Clinical and Experimental Nephrology

, Volume 14, Issue 6, pp 584–588 | Cite as

Association between cystatin C and inflammation in patients with essential hypertension

  • Takafumi Okura
  • Masanori Jotoku
  • Jun Irita
  • Daijiro Enomoto
  • Tomoaki Nagao
  • Veena Rasika Desilva
  • Shiho Yamane
  • Zuowei Pei
  • Shiho Kojima
  • Yasuyuki Hamano
  • Shinichi Mashiba
  • Mie Kurata
  • Ken-ichi Miyoshi
  • Jitsuo Higaki
Original Article

Abstract

Background

Serum cystatin C is not only a marker of renal function but also acts as an independent risk factor for cardiovascular damage, heart failure, and death. It is known that the initiation and progression of these cardiovascular events contributes to renal dysfunction and chronic inflammation. In this study, we investigated the relationship between cystatin C and proinflammatory cytokines.

Methods

Eighty-eight patients with essential hypertension participated in the study, which involved measuring proinflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and C reactive protein (CRP).

Results

Positive correlations were detected between cystatin C and estimated glomerular filtration rate (eGFR) (r = −0.503, p < 0.001), systolic blood pressure (r = −0.246, p = 0.034), and pulse pressure (r = −0.295, p = 0.010). In contrast, serum creatinine correlated only with eGFR (r = −0.755, p < 0.001) and eGFR correlated only with age (r = −0.339, p = 0.001) and not with the other clinical parameters, whereas cystatin C also correlated with log natural (ln) IL-6 (r = −0.247, p = 0.033) and ln TNF-α (r = −0.405, p < 0.001) but not with CRP (r = −0.188, p = 0.108). In contrast, plasma creatinine and eGFR did not correlate with any of these proinflammatory cytokines. Stepwise regression analysis showed that ln TNF-α, eGFR and pulse pressure were independent determinants of serum cystatin C concentration.

Conclusion

This study showed that cystatin C is a marker of inflammation as well as renal function.

Keywords

Cystatin C Creatinine Estimated glomerular filtration rate Tumor necrosis factor-α Interleukin-6 

Introduction

There is increasing evidence that serum levels of cystatin C are a better marker of renal function than serum creatinine [1, 2]. Cystatin C, a nonglycosylated 13-kD basic protein, is a member of the cystatin superfamily of endogenous cysteine proteinase inhibitors [3]. Cystatin C is a small molecular-weight protein produced by all nucleated cells and is filtrated freely by the glomerulus and almost completely reabsorbed and catabolized in the renal tubules [4, 5]. The level of cystatin C in serum is therefore determined primarily by the GFR [4, 6]. Inflammation may contribute to the initiation and progression of atherosclerosis [7, 8]. High blood pressure may also promote vascular oxidative stress and exert a proinflammatory influence on the arterial wall via redox-sensitive mechanisms [9]. Indeed, hypertensive patients who are free from any other important medical conditions have been shown to have high circulating levels of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) [10], interleukin-6 (IL-6) [10, 11], and C-reactive protein (CRP) [12]. There is also evidence these cytokines may cause endothelium inflammation and elevated blood pressure.

Several studies have reported that serum cystatin C not only is a marker of renal function but also is an independent risk factor for cardiovascular damage [13, 14], heart failure [15], and death [16]. In an earlier paper on patients with essential hypertension (EHT), we reported that serum cystatin C levels were a sensitive marker of the severity of end-organ damage, as well as albumin excretion rate, left ventricular hypertrophy. and carotid artery thickness [17]. Recently, cystatin C was shown to correlate with TNF-α, IL-6, and CRP levels in elderly patients [18]. In the study presented here, we measured these proinflammatory cytokines and examined their relationship with cystatin C in patients with EHT.

Patients and methods

Patients

We studied 88 patients attending Ehime University Hospital who were diagnosed as having EHT without known renal disease. Hypertension was defined as a systolic blood pressure (SBP) ≥140 mmHg and/or diastolic blood pressure (DBP) ≥90 mmHg, measured three times in the sitting position using a brachial sphygmomanometer, or those on treatment with an antihypertensive drug. Exclusion criteria included either a previous history of symptomatic cardiovascular disease (CVD), myocardial infarction, atrial fibrillation, valvular heart disease, symptomatic cerebrovascular events, thyroid disease, glomerulonephritis, estimated GFR<30 ml/min/1.73 m2 (eGFR for Japanese formula 194 × age−0.287 × Cr−1.094, if female ×0.739), renovascular hypertension, or abnormalities in kidney structure.

This study was approved by the ethical committee of Ehime University Hospital, and informed consent was obtained from each patient prior to enrolment.

Blood sampling and proinflammatory cytokine measurement

A fasting blood sample was taken from the brachial vein after the patient was in the supine position for 20 min. Serum cystatin C was determined using latex turbidimetric immunoassay kits [within-run precision: CV 0.6–1.0% (0.57–1.93 mg/l) n = 10, between-day precision: CV 0.7–2.0% (0.57–0.88 mg/l) n = 5, Ikagaku Co., Ltd. Japan] [19]. For this assay, a rabbit polyclonal antihuman cystatin C antibody (Dakopatts, Glostrup, Denmark) was dissolved in a Tris–hydrochloride buffer solution. Measurements of serum creatinine, low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and CRP were carried out using an automatic analyzer (model TBA-60S; Toshiba Inc., Tokyo, Japan). IL-6 was measured by chemiluminescent enzyme immunoassay and TNF-α by enzyme-linked immunosorbent assay (ELISA) (SRL Inc., Tokyo, Japan). Hemoglobin A1c (HbA1c) was analyzed by high-performance liquid chromatography (HPLC) on an ADAMS-A1c HA-8160 (ARKRAY Inc., Kyoto, Japan).

Statistical analysis

All the analyses were performed using SPSS14.0 (SPSS, Inc., IL, USA). All values in the text and tables are expressed as mean ± standard deviation (SD). Pearson’s correlation coefficient was used to examine the association between variables. IL-6, TNF-α, and CRP data were logarithmically transformed to normalize distribution. Stepwise regression analysis was performed to identify the determining factors for cystatin C level, with ln-IL-6, ln-TNF-α, eGFR, pulse pressure, and age as the independent variables. A p value <0.05 was considered statistically significant. Because the levels of proinflammatory cytokines were not normally distributed, these parameters were expressed as median values and 25% and 75% interquartile ranges.

Results

Characteristics of study participants

Clinical characteristics of study participants are summarized in Table 1. Seventy-four patients (84%) were treated with antihypertensive drugs. Of these, 36 (40%) were taking an angiotensin II receptor blocker, 58 (65%) a calcium-channel blocker, eight (9.0%) a β-blocker, and eight (9.0%) a diuretic. Fourteen patients (16%) also had diabetes mellitus. Mean age of the 88 participants, 48 of whom were women, was 62 ± 13 years. There were significant differences between men and women for blood pressure level, cystatin C, creatinine, eGFR, HDL-C, LDL-C, and proinflammatory cytokines.
Table 1

Patients characteristics

Number (women) 88 (54%)

Number  ± standard deviation (range)

Age (years)

62 ± 13

BMI (kg/m2)

25.0 ± 4.0

Systolic BP (mmHg)

151 ± 21

Diastolic BP (mmHg)

89 ± 16

Pulse pressure (mmHg)

69 ± 12

Creatinine (mg/dl)

0.74 ± 0.19

Estimated GFR

74.7 ± 16.7

HbA1c (%)

5.61 ± 0.70

LDL-C (mg/dl)

119 ± 26.2

HDL-C (mg/dl)

54 ± 12

Triglyceride (mg/dl)

116 ± 64

TNF-α (pg/ml)a

1.25 (0.8–1.9)

IL-6 (pg/ml)a

1.60 (1.20–2.40)

CRP (mg/ml)a

0.08 (0.05–0.15)

Diabetes (%)

14 (16)

ARB already receiving (%)

36 (40)

CCB already receiving (%)

58 (65)

β blocker already receiving (%)

8 (9)

Diuretic already receiving (%)

8 (9)

Statin (%)

8 (9)

Antiplatelet agent (%)

9 (10)

BMI body mass index, GFR glomerular filtration rate, HbA1c hemoglobin A1c, LDL-C low-density-lipoprotein cholesterol, HDL-C high-density-lipoprotein cholesterol, TNF-α tumor necrosis factor-α, IL-6 interleukin-6, CRP C-reactive protein, ARB angiotensin II receptor blocker, CCB calcium channel blocker

aExpressed as median (25–75%)

Relationship between cystatin C and proinflammatory cytokines

We first estimated the relationship between cystatin C or creatinine or eGFR and conventional risk factors for atherosclerosis and proinflammatory cytokines (Table 2). We found a positive correlation between cystatin C and eGFR (r = −0.503, p < 0.001), creatinine (r = 0.494, p < 0.001), SBP (r = −0.246, p = 0.034), and pulse pressure (r = −0.295, p = 0.010). However, creatinine and eGFR did not correlate with any of these parameters except for eGFR (r = −0.755, p < 0.001) and age (r = −0.339, p = 0.001), respectively. Cystatin C also correlated with ln-IL-6 (r = −0.247, p = 0.033) (Fig. 1a) and ln-TNF-α (r = −0.405, p < 0.001) (Fig. 1b) but not with ln-CRP (r = −0.188, p = 0.108). In contrast, creatinine and eGFR did not correlate with any of these proinflammatory cytokines. We then performed stepwise regression analysis to identify the independent determinant factors of cystatin C. This showed that ln-TNF-α, eGFR, and pulse pressure were independently associated with cystatin C levels (Table 3).
Table 2

Correlation between cystatin C, creatinine or estimated glomerular filtration rate (eGFR) and proinflammatory cytokines and clinical parameters

 

Cystatin C

P value

Creatinine

P value

eGFR

P value

Age

0.213

0.067

−0.047

0.690

−0.339

0.001

BMI

0.051

0.666

−0.096

0.417

0.108

0.323

Systolic blood pressure

0.246

0.034

−0.032

0.788

0.024

0.824

Diastolic blood pressure

0.081

0.493

0.058

0.626

0.124

0.262

Pulse pressure

0.295

0.010

0.137

0.244

−0.116

0.295

HbA1c

0.008

0.948

−0.206

0.077

0.198

0.068

LDL-C

−0.093

0.433

−0.153

0.194

0.021

0.851

HDL-C

−0.010

0.932

0.001

0.923

−0.114

0.294

eGFR

−0.503

<0.001

−0.755

<0.001

  

ln-TNF-α

0.405

<0.001

0.172

0.142

−0.073

0.498

ln-IL-6

0.247

0.033

−0.099

0.403

0.009

0.936

ln-CRP

0.188

0.108

−0.195

0.096

0.095

0.384

BMI body mass index, HbA1c hemoglobin A1c, LDL-C low-density-lipoprotein cholesterol, HDL-C high-density-lipoprotein cholesterol, eGFR estimated glomerular filtration rate, TNF-α tumor necrosis factor-α, IL-6 interleukin-6, CRP C-reactive protein, ln log natural

Fig. 1

Correlation between cystatin C and proinflammatory cytokines. a Relationship between cystatin C and tumor necrosis factor (TNF)-α. b Relationship between cystatin C and interleukin (IL)-6

Table 3

Independent determinant factor of cystatin C

 

Beta

P value

Pulsed pressure

0.228

0.008

Estimated GFR

−0.436

<0.001

ln-TNF-α

0.402

<0.001

Ln TNF-α, eGFR, pulse pressure and age were used as the independent variables

GFR glomerular filtration rate, TNF-α tumor necrosis factor-α

Discussion

In this cross-sectional study in patients with EHT, we demonstrated that cystatin C was associated independently with the proinflammatory cytokine TNF-α, pulse pressure, and renal function assessed by eGFR. Plasma creatinine and eGFR did not show similar associations. These findings suggest that cystatin C is a marker of both inflammation and renal function, whereas creatinine and eGFR are not. Several studies have reported that serum cystatin C not only is a marker of renal function but also represents an independent risk factor for cardiovascular damage, heart failure, and death [13, 14, 15, 16]. It is now well established that inflammation plays a central role in the development of atherosclerosis and related complications [7, 8]. A number of studies have shown that increased blood pressure has a major influence on increasing levels of circulating TNF-α, IL-6, and CRP [10, 11, 12]. These proinflammatory cytokines and biomarkers induce arterial wall inflammation [20, 21], which in turn causes further damage to the endothelium and elevation of blood pressure, leading to a vicious cycle and promotion of atherosclerosis. We previously reported that cystatin C shows an age, body mass index, and lipid status-independent association with mean 24-h SBP estimated by ambulatory blood pressure monitoring [17]. These results are consistent with those of the study reported here. Elevated SBP and pulse pressure indicate increased peripheral vascular resistance [22]. We previously showed there is a relationship between cystatin C and hypertensive end-organ damage assessed by left ventricular mass index, carotid intima media thickness, and albumin excretion rate [17]. Taken together these earlier results and the findings of this study suggest that cystatin C levels in hypertensive patients may reflect pressure overload in the vascular wall or atherosclerosis. Interestingly, Kestenbaum et al. [23] reported that cystatin C levels were associated with incident hypertension in individuals without clinical kidney disease or CVD, indicating that cystatin C may be involved in the pathogenesis of hypertension as well as in end-organ damage associated with elevated blood pressure. Elevated proinflammatory cytokines, especially TNF-α related to cystatin C, may involve endothelial dysfunction and induce high blood pressure in the future.

Renal dysfunction predicts adverse CVD and death in a variety of clinical settings [24]. One reason decreased GFR increases CVD rate may be accumulation of proinflammatory cytokines, as there is evidence that elevated levels of these cytokines in patients with chronic kidney disease are dependent on a decline in kidney filtration [18]. In the study reported here, cystatin C was associated independently with TNF-α but not with IL-6 or CRP. TNF-α is cleared mainly by the kidney, whereas IL-6 and CRP are cleared primarily by the liver [25, 26, 27]. Although we showed cystatin C was associated with TNF-α independent of eGFR, it is possible this association may have been due to renal clearance.

Recently, an in vitro study carried out by Frendéus et al. [28] reported that addition of cystatin C to the culture medium enhanced TNF-α expression in murine peritoneal macrophages induced by interferon (IFN)-γ. These results suggest that a consequence of increased expression of cystatin C may enhance proinflammatory responses by IFN-γ-primed macrophages. Increased cystatin C in CKD patients may therefore stimulate proinflammatory cytokines, especially TNF-α.

Keller et al. [18] reported that cystatin C concentrations in adults aged 70–79 years showed stronger associations with inflammatory biomarkers, particularly TNF-α, than either creatinine level or eGFR. These results are similar to those of this study. Although our patients had a mean age of 62 years and were therefore younger than the patients in Keller’s study, they had EHT and therefore may have had advanced organ damage and atherosclerosis similar to that observed in older patients. Previous studies have also reported CRP may influence serum cystatin C levels [29, 30], although we could not confirm this association in our study. It is possible this lack of association may have been due to the small size of our study group.

In addition to the limitation of a relatively small study population, we were also unable to examine the effects of medication on cystatin C and proinflammatory cytokines. Stevens et al. [31] demonstrated recently that diabetes mellitus also has an impact on cystatin C levels, with a mean increase of 8.5% in these levels. Sixteen of the patients in our study had diabetes mellitus.

In summary, serum cystatin C was associated with SBP, pulse pressure, eGFR, and serum levels of IL-6 and TNF-α. In comparison, serum creatinine was only associated with eGFR, and eGFR was only associated with age. Cystatin C showed independent relationships with pulse pressure, eGFR, and TNF-α. These results suggest that cystatin C is a useful marker of future cardiovascular events in hypertensive patients. A further study is needed to clarify whether cystatin C is a useful marker for determining the prognosis of future cardiovascular events or death in hypertensive patients.

Notes

Acknowledgments

This work was supported by a Grant-in-Aid Scientific Research (C) awarded to TO by the Japan Society for the Promotion of Science (22590912).

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Copyright information

© Japanese Society of Nephrology 2010

Authors and Affiliations

  • Takafumi Okura
    • 1
  • Masanori Jotoku
    • 1
  • Jun Irita
    • 1
  • Daijiro Enomoto
    • 1
  • Tomoaki Nagao
    • 1
  • Veena Rasika Desilva
    • 1
  • Shiho Yamane
    • 1
  • Zuowei Pei
    • 1
  • Shiho Kojima
    • 2
  • Yasuyuki Hamano
    • 2
  • Shinichi Mashiba
    • 2
  • Mie Kurata
    • 3
  • Ken-ichi Miyoshi
    • 1
  • Jitsuo Higaki
    • 1
  1. 1.Department of Integrated Medicine and InformaticsEhime University Graduate School of MedicineToonJapan
  2. 2.Department of Research and Development, Ikagaku Co., Ltd.KyotoJapan
  3. 3.Department of Pathology, Division of PathogenomicsEhime University Graduate School of MedicineToonJapan

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