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

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

Identification of novel translational urinary biomarkers for acetaminophen-induced acute liver injury using proteomic profiling in mice

Contains fulltext : 108207.pdf (publisher's version ) (Open Access)Drug-induced liver injury (DILI) is the leading cause of acute liver failure. Currently, no adequate predictive biomarkers for DILI are available. This study describes a translational approach using proteomic profiling for the identification of urinary proteins related to acute liver injury induced by acetaminophen (APAP). Mice were given a single intraperitoneal dose of APAP (0-350 mg/kg bw) followed by 24 h urine collection. Doses of >/=275 mg/kg bw APAP resulted in hepatic centrilobular necrosis and significantly elevated plasma alanine aminotransferase (ALT) values (p<0.0001). Proteomic profiling resulted in the identification of 12 differentially excreted proteins in urine of mice with acute liver injury (p<0.001), including superoxide dismutase 1 (SOD1), carbonic anhydrase 3 (CA3) and calmodulin (CaM), as novel biomarkers for APAP-induced liver injury. Urinary levels of SOD1 and CA3 increased with rising plasma ALT levels, but urinary CaM was already present in mice treated with high dose of APAP without elevated plasma ALT levels. Importantly, we showed in human urine after APAP intoxication the presence of SOD1 and CA3, whereas both proteins were absent in control urine samples. Urinary concentrations of CaM were significantly increased and correlated well with plasma APAP concentrations (r = 0.97; p<0.0001) in human APAP intoxicants, who did not present with elevated plasma ALT levels. In conclusion, using this urinary proteomics approach we demonstrate CA3, SOD1 and, most importantly, CaM as potential human biomarkers for APAP-induced liver injury

Inhibition of Nrf2 alters cell stress induced by chronic iron exposure in human proximal tubular epithelial cells

Iron can catalyze reactive oxygen species (ROS) formation, causing cellular injury. In systemic iron overload, renal tubular epithelial cells are luminally exposed to high iron levels due to glomerular filtration of increased circulating iron. Reports of tubular dysfunction and iron deposition in β-thalassemia major support an association between increased chronic iron exposure and renal tubular injury. In acute iron exposure, Nuclear factor-erythroid 2-related factor 2 (Nrf2) may protect from iron-induced injury, whereas chronic renal stress may lead to Nrf2 exhaustion. We studied the cytotoxic mechanisms of chronic iron exposure using human conditionally immortalized proximal tubular epithelial cells (ciPTECs). Long-term iron exposure resulted in iron accumulation, cytosolic ROS formation and increased heme oxygenase 1 (HMOX-1) mRNA expression (all p < 0.001). This was accompanied by nuclear translocation of Nrf2 and induction of its target protein NQO1, which both could be blocked by the Nrf2 inhibitor trigonelline. Interestingly, iron and trigonelline incubation reduced ROS production, but did not affect HMOX-1 mRNA levels. Moreover, ferritin protein and CHOP mRNA expression were induced in combined iron and trigonelline incubated cells (p < 0.05). Together, these findings suggest that chronic iron exposure induces oxidative stress and that exhaustion of the antioxidant Nrf2 pathway may lead to renal injury

Application of urine proteomics for biomarker discovery in drug-induced liver injury

Abstract The leading cause of hepatic damage is drug-induced liver injury (DILI), for which currently no adequate predictive biomarkers are available. Moreover, for most drugs related to DILI, the mechanisms underlying the adverse reaction have not yet been elucidated. Urinary protein biomarker candidates for DILI have emerged in the past few years and correlate well with clinical studies for serum DILI biomarkers. The goal of this review was to investigate the use of urine as a source of protein biomarkers for drug-induced liver injury. Finally, we discuss some of the current strategies required to advance the field of biomarker discovery for DILI with respect to appropriate clinical biobanking and adequate translational research

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

Probst S, Fels J, Scharner B, et al. Role of hepcidin in oxidative stress and cell death of cultured mouse renal collecting duct cells: protection against iron and sensitization to cadmium. Archives of toxicology. 2021.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+ (0.5-5mumol/l) and/or Fe2+ (50-100mumol/l) for 4-24h. 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

N-acetyl-meta-aminophenol, the alleged nontoxic isomer of acetaminophen, is toxic in both rat and human precision-cut liver slices

N-acetyl-meta-aminophenol (AMAP) is generally considered as a non-toxic regioisomer of the well-known hepatotoxicant acetaminophen (APAP). However, so far AMAP has only been shown to be non-toxic in mice and hamsters. To investigate whether AMAP could also be used as non-toxic analog of APAP in studies in rat and human, the toxicity of APAP and AMAP was tested ex vivo in precision-cut liver slices (PCLS) of mouse, rat and human. Based on ATP content and histomorphology, APAP was most toxic in mouse and least toxic in human PCLS. Surprisingly, although AMAP showed a much lower toxicity than APAP in mouse PCLS, AMAP was more toxic than APAP at all concentrations tested in both rat and human PCLS. In HepG2 cells, AMAP caused similar apoptosis and induced a higher Nrf2-mediated oxidative stress signal than APAP. The protein profile in the medium of AMAP-treated rat PCLS was similar to that of APAP, whereas in the medium of mouse PCLS it was similar to the control. Metabolite profiling indicated that mouse PCLS produced the highest amount of glutathione conjugate of APAP, while no glutathione conjugate of AMAP was detected in all three species. Mouse also produced ten times more hydroquinone metabolites of AMAP, the assumed proximate reactive metabolites, than rat or human. In conclusion, AMAP can be used as non-toxic isomer of APAP in mouse but not in rat and human. The marked species differences in APAP and AMAP toxicity and metabolism underline the importance of using human tissues for better prediction of toxicity in man

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Drug-induced liver injury is one of the leading causes of drug withdrawal from the market. In this study, we investigated the applicability of protein profiling of the incubation medium of human, mouse and rat precision-cut liver slices (PCLS) exposed to liver injury-inducing drugs for biomarker identification, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. PCLS were incubated with acetaminophen (APAP), 3-acetamidophenol, diclofenac and lipopolysaccharide for 24-48 h. PCLS medium from all species treated with APAP demonstrated similar changes in protein profiles, as previously found in mouse urine after APAP-induced liver injury, including the same key proteins: superoxide dismutase 1, carbonic anhydrase 3 and calmodulin. Further analysis showed that the concentration of hepcidin, a hepatic iron-regulating hormone peptide, was reduced in PCLS medium after APAP treatment, resembling the decreased mouse plasma concentrations of hepcidin observed after APAP treatment. Interestingly, comparable results were obtained after 3-acetamidophenol incubation in rat and human, but not mouse PCLS. Incubation with diclofenac, but not with lipopolysaccharide, resulted in the same toxicity parameters as observed for APAP, albeit to a lesser extent. In conclusion, proteomics can be applied to identify potential translational biomarkers using the PCLS system

Effect of Deferoxamine on Post-Transfusion Iron, Inflammation, and In Vitro Microbial Growth in a Canine Hemorrhagic Shock Model: A Randomized Controlled Blinded Pilot Study

Red blood cell (RBC) transfusion is associated with recipient inflammation and infection, which may be triggered by excessive circulating iron. Iron chelation following transfusion may reduce these risks. The aim of this study was to evaluate the effect of deferoxamine on circulating iron and inflammation biomarkers over time and in vitro growth of Escherichia coli (E. coli) following RBC transfusion in dogs with atraumatic hemorrhage. Anesthetized dogs were subject to atraumatic hemorrhage and transfusion of RBCs, then randomized to receive either deferoxamine or saline placebo of equivalent volume (n = 10 per group) in a blinded fashion. Blood was sampled before hemorrhage and then 2, 4, and 6 h later. Following hemorrhage and RBC transfusion, free iron increased in all dogs over time (both p \u3c 0.001). Inflammation biomarkers interleukin-6 (IL6), CXC motif chemokine-8 (CXCL8), interleukin-10 (IL10), and keratinocyte-derived chemokine (KC) increased in all dogs over time (all p \u3c 0.001). Logarithmic growth of E. coli clones within blood collected 6 h post-transfusion was not different between groups. Only total iron-binding capacity was different between groups over time, being significantly increased in the deferoxamine group at 2 and 4 h post-transfusion (both p \u3c 0.001). In summary, while free iron and inflammation biomarkers increased post-RBC transfusion, deferoxamine administration did not impact circulating free iron, inflammation biomarkers, or in vitro growth of E. coli when compared with placebo