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Abstract: TH-PO1091

ARHGEF7 (β-PIX) Protects Podocytes from Cell Apoptosis via Cdc42 Activation

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Matsuda, Jun, McGill University Health Centre, Montreal, Quebec, Canada
  • Maier, Mirela, McGill University Health Centre, Montreal, Quebec, Canada
  • Aoudjit, Lamine, McGill University Health Centre, Montreal, Quebec, Canada
  • Takano, Tomoko, McGill University Health Centre, Montreal, Quebec, Canada
Background

Rho-family small GTPases (Rho GTPases), including Rac1 and Cdc42, are important for podocyte functions. β-PIX, encoded by ARHGEF7, is a guanine nucleotide exchange factors (GEF) that activates Rac1 and Cdc42. Recently, we established that podocyte-specific β-PIX deficient (KO) mice develop progressive proteinuria starting at ~8 weeks of age, and glomerulosclerosis and podocyte loss by 13 weeks of age. Here, we investigated the mechanism of podocyte loss induced by β-PIX deficiency, with a particular focus on the pro-survival transcriptional co-activator, Yes-associated protein (Yap), which is known to be activated by Cdc42.

Methods

Cultured mouse podocytes (MP) with β-PIX knockdown (KD) and their controls were established using shRNAs. Rac1/Cdc42 activity was assessed by pull-down assay. Rho/Rac/Cdc42 Activator I (Cytoskeleton, CN04) and Adriamycin (ADR) were used as a general Rho GTPase activator and a podocyte toxin, respectively. Apoptosis was studied by TUNEL staining and immunoblotting for cleaved caspase 3 (CC3). Nuclear translocation and activity of Yap were assessed by immunostaining and the TEA domain (TEAD)-luciferase assay. Data are provided as the mean ± SE.

Results

Cdc42, but not Rac1 activity, was significantly decreased by 34% in isolated glomeruli from 5-week-old β-PIX KO mice, compared with controls (n=7-10, p<0.05). Similarly, Cdc42, but not Rac1, activation in β-PIX KD MP was significantly blunted by 46%, compared with control MP (n=4, p<0.01). At 6 hrs after ADR treatment, KD MP showed 9.4±2.5 % TUNEL-positive cells, as compared with 3.8±0.7 % in control MP (n=3, p<0.05). CC3 was also increased in KD MP. Correspondingly, there were less adherent KD MP at 8 hrs of ADR treatment, compared with control MP. Control MP showed predominant nuclear localization of Yap, while KD MP exhibited partial cytoplasmic retention (Nuclear/Total Yap ratio; Control: 0.90±0.02; KD: 0.80±0.04, n=10, p<0.05). The TEAD-luciferase activity was significant lower in KD MP than in control MP (Control: 10.3±1.3; KD: 6.9±0.76 a.u., n=9, p<0.05). mRNA expression of the Yap target genes by RNA-seq were significantly reduced in the glomeruli of KO mice.

Conclusion

Loss of β-PIX in podocytes resulted in increased apoptosis and detachment from matrix. This may be mediated by the cytoplasmic retention and decreased transcriptional activity of Yap via decreased Cdc42 activity.