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Comparative Study
. 2012 Apr;23(4):641-51.
doi: 10.1681/ASN.2011080829. Epub 2012 Jan 26.

Disruption of IFT Complex A Causes Cystic Kidneys Without Mitotic Spindle Misorientation

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Free PMC article
Comparative Study

Disruption of IFT Complex A Causes Cystic Kidneys Without Mitotic Spindle Misorientation

Julie A Jonassen et al. J Am Soc Nephrol. .
Free PMC article

Abstract

Intraflagellar transport (IFT) complexes A and B build and maintain primary cilia. In the mouse, kidney-specific or hypomorphic mutant alleles of IFT complex B genes cause polycystic kidneys, but the influence of IFT complex A proteins on renal development is not well understood. In the present study, we found that HoxB7-Cre-driven deletion of the complex A gene Ift140 from collecting ducts disrupted, but did not completely prevent, cilia assembly. Mutant kidneys developed collecting duct cysts by postnatal day 5, with rapid cystic expansion and renal dysfunction by day 15 and little remaining parenchymal tissue by day 20. In contrast to many models of polycystic kidney disease, precystic Ift140-deleted collecting ducts showed normal centrosomal positioning and no misorientation of the mitotic spindle axis, suggesting that disruption of oriented cell division is not a prerequisite to cyst formation in these kidneys. Precystic collecting ducts had an increased mitotic index, suggesting that cell proliferation may drive cyst expansion even with normal orientation of the mitotic spindle. In addition, we observed significant increases in expression of canonical Wnt pathway genes and mediators of Hedgehog and tissue fibrosis in highly cystic, but not precystic, kidneys. Taken together, these studies indicate that loss of Ift140 causes pronounced renal cystic disease and suggest that abnormalities in several different pathways may influence cyst progression.

Figures

Figure 1.
Figure 1.
HoxB7-Cre efficiently deletes the Ift140flox allele. (A) Diagram of targeting vector. Exons are displayed as boxes, whereas the coding region is shaded in black. frt, FlpE recombinase sites; loxP, Cre recombinase sites; neo, β-galactosidase-neomycin resistance gene fusion. (B) Affinity-purified anti-MmIFT140 detects a single band in protein extracts from the mouse cell lines 488 and IMCD3 but nothing in the human cell line hTert-RPE. This band is observed in extracts from mouse epithelial kidney (MEK) cells derived from a control kidney (+/+) but not from an experimental Ift140null2/null1 kidney. The MEK Western was also probed with a tubulin antibody (B-5-1-2) as a loading control. (C) In interphase control primary kidney cells, IFT140 (red) is found most strongly at the base (arrow) of the cilium (green, 6-11B-1) and is also found along the ciliary shaft and at the tip. In experimental cells (Ift140null2/null1), the cilia are very short or not present, and no IFT140 is associated with the remaining centrioles (arrow) or shortened cilium (arrowhead). These cells were also labeled with Dolichos biflorus agglutinin (DBA) in green to mark collecting duct cells. Note that DBA staining is far weaker than the 6-11B-1 staining. Asterisk marks an unidentified structure labeled by the IFT140 antibody in both experimental and control cells. Scale bar, 5 μm for both images. Bottom panels are three-time enlargements of the ciliary region shown as merged images and green (6-11B-1) and red (IFT140) channels separately. (D) In mitotic IMCD3 cells, IFT140 (green) is found in puncta in the cytoplasm but is not associated with the spindle pole bodies (arrows). Arrowheads mark cilia stained by IFT140 in neighboring cells. In contrast, IFT20 (green) is found at the spindle pole bodies (arrows) and in the cytoplasm. The arrowhead marks IFT20 staining the Golgi complex in a neighboring cell. In both images, red is anti–α-tubulin. Scale bar, 5 μm for both images. (E) Kidneys from control and experimental p5 animals were stained for cilia (green, 6-11B-1), collecting ducts (green, DBA), IFT140 (red), and nuclei (blue, 4',6-diamidino-2-phenylindole [DAPI]). Note that the DBA staining is weaker than the 6-11B-1 staining and does project in these images but was visible to identify collecting ducts. Arrows mark centrosomes in collecting ducts. Insets are three-time enlargements of cilia/centrosomes at arrows. Size bar, 10 μm for all images. Images are maximum projections of 13 confocal Z-images taken 0.25 μm apart. (F) Kidneys from control and experimental p5 animals were stained for centrosomes (green, γ-tubulin), collecting ducts (green, DBA), IFT140 (red), and nuclei (blue, DAPI). Note that the DBA staining is weaker than the γ-tubulin staining and does project in these images, but it was visible to identify collecting ducts. Arrows mark centrosomes in collecting ducts. Insets are three-time enlargements of centrosomes at arrows. Size bar, 10 μm for all images. Images are maximum projections of 13 confocal Z-images taken 0.25 μm apart.
Figure 2.
Figure 2.
Deletion of Ift140 in mouse collecting ducts causes renal cysts and renal failure. (A) Gross morphology of experimental kidneys (top pair) and control kidneys at p20. (B) Hematoxylin/eosin (H&E) -stained sections of control and mutant kidneys at p0, p5, p10, and p20. Experimental kidneys at p0 are normal except for some minor dilation of the medullary collecting ducts (arrows) but become cystic (arrows) with age. Scale bars, 200 μm for both images in a pair. (C) Mean ± SEM individual kidney weights of control (open bars) and experimental (filled bars) animals at 5-day intervals between p0 and p20. **P<0.01 versus age-matched control (5–12 animals per time point). (D) BUN levels (mean ± SEM) in control and experimental animals at p15 and p20. n=3 animals at p15, and n=4 animals at p20 for each genotype. **P<0.01 comparing control and experimental animals at each time point. +P<0.05 comparing experimental animals between p15 and p20.
Figure 3.
Figure 3.
Effects of Ift140 deletion on cilia. (A) Kidneys from control and experimental animals were stained for cilia (red, 6-11B-1), collecting ducts (green, DBA), and nuclei (blue, DAPI) at the day of birth (p0), and postnatal days 5, 10, and 20. Arrows mark collecting ducts, and arrowheads mark ciliated nephrons near collecting ducts; cysts and dilated ducts are marked with a C. Size bar, 10 μm. Insets are four-time enlargements of areas marked by the arrow. Images are maximum projections of 16 confocal Z-images taken 0.5 μm apart. (B) IFT88 accumulates in mutant cilia. Kidneys from p5 control and experimental animals were stained for IFT88 (red), acetylated tubulin (green, cilia), DBA (green, collecting ducts), and nuclei (blue, DAPI). Size bar, 5 μm for all images in C. Images are maximum projections of 26 confocal Z-images taken 0.2 μm apart. (C) Mouse embryonic fibroblasts derived from wild-type (WT) and Ift140null2/null2 (−/−) animals were stained for acetylated tubulin (red) to mark cilia (arrows) and IFT88 (green) or IFT27 (green) along with DAPI (blue). Note that Ift140 experimental cells accumulate IFT88 and IFT27 in the cilia. Size bar, 10 μm for all images.
Figure 4.
Figure 4.
Deletion of Ift140 does not lead to mitotic spindle misorientation or centrosomal abnormalities in collecting duct cells. (A) In normal p5 kidney tubules, mitotic spindles (red, phospho-histone H3) typically orient parallel to the long axis of the collecting duct (green, DBA; A, left and B, open bars). The absence of IFT140 does not alter this relationship in p5 collecting ducts (A, right and B, filled bars). Size bar, 10 μm for all four images. Images are maximum projections of 15 confocal Z-images taken 0.5 μm apart. (B) Mitotic spindle orientation quantitation. Mitotic collecting duct cells were photographed, and the angle between the long axis of the tubule and the spindle was measured as depicted in the schematic diagram in left; angles were grouped into 10° bins. Inset bar graph shows circular mean mitotic spindle orientation and the 95% confidence interval about the mean in collecting ducts of control (open bars, n=41) and experimental (filled bars, n=51) kidneys. (P=0.479, NS, Kolmogorov–Smirov test). (C) Centrosome phenotypes. In normal kidneys, centrosomes (red, GTU-88) are normally found at the center of apical surface of the cell. This location is not altered when Ift140 is deleted. Scale bar, 5 μm for all images in C. Images are maximum projections of three confocal Z-images taken 0.5 μm apart.
Figure 5.
Figure 5.
Proliferation and apoptosis in Ift140-defective collecting ducts. (A) p5 kidneys labeled with phospho-histone H3 (red) and DBA (green) to identify mitotic collecting duct cells (arrows). Size bar, 10 μm. Mitotic (phospho-histone H3-positive) cells were counted in >1000 cells from cortical collecting ducts or cortical cysts in p5 and p20 Ift140 control (open bars) and experimental (filled bars) kidneys. Bars depict mean percent ± SEM; n=4–5 animals of each genotype and age. **P<0.01 comparing control and experimental animals. (B) p5 and p20 kidneys labeled with cleaved caspase-3 (red) and DBA (green) to identify apoptotic collecting duct cells (arrow). At p20, most apoptotic cells (arrows) were outside of the cysts (C). Images are maximum projections of three wide-field images taken 2 μm apart. Size bar, 10 μm for all images in B. Apoptotic (cleaved caspase-3–positive) cells were counted in 1000 cells from cortical collecting ducts or cortical cysts in p5 and p20 Ift140 control (open bars) and experimental (filled bars) kidneys. Bars depict mean percent ± SEM; n=4–5 animals of each genotype and age. The differences were NS (P was not <0.05).
Figure 6.
Figure 6.
Altered canonical Wnt signaling and selected gene expression in collecting duct Ift140-deleted kidneys. (A) Western blot analysis of β-catenin. Control (Con) and experimental (Exp) kidneys were fractionated into cytosol and nuclear fractions and analyzed by Western blots with antibodies to β-catenin and dephosphorylated (active) β-catenin. RelB and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are loading controls. Each lane is a different animal. (B) Quantitative PCR analysis of 12 genes in experimental (filled bars) and control (open bars) kidneys at selected postnatal times. Bars depict mean ± SEM of 5–11 individual mouse kidneys in each treatment and age group. Gene expression data are normalized to GAPDH expression. **P<0.01, *P<0.05; Tukey HSD post hoc test. Raw data and statistical analysis of temporal changes of gene expression are included in Supplemental Tables 1–3.
Figure 7.
Figure 7.
Fibrosis accompanies the development of cystic disease. (A) Interstitial cells in experimental p20 animals show increased staining with smooth muscle actin (arrow) compared with controls. Arrowheads depict vascular structures in both genotypes. Scale bar, 50 μm. (B) Trichrome blue staining reveals increased collagen deposition (arrow) in experimental kidneys at p20. Scale bar, 100 μm.

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