UB branching and patency in Cited1^C57Bl/6 null mice. (A and B) Whole-mount preparations of cultured metanephroi isolated from two E12.5 wild-type (A) and two Cited1^C57Bl/6 null (B) embryos, cultured for 3 d and stained using anti–Calbindin D28K antibodies (a UB marker). (C and D) Calbindin D28K staining of E15.5 kidneys from two wild-type (C) and two Cited1^C57Bl/6 null (D) embryos. Dotted blue lines in C illustrate outer nephrogenic zone areas used to count Calbindin D28K–positive UB tips. (E and F) Sections through the renal pelvis of E16.5 kidneys from wild-type (E) and Cited1^C57Bl/6 null (F) mice stained for α-smooth muscle actin. (G and H) Representative images illustrating methylene blue dye passage down the ureters of E18.5 wild-type (G) and Cited1^C57Bl/6 (H) null mice after injection into the renal pelvis. Images obtained using a dissecting microscope. Magnification, ×100 in A through F; ×10 in G and H.

Renal patterning defects in female Cited1^C57Bl/6 heterozygotes. Hematoxylin- and eosin-stained sections through the renal pelvis of E18.5 embryonic kidneys from female Cited1^C57Bl/6 mutants. Female Cited1^C57Bl/6 heterozygotes with P+/− (with placental insufficiency) but not M+/− (without placental insufficiency) wild-type X chromosomes have abnormal organization and expansion of the renal medulla into the pelvis (arrows). Wild-type female littermate kidneys are shown from comparison. Magnification, ×100.

Renal hypoxia in Cited1^C57Bl/6 mutant mice with placental insufficiency. (A through C) Representative immunohistochemical staining for pimonidazole adducts in transverse sections through E17.5 kidneys from Cited1^C57Bl/6 null mice (−/−) and Cited1^P+/− female heterozygotes with placental insufficiency (P+/−), compared with wild-type female (+/+) mice derived from the same litter. Tissue preparation and immunohistochemical staining were performed simultaneously under identical conditions on all three sections. Arrows indicate staining for pimonidazole adducts in inner and outer medullas. (D) Immunoblot for pimonidazole adducts in E17.5 kidney lysates obtained from wild-type embryos with or without maternal pimonidazole 2 h before killing, as indicated. Lanes 1 and 3, 10 mg of protein; lane 2, 5 mg of protein. (E) Representative Western blot illustrating changes in density of pimonidazole adduct protein bands in E17.5 kidney lysates obtained from wild-type and Cited1^C57Bl/6 null embryos derived from the same litter. Arrows indicate the distinctive approximately 80-kD band that is used for quantitative densitometry. β-Actin control shown. (F) Semiquantitative analysis of E17.5 kidney pimonidazole adduct immunoblots from wild type (WT), Cited1^C57Bl/6 null, and Cited1^C57Bl/6 heterozygotes with M+/− and P+/− wild-type X chromosomes. Densitometry of conserved 80-kD pimonidazole adduct band corrected for β-actin loading. Each litter (n = 5) was evaluated on individual blots, and results were normalized to WT controls to correct for between-litter variations. Data are means ± SEM (mouse numbers). Kruskal-Wallis ANOVA, *P < 0.05 versus WT and Cited1^M+/− controls. Magnification, ×200.

Renal medullary dysplasia in E18.5 Cited1^C57Bl/6 null mice. (A through C) Cited1^C57Bl/6 null kidneys have renal medullary dysplasia associated with disorganized expansion of the medulla into the renal pelvis (arrows). (D through F) Wild-type kidneys are shown for comparison. Boxes in A and D indicate enlarged insets. (A′ and D′) Insets showing normal cortical thickness and nephronic patterning in Cited1^C57Bl/6 null and wild-type kidneys. (G) Frequency of renal medullary dysplasia in E18.5 kidneys from five Cited1^C57Bl/6 null and eight wild-type male littermates (kidney numbers). Magnification, ×100.

Renal function in adult Cited1^C57Bl/6 mutant mice. (A through C) GFR (measured by FITC-inulin clearance in μl/min per mouse; A), glomerular counts (direct counting of glomeruli after acid digestion of the kidneys; B), and urine-concentrating capacity (spot urine osmolality in mOsm/kg; C) were determined in adult male wild-type (WT) and Cited1^C57Bl/6 null mice. Samples were obtained from controls with free access to water, after 23 h of water deprivation and after intraperitoneal injection with 1 ng/g desmopressin intraperitoneally. Individual data points with mean vales shown. Kruskal-Wallis ANOVA, *P < 0.05 versus WT controls. No significant differences were detected between glomerular counts or GFR between genotypes.

Apoptosis in renal medullas of Cited1^C57Bl/6 mutant mice with placental insufficiency. (A) Representative images showing TUNEL staining for apoptotic nuclei in E17.5 embryonic kidneys from wild type (+/+) and Cited1 null (−/−) embryonic kidneys. (B) Graph indicates mean ± SEM apoptotic indices in embryonic kidneys isolated at E16.5, E17.5, and E18.5 from wild type and Cited1 null embryos (mouse numbers). Kruskal-Wallis ANOVA, *P < 0.05 versus wild-type controls. (C) Representative images showing TUNEL staining of E17.5 embryonic kidneys from wild-type, Cited1^C57Bl/6 null, and Cited1^C57Bl/6 heterozygotes with M+/− (no placental insufficiency) and P+/− (with placental insufficiency) wild-type X chromosomes. (D) Graph illustrates mean ± SEM apoptotic indices in the renal medullas of E17.5 kidneys from the same genotypes (mouse numbers). ANOVA, *P < 0.05 versus WT controls. Magnification, ×400.