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C-reactive Protein and Hepcidin in Non-Dialysis Chronic Kidney Disease

Complications such as anemia and its clinical consequences arise as chronic kidney diseases progress,. One renal anemia pathophysiology is a disruption of iron metabolism, regulated by the main iron exporter hormone, hepcidin. Chronic kidney disease patients were constantly in an infl ammatory state, represented by an increased in C-reactive protein. This infl ammatory state would facilitate the liver to secrete hepcidin, which would subsequently follow a decrease of iron circulation, thus resulting in functional iron defi ciency. Both acute phase reactants which used thoroughly as markers in tropical and infectious diseases, had their own roles in chronic kidney disease. The correlation of c-reactive protein and hepcidin in chronic kidney disease patients was still controversial. To analyse the relationship between c-reactive protein and hepcidin in non-dialysis chronic kidney disease patients. We conducted an observational cross-sectional study with 40 non-dialysis chronic kidney disease patients who met the inclusion and exclusion criteria. Patients were enrolled with consecutive sampling and were examined for serum c-reactive protein and hepcidin levels.A total of forty subjects (67.5% male with mean age of 50.23 ± 1.04 years) were eligible for enrolment in this study. The most comorbid factor was hypertension (62.5%). The common stage for chronic kidney disease was stage 3 (40%). The mean hemoglobin value was 10.74 ± 0.36 g/dL, mean blood urea nitrogen was 39.98 ± 29.59 mg/dL, and serum creatinine of 4.12 ± 3.39 mg/dL. Mean serum c-reactive protein levels were 3.52 ± 5.13 mg/l. Mean hepcidin level were 94,03 ± 95,39 ng/ml. Serum C-reactive protein levels correlated positively (r=0.487) and signifi cantly (p-value=0.001) with serum hepcidin value. C-reactive protein and hepcidin was signifi cantly correlated in non-dialysis chronic kidney disease patients.


INTRODUCTION
Hepcidin and c-reactive protein (CRP) had their roles in infectious diseases for a period of time. Hepcidin lowered mammal's blood iron levels at the time the pathogen-infected the hosts. Low blood iron level hindered pathogen's growth so that infections might be stopped. C-reactive protein had its own roles in activating platelets, leukocytes, endothelial growth factors, complements and chemokines during infections to cease infection. However, the roles of these two markers in renal anemia in chronic kidney disease (CKD) have not been elucidated yet.
C-reactive protein has been one of the sensitive inflammation markers which correlate with hepcidin in CKD. There have been substantial studies to backed up and come against it. Chronic kidney disease, stated as a chronic state of lowgrade infl ammation, could initiate a chain of sequences that lead to secretion of CRP and hepcidin. However, hepcidin was fi rst recognized by Ganz, et al. as liver expressed antimicrobial peptide-1 (LEAP-1) secreted during infection or high-grade infl ammation, putting hepcidin into lower place in this chain of sequences than CRP.
C-reactive protein was proven to be inversely correlated with the estimated glomerular fi ltration rate (eGFR) and stage in CKD. CRP also correlated with other infl ammation markers such as interleukin-6 (IL-6). Interleukin-6 was directly correlated with the secretion of CRP and hepcidin in the human liver.
Hepcidin is a major iron exporter hormone in mammals. It interacts with its receptor, ferroportin in gastrointestinal tracts and reticuloendothelial systems. Degradation and internalizing process of ferroportin inhibits daily iron intake entering circulation from duodenum and traps intracellular storage iron. These processes create a hypoferremia state which results in functional iron defi ciency anemia. Anemia brings clinical consequences such as a decrease of quality of life, deterioration of eGFR, increased cardiovascular events, increased mortality rate, and even increased economical burden. High infl ammation state and other confounding factors (anemia, duration of dialysis) was seen in CKD patients on dialysis which lead to sample selection of non-dialysis patients. Serum hepcidin level was measured using the Enzyme Linked Immunosorbent Assay (ELISA) method. Serum were stored in a deep freezer at a temperature of -80°C until the hepcidin measurement was performed. The reagent used was DRG Hepcidin-25 (bioactive) ELISA from CAT No EIA-5782, an enzyme immunoassay for in-vitro quantitative examination for hepcidin-25 peptide in serum and plasma. 3,4 Statistical analysis: All data was analysed by Statistical Package for the Social Sciences (SPSS ver 23). Data was delivered in the form of analytic statistics. Data analysis was provided in mean ± standard error of mean (SE). Correlation of serum hepcidin with CKD stage was calculated by Pearson parametric test if it had normal distribution or Spearman parametric test if the data distribution was not normal. It was said to be signifi cant if the p-value is <0.05.

Patient Characteristics
A total of forty subjects (67.5% male with mean age of 50.23 ± 1.04 years) were eligible for enrollment in this study. This study was done in Nephrology Outpatient Clinic, Dr Soetomo General Hospital, Surabaya, Indonesia within the period of 1 June 2018 -31 August 2018. The results of demographic and clinical characteristics of this study subjects were described in Table 1 and Table 2.
Twenty seven of 40 subjects were male (67.5%), the youngest is 27 years old and the oldest is 58 years old with mean age of 50.23 years old (SE 1.04), mean of body mass index (BMI) was 22.54 (SE 0.57). Based on CKD stage, 16 patients (40%) of the total sample had stage 3 CKD. (Table 1)

Clinical Characteristics
Mean hemoglobin level of 40 subjects was 10.74 g/dL with SE of 0.36. Mean of blood urea nitrogen (BUN) levels was 39.98 mg/dL with SE of 4.68. Mean level of serum creatinine was 4.12 with SE of 0.54. The most frequent comorbid factor was hypertension (62.5%) ( Table 2).

Signifi cance and Strength of CRP and Hepcidin Value Correlation in Non-Dialysis CKD Patients.
Distribution of CRP and hepcidin level data were used to analyze the correlation between CRP and hepcidin in CKD patients. Kolmogorov-Smirnov normality test showed that the distribution of CRP and hepcidin level data is abnormal (both p-value < 0.05). Spearman correlation test was used to further analyze the correlation between CRP and hepcidin levels. (Table 5) Analysis of CRP and hepcidin value showed an association with a positive correlation coeffi cient of 0.487. The correlation of CRP and hepcidin value in this study was signifi cant, indicated by the p-value = 0.001. The meaning of this positive correlation coeffi cient showed a unidirectional relation, if the CRP level increase, the hepcidin level would be increased consequently.

DISCUSSION
Chronic kidney diseases progressed alongside complications such as anemia and its clinical consequences. One of the renal anemia pathophysiologies was disruption of iron metabolism, regulated by main iron exporter hormone, hepcidin. Chronic kidney disease patients were constantly in an inflammatory state, represented by increased of CRP. This infl ammatory state results in the liver secreting hepcidin, which subsequently followed a decrease in iron circulation, thus resulting in functional iron defi ciency. Inclusion of stage 3 to 5 CKD patients was based on earlier studies that stated complications of CKD, particularly anemia, were more commonly seen in stage 3 to 5 CKD patients. Non-dialysis CKD patients were selected to reduce confounding factor such as duration of dialysis in CKD patients. 5,6 Most of the study subjects were men with a percentage of 67.5%, similar to studies by Toima, et al., Mercadel,et al,Elmenyawi,et al. 5,6,7 Higher male prevalence than female could be infl uenced by numerous factors like hypertension, hyperglycemia, lifestyle, kidney structure and hormonal diff erences. 8 The mean age in this study was 50.23 ± 1.04 years old, similar to studies by Mercadel, et al,   5,6,7 Aging process infl uenced CKD progression and lesser function was expected from older nephrons. 9 Mean hemoglobin result in this study was 10.74 g/dL with SE of 0.36. The results of this study were similar to other studies by Toima, et al., Peters,et al.,and Goyal,et al. 5,10,11 Mean results of BUN value were 39.98 mg/dL with SE of 4.8. BUN and creatinine serum levels found in this study were similar to study by Toima, et al. 5 The most frequent comorbid disease in this study was hypertension, at 62.5% of the total subjects. Study by Toima, et al., Peters, et al., and Goyal, et al., also mentioned hypertension as the most frequent comorbid disease found in CKD patient. 5,10,11 Hypertension was the highest prevalent chronic disease in Indonesia based on 2013 RISKESDAS study. 12 Hypertension risk factors were age, race, family history, obesity, high sodium intake, and smoking. 8 In this study, higher mean CRP levels was seen in more advanced CKD stage. The mean total CRP level in this study was 3.52 mg/L with a SE of 0.81. This was similar to previous studies by Toima et al. who found CRP levels of 6.0 mg/L with a standard deviation of 0.9, Elmenyawi et al. who found mean CRP level was 4.28 mg/L with a standard deviation of 3.7, Rasheed et al. who found CRP mean levels were 7.59 mg/L in all CKD stages, and. 5,7,13 Fluctuations in CRP levels may also have been due to the highest staging diff erences in the population study. In a study by Elmenyawi et al the most frequent CKD stage in the population was stage 3 with mean CRP level at 3.52 and 4.28 mg/L. 7 In the study of Toima et al. and Rasheed et al. the highest staging in the population was stage 5 and mean CRP levels were, 6.0 and 7.59 mg/L. 5,13 This study found that CRP level increased along with a decrease in eGFR, which were consistent with other studies. 14,16 Total mean hepcidin found in this study was 94.03 ng/ml with SE of 15.08. While Toima, et al. found mean hepcidin level of 84 ng/mL with a standard deviation of 18.6, Goyal, et al. and Uehata, et al. found mean hepcidin levels of 65.0 ng/mL and 15.4 ng/mL respectively. 5,11,17 Analysis of CRP and hepcidin levels in nondialysis CKD patients revealed a moderate to signifi cant relationship (correlation coeffi cient 0.487; p-value 0.001). This result indicated that an increase in CRP levels would lead to a directly proportional increase of hepcidin value. The results of this study were in accordance with studies by Toima et al., Peters et al., and Lee et al., who inferred a positive relationship between CRP and hepcidin levels. 5,10,15 Toima et al. organized a study in Egypt regarding the importance of hepcidin role as a novel biomarker which refl ected iron status in CKD patients and its relationship with CRP levels. Thirty CKD patients and 10 healthy subjects, used as controls, were enrolled. The result showed a correlation in CRP and hepcidin value with R of 0.68 (p = 0.001). 5 Patients who had iron or erythropoietin therapy for the previous 21 days were excluded. Inclusion of diabetes mellitus patients might lead to a strong correlation found in this study. This study used the same method in CRP and hepcidin level measurement as Toima, et al. 5 Peters, et al. conducted an observational crosssectional study of factors aff ecting hepcidin in 83 non-dialysis CKD patients and 48 dialysis CKD patients in the Netherlands. There was a weak positive relationship (r=0.21, p <0.001) between CRP and hepcidin levels which was probably related to inclusion of patients who were under erythropoietin therapy. The method used to measure CRP level was the same as in our study, but a diff erent method (light chromatography mass spectrometry / LC-MS) was used in measuring hepcidin level. 10 Lee, et al. in Korea analysed whether hepcidin was a novel uremic toxin using multivariate analysis of various variables aff ecting hepcidin in 2090 non-dialysis CKD patients. They found a positive correlation between CRP and hepcidin with r=0.23 (p <0.001). Patients with intravenous iron, oral iron, and erythropoietin therapy were not excluded. These factors might have played a role as confounding factors to the weak correlation. This study used the same method in CRP and hepcidin level measurement as Lee, et al. 15 The included 505 samples of non-dialysis CKD patients and found no association between CRP and hepcidin levels (r = 0.03 and p = 0.4). Patients with liver cirrhosis were not excluded, while liver cirrhosis can induce negative feedback on hepcidin and CRP. The Level of CRP was measured using immunoagglutination detection method and hepcidin level was determined using LC-MS. 17 Another study by Goyal, et al. in India analyzed the relation between CRP and hepcidin levels in 100 non-dialysis CKD patients. They found similar results with this study, the correlation coeffi cient of 0.0001 and p = 0.896 between CRP and hepcidin levels. Patients having oral iron therapy were not excluded, while oral iron could induce positive feedback on hepcidin. C-reactive protein levels were measured using different EIA kits which could influence the absence of association of CRP and hepcidin. 11 Study by Wagner, et al., which analysed predictive factors of mortality in patients with non-dialysis CKD patients, showed contradiction to this study by stating that CRP level was not associated with hepcidin levels (correlation coeffi cient of 0.01 and p <0.001). Their case control study stated that CRP and hepcidin (measured hepcidin using RIA method) were infl uenced by factors that change over time. The mean hemoglobin in their study was higher than this study (13.1 g/dL) while anemia could induce negative feedback on hepcidin. 18 These factors might have played a role in the absence of a correlation. Another study contrasting this study results was Macdougall et al. in the Netherlands who used a random sampling system and including patients with erythropoietin therapy which caused positive feedback on hepcidin. 19 There were diff erences in the results of this study compared to previous studies. Diff erent methods in measuring CRP and hepcidin levels could have contributed to this result. In earlier studies, the CRP level was measured using the immunoturbidimetric assay, immunoagglutination, or EIA method. 5,10,15 In previous studies, hepcidin level was measured by ELISA and LC-MS methods. 20 Studies conducted by Mercadel, et al., Macdougall, et al. used diff erent methods to determine hepcidin levels. 6,21 Hepcidin could be measured using RIA, ELISA, and mass spectrometry-based methods. 22 Measurement using RIA detects hepcidin-25 greater than actual condition. Measuring hepcidin-25 using ELISA were accurate and cheap. 3,23 Mass spectrometrybased was indeed more accurate but not practical, requiring more instruments and too expensive. Besides diff erences in measurement methods, there were also diff erences in this study subjects' characteristics compared to previous studies. 24 History of erythropoietin therapy, a history of blood transfusion, iron therapy, diabetes mellitus, and other factors infl uencing CRP and hepcidin level were not excluded in previous studies, whereas it could have affected the results. 6,11,14,25 This was a novel study of hepcidin and inflammation marker in CKD in Surabaya, although we were aware that the small number of subjects might interfere with the study results. Study with a larger, more homogenous sample, more markers of infl ammation and iron might be needed in the future.

CONCLUSION
A signifi cant positive correlation with r s = 0.487, p = 0.001) was found between CRP and hepcidin levels in non-dialysis CKD patients. If there was an increase in serum CRP levels in non-dialysis CKD patients there was a tendency for an increase in serum hepcidin levels.

CONFLICT OF INTEREST
There is no confl ict of interest of this paper.

ACKNOWLEDGEMENT
This project would have been impossible without the support of all the participants in this study, all Nephrology Division staff s and Internal Medicine Department functional staff s of Dr. Soetomo General Hospital Surabaya. The authors would like to thank Prodia Laboratory Surabaya