Fig 1.
PIO did not cause morphologic kidney damage, but affected the spleen.
Fixed kidney and spleen sections from control and iron-loaded mice were histologically stained (H&E) and imaged. No damage was observed in kidney sections of iron-loaded mice (B and D) compared to the control sections (A and C). However, iron accumulation caused damage to the spleen as can be evaluated by moderate degree of hemosiderosis (seen as brownish pigment in the red pulp of section F compared to E), and by a mild depletion of lymphocytes in the white pulp (indicated by red arrowheads); the inserts in the left corner of E and F are higher magnifications of these sections. Scale bar 100 μm, n = 3 for control and iron-loaded mice each.
Fig 2.
Cubilin expression in iron loaded kidneys is elevated both in the cortex and the medulla.
The fixed kidney sections were incubated with cubilin or TfR1 antibodies and stained by IF or IHC. (A-F) Cubilin was observed apically in the cortex, as expected, with increased staining in kidneys from iron overloaded mice (compare A to D). Interestingly, in PIO, significant cubilin expression was also observed in the medulla (compare B to E, and C to F). Inserts are negative controls (N.C) for cubilin staining; (G-I) Visualization of TfR1 by IHC (G-H) and by IF (I). (G) TfR1 staining was seen mainly in the medulla, (the dashed line separates the cortex from the medulla. In the medulla, TfR1 was seen apically (H-I) (indicated by arrows). Inserts are negative controls (N.C) for TfR1 staining; scale bar of 50μm, n = 3 for control and iron-loaded mice each.
Fig 3.
Cubilin and Tfr1 are regulated by iron in opposite directions.
TfR1 (A) and cubilin (B) protein levels were tested by Western-blot analysis of kidney lysates. Membranes were probed with either cubilin, TfR1 or actin antibodies. TfR1 levels in iron over-loaded mice were decreased. Cubilin levels were increased in kidneys from iron–loaded mice. (C, D and E) Kidney mRNA levels of TfR1, cubilin and megalin were evaluated by qPCR. (C) TfR1 mRNA levels correlated with protein levels (n = 5, *P<0.05), (D) no significant difference in cubilin mRNA levels was observed (n = 6). (E) Megalin mRNA expression increased in iron overloaded kidneys (n = 7, **p<0.005).
Fig 4.
Ferritin distribution in renal epithelial cells is regulated by the iron status: Kidney sections from control, iron-loaded and Irp2-/- mice were stained with H-ferritin antibody.
Ferritin was apically polarized in kidneys from Irp2-/- mice and to some extent also in wild-type control mice (arrow heads). In contrast, in iron overloaded mice ferritin was distributed throughout the cells and also found in basolateral regions (arrows). Scale bar represents 50μm.
Fig 5.
FPN levels are not reduced in kidneys of iron overloaded mice.
Spleens and kidneys were lysed in 1% triton buffer and 40μg protein was submitted to Western blot analysis following protein separation by 10% SDS-PAGE. Proteins were electro-transferred to a nitrocellulose membrane, which was incubated with antibodies against FPN or actin. FPN was detected as a 60kDa band.
Fig 6.
Iron accumulates in the kidney-interstitium of PIO mice.
Fixed kidney sections from PIO mice (A-B and D-F) and from dietary iron overloaded mice (C) were stained and imaged. (A-C) Ferric iron was visualized with Prussian blue staining in cortex and medulla, respectively. In PIO mice, most iron accumulated in and around glomeruli and in the interstitium, and markedly little iron was detected in renal epithelial cells. In contrast, following dietary iron overload, most iron accumulated in proximal tubule epithelium of the cortex. (D) Light microscope; the black square indicates a sub-region of the medulla, which is enlarged in (E and F). (E) AirSEM analysis of the sub-region followed by EDX [Fe] mapping (F) indicated iron accumulation (marked with arrows) in the interstitium of the medulla. (G) Regions of interest in tubules and interstitium of cortex and medulla were selected and iron levels were quantified using airSEM. At least 2-fold increase of interstitial iron in the medulla compared to cortex was measured. ** P< 0.0001.
Fig 7.
Ferritin accumulated in medullary interstitial macrophages: Kidney sections from PIO mice were co-stained with anti-CD-68 and L-ferritin antibodies.
Co-localization can be observed as yellow regions in the right panel. Scale bar represents 50μm.