Abstract
Hepcidin is an essential regulator of iron homeostasis in chronic kidney disease (CKD) anemia, reticulocyte hemoglobin equivalent (RET-He) can be used to evaluate the availability of iron for erythropoiesis. Previous research has found that hepcidin indirectly regulates RET-He. This study aimed to investigate the association of hepcidin, RET-He and anemia-related indicators on anemia in chronic kidney disease. A total of 230 individuals were recruited, including 40 CKD3-4 patients, 70 CKD5 patients without renal replacement therapy, 50 peritoneal dialysis patients, and 70 hemodialysis patients. The serum levels of hemoglobin (Hb), reticulocyte, RET-He, serum iron, serum creatinine, serum ferritin, total iron binding capacity, hepcidin-25, high sensitivity C-reactive protein, transferrin, erythropoietin, intrinsic factor antibody, soluble transferrin receptor and interleukins-6 (IL-6) were measured. Hepcidin-25 was positively associated with IL-6, and negatively with total iron binding capacity, intrinsic factor antibody, and transferrin. Reticulocyte Hb equivalent was associated positively with Hb, serum ferritin, serum iron, transferrin saturation, and negatively with serum creatinine, reticulocyte, IL-6, STfR. Hepcidin-25 was not associated with RET-He, while IL-6 was independently associated with hepcidin-25 and RET-He, suggesting that hepcidin has no effffect on the iron dynamics of reticulocytes in CKD, may be related to IL-6, indicate a likelihood of a threshold for stimulation of hepcidin-25 expression by IL-6 in order to indirectly regulates RET-He.
Keywords: anemia, chronic kidney disease, hemodialysis, hepcidin-25, peritoneal dialysis, reticulocyte hemoglobin equivalent
1. Introduction
Chronic kidney disease (CKD) is a widespread condition with increasing number of incidences every year.[1] Anemia is one of the common complications in patients with CKD. Researchers found that approximately 10% of CKD stage 1 to 2 patients and 50% of CKD stage 3 to 5 patients developed anemia during the course of CKD,[2] while a higher proportion of anemia was observed in hemodialysis patients. If not actively treated, anemia can accelerate the deterioration of renal function, causing complications of the respiratory and digestive systems and increasing the risk of cardiovascular events.[3] Iron deficiency often reduces the patients sensitivity towards erythropoietin treatment, resulting in an increase in the dosage of erythropoietin and thereby increasing the cost of treatment.[4] Therefore, CKD anemia patients should be provided supplementation with adequate dose of iron along with erythropoietin (EPO) treatment.[5,6] However, with the emergence of safety issues associated with ESA and the increasing cost, the role of reduced iron availability in the pathogenesis of CKD anemia is attracting more and more attention.
Hepcidin-25 (2.5 kDa) is a negative regulatory hormone of iron metabolism that downregulates the level of serum iron and plays an essential role in CKD anemia.[7–11] Some studies have shown that the expression of hepcidin is regulated by inflammation and iron load.[12,13] Patients with CKD are in a chronic inflammatory state, resulting in elevated levels of inflammatory factors, such as high sensitivity C-reactive protein (hs-CRP), interleukins-1, interleukins-6 (IL-6), tumor necrosis factor-α, and interferon-γ.[14]
The absolute or functional deficiency of iron is related to the impairment of hemoglobin synthesis, which leads to anemia and reduced tissue oxygen. Serum ferritin (SF) and transferrin saturation (TSAT) are routine biochemical examination projects for diagnosing iron deficiency, which reflects the condition of stored iron and has high accuracy. However, these indicators are easily affected by inflammation; therefore, they have some limitations. As a result, these tests should be replaced by more stable iron parameters to assess the iron status in patients with CKD.As per recent studies, the reticulocyte hemoglobin equivalent (RET-He) is the reflection of iron content in reticulocytes and can be used to evaluate the availability of iron for erythropoiesis.[15–19] Since the National Kidney Foundation Kidney Disease Outcomes Quality Initiative recommended the use of RET-He in 2006, RET-He has been regarded as important early indicators for assessing the iron status in patients with CKD.[20]
A previous study found there was no correlation between serum hepcidin levels and their RET-He levels on peritoneal dialysis patients,[21] while another study has also shown that RET-He depends on available iron in blood, indirectly regulated by hepcidin.[22] The association of hepcidin and RET-He on anemia in CKD has not been studied before; in this study, anemia related indicators were measured to study their association with hepcidin and RET-He on anemia in CKD, in order to improve anemia and the quality of life of patients with CKD.
2. Materials and methods
2.1. Subjects
This study recruited CKD patients hospitalized in Qilu Hospital of Shandong University (Qingdao) from January 2019 to December 2020. Inclusion criteria were stable adult CKD patients. Exclusion criteria included acute-phase inflammation, bleeding, blood transfusion or surgical procedures within 3 months, malignant tumor, hematological diseases, nonrenal anemia, or liver dysfunction were also excluded from the study. This study protocol was reviewed and approved by the Medical Ethics Committee, Qilu Hospital of Shandong University (Qingdao, Shandong, China), approval number KYLL-KS-2018022. All patients participating in this study were informed, signed informed consent, and voluntarily participated. All methods were carried out in accordance with Declaration of Helsinki.
2.2. Data collection
Blood samples were collected in CKD3-4, CKD5 and peritoneal dialysis (PD) patients during the hospitalization, and at the start of hemodialysis (HD) treatment after a 2 days interval from the last treatment in HD patients. The data of each patient was collected from our hospital information management system. The hemoglobin (Hb), reticulocyte (RET), RET-He, serum iron (SI) serum creatinine (Scr), SF, total iron binding capacity (TIBC), hepcidin-25, hs-CRP, transferrin (TRF), EPO, intrinsic factor antibody (IFAb), soluble transferrin receptor (sTfR), IL-6 were analyzed in each group to investigate their association with hepcidin and RET-He on anemia in chronic kidney disease.
2.3. Disease definition
Chronic kidney disease was defined as abnormalities of kidney structure or function, present for more than 3 months, with implications for health according to the Kidney Disease: Improving Global Outcomes CKD Work Group,[23] and the estimated glomerular filtration rate was calculated using the simplified prediction equation derived from the modified version in the Modification of Diet in Renal Disease study. The abbreviated Modification of Diet in Renal Disease estimate of kidney function was calculated as 175 × (SCr/88.4) -1.154 × age -0.203 × 0.742 (if female).[24] In consideration of good compliance and convenient design, the inclusion criteria for CKD3-4, CKD5, and PD patients were selected hospitalized patients. All patients provided written informed consent to participate in the study. GFR in CKD3 to 4 group was 15 to 59 mL/minutes/1.73m2, and ≤ 15 mL/minutes/1.73 m2 in CKD 5 group.
2.4. Detection method
The data of Hb, RET-He hs-CRP, Scr, SI, TRF, SF, EPO, IFAb, sTfR was collected from our hospital information management system. The levels of Hb, RET-He (Blood cell analyzer Sysmex XN-3000), hs-CRP(DM79-2), Scr (Roche c701 biochemical analyzer), SI, TRF (autobio TBA-FX8), SF, EPO, IFAb, sTfR (Beckman DX1800) in the laboratory department. TIBC was measured using the TIBC test kit (wuhan Elabscience Biotechnology Co., Ltd.), serum IL-6 was measured using the human IL-6 enzyme-linked immunosorbent Kit (wuhan Elabscience Biotechnology Co., Ltd.), hepcidin-25 was measured using the human hepcidin-25 enzyme-linked immunosorbent Kit (wuhan Elabscience Biotechnology Co., Ltd.)
2.5. Statistics
Statistical analysis of the data was performed using the SPSS software (version 21.0, IBM Corp., Armonk, NY). Data are presented as mean ± SD. Serum ferritin, SI, EPO, IFAb, IL-6, and hs-CRP levels were log-transformed to reduce the skewness of the data. Differences among the NC group, CKD3-4, CKD5, PD, and HD patients were analyzed by 1-way ANOVA. The associations between each clinical parameter were calculated using Pearson correlation coefficients. Factors affecting the levels of hepcidin-25 and the RET-He were analyzed by multiple linear regression model, and model was adjusted by age, sex, and ESA use. P values < .05 were regarded as statistically significant.
3. Results
In this study, we enrolled 230 CKD patients (men, n = 153 59%), with a range age of 26 to 88 years, the mean age of subjects was 59 ± 12 years, and the median was 62 years at Qilu Hospital (Qingdao) of Shandong University. Among them, 110 patients did not undergo dialysis (40 CKD3-4 patients, 70 CKD5 patients without renal replacement therapy), 50 patients underwent PD (PD group), and 70 patients underwent HD (HD group). The median duration of dialysis was 23 months (range: 2–144 months) in HD patients and 12 months (range: 2–48 months) in PD patients. Diabetic nephropathy (accounted for 38% of the CKD patients) was the most common underlying disease, followed by chronic nephritis (accounted for 34% of the CKD patients), hypertensive renal disease (accounted for 11% of the CKD patients) and chronic kidney disease caused by other conditions (accounted for 17% of the CKD patients). Eighteen (45%) of CKD 3 to 4, 32 (46%) of CKD5 and 37 (74%) of PD patients received ESA (Epogen, 10,000 IU/week) by subcutaneous injection, and 64 (92%) of HD patients did via intravenous injection. Twenty-eight (70 %) of CKD3 to 4, 38 (54%) of CKD5, and 46 (92%) of PD patients received oral iron supplementation, while 18 (26%) of HD patients were administered with intravenous iron preparation.
Table 1 shows the biochemical characteristics of each group. As expected, Scr levels in CKD5, HD and PD groups were significantly higher (P < .05) than in CKD3-4. RET-He level was lower in HD group compared with the other groups (P < .05). The hs-CRP levels in CKD5, PD and HD group were significantly different (P < .05) compared with that in CKD3 to 4 group. Compared with the Hepcidin-25 and IL-6 level in CKD3 to 4 and CKD5 group, there was significant difference in HD group (P < .05). Total iron binding capacity levels were higher in the HD group than in the PD group (P < .05), and there was no significant difference among the other groups (P > .05). Serum ferritin, SI, TSAT, and TRF levels were no significant difference among each group (P > .05). Intrinsic factor antibody level was higher in the CKD5 group than in the HD groups (P < .05). Soluble transferrin receptor level was higher in the HD group than in the other groups (P < .05).
Table 1.
Various indicators of the groups.
| Total (n = 230) | CKD3–4 (n = 40) | CKD5 (n = 70) | PD (n = 50) | HD (n = 70) | P value | |
|---|---|---|---|---|---|---|
| Age (yr) | 59.77 ± 12.35 | 61.70 ± 11.65 | 59.94 ± 13.63 | 56.90 ± 11.28 | 60.56 ± 12.01 | .265 |
| Male sex, n (%) | 138 (60.0) | 24 (60.0) | 35 (50.0) | 27 (54.0) | 52 (74.2) | .022 |
| Hb (g/L) | 95.95 ± 21.78 | 103.03 ± 18.18 | 85.40 ± 18.45* | 96.30 ± 22.83† | 102.21 ± 22.25† | <.001 |
| Scr (umol/L) | 656.67 ± 320.98 | 217.25 ± 75.87 | 613.57 ± 257.86* | 893.74 ± 285.32*,† | 781.54 ± 209.78*,† | <.001 |
| RET (%) | 1.85 ± 0.58 | 1.93 ± 0.42 | 1.83 ± 0.63 | 1.65 ± 0.57 | 1.96 ± 0.59‡ | <.001 |
| RET-He (pg) | 31.04 ± 2.51 | 32.22 ± 2.08 | 31.08 ± 2.42* | 31.24 ± 2.29 | 30.18 ± 2.70*,†,‡ | <.001 |
| Hs-CRP (mg/L) | 3.81 ± 2.87 | 2.64 ± 2.33 | 3.99 ± 2.88* | 3.98 ± 3.02* | 4.17 ± 2.92* | 0.041 |
| Hepcidin-25 (ng/mL) | 9.53 ± 2.82 | 8.87 ± 2.33 | 9.04 ± 2.71 | 9.50 ± 2.74 | 10.41 ± 3.08*,† | 0.01 |
| IL-6 (pg/mL) | 44.03 ± 24.85 | 36.65 ± 14.59 | 39.06 ± 20.88 | 40.78 ± 19.85 | 55.53 ± 31.86 *,†,‡ | <.001 |
| SF (ng/mL) | 112.10 ± 75.73 | 91.49 ± 51.46 | 107.17 ± 70.89 | 116.92 ± 84.02 | 125.36 ± 83.85 | .132 |
| SI (umol/L) | 11.58 ± 4.41 | 11.02 ± 3.07 | 11.16 ± 4.76 | 11.34 ± 4.89 | 12.50 ± 4.29 | .214 |
| TIBC (umol/L) | 31.21 ± 7.98 | 30.67 ± 7.03 | 31.47 ± 8.33 | 29.52 ± 7.17 | 32.47 ± 8.58‡ | .238 |
| TSAT (100%) | 37.98 ± 13.18 | 37.66 ± 13.68 | 35.88 ± 12.95 | 38.79 ± 13.83 | 39.67 ± 12.63 | .374 |
| IFAb (AU/mL) | 1.19 ± 0.12 | 1.18 ± 0.10 | 1.23 ± 0.14 | 1.18 ± 0.11 | 1.16 ± 0.11† | .003 |
| STfR (nmol/L) | 17.74 ± 6.48 | 15.98 ± 4.09 | 16.60 ± 5.93 | 15.78 ± 6.65 | 21.27 ± 6.67*,†,‡ | <.001 |
| TRF(g/L) | 1.86 ± 0.44 | 1.95 ± 0.28 | 1.82 ± 0.33 | 1.92 ± 0.43 | 1.80 ± 0.59 | .196 |
| EPO (mIU/mL) | 28.18 ± 46.19 | 16.40 ± 16.09 | 25.46 ± 37.24 | 35.23 ± 42.17* | 32.60 ± 64.36 | .199 |
| ESA use, n (%) | 151 (65.7) | 18 (45.0) | 32 (46.0) | 37 (74.0) *,† | 64 (92.0) *,† | <.001 |
P values mean the overall differences between CKD3-4, CKD5, PD and HD groups. Data were presented as mean ± SD.
CKD3-4 = CKD3-4 patients, CKD5 = CKD5 patients without renal replace treatment, EPO = erythropoietin, Hb = hemoglobin, HD = hemodialysis patients, hs-CRP = high sensitivity C-reactive protein, IFAb = intrinsic factor antibody, IL-6 = interleukins-6, PD = peritoneal dialysis patients, RET = reticulocyte, RET-He = reticulocyte hemoglobin equivalent, Scr = serum creatinine, SF = serum ferritin, SI = serum iron, sTfR = soluble transferrin receptor, TIBC = total iron binding capacity, TRF = transferrin, TSAT = transferrin saturation.
Statistically significant difference compared with the CKD3-4 group (P < .05).
Statistically significant difference compared with the CKD5 group (P < .05),
Statistically significant difference compared with the PD group (P < .05).
3.1. Pearson correlation analysis of hepcidin-25 and other indicators. SF, SI, EPO, IFAb, IL-6, and hs-CRP levels were log-transformed (Table 2).
Table 2.
Pearson correlation analysis of hepcidin-25 and other indicators.
| Whole | CKD3–4 | CKD5 | PD | HD | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| r | P value | r | P value | r | P value | r | P value | r | P value | |
| Hb (g/L) | −0.024 | .718 | 0.294 | .066 | −0.286 | .016 | 0.078 | .590 | −0.135 | .266 |
| Scr (umol/L) | 0.124 | .059 | −0.148 | .361 | 0.069 | .572 | −0.04 | .782 | 0.107 | .377 |
| RET (%) | 0.013 | .844 | 0.127 | .433 | 0.058 | .636 | −0.018 | .903 | −0.086 | .478 |
| Hs-CRP (mg/L) | 0.082 | .218 | −0.125 | .443 | 0.102 | .401 | −0.095 | .510 | 0.198 | .101 |
| IL-6 (pg/mL) | 0.313 | <.001 | 0.127 | .434 | 0.217 | .071 | 0.393 | .005 | 0.290 | .015 |
| SF (ng/mL) | 0.125 | .059 | 0.170 | .295 | −0.012 | .920 | 0.026 | .857 | 0.234 | .051 |
| SI (umol/L) | −0.060 | .363 | 0.031 | .852 | −0.070 | .563 | −0.168 | .245 | −0.119 | .326 |
| TIBC (umol/L) | −0.169 | .010 | −0.202 | .211 | −0.208 | .084 | −0.453 | .001 | −0.038 | .754 |
| TSAT (100 %) | 0.067 | .309 | 0.221 | .170 | 0.098 | .419 | 0.080 | .578 | −0.101 | .407 |
| IFAb (AU/mL) | −0.193 | .003 | 0.118 | .468 | −0.114 | .349 | −0.377 | .007 | −0.192 | .111 |
| STfR (nmol/L) | 0.044 | .505 | −0.389 | .013 | −0.016 | .896 | 0.126 | .382 | −0.051 | .673 |
| TRF (g/L) | −0.141 | .033 | 0.103 | .525 | −0.119 | .326 | −0.143 | .323 | −0.177 | .142 |
| EPO (mIU/mL) | 0.192 | .164 | −0.021 | .900 | 0.203 | .092 | 0.160 | .268 | −0.061 | .618 |
SF, SI, EPO, IFAb, IL-6, hs-CRP levels were log-transformed.
CKD3-4 = CKD3-4 patients, CKD5 = CKD5 patients without renal replace treatment, EPO = erythropoietin, Hb = hemoglobin, HD = hemodialysis patients, hs-CRP = high sensitivity C-reactive protein, IFAb = intrinsic factor antibody, IL-6 = interleukins-6, PD = peritoneal dialysis patients, RET = reticulocyte, RET-He = reticulocyte hemoglobin equivalent, Scr = serum creatinine, SF = serum ferritin, SI = serum iron, sTfR = soluble transferrin receptor, TIBC = total iron binding capacity, TRF = transferrin, TSAT = transferrin saturation.
Hepcidin-25 was positively associated with IL-6 (R = 0.313, P < .001) in the whole group, while in the CKD3-4 group, hepcidin-25 was associated negatively with STfR (r = −0.389, P = .013); in the CKD5 group, hepcidin-25 was associated negatively with Hb (r = −0.286, P = .016); in the PD group, hepcidin-25 was associated positively with IL-6 (R = 0.393, P = .005), negatively with TIBC (r = −0.453, P = .001), and IFAb (r = −0.377, P = .007); in the HD group, hepcidin-25 was associated positively with IL-6 (R = 0.290, P = .015).
3.2. Pearson correlation analysis of RET-He and other indicators in each group. SF, SI, EPO, IFAb, IL-6, and hs-CRP levels were log-transformed (Table 3).
Table 3.
Pearson correlation analysis of reticulocyte hemoglobin equivalent and other indicators in each group.
| Whole | CKD3–4 | CKD5 | PD | HD | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| r | P value | r | P value | r | P value | r | P value | r | P value | |
| Hb (g/L) | 0.196 | .003 | 0.286 | .074 | 0.203 | .091 | 0.147 | .307 | 0.265 | .026 |
| Scr (umol/L) | −0.234 | <.001 | −0.046 | .780 | −0.217 | .071 | −0.074 | .611 | −0.136 | .263 |
| RET (100%) | −0.150 | .023 | −0.246 | .126 | −0.148 | .221 | −0.193 | .178 | −0.082 | .499 |
| Hs-CRP (mg/L) | −0.119 | .072 | −0.083 | .611 | −0.188 | .118 | −0.082 | .573 | 0.065 | .595 |
| IL-6 (pg/mL) | −0.185 | .005 | −0.027 | .868 | −0.078 | .519 | −0.106 | .466 | −0.210 | .081 |
| SF (ng/mL) | 0.144 | .028 | −0.213 | .187 | 0.381 | .001 | 0.097 | .504 | 0.205 | .089 |
| SI (umol/L) | 0.256 | <.001 | 0.321 | .043 | 0.371 | .002 | 0.402 | .004 | 0.158 | .191 |
| TIBC (umol/L) | 0.029 | .657 | 0.189 | .242 | 0.150 | .215 | 0.044 | .763 | −0.061 | .613 |
| TSAT (100%) | 0.227 | .001 | 0.124 | .445 | 0.276 | .021 | 0.394 | .005 | 0.209 | .083 |
| IFAb (Au/mL) | −0.093 | .158 | −0.085 | .601 | −0.126 | .297 | −0.164 | .255 | −0.116 | .341 |
| STfR (nmol/L) | −0.238 | <.001 | −0.259 | .106 | −0.275 | .021 | −0.262 | .066 | −0.010 | .938 |
| TRF (g/L) | −0.125 | .058 | 0.142 | .382 | −0.159 | .188 | −0.352 | .012 | −0.163 | .179 |
| EPO (mIU/mL) | −0.101 | .128 | −0.382 | .015 | −0.154 | .204 | −0.030 | .835 | 0.017 | .888 |
SF, SI, EPO, IFAb, IL-6, hs-CRP levels were log-transformed.
CKD3-4 = CKD3-4 patients, CKD5 = CKD5 patients without renal replace treatment, EPO = erythropoietin, Hb = hemoglobin, HD = hemodialysis patients, hs-CRP = high sensitivity C-reactive protein, IFAb = intrinsic factor antibody, IL-6 = interleukins-6, PD = peritoneal dialysis patients, RET = reticulocyte, RET-He = reticulocyte hemoglobin equivalent, Scr = serum creatinine, SF = serum ferritin, SI = serum iron, sTfR = soluble transferrin receptor, TIBC = total iron binding capacity, TRF = transferrin, TSAT = transferrin saturation.
In the whole group, reticulocyte hemoglobin equivalent was associated positively with SI (R = 0.256, P < .001), TSAT (R = 0.227, P = .001) and negatively with Scr (r = −0.234, P < .001) and STfR (r = −0.238, P < .001).
In the CKD3 to 4 group, RET-He was associated positively with SI (R = 0.321, P = .043) and negatively with EPO (r = −0.382, P = .015). In the CKD5 group, RET-He was associated positively with SF (R = 0.381, P = .001), SI (R = 0.371, P = .002), and TSAT (R = 0.276, P = .021) while negatively associated with STfR (r = −0.275, P = .021). In the PD group, RET-He was associated positively with SI (R = 0.402, P = .004) and TSAT (R = 0.394, P = .005), negatively with TRF (r = −0.352, P = .012). In the HD group, RET-He was associated positively with Hb (R = 0.265, P = .026).
3.3. The association between the levels of hepcidin-25 and RET-He in each group (Table 4). SF, SI, EPO, IFAb, IL-6, hs-CRP levels were log-transformed.
Table 4.
Pearson correlation analysis of reticulocyte hemoglobin equivalent and hepcidin-25 in the whole group and in each group.
| Whole | CKD3–4 | CKD5 | PD | HD | |
|---|---|---|---|---|---|
| r | −0.079 | −0.049 | −0.227 | 0.217 | 0.005 |
| P value | .232 | .763 | .058 | .130 | .968 |
CKD3-4 = CKD3-4 patients, CKD5 = CKD5 patients without renal replace treatment, HD = hemodialysis patients, PD = peritoneal dialysis patients.
Hepcidin-25 was not correlated with RET-He (r = −0.079, P = .232); and there was no correlation between hepcidin-25 and RET-He in each group; the details are as follows: CKD3 to 4 (r = −0.049, P = .763), CKD5(r = −0.227, P = .058), PD (R = 0.217, P = .130), HD (R = 0.005, P = .968).
3.4. The multiple regression model for serum hepcidin-25 (Table 5) and RET-He (Table 6).
Table 5.
Multiple regression model for serum hepcidin-25.
| Univariate model | P value | Adjusted model* | ||
|---|---|---|---|---|
| Nonstandardized coefficient (95% CI) | Nonstandardized coefficient (95% CI) | P value | ||
| Hb (g/L) | −0.003 (−0.020, 0.014) | .718 | −0.003 (−0.020, 0.014) | .727 |
| Scr (umol/L) | 0.001 (0.000, 0.002) | .059 | 0.001 (0.000, 0.002) | .062 |
| RET (%) | 0.063 (−0.570, 0.696) | .844 | 0.045 (−0.582, 0.672) | .887 |
| RET-He (pg) | −0.089 (−0.236, 0.057) | .232 | −0.082 (−0.229, 0.065) | .274 |
| Hs-CRP (mg/L) | 0.637 (−0.379, 1.654) | .218 | 0.397 (−0.627, 1.421) | .446 |
| IL-6 (pg/mL) | 4.035 (3.439, 5.631) | <.001 | 3.685 (2.040, 5.329) | <.001 |
| SF (ng/mL) | 1.000 (−0.039, 2.039) | .059 | 0.785 (−0.269, 1.839) | .144 |
| SI (umol/L) | −0.983 (−3.108, 1.141) | .363 | −1.113 (−3.265, 1.038) | .309 |
| TIBC (umol/L) | −0.060 (−0.105, −0.014) | .010 | −0.062 (−0.108, −0.016) | .008 |
| TSAT (100%) | 0.014 (−0.013, 0.042) | .309 | 0.013 (−0.015, 0.041) | .352 |
| IFAb (AU/mL) | −12.602 (−20.955, −4.250) | .003 | −11.612 (−19.958, −3.266) | .007 |
| STfR (nmol/L) | 0.019 (−0.038, 0.076) | .505 | 0.006 (−0.051, 0.063) | .831 |
| TRF (g/L) | −0.905 (−1.734, −0.076) | .033 | −0.655 (−1.516, 0.206) | .135 |
| EPO (mIU/mL) | 0.617 (−0.253, 1.486) | .164 | 0.423 (−0.454, 1.301) | .343 |
SF, SI, EPO, IFAb, IL-6, hs-CRP levels were log-transformed.
EPO = erythropoietin, Hb = hemoglobin, hs-CRP = high sensitivity C-reactive protein, IFAb = intrinsic factor antibody, IL-6 = interleukins-6, RET = reticulocyte, RET-He = reticulocyte hemoglobin equivalent, Scr = serum creatinine, SF = serum ferritin, SI = serum iron, sTfR = soluble transferrin receptor, TIBC = total iron binding capacity, TRF = transferrin, TSAT = transferrin saturation.
Model was adjusted by age, sex, and ESA use.
Table 6.
Multiple regression model for reticulocyte hemoglobin equivalent.
| Univariate model | P value | Adjusted model* | ||
|---|---|---|---|---|
| Nonstandardized coefficient (95% CI) | Nonstandardized coefficient (95% CI) | P value | ||
| Hb (g/L) | 0.023 (0.008, 0.037) | .003 | 0.021 (0.007, 0.036) | .005 |
| Scr (umol/L) | −0.002 (−0.003, −0.001) | <.001 | −0.002 (−0.003, −0.001) | .003 |
| RET (%) | −0.645 (−1.201, −0.089) | .023 | −0.642 (−1.194, −0.090) | .023 |
| Hepcidin-25 (ng/mL) | −0.070 (−0.186, 0.045) | .232 | −0.065 (−0.182, 0.052) | .274 |
| Hs-CRP (mg/L) | −0.827 (−1.726, 0.073) | .072 | −0.846 (−1.753, 0.061) | .067 |
| IL-6 (pg/mL) | −2.113 (−3.580, −0.647) | .005 | −2.150 (−3.651, −0.649) | .005 |
| SF (ng/mL) | 1.030 (0.109, 1.950) | .028 | 1.032 (0.098, 1.966) | .030 |
| SI (umol/L) | 3.710 (1.883, 5.537) | <.001 | 4.188 (2.347, 6.029) | <.001 |
| TIBC (umol/L) | 0.009 (−0.032, 0.050) | .657 | 0.021 (−0.021, 0.062) | .331 |
| TSAT (100%) | 0.043 (0.019, 0.067) | .001 | 0.044 (0.020, 0.068) | <.001 |
| IFAb (AU/mL) | −5.407 (−12.933, 2.120) | .158 | −4.985 (−12.517, 2.547) | .194 |
| STfR (nmol/L) | −0.092 (−0.141, −0.043) | <.001 | −0.089 (−0.139, −0.040) | <.001 |
| TRF (g/L) | −0.713 (−1.451, 0.025) | .058 | −0.707 (−1.472, 0.059) | .070 |
| EPO (mIU/mL) | −0.599 (−1.370, 0.173) | .128 | −0.647 (−1.426, 0.131) | .103 |
SF, SI, EPO, IFAb, IL-6, hs-CRP levels were log-transformed.
EPO = erythropoietin, Hb = hemoglobin, hs-CRP = high sensitivity C-reactive protein, IFAb = intrinsic factor antibody, IL-6 = interleukins-6, RET = reticulocyte, RET-He = reticulocyte hemoglobin equivalent, Scr = serum creatinine, SF = serum ferritin, SI = serum iron, sTfR = soluble transferrin receptor, TIBC = total iron binding capacity, TRF = transferrin, TSAT = transferrin saturation.
Model was adjusted by age, sex, and ESA use.
Multiple regression model was adjusted by age, sex, and ESA use. Table 5 shows that among these factors, IL-6 (B = 3.685; P < .001), TIBC (B = −0.062; P = .008), and IFAb (B = −11.612; P = .007) were independently associated with hepcidin-25 levels. Table 6 shows that among these factors, Hb (B = 0.021; P = .005), Scr (B = −0.002; P = .003), RET (B = −0.642, P = .023), IL-6 (B = −2.150; P = .005),SF (B = 1.032; P = .030), SI (B = 4.188; P < .001), TSAT (B = 0.044; P < .001), and STfR (B = −0.089; P < .001)were independently associated with RET-He levels. Interleukins-6 was independently associated with hepcidin-25 and RET-He.
4. Discussion
Anemia is a major complication in patients with chronic kidney disease. The pathogenesis of anemia in CKD is multifactorial, and the inadequate production of EPO is the leading pathogenetic cause. Other factors such as shortened erythrocyte survival, iron deficiency, bleeding, and subclinical chronic inflammation may also play a role and contribute to anemia in CKD. Erythropoietin is effective if iron is available; however, unnecessary iron supplementation results in iron overload. In CKD, both iron deficiency and chronic inflammation are 2 major factors that contribute to the hypo responsiveness of erythropoietin treatment.[25] Accumulating evidence shows that hepcidin has emerged as a central molecule that regulates iron metabolism in CKD. Hepcidin reduces iron absorption and efflux and could be predictive of iron status and the response to iron supplementation or erythropoietin-stimulating agents.[26] Patients with CKD frequently suffer from a chronic inflammatory state which is related to several inflammatory mediators, crucially IL-6, which contributes in renal anemia and resistance to ESA. Interleukins-6 levels are increased in patients with CKD, possibly due to the effects of uremic toxins, and inversely correlate with GFR.[27]
Hepcidin-25 is induced by inflammatory stimuli such as IL-6 in inflammation anemia, such as CKD anemia.[28] Higher circulating levels of IL-6 increase the expression of hepcidin, leading to iron sequestration, decreased iron absorption, and lack of iron availability for erythropoiesis. In this study, we measured hepcidin-25 and IL-6, which had a higher level in HD patients compared with non-dialysis patients. In the multiple regression model IL-6 was independently associated with hepcidin-25 levels. Pearson correlation analysis revealed that hepcidin-25 was positively associated with IL-6, and negatively with TIBC, IFAb, and TRF, but not associated with Hb, Scr, RET, hs-CRP, SF, SI, TSAT, STfR, and EPO. In the non-dialysis group, hepcidin-25 had no significant correlation with IL-6, whereas in the dialysis group, hepcidin-25 was positively associated with IL-6, which may be related to our small sample size in each group. This may indicate a likelihood of a threshold for stimulation of hepcidin-25 expression by IL-6.
In recent years, RET-He, whose values are equivalent to reticulocyte hemoglobin content, has emerged as an additional parameter that is helpful in identifying iron-deficient erythropoiesis.[29] Reticulocyte hemoglobin equivalent is advantageous as it does not interfere with inflammatory conditions and can be easily measured with a widely available and popular blood cell counter.[30] Previous research has found that hepcidin indirectly regulates the content of hemoglobin in reticulocytes, and RET-He measurements serve as an indicator of iron deficiency secondary to an increase in hepcidin since hepcidin inhibits the efflux of iron and decreases available iron.[22] In our study, we also studied the RET-He and its correlation with other indicators. Pearson correlation analysis revealed that RET-He was associated positively with Hb, SF, SI, TSAT, and negatively with Scr, RET, IL-6, STfR, and there was no association with hs-CRP, TIBC, IFAb, TRF, and EPO. In order to prove whether there is correlation between hepcidin-25 and RET-He in CKD, we conducted a Pearson correlation analysis for hepcidin-25 and RET-He in the whole group and each group; the results showed that hepcidin-25 was not correlated with RET-He, and there was no correlation between hepcidin-25 and RET-He in each group. Previous research showed that RET-He values decreased in response to increased hepcidin during septic shock[22] and community-acquired pneumonia (CAP)[31] which was not consistent with our results. We consider that it may be related to the degree of inflammatory response, unlike septic shock and CAP, CKD belong to chronic microinflammatory condition.
In this study, multiple regression analyses also revealed that IL-6 and TIBC were associated with hepcidin-25 levels. Serum creatinine, SI, SF, TSAT, RET, STfR, Hb, and IL-6 levels were associated with RET-He levels. IL-6 is related to both hepcidin-25 and RET-He. Chronic Kidney Disease patients suffer from chronic inflammation due to high level of IL-6; while higher circulating levels of IL-6 increase the expression of hepcidin-25, promote the synthesis and secretion of hepcidin-25 in CKD. We infer that in acute and severe inflammation, increase in hepcidin secretion decreases the release of recycled heme iron by macrophages, increases the sequestration of iron stores in hepatocytes, and reduces circulating free iron, leading to a decrease in RET-He, severe deficiency of iron absorption in the body, and aggravation of anemia. In this study, hepcidin-25 was not associated with RET-He, these results indicated that hepcidin-25 could not regulate the expression of RET-He in the CKD which may related to IL-6 level.
The limitation of this study is the lack of longitudinal analysis; we will collect longitudinal data and complete the following study. Some patients with CKD were found for the first time and had not been treated in the early stage, so patients on CKD5 non-dialysis have hemoglobin concentration below current guideline recommendations.
5. Conclusion
No correlation was found between the serum hepcidin levels and reticulocyte hemoglobin levels in this study, suggesting that hepcidin does not directly regulate iron dynamics of reticulocytes in CKD, which may relate to IL-6 level. The specific mechanism needs to be further studied.
Author contributions
Conceptualization: Zhaoli Gao, Zhao Hu.
Data curation: Zhaoli Gao, Xiaotian Ma.
Formal analysis: Zhaoli Gao, Xiaotian Ma.
Writing – original draft: Zhaoli Gao, Yingying Hu, Yanxia Gao, Zhao Hu.
Writing – review & editing: Zhaoli Gao, Zhao Hu.
Abbreviations:
- CKD
- chronic kidney disease
- EPO
- erythropoietin
- Hb
- hemoglobin
- HD
- hemodialysis
- hs-CRP
- high sensitivity C-reactive protein
- IFAb
- intrinsic factor antibody
- IL-6
- interleukins-6
- PD
- peritoneal dialysis
- RET
- reticulocyte
- RET-He
- reticulocyte hemoglobin equivalent
- Scr
- serum creatinine
- SF
- serum ferritin
- SI
- serum iron
- sTfR
- soluble transferrin receptor
- TIBC
- total iron binding capacity
- TRF
- transferrin
- TSAT
- transferrin saturation
This study protocol was reviewed and approved by the Medical Ethics Committee, Qilu Hospital of Shandong University (Qingdao, Shandong, China), approval number KYLL-KS-2018022. All patients participating in this study were informed, signed informed consents, and voluntarily participated. All methods were carried out in accordance with Declaration of Helsinki.
All data generated or analyzed during this study are included in this published article [and its supplementary information files].
This study was supported by the Scientific Research Foundation of Qilu Hospital (Qingdao) (QDKY2018QN13).
The authors have no conflicts of interest to disclose.
How to cite this article: Gao Z, Hu Y, Gao Y, Ma X, Hu Z. The association of hepcidin, reticulocyte hemoglobin equivalent and anemia-related indicators on anemia in chronic kidney disease. Medicine 2023;102:17(e33558).
Contributor Information
Zhaoli Gao, Email: gaoyanxia31@163.com.
Yingying Hu, Email: sdhuzhao@vip.126.com.
Yanxia Gao, Email: gaoyanxia31@163.com.
Xiaotian Ma, Email: mxt_qlqd@sina.com.
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