Evaluation of serum NGAL and hepcidin levels in chronic kidney disease patients

Abstract

Cardiovascular disease is the major cause of death in chronic kidney disease (CKD) patients. The main underlying reason is inflammation. In CKD, interleukin-6 and hypersensitive C-reactive protein are known to be used for the evaluation of inflammation and serum levels increase with decreased creatinine clearance. Neutrophil gelatinase-associated lipocalin (NGAL) and hepcidin are also considered to be effective in the assessment of inflammatory conditions. The possible interactions of NGAL and hepcidin with inflammatory markers in CKD patients including the kidney transplants, which have not been thoroughly explained up to date wereevaluated in this study. Serum creatinine, iron, unsaturated iron binding capacity, interleukin-6, hypersensitive C-reactive protein, NGAL, hepcidin and pro-hepcidin levels were measured in a cohort of 163 CKD patients including transplant patients and 82 healthy volunteers. Clinical evaluation and classification of the patients were done according to the NFK/KDOQI guideline. Serum hepcidin, Prohepcidin, NGAL, hypersensitive C-reactive protein and interleukin-6 levels were higher in patient groups compared to the control group. In patient groups, while hepcidin, NGAL, interleukin-6, hypersensitive C-reactive protein levels were correlated with creatinine and glomerular filtration rate, iron metabolism parameters were not correlated with the inflammation biomarkers. Inflammation related hepcidin and NGAL weakly correlated with creatinine clearance. Our results demonstrated that serum NGAL and hepcidin levels might be valuable for the evaluation of inflammation in CKD, and these new inflammation parameters are not related through iron metabolism.

• Inflammation
• NGAL
• hepcidin
• CKD
• renal transplant

Introduction

Chronic kidney disease (CKD) is a general term for heterogeneous disorders affecting kidney structure and functions1 and is a major worldwide public health problem in which factors such as diabetes, hypertension, autoimmune diseases, urinary stones, drug toxicity directly initiate kidney damage.2 The most serious outcome of CKD is considered as kidney failure. In these cases, severely decreased kidney function results with complications and treatment can be achieved only by dialysis or transplantation.1 In CKD, irreversible abnormalities in kidney structure (damage) are due to ischemic, toxic or metabolic damage.3 According to KDIGO 2012 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease, because of the graft failure and repression of immunity caused by the drugs used after transplantation, kidney transplant recipients are considered as CKD patients.1,2

The major cause of mortality in CKD patients is cardiovascular disease.2 The complications seen in these patients are the results of uremia, oxidative stress and fluid overload, which are based on inflammation. Inflammation increases directly related with the progression of the disease starting from the initiation of the disease.1,4 Inflammatory markers hs-CRP and IL-6 are known to be valuable in the evaluation of inflammation4,5 and serum IL-6 levels increase depending on the reduction of renal function.4,6 In addition to the known inflammation markers, neutrophil gelatinase-associated lipocalin (NGAL) and hepcidin are considered to be effective in the assessment of inflammatory conditions.7 NGAL also known as lipocalin-2 (lcn 2) is a 25-kDa transporter belonging to the lipocalin superfamily8,9 contains 178 amino acids.10 NGAL is expressed and secreted by immune cells, hepatocytes and renal tubular cells in various pathologic states. NGAL exerts bacteriostatic effects by capturing and depleting small iron binding molecules “siderophores” that are synthesized by certain bacteria.9 NGAL can be freely filtrated through glomerulus and reabsorbed from proximal tubules.11 It has been reported that serum levels of NGAL increase in acute and chronic renal failure.9,10

Hepcidin is a peptid hormone that was discovered simultaneously as an antibacterial protein found in human urine and as a protein preferentially expressed in iron-loaded murine liver. This is a circulating antimicrobial peptide mainly synthesized in the liver, which has been recently proposed as a factor regulating the uptake of dietary iron and its mobilization from macrophages and hepatic stores. Hepcidin was also considered as an acute phase protein.12 Hepcidin synthesis is induced by IL-6.13,14

The role of NGAL and hepcidin in inflammation is also a matter of debate whether they act through the loss of renal function, iron metabolism or another mechanism.7 There are very few studies investigating the interactions between NGAL and hepcidin in CKD patients. However, to the best of our knowledge, there is no data on this interaction in CKD patients including transplant patients. Our study is based on the hypothesis that this interaction may explain the inflammation in CKD.

Materials and methods

Study group

The study protocol was approved by the institutional ethical committee and a written informed consent was obtained from the recipients before the enrollment.

Between February and June 2012, a total of 163 patients stages I–V CKD, diagnosed in the last 5 years in Pamukkale University Hospital, were included to the study. Eighty-one of them were without dialysis treatment (43 males, 38 females, 18–75 years) and 82 were treated with kidney transplantation (44 males, 38 females, 18–75 years).

Clinical evaluation and classification of the patients with CKD were done according to the NFK/KDOQI guideline.1,2 Control group was consisted of 82 healthy known individuals without malignancy, active infection, chronic/metabolic disease and diabetes.

Samples and the measurement of the parameters

Fasting venous blood samples were collected in the morning and 24 h urine collection was demanded from all the participants. Serum obtained by the centrifugation of the blood samples was aliquated and stored in −20 °C until serum NGAL, IL-6, hepcidin and pro-hepcidin levels was measured. Serum hs-CRP, iron, UIBC, creatinine and urine creatinine levels were measured the same day the samples obtained.

Serum creatinine, iron, unsaturated iron binding capacity (UIBC) and urine creatinine levels were measured by using colorimetric, hsCRP immunoturbidimetric on a Cobas 6000 autoanalyzer (Roche Diagnostics, Tokyo, Japan). Serum NGAL (Biovendor, Czech Republic), IL-6 (eBioscience, Austria), hepcidin (DRG, Germany) and pro-hepcidin (DRG) levels were measured by using commercial kits according to the manufacturer’s instructions. All these four parameters were measured by enzyme-linked immunosorbent method.

Total iron binding capacity (TIBC) was calculated as the total of serum iron and UIBC. TIBC is used in calculation of % transferrin saturation. Transferrin saturation (%) was calculated as the rate of iron level to the TIBC (Tfr sat = (serum iron level × 100)/TIBC) and referents intervals are as 15–45%.

Creatinine clearance (GFR) was calculated by the formula: 24 h urine vol/1440 × serum creatinine level. Referent values are 88–128 mL/min (female) and 97–137 mL/min (male).1,2

Statistical analysis

SPSS software, version 17.0 (SPSS, Chicago, IL) was used for statistical analyses. The distribution of continuous variables was analyzed using the Kolmogorov–Smirnov test, in order to assess significant departures from normality and for homogenicity of variants Levene test was used. For non-normally distributed parameters, results were presented as median (interquartile range).

For samples with normal distribution statistical analysis for the differences between patient group (CKD + transplanted group) and the control group were performed with t-test and expressed as mean ± SD.

For samples with skewed distribution statistical analysis for the differences between patient group (CKD + transplanted group) and the control group were performed with Mann–Whitney U test and expressed as median (interquartile range).

Whenever the parameters presented normal distribution multiple comparisons between groups were performed by one-way ANOVA. For analysis of variance Kruskal–Wallis test was used for samples with skewed distribution.

Correlations were evaluated by correlation and multiple regression analyses where appropriate (y = ax+b). In all three groups, while the distributions are non-parametric and study groups are small spearmen correlation analyses was performed and CKD and renal transplant groups are considered as one group. In correlation analysis, rho (spearmen correlation coefficient) values are accepted as 0.000–0.49 weak correlation, 0.50–0.69 moderate correlation, ≥0.70 strong correlation. For the other statistical analyses, significance was accepted at p less than 0.05.

Results

All the variables of the groups are given in Table 1. CKD compared to the control group had significantly higher hs-CRP levels (p = 0.000), whereas renal transplant group compared to the control group and CKD group compared to the transplant group there were no significant differences (p = 0.052, p = 0.107, respectively). Significant differences were determined in terms of hepcidin between all groups (p = 0.000). In terms of Prohepcidin, while there was a significant difference between control group and both CKD and renal transplant group (p = 0.000, p = 0.000 respectively), no significant difference was determined between CKD and renal transplant group (p = 1.000). Serum IL-6 and NGAL levels were significantly different between all three groups (p = 0.000).

Table 1. Clinical and biochemical features of the study groups.

Variable	Control (n = 81)	CKD (n = 81)	Renal transplant (n = 82)
Age (years)	46.73 ± 10.50^f	50.29 ± 15.68^h	41.79 ± 11.66
BMI (kg/cm^2)	23.33 ± 0.24^d	23.11 ± 0.22^i	24.22 ± 0.22
Creatinine (mg/dL)	0.74 (0.25)^a^,^g	2.47 (0.90)^h	1.01 (0.10)
GFR (mL/min)	108.79 (29.62)^a^,^c	36.6 (29)^h	55.94 (35.10)
Iron (μg/dL)	89.49 ± 33.17^b	68.53 ± 28.53^h	90.76 ± 45.37
UIBC (μg/dL)	245 (98.2)^a^,^e	188.5 (91.3)	212 (114.6)
TIBC (μg/dL)	345.53 ± 62.12^a^,^c	262.74 ± 41.90^h	308.12 ± 65.04
Tfr sat (%)	27.34 ± 12.20	27.24 ± 12.49	31.64 ± 19.40
hs-CRP (mg/L)	1.02 (1.06)^a	3.94 (7.46)	1.61 (3.81)
IL-6 (pg/mL)	1.75 (1.88)^a^,^c	48.6 (27.52)^h	10.33 (4.64)
Hepcidin (ng/mL)	3.28 (5.12)^a^,^c	81 (53.03)^h	30.18 (14.57)
Pro-hepcidin (ng/mL)	43.65 (7.04)^a^,^c	773.72 (536.07)	581.4 (490.95)
NGAL (ng/mL)	1.66 (1.15)^a^,^c	4.01 (1.32)^h	2.96 (1.96)

Notes: Values are expressed as mean ± SD and median (interquartile range). Difference between control and CKD groups

^ap = 0.000,

^b p = 0.001.

Difference between control and renal transplant groups

^c p = 0.000,

^d p = 0.002,

^e p = 0.005,

^f p = 0.037,

^g p = 0.043.

Difference between CKD and renal transplant groups

^h p = 0.000,

ⁱ p = 0.018.

In CKD group, as the patients were under iron replacement therapy, there was no significant difference among control and patient groups in Tfr sat (p = 0.107). No significant differences were detected among control and CKD group in regard to BMI (p = 0.766) and age (p = 0.182), control and renal transplant group in regard to hs-CRP (p = 0.052) and iron (p = 0.973), CKD and renal transplant group in regard to hs-CRP (p = 0.107), UIBC (p = 0.149) and Prohepcidin (p = 1.000).

In Table 2, correlations among inflammation parameters and iron, UIBC, TIBC, Tfr sat, in Table 3 correlations among inflammation parameters and renal function markers in patient groups were given.

Table 2. Correlations among inflammation parameters and iron, UIBC, TIBC, Tfr sat in patient group.

	Hepcidin	Pro- hepcidin	NGAL	IL-6	hs-CRP
Iron	r = −0.128p = 0.106	r = −0.003p = 0.965	r = −0.176p = 0.025	r = 0.250p = 0.001	r = −0.293p = 0.000
UIBC	r = −0.198p = 0.011	r = −0.146p = 0.063	r = 0.039p = 0.624	r = −0.101p = 0.199	r = −0.171p = 0.029
TIBC	r = −0.337p = 0.000	r = −0.214p = 0.006	r = −0.089p = 0.262	r = −0.309p = 0.000	r = −0.241p = 0.002
Tfr sat.	r = −0.005p = 0.947	r = 0.069p = 0.383	r = −0.128p = 0.106	r = −0.111p = 0.159	r = 0.021p = 0.787

Table 3. Correlations among inflammation parameters and renal function markers in patient group.

	Hepcidin	Prohepcidin	NGAL	IL-6	hs-CRP
Creatinine	r = 0.432p = 0.000	r = 0.080p = 0.314	r = 0.347p = 0.000	r = 0.528p = 0.000	r = 0.261p = 0.001
GFR	r = −0.342p = 0.000	r = −0.053p = 0.503	r = 0.295p = 0.000	r = −0.398p = 0.000	r = −0.392p = 0.000

In our study, we did not establish any correlation between markers of inflammation and both renal function and iron status indicators in healthy participants.

Discussion

Inflammation is the most important underlying mechanism in cardiovascular disease and other systemic diseases, which are the major causes of morbidity and mortality in chronic renal failure1,15

In chronic renal disease, the toxicity caused by increased oxidative stress, decreased cytokine clearance, chronic infections, hypertension, fluid overload, metabolic dysfunction (hyperglycemia) and uremia induce some signal pathways which results with increased serum IL-6 level. Increased IL-6 leads to chronic inflammatory conditions such as the synthesis of acute phase proteins like CRP, hypercoagulability and accelerated atherosclerosis.16,17 In our study, we demonstrated that serum IL-6 level is highly related with the kidney function parameters compared to hsCRP level. This result might suggest that IL-6 is the main contributor in chronic inflammation and is related with the loss of renal function. Serum hsCRP level of the CKD group was significantly (p = 0.000) higher than the control group, that might support the concept of higher risk of morbidity and mortality from CVD in CKD patients. There was a negative correlation in serum hsCRP, IL-6 levels with GFR in both CKD and renal transplant patients in our study. Serum IL-6 showed a stronger relation with the renal function parameters (serum creatinine level, GFR) than hsCRP level. These results support those inflammation parameters in serum increase depending on the decrease of the creatinine clearance or the loss of renal function.

Hepcidin’s molecular structure and control mechanisms are better understood by the studies done in the last 5 years, and is clearly demonstrated that hepcidin is the basic regulator of systemic iron metabolism.7,14,18 Hepcidin is eliminated from the body through the kidneys.7 During inflammation and infections, hepcidin level increases and extracellular iron levels decrease leading to the inhibition of iron acquisition, which is essential necessary for the microorganisms.14 Studies since the discovery of hepcidin are generally conducted in animals and in vitro experiments.19 Small and compact peptide structure, the tight integration between components and antigen-compatibility of hepcidin is hindering factors for development of a suitable immunochemical method.20 For this reason, many clinical trials are carried out with Prohepcidin consisting of 60 aa, which is the precursor protein of hepcidin although biological relation and relevance of Prohepcidin with hepcidin is not clear20 and Prohepcidin is now believed to be poor marker of hepcidin bioactivity.14 Prohepcidin levels differ by stimulation of inflammation rather than changes in iron metabolism.19,21 Due to the technical problems and roles of both Prohepcidin and hepcidin in inflammation course and control of iron metabolism, additionally the determination of Prohepcidin is important for providing information for the evaluation of inflammation.21

In our study, because of the above-mentioned reasons in addition to serum hepcidin level, serum prohepcidin level was also measured and no significant correlation was determined among them. This finding supports the weak biological relationship between each other. The weak correlation of Prohepcidin with both iron parameters and cytokines in patient groups suggests that it may not be an advantageous biomarker in clinical surveillance and researches.

While hepcidin levels were found to be significantly different in all groups, Prohepcidin levels were higher in CKD group compared to control and renal transplant groups and not significantly different in CKD and renal transplant groups The increase in serum hepcidin level in CKD group compared to control group might be a result of not only the increased production due to inflammation, but also to the participation of kidneys in elimination of hepcidin as suggested by Kulaksız et al.22 In CKD group depending on the damage of kidney, the elimination of hepcidin is limited and results with the increase in serum level.

In our study, another inflammation parameter NGAL was found to be negatively correlated with iron. It can be suggested that this correlation is a result of NGAL binding small iron binding molecules and load the cell with iron23 demonstrating the importance of iron load during bacterial infections and kidney damage. It was stated that in various pathologic conditions NGAL expressed in immune cells and secreted limits tubular injury and which is an independent affect from its bacteriostatic action. NGAL is also accepted as modulating various cellular responses, such as proliferation, apoptosis and differentiation with an unknown mechanism.9,23,24 Recent studies demonstrate the involvement of NGAL in adaptive stage of CKD.7 It was hypothesized that in CKD, the change in the physiology of this protein is comparable to the acute injury. Chronic injured kidney tubules produce enhanced NGAL. In conclusion, increased NGAL level is not only a result of decreased clearance, but also increased production.14

In our study, serum NGAL levels are significantly different when compared in all groups. CKD group has the highest mean level of NGAL. This result indicates that chronic injury might enhance the production and/or reduce the clearance of NGAL. Increased serum NGAL level in renal transplant group compared to healthy group might be due to toxic injury caused by the calcineurin inhibitors used.25 NGAL positively correlated with serum creatinine levels in patient groups is an evidence for association with the elimination. There are other studies also demonstrating the correlation of NGAL with creatinine levels in CKD patients with higher levels than the controls.26 Both NGAL and hepcidin possessing antimicrobial effects are acute phase proteins, which are related with inflammation and restrict iron uptake and usage. While NGAL express this activity by stimulating the synthesis of iron binding proteins (transferrin), decreasing the absorption of dietary iron and enhancing the iron storing protein levels, hepcidin an antibacterial defensin induced in the liver as a response to IL-6 stimulation, expresses by decreasing the absorption of dietary iron and preventing iron release from macrophages.7,14,19

In our study, hepcidin and NGAL, regulator in iron metabolism and inducing kidney injury along with inflammation were demonstrated to be related with the kidney function parameters. The negative correlation of these parameters with GFR supports the estimate that these parameters might reflect the progression of inflammation in CKD.26 In conclusion, the relation of both serum NGAL and hepcidin levels with the inflammatory parameters and renal function indicators supports the point of view that both parameters are effective in inflammation possibly by similar mechanisms.

Depending on the correlations the relation of the inflammatory parameters with the renal function indicators is more prominent than the relations with the iron metabolism parameters. Also, the correlation of GFR related IL-6 with hepcidin and NGAL is also an evidence for the relevance with inflammation in CKD.

According to our results, we conclude that serum hepcidin, Prohepcidin and NGAL levels can be used for the evaluation of inflammation in CKD patients including renal transplant patients together with the serum IL-6 and hsCRP levels. The data of our study can be used as preliminary information in the new studies regarding hepcidin and NGAL related with inflammation in CKD patients including the renal transplant patients.

Declaration of interest

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