Crk1/2 and CrkL form a hetero-oligomer and functionally complement each other during podocyte morphogenesis

Abstract

Activation of the slit diaphragm protein nephrin induces actin cytoskeletal remodeling, resulting in lamellipodia formation in podocytes in vitro in a phosphatidylinositol-3 kinase-, focal adhesion kinase-, Cas-, and Crk1/2-dependent fashion. In mice, podocyte-specific deletion of Crk1/2 prevents or attenuates foot process effacement in two models of podocyte injury. This suggests that cellular mechanisms governing lamellipodial protrusion in vitro are similar to those in vivo during foot process effacement. As Crk1/2-null mice developed and aged normally, we tested whether the Crk1/2 paralog, CrkL, functionally complements Crk1/2 in a podocyte-specific context. Podocyte-specific CrkL-null mice, like podocyte-specific Crk1/2-null mice, developed and aged normally but were protected from protamine sulfate-induced foot process effacement. Simultaneous podocyte-specific deletion of Crk1/2 and CrkL resulted in albuminuria detected by 6 weeks postpartum and associated with altered podocyte process architecture. Nephrin-induced lamellipodia formation in podocytes in vitro was CrkL-dependent. CrkL formed a hetero-oligomer with Crk2 and, like Crk2, was recruited to tyrosine phosphorylated nephrin. Thus, Crk1/2 and CrkL are physically linked, functionally complement each other during podocyte foot process spreading, and together are required for developing typical foot process architecture.

Figure 1

CrkL and Crk1/2 co-localize with Nephrin at the podocyte slit-diaphragm and podocyte-specific CrkL^f/f;Podocin-Cre^Tg/+ mice develop and age normally. (A) Paraffin-embedded wild-type kidney sections were stained for Nephrin, Crk1/2 and CrkL (upper panel). Merged images of either CrkL (red) and Crk1/2 (green), Nephrin (red) and CrkL (green) or Nephrin (red) and Crk1/2 (green) are shown in the lower panel. A merged image of Nephrin (red), Crk1/2 (green), CrkL (purple), and DAPI (blue) staining is presented in the far right panel. Scale bars: 20 μm. (B and C) Control, podocyte-specific Crk1/2^f/f;Podocin-Cre^Tg/+ (Crk1/2 KO) or podocyte-specific CrkL^f/f;Podocin-Cre^Tg/+ (CrkL KO) mice were stained for Crk1/2 (green, B) or CrkL (green, C), Nephrin (red) and DAPI (blue). Higher magnifications of part of a glomerulus are shown in the far right panel. Scale bars: 20 μm. (D) Left: Boxplot showing the median, first and third interquartile ranges as well as the minimum and maximum of urinary albumin/creatinine ratios (a/c) of control (CrkL^f/f) and podocyte-specific CrkL^f/f;Podocin-Cre^Tg/+ mice at the age of 6 months (albumin:creatinine values in μg/mg of 4-5 mice). Middle: Statistical analysis of serum creatinine levels of control and podocyte-specific CrkL^f/f;Podocin-Cre^Tg/+ mice at the age of 6 months (means +/− SEM of blood creatinine values in mg/dL of 6-8 mice). Right: Statistical analysis of blood urea nitrogen (BUN) levels of control and podocyte-specific CrkL^f/f;Podocin-Cre^Tg/+ mice at the age of 6 months (means +/− SEM of BUN values in mg/dL of 6-8 mice). (n.s.: not significant).

Figure 2

Podocyte-specific CrkL^f/f;Podocin-Cre^Tg/+ mice are protected from protamine sulfate-induced podocyte injury. (A and B) Scanning or transmission electron microscopy (EM) images of podocytes of control (CrkL^f/f) or podocyte-specific CrkL^f/f;Podocin-Cre^Tg/+ (CrkLko) mice perfused with HBSS or protamine sulfate (PS). Results are representative of 3-4 mice per group. At the bottom the number of podocyte intercellular junctions per micron glomerular basement membrane (GBM) as seen by transmission EM is shown (B). Data represent means +/− SEM. Scale bars: 10 μm (left column, A), 5 μm (middle column, A), 2 μm (right column, A); 1 μm (B).

Figure 3

Podocyte-specific Crk1/2^f/f;CrkL^f/f;Podocin-Cre^Tg/+ mice develop a progressive albuminuria. (A) Paraffin-embedded kidney sections of control (Crk1/2^f/f;CrkL^f/f) or podocyte-specific Crk1/2^f/f;CrkL^f/f;Podocin-Cre^Tg/+ (Crk1/2/L KO) mice were stained for Nephrin, Crk1/2, CrkL and DAPI. Merged images of Nephrin (red) and Crk1/2 (green), Nephrin (red) and CrkL (green) or CrkL (red) and Crk1/2 (green) are shown in the second and fourth row. Merged images of Nephrin (red), Crk1/2 (green), CrkL (purple) and DAPI (blue) are shown in the far right panel. Scale bars: 20 μm. (B) Boxplot showing the median, first and third interquartile ranges as well as the minimum and maximum of urinary albumin/creatinine ratios of control (Crk1/2^f/f;CrkL^f/f) and podocyte-specific Crk1/2^f/f;CrkL^f/f;Podocin-Cre^Tg/+ mice at the age of 6 weeks, 3 months and 7 months in μg/mg (albumin/creatinine values of 6-12 mice per group). (C) Upper graph: Statistical analysis of blood urea nitrogen (BUN) levels of control and podocyte-specific Crk1/2^f/f;CrkL^f/f;Podocin-Cre^Tg/+ mice at the age of 7 months in mg/dL (means +/− SEM of BUN values of 9-11 mice per group). Lower graph: Statistical analysis of serum creatinine levels of control and podocyte-specific Crk1/2^f/f;CrkL^f/f;Podocin-Cre^Tg/+ (dko) mice at the age of 7 months in mg/dL (means +/− SEM of serum creatinine values of 9-11 mice per group). P value as indicated; n.s.: not significant.

Figure 4

Podocyte-specific Crk1/2^f/f;CrkL^f/f;Podocin-Cre^Tg/+ mice exhibit abnormally long foot processes and a decreased number of secondary podocyte processes per glomerulus. (A) Paraffin-embedded kidney sections of control (Crk1/2^f/f;CrkL^f/f) or podocyte-specific Crk1/2^f/f;CrkL^f/f;Podocin-Cre^Tg/+ (Crk1/2/Lko) mice at the age of 7 months were stained with hematoxilin&eosin (H&E) or Periodic Acid Schiff (PAS). Scale bars: 60 μm (first and third column from left) and 20 μm (second and forth column from left). (B) Transmission electron microscopy (EM) images of control or podocyte-specific Crk1/2^f/f;CrkL^f/f;Podocin-Cre^Tg/+ mouse glomeruli at the age of 7 months. Scale bars: 5 μm (upper panel), 1 μm (lower panel). (C and D) Scanning EM images of control or podocyte-specific Crk1/2^f/f;CrkL^f/f;Podocin-Cre^Tg/+ mouse glomeruli at the age of 6 weeks (C) or 7 months (D). Scale bars: 4 μm (left column, C), 2 μm (right column, C); 10 μm (left column, D), 5 μm (middle column, D), 2 μm (right column, D). (E) Statistical analysis of relative tertiary process length of podocyte-specific Crk1/2^f/f;CrkL^f/f;Podocin-Cre^Tg/+ mouse podocytes normalized to controls as seen by transmission EM. (F) Statistical analysis of relative glomerular capillary widths of podocyte-specific Crk1/2^f/f;CrkL^f/f;Podocin-Cre^Tg/+ mice normalized to controls as seen by transmission EM. (G) Podocyte number per glomerulus of control mice or podocyte-specific Crk1/2^f/f;CrkL^f/f; Podocin-Cre^Tg/+ mice. (F) Statistical analysis of the number of secondary podocyte processes per glomerulus of control or podocyte-specific Crk1/2^f/f;CrkL^f/f;Podocin-Cre^Tg/+ mice as seen by scanning EM. Shown are means +/− SEM (n.s., not significant; P value as indicated). Results are representative of at least 3 mice per group.

Figure 5

CrkL associates with Nephrin and forms a hetero-oligomer with Crk2. (A) Cultured human podocytes expressing CD16/7-NephrinCD (CD16NCD) or CD16/7-HA (CD16HA) and CrkL-myc were activated by addition of monoclonal anti-CD16 primary antibody (1°) and goat anti-mouse IgG Texas Red-conjugated secondary antibody (2°) to the media of live cells. CrkL-myc was detected with rabbit polyclonal anti-myc primary antibody and Alexa Fluor 488-labeled secondary antibody. Cells were analyzed by confocal microscopy. Note that CD16/7-NephrinCD (red) and CrkL-myc (green) co-localize. Scale bars: 20 μm. yz plane reconstructions are shown at the far right. (B) Lysates of cultured HEK293 cells expressing CD16/7-NephrinCD and/or CrkL-His-myc were incubated with Nickel beads. (C and D) Co-immunoprecipitation and immunoblots using indicated antibodies demonstrated that Crk2-GFP and Myc-CrkL co-immunoprecipitated (C) while Myc-CrkL and GFP alone (C) or Myc-CrkL and Crk1-GFP (D) did not co-immunoprecipitate. (E) Crk2-His, GST and GST-CrkL were expressed in BL21 E. coli. Purified recombinant Crk2-His was incubated with either GST or GST-CrkL and pulled down using Nickel beads demonstrating that Crk2-His and GST-CrkL interacted directly. Results are representative of at least 3 independent experiments.

Figure 6

CrkL like Crk2 is required for Nephrin-induced lamellipodia formation. (A) Immunoblot demonstrating specific attenuation by knockdown of CrkL, Crk2, or CrkL and Crk2 expression in human podocyte cell lines. Scrambled shRNA was used as control. (B) Podocytes expressing plasmid encoding CrkL or Crk2 shRNA or both were transfected with CD16/7-NephrinCD, and mouse Crk2 (mCrk2) or mouse CrkL (mCrkL) or both as indicated and activated by clustering as described in Figure 5. The fraction of cells exhibiting lamellipodial protrusions was evaluated after fixation. (C) Crk2 and CrkL function in a synergistic fashion. The Crk1/2;CrkL double knockdown human podocytes were rescued with different amounts of Crk2, CrkL, or Crk2 + CrkL by transiently transfecting the human podocytes stably expressing Crk2 shRNA (stable Crk2 KD cell) with plasmids encoding CD16/7-NephrinCD, CrkL shRNA and indicated quantities of plasmids (x-axis) encoding mouse Crk2 and/or CrkL. Cells were activated by clustering. The fraction of cells showing lamellipodia protrusions were determined after fixation and are represented as bars. The synergistic enhancement of lamellipodia formation was noted when either 375ng or 625ng of each of Crk2 and CrkL plasmids were used in this assay. The line graph presented in D further confirms the observations from figure C. (D) *P<0.05, Crk2 + CrkL vs. Crk2 or CrkL. (B and C) Note that total quantity of plasmid used for each condition was maintained equal by addition of appropriate amounts of empty expression plasmid. Results are representative of 3 independent experiments and are shown as means +/− SEM. P values as indicated and were significant in each case.