Protein Tyrosine Phosphatase 1B Inhibition Protects against Podocyte Injury and Proteinuria  Takanori Kumagai, Cindy Baldwin, Lamine Aoudjit, Lisa Nezvitsky, Richard Robins, Ruihua Jiang, Tomoko Takano  The American Journal of Pathology  Volume 184, Issue 8, Pages 2211-2224 (August 2014) DOI: 10.1016/j.ajpath.2014.05.005 Copyright © 2014 American Society for Investigative Pathology Terms and Conditions

Figure 1 PTP1B is up-regulated in podocytes in the models of podocyte injury. A: Sprague-Dawley rats were injected with PAN or vehicle (control) and analyzed after 1 week. Immunoblot analysis of the glomerular lysates shows up-regulation of PTP1B in PAN rats. Kidney sections were stained with PTP1B (red); the podocyte foot process marker, synaptopodin (green); the podocyte nuclear marker, WT1 (green); and a nuclear dye, DAPI (blue). Arrows indicate likely podocyte staining; double arrows, podocyte nuclei double-stained with WT1 and DAPI. B: Mice were injected with vehicle (control), 12.5 mg/kg of ADR, or 200 μg of LPS, and the kidneys were stained with PTP1B (red) and synaptopodin (green) after 1 week (control and ADR) or 24 hours (LPS). Arrows indicate likely podocyte staining. The American Journal of Pathology 2014 184, 2211-2224DOI: (10.1016/j.ajpath.2014.05.005) Copyright © 2014 American Society for Investigative Pathology Terms and Conditions

Figure 2 Establishment of PTP1Bpod−/− mice. A: Genomic DNA analysis by PCR. Deletion of the loxP-flanked sequence in the PTP1B gene resulted in a 458-bp amplicon, which was detected in the glomerular (glom) DNA, but not in the tail DNA, of PTP1Bpod−/− mice. The band was not detected in control mice. B: Glomerular lysates of control and PTP1Bpod−/− mice, by immunoblot (IB) analysis for phosphorylation of nephrin at Y1232 (pNephrinY1232) and total Nephrin. C: Podocytes outgrowing from decapsulated glomeruli were obtained as described previously,4 and cell lysates were subjected to immunoblot analysis for PTP1B and tubulin (loading). Each lane represents cell lysates from different mice. ∗∗P < 0.01 versus controls. n = 4 per group (B). GADPH, glyceraldehyde-3-phosphate dehydrogenase; a.u., arbitrary unit. The American Journal of Pathology 2014 184, 2211-2224DOI: (10.1016/j.ajpath.2014.05.005) Copyright © 2014 American Society for Investigative Pathology Terms and Conditions

Figure 3 PTP1Bpod−/− mice are resistant to proteinuria. A: Urinary ACRs were not statistically significantly different between PTP1Bpod−/− mice and control littermates, in both sexes at up to 7.5 months of age. B: Urinary ACRs of PTP1Bpod−/− mice and control littermates injected with LPS at the indicated times. Urinary ACR is significantly reduced in PTP1Bpod−/− mice compared to that in control mice at 24 hours after LPS injection. C: Electron microscopy shows that control mice injected with LPS demonstrated diffuse foot process effacement, which was reduced in PTP1Bpod−/− mice. D: Quantification of foot process widths (FPWs) of PTP1Bpod−/− and control mice with (+) and without (−) LPS. E: PTP1Bpod−/− and control mice were injected with 12.5 mg/kg of ADR, and urinary ACRs were quantified at the baseline and 1 week after the injection. PTP1Bpod−/− mice (inverted triangles) were resistant to proteinuria induced by ADR, whereas control (Cntl) mice (triangles) were not. Data are expressed as means ± SEM (B and D). Bar and error bars indicate means ± SEM (E). n = 5 per group (B); n = 3 per group (D); n = 16 in the PTP1Bpod−/− group, n = 12 in the control group (E). ∗P < 0.05, ∗∗P < 0.01 versus control; ††P < 0.01 versus control + LPS. Original magnification: ×6800 (C). The American Journal of Pathology 2014 184, 2211-2224DOI: (10.1016/j.ajpath.2014.05.005) Copyright © 2014 American Society for Investigative Pathology Terms and Conditions

Figure 4 PTP1B inhibitor ameliorates LPS-induced proteinuria and foot process effacement. A: In WT mice, proteinuria induced by LPS (as in Figure 3) was significantly reduced by 10 mg/kg PTP1B inhibitor. B: Representative electron micrographs show LPS-induced foot process effacement (arrow), which is reversed by the PTP1B inhibitor. C: Quantification of foot process widths (FPWs) of the three groups. Data are expressed as means ± SEM (A and C). n = 4 in the LPS + vehicle group, n = 5 in the LPS + PTP1B inhibitor group (A); n = 3 per group (C). ∗P < 0.05; ∗∗P < 0.01 versus normal; ††P < 0.01 versus LPS + vehicle. Original magnification: ×6800 (B). The American Journal of Pathology 2014 184, 2211-2224DOI: (10.1016/j.ajpath.2014.05.005) Copyright © 2014 American Society for Investigative Pathology Terms and Conditions

Figure 5 PTP1B transgene expression in podocytes induces proteinuria and foot process effacement in male mice. A: Kidney sections stained with FLAG (for PTP1B) (red) and synaptopodin (green) show expression of the transgene (FLAG-PTP1B) in podocytes in transgenic mice (PTP1BpodTG). PTP1B expression was confirmed in transgenic mice. Expression of FLAG-PTP1B in the transgenic mice was also confirmed by immunoblot analysis of the glomerular lysates. B: Urinary ACR was significantly increased in male transgenic mice compared to that in control littermates at 3 months of age. C: Electron micrographs show area of increased foot process effacement (arrow) in male transgenic mice. D: Quantification of foot process widths (FPWs). n = 4 per group (B); n = 3 per group (D). ∗∗P < 0.01 versus control littermates. Original magnification: ×6800 (C). The American Journal of Pathology 2014 184, 2211-2224DOI: (10.1016/j.ajpath.2014.05.005) Copyright © 2014 American Society for Investigative Pathology Terms and Conditions

Figure 6 PTP1B knockdown and inhibition in cultured mouse podocytes decreases cell migration. A: Mouse podocytes were stably transfected with a PTP1B shRNA (PTP1B-KD) or a control shRNA (control) and treated with 20 μg/mL of LPS (+) or left untreated (−) for 24 hours. Cell lysates were blotted for pFAKY397, total FAK, PTP1B, and tubulin. Densitometric analyses (PTP1B/tubulin and pFAKY397/total FAK) of the four experiments are shown. B: Wound healing was compared between control and PTP1B knockdown podocytes with (+) or without (−) LPS. C: Control podocytes were (+) or were not (−) stimulated with LPS for 24 hours in the presence of 50 μmol/L of PTP1B inhibitor or inactive vehicle, and cell lysates were blotted for pFAKY397 and total FAK. D: Control podocytes were stimulated with LPS with (+) and without (−) PTP1B inhibitor, and wound healing was compared. Data are expressed as means ± SEM (A–D). n = 3 per group (B and D); n = 4 per group (A and C). ∗P < 0.05, ∗∗P < 0.01 versus control/LPS−; †P < 0.05, ††P < 0.01 versus control/LPS+. The American Journal of Pathology 2014 184, 2211-2224DOI: (10.1016/j.ajpath.2014.05.005) Copyright © 2014 American Society for Investigative Pathology Terms and Conditions

Figure 7 Activation of FAK by LPS is reduced in vivo by podocyte-specific PTP1B deletion or by the PTP1B inhibitor. PTP1Bpod−/− mice and control littermates were (+) or were not (−) injected with LPS, with (+) or without (−) PTP1B inhibitor. Glomerular lysates were blotted for pFAKY397 and total FAK. LPS increased pFAKY397 in control mice, but the response was blunted in PTP1Bpod−/− mice and in mice treated with the PTP1B inhibitor. PTP1B inhibitor does not affect the basal pFAKY397 in the glomerulus. Data are expressed as means ± SEM. n = 4 per group (left panel); n = 3 per group (right panel). ∗∗P < 0.01 versus control/LPS−/PTP1B inhibitor−; ††P < 0.01 versus control/LPS+/PTP1B inhibitor−. The American Journal of Pathology 2014 184, 2211-2224DOI: (10.1016/j.ajpath.2014.05.005) Copyright © 2014 American Society for Investigative Pathology Terms and Conditions

Figure 8 PTP1B activates SFK by dephosphorylating SrcY527. HEK293T cells were transfected with Fyn (SFK expressed in podocytes) and glutathione S-transferase (GST)-PTP1B-WT or GST-PTP1B-DA (substrate-trapping mutation), and GST pulldown (PD) was performed. A: The DA mutation, but not the WT PTP1B, pulls down Fyn. Fyn that was pulled down was phosphorylated at Y527. B: Mouse podocytes were (+) or were not (−) stimulated with LPS and with (+) or without (−) 50 μmol/L of PTP1B inhibitor for 24 hours, and cell lysates were blotted for the indicated antibodies. C: Mouse podocytes were (+) or were not (−) stimulated with LPS and with or without SU6656 (2.5 μmol/L of SFK inhibitor) for 24 hours, and cell lysates were blotted for the indicated antibodies. D: PTP1Bpod−/− mice and control littermates were (+) or were not (−) injected with LPS and with or without PTP1B inhibitor. Glomerular lysates were blotted for nonphosphoSrcY527 and Fyn. Data are expressed as means ± SEM. n = 3 per group (B and C); n = 3 or 4 (control/LPS+/PTP1B inhibitor+) per group (D). ∗P < 0.05, ∗∗P < 0.01 versus control; ††P < 0.01 versus LPS+/PTP1B inhibitor− (B); †P < 0.05 versus LPS+/SU6656− (C); ∗P < 0.05 versus control/LPS−/PTP1B inhibitor−, ††P < 0.01 versus control/LPS+/PTP1B inhibitor− (D). IB, immunoblot analysis; TL, total lysates; n.s., non-specific band. The American Journal of Pathology 2014 184, 2211-2224DOI: (10.1016/j.ajpath.2014.05.005) Copyright © 2014 American Society for Investigative Pathology Terms and Conditions

Figure 9 PTP1B transgene expression in podocytes in mice induces activation of SFK and FAK and decreases nephrin phosphorylation. A: Glomerular lysates of PTP1BpodTG male mice and control littermates were analyzed by immunoblot (IB) analysis for nonphosphoSrcY527 and Fyn. PTP1BpodTG mice had increased nonphosphoSrcY527, corresponding to the increased SFK activity. B: Glomerular lysates were analyzed by immunoblot analysis for pFAKY397 and total FAK. PTP1BpodTG mice had increased pFAKY397, corresponding to the increased FAK activity. C: Glomerular lysates were analyzed by immunoblot analysis for phosphorylation of nephrin at Y1232 (pNephrinY1232) and total Nephrin. PTP1BpodTG mice had decreased pNephrinY1232. Data are expressed as means ± SEM. n = 3 per group (A–C). ∗∗P < 0.01. The American Journal of Pathology 2014 184, 2211-2224DOI: (10.1016/j.ajpath.2014.05.005) Copyright © 2014 American Society for Investigative Pathology Terms and Conditions

Figure 10 Proposed mechanisms for the contribution of PTP1B to proteinuria. The carboxy terminal Src tyrosine kinase (Csk) phosphorylates the inhibitory phosphorylation site of the SFK, SrcY527, thereby inhibiting its activity. In proteinuric kidney disease, PTP1B is up-regulated in podocytes, which results in dephosphorylation of SrcY527 and activation of SFKs. Activated SFK mediates phosphorylation of the activation loop of FAK, leading to its activation. FAK activation in podocytes results in migratory phenotype, leading to foot process effacement and proteinuria. The American Journal of Pathology 2014 184, 2211-2224DOI: (10.1016/j.ajpath.2014.05.005) Copyright © 2014 American Society for Investigative Pathology Terms and Conditions