism of this therapeutic effect.