SLIT2/ROBO2 signaling pathway inhibits nonmuscle myosin IIA activity and destabilizes kidney podocyte adhesion
Xueping Fan, Hongying Yang, Sudhir Kumar, Kathleen E. Tumelty, Anna Pisarek-Horowitz, Hila Milo Rasouly, Richa Sharma, Stefanie Chan, Edyta Tyminski, Michael Shamashkin, Mostafa Belghasem, Joel M. Henderson, Anthony J. Coyle, David J. Salant, Stephen P. Berasi, Weining Lu
Xueping Fan, Hongying Yang, Sudhir Kumar, Kathleen E. Tumelty, Anna Pisarek-Horowitz, Hila Milo Rasouly, Richa Sharma, Stefanie Chan, Edyta Tyminski, Michael Shamashkin, Mostafa Belghasem, Joel M. Henderson, Anthony J. Coyle, David J. Salant, Stephen P. Berasi, Weining Lu
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Research Article Nephrology

SLIT2/ROBO2 signaling pathway inhibits nonmuscle myosin IIA activity and destabilizes kidney podocyte adhesion

Abstract

The repulsive guidance cue SLIT2 and its receptor ROBO2 are required for kidney development and podocyte foot process structure, but the SLIT2/ROBO2 signaling mechanism regulating podocyte function is not known. Here we report that a potentially novel signaling pathway consisting of SLIT/ROBO Rho GTPase activating protein 1 (SRGAP1) and nonmuscle myosin IIA (NMIIA) regulates podocyte adhesion downstream of ROBO2. We found that the myosin II regulatory light chain (MRLC), a subunit of NMIIA, interacts directly with SRGAP1 and forms a complex with ROBO2/SRGAP1/NMIIA in the presence of SLIT2. Immunostaining demonstrated that SRGAP1 is a podocyte protein and is colocalized with ROBO2 on the basal surface of podocytes. In addition, SLIT2 stimulation inhibits NMIIA activity, decreases focal adhesion formation, and reduces podocyte attachment to collagen. In vivo studies further showed that podocyte-specific knockout of Robo2 protects mice from hypertension-induced podocyte detachment and albuminuria and also partially rescues the podocyte-loss phenotype in Myh9 knockout mice. Thus, we have identified SLIT2/ROBO2/SRGAP1/NMIIA as a potentially novel signaling pathway in kidney podocytes, which may play a role in regulating podocyte adhesion and attachment. Our findings also suggest that SLIT2/ROBO2 signaling might be a therapeutic target for kidney diseases associated with podocyte detachment and loss.

Authors

Xueping Fan, Hongying Yang, Sudhir Kumar, Kathleen E. Tumelty, Anna Pisarek-Horowitz, Hila Milo Rasouly, Richa Sharma, Stefanie Chan, Edyta Tyminski, Michael Shamashkin, Mostafa Belghasem, Joel M. Henderson, Anthony J. Coyle, David J. Salant, Stephen P. Berasi, Weining Lu

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Figure 2

ROBO2, SRGAP1, and nonmuscle myosin IIA (NMIIA) are expressed in mouse podocytes.

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ROBO2, SRGAP1, and nonmuscle myosin IIA (NMIIA) are expressed in mouse p...
(A–C) Differentiated mouse podocytes express SRGAP1, myosin regulatory light chain (MRLC), and NMIIA heavy chain (NMHC IIA). ×400 magnification. (D) LacZ staining shows that SRGAP1 is expressed in embryonic day 16.5 mouse glomeruli (white arrow) of a Srgap1-LacZ reporter mouse kidney. ×50 magnification. (E and F) SRGAP1 protein is detected by immunofluorescence staining in mouse kidney glomeruli at postnatal day 1 (P1) and 2 weeks of age (2W). ×400 magnification. (G–I) SRGAP1 (red) is colocalized with ROBO2 (green) in the P1 mouse glomeruli as detected by coimmunolabeling. ×400 magnification. (J–L) SRGAP1 (red) is colocalized with nephrin (green) in the P1 mouse glomeruli as detected by coimmunolabeling. ×400 magnification. (M–O) SRGAP1 (red) is colocalized with podocin (green) in the P1 mouse glomeruli as detected by coimmunolabeling. ×400 magnification.