title: Iron-dependent turnover of IRP-1/c-aconitase in kidney cells.
abstract: The kidney plays an important role in iron homeostasis and actively reabsorbs citrate. The bifunctional iron-regulatory protein IRP-1 potentially regulates iron trafficking and participates in citrate metabolism as a cytosolic (c-) aconitase. We investigated the role of cellular iron status in determining the expression and dynamics of IRP-1 in two renal cell types, with the aim of identifying a role of the protein in cellular ROS levels, citrate metabolism and glutamate production. The effects of iron supplementation and chelation on IRP-1 protein and mRNA levels and protein turnover were compared in cultured primary rat mesangial cells and a porcine renal tubule cell line (LLC-PK1). Levels of ROS were measured in both cell types, and c-aconitase activity, glutamate, and glutathione were measured in LLC-PK1 cells, with and without IRP-1 silencing and in glutamine-supplemented or nominally glutamine-free medium. Iron supplementation decreased IRP-1 levels (e.g., approx. 40% in mesangial cells treated with 10 μg ml(-1) iron for 16 h) and increased ubiquitinated IRP-1 levels in both cells types, with iron chelation having the opposite effect. Although iron increased ROS levels (three-fold with 20 μg ml(-1) iron in mesangial cells and more modestly by about 30% with 50 μg ml(-1) in LLC-PK1 cells, both after 24 h), protein degradation was not ROS-dependent. In LLC-PK1 cells, 10 μg ml(-1) iron (24 h) increased both aconitase activity (30%) and secreted glutamate levels (65%). Silencing did not remove the glutamate response to iron but decreased the c-aconitase activity of the residual protein independent of iron loading (37% and 46% of control levels, without and with iron treatment, respectively). However, in glutamine-free medium, glutamate was still increased by iron, even in IRP-1-silenced cells, and did not correspond to c-aconitase. Silencing decreased the amount of ferritin measured in response to iron loading, decreased the affect of iron on total glutathione by 48%, and increased the response of ROS to iron loading by 38%. We conclude that iron increases turnover of IRP-1 in kidney cells, while increasing aconitase activity, suggesting that the apoprotein (aconitase-inactive) form is not exclusively responsible for turnover. Iron increases glutamate levels in tubule epithelial cells, but this appears to be independent of c-aconitase activity or the availability of extracellular glutamine. IRP-1 protein levels are not regulated by ROS, but IRP-1-dependent ferritin expression may decrease ROS and increase total glutathione levels, suggesting that ferritin levels are more important than citrate metabolism in protecting renal cells against iron.
authors: University of Toronto, Laboratory Medicine and Pathobiology, 1 King's College Circle, Toronto, Ont. M5S 1A8, Canada. doug.templeton@utoronto.ca.
authors: Ying
authors: Y
authors: Liu
authors: Douglas M
authors: DM
authors: Templeton
mesh_terms: D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D020868:Gene Silencing; D018698:Glutamic Acid / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D035941:Iron Regulatory Protein 1 / Q000235:genetics / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D018374:LLC-PK1 Cells; D059748:Proteolysis; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D013552:Swine
chemical_list: D017382:Reactive Oxygen Species; D018698:Glutamic Acid; D007501:Iron; D035941:Iron Regulatory Protein 1; D005978:Glutathione