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. 2000 Feb 15;97(4):1731-6.
doi: 10.1073/pnas.040550097.

Polycystin 1 is required for the structural integrity of blood vessels

K Kim  1 , I Drummond, O Ibraghimov-Beskrovnaya, K Klinger, M A Arnaout
Affiliations

Polycystin 1 is required for the structural integrity of blood vessels

K Kim et al. Proc Natl Acad Sci U S A. .

Abstract

Autosomal dominant polycystic kidney disease (ADPKD), often caused by mutations in the PKD1 gene, is associated with life-threatening vascular abnormalities that are commonly attributed to the frequent occurrence of hypertension. A previously reported targeted mutation of the mouse homologue of PKD1 was not associated with vascular fragility, leading to the suggestion that the vascular lesion may be of a secondary nature. Here we demonstrate a primary role of PKD1 mutations in vascular fragility. Mouse embryos homozygous for the mutant allele (Pkd1(L)) exhibit s.c. edema, vascular leaks, and rupture of blood vessels, culminating in embryonic lethality at embryonic day 15.5. Kidney and pancreatic ductal cysts are present. The Pkd1-encoded protein, mouse polycystin 1, was detected in normal endothelium and the surrounding vascular smooth muscle cells. These data reveal a requisite role for polycystin 1 in maintaining the structural integrity of the vasculature as well as epithelium and suggest that the nature of the PKD1 mutation contributes to the phenotypic variance in ADPKD.

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Figures

Figure 1
Figure 1
Targeted disruption of mouse Pkd1. (A) The genomic segment of WT and recombinant murine Pkd1. Exons (black boxes) are numbered. The position of the recombinations, the expected sizes of WT and mutant (Mut.) fragments, and the position of the external 5′ and 3′ probes are indicated. (B) Southern blots of genomic DNA extracted from ES clones, KpnI-restricted, and probed with the 5′ external probe. A recombinant mutant fragment (8.1 kb) replaces the normal allele (6.8 kb) in several clones. DNA from clone #282 and #130 is shown in lanes 2 and 6. (C and D) Germ-line transmission of the mutant allele. Yolk sac DNA obtained from the offspring of F1 intercrosses was extracted, digested with KpnI or XhoI, and probed, respectively, with the 5′ (C) or 3′ (TSC2 exon 33, 430 bp, D) probes. Fragment size is indicated by arrows. Southern blots of DNA from clone #282 (lanes 1–4 in C and 1–3 in D) and #130 (lanes 5–7, C) F2 embryos is shown. +/+, +/−, and −/− indicate WT, Pkd1+/L, and Pkd1L/L, respectively. (E--G) Northern blot analysis. The mutant allele (15.4 kb) is found in Pkd1L/L (lane 3) and Pkd1+/L (lane 2) but not WT animals, who express only the WT mRNA of 14.1 kb (lane 1). The mutant but not the WT allele hybridized with the neo probe (F). (G) The normal-sized mRNA for mouse TSC2 (5.6 kb, *) in Pkd1L/L embryos. (H) Primary structure of polycystin 1 and the site of the predicted truncation (arrow). Mutant mouse polycystin 1 is expected to terminate after L3946 (equivalent to L3955 in human).
Figure 2
Figure 2
Gross phenotype of PkdL/L embryos at different stages of development. Whole mounts of PkdL/L (B, D, F, H, J, and K) and age-matched WT embryos (A, C, E, G, and I) are shown. Localized hemorrhages and s.c. edema (arrows) distributed over the body surface, with punctate and localized hemorrhages seen in the head (B, D, and J) and toes (H and K) of Pkd1L/L (J, H, and K) but not WT (A, C, E, G, and I) unstained embryos. At E15.5, fatal hemorrhages and edema are seen in PkdL/L embryos (H). Staged embryos were dissected in PBS and photographed by using a Leica MZ12 microscope.
Figure 3
Figure 3
Histological analysis of tissues from Pkd1L/L embryos. (A) Hematoxylin and eosin stain showing red blood cells (arrowheads) outside a blood vessel in the neck region at E13.5. (B) A skin capillary at E13.5 from a region not showing gross hemorrhage. A red blood cell (arrowhead and Inset) is seen traversing the endothelial cell lining between two adjacent cells. (C–F) Tissue sections revealing the leak of intravascularly injected fluorescent dextran into the extravascular interstitium (D, arrows) at distant sites in a E12.5 PkdL/L embryo (D and F). In the age-matched WT (C and E), dextran is retained within the vasculature (arrows). No fluorescence is seen on the basolateral side of the lining endothelium in WT embryos (E, arrows). In contrast, fluorescent dextran is seen basolaterally (F, arrows) and at intercellular junctions in the mutant. (G–I) Hematoxylin and eosin stain of a section of a normal developing kidney at E14.5 (G), showing two normal glomeruli (arrowheads). An age-matched PkdL/L kidney (H) shows two large glomerular cysts (arrows), and a tubular cyst (arrowhead). (I) A section of a pancreas from this PkdL/L embryo shows two tubular cysts (arrows) adjacent to normal-sized ducts (arrowhead). Sections shown in A, B, G, H, and I are 5 μm thick, and those in C–F are 4 μm thick. (Scale bars = 10 μm, except in E and F where the scale bar = 5 μm.)
Figure 4
Figure 4
Expression of polycystin 1 in WT and Pkd1L/L E14.5 mouse embryos. Polycystin 1 is detected in endothelial cells (arrowheads in A and B) as well as in vascular smooth muscle cells (arrows in A and B) by using the anti-LRR antibody. Polycystin 1 expression (horseradish peroxidase reaction product) is similar in small vessels in the region of the hind limb in WT (A) and Pkd1L/L mutant (B) embryos. In fetal kidneys, polycystin 1 expression is observed mainly on the apical (arrows in C and E) surfaces of WT and Pkd1L/L renal tubules. No staining of renal tubules (arrows) from either WT or Pkd1L/L embryos is observed in the presence of a 20-fold excess of the LRR fusion protein (D and F, respectively). * in F points to a tubular cyst. (Scale bars = 10 μm.)
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