Initial stages of renal development were preserved in TfR1^−/− mice. (A) Pax2⁺ Wolffian ducts and mesonephros and (B) Pax2⁺ ureteric buds surrounded by mesenchymal cells were detected in TfR1^−/− E11 embryos. Crosses with Hoxβ7-GFP mice distinguished the ureteric bud (GFP⁺Pax-2⁺) from mesenchyme (GFP⁻Pax-2⁺). Bar=10 μm.

Ferritin capture, cell proliferation and Scara5 expression in the capsule of the E15 kidney. (A, B) Both wild type and GFP-TfR1^−/− capsular cells captured rhodamine coupled “Exogenous L-Ferritin”. Bars=20 μm. (C) Holoferritin (+HF; 1.25 μg/ml) induced phosphohistone⁺ nuclei and expanded layers of capsular cells, (D) whereas culture with apoferritin (−HF; 1.25 μg/ml) failed to preserve the capsule. (E) “Endogenous Ferritin” was present throughout the kidney, particularly in the capsule. E-cadherin marked the UB. (F) Scara5 RNA was expressed by spindle-shaped cells encapsulating the kidney (arrowheads) and more faintly by cortical stroma.

Interaction between Scara5 and L-ferritin. (A) Transient transfection of Scara5-V5 (anti-V5, green) and (B) stable expression of Scara5-V5 resulted in the capture of Alexa568-ferritin (A, B right panel) whereas parental Trvb, which are Scara5⁻TfR1⁻ cells, transfected with an empty vector were negative (B left panel). Tris-quenched Alexa568 dye (no ferritin) was an additional control (middle panel). (C) Expression of Scara5-V5 (59KDa, 70KDa) was detected by V5 monoclonal antibody. (D) Endocytosis of Alexa568-ferritin was analyzed in Scara5⁻ (Trvb, B2 clones) and Scara5⁺ (D2 and H2) stable clones. Nearly all D2 and H2 cells captured apo- and L-ferritins, but not H-ferritin. Bar=10 μm.

Scara5 mediates cellular uptake of ferritin-bound iron and represents an endogenous pathway in MSC-1 cells. (A) Capture of ⁵⁹Fe-ferritin by Scara5⁺ at 37°C (red line) but not at 4°C (black line) nor by parental Trvb cells (blue line). (B) Lysosomes of Scara5⁺ Trvb cells were marked with fluorescein-dextran (green). They contained endocytosed Alexa568-ferritin (red). Original magnification 40X. (C) Transfer of iron to the cytoplasm was detected using two IRE-based iron reporters and FACS analysis in MSC-1 cells. The maximal 5′ IRE-CFP signal (designated 100%-black bars) was generated by iron loading with holotransferrin (20 μg/ml) and ferric ammonium citrate (20 μM) (HT+Fe) and the maximal 3′ IRE-YFP signal (designated 100%-stippled bars) was generated by DFO (20 μM). These treatments produced a ~7-fold range in the ratio of 5′/3′ IRE signals. Treatment with holoferritin (HF) increased 5′ IRE-CFP but decreased the 3′ IRE-YFP signal. (D) Holoferritin induces cell growth. Scara5⁺ clone H responded to holo- but not to apo-ferritin, whereas Trvb cells failed to respond. Cell protein was detected by GAPDH immunoblots. (E) Alexa488-ferritin endocytosis was blocked by Poly I >Poly G >Poly C (50 μM). Similar data were obtained in Scara5⁺ Trvb and MSC1 cells. Bar=20 μM.

Development of GFP-TfR1^−/− cells in E15 kidney. (A) GFP-TfR1^−/− cells were readily detected in capsule and stroma, mesenchyme, renal vesicles, and S-shaped bodies, but detected with limited frequency in UB tips (bar=10 μm). (B) GFP-TfR1^−/− cells (42%) reside in the capsule, interstitium, and podocalyxin⁺ vascular progenitors (confocal section; bar=10 μm). (C, D) GFP-TfR1^−/− cells (58%) also resided in proximal and distal nephrons (Ecadherin⁺; dark blue), in podocytes (podocalyxin⁺; red, Glm=glomerulus) and in the UB stalk (Ecadherin⁺, dark blue; Troma⁺, pink). In some cases, entire segments of the S-shaped body were composed of GFP-TfR1^−/− cells (confocal section; bar=10 μm). (E, F) Genes specific for UB tip or mesenchymal-stromal compartments were assayed in GFP-TfR1^−/− and wild type cells and presented as the log₂ ratio of microarray values. UB tip specific genes were under represented in GFP-TfR1^−/− cells, whereas mesenchymal-stromal genes were expressed. (G) Both GFP-TfR1^−/− and wild type cells (two sections are shown) contained cytoplasmic ferritin. (H) Wild type cells captured Alexa568-transferrin, but neighboring GFP-TfR1^−/− were unlabeled. Bar=10 μm.

Localization of the transferrin pathway. (A, B, C) TfR1 was initially localized to UB tips and cap mesenchyme. By E13, renal vesicles also expressed TfR1 (*B, C). TfR1 expression was reduced or absent in glomeruli (“G”), stroma and capsule. (D) Rhodamine- and (E) fluorescein-transferrin endocytosis paralleled TfR1 expression. Stroma and capsule were weakly labeled despite active fluid-phase endocytosis (rhodamine-dextran; E). (*Presumptive distal nephron; Bar= 10 μm).

Interaction between Scara5 and ferritin at the cell surface. (A) Scara5-V5 (anti-V5; Cy2; green) and ferritin (anti-ferritin; Cy3; red) co-localized at the surface (Z-stack; inset: single confocal section) of Scara5-V5 transfected MSC-1 cells incubated with untagged ferritin (2hrs; 4°C). Toto3 marked nuclei. Bar=10 μm. (B) Endocytosis of rhodamine-ferritin (30hrs; 37°C; top panel) was blocked by a 100 fold excess untagged ferritin (bottom panel), Bar=20 μm. (C) V5 antibodies precipitated Scara5-V5 with avidin tetramer-ferritin monomer (85KDa) or multimers (200KDa, >250KDa), but not with unconjugated avidin. Irrelevant isotype matched antibodies did not immunoprecipitate ferritin-avidin nor were ferritin-avidin complexes recovered without cell extracts. (D) Scara5-V5 was detected in cell extracts (clone D2) and in pull-downs with ferritin-avidin-biotin-agarose beads but not with avidin-biotin-agarose-beads.