Kidney tubule iron loading in experimental focal segmental glomerulosclerosis.

Kidney iron deposition may play a role in the progression of tubulointerstitial injury during chronic kidney disease. Here, we studied the molecular mechanisms of kidney iron loading in experimental focal segmental glomerulosclerosis (FSGS) and investigated the effect of iron-reducing interventions on disease progression. Thy-1.1 mice were injected with anti-Thy-1.1 monoclonal antibody (mAb) to induce proteinuria. Urine, blood and tissue were collected at day (D)1, D5, D8, D15 and D22 after mAb injection. Thy-1.1 mice were subjected to captopril (CA), iron-deficient (ID) diet or iron chelation (deferoxamine; DFO). MAb injection resulted in significant albuminuria at all time points (p < 0.01). Kidney iron loading, predominantly in distal tubules, increased in time, along with urinary kidney injury molecule-1 and 24p3 concentration, as well as kidney mRNA expression of Interleukin-6 (Il-6) and Heme oxygenase-1 (Ho-1). Treatment with CA, ID diet or DFO significantly reduced kidney iron deposition at D8 and D22 (p < 0.001) and fibrosis at D22 (p < 0.05), but not kidney Il-6. ID treatment increased kidney Ho-1 (p < 0.001). In conclusion, kidney iron accumulation coincides with progression of tubulointerstitial injury in this model of FSGS. Reduction of iron loading halts disease progression. However, targeted approaches to prevent excessive kidney iron loading are warranted to maintain the delicate systemic and cellular iron balance.

Role of hepcidin in oxidative stress and cell death of cultured mouse renal collecting duct cells: protection against iron and sensitization to cadmium.

The liver hormone hepcidin regulates systemic iron homeostasis. Hepcidin is also expressed by the kidney, but exclusively in distal nephron segments. Several studies suggest hepcidin protects against kidney damage involving Fe2+ overload. The nephrotoxic non-essential metal ion Cd2+ can displace Fe2+ from cellular biomolecules, causing oxidative stress and cell death. The role of hepcidin in Fe2+ and Cd2+ toxicity was assessed in mouse renal cortical [mCCD(cl.1)] and inner medullary [mIMCD3] collecting duct cell lines. Cells were exposed to equipotent Cd2+ (0.5-5 µmol/l) and/or Fe2+ (50-100 µmol/l) for 4-24 h. Hepcidin (Hamp1) was transiently silenced by RNAi or overexpressed by plasmid transfection. Hepcidin or catalase expression were evaluated by RT-PCR, qPCR, immunoblotting or immunofluorescence microscopy, and cell fate by MTT, apoptosis and necrosis assays. Reactive oxygen species (ROS) were detected using CellROX™ Green and catalase activity by fluorometry. Hepcidin upregulation protected against Fe2+-induced mIMCD3 cell death by increasing catalase activity and reducing ROS, but exacerbated Cd2+-induced catalase dysfunction, increasing ROS and cell death. Opposite effects were observed with Hamp1 siRNA. Similar to Hamp1 silencing, increased intracellular Fe2+ prevented Cd2+ damage, ROS formation and catalase disruption whereas chelation of intracellular Fe2+ with desferrioxamine augmented Cd2+ damage, corresponding to hepcidin upregulation. Comparable effects were observed in mCCD(cl.1) cells, indicating equivalent functions of renal hepcidin in different collecting duct segments. In conclusion, hepcidin likely binds Fe2+, but not Cd2+. Because Fe2+ and Cd2+ compete for functional binding sites in proteins, hepcidin affects their free metal ion pools and differentially impacts downstream processes and cell fate.

Sustained Exposure to High Carbohydrate Availability Does Not Influence Iron-Regulatory Responses in Elite Endurance Athletes.

This study implemented a 2-week high carbohydrate (CHO) diet intended to maximize CHO oxidation rates and examined the iron-regulatory response to a 26-km race walking effort. Twenty international-level, male race walkers were assigned to either a novel high CHO diet (MAX = 10 g/kg body mass CHO daily) inclusive of gut-training strategies, or a moderate CHO control diet (CON = 6 g/kg body mass CHO daily) for a 2-week training period. The athletes completed a 26-km race walking test protocol before and after the dietary intervention. Venous blood samples were collected pre-, post-, and 3 hr postexercise and measured for serum ferritin, interleukin-6, and hepcidin-25 concentrations. Similar decreases in serum ferritin (17-23%) occurred postintervention in MAX and CON. At the baseline, CON had a greater postexercise increase in interleukin-6 levels after 26 km of walking (20.1-fold, 95% CI [9.2, 35.7]) compared with MAX (10.2-fold, 95% CI [3.7, 18.7]). A similar finding was evident for hepcidin levels 3 hr postexercise (CON = 10.8-fold, 95% CI [4.8, 21.2]; MAX = 8.8-fold, 95% CI [3.9, 16.4]). Postintervention, there were no substantial differences in the interleukin-6 response (CON = 13.6-fold, 95% CI [9.2, 20.5]; MAX = 11.2-fold, 95% CI [6.5, 21.3]) or hepcidin levels (CON = 7.1-fold, 95% CI [2.1, 15.4]; MAX = 6.3-fold, 95% CI [1.8, 14.6]) between the dietary groups. Higher resting serum ferritin (p = .004) and hotter trial ambient temperatures (p = .014) were associated with greater hepcidin levels 3 hr postexercise. Very high CHO diets employed by endurance athletes to increase CHO oxidation have little impact on iron regulation in elite athletes. It appears that variations in serum ferritin concentration and ambient temperature, rather than dietary CHO, are associated with increased hepcidin concentrations 3 hr postexercise.

Serum hepcidin concentrations in relation to iron status in children with type 1 diabetes.

Chronic low-grade inflammation in type 1 diabetes (T1D) might increase hepcidin synthesis, possibly resulting in functional iron deficiency (FID). We hypothesized that in T1D children with FID, hepcidin concentrations are increased compared to those with normal iron status and those with absolute iron deficiency (AID). We evaluated hepcidin concentrations in T1D children in relation to iron status, and investigated whether hepcidin is useful in assessing FID. A cross-sectional study was conducted. FID was defined as elevated zinc protoporphyrin/heme ratio and/or red blood cell distribution width, and AID as low serum ferritin concentration. Post-hoc analyses with different definitions of FID were performed, using transferrin saturation and reticulocyte hemoglobin content. Serum hepcidin concentrations were measured using mass-spectrometry. The IRODIAB-study is registered at www.trialregister.nl (NTR4642). This study included 215 T1D children with a median age of 13.7 years (Q1-Q3: 10.1-16.3). The median (Q1-Q3) hepcidin concentration in patients with normal iron status was 1.8 nmol/l (0.9-3.3), in AID-patients, 0.4 nmol/l (0.4-0.4) and in FID-patients, 1.6 nmol/l (0.7-3.5). Hepcidin concentrations in FID-patients were significantly higher than in AID-patients (p < 0.001). Irrespective of FID-definition used, hepcidin concentrations did not differ between FID-patients and patients with normal iron status. This might be explained by the influence of various factors on hepcidin concentrations, and/or by differences in response of iron parameters over time. Single hepcidin measurements do not seem useful in assessing FID in T1D children. Multiple hepcidin measurements over time in future studies, however, might prove to be more useful in assessing FID in children with T1D.

The multifaceted role of iron in renal health and disease.

Iron is an essential element that is indispensable for life. The delicate physiological body iron balance is maintained by both systemic and cellular regulatory mechanisms. The iron-regulatory hormone hepcidin assures maintenance of adequate systemic iron levels and is regulated by circulating and stored iron levels, inflammation and erythropoiesis. The kidney has an important role in preventing iron loss from the body by means of reabsorption. Cellular iron levels are dependent on iron import, storage, utilization and export, which are mainly regulated by the iron response element-iron regulatory protein (IRE-IRP) system. In the kidney, iron transport mechanisms independent of the IRE-IRP system have been identified, suggesting additional mechanisms for iron handling in this organ. Yet, knowledge gaps on renal iron handling remain in terms of redundancy in transport mechanisms, the roles of the different tubular segments and related regulatory processes. Disturbances in cellular and systemic iron balance are recognized as causes and consequences of kidney injury. Consequently, iron metabolism has become a focus for novel therapeutic interventions for acute kidney injury and chronic kidney disease, which has fuelled interest in the molecular mechanisms of renal iron handling and renal injury, as well as the complex dynamics between systemic and local cellular iron regulation.

Influence of Periodizing Dietary Carbohydrate on Iron Regulation and Immune Function in Elite Triathletes.

Sleeping with low carbohydrate (CHO) availability is a dietary strategy that may enhance training adaptation. However, the impact on an athlete's health is unclear. This study quantified the effect of a short-term "sleep-low" dietary intervention on markers of iron regulation and immune function in athletes. In a randomized, repeated-measures design, 11 elite triathletes completed two 4-day mixed cycle run training blocks. Key training sessions were structured such that a high-intensity training session was performed in the field on the afternoon of Days 1 and 3, and a low-intensity training (LIT) session was performed on the following morning in the laboratory (Days 2 and 4). The ingestion of CHO was either divided evenly across the day (HIGH) or restricted between the high-intensity training and LIT sessions, so that the LIT session was performed with low CHO availability (LOW). Venous blood and saliva samples were collected prior to and following each LIT session and analyzed for interleukin-6, hepcidin 25, and salivary immunoglobulin-A. Concentrations of interleukin-6 increased acutely after exercise (p < .001), but did not differ between dietary conditions or days. Hepcidin 25 increased 3-hr postexercise (p < .001), with the greatest increase evident after the LOW trial on Day 2 (2.5 ± 0.9 fold increase ±90% confidence limit). The salivary immunoglobulin-A secretion rate did not change in response to exercise; however, it was highest during the LOW condition on Day 4 (p = .046). There appears to be minimal impact to markers of immune function and iron regulation when acute exposure to low CHO availability is undertaken with expert nutrition and coaching input.

Acute carbohydrate ingestion does not influence the post-exercise iron-regulatory response in elite keto-adapted race walkers.

OBJECTIVES: Adhering to a low carbohydrate (CHO) high fat (LCHF) diet can alter markers of iron metabolism in endurance athletes. This investigation examined the re-introduction of CHO prior to, and during exercise on the iron-regulatory response to exercise in a homogenous (in regard to serum ferritin concentration) group of athletes adapted to a LCHF diet. DESIGN: Parallel groups design. METHODS: Three weeks prior to the exercise trials, twenty-three elite race walkers adhered to either a CHO-rich (n=14) or LCHF diet (n=9). A standardised 19-25km race walk was performed while athletes were still adhering to their allocated dietary intervention (Adapt). A second test was performed three days later, where all athletes were placed on a high CHO diet (CHO Restoration). Venous blood samples were collected pre-, post- and 3h post-exercise and measured for interleukin-6 (IL-6) and hepcidin-25. RESULTS: The post-exercise IL-6 increase was greater in LCHF (p<0.001) during both the Adapt (LCHF: 13.1-fold increase; 95% CI: 5.6-23.0, CHO: 8.0-fold increase; 5.1-11.1) and CHO Restoration trials (LCHF: 18.5-fold increase; 10.9-28.9, CHO: 6.3-fold increase; 3.9-9.5); outcomes were not different between trials (p=0.84). Hepcidin-25 concentrations increased 3h post-exercise (p<0.001), however, they did not differ between trials (p=0.46) or diets (p=0.84). CONCLUSIONS: The elevated IL-6 response in athletes adapted to a LCHF diet was not attenuated by an acute increase in exogenous CHO availability. Despite diet-induced differences in IL-6 response to exercise, post-exercise hepcidin levels were similar between diets and trials, indicating CHO availability has minimal influence on post-exercise iron metabolism.

Iron handling by the human kidney: glomerular filtration and tubular reabsorption both contribute to urinary iron excretion.

In physiological conditions, circulating iron can be filtered by the glomerulus and is almost completely reabsorbed by the tubular epithelium to prevent urinary iron wasting. Increased urinary iron concentrations have been associated with renal injury. However, it is not clear whether increased urinary iron concentrations in patients are the result of increased glomerular iron filtration and/or insufficient tubular iron reabsorption and if these processes contribute to renal injury. We measured plasma and urine iron parameters and urinary tubular injury markers in healthy human subjects ( n = 20), patients with systemic iron overload ( n = 20), and patients with renal tubular dysfunction ( n = 18). Urinary iron excretion parameters were increased in both patients with systemic iron overload and tubular dysfunction, whereas plasma iron parameters were only increased in patients with systemic iron overload. In patients with systemic iron overload, increased urinary iron levels were associated with elevated circulating iron, as indicated by transferrin saturation (TSAT), and increased body iron, as suggested by plasma ferritin concentrations. In patients with tubular dysfunction, enhanced urinary iron and transferrin excretion were associated with distal tubular injury as indicated by increased urinary glutathione S-transferase pi 1-1 (GSTP1-1) excretion. In systemic iron overload, elevated urinary iron and transferrin levels were associated with increased injury to proximal tubules, indicated by increased urinary kidney injury marker 1 (KIM-1) excretion. Our explorative study demonstrates that both glomerular filtration of elevated plasma iron levels and insufficient tubular iron reabsorption could increase urinary iron excretion and cause renal injury.

Sustained plasma hepcidin suppression and iron elevation by Anticalin-derived hepcidin antagonist in cynomolgus monkey.

BACKGROUND AND PURPOSE: Anaemia of chronic disease (ACD) has been linked to iron-restricted erythropoiesis imposed by high circulating levels of hepcidin, a 25 amino acid hepatocyte-derived peptide that controls systemic iron homeostasis. Here, we report the engineering of the human lipocalin-derived, small protein-based anticalin PRS-080 hepcidin antagonist with high affinity and selectivity. EXPERIMENTAL APPROACH: Anticalin- and hepcidin-specific pharmacokinetic (PK)/pharmacodynamic modelling (PD) was used to design and select the suitable drug candidate based on t1/2 extension and duration of hepcidin suppression. The development of a novel free hepcidin assay enabled accurate analysis of bioactive hepcidin suppression and elucidation of the observed plasma iron levels after PRS-080-PEG30 administration in vivo. KEY RESULTS: PRS-080 had a hepcidin-binding affinity of 0.07 nM and, after coupling to 30 kD PEG (PRS-080-PEG30), a t1/2 of 43 h in cynomolgus monkeys. Dose-dependent iron mobilization and hepcidin suppression were observed after a single i.v. dose of PRS-080-PEG30 in cynomolgus monkeys. Importantly, in these animals, suppression of free hepcidin and subsequent plasma iron elevation were sustained during repeated s.c. dosing. After repeated dosing and followed by a treatment-free interval, all iron parameters returned to pre-dose values. CONCLUSIONS AND IMPLICATIONS: In conclusion, we developed a dose-dependent and safe approach for the direct suppression of hepcidin, resulting in prolonged iron mobilization to alleviate iron-restricted erythropoiesis that can address the root cause of ACD. PRS-080-PEG30 is currently in early clinical development.

Dietary hemoglobin rescues young piglets from severe iron deficiency anemia: Duodenal expression profile of genes involved in heme iron absorption.

Heme is an efficient source of iron in the diet, and heme preparations are used to prevent and cure iron deficiency anemia in humans and animals. However, the molecular mechanisms responsible for heme absorption remain only partially characterized. Here, we employed young iron-deficient piglets as a convenient animal model to determine the efficacy of oral heme iron supplementation and investigate the pathways of heme iron absorption. The use of bovine hemoglobin as a dietary source of heme iron was found to efficiently counteract the development of iron deficiency anemia in piglets, although it did not fully rebalance their iron status. Our results revealed a concerted increase in the expression of genes responsible for apical and basolateral heme transport in the duodenum of piglets fed a heme-enriched diet. In these animals the catalytic activity of heme oxygenase 1 contributed to the release of elemental iron from the protoporphyrin ring of heme within enterocytes, which may then be transported by the strongly expressed ferroportin across the basolateral membrane to the circulation. We hypothesize that the well-recognized high bioavailability of heme iron may depend on a split pathway mediating the transport of heme-derived elemental iron and intact heme from the interior of duodenal enterocytes to the bloodstream.

Cardiac Hepcidin Expression Associates with Injury Independent of Iron.

BACKGROUND: Hepcidin regulates systemic iron homeostasis by downregulating the iron exporter ferroportin. Circulating hepcidin is mainly derived from the liver but hepcidin is also produced in the heart. We studied the differential and local regulation of hepcidin gene expression in response to myocardial infarction (MI) and/or chronic kidney disease (CKD). We hypothesized that cardiac hepcidin gene expression is induced by and regulated to severity of cardiac injury, either through direct (MI) or remote (CKD) stimuli, as well as through increased local iron content. METHODS: Nine weeks after subtotal nephrectomy (SNX) or sham surgery (CON), rats were subjected to coronary ligation (CL) or sham surgery to realize 4 groups: CON, SNX, CL and SNX + CL. In week 16, the gene expression of hepcidin, iron and damage markers in cardiac and liver tissues was assessed by quantitative polymerase chain reaction and ferritin protein expression was studied by immunohistochemistry. RESULTS: Cardiac hepcidin messenger RNA (mRNA) expression was increased 2-fold in CL (p = 0.03) and 3-fold in SNX (p = 0.01). Cardiac ferritin staining was not different among groups. Cardiac hepcidin mRNA expression correlated with mRNA expression levels of brain natriuretic peptide (ß = 0.734, p < 0.001) and connective tissue growth factor (ß = 0.431, p = 0.02). In contrast, liver hepcidin expression was unaffected by SNX and CL alone, while it had decreased 50% in SNX + CL (p < 0.05). Hepatic ferritin immunostaining was not different among groups. CONCLUSIONS: Our data indicate differences in hepcidin regulation in liver and heart and suggest a role for injury rather than iron as the driving force for cardiac hepcidin expression in renocardiac failure.

Urinary Hepcidin Levels in Iron-Deficient and Iron-Supplemented Piglets Correlate with Hepcidin Hepatic mRNA and Serum Levels and with Body Iron Status.

Among livestock, domestic pig (Sus scrofa) is a species, in which iron metabolism has been most intensively examined during last decade. The obvious reason for studying the regulation of iron homeostasis especially in young pigs is neonatal iron deficiency anemia commonly occurring in these animals. Moreover, supplementation of essentially all commercially reared piglets with iron entails a need for monitoring the efficacy of this routine practice followed in the swine industry for several decades. Since the discovery of hepcidin many studies confirmed its role as key regulator of iron metabolism and pointed out the assessment of its concentrations in biological fluids as diagnostic tool for iron-related disorder. Here we demonstrate that urine hepcidin-25 levels measured by a combination of weak cation exchange chromatography and time-of-flight mass spectrometry (WCX-TOF MS) are highly correlated with mRNA hepcidin expression in the liver and plasma hepcidin-25 concentrations in anemic and iron-supplemented 28-day old piglets. We also found a high correlation between urine hepcidin level and hepatic non-heme iron content. Our results show that similarly to previously described transgenic mouse models of iron disorders, young pigs constitute a convenient animal model to explore accuracy and relationship between indicators for assessing systemic iron status.

Biomarkers for methotrexate-induced liver injury: urinary protein profiling of psoriasis patients.

Hepatic fibrosis is an adverse drug reaction of methotrexate (MTX) seen after long-term use in psoriasis patients. Currently, patients are monitored for MTX-induced hepatic fibrosis by performing liver biopsy, which is risky and burdensome for the patient, or by measuring plasma procollagen type III aminopeptide (PIIINP), which is not conclusive. The objective of this study was to identify novel predictive and preferably non-invasive biomarkers to monitor psoriasis patients for MTX-induced hepatic fibrosis. Urine samples were collected from 60 psoriasis patients treated with MTX and divided into two categories: low cumulative dose (< 1500 mg MTX) and high cumulative dose (> 1500 mg). Urinary proteins were profiled using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and identified using electrospray ionization LTQ. In urine of psoriasis patients with high cumulative MTX dose multiple proteins were identified that are associated with hepatic fibrosis, such as N-cadherin, inter-alpha-trypsin inhibitor heavy chain H4, haptoglobin and serotransferrin. These proteins may be candidate urinary biomarkers to monitor MTX-induced hepatic fibrosis. In conclusion, urinary proteome analysis identified a profile of potentially predictive biomarkers for MTX-induced hepatic fibrosis in psoriasis patients with high cumulative dose of MTX.

Urinary proteomic profiling reveals diclofenac-induced renal injury and hepatic regeneration in mice.

Diclofenac (DF) is a widely used non-steroidal anti-inflammatory drug for the treatment of rheumatic disorders, but is often associated with liver injury. We applied urinary proteomic profiling using MALDI-TOF MS to identify biomarkers for DF-induced hepatotoxicity in mice. Female CH3/HeOUJIco mice were treated with 75mg/kg bw DF by oral gavage and 24h urine was collected. Proteins identified in urine of DF-treated mice included epidermal growth factor, transthyretin, kallikrein, clusterin, fatty acid binding protein 1 and urokinase, which are related to liver regeneration but also to kidney injury. Both organs showed enhanced levels of oxidative stress (TBARS, p<0.01). Kidney injury was confirmed by histology and increased Kim1 and Il-6 mRNA expression levels (p<0.001 and p<0.01). Liver histology and plasma ALT levels in DF-treated mice were not different from control, but mRNA expression of Stat3 (p<0.001) and protein expression of PCNA (p<0.05) were increased, indicating liver regeneration. In conclusion, urinary proteome analysis revealed that DF treatment in mice induced kidney and liver injury. Within 24h, however, the liver was able to recover by activating tissue regeneration processes. Hence, the proteins found in urine of DF-treated mice represent kidney damage rather than hepatic injury.

Acute acetaminophen intoxication leads to hepatic iron loading by decreased hepcidin synthesis.

Acetaminophen (APAP), a major cause of acute liver injury in the Western world, is mediated by metabolism and oxidative stress. Recent studies have suggested a role for iron in potentiating APAP-induced liver injury although its regulatory mechanism is not completely understood. The current study was designed to unravel the iron-regulating pathways in mice after APAP-induced hepatotoxicity. Mice with severe injury showed a significant increase in liver iron concentration and oxidative stress. Concurrently, the plasma concentration of hepcidin, the key regulator in iron metabolism, and hepatic hepcidin antimicrobial peptide (Hamp) mRNA expression levels were significantly reduced. We showed that hepcidin transcription was inhibited via several hepcidin-regulating factors, including the bone morphogenetic protein/small mother against decapentaplegic (BMP/SMAD) pathway, CCAAT/enhancer-binding protein α (C/EBPα), and possibly also via erythropoietin (EPO). Downregulation of the BMP/SMAD signaling pathway was most likely caused by hypoxia-inducible factor 1α (HIF-1α), which was increased in mice with severe APAP-induced liver injury. HIF-1α stimulates cleaving of hemojuvelin, the cofactor of the BMP receptor, thereby blocking BMP-induced signaling. In addition, gene expression levels of C/ebpα were significantly reduced, and Epo mRNA expression levels were significantly increased after APAP intoxication. These factors are regulated through HIF-1α during oxidative stress and suggest that HIF-1α is a key modulator in reduced hepcidin transcription after APAP-induced hepatotoxicity. In conclusion, acute APAP-induced liver injury leads to activation of HIF-1α, which results in a downregulation in hepcidin expression through a BMP/SMAD signaling pathway and through C/EBPα inhibition. Eventually, this leads to hepatic iron loading associated with APAP cytotoxicity.

Mass spectrometry analysis of hepcidin peptides in experimental mouse models.

The mouse is a valuable model for unravelling the role of hepcidin in iron homeostasis, however, such studies still report hepcidin mRNA levels as a surrogate marker for bioactive hepcidin in its pivotal function to block ferroportin-mediated iron transport. Here, we aimed to assess bioactive mouse Hepcidin-1 (Hep-1) and its paralogue Hepcidin-2 (Hep-2) at the peptide level. To this purpose, Fourier transform ion cyclotron resonance (FTICR) and tandem-MS was used for hepcidin identification, after which a time-of-flight (TOF) MS-based methodology was exploited to routinely determine Hep-1 and -2 levels in mouse serum and urine. This method was biologically validated by hepcidin assessment in: i) 3 mouse strains (C57Bl/6; DBA/2 and BABL/c) upon stimulation with intravenous iron and LPS, ii) homozygous Hfe knock out, homozygous transferrin receptor 2 (Y245X) mutated mice and double affected mice, and iii) mice treated with a sublethal hepatotoxic dose of paracetamol. The results showed that detection of Hep-1 was restricted to serum, whereas Hep-2 and its presumed isoforms were predominantly present in urine. Elevations in serum Hep-1 and urine Hep-2 upon intravenous iron or LPS were only moderate and varied considerably between mouse strains. Serum Hep-1 was decreased in all three hemochromatosis models, being lowest in the double affected mice. Serum Hep-1 levels correlated with liver hepcidin-1 gene expression, while acute liver damage by paracetamol depleted Hep-1 from serum. Furthermore, serum Hep-1 appeared to be an excellent indicator of splenic iron accumulation. In conclusion, Hep-1 and Hep-2 peptide responses in experimental mouse agree with the known biology of hepcidin mRNA regulators, and their measurement can now be implemented in experimental mouse models to provide novel insights in post-transcriptional regulation, hepcidin function, and kinetics.