Iron uptake by ZIP8 and ZIP14 in human proximal tubular epithelial cells.

In patients with iron overload disorders, increasing number of reports of renal dysfunction and renal iron deposition support an association between increased iron exposure and renal injury. In systemic iron overload, elevated circulating levels of transferrin-bound (TBI) and non-transferrin-bound iron (NTBI) are filtered to the renal proximal tubules, where they may cause injury. However, the mechanisms of tubular iron handling remain elusive. To unravel molecular renal proximal tubular NTBI and TBI handling, human conditionally immortalized proximal tubular epithelial cells (ciPTECs) were incubated with 55Fe as NTBI and fluorescently labeled holo-transferrin as TBI. Ferrous iron importers ZIP8 and ZIP14 were localized in the ciPTEC plasma membrane. Whereas silencing of either ZIP8 or ZIP14 alone did not affect 55Fe uptake, combined silencing significantly reduced 55Fe uptake compared to control (p < 0.05). Furthermore, transferrin receptor 1 (TfR1) and ZIP14, but not ZIP8, colocalized with early endosome antigen 1 (EEA1). TfR1 and ZIP14 also colocalized with uptake of fluorescently labeled transferrin. Furthermore, ZIP14 silencing decreased 55Fe uptake after 55Fe-Transferrin exposure (p < 0.05), suggesting ZIP14 could be involved in early endosomal transport of TBI-derived iron into the cytosol. Our data suggest that human proximal tubular epithelial cells take up TBI and NTBI, where ZIP8 and ZIP14 are both involved in NTBI uptake, but ZIP14, not ZIP8, mediates TBI-derived iron uptake. This knowledge provides more insights in the mechanisms of renal iron handling and suggests that ZIP8 and ZIP14 could be potential targets for limiting renal iron reabsorption and enhancing urinary iron excretion in systemic iron overload disorders.

Intronic SNP rs3811647 of the human transferrin gene modulates its expression in hepatoma cells.

INTRODUCTION: Transferrin (Tf) exerts a crucial function in the maintenance of systemic iron homeostasis. The expression of the Tf gene is controlled by transcriptional mechanism, although little is known about genetic factors influence. OBJECTIVE: To study the role of rs3811647 in Tf expression using an in-vitro assay on hepatoma cells. DESIGN AND METHODS: Hep3B cells were co-transfected with constructs containing A (VarA-Tf-luc) and G (VarG-Tf-luc) variants of rs3811647, using luciferase as a surrogate reporter of Tf expression. RESULTS: Luciferase assays showed a higher intrinsic enhancer activity (p < 0.05) in the A compared with the G variant. In silico analysis of SNP rs3811647 showed that the A allele might constitute a binding site for the transcription factor glucocorticoid receptor (GR). CONCLUSION: The A allele of SNP rs3811647 increases Tf expression in a manner that might underlie inter-individual variation in serum transferrin levels observed in different population groups.

Secondary structure of the MiRP1 (KCNE2) potassium channel ancillary subunit.

MiRP1 (encoded by the KCNE2 gene) is one of a family of five single transmembrane domain voltage-gated potassium (Kv) channel ancillary subunits currently under intense scrutiny to establish their position in channel complexes and elucidate alpha subunit contact points, but its structure is unknown. MiRP1 mutations are associated with inherited and acquired cardiac arrhythmia. Here, synthetic peptides corresponding to human MiRP1 (full-length and separate domains) were structurally analyzed using FTIR and CD spectroscopy. The N-terminal (extracellular) domain was soluble and predominantly non-ordered in aqueous media, but predominantly alpha-helical in L-alpha-lysophosphatidylcholine (LPC) micelles. The MiRP1 transmembrane domain was predominantly a mixture of alpha-helix and non-ordered structure in LPC micelles, with a minor contribution from non-aggregated beta-strand. The intracellular C-terminal domain was insoluble in aqueous solution; reconstitution into non-aqueous environments resulted in solubility and adoption of increasing amounts of alpha-helix, with the solvent order sodium dodecyl sulphate < dimyristoyl L-alpha-phosphatidylcholine (DMPC) < LPC < trifluoroethanol. Correlation of secondary structure changes with lipid transition temperature during heating suggested that the MiRP1 C-terminus incorporates into DMPC bilayers. Full-length MiRP1 was soluble in SDS micelles and calculated to contain 34% alpha-helix, 23% beta-strand and 43% non-ordered structure in this environment, as determined by CD spectroscopy. Thus, MiRP1 is highly dependent upon hydrophobic interaction via lipid and/or protein contacts for adoption of ordered structure without nonspecific aggregation, consistent with a role as a membrane-spanning subunit within Kv channel complexes. These data will provide a structural framework for ongoing mutagenesis-based in situ structure-function studies of MiRP1 and its relatives.

Altered ATP-sensitive P2 receptor subtype expression in the Han:SPRD cy/+ rat, a model of autosomal dominant polycystic kidney disease.

The effects of extracellular ATP on fluid secretion and reabsorption by renal epithelial cells, as well as its known effects on cell proliferation and death, are potentially important contributory factors in the development and growth of renal cysts. In this study, we have investigated the protein and mRNA expression of several P2Y receptor subtypes (P2Y(1,2,4,6)), as well as the P2X(5) and P2X(7) receptors, in kidney tissue from the Han:SPRD (cy/+) rat model of polycystic kidney disease. All of the P2Y receptors tested for, and the P2X(5) and P2X(7) subtypes, were located on the cyst-lining cells of Han:SPRD (cy/+) rat polycystic kidneys; most immunostaining was cytosolic and we could not confidently localize it to one or other membrane. However, the staining pattern for P2Y(6) was uniquely granular when compared with the other P2 receptors. P2Y(2) and P2Y(6) receptor mRNA was increased in both homozygote (cy/cy) and heterozygote (cy/+) rat kidneys when compared with unaffected littermates. The protein levels of P2Y(2) and P2Y(6) receptors were also increased, being undetectable or at a low level, respectively, in control tissue. Finally, P2X(7) receptor mRNA was increased in cy/+, but not in cy/cy rat kidneys. Our results show that a number of P2Y receptor subtypes, as well as the P2X(5) and P2X(7) receptors, are clearly expressed in cyst-lining cells in the Han:SPRD (cy/+) rat model of renal cystic disease. Furthermore, P2Y(2) and P2Y(6) receptor mRNA and protein levels are markedly increased in cystic rat kidneys compared with normal rats of the same genetic background. Thus, the most consistent findings were an increase in the expression of P2Y(2), P2Y(6) and P2X(7) receptors in cystic tissue. Given the widely reported effects of stimulating these P2 receptor subtypes in epithelial and other renal cells, they could contribute to the development and growth of renal cysts: extracellular ATP and its products 'trapped' in cyst fluid may activate P2 receptors expressed by cyst-lining cells, causing cyst expansion from increased fluid secretion and/or reduced reabsorption, as well as an increase in cell turnover (re-modeling).

Duodenal mucosal reductase in wild-type and Hfe knockout mice on iron adequate, iron deficient, and iron rich feeding.

BACKGROUND: Genetic haemochromatosis is a common hereditary iron loading disorder in humans. The disease is associated with loss of function mutations in the HFE gene. This is thought to change iron stores via increased iron absorption. AIMS: In this study we investigated how adaptation of mucosal reductase activity is engaged in this process and how the changes compare with adaptation seen when an iron deficient diet is fed. METHODS: Duodenal mucosal surface reductase was measured with nitroblue tetrazolium in age matched groups of male Hfe knockout mice (Hfe) and wild- type mice fed a purified diet containing normal (iron adequate), high (iron rich), or low (iron deficient) iron concentrations. RESULTS: Reductase activity increased when mice were fed an iron deficient diet and decreased when they were fed an iron rich diet. Total villus activity, as measured by the average area under the activity curve along the crypt-villus axis, was increased 2.8-2.9-fold by iron deficiency in both genotypes. Approximately half of this difference was attributable to the significantly increased length of the villi in mice on an iron deficient diet (p<0.05). Hfe knockout did not affect villus length but increased mucosal reductase activity near the villus tips. Similar increases (1.3-1.6-fold) were seen on all diets but the increase was significant for iron deficient and iron loaded diets only (p<0.05). CONCLUSION: Hfe gene product and dietary iron downregulate villus reductase activity in mice.

Iron transport across cell membranes: molecular understanding of duodenal and placental iron uptake.

Iron is an essential element playing a vital role in many cellular processes. This requirement is complicated by the fact that environmental iron is invariably present as insoluble Fe(3+) leading to poor bioavailability and toxicity, since even low concentrations of iron catalyse the production of damaging reactive oxygen species. As a result organisms have evolved efficient uptake and transport systems to extract iron from their environment as well as ferritins that store iron in a non-toxic form. In higher organisms, the first membrane barrier encountered is the apical surface of the duodenal enterocyte, a specialized absorptive cell of the intestinal epithelium that undertakes vectorial transport of iron. Iron is initially solubilized by reduction and Fe(2+) is transported across the cell membrane by a carrier-mediated transport process. This is followed by intracellular transfer of iron to the basolateral enterocyte membrane with subsequent transfer and release of iron to transferrin in the portal blood. A second site of iron transport is at the placento-fetal barrier where similar principles operate. In this review we describe recently identified transmembrane transporters and associated accessory proteins responsible for iron transport at these two sites.

Long-term sequelae of HFE deletion in C57BL/6 x 129/O1a mice, an animal model for hereditary haemochromatosis.

BACKGROUND: HFE knockout mice (C57BL/6 x 129/Ola strain) mimic the functional aberrations of human hereditary haemochromatosis (HH) in short-term experiments. The present study investigates functional and morphological long-term changes. METHODS: HFE(o/o), HFE(+/o) and HFE(+/+) mice were maintained on iron-rich and control diets for 2 weeks, 3, 12 and 18 months. Light microscopic tissue iron distribution, pathomorphological alterations, tissue iron content and oxidative stress were analysed in liver, pancreas, spleen, gastrointestinal tract, kidneys and myocardium. Additionally, duodenal 59Fe absorption and 59Fe whole body loss were measured. RESULTS: Iron distribution between organs and microscopic iron deposition in the tissues resembled the patterns described in HH. After 3 months of iron-rich feeding duodenal 59Fe absorption decreased to approximately 15% of iron-adequate controls but remained about twice as high in HFE(o/o) as in HFE(+/+) mice. Hepatic iron concentrations reached only half the values known to induce hepatic fibrosis in rats and humans, while whole body 59Fe loss was about twice as high. Consequently no hepatic fibrosis developed, although massive hepatocellular iron deposition and indication for oxidative stress were observed. CONCLUSION: C57BL/6 x 129/O1a HFE(o/o) mice mimic HH iron distribution and the regulation of intestinal iron absorption after long-term feeding. However, characteristic morphological late changes in untreated HH are not modelled.