Abstract: SA-PO307

Module IV-Defected Mutant CCN2 Knock-In Transgenic Mice Grow and Develop Normally, but Fibrotic Properties Are Attenuated in a Number of Kidney Diseases

Session Information

Category: Cell Biology

  • 204 Extracellular Matrix Biology, Fibrosis, Cell Adhesion

Authors

  • Inoue, Tsutomu, Saitama Medical University, Iruma-gun, Saitama, Japan
  • Atsushi, Ono, Saitama Medical University, Iruma-gun, Saitama, Japan
  • Okada, Hirokazu, Saitama Medical University, Iruma-gun, Saitama, Japan
Background

CCN2 has been considered as important therapeutic target for CKD. It also plays a part in wound healing and metabolism in hard tissues, thus inhibition of the overall function of CCN2 is not practical as a treatment. Therefore, we are focusing on the modules that are responsible for fibrosis in the kidney.

Methods

As previously reported, we found that module IV-defective CCN2 expressed in tubular epithelial cells did not induce fibronectin synthesis in other cells. A CCN2 knockout mouse is potentially lethal. We next generated exon 5-deleted CCN2 gene knock-in mice (CCN2Ex5-/-), in which mutant CCN2 was expressed under the same control as the wild type. This experimental strategy allows us to investigate the fibrotic properties of CCN2 in detail. Unilateral ureter obstruction (UUO), subtotal nephrectomy (5/6Nx) and ischemic reperfusion (IRI) models were employed.

Results

In the UUO model, interstitial fibrosis was progressed at 8 and 24 hours, and 7 days in a time-dependent manner. The fibrotic area detected by Masson trichrome staining was significantly reduced in CCN2Ex5-/- mice. Expression of mutant CCN2 was elevated in CCN2Ex5-/- mice in comparison to CCN2Ex5+/+ mice. On the other hand, the expression of type I collagen (0.86 ± 0.28 vs. 0.52 ± 0.18), TGF-β1 (0.35 ± 0.07 vs. 0.23 ± 0.06), PAI-1 (0.13 ± 0.05 vs. 0.06 ± 0.04) were significantly decreased in CCN2Ex5-/- mice (p<0.05, Mann-Whitney U test). In the IRI model, although there was no significant difference in fibrosis or cytokine expression in the acute phase (at 3 days), both the progression of fibrosis and expression of related cytokines were significantly suppressed in CCN2Ex5-/- mice in the chronic phase (2 weeks). The same results were obtained with the 5/6Nx model. Western blot analysis using UUO 24-hour whole kidney samples revealed that there were no significant differences in signaling alterations in previously-reported cascades, including MAPK (ERK, p38, JNK) and Wnt/β-cat, between CCN2Ex5+/+ and CCN2Ex5-/-.

Conclusion

As module IV-defective CCN2 probably acts in a dominant-negative manner, the progression of interstitial fibrosis was suppressed in our transgenic mice. Also, this study indicates that module IV specifically contributes to the progression of CKD regardless of the type of primary disease process.

Funding

  • Government Support - Non-U.S.