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Kidney Week

Abstract: TH-PO477

CCN2 Module IV-Derived Decoy Peptides Attenuate Renal Fibrogenesis Through Inhibition of FAK Pathway in the Tubular Epithelium

Session Information

  • CKD: Mechanisms - I
    November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
    Abstract Time: 10:00 AM - 12:00 PM

Category: CKD (Non-Dialysis)

  • 2103 CKD (Non-Dialysis): Mechanisms

Authors

  • Amano, Hiroaki, Saitama Medical University, Saitama, Japan
  • Inoue, Tsutomu, Saitama Medical University, Saitama, Japan
  • Okada, Hirokazu, Saitama Medical University, Saitama, Japan

Group or Team Name

  • Department of Nephrology, Saitama Medical University
Background

CCN2 mediates the renal fibrotic action of TGF-b. We focused on CCN2 module-IV (M4) and its contribution to fibrogenesis. We previously demonstrated that M4 activated FAK (Focal Adhesion Kinase), PI3K, Akt, GSK-3b (Glycogen Synthase Kinase-3b) and b-Catenin using knock-in mice bearing M4-deleted, mutant CCN2 gene (mCCN2 mice). Furthermore, Erk1/2 and p38 were also phosphorylated by M4. Since FAK is directly activated by integrin, we considered that M4 interacts with integrin. Thus, we created decoy peptides (DCs) that mimic partial sequences of M4 and tested their anti-fibrotic effects.

Methods

Six types of DCs were designed as unmodified peptides that divide M4 into 10-14 aa and purified by HPLC. Each DCs was administered to wild-type mice with unilateral ureter obstruction (UUO) model, and histological examination and immunohistochemistry (IHC) were performed. Next, the human proximal tubular cell line, HK-2, was cultured for 24 hours in Ham's F-12/DMEM supplemented with 5% FCS, after which the medium was replaced with serum free medium, and added dissolved DCs directly to the medium. Activation of signal pathways was assayed with Western blots.

Results

Two of six DCs, DC2 and DC5, inhibited the progression of renal fibrosis in the UUO model. IHC revealed that accumulation of p-FAK in tubular epithelium cells (TECs) was suppressed in the DC2- and DC5-groups. Next, DC5 was selected for the in-vitro experiment because its aa sequence has been conserved in mouse and human. The levels of p-FAK were significantly lowered in the DC5-treated TECs compared to the control (p-FAK/FAK: 1.0+/-0.1 vs. 0.5+/-0.1, p<0.01). The levels of p-Akt, and p-GSK-3b were also lowered (p-Akt/ Akt: 7.0+/-1.4 vs. 2.5+/-0.3, p<0.05), (p-GSK-3b/ GSK-3b: 3.2+/-0.3 vs. 1.6+/-0.6, p<0.05). Among MAPKs, only the levels of p-p38 were lowered by DC5.

Conclusion

DCs likely suppressed FAK-mediated renal fibrogenesis in mice. This finding is the same as that observed in the mCCN2 mice, suggesting that these DCs may have inhibited the interaction between M4 and integrin in TEC. The in-vitro experiments showed that down-stream signals activated by CCN2M4-Integrin-FAK pathway were PI3K-Akt-mediated phosphorylation of GSK-3b and p38 in TECs. The CCN2M4-Integrin-FAK pathway seems to be a promising, therapeutic target for attenuating renal fibrogenesis.