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

Abstract

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.

Keywords: Iron; Non-transferrin-bound iron; Proximal tubular epithelial cell; Transferrin; ZIP14; ZIP8.

Fig. 1

Uptake of non-transferrin-bound iron and transferrin-bound iron in ciPTECs. Intracellular iron concentration (a), total ferritin and transferrin receptor 1 (TfR1) protein levels (b) in ciPTECs after incubation with different concentrations of ferric citrate (NTBI). Zona occludens 1 (ZO-1) immunostaining (in green) confirming monolayer integrity in polarized ciPTECs. ZO-1 tight junctions indicated by arrows (c). ⁵⁵Fe content in apical compartment, basolateral compartment and cell lysate after ⁵⁵Fe exposure from the apical (A to BL) or basolateral (BL to A) cellular side (d). Alexa546-Transferrin (546Tf, in red) internalization alone, combined with holo-transferrin (Holo-Tf) or at 4 °C (e). 546Tf internalization in iron deplete or iron replete conditions, from apical or basolateral cellular side (f). Nuclei counterstained with DAPI (in blue). Representative images and graphs showing mean of three independent experiments for each time point or FeC concentration except n = 6 for panel c. Scale bar 5 µM. One-way ANOVA with Dunnett’s post test compared to control at each time point was used in a; *p < 0.05; **p < 0.01; ***p < 0.001. (Color figure online)

Fig. 2

Ferroportin-mediated iron export in ciPTECs. Ferroportin immunostaining (in green) in polarized ciPTECs (a). Nuclei counterstained with DAPI (in blue). Confocal images taken in x–y and y–z axis showing apical (A) and basolateral (BL) cellular side. CiPTEC ferroportin mRNA expression (b), pmol ⁵⁵Fe in cell lysate or solution (c), total ferritin protein expression and transferrin receptor 1 (TfR1) mRNA expression (d) after transfection with scrambled control (Scr) or ferroportin (FPN1) small interfering RNAs (siRNA). Representative images and graphs showing mean of at least three independent experiments (a, n = 3; b, n = 9; c, n = 4; d, both n = 3). Scale bar 5 µM. Student’s t-test was used in b, c and d; *p < 0.05; **p < 0.01. (Color figure online)

Fig. 3

Presence of ZIP8, ZIP14 and DMT1 in ciPTECs. ZIP8, ZIP14 and divalent metal transporter 1 (DMT1) immunostaining (in green) in polarized ciPTECs. Nuclei counterstained with DAPI (in blue). Confocal images taken in x–y and y–z axis showing apical (A) and basolateral (BL) cellular side (a). Cell surface biotinylation and immunoblotting of ZIP8, ZIP14 and DMT1 in both membrane fraction and total lysate fraction. In addition, Na K ATPase was used as positive control for cellular membrane proteins, early endosome antigen 1 (EEA1) as negative control for endosomal proteins and β-actin as negative control for cytosolic proteins. Similar Na K ATPase protein levels were loaded in both membrane fraction and total lysate fraction. Prolonged chemiluminescence confirmed depicted findings (data not shown) (b). Representative images showing three independent experiments. Scale bar 5 µM. (Color figure online)

Fig. 4

ZIP8 and ZIP14 mediate NTBI uptake in ciPTECs. ZIP8 and ZIP14 mRNA and protein expression after transfection with scrambled control (Scr) and either ZIP8 (a) or ZIP14 small interfering RNA (siRNA) (b). Pmol ⁵⁵Fe uptake in cell lysate after either ZIP8 (c) or ZIP14 siRNA transfection (d). ZIP8 and ZIP14 mRNA and protein expression (e) and pmol ⁵⁵Fe uptake in cell lysate (f) after transfection with scrambled control or combined ZIP8 + ZIP14 siRNAs. Representative graphs. Number of experiments depicted in each panel. Student’s t-test was used in a–e; *p < 0.05; ***p < 0.001

Fig. 5

Transferrin receptor 1 mediates Alexa546-Transferrin uptake in ciPTECs. Transferrin receptor 1 (TfR1) immunostaining (in green) in polarized ciPTECs (a). TfR1 immunostaining (in green) colocalization with Alexa546-Transferrin (546Tf) internalization (in red) (b) or early endosome antigen 1 (EEA1) immunostaining (in red) (c). Nuclei counterstained with DAPI (in blue). Confocal images taken in x–y and y–z axis showing apical (A) and basolateral (BL) cellular side. Representative images of three experiments. Scale bar 5 µM. (Color figure online)

Fig. 6

ZIP14 mediates Alexa546-Transferrin uptake in ciPTECs. Double immunostaining of ZIP8 or ZIP14 (in green) and early endosome antigen 1 (EEA1, in red) in unstimulated conditions (a) or after 48 h iron overload exposure (b). ZIP14 immunostaining (in green) colocalization with Alexa546-Transferrin (546Tf) internalization (in red) (c). 546Tf internalization (d) or pmol ⁵⁵Fe in cell lysate after ⁵⁵Fe-TBI exposure (e) after transfection with scrambled control or ZIP14 small interfering RNA (siRNA). Double immunostaining of divalent metal transporter 1 (DMT1, in green) and EEA1 (in red) (f). DMT1 mRNA expression (g) after transfection with ZIP14 or scrambled control siRNAs. Nuclei counterstained with DAPI (in blue). Confocal images show x–y and y–z axis. Representative images and graphs of three experiments. Scale bar 5 µM. Student’s t-test was used in e, g; *p < 0.05. (Color figure online)

Fig. 7

Proposed NTBI and TBI handling in proximal tubular epithelial cells. Non-transferrin-bound iron (NTBI) and transferrin-bound iron (TBI) present in the systemic circulation are filtered by the glomerulus into the tubular lumen and are subsequently reabsorbed by proximal tubular cells. NTBI uptake at the plasma membrane involves both ZIP8 and ZIP14 that show redundancy. TBI uptake involves TfR1-mediated endocytosis. Subsequently, iron is transported from the endosome towards the cytosol via ZIP14 and potentially also divalent metal transporter 1 (DMT1). Once in the cytosol, in the labile iron pool, iron is utilized, stored in ferritin or exported back into the circulation by ferroportin at the basolateral membrane. (Color figure online)