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
View: Text | PDF
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

×

Figure 5

Robo2 deficiency protects mice from DOCA-salt-uninephrectomy–induced podocyte loss.

Options: View larger image (or click on image) Download as PowerPoint
Robo2 deficiency protects mice from DOCA-salt-uninephrectomy–induced po...
(A) Representative images of periodic acid-Schiff (PAS) staining of mouse kidney tissues show proteinaceous casts (asterisks), glomerulosclerosis, and increased matrix expression after 5-week deoxycorticosterone acetate (DOCA)-salt-uninephrectomy hypertension injury in wild-type, Myh9 conditional knockout (cKO), Robo2 cKO, and Myh-Robo2 double KO mice. Magnification: ×400. g, glomerulus. (B) Quantification of percentage of sclerotic glomeruli in kidney samples from A. Data are presented as the mean ± SEM, n = 3 animals per group, minimum 100 glomeruli counted in each animal. P = 0.028 compared with the Myh9 cKO group by 1-way ANOVA. (C) Representative images of WT1-positive podocytes in the glomeruli isolated from 4 groups of mice with Myh9/Robo2 single cKO, Myh9-Robo2 double cKO (dKO), and wild-type (WT) genotype after 5-week DOCA-salt-uninephrectomy injury. Magnification: ×400. g, glomerulus. (D) Quantification of podocyte number per glomerulus. Data are presented as the mean ± SEM, n = 3 animals per group, minimum 50 glomeruli counted in each animal. *P < 0.05, **P < 0.01 by 1-way ANOVA. (E) Albuminuria from the mice 5 weeks after DOCA-salt-uninephrectomy injury detected by acrylamide gel and Coomassie blue staining. WT, wild-type; Robo2 cKO, Robo2fl/fl;Nphs2-Cre+ single knockout mice; Myh9 cKO, Myh9 fl/fl;Nphs2-Cre+ single knockout mice; dKO, Robo2fl/fl;Myh9 fl/fl;Nphs2-Cre+ double knockout mice. (F) Quantification of urine albumin/creatinine ratios from each group of mice. Data are presented as the mean ± SEM, n = 4 animals. *P < 0.01, **P < 0.05 by 1-way ANOVA.