Relationship of Serum Cystatin C with C-Reactive Protein and Apolipoprotein A1 in Patients on Hemodialysis

Abstract

Cystatin C is considered an indicator of acute renal failure and also a risk factor of cardiovascular disease. This study was undertaken to examine the relationship of serum cystatin C with C-reactive protein (CRP), lipids, and lipid-related compounds in patients on hemodialysis (HD). Cystatin C, CRP, total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and apolipoprotein A1 and B were analyzed in serum of 30 patients on HD for 118 ± 18 months with low-flux dialyzers, before and after HD. The results were compared with those obtained by 21 healthy individuals (NC). Multiple regression analysis was performed to evaluate the association of cystatin C concentration before HD with clinical and laboratory parameters. The results showed that cystatin C before HD was not associated with age, body mass index (BMI), or duration of HD. However, it was significantly correlated with creatinine (r = 0.435, p = 0.021) and albumin (r = 0.483, p = 0.009) concentrations. Moreover, a highly significant association was shown with logCRP (r = 0.692, p < 0.0001). Among the lipid and lipid-related compounds studied, a significant correlation was found between cystatin C and apolipoprotein A1 concentrations (r = 0.402, p = 0.034). None of those correlations were observed in the NC group. In conclusion, it seems that cystatin C levels before HD are related with CRP, an important inflammatory factor, and also with apolipoprotein A1, which has been proved to accelerate the atherosclerosis process. However more studies are needed to confirm these findings.

Keywords:

• cystatin C
• C-reactive protein
• apolipoprotein A1
• hemodialysis

INTRODUCTION

Cystatin C is a low molecular weight, non-glycosylated protein and cysteine protease inhibitor. It is produced at a stable rate by most nucleated cells types,[1–4] almost freely filtered by the glomeruli and catabolized by the proximal tubules.[5],[6] Recently, serum cystatin C has been proven to be one of the more promising endogenous proteins for determining glomerular filtration rate (GFR) and assessing renal function.[7–10] Recent studies have proved that serum cystatin C may be a useful marker in the early detection of acute renal failure[11] as well as in the diagnosis of chronic kidney disease in older people, and that is more sensitive than serum creatinine for the management of patients with moderately reduced GFR.[12],[13] On the other hand, it has been shown that cystatin C may also participate in the inflammatory process observed in patients with coronary heart disease.[14] The levels of serum cystatin C are independently associated with an increased risk of cardiovascular heart events,[15],[16] a fact that demonstrates that cystatin C is not simply a marker of renal failure.

There is limited evidence in the literature regarding the role of cystatin C in end-stage renal failure patients.[17],[18] In this study, we sought to evaluate the relationship of serum cystatin C with the acute phase protein CRP and atherosclerosis factors such as total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and apolipoprotein A1 and B.

MATERIALS AND METHODS

The study was carried out with 30 renal failure patients on stable hemodialysis (HD) (age 61 ± 3 years, 16 males, and 14 females) for a mean period of 118 ± 8 months. Smokers and patients with cancer, chronic hepatitis, or hematological or inflammatory disorders were excluded. The underlying renal disorders were interstitial nephritis (n = 7), glomerulonephritis (n = 7), polycystic kidney disease (n = 7), hypertension (n = 2), diabetes (n = 2), amyloidosis (n = 1), cardiac failure (n = 3), and IgA nephropathy (n = 1). None of the patients was taking lipid-lowering drugs or glucocorticoid therapy. Hemodialysis was performed using low-flux dialyzers. Heparin low-molecular weight was used as an anticoagulant. Patients were dialyzed three times a week, for four hours each session, using bicarbonate-buffered dialysate. The quality of the dialysate and water and the bacteriological testing of central venous catheters were under frequent surveillance. All patients were anuric, and the natural creatinine clearance was zero. The Kt/V index of adequacy of HD was 1.28 ± 0.03. The control group (NC) consisted of 21 healthy non-smoking individuals (11 males and 10 females, age 56 ± 6 years), who were recruited in the same center and were not on any kind of prescribed medication or dietary restrictions. All patients and controls gave their informed consent.

Blood samples (5 ml) were taken after overnight fasting from intravenous fistula of patients, just before and immediately after the second weekly dialysis session. They were allowed to clot for 30 min, centrifuged at 2000 × g for 15 min for the separation of serum, and stored at −80°C until analysis. C-reactive protein (CRP) was measured by nephelometry with a highly sensitive assay (Dade Behring, Marburg, Germany). Cystatin C was determined by the particle-enhanced turbidimetric immunoassay[19] of Dako Cytomation (Glostrup, Denmark) modified for Integra 800 analyzing system (Roche Diagnostics GmbH, Mannheim, Germany). Creatinine concentration was measured by kinetic Jaffe method without deproteinization. LDL cholesterol was determined by a direct enzymatic colorimetric assay, while apolipoproteins A1 and B were measured with immunoturbidimetric methods.

Statistical analysis of data was performed by one-way analysis of variance (ANOVA) test in order to estimate differences between pre-HD, post-HD, and NC values. Multiple regression analysis was assessed to evaluate associations between cystatin C concentration and the studied parameters before HD. As the distribution of the CRP variable was skewed, logarithmic (log) transformation was used to gain normal distribution. Data were expressed as mean values ± SEM. All tests were considered significant at p < 0.05.

RESULTS

Table 1 shows the demographic data of hemodialysis (HD) patients and healthy individuals (NC). The biochemical data in patients before and after HD are presented in Table 2. It can be seen that the levels of cystatin C in patients were higher than in healthy individuals (NC: 0.65 ± 0.04, HD before: 6.27 ± 0.25, HD after: 7.34 ± 0.30 mg/L) and significantly increased after HD session compared to pre-dialysis (p < 0.01) and NC (p < 0.001) values. Albumin, total cholesterol, triglycerides, LDL cholesterol, and apo-A1 concentrations were higher after HD than before HD (p < 0.05). Demographic and biochemical data before HD were tested in relation to serum cystatin C concentration by multiple regression analysis (see Table 3). It was found that the concentration of cystatin C was significantly correlated with serum creatinine (r = 0.435, p = 0.021) and albumin (r = 0.483, p = 0.009) before hemodialysis. Among the lipid and lipid-related compounds studied, apolipoprotein A1 was the only associated with cystatin C before hemodialysis (r = 0.402, p = 0.034). Moreover, a highly significant correlation was found between logCRP and cystatin C concentration (r = 0.692, p < 0.0001; see Figure 1). In healthy individuals, none of the above correlations of cystatin C was significant.

Table 1 Demographic data in patients on hemodialysis (HD) and in healthy individuals (NC)

	NC	HD
N	21	30
Sex (M:F )	11:10	16:14
Age (years)	56 ± 6	61 ± 3
Dialysis duration (months)	—	118 ± 18
Weight (kg)	67.2 ± 6.1	70.5 ± 3.2
BMI (kg/m^2)	23.0 ± 1.0	24.7 ± 1.0

Table 2 Biochemical data of patients on hemodialysis (HD) and of healthy individuals (NC)

	NC	Before HD	After HD
Urea (mmol/L)	4.2 ± 0.4	28.6 ± 1.6*	8.9 ± 0.7^†
Creatinine (μmol/L)	71 ± 18	886 ± 49*	350 ± 21^‡
Albumin (g/dL)	4.5 ± 0.1	3.9 ± 0.1*	4.4 ± 0.1
Total cholesterol (mmol/L)	4.66 ± 0.21	4.05 ± 0.16^§	4.70 ± 0.19
Triglycerides (mmol/L)	0.88 ± 0.03	1.73 ± 0.17*	2.50 ± 0.24^‡
HDL cholesterol (mmol/L)	2.00 ± 0.18	1.10 ± 0.07^‡	1.29 ± 0.07^‡
LDL cholesterol (mmol/L)	2.80 ± 0.21	2.40 ± 0.17^§	2.89 ± 0.02
Apolipoprotein A1 (mg/dL)	128 ± 2	149 ± 7*	174 ± 4^‡
Apolipoprotein B (mg/dL)	75 ± 8	75 ± 3	88 ± 5
CRP (mg/L)	2.00 ± 0.70	4.76 ± 0.40^‡	5.85 ± 0.40^‡
Cystatin C (mg/L)	0.65 ± 0.04	6.27 ± 0.25*	7.34 ± 0.0.30^‡

*p < 0.01 vs. after HD and NC, ^†p < 0.05 vs. NC, ^‡p < 0.001 vs. NC, ^§p < 0.05 vs. afer HD.

Table 3 Multiple regression coefficients between serum cystatin C concentration and age, BMI, duration of HD, creatinine, albumin, lipids, lipoproteins, apolipoproteins, and CRP concentration in HD patients

Variable	Beta coefficient	p
Age	0.082	0.156
BMI	0.243	0.756
Duration of HD	0.144	0.674
Creatinine	0.435	0.021
Albumin	0.483	0.009
Total cholesterol	0.027	0.889
Triglycerides	0.233	0.232
HDL cholesterol	0.099	0.614
LDL cholesterol	0.061	0.757
Apolipoprotein A1	0.402	0.034
Apolipoprotein B	0.031	0.876
log CRP	0.692	<0.0001

Figure 1. Relationship between serum concentration of cystatin C and logCRP before hemodialysis (r = 0.692, p < 0.0001, n = 30).

DISCUSSION

The results of this study demonstrate that cystatin C levels in serum of patients with end-stage renal failure are remarkably higher than in healthy individuals. Although there is limited evidence about the range of cystatin C concentration during hemodialysis therapy, the levels before dialysis are in agreement with those measured by other authors and significantly higher post-dialysis than pre-dialysis.[17],[20] This increase can be attributed to the low-flux membranes used, which retain cystatin C and other middle molecular weight molecules, in contrast to high- flux hemodialyzers or hemodiafiltration, which allow the removal of the above compounds and result in a substantial decrease in the post-dialysis levels of cystatin C.[21],[22]

We intended to determine if the increased concentration of serum cystatin C in hemodialysis patients is connected to the inflammatory factor CRP as well as to markers of atherosclerosis. As has been previously reported, the inflammatory process is related to atherosclerosis in chronic kidney disease[23],[24] and is revealed by a release of CRP and inflammatory cytokines (i.e., Il-6) secreted by inflammatory cells present in atherosclerotic lesions[14],[25] and also by leukocytes in contact with dialysis membranes.[26] Moreover, the reduced CRP renal clearance[27] also contributes to the development of inflammatory status in pre-dialysis patients. Among the markers associated with atherosclerosis that we studied, it is found that age and BMI are not correlated to cystatin C in HD patients. This is in agreement with other studies that refer to healthy populations[28],[29]; however, it opposes other findings referring to healthy elderly individuals[30] or individuals randomly selected for cross-sectional analysis, after adjusting for creatinine clearance.[31] It seems thus that in end-stage renal insufficiency, age and muscle mass do not influence the production and/or the catabolism of cystatin C. The well-documented relationship between cystatin C and creatinine[32],[33] has been confirmed in our study. However, a new finding for HD patients arises from its association with apolipoprotein A1 concentration. In elderly men, apolipoprotein A1 has been characterized as a better predictor for atherosclerosis than apoB, apoB/apoA1 ratio, HDL-C, and LDL-C, having significant correlation with cystatin C and CRP.[34] The increase of apolipoprotein A1 in patients with renal failure has been attributed to reduced renal clearance and/or homocysteine releasing apo-A from Lp(a), and has been suggested to be one of the reasons contributing to the incidence of vascular diseases.[35],[36] We found also that cystatin C values were associated with the observed reduced levels of albumin before HD. Recent studies show that hypoalbuminemia may result from inflammation and contribute to morbidity in chronic renal patients, constituting an important predictor of coronary heart disease.[37],[38] A possible implication of cystatin C in the inflammation procedure was also seen by the highly significant correlation with CRP. The mechanism that could explain this correlation may be that during vascular injury in HD patients, the increased production of inflammatory CRP and cytokines stimulate the production of elastolytic cysteine proteases. This might be counterbalanced by the increase of cystatin C in cells outside the artery wall, resulting in an increase in serum cystatin C levels.

In conclusion, our results show a significant association of cystatin C concentration before hemodialysis with the levels of CRP and apolipoprotein A1. The above associations may lead to an acceleration of the inflammatory and atherosclerosis process in those patients. However, further studies are needed to confirm this possibility.

DECLARATION OF INTEREST

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.