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. 2013 Oct-Dec;4(4):221-30.
doi: 10.4161/sgtp.27430. Epub 2013 Dec 23.

The Rac-specific exchange factors Dock1 and Dock5 are dispensable for the establishment of the glomerular filtration barrier in vivo

Mélanie Laurin  1 , Annie Dumouchel  2 , Yoshinori Fukui  3 , Jean-François Côté  4
Affiliations

The Rac-specific exchange factors Dock1 and Dock5 are dispensable for the establishment of the glomerular filtration barrier in vivo

Mélanie Laurin et al. Small GTPases. 2013 Oct-Dec.

Abstract

Podocytes are specialized kidney cells that form the kidney filtration barrier through the connection of their foot processes. Nephrin and Neph family transmembrane molecules at the surface of podocytes interconnect to form a unique type of cell-cell junction, the slit diaphragm, which acts as a molecular sieve. The cytoplasmic tails of Nephrin and Neph mediate cytoskeletal rearrangement that contributes to the maintenance of the filtration barrier. Nephrin and Neph1 orthologs are essential to regulate cell-cell adhesion and Rac-dependent actin rearrangement during Drosophila myoblast fusion. We hypothesized here that molecules regulating myoblast fusion in Drosophila could contribute to signaling downstream of Nephrin and Neph1 in podocytes. We found that Nephrin engagement promoted recruitment of the Rac exchange factor Dock1 to the membrane. Furthermore, Nephrin overexpression led to lamellipodia formation that could be blocked by inhibiting Rac1 activity. We generated in vivo mouse models to investigate whether Dock1 and Dock5 contribute to the formation and maintenance of the kidney filtration barrier. Our results indicate that while Dock1 and Dock5 are expressed in podocytes, their functions are not essential for the development of the glomerular filtration barrier. Furthermore, mice lacking Dock1 were not protected from LPS-induced podocyte effacement. Our data suggest that Dock1 and Dock5 are not the important exchange factors regulating Rac activity during the establishment and maintenance of the glomerular barrier.

Keywords: Dock1; Dock180; Dock5; Elmo; Rac; Rho GTPase; podocyte.

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Figures

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Figure 1. The Drosophila myoblast fusion machinery is expressed in mouse podocytes. (A) Mouse podocytes were differentiated in vitro for 7 or 14 d or left undifferentiated and expression of components of the Drosophila myoblast fusion machinery was evaluated by RT-PCR. (B) Dock1 is expressed in the kidney glomerulus. IHC analyses showing Dock1 (left), Wt1 (middle) and merge (right) expression in E16.5 Dock1+/+ and Dock1−/− glomerulus (Scale bar: 20 μm, 40x).
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Figure 2. Dock1 is recruited to actin comet tails upon clustering of Nephrin molecules. (A) DOCK1 is recruited to actin comet tails upon clustering of chimeric CD16-Nephrin molecules. CHO.KI cells were transfected with CD16-NephrinIC-Myc and Flag-DOCK1 and clustering of Nephrin molecules was induced by an anti-CD16 antibody treatment. Immunofluorescence analyses showing the distribution of CD16-NephrinIC-Myc (blue), Flag-DOCK1 (green) and actin (red; phalloidin) molecules in non-treated and CD16-treated cells (Scale bar: 20 μm, 60x). (B) Expression of CD16-NephrinIC-Myc induces lamellipodia formation in cells through Rac activation. CHO.KI cells were transfected with CD16-NephrinIC-Myc and left untreated or treated with a Rac1 inhibitor. Immunofluorescence analyses showing expression of CD16-NephrinIC-Myc (red) and actin (green; phalloidin) (Scale bar: 20 μm, 60x). (C) Quantification of the % of cells with lamellipodia in B (n = 3).
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Figure 3. Podocyte-specific ablation of Dock1 expression. (A) Partial schematic representation of the Dock1 wt allele, the Dock1 flx allele and the Dock1 recombined (ko) allele after exposure to the Cre recombinase. Positions of PCR primers used for genotyping analyses are indicated. (B) PCR analyses using P1, P2, P3 and Cre primer sets on genomic DNA extracted from mice kidneys or tails of the indicated genotype. A recombined Dock1 flx allele (ko allele) is detected in the kidney of transgenic Pod-Cre+ mice. (C) IHC analyses showing the absence of Dock1 expression in the podocytes of Pod-Cre+Dock1flx/flx mice (Scale bar: 20 μm, 100x).
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Figure 4.Pod-Cre+Dock1flx/flx mice display normal glomerular structure and function. (A) H&E-staining showing the normal glomerular development of 3 mo old Pod-Cre-Dock1flx/flx and Pod-Cre+Dock1flx/flx mice (Scale bar 20 μm, 40x). (B) Absence of proteinuria in the urine of 3-mo old Pod-Cre-Dock1flx/flx and Pod-Cre+Dock1flx/flx mice. Coomassie stained protein gel showing BSA standards (left) and the albumin content from the urine (right) of 3 mo old Pod-Cre-Dock1flx/flx and Pod-Cre+Dock1flx/flx mice. (C) Normal renal function in the absence of Dock1 expression in podocytes. Quantification of the average albumin-to-creatinine ratios in the urine of Pod-Cre-Dock1flx/flx and Pod-Cre+Dock1flx/flx mice at 3 and 6 mo after their birth (n = 5).
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Figure 5.Dock5 is not essential to glomerular function. (A) Dock2 is not expressed in the kidney. Dock2-GFP expression is absent in the kidney (top), but can be detected in the spleen (bottom) (Scale bar: 100 μm, 20x). (B) IHC analyses showing the expression of Dock5 and Wt1 in the podocytes of Pod-Cre+Dock1+/+Dock5+/+ mice (Scale bar: 25 μm, 100x). (C) Normal renal function in the absence of Dock5 expression in mice. Quantification of the average albumin-to-creatinine ratio in the urine of Pod-Cre+Dock1+/+Dock5+/+ and Pod-Cre+Dock1+/+Dock5−/− 2 mo old mice (n = 3). (D) Absence of proteinuria in the urine of P21 Pod-Cre-Dock1flx/flxDock5+/+ and Pod-Cre+Dock1flx/flxDock5−/− mice. Coomassie stained protein gel showing BSA standards (left) and the albumin content from the urine of a control mouse and 4 Pod-Cre+Dock1flx/flxDock5−/− mutant mice.
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Figure 6. Loss of Dock1 expression in podocytes does not protect against LPS-induced proteinuria. (A) H&E-staining showing the glomerulus of 6 wk old Control and Pod-Cre+Dock1flx/flx female mice 28 h (t = 28) after LPS injection (Scale bar 20 μm, 40x). (B) LPS injection induces significant proteinuria in 6 wk old Pod-Cre+Dock1+/+ and Pod-Cre+Dock1flx/flx mice. Coomassie stained protein gel showing BSA standards (left) and the albumin content in the urine (right) collected from Pod-Cre+Dock1+/+ and Pod-Cre+Dock1flx/flx mice before (t = 0) and 6, 24 and 28 h (t = 6, t = 24, t = 28) after LPS injection. (C) Quantification of the average albumin-to-creatinine ratio in the urine of Pod-Cre-Dock1flx/flx and Pod-Cre+Dock1flx/flx before (t = 0) and 28 h (t = 28) after LPS injection.
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