Fig 1.
Expression pattern of Kif5b in newborn mouse kidney.
(A) Overall view of Kif5b expression in the kidney: strong expression in tubular structures in both cortex and medulla, and low levels of expression in the nephrogenic zone. (B) In the nephrogenic zone, Kif5b was expressed in the ureteric bud, but exhibited a very low expression in the cap mesenchyme, comma-shaped body, S-shaped body and advanced S-shaped body. (C, D) Kif5b was widely expressed in the renal tubules of the cortex and the medulla, but expression in glomeruli and in interstitial cells was quite rare. G, glomerulus; PCT, proximal convoluted tubules; TAL, thick ascending limbs of loops of Henle; DCT, distal convoluted tubule; CD, collecting duct. Images are representative of kidney tissue sections from five mice. Scale bar: (A) 500 μm, (B-D) 50 μm.
Fig 2.
Embryonic developmental expression of Kif5b in mouse kidney.
(A-D) Immunolocalization of Kif5b protein in the fetal mouse kidney at embryonic days (E) 12.5, 16.5. (A, B) Kif5b was not detectable in the E12.5 fetal kidney. (C, D) At E16.5, Kif5b was highly expressed in the ureteric bud (UB) and tubules (T). Images are representative of kidney tissue sections from three mice. (E) Quantitative real-time RT-PCR analysis showing the expression of Kif5 members: Kif5a, Kif5b and Kif5c mRNAs during kidney development. Br E18.5, brain of E18.5 embryo; KY E12.5, kidney of E12.5 embryo. mRNAs of brain tissues from E18.5 embryos were used as positive controls in the real-time PCR analysis. Bars represent the means ± S.D. (n = 3). One-way ANOVA was performed to compare specific gene expression levels in the kidneys between different stages. * P<0.05 versus KY E12.5. Scale bar = 25 μm.
Fig 3.
Spatiotemporal expression pattern of Kif5b in mouse kidney.
(A, B) Kif5b was widely distributed inside renal tubular epithelial cells of newborn mice. (C-H) Kif5b was gradually restricted to epithelial cells in DCT and TAL during postnatal kidney development. (I) Quantitative analysis of the relative optical density values for cells in renal tubules. Kidney sections were analyzed using ImageJ software to determine the mean optical density values per cell of positive-labeled tubules in the kidney. The relative mean optical densities of cells in the tubular structures compared with that in the DCT were calculated. Bars represent the means ± S.D. (n = 10 for each tubular structure from three mice were measured and analyzed). One-way ANOVA was performed to compare relative intensity levels of different tubular structures at indicated stages. * P<0.05 versus DCT. (J) Kif5b was evenly distributed in the cytoplasm of renal epithelial cells in newborn mice. (K) Kif5b was asymmetrically distributed in the basolateral domain in renal tubular cells of adult mice. Lu, lumen; PCT, proximal convoluted tubules; TAL, thick ascending limbs of loops of Henle; DCT, distal convoluted tubule; CD, collecting duct. Images are representative of kidney tissue sections from three mice. Scale bar: (A-H) 50 μm, (I, J) 10 μm.
Fig 4.
Immunolocalization of Kif5b in mouse kidney PT (A-C), TAL (D-F), CD (G-I) and DCT (J-K), using antibodies against specific markers for each segment.
Kif5b was expressed in TAL (THP-positive tubules) and DCT (NCC-positive tubules), but not in PT (LTL-positive tubules) or CD (AQP2-positive tubules). (L) The diagram shows the selective and asymmetric expression pattern of Kif5b in renal tubules of adult mice. Red, Kif5b; orange, LTL; yellow, THP; green, NCC; light blue, AQP2; blue, nucleus. Only cells from one side of the tubules are presented and the other side is represented by a dotted line. Arrows represent the direction of urinary flow. Cortex and medulla are separated by a dashed line. G, glomerulus; PT, proximal tubule; TDL, thick descending limbs of Henle; TS, thin segment; TAL, thick ascending limbs of Henle; DCT: distal convoluted tubule; CD, collecting duct. Images are representative of kidney tissue sections from three mice. Scale bar = 50 μm.
Fig 5.
Immunolocalization of Na+/K+-ATPase in mouse kidney PT (A-C), TAL (D-F), DCT (G-I) and CD (J-L) using antibodies against specific markers for each segment.
Kif5b was expressed in TAL (THP-positive tubules) and DCT (NCC-positive tubules), but not in PT (LTL-positive tubules) or CD (DBA-positive tubules). (M-O) Co-localization of Kif5b and Na+/K+-ATPase in renal tubules of adult mice. PT, proximal tubule; TAL, thick ascending limbs of Henle; DCT: distal convoluted tubule; CD, collecting duct. Images are representative of kidney tissue sections from three mice. Scale bar = 50 μm.
Fig 6.
Kidneys from Kif5b-Pax2KD (Kif5bfl/-:Pax2-Cre) embryos show normal gross histological morphology.
(A) Anti-β-galactosidase immunostaining of E19.5 R26R+/+:Pax2-Cre mouse kidney. (B) Western blot analysis of Kif5b levels in kidneys from E19.5 mutant (Kif5bfl/-:Pax2-Cre), heterozygous (Kif5bfl/- and Kif5bfl/+:Pax2-Cre) and wild-type (Kif5bfl/+) embryos. Actin was used as a loading control. (C-D) Immunohistochemical analysis of Kif5b expression patterns/levels in kidneys from E19.5 control (Kif5bfl/+) and mutant (Kif5bfl/-:Pax2-Cre) embryos. (E-H) H&E staining showing that the organization of the nephrogenic zone, cortex and medulla is similar between control (Kif5bfl/+) and mutant (Kif5bfl/-:Pax2-Cre) kidneys. G, glomerulus; NZ, nephrogenic zone. Images are representative of kidney tissue sections from ten E19.5 embryos. Scale bar = 50 μm.
Fig 7.
Kidneys from Kif5b-Pax2KD (Kif5bfl/-:Pax2-Cre) embryos have a reduced cell proliferation rate.
(A) In situ analysis of BrdU incorporation at E18.5. Pregnant mice were injected with BrdU and sacrificed 4 hours later. BrdU incorporation was detected by an in situ BrdU incorporation assay kit. Brown-stained nuclei are BrdU-positive. Tissues were counterstained with hematoxylin. Images are representative of kidney tissue sections from three embryos. Arrows, BrdU positive cells. Scale bar = 100 μm. (B) Quantitative analysis of BrdU incorporation at E18.5. BrdU incorporation is expressed as the percentage of the total number of cells in the ureteric bud and its branches (UB), in the metanephric mesenchyme (MM) or in the interstitial cells (IC). Kif5b deficiency significantly decreased BrdU incorporation in both ureteric bud and mesenchyme-derived epithelial structures, compared to wild type. Bars represent the means ± S.D. Six sections from three wild type and three mutant embryos were imaged and counted. P values are indicated in the figure. * P<0.05.
Fig 8.
Kif5b deficient kidneys (Kif5bfl/-:Pax2-Cre) show normal localization of apical markers.
Co-immunostaining of Kif5b (red) and apical markers LTL, THP, NCC and DBA (green) in kidney sections of E18.5 embryos. Images are representative of kidney tissue sections from three mice. Scale bar = 25 μm.
Fig 9.
Mislocalization of Na+/K+-ATPase in Kif5b knock-down kidneys.
Na+/K+-ATPase was basolaterally localized in renal epithelial cells of control E18.5 embryos (Kif5bfl/+), but it was mislocalized to the apical surface in renal epithelial cells of mutant E18.5 embryos (Kif5bfl/-:Pax2-Cre). Images are representative of kidney tissue sections from three mice. Green, Kif5b; Red, Na+/K+-ATPase.