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Abstract: PO2466

CCN2/CTGF Causes Renal Fibrosis Progression Through the Integrin/FAK Signal Pathway

Session Information

Category: CKD (Non-Dialysis)

  • 2103 CKD (Non-Dialysis): Mechanisms

Authors

  • Fukaya, Daichi, Saitama Ika Daigaku, Iruma-gun, Saitama, Japan
  • Inoue, Tsutomu, Saitama Ika Daigaku, Iruma-gun, Saitama, Japan
  • Amano, Hiroaki, Saitama Ika Daigaku, Iruma-gun, Saitama, Japan
  • Watanabe, Yusuke, Saitama Ika Daigaku, Iruma-gun, Saitama, Japan
  • Okada, Hirokazu, Saitama Ika Daigaku, Iruma-gun, Saitama, Japan
Background

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that accumulates with integrins at focal adhesions and is involved in intracellular signal transduction. We have focused on CCN2, and demonstrated that FAK phosphorylation is also reduced when the progression of fibrosis is suppressed in a tubular cell-specific CCN2 knockdown mouse. In this study, we examined in greater detail the relationship between CCN2 and FAK.

Methods

A mouse ischemia-reperfusion (IRI) model, cultured human renal tubular epithelial cells (HK-2), 3 types of anti-phosphorylated FAK antibody (Y397, Y576 / 577, Y925), and anti-total FAK antibody were used to perform Western blotting. Furthermore, we examined what subunits of the integrin were expressed in HK-2 by using RT-PCR. A specific neutralizing antibody against integrin was also used to suppress the binding of CCN2 to integrins.

Results

A significant increase in total FAK was observed in the chronic phase (day 12) as fibrosis progressed (total FAK/GAPDH; control 0.81 ± 0.10 vs. day 12 1.96 ± 0.20). Among the phosphorylated FAK (pFAK), Y397 was particularly significant (pFAK/FAK: control 0.39 ± 0.01 vs. day 12 0.75 ± 0.17). Positive staining for pFAK was observed in tubular epithelial cells. In serum-stimulated HK-2, FAK was phosphorylated, but the addition of the decoy peptide of the CCN2 VI-module decreased the amount of Y397. Results of RT-PCR confirmed the expression of several integrin subunits. The results of studies using neutralizing antibodies revealed that the decrease in pFAK was most remarkable when the anti-integrin αv antibody was added (pFAK/FAK: control 0.99 ± 0.13 vs. 0.48 ± 0.06 after addition of the antibody).

Conclusion

By using the IRI model, we found that not only the expression of FAK was increased but also its phosphorylation was promoted in the injured kidney. CCN2 produced in tubular epithelial cells acts via cellular integrin αv in autocrine/paracrine manner, and promotes renal fibrosis through phosphorylation of the tyrosine 397 residue of FAK. CCN2 has been previously shown to activate Wnt/β-catenin and TGF-β/Smad pathways. However, here we identified another pathway for CCN2 in relation to kidney fibrosis. Several FAK inhibitors have already been investigated as anticancer agents. Further clarification of the pathways may prove the therapeutic effects of these inhibitors on CKD.

Funding

  • Government Support – Non-U.S.