Kidney Week

Abstract: FR-PO747

GIT2 Maintains Podocyte Architecture through Suppressing Rac1 Activity and Focal Adhesion Turnover

Session Information

• Glomerular Diseases: Mechanisms and Podocyte Biology
  October 25, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology

Authors

• Shimada, Naoyuki, Department of Nephrology, Osaka University Graduate School of Medicine, Suita, Japan

Naoyuki Shimada, MD

• Matsuda, Jun, Department of Nephrology, Osaka University Graduate School of Medicine, Suita, Japan

Jun Matsuda, MD, PhD

• Tokuchi, Maho, Department of Nephrology, Osaka University Graduate School of Medicine, Suita, Japan

Maho Tokuchi, MD

• Asano-Matsuda, Kana, Department of Nephrology, Osaka University Graduate School of Medicine, Suita, Japan

Kana Asano-Matsuda, MD

• Takano, Tomoko, Division of Nephrology, McGill University Health Centre, Montreal, Quebec, Canada

Tomoko Takano, MD, PhD

• Isaka, Yoshitaka, Department of Nephrology, Osaka University Graduate School of Medicine, Suita, Japan

Yoshitaka Isaka, MD, PhD

Background

Podocyte foot processes are supported by the actin cytoskeleton which is precisely organized by Rho GTPases including Rac1. Overactivation of Rac1 in podocytes causes structural changes in foot processes and proteinuria. To elucidate the regulatory mechanism of Rac1, we performed proximity-based ligation assay and identified GIT2 as a Rac1-interacting protein in immortalized human podocytes (HP).

Methods

Subcellular localization of endogenous GIT2 in HP was examined by immunocytochemistry. HP with GIT2 knockdown (KD), GIT2 overexpression (OE) and control (CTRL) cells were established using lentiviral transduction. Rac1 activity was assessed by pull-down assay. Cell areas were quantified in fixed cells stained by phalloidin. Focal adhesion dynamics were analyzed using time-lapse fluorescence microscopy in cells transfected with mRFP-paxillin. Subcellular localization of the tyrosine phosphatase, PTP1B, was monitored using time-lapse fluorescence microscopy.

Results

GIT2 localized with paxillin in focal adhesions. GTP-bound (active) Rac1 levels were significantly higher in GIT2 KD HP than in CTRL. GIT2 KD elicited cell spreading with marked lamellipodial protrusions, which was significantly attenuated by the Rac1 inhibitor NSC23766. GIT2 KD HP had smaller focal adhesions than CTRL. Focal adhesion disassembly rate was significantly higher in GIT2 KD HP, resulting in shorter focal adhesion lifetime. Among the major focal adhesion proteins, tyrosine phosphorylation of p130Cas protein was most prominently increased in GIT2 KD HP, as compared with CTRL. PTP1B localization to focal adhesions was impaired in GIT2 KD HP. The phenotypes observed in GIT2 KD HP shown above were reversed by GIT2 OE.

Conclusion

We demonstrated that GIT2 suppresses Rac1 activity in focal adhesions and contributes to maintaining podocyte morphology and function. Furthermore, we showed that GIT2 facilitates translocation of the PTP1B to focal adhesions where it dephosphorylates p130Cas, thereby suppressing local Rac1 activity.