Congenital nephrotic syndrome in mice lacking CD2-associated protein

Science

Volumn 286, Issue 5438, 1999, Pages 312-315

  Shih, Neng Yao ^a   Li, Jun ^a   Karpitskii, Vladimir ^a   Nguyen, Ancho ^a   Dustin, Michael L ^a   Kanagawa, Osami ^a   Miner, Jeffrey H ^a   Shaw, Andrey S ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

CD2 ANTIGEN; MEMBRANE PROTEIN;

ANIMAL EXPERIMENT; ANIMAL MODEL; ANTIGEN EXPRESSION; ANTIGEN PRESENTING CELL; ARTICLE; CELL HYPERPLASIA; EXTRACELLULAR MATRIX; IMMUNOBLOTTING; KIDNEY FAILURE; MESANGIUM CELL; MOUSE; NEPHROTIC SYNDROME; NONHUMAN; PATHOGENESIS; PRIORITY JOURNAL;

ADAPTOR PROTEINS, SIGNAL TRANSDUCING; ANIMALS; BASEMENT MEMBRANE; CYTOSKELETAL PROTEINS; EPITHELIAL CELLS; EXTRACELLULAR MATRIX PROTEINS; GLOMERULAR MESANGIUM; INTERCELLULAR JUNCTIONS; KIDNEY GLOMERULUS; LYMPHOCYTE ACTIVATION; MEMBRANE PROTEINS; MICE; MICE, KNOCKOUT; MICROSCOPY, ELECTRON; NEPHROTIC SYNDROME; PROTEINS; RECOMBINANT FUSION PROTEINS; T-LYMPHOCYTES;

ANIMALIA;

EID: 0033536599     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5438.312     Document Type: Article

References (22)

1. M. Dustin et al., Cell 94, 667 (1998).
2. Hybridization with mouse CD2AP cDN A was used to clone a genomic fragment from, a 129/Svj phage library. A 6-kb fragment 5′ of the exon encoding the first SH3 domain was generated by polymerase chain reaction (PCR) with the following primers: 5′-GCGG-CCGCTGTATATGATATGAGTACACTGTAG-3′ and 5′-GCGGCCGCACATTCATTACATACTGCATTC3′. After digestion with Not I, it was cloned into the Not I site of the targeting vector pTK.NEO.UMS [L. F. Reis, H. Ruffner, G. Stark, M. Aguet, C. Weissman, EMBO J. 13, 4798 (1994)]. A 1.1-kb fragment 3′ of the exon encoding the first SH3 domain was generated with the two primers 5′-TAGAACATCGATGTCAAGAAA-TAAATGCATATG-3′ and 5′-AATCTAATCGATTCCCA-GCATCCACAGCTC-3′ and cloned into the Cla I site of the targeting vector.
3. Transfection of RW-4 embryonic stem (ES) cells was performed by the Washington University ES Cell Core. Homologous recombinants were screened by Southern blotting with a 500-base pair fragment (encompassing the Bam HI-Kpn I fragment) generated with the primers 5′-GGATCCCCTGGAGCTGTG-GATG-3′ and 5′-GGTACCATTTCCATTTCTGCTAGG-3′, which is external to the 3′ arm of the targeting vector. Three positive clones were identified, and two clones were microinjected into C57/Bl6 blastocysts with standard methods [S. L. Mansour, K. R. Thomas, M. R. Capecchi, Nature 336, 348 (1988)].
4. For immunoblotting, tissues were homogenized in a buffer containing 1% NP-40, 40 mM Hepes (pH 7.6), 100 mM NaCl, 1 mM sodium vanadate, and protease inhibitors. Equal amounts of tissue samples were loaded and resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose filters, and blotted with a rabbit antiserum to CD2AP as described (1).
5. Tissues from CD2AP KO mice were fixed in 4% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin by the Research Histology Facility in the Washington University Department of Pathology.
6. N.-Y. Shin et al., data not shown.
7. One-, two-, and three-week-old animals were killed, and urine was collected. Proteinuria was analyzed by SDS-PAGE and Coomasie staining. By 3 weeks of age, proteinuria in KO animals was quite substantial, measuring over 1000 mg/dl.
8. Kidneys from wild-type or KO animals were fixed in gluteraldehyde and embedded in epoxy resin. Thin sections stained with uranyl acetate were examined in the Diagnotic EM Facility of the Washington University Department of Pathology.
9. Freshly dissected kidneys were frozen in OCT compound and sectioned at 7 μm. After fixation with 2% paraformaldehyde in phosphate-buffered saline (PBS) and clearing in 100 mM glycine in PBS, sections were incubated with primary antibodies for 1 hour, washed in PBS, and incubated with Cy3- or fluorescein isothiocyanate-conjugated second antibodies (or with both). After rinsing in PBS, sections were viewed and digitally imaged under epifluorescent illumination. Primary antibodies were as follows: rabbit antibodies to CD2AP (1), laminin α2 [Y.-S. Cheng et al., J. Biol. Chem. 272, 31525 (1997)], integrin α3 [C. M. DiPersio, S. Shah, R. O. Hynes, J. Cell Sci. 108, 2321 (1995)] , and collagen α4(IV) [J. H. Miner and J. R. Sanes, J. Cell Biol. 127, 879 (1994)]; mouse antibody to synaptopodin [P. Mundel et al., J. Histochem. Cytochem. 39, 1047 (1991)]; and rat antibodies to laminin γ1 (MAB1914; Chemicon) and pertecan [M. Kato, Y. Koike, S. Suzuki, K. Kimata, J. Cell Biol. 106, 2203 (1988)].
10. Freshly dissected kidneys were frozen in OCT compound and sectioned at 7 μm. After fixation with 2% paraformaldehyde in phosphate-buffered saline (PBS) and clearing in 100 mM glycine in PBS, sections were incubated with primary antibodies for 1 hour, washed in PBS, and incubated with Cy3- or fluorescein isothiocyanate-conjugated second antibodies (or with both). After rinsing in PBS, sections were viewed and digitally imaged under epifluorescent illumination. Primary antibodies were as follows: rabbit antibodies to CD2AP (1), laminin α2 [Y.-S. Cheng et al., J. Biol. Chem. 272, 31525 (1997)], integrin α3 [C. M. DiPersio, S. Shah, R. O. Hynes, J. Cell Sci. 108, 2321 (1995)] , and collagen α4(IV) [J. H. Miner and J. R. Sanes, J. Cell Biol. 127, 879 (1994)]; mouse antibody to synaptopodin [P. Mundel et al., J. Histochem. Cytochem. 39, 1047 (1991)]; and rat antibodies to laminin γ1 (MAB1914; Chemicon) and pertecan [M. Kato, Y. Koike, S. Suzuki, K. Kimata, J. Cell Biol. 106, 2203 (1988)].
11. Freshly dissected kidneys were frozen in OCT compound and sectioned at 7 μm. After fixation with 2% paraformaldehyde in phosphate-buffered saline (PBS) and clearing in 100 mM glycine in PBS, sections were incubated with primary antibodies for 1 hour, washed in PBS, and incubated with Cy3- or fluorescein isothiocyanate-conjugated second antibodies (or with both). After rinsing in PBS, sections were viewed and digitally imaged under epifluorescent illumination. Primary antibodies were as follows: rabbit antibodies to CD2AP (1), laminin α2 [Y.-S. Cheng et al., J. Biol. Chem. 272, 31525 (1997)], integrin α3 [C. M. DiPersio, S. Shah, R. O. Hynes, J. Cell Sci. 108, 2321 (1995)] , and collagen α4(IV) [J. H. Miner and J. R. Sanes, J. Cell Biol. 127, 879 (1994)]; mouse antibody to synaptopodin [P. Mundel et al., J. Histochem. Cytochem. 39, 1047 (1991)]; and rat antibodies to laminin γ1 (MAB1914; Chemicon) and pertecan [M. Kato, Y. Koike, S. Suzuki, K. Kimata, J. Cell Biol. 106, 2203 (1988)].
12. Freshly dissected kidneys were frozen in OCT compound and sectioned at 7 μm. After fixation with 2% paraformaldehyde in phosphate-buffered saline (PBS) and clearing in 100 mM glycine in PBS, sections were incubated with primary antibodies for 1 hour, washed in PBS, and incubated with Cy3- or fluorescein isothiocyanate-conjugated second antibodies (or with both). After rinsing in PBS, sections were viewed and digitally imaged under epifluorescent illumination. Primary antibodies were as follows: rabbit antibodies to CD2AP (1), laminin α2 [Y.-S. Cheng et al., J. Biol. Chem. 272, 31525 (1997)], integrin α3 [C. M. DiPersio, S. Shah, R. O. Hynes, J. Cell Sci. 108, 2321 (1995)] , and collagen α4(IV) [J. H. Miner and J. R. Sanes, J. Cell Biol. 127, 879 (1994)]; mouse antibody to synaptopodin [P. Mundel et al., J. Histochem. Cytochem. 39, 1047 (1991)]; and rat antibodies to laminin γ1 (MAB1914; Chemicon) and pertecan [M. Kato, Y. Koike, S. Suzuki, K. Kimata, J. Cell Biol. 106, 2203 (1988)].
13. Freshly dissected kidneys were frozen in OCT compound and sectioned at 7 μm. After fixation with 2% paraformaldehyde in phosphate-buffered saline (PBS) and clearing in 100 mM glycine in PBS, sections were incubated with primary antibodies for 1 hour, washed in PBS, and incubated with Cy3- or fluorescein isothiocyanate-conjugated second antibodies (or with both). After rinsing in PBS, sections were viewed and digitally imaged under epifluorescent illumination. Primary antibodies were as follows: rabbit antibodies to CD2AP (1), laminin α2 [Y.-S. Cheng et al., J. Biol. Chem. 272, 31525 (1997)], integrin α3 [C. M. DiPersio, S. Shah, R. O. Hynes, J. Cell Sci. 108, 2321 (1995)] , and collagen α4(IV) [J. H. Miner and J. R. Sanes, J. Cell Biol. 127, 879 (1994)]; mouse antibody to synaptopodin [P. Mundel et al., J. Histochem. Cytochem. 39, 1047 (1991)]; and rat antibodies to laminin γ1 (MAB1914; Chemicon) and pertecan [M. Kato, Y. Koike, S. Suzuki, K. Kimata, J. Cell Biol. 106, 2203 (1988)].
14. P. Mundel et al., J. Cell Biol. 139, 193 (1997).
15. M. Kestila et al., Mol. Cell 1, 575 (1998).
16. V. Ruotsalainen et al., Proc. Natl. Acad. Sci. U.S.A. 96, 7962 (1999).
17. G/Nephrin was generated by replacing the Bam HI-Hind III fragment of pGem G/epsilon [L. T. Gauen, A. N. Kong, L. E. Samelson, A. S. Shaw, Mol. Cell. Biol. 38, 5438 (1992)] with a PCR-generated Bgl II-Hind III fragment encoding residues 1086 to 1241 of the nephrin cytoplasmic domain with an expressed sequence tag done (AA678622) as a template. Myc-CD2AP (1) was coexpressed with VSV G fusion proteins in HeLa cells and analyzed as described previously (1).
18. N. Shin and A. Shaw, unpublished data.
19. 3H]thymidine incorporation 48 and 72 hours after stimulation. KO T cells proliferated about 20 to 30% compared with wild-type T cells.
20. 7) from 129Sv/JxCD2AP KO mice were injected into irradiated (950 rads) C57/BL6 mice. Graft reconstitution was confirmed with antibody Ly9.1 staining, which recognizes the 129/SvJ T cells but not the C57/BL6 T cells. No kidney abnormality was detected up to 1 year after transplant. T cells from mice reconstituted with KO bone marrow exhibited proliferative responses of about 60 to 70% compared with T cells from animals reconstituted with wild-type bone marrow when stimulated with antibody to CD3.
21. K. H. Kirsch, M. M. Georgescu, S. Ishimaru, H. Hanafusa, Proc. Natl. Acad. Sci. U.S.A. 96, 6211 (1999).
22. N. Shih and J. Li contributed equally to this manuscript. We thank E. Unanue and R. Cotran for invaluable help and suggestions; J. Kissane, J. Saffitz, and M. LaRegina for help with pathological analysis; M. White for ES cell microinjections; A. Chan for help with isolation of the genomic clone; C. Li for antibody staining; and M. A. Arnaout, P. Mundel, P. Yurchenco, M. Dipersio, and M. Yamagata for providing antibodies.