Volume 92, Issue 2, Pages 349-364 (August 2017) Rac1 activation in podocytes induces the spectrum of nephrotic syndrome  Richard Robins, Cindy Baldwin, Lamine Aoudjit, Jean-François Côté, Indra R. Gupta, Tomoko Takano  Kidney International  Volume 92, Issue 2, Pages 349-364 (August 2017) DOI: 10.1016/j.kint.2017.03.010 Copyright © 2017 International Society of Nephrology Terms and Conditions

Figure 1 Podocyte-specific hyperactivation of Ras-related C3 botulinum toxin substrate 1 (Rac1) induces a variable phenotype depending on the dose of active Rac1. (a) Detection of doxycycline (Dox)-induced proteinuria (day 5) by Coomassie-stained gel of urine samples separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. High-responder mice showed an intense band at the molecular weight of albumin (67 kDa), whereas in low-responder mice, proteinuria was barely detectable. Lower panel shows quantification of the urine albumin:creatinine ratio (ACR). *P < 0.05 versus low responder, **P < 0.01 versus Dox−, n = 4 mice per group. Control mice did not respond to Dox treatment. (b) Transmission electron micrographs of the kidneys after 5 days of Dox treatment. Control mice showed intact podocyte foot processes along the glomerular basement membrane (11k ×). Foot process effacement was readily observable in the high-responder mice but was less extensive in the low-responder mice. Bar = 100 μm. Lower panel shows the quantification of the foot process width (FPW), which was inversely correlated with the degree of foot process effacement. *P < 0.05 versus low responder, **P < 0.01 versus control, n = 3 capillaries from 3 mice per treatment. (c) Immunofluorescence staining of FLAG (transgene) expression in the glomeruli after 5 days of Dox treatment. The FLAG signal (red) colocalized with synaptopodin (green) and was more intense and diffuse in high-responder mice than in low-responder mice. Bars = 10 μm. (d) Rac1-guanosine triphosphate (GTP; active Rac1, green) colocalized with FLAG (red), confirming the biological activity of the transgene. Rac1-GTP staining was more intense and diffuse in high-responder mice than in low-responder mice. *Nonspecific staining. Bars = 10 μm. CA-Rac1, constitutively active form of Rac1; DAPI, 4′,6-diamidino-2-phenylindole. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2017 92, 349-364DOI: (10.1016/j.kint.2017.03.010) Copyright © 2017 International Society of Nephrology Terms and Conditions

Figure 1 Podocyte-specific hyperactivation of Ras-related C3 botulinum toxin substrate 1 (Rac1) induces a variable phenotype depending on the dose of active Rac1. (a) Detection of doxycycline (Dox)-induced proteinuria (day 5) by Coomassie-stained gel of urine samples separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. High-responder mice showed an intense band at the molecular weight of albumin (67 kDa), whereas in low-responder mice, proteinuria was barely detectable. Lower panel shows quantification of the urine albumin:creatinine ratio (ACR). *P < 0.05 versus low responder, **P < 0.01 versus Dox−, n = 4 mice per group. Control mice did not respond to Dox treatment. (b) Transmission electron micrographs of the kidneys after 5 days of Dox treatment. Control mice showed intact podocyte foot processes along the glomerular basement membrane (11k ×). Foot process effacement was readily observable in the high-responder mice but was less extensive in the low-responder mice. Bar = 100 μm. Lower panel shows the quantification of the foot process width (FPW), which was inversely correlated with the degree of foot process effacement. *P < 0.05 versus low responder, **P < 0.01 versus control, n = 3 capillaries from 3 mice per treatment. (c) Immunofluorescence staining of FLAG (transgene) expression in the glomeruli after 5 days of Dox treatment. The FLAG signal (red) colocalized with synaptopodin (green) and was more intense and diffuse in high-responder mice than in low-responder mice. Bars = 10 μm. (d) Rac1-guanosine triphosphate (GTP; active Rac1, green) colocalized with FLAG (red), confirming the biological activity of the transgene. Rac1-GTP staining was more intense and diffuse in high-responder mice than in low-responder mice. *Nonspecific staining. Bars = 10 μm. CA-Rac1, constitutively active form of Rac1; DAPI, 4′,6-diamidino-2-phenylindole. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2017 92, 349-364DOI: (10.1016/j.kint.2017.03.010) Copyright © 2017 International Society of Nephrology Terms and Conditions

Figure 2 Prolonged Ras-related C3 botulinum toxin substrate 1 (Rac1) hyperactivation causes persistent proteinuria and histologic changes similar to focal segmental glomerulosclerosis. (a) The urine albumin:creatinine ratio (ACR) time course for 4 high-responder mice treated with doxycycline (Dox) for 30 days demonstrated heavy and persistent proteinuria. (b) Immunostaining of a high-responder mouse clearly showed FLAG expression (red) on day 5 of Dox treatment, which remained until day 14 when synaptopodin expression (green) started to decrease. By days 21 and 30 of Dox treatment, there was an obvious decrease in both FLAG and synaptopodin. Bars = 10 μm. (c) Periodic acid–Schiff (PAS) staining of a high-responder mouse treated with Dox for up to 30 days. Glomeruli showed a time-dependent progression of glomerulosclerosis. Bars = 60 μm (top) and 10 μm (bottom). (d) PAS staining of a high-responder mouse at day 30 showing pathologic changes, including glomerulosclerosis (*), cast formation (**), and tubular dilatation (arrow). Bar = 30 μm. (e) Sclerosis score for glomeruli from high-responder mice treated with Dox for 30 days. The glomeruli were scored using a numerical system (see Materials and Methods). Control: 0.10 ± 0.07; high responder: 1.22 ± 0.35. Further, 10–20 glomeruli were scored per section, n = 5 mice, *P < 0.05. DAPI, 4′,6-diamidino-2-phenylindole. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2017 92, 349-364DOI: (10.1016/j.kint.2017.03.010) Copyright © 2017 International Society of Nephrology Terms and Conditions

Figure 2 Prolonged Ras-related C3 botulinum toxin substrate 1 (Rac1) hyperactivation causes persistent proteinuria and histologic changes similar to focal segmental glomerulosclerosis. (a) The urine albumin:creatinine ratio (ACR) time course for 4 high-responder mice treated with doxycycline (Dox) for 30 days demonstrated heavy and persistent proteinuria. (b) Immunostaining of a high-responder mouse clearly showed FLAG expression (red) on day 5 of Dox treatment, which remained until day 14 when synaptopodin expression (green) started to decrease. By days 21 and 30 of Dox treatment, there was an obvious decrease in both FLAG and synaptopodin. Bars = 10 μm. (c) Periodic acid–Schiff (PAS) staining of a high-responder mouse treated with Dox for up to 30 days. Glomeruli showed a time-dependent progression of glomerulosclerosis. Bars = 60 μm (top) and 10 μm (bottom). (d) PAS staining of a high-responder mouse at day 30 showing pathologic changes, including glomerulosclerosis (*), cast formation (**), and tubular dilatation (arrow). Bar = 30 μm. (e) Sclerosis score for glomeruli from high-responder mice treated with Dox for 30 days. The glomeruli were scored using a numerical system (see Materials and Methods). Control: 0.10 ± 0.07; high responder: 1.22 ± 0.35. Further, 10–20 glomeruli were scored per section, n = 5 mice, *P < 0.05. DAPI, 4′,6-diamidino-2-phenylindole. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2017 92, 349-364DOI: (10.1016/j.kint.2017.03.010) Copyright © 2017 International Society of Nephrology Terms and Conditions

Figure 3 Prolonged Ras-related C3 botulinum toxin substrate 1 (Rac1) hyperactivation causes urinary podocyte shedding. (a) Immunofluorescence showed that the number of Wilms tumor 1 (WT1)–positive nuclei (green) decreased after 30 days of doxycycline (Dox) treatment in high-responder mice compared with control mice. Bars = 10 μm. (b) Podocyte densities in control and high-responder mice after 5 and 30 days of Dox treatment. Values are the average of 10 glomeruli per kidney section, n = 3–7 mice, *P < 0.05 versus control. (c) Urinary protein on day 30 of Dox treatment was precipitated with trichloroacetic acid and immunoblotted for WT1 and FLAG. Both were detected in high-responder mice but not in control mice. DAPI, 4′,6-diamidino-2-phenylindole. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2017 92, 349-364DOI: (10.1016/j.kint.2017.03.010) Copyright © 2017 International Society of Nephrology Terms and Conditions

Figure 4 High- and low-responder mice display variable rates of recovery from proteinuria. (a) Three high-responder mice were treated with doxycycline (Dox) for 5 days, following which Dox was withdrawn. The urine albumin:creatinine ratio (ACR) was then monitored for 4 additional weeks after Dox withdrawal. All 3 mice showed a sharp decline in ACR, with 1 mouse completely recovering and the remaining 2 mice recovering to near basal proteinuria levels. (b) Four low-responder mice were treated with Dox for 5 days, following which Dox was withdrawn. The urine ACR was then monitored for 2 additional weeks. All 4 mice showed a sharp decline in ACR, with 3 mice completely recovering after 1 week. (c,d) Representative periodic acid–Schiff staining of high-responder mice (c; at day 33 from a) and low-responder mice (d; at day 19 from b). Despite the near-complete recovery of proteinuria, high-responder mice showed some glomerulosclerosis, whereas the histology of low-responder mice was normal. Bar = 20 μm. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2017 92, 349-364DOI: (10.1016/j.kint.2017.03.010) Copyright © 2017 International Society of Nephrology Terms and Conditions

Figure 5 Treatment of constitutively active form of Ras-related C3 botulinum toxin substrate 1 (CA-Rac1) high-responder mice with the p38 inhibitor SB-203580 protects against the development of proteinuria and associated pathologic changes. (a) Immunoblotting of glomerular lysates from high-responder mice (days 14–28 on doxycycline [Dox]) showed a significant increase in the phosphorylated p38 (p-p38):p38 ratio, reflecting the activation of p38 in the glomeruli (control: 1.0 ± 0.1, high responder: 4.8 ± 1.6, *P < 0.05 vs. control, n = 5 mice). (b) Immunofluorescence staining showed increased p-p38 in high-responder mice (second from the top) and focal segmental glomerulosclerosis (FSGS) patients, mostly colocalizing with Wilms tumor 1 (WT1), indicating the activation of p38 in podocytes. Bar = 10 μm. (c). High-responder mice were treated with SB-203580 (10 mg/kg/d, i.p.) or the vehicle (phosphate-buffered saline) from day 3 until day 14 of Dox treatment. The urine albumin:creatinine ratio (ACR) on day 14 was significantly reduced in SB-203580-treated mice. *P < 0.05 versus vehicle, n = 3–4 mice. (d) Podocyte densities in control and high-responder mice treated with or without SB-203580. *P < 0.01 versus control, **P < 0.05 versus vehicle, n = 20 glomeruli from 2 mice per group. (e) Periodic acid–Schiff staining showed that SB-203580-treated mice had less glomerulosclerosis than vehicle-treated mice. Left: representative images; right: quantification of sclerosis score. *P < 0.01 versus control, **P < 0.01 versus vehicle, n = 50–58 glomeruli from 2 mice per group. Bar = 50 μm. (f) Immunoblots of the glomerular lysates showed that high-responder mice (vehicle treated, at day 14 on Dox) had a decreased level of synaptopodin, which was inhibited by SB-203580, but a comparable expression level of the transgene (FLAG). The level of phosphorylated mitogen-activated protein kinase-activated protein kinase 2 (p-MAPKAPK2), a downstream effector of p38, was markedly diminished, demonstrating the effective inhibition of p38 by SB-203580. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2017 92, 349-364DOI: (10.1016/j.kint.2017.03.010) Copyright © 2017 International Society of Nephrology Terms and Conditions

Figure 5 Treatment of constitutively active form of Ras-related C3 botulinum toxin substrate 1 (CA-Rac1) high-responder mice with the p38 inhibitor SB-203580 protects against the development of proteinuria and associated pathologic changes. (a) Immunoblotting of glomerular lysates from high-responder mice (days 14–28 on doxycycline [Dox]) showed a significant increase in the phosphorylated p38 (p-p38):p38 ratio, reflecting the activation of p38 in the glomeruli (control: 1.0 ± 0.1, high responder: 4.8 ± 1.6, *P < 0.05 vs. control, n = 5 mice). (b) Immunofluorescence staining showed increased p-p38 in high-responder mice (second from the top) and focal segmental glomerulosclerosis (FSGS) patients, mostly colocalizing with Wilms tumor 1 (WT1), indicating the activation of p38 in podocytes. Bar = 10 μm. (c). High-responder mice were treated with SB-203580 (10 mg/kg/d, i.p.) or the vehicle (phosphate-buffered saline) from day 3 until day 14 of Dox treatment. The urine albumin:creatinine ratio (ACR) on day 14 was significantly reduced in SB-203580-treated mice. *P < 0.05 versus vehicle, n = 3–4 mice. (d) Podocyte densities in control and high-responder mice treated with or without SB-203580. *P < 0.01 versus control, **P < 0.05 versus vehicle, n = 20 glomeruli from 2 mice per group. (e) Periodic acid–Schiff staining showed that SB-203580-treated mice had less glomerulosclerosis than vehicle-treated mice. Left: representative images; right: quantification of sclerosis score. *P < 0.01 versus control, **P < 0.01 versus vehicle, n = 50–58 glomeruli from 2 mice per group. Bar = 50 μm. (f) Immunoblots of the glomerular lysates showed that high-responder mice (vehicle treated, at day 14 on Dox) had a decreased level of synaptopodin, which was inhibited by SB-203580, but a comparable expression level of the transgene (FLAG). The level of phosphorylated mitogen-activated protein kinase-activated protein kinase 2 (p-MAPKAPK2), a downstream effector of p38, was markedly diminished, demonstrating the effective inhibition of p38 by SB-203580. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2017 92, 349-364DOI: (10.1016/j.kint.2017.03.010) Copyright © 2017 International Society of Nephrology Terms and Conditions

Figure 6 Chemical activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) in podocytes causes cell detachment in vitro. (a) Mouse podocytes grown on laminin-521–coated plates were treated with the indicated chemicals for 16 hours and the remaining cells were then counted, as detailed in the Materials and Methods. *P < 0.05 versus ML141 + C3, **P < 0.05 versus ML141 + C3 + CN04, n = 5. (b) Mouse podocytes with doxycycline (Dox)-inducible green fluorescent protein–constitutively active form of Rac1 (GFP-CA-Rac1) were grown on laminin-521–coated coverslips and treated with Dox (0.1 μg/ml) or left untreated (control) in the presence or absence of SB-203580 (10 μM) for 16 hours. Immunostaining and quantification of β1 integrin at the bottom of the cells were performed as outlined in the Materials and Methods. For Dox-treated conditions, only green cells expressing GFP-CA-Rac1 were quantified. *P < 0.05 versus the other treatments, n = 15 cells each pooled from 3 independent experiments. Kidney International 2017 92, 349-364DOI: (10.1016/j.kint.2017.03.010) Copyright © 2017 International Society of Nephrology Terms and Conditions

Figure 7 Ras-related C3 botulinum toxin substrate 1 (Rac1) is activated in glomeruli and podocytes of patients with minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). (a) Kidney biopsy specimens were immunostained for Rac-guanosine triphosphate (GTP) (active Rac, red), nephrin (green), and 4′,6-diamidino-2-phenylindole (DAPI; blue). The normal control showed minimal staining for Rac-GTP in glomeruli, which is consistent with the low degree of Rac1 activity. In MCD and idiopathic FSGS patients, Rac-GTP staining was markedly increased, and most of the staining overlapped with nephrin, suggesting Rac1 activity in podocytes. Bar = 40 μm. (b) Cultured human podocytes were incubated with sera (10% in culture medium) from a healthy individual (healthy control) or 2 FSGS patients (FSGS1 and FSGS 2) for 15 minutes, and the Rac1 activity in the cell lysates was determined by glutathione S-transferase (GST)–Cdc42- and Rac-interactive binding motif (CRIB) pulldown, as outlined in the Materials and Methods. Sera from FSGS patients significantly increased the Rac1 activity compared with those from the healthy control. P < 0.05 versus healthy control, n = 4 repeated assays. (c) Similarly to (b), cultured human podocytes were incubated with sera (10% in culture medium) from a healthy control or 2 MCD patients in relapse (MCD1 and MCD2) for 15 minutes, and phosphorylated p21-activated kinase (p-PAK) was quantified as a surrogate marker of Rac1 activation. The p-PAK:total PAK ratio was significantly increased by sera from MCD patients. *P < 0.05 versus healthy control, n = 3 repeated assays. (d) Cultured human podocytes were incubated with sera from FSGS patients, as outlined in (c) and (d), and phosphorylated p38 (p-p38) and p38 in the cell lysates were detected and quantified using immunoblotting. The p-p38:total p38 ratio was significantly increased by sera from FSGS patients. *P < 0.05 versus healthy control; **P < 0.01 versus healthy control; values are the means of 3 replicates. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International 2017 92, 349-364DOI: (10.1016/j.kint.2017.03.010) Copyright © 2017 International Society of Nephrology Terms and Conditions