Judit Kiss

The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice

Systemic iron homeostasis is mainly controlled by the liver through synthesis of the peptide hormone hepcidin (encoded by Hamp), the key regulator of duodenal iron absorption and macrophage iron release. Here we show that the liver-specific microRNA miR-122 is important for regulating Hamp mRNA expression and tissue iron levels. Efficient and specific depletion of miR-122 by injection of a locked-nucleic-acid-modified (LNA-modified) anti-miR into WT mice caused systemic iron deficiency, characterized by reduced plasma and liver iron levels, mildly impaired hematopoiesis, and increased extramedullary erythropoiesis in the spleen. Moreover, miR-122 inhibition increased the amount of mRNA transcribed by genes that control systemic iron levels, such as hemochromatosis (Hfe), hemojuvelin (Hjv), bone morphogenetic protein receptor type 1A (Bmpr1a), and Hamp. Importantly, miR-122 directly targeted the 3′ untranslated region of 2 mRNAs that encode activators of hepcidin expression, Hfe and Hjv. These data help to explain the increased Hamp mRNA levels and subsequent iron deficiency in mice with reduced miR-122 levels and establish a direct mechanistic link between miR-122 and the regulation of systemic iron metabolism.

Hfe acts in hepatocytes to prevent hemochromatosis.

Hereditary hemochromatosis (HH) is a prevalent, potentially fatal disorder of iron metabolism hallmarked by intestinal hyperabsorption of iron, hyperferremia, and hepatic iron overload. In both humans and mice, type I HH is associated with mutations in the broadly expressed HFE/Hfe gene. To identify where Hfe acts to prevent HH, we generated mice with tissue-specific Hfe ablations. This work demonstrates that local Hfe expression in hepatocytes serves to maintain physiological iron homeostasis, answering a long-standing question in medicine and explaining earlier clinical observations.

Ca2+ channel blockers reverse iron overload by a new mechanism via divalent metal transporter-1

Hereditary hemochromatosis and transfusional iron overload are frequent clinical conditions associated with progressive iron accumulation in parenchymal tissues, leading to eventual organ failure. We have discovered a new mechanism to reverse iron overload—pharmacological modulation of the divalent metal transporter-1 (DMT-1). DMT-1 mediates intracellular iron transport during the transferrin cycle and apical iron absorption in the duodenum. Its additional functions in iron handling in the kidney and liver are less well understood. We show that the L-type calcium channel blocker nifedipine increases DMT-1–mediated cellular iron transport 10- to 100-fold at concentrations between 1 and 100 μM. Mechanistically, nifedipine causes this effect by prolonging the iron-transporting activity of DMT-1. We show that nifedipine mobilizes iron from the liver of mice with primary and secondary iron overload and enhances urinary iron excretion. Modulation of DMT-1 function by L-type calcium channel blockers emerges as a new pharmacological therapy for the treatment of iron overload disorders.

Loss or Silencing of the PHD1 Prolyl Hydroxylase Protects Livers of Mice Against Ischemia/Reperfusion Injury

BACKGROUND & AIMS Liver ischemia/reperfusion (I/R) injury is a frequent cause of organ dysfunction. Loss of the oxygen sensor prolyl hydroxylase domain enzyme 1 (PHD1) causes tolerance of skeletal muscle to hypoxia. We assessed whether loss or short-term silencing of PHD1 could likewise induce hypoxia tolerance in hepatocytes and protect them against hepatic I/R damage. METHODS Hepatic ischemia was induced in mice by clamping of the portal vessels of the left lateral liver lobe; 90 minutes later livers were reperfused for 8 hours for I/R experiments. Hepatocyte damage following ischemia or I/R was investigated in PHD1-deficient (PHD1(-/-)) and wild-type mice or following short hairpin RNA-mediated short-term inhibition of PHD1 in vivo. RESULTS PHD1(-/-) livers were largely protected against acute ischemia or I/R injury. Among mice subjected to hepatic I/R followed by surgical resection of all nonischemic liver lobes, more than half of wild-type mice succumbed, whereas all PHD1(-/-) mice survived. Also, short-term inhibition of PHD1 through RNA interference-mediated silencing provided protection against I/R. Knockdown of PHD1 also induced hypoxia tolerance of hepatocytes in vitro. Mechanistically, loss of PHD1 decreased production of oxidative stress, which likely relates to a decrease in oxygen consumption as a result of a reprogramming of hepatocellular metabolism. CONCLUSIONS Loss of PHD1 provided tolerance of hepatocytes to acute hypoxia and protected them against I/R-damage. Short-term inhibition of PHD1 is a novel therapeutic approach to reducing or preventing I/R-induced liver injury.

Loss of the Oxygen Sensor PHD3 Enhances the Innate Immune Response to Abdominal Sepsis

Hypoxia and HIFs (HIF-1α and HIF-2α) modulate innate immune responses in the setting of systemic inflammatory responses and sepsis. The HIF prolyl hydroxylase enzymes PHD1, PHD2 and PHD3 regulate the mammalian adaptive response to hypoxia; however, their significance in the innate immune response has not been elucidated. We demonstrate in this study that deficiency of PHD3 (PHD3−/−) specifically shortens the survival of mice subjected to various models of abdominal sepsis because of an overwhelming innate immune response, leading to premature organ dysfunction. By contrast, this phenotype was absent in mice deficient for PHD1 (PHD1−/−) or PHD2 (PHD2+/−). In vivo, plasma levels of proinflammatory cytokines were enhanced, and recruitment of macrophages to internal organs was increased in septic PHD3-deficient mice. Reciprocal bone marrow transplantation in sublethally irradiated mice revealed that enhanced susceptibility of PHD3-deficient mice to sepsis-related lethality was specifically caused by loss of PHD3 in myeloid cells. Several in vitro assays revealed enhanced cytokine production, migration, phagocytic capacity, and proinflammatory activation of PHD3-deficient macrophages. Increased proinflammatory activity of PHD3-deficient macrophages occurred concomitantly with enhanced HIF-1α protein stabilization and increased NF-κB activity, and interference with the expression of HIF-1α or the canonical NF-κB pathway blunted their proinflammatory phenotype. It is concluded that impairment of PHD3 enzyme function aggravates the clinical course of abdominal sepsis via HIF-1α– and NF-κB–mediated enhancement of the innate immune response.

SELDI-TOF MS detection of urinary hepcidin.

Hepcidin is a 25-residue hepatic peptide that regulates iron absorption from the diet and tissue iron distribution. Inappropriately low Hepcidin expression is implicated in the pathogenesis of hereditary hemochromatosis and iron-loading anemias, like the thalassemias. Increased hepcidin expression mediates iron retention in the anemias of inflammation and plays a pathogenic role in iron-refractory iron-deficiency anemia (IRIDA). Because of its clinical importance, Hepcidin is expected to be a useful biomarker for diagnosis and management of iron-related disorders. So far an ELISA for human hepcidin and SELDI-TOF-MS based approaches have been applied to monitor urinary and/or serum hepcidin levels. Here we report a modified protocol for SELDI-TOF based detection of human, urinary hepcidin. We show that CM10 Proteinchips are superior to NP20 Proteinchips commonly used in previously reported protocols to sensitively and accurately detect urinary hepcidin. Application of this modified hepcidin assay accurately detects increased hepcidin levels in the urine of sepsis patients.

Molecular oxygen sensing: implications for visceral surgery.

BackgroundSince mammalian cells rely on the availability of oxygen, they have devised mechanisms to sense environmental oxygen tension, and to efficiently counteract oxygen deprivation (hypoxia). These adaptive responses to hypoxia are essentially mediated by hypoxia inducible transcription factors (HIFs). Three HIF prolyl hydroxylase enzymes (PHD1, PHD2 and PHD3) function as oxygen sensing enzymes, which regulate the activity of HIFs in normoxic and hypoxic conditions. Many of the compensatory functions exerted by the PHD–HIF system are of immediate surgical relevance since they regulate the biological response of ischemic tissues following ligation of blood vessels, of oxygen-deprived inflamed tissues, and of tumors outgrowing their vascular supply.PurposeHere, we outline specific functions of PHD enzymes in surgically relevant pathological conditions, and discuss how these functions might be exploited in order to support the treatment of surgically relevant diseases.