Hepatokine FGL1 and hepcidin in anemia of chronic kidney disease: A cross sectional study

1. Introduction

Chronic kidney disease (CKD) is a major global health concern, characterized by persistent kidney dysfunction [estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m²] or structural damage lasting over 3 months. Affecting more than 850 million of the global population, CKD is more prevalent than diabetes or cancer, with hypertension (HTN) and type 2 diabetes mellitus (DM) being its leading causes (Jager et al., 2019). The disease advances to end-stage renal disease (ESRD) and requires dialysis or kidney transplantation. Its complex etiology involves metabolic, vascular, and genetic factors, which create a strong rationale for early detection and timely management (Kovesdy, 2022).

Anemia is one of the most prevalent and debilitating complications of CKD. Its severity increases as the disease advances to end-stage renal failure. Several interrelated mechanisms contribute to the pathogenesis of anemia in CKD, including insufficient erythropoietin (EPO) production, iron-restricted erythropoiesis secondary to iron sequestration within macrophages, and chronic inflammation that disrupts iron homeostasis (Portolés et al., 2021).

Hepcidin is a 25-amino acid peptide hormone that serves as the central regulator of systemic iron homeostasis. Elevated iron levels stimulate bone morphogenetic protein (BMP-6), which activates the SMAD (SMA and MAD homologs) signaling pathway to upregulate hepcidin transcription. Inflammation, primarily mediated by IL-6, promotes hepcidin synthesis through activation of the JAK-STAT3 pathway, which impairs systemic iron availability (Nemeth and Ganz, 2021). Hepcidin exerts its effect by binding to ferroportin, the sole known iron exporter expressed on enterocytes, macrophages, and hepatocytes. This interaction leads to the internalization and degradation of ferroportin, thereby reducing dietary iron absorption, recycling from macrophages, and iron release from hepatic stores. Conversely, erythropoietic demand or hypoxia suppresses hepcidin expression to ensure sufficient iron availability (Camaschella et al., 2020; Saboor, 2021).

Fibrinogen-like protein 1 (FGL1) is a hepatokine primarily secreted by hepatocytes. It belongs to the fibrinogen superfamily and plays pivotal roles in liver regeneration, immune modulation, and metabolic regulation, including glucose and lipid homeostasis and metabolism (Chen et al., 2024; Liu et al., 2022). More recently, FGL1 has been implicated in the regulation of systemic iron metabolism. It attenuates hepcidin expression by antagonizing the BMP/SMAD signaling pathway (Sardo et al., 2024). While the erythroid regulator erythroferrone (ERFE) has been extensively studied in the context of hepcidin suppression, the contribution of hepatic FGL1, particularly in CKD, where inflammatory and metabolic derangements are common, remains insufficiently characterized. Understanding its role could shed light on potential disruptions in the liver-iron axis in chronic renal disease.

ERFE is a soluble glycoprotein hormone secreted by erythroid precursors in response to EPO (Arezes et al., 2018). This hormone acts directly on liver to suppress hepcidin synthesis to enhance iron bioavailability. ERFE binds to BMP ligands, particularly BMP6 and BMP2, in the liver and inhibits their interaction with BMP receptors. This inhibition reduces SMAD1/5/8 phosphorylation, leading to diminished transcriptional activation of the HAMP gene and suppression of hepatic hepcidin synthesis (Babar and Saboor, 2024; Srole and Ganz, 2021).

IL-6 is a multifunctional cytokine that plays key roles in immune regulation and hematopoiesis. In CKD, IL-6 contributes to persistent hepcidin upregulation through sustained inflammatory signaling. IL-6 activates the hepatic JAK2/STAT3 signaling pathway. This activation leads to iron-restrictive anemia by impairing iron mobilization and diminishing the erythropoietic response (Grebenciucova and VanHaerents, 2023).

Disordered iron metabolism in CKD results from a complex interplay between systemic inflammation, impaired erythropoietic signaling, and hepatic hepcidin dysregulation. IL-6-mediated induction of hepcidin is a well-established mechanism contributing to functional iron deficiency and anemia in CKD (Goyal et al., 2018). Recent evidence suggests that FGL1 may modulate iron metabolism by antagonizing the BMP/SMAD signaling pathway, thereby suppressing hepcidin expression (Sardo et al., 2024). However, the relative contribution of FGL1, particularly in conjunction with hepcidin, ERFE, and IL-6, remains uncharacterized in the CKD patients on dialysis. This study aimed to examine the role of FGL1 in the regulation of hepcidin and its relationship with ERFE and IL-6 in patients with CKD on dialysis. The objective was to determine whether FGL1 levels contribute to inadequate control of hepcidin, which may impair iron homeostasis and lead to persistent anemia in this patient group.

2. Material and Methods

This was a comparative, cross-sectional observational study conducted using a purposive sampling technique between January 2025 and May 2025. All patients with CKD on hemodialysis (n = 42) from University Hospital Sharjah, Sharjah, UAE, were enrolled. Inclusion criteria for included adult patients (aged 18-70 years) with stage V CKD receiving regular hemodialysis for >3 months and eGFR <60 mL/min/1.73 m². Healthy controls (n = 42) were recruited from hospital staff with no known history of acute or chronic illness. With the help of the treating physician and on-duty nurse, a questionnaire was filled out, including demographics, co-morbidities, and patient disease status. Blood samples were collected in EDTA anticoagulated vacutainer tubes and plain tubes from both cohorts. Whole blood collected in EDTA tubes was analyzed for complete blood count (CBC). Samples collected in plain tubes were allowed to clot, then centrifuged to separate serum. The serum was divided into two aliquots: one was used for serum iron and ferritin measurements, and the other was stored at –80°C for later enzyme-linked immunosorbent assay (ELISA)-based quantification of FGL1, ERFE, and hepcidin. Written informed consent was obtained from healthy controls and patients after explaining the purpose of the study. This study received ethical approval from the Research Ethics Committee at the University of Sharjah (REC-23-12-16-01-F) and the Ethics and Research Committee of the University Hospital Sharjah (UHS-HERC-155-29022024). All procedures were conducted in accordance with the ethical standards outlined in the Declaration of Helsinki.

CBC analyses were performed using Mindray BC-6800 Plus hematology analyzer (China). Serum iron and serum ferritin were determined using ARCHITECT c4000 clinical chemistry analyzer (Illinois, USA). Serum levels of FGL1, ERFE, hepcidin, and IL-6 were quantified using ELISA with Assay Genie ELISA kits (Dublin, Ireland).

3. Results

This study compared 42 patients with CKD undergoing dialysis and 42 healthy controls to evaluate iron-regulatory parameters. The mean age of CKD patients was slightly higher (67.26 ± 11.77 years) compared to the control group (47.97 ± 8.56 years). Among the CKD cohort, 97.6% were diagnosed with ESRD (Table 1). DM and HTN were the most prevalent comorbidities, each present in 73.8% of the patients (Table 1). Additionally, ischemic heart disease (IHD) was noted in 38% and dyslipidemia (DSL) in 28.6% of the cases (Table 1). The cohort was nearly balanced between the sexes, with 48% males and 52% females. Additionally, more than 80% of patients in this study received darbepoetin alfa, a long-acting erythropoiesis-stimulating agent (ESA).

3.3. Interrelationships among FGL1, ERFE, hepcidin, and IL-6

The correlation analysis between key iron-regulatory and inflammatory markers (hepcidin, FGL1, ERFE, and IL-6) was performed for both cohorts. The Pearson correlation test, applied to the normally distributed ERFE data, showed a moderate positive correlation between ERFE and FGL1 (r = 0.41, p <0.05). For non-normally distributed variables, i.e., FGL1, hepcidin, and IL-6, Spearman correlation analysis was used. Spearman’s results indicated a moderate positive correlation between FGL1 and hepcidin (ρ = 0.29, p < 0.05), and a weak positive correlation between IL-6 and hepcidin (ρ = 0.20, p > 0.05). No significant correlations were observed between IL-6 and either FGL1 or ERFE, nor between hepcidin and ERFE. Pearson and Spearman correlations have been depicted in Figs. 1(a, b).

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Fig. 1.

(a,b) Correlation matrix of iron-regulatory markers and IL-6 in CKD. Pearson/Spearman correlation heatmap showing the relationships among serum levels of IL-6, hepcidin, FGL1, and ERFE in combined healthy control and CKD patient groups. The color scale represents the strength and direction of correlations, with warmer colors indicating positive correlations and cooler colors indicating negative correlations.

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3.4 Pairwise correlation of the analyzed parameters among CKD patients

A pairwise scatter plot matrix was constructed to visualize the distribution and linear relationships between FGL1, ERFE, hepcidin, and IL-6 in CKD patients (Fig. 2). The regression lines indicated a positive correlation between hepcidin and IL-6, which suggests that hepcidin increases as IL-6 levels rise. Likewise, FGL1 demonstrated a moderate positive association with both hepcidin and ERFE. The strongest positive correlation appeared between FGL1 and ERFE, supporting their linked roles in iron regulation and erythropoiesis. In contrast, IL-6 and ERFE showed a weak positive correlation, while the association between ERFE and hepcidin remains minimal.

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Fig. 2.

Pairwise scatter plot matrix of FGL1, ERFE, hepcidin IL-6 with regression trends in CKD patients. This pairwise plot illustrates the pairwise relationships among FGL1, ERFE, hepcidin, and IL-6 levels in CKD patients. Each scatterplot includes a linear regression line with a 95% confidence interval (shaded). Diagonal panels show the kernel density estimate of each variable. The strongest visual association is observed between FGL1 and ERFE, suggesting a potential link in their regulatory roles related to iron metabolism.

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4. Discussion

The present study explored the associations of hematological, biochemical key biomarkers involved in iron metabolism (FGL1, ERFE, hepcidin) and IL-6 in patients with CKD. CKD patients exhibited significant alterations in hematological and biochemical parameters compared to healthy controls (Table 1). The patient cohort presented with significantly lower levels of hemoglobin, RBC count, serum iron, and MCHC. These findings are consistent with prior studies highlighting anemia and altered iron homeostasis as prevalent complications in CKD, primarily attributed to impaired EPO synthesis and disturbed iron metabolism (Atkinson and Warady, 2018, Honda et al., 2019)

The current study demonstrated significantly reduced levels of FGL1 in CKD patients on dialysis compared to healthy controls. To our knowledge, no previous human or clinical study has specifically evaluated circulating FGL1 levels in CKD in relation to iron regulation, making our findings novel in this context. The only mechanistic evidence available derives from preclinical models, notably the study by Sardo et al., which reported that FGL1 is upregulated during acute anemia or hemorrhage in mice and acts as a BMP6 antagonist to suppress hepcidin via the SMAD signaling pathway (Sardo et al., 2024). In contrast, the lower FGL1 levels observed in our CKD cohort suggest that chronic disease settings may impair hepatic FGL1 expression or release, potentially due to sustained inflammation, uremic toxins, or hepatic dysfunction [evident by high mean alkaline phosphatase levels (data not shown) in CKD patients]. This divergence indicates that FGL1-mediated hepcidin suppression, which is beneficial in acute anemia, may become blunted or dysregulated in CKD. These findings point to a possible maladaptive failure of FGL1 response in chronic disease and highlight the need for further research to clarify its regulatory role in human CKD.

In contrast to the expected compensatory elevation during anemia, ERFE levels in CKD patients did not show statistically significant difference compared to healthy controls. This finding is notable given that 80% of the patients were receiving ESA, which are known to enhance ERFE production. The reduced ERFE levels may reflect impaired erythropoietic activity due to bone marrow suppression, chronic inflammation, or uremic milieu commonly seen in advanced CKD. These factors may reduce the erythroid response even in the presence of ESA therapy, limiting ERFE synthesis. Our results align with the findings of Honda et al., who reported no significant baseline difference in ERFE levels between hemodialysis patients and healthy controls, though a modest increase was observed post-ESA administration (Honda et al., 2016). This suggests that ERFE upregulation in CKD may require more robust or sustained erythropoietic stimuli than what is provided during routine ESA use.

In contrast, some other studies have demonstrated significantly increased ERFE levels following exogenous EPO administration. Hanudel et al. showed an acute increase in ERFE in both normal and CKD mice after EPO treatment (Hanudel et al., 2018), while another study reported similar findings in healthy individuals and anemic patients exposed to high altitude (Robach et al., 2021). The discrepancy between these studies and our findings may be due to differences in study design, population characteristics, post-ESA, and baseline bone marrow responsiveness. Unlike controlled interventional studies that measure ERFE shortly after EPO administration, our cross-sectional design reflects basal ERFE levels without standardized ESA timing, potentially underestimating its transient induction.

Further insights into ERFE physiology are provided by Coffey et al., who demonstrated that chronic ERFE overexpression in transgenic mice suppressed hepcidin in a dose-dependent manner and led to systemic iron overload, growth impairment, renal dysfunction, and neurological deficits (Coffey et al., 2022). These findings underscore the critical need for tightly regulated ERFE activity, as both deficiency and excess carry pathological consequences. These findings suggest that while ERFE is a key regulator during stress erythropoiesis, as observed in β thalassemia and stress erythropoiesis models (Kautz et al., 2014), its expression in CKD may be attenuated or dependent on ESA exposure, aligning with our current study, which showed no significant difference in ERFE levels between CKD patients and controls.

Collectively, while ERFE is a well-established mediator of hepcidin suppression and iron mobilization during stress erythropoiesis, its role in CKD appears context dependent. Factors such as chronic inflammation, ESA responsiveness, and marrow reserve may modulate its expression, explaining the variable results observed across studies and supporting the nuanced interpretation of ERFE behavior in CKD patients.

This study demonstrated significantly elevated serum hepcidin levels in patients with CKD compared to healthy controls. These findings are consistent with previous research reporting up to a 20-fold increase in hepcidin concentrations among CKD patients (Troutt et al., 2013). Elevated hepcidin has also been observed in early stages of diabetic CKD, particularly stages II and III (Wagner et al., 2015), and was notably higher in patients with ESRD undergoing hemodialysis compared to non-dialyzed CKD patients and healthy individuals (Hammad et al., 2025). Additionally, impaired renal clearance contributes to hepcidin accumulation, as reflected by the inverse association between hepcidin levels and eGFR (Troutt et al., 2013). These findings highlight the pathogenic role of hepcidin in CKD-associated anemia and support the rationale for therapeutic interventions that target hepcidin regulation.

In the current study cohort, the mean IL-6 level in CKD patients was higher than that in the control group. However, this difference did not reach statistical significance (p = 0.25). Although CKD is typically associated with persistent low-grade inflammation, often marked by elevated IL-6 levels, our results revealed only a modest and statistically nonsignificant increase in IL-6 among the patient group. This finding contrasts with some earlier studies that have consistently reported significantly elevated IL-6 levels in CKD, particularly in advanced stages and dialysis-dependent patients (Goyal et al., 2018; Hammad et al., 2025).

Several factors could explain the statistically nonsignificant IL-6 levels in the CKD cohort. First, the variability within the CKD group, as reflected by the wide standard deviation and narrow interquartile range, suggests heterogeneous inflammatory status, possibly influenced by differences in comorbid conditions, dialysis status, or medication use (e.g., anti-inflammatory drugs or ESA therapy). Second, the cross-sectional design and single time-point measurement may not capture the dynamic nature of IL-6 fluctuations in CKD patients, particularly those experiencing intermittent inflammation or subclinical infections. Additionally, methodological differences in IL-6 assays, pre-analytical variability, or limited sample size could contribute to reduced statistical power and inter-group discrimination. It is also possible that non-IL-6 mediated stimuli, such as TNF-α or uremic toxins, are more relevant drivers of inflammation and anemia in this cohort. These findings suggest the need for comprehensive profiling of inflammatory mediators and repeated longitudinal assessments to better understand the role of IL-6 in CKD-related anemia and iron metabolism.

Correlation analyses revealed moderate, statistically significant positive associations between FGL1 and ERFE and between FGL1 and hepcidin, highlighting interconnected regulation among these proteins in CKD. The observed correlations support the hypothesis that FGL1 (Sardo et al., 2024) and ERFE collectively influence hepcidin expression and systemic iron metabolism (Kautz et al., 2015, Kautz et al., 2014). Hepcidin has been identified as an independent predictor of disease progression, with a moderate discriminative power for identifying progression to hemodialysis-ESRD (Hammad et al., 2025). Additionally, the weak but positive correlation between IL-6 and hepcidin substantiates existing literature explaining the regulatory relationship between IL-6 and hepcidin in CKD, with IL-6 acting as a principal pro-inflammatory cytokine that induces hepatic hepcidin synthesis (Yacoub et al., 2020). A recent study reported significantly elevated IL-6 and hepcidin levels in hemodialysis-dependent ESRD patients compared to non-dialyzed CKD patients and healthy controls (Hammad et al., 2025). Both biomarkers independently predicted CKD progression, emphasizing their role in inflammation-mediated iron dysregulation and anemia. These findings support the role of IL-6-driven hepcidin upregulation in the pathogenesis and progression of CKD. In support of our findings, a previous study in pediatric non-dialyzed CKD patients reported a strong positive correlation between serum hepcidin and IL-6. Hepcidin also showed significant inverse correlations with hemoglobin, total iron binding capacity (TIBC) and eGFR, indicating its role in inflammation-induced iron restriction and anemia even in patients without overt iron deficiency (Goyal et al., 2018).

Interestingly, despite these correlations among biomarkers, regression analysis evaluating clinical predictors (ESA use, HTN, DM) did not demonstrate significant associations with IL-6, ERFE, hepcidin, or FGL1. IL-6 emerged as a relatively stronger predictor for ESA use and diabetes, while ERFE showed a mild predictive trend for HTN. However, the lack of statistical significance and modest explanatory power (low R² values) underscores the complexity and multifactorial nature of CKD-related comorbidities. Previous research similarly emphasizes the challenges in identifying singular biomarkers for these clinical outcomes due to intricate pathophysiological interplays (Spoto et al., 2019).

The absence of significant associations between hematological parameters and iron regulatory markers may reflect the multifaceted mechanisms governing iron dysregulation and erythropoiesis in CKD. Although hepcidin levels correlate positively with markers of inflammation, direct relationships with standard hematological indices remain weak, suggesting indirect or multifactorial influences. Prior studies have also noted that alterations in iron homeostasis biomarkers do not always directly correlate with traditional red blood cell parameters in CKD, partly due to underlying chronic inflammation and erythropoietic dysfunction (Ganz and Nemeth, 2016).

5. Conclusions

This study provides novel insights into the complex interplay of hepatic and erythroid-derived regulators of iron metabolism in CKD. Significantly reduced FGL1 levels were found in CKD patients on dialysis, alongside non-significant alterations in ERFE and IL-6, and elevated hepcidin levels. A positive correlation between FGL1 and hepcidin suggests a possible compensatory or dysregulated hepatic response under chronic inflammatory conditions. Further longitudinal studies are needed to delineate the role of FGL1 in iron homeostasis and to assess its potential as a diagnostic or therapeutic target in CKD-related anemia.