Hepcidin regulation in a mouse model of acute hypoxia.

OBJECTIVE: During hypoxia, hepcidin expression is inhibited to allow iron mobilization to sustain erythropoietic expansion. We analyzed molecular mechanisms underlying hypoxia-induced hepcidin inhibition in an in vivo model of acute hypoxia. METHODS: Mice were kept under normal or hypoxic conditions for 6 hours and 15 hours and treated with α-PDGF-BB antibody or PDGF-BB receptor inhibitor. Blood, liver, spleen, and bone marrow were collected to extract RNA and protein or to quantify EPO and PDGF-BB. mRNA and protein levels were assessed by RT-PCR and Western blot. RESULTS: Hepcidin was strongly inhibited at 15 hours, and this downregulation followed erythropoiesis activation and upregulation of several growth factors. PDGF-BB, erythroferrone, GDF15, and TWSG1 were upregulated by hypoxia in the bone marrow, but not in spleen or liver. Inactivation of PDGF-BB or its receptor suppressed the hypoxia-induced hepcidin inhibition. CONCLUSION: Spleen and liver are not involved in the early stages of hypoxia-induced hepcidin downregulation. Our data support the role of PDGF-BB and probably also of erythroferrone in the recruitment of iron for erythropoiesis in the hypoxia setting. The rapid normalization of all the erythroid factors against persistent hepcidin suppression suggests that other signals are involved that should be clarified in future studies.

Unexplained isolated hyperferritinemia without iron overload.

Although hyperferritinemia may be reflective of elevated total body iron stores, there are conditions in which ferritin levels are disproportionately elevated relative to iron status. Autosomal dominant forms of hyperferritinemia due to mutations in the L-ferritin IRE or in A helix of L-ferritin gene have been described, however cases of isolated hyperferritinemia still remain unsolved. We describe 12 Italian subjects with unexplained isolated hyperferritinemia (UIH). Four probands have affected siblings, but no affected parents or offspring. Sequencing analyses did not identify casual mutations in ferritin gene or IRE regions. These patients had normal levels of intracellular ferritin protein and mRNA in peripheral blood cells excluding pathological ferritin production at transcriptional and post-transcriptional level. In contrast with individuals with benign hyperferritinemia caused by mutations affecting the ferritin A helix, low rather than high glycosylation of serum ferritin was observed in our UIH subjects compared with controls. These findings suggest that subjects with UIH have a previously undescribed form of hyperferritinemia possibly attributable to increased cellular ferritin secretion and/or decreased serum ferritin clearance. The cause remains to be defined and we can only speculate the existence of mutations in gene/s not directly implicated in iron metabolism that could affect ferritin turnover including ferritin secretion.

GNPAT rs11558492 is not a Major Modifier of Iron Status: Study of Italian Hemochromatosis Patients and Blood Donors.

BACKGROUND AND AIM: HFE-related Hemochromatosis (HH) is characterized by marked phenotype heterogeneity, probably due to the combined action of acquired and genetic factors. Among them, GNPAT rs11558492 was proposed as genetic modifier of iron status, but results are still controversial. To shed light on these discrepancies, we genotyped 298 Italian p.C282Y homozygotes and 169 healthy controls. MATERIAL AND METHODS: Allele and genotype frequencies were analysed and compared with those reported in Exome Variant Server (EVS). To explore the role of rs11558492 as a potential modifier of iron status, serum ferritin (SF), liver iron concentration (LIC) and iron removed (IR) were studied according to allele and genotype frequencies. In addition, the effect of the SNP on liver fibrosis was examined comparing patients with absent/mild-moderate fibrosis to those with severe fibrosis-cirrhosis. RESULTS: GNPAT rs11558492 minor allele (G) frequency (MAF) was 20.3% in HFE-HH, 17.2% in controls and 20.6% in EVS database. Genotype frequencies were 64% and 69.2% (AA), 31.2% and 27.2% (AG), 4.8% and 3.6% (GG) in HFE-HH and controls, respectively. No significant differences were found comparing genotype and allele frequencies even selecting subgroups of only-males with extreme phenotypes and low alcohol intake. SF, IR and LIC levels did not significantly differ according to rs11558492 genotypes. Also, MAF did not differ between patients with absent/mild fibrosis and severe fibrosis/cirrhosis. CONCLUSIONS: Our findings indicate that GNPAT rs11558492 is not a major modifier of iron status and is not associated with liver fibrosis in HFE-HH patients.

Alterations in sympathetic nerve traffic in genetic haemochromatosis before and after iron depletion therapy: a microneurographic study.

AIMS: Haemochromatosis (HH) displays a number of circulatory alterations concurring at increase cardiovascular risk. Whether these include sympathetic abnormalities in unknown. METHODS AND RESULTS: In 18 males with primary HH (age: 42.3 ± 10.4 years, mean ± SD), clinic and beat-to-beat blood pressure (BP, Finapres), heart rate (HR, EKG), and muscle sympathetic nerve activity (MSNA, microneurography) traffic were measured in the iron overload state and after iron depletion therapy. Haemochromatosis patients displayed elevated serum iron indices while other haemodynamic and metabolic variables were superimposable to ones seen in 12 healthy subjects (C). Muscle sympathetic nerve activity was significantly greater in HH than C (64.8 ± 13.3 vs. 37.8 ± 6.7 bs/100 hb, P < 0.01). Iron depletion caused a significant reduction in serum ferritin, transferrin saturation, and MSNA (from 64.8 ± 13.3 to 39.2 ± 9.2 bs/100 hb, P < 0.01) and a significant improvement in baroreflex-MSNA modulation. This was paralleled by a significant increase in the high-frequency HR variability and by a significant reduction in the low-frequency systolic BP variability components. Before after iron depletion therapy, MSNA was significantly and directly related to transferrin saturation, liver iron concentration, and iron removed, while the MSNA reductions observed after the procedure were significantly and inversely related to the baroreflex-MSNA increases detected after iron depletion. In C, all variables remained unchanged following 1 month observation. CONCLUSION: These data provide the first evidence that in HH iron overload is associated with an hyperadrenergic state and a baroreflex alteration, which are reversed by iron depletion. These findings underline the importance of iron overload in modulating sympathetic activation, possibly participating at the elevated cardiovascular risk reported in HH.

Proprotein convertase 7 rs236918 associated with liver fibrosis in Italian patients with HFE-related hemochromatosis.

BACKGROUND AND AIM: p.Cys282Tyr homozygosity is the prevalent genotype in (HFE)-related Hereditary Hemochromatosis with low penetrance and variable expression. However, liver cirrhosis and hepatocellular carcinoma remain the main causes of mortality in these patients. Detection of genetic modifiers identifying patients at risk for liver damage would be relevant for their clinical management. We evaluated proprotein convertase 7 (PCSK7) rs236918 as genetic marker of risk of liver fibrosis in an Italian cohort of p.Cys282Tyr homozygotes. METHODS: Liver fibrosis was histologically assessed by Ishak score. We evaluated PCSK7 alleles and genotypes frequencies according to single or grouped staging scores: absent/mild fibrosis (stage: 0-2), moderate (stage: 3-4), and severe fibrosis/cirrhosis (stage: 5-6). Single nucleotide polymorphism genotyping was performed by restriction fragment length polymorphism or Taqman 5'-nuclease assays. RESULTS: The rs236918 allele C frequency increased from stages 0-2 to 5-6 (7.1% vs 13.6%, vs 21.9%, P = 0.003). The wild-type genotype was significantly more frequent in the absent/mild fibrosis group (54.2%) compared with only 17% in patients with severe fibrosis/cirrhosis. At univariate proportional odds model, patients with GC + CC genotypes were 2.77 times (P = 0.0018) more likely to have worse liver staging scores than wild-type patients. In the adjusted analysis, odds ratio was 2.37 (P = 0.0218), and 2.56 (P = 0.0233) when the analysis was restricted to males. An exploratory mediation analysis suggested a direct effect of genotype on severe fibrosis/cirrhosis (odds ratio = 3.11, P = 0.0157), and a mild non-significant indirect effect mediated through iron accounting for 28%. CONCLUSIONS: These findings confirm that PCSK7 rs236918 C allele is a risk factor for cirrhosis development in Italian patients with HFE-Hemochromatosis.

Renal hemodynamic response to L-arginine in uncomplicated, type 1 diabetes mellitus: the role of buffering anions and tubuloglomerular feedback.

According to the "tubulocentric" hypothesis of the glomerular hyperfiltration of diabetes mellitus (DM), tubuloglomerular feedback (TGF) is the critical determinant of the related renal hemodynamic dysfunction. To examine the role of TGF in human type 1 DM, 12 salt-replete healthy (C) and 11 uncomplicated DM individuals underwent measurements of glomerular filtration rate (GFR), renal blood flow (RBF), and lithium-derived absolute "distal" sodium delivery (DDNa). Measurements were made during two 3-h infusions of 0.012 mmol·kg(-1)·min(-1) l-arginine (ARG) buffered with either equimolar HCl (ARG.HCl) or citric acid (ARG.CITR). Our hypothesis was that changes in TGF signaling would be directionally opposite ARG.HCl vs. ARG.CITR according to the effects of the ARG-buffering anion on DDNa. Similar changes in C and DM followed ARG.CITR, with declines in DDNa (-0.26 ± 0.07 mmol/min C vs. -0.31 ± 0.07 mmol/min DM) and increases in RBF (+299 ± 25 vs. +319 ± 29 ml·min(-1)·1.73 m(-2)) and GFR (+6.6 ± 0.8 vs. +11.6 ± 1.2 ml·min(-1)·1.73 m(-2)). In contrast, with ARG.HCl, DDNa rose in both groups (P = 0.001), but the response was 73% greater in DM (+1.50 ± 0.15 mmol/min C vs. +2.59 ± 0.22 mmol/min DM, P = 0.001). RBF also increased (P = 0.001, +219 ± 20 ml·min(-1)·1.73 m(-2) C, +105 ± 14 DM), but ΔRBF after ARG.HCl was lower vs. ARG.CITR in both groups (P = 0.001). After ARG.HCl, ΔRBF also was 50% lower in DM vs. C (P = 0.001) and GFR, unchanged in C, declined in DM (-7.4 ± 0.9 ml·min(-1)·1.73 m(-2), P = 0.02 vs. C). After ARG.HCl, unlike ARG.CITR, DDNa increased in C and DM, associated with less ΔRBF and ΔGFR vs. ARG.CITR. This suggests that the renal hemodynamic response to ARG is influenced substantially by the opposite actions of HCl vs. CITR on DDNa and TGF. In DM, the association of ARG.HCl-induced exaggerated ΔDDNa, blunted ΔRBF, and the decline in GFR vs. C shows an enhanced TGF dependence of renal vasodilatation to ARG, in agreement with a critical role of TGF in DM-related renal hemodynamic dysfunction.

Contribution of bradykinin B2 receptors to the inhibition by valsartan of systemic and renal effects of exogenous angiotensin II in salt-repleted humans.

To investigate whether bradykinin (BK) participates in the inhibition of renal effects of exogenous angiotensin II (AngII) by AngII type 1 receptor (AT1R) blockade, eight salt-repleted volunteers underwent four p-aminohippurate- and inulin-based renal studies of AngII infusion at increasing rates of 0.625, 1.25, and 2.5 ng.kg.min(-1) for 30 min. Studies 1 and 2 were preceded by 3 days of placebo, whereas studies 3 and 4 used 240 to 320 mg.day(-1) valsartan. Bradykinin B2-type receptor (BKB2R) antagonist icatibant (50 mug.kg(-1)) was coinfused in studies 2 and 4. Mean blood pressure (MBP), glomerular filtration rate (GFR), renal blood flow (RBF), and renal sodium excretion (UNaV) were measured. In study 1, MBP rose by 12.8%, UNaV decreased by 68%, and GFR and RBF also fell (p < 0.001 for all). In study 2, GFR and RBF fell as in study 1, but the rise in MBP and the fall in UNaV were accentuated [+20.0%, analysis of variance (ANOVA), p < 0.02 versus study 1 and -80.0%, p < 0.05, respectively]. In study 3, AngII had no effects, and in study 4, renal hemodynamics remained unaffected, but MBP still rose and UNaV fell (ANOVA, p < 0.02 and 0.005 versus study 3, respectively). Icatibant accentuated AngII-induced changes in MBP and UNaV. Previous AT1R blockade prevented any systemic and renal effects of AngII, but significant changes in MBP and UNaV still followed AngII plus icatibant even after AT1R blockade. BK, through BKB2Rs, participates in the inhibitory action of AT1R blockers toward actions of exogenous AngII on MBP and UNaV in healthy humans.

Phenotypic heterogeneity in seven Italian cases of aceruloplasminemia.

INTRODUCTION: Aceruloplasminemia is an ultra-rare hereditary disorder characterized by iron-restricted microcytic anemia and tissue iron overload associated with diabetes, retinal and progressive neurological degeneration. We describe genotypes and phenotypes at diagnosis, and disease evolution of seven Italian patients. METHODS: Anagraphical, biochemical, genetic, clinical and instrumental data were collected at diagnosis and during a long-term follow-up. Mutations, ferroxidase activity and Western Blot analysis of ceruloplasmin were performed according to standard protocols. RESULTS: Three mutations were already described (p.Phe217Ser, deletions of exon 11 and 12), p.Ile991Thr is a very rare variant, p.Cys338Ser and IVS6+1G > A were novel mutations. In silico analyses suggested they were highly likely or likely to be damaging. At diagnosis, 100% had microcytosis, 86% had mild-moderate anemia, low serum iron and high serum ferritin. Four (57%) had type 1 diabetes or glucose intolerance, 3/7 had neurological manifestations, and only one had early diabetic retinopathy. All but one underwent iron chelation therapy requiring temporary discontinuation because of anemia worsening. At the end of follow-up, three patients aggravated and 2 developed neurological symptoms; only two patients were free of neurological manifestations and showed mild or absent brain iron. CONCLUSION: Aceruloplasminemia phenotypes ranged from classical characterized by progressive neurologic derangement to milder in which signs of systemic iron overload prevailed over brain iron accumulation. Within this large heterogeneity, microcytosis with or without anemia, low serum iron and high serum ferritin were the early hallmarks of the disease. Therapeutic approaches other than iron chelation should be explored to reduce morbidity and improve life expectancy.

GNPAT rs11558492 is not a Major Modifier of Iron Status: Study of Italian Hemochromatosis Patients and Blood Donors

ABSTRACT Background and Aim. HFE-related Hemochromatosis (HH) is characterized by marked phenotype heterogeneity, probably due to the combined action of acquired and genetic factors. Among them, GNPATrs11558492 was proposed as genetic modifier of iron status, but results are still controversial. To shed light on these discrepancies, we genotyped 298 Italian p.C282Y homozygotes and 169 healthy controls. Material and methods. Allele and genotype frequencies were analysed and compared with those reported in Exorne Variant Server (EVS). To explore the role of rs11558492 as a potential modifier of iron status, serum ferritin (SF), liver iron concentration (LIC) and iron removed (IR) were studied according to allele and genotype frequencies. In addition, the effect of the SNP on liver fibrosis was examined comparing patients with absent/mild-moderate fibrosis to those with severe fibrosis-cirrhosis. Results. GNPAT rs11558492 minor allele (G) frequency (MAF) was 20.3% in HFE- HH, 17.2% in controls and 20.6% in EVS database. Genotype frequencies were 64% and 69.2% (AA), 31.2% and 27.2% (AG), 4.8% and 3.6% (GG) in HFE-HH and controls, respectively. No significant differences were found comparing genotype and allele frequencies even selecting subgroups of only-males with extreme phenotypes and low alcohol intake. SF, IR and LIC levels did not significantly differ according to rs11558492 genotypes. Also, MAF did not differ between patients with absent/mild fibrosis and severe fibrosis/cirrhosis. Conclusions. Our findings indicate that GNPAT rs11558492 is not a major modifier of iron status and is not associated with liver fibrosis in HFE- HH patients.(AU)