Abstract: TH-PO510

A Novel I-Body AD-114 Suppressed TGFβ1-Induced Fibronectin and Collagen 4 in Renal Proximal Tubular Cells via Smad and p38 Pathways

Session Information

Category: Chronic Kidney Disease (Non-Dialysis)

  • 308 CKD: Mechanisms of Tubulointerstitial Fibrosis

Authors

  • Cao, Qinghua, Kolling Institute, Sydney, New South Wales, Australia
  • Huang, Chunling, University of Sydney , Sydney, New South Wales, Australia
  • Yi, Hao, Kolling Institute, Sydney, New South Wales, Australia
  • Stangenberg, Stefanie, None, Naremburn, New South Wales, Australia
  • Pollock, Carol A., The University of Sydney, Sydney, New South Wales, Australia
  • Chen, Xinming, University of Sydney , Sydney, New South Wales, Australia
Background

Fibrosis is the final common pathway of chronic kidney disease (CKD). CXCR4 has been demonstrated to be a central player in the development of tissue fibrosis. However, the only approved CXCR4 antagonist was terminated due to its off-target cardiotoxicity.
Our collaborator (Adalta Limited, Australia) has developed a fully human single-domain antibody-like scaffold termed i-body AD-114 with specific high binding affinity to CXCR4. AD-114 selectively blocks CXCR4 signaling and has shown anti-fibrotic effects in lung, liver and eye fibrosis. However, the role of AD-114 in renal fibrosis has not been studied.

Methods

To detect CXCR4 expression in the kidney, biopsies from patients with diabetic nephropathy (DN) and kidneys from three mouse models of CKD were collected and CXCR4 expression was detected using immunohistochemistry.
To determine the role of TGFβ1 in AD-114 binding with CXCR4 in human renal proximal tubular cells (PTCs), PTCs were incubated with/without TGFβ1 (2ng/ml) in absence or presence of AD-114 for 48 hours, and then AD-114 binding with PTC was detected using immunocytochemistry.
To examine whether AD-114 blocks TGFβ1-induced fibrotic responses in PTCs, PTCs were incubated with/without TGFβ1 (2ng/ml) in absence or presence of AD-114 (1mM or 3mM) for 48 hours and the supernatant and cell lysis protein were collected for Western blot analysis. Fibronectin (FN) and collagen 4 (Col 4) in the supernatant and phosphorylation of Smad2/3, p38 and NF-κB(p65) in cell lysis were measured by Western blot.

Results

CXCR4 expression was significantly upregulated in patients with DN and fibrotic kidneys of three mouse models of CKD compared to control groups (P<0.001, n=6). TGFβ1 significantly increased AD-114 binding to renal PTCs compared to negative control i-body and normal controls (P<0.001, n=4). AD-114 (3µM) suppressed TGFβ1-induced overexpression of FN and Col 4 via decreasing Smad2/3 and p38 phosphorylation compared to a lower concentration of AD-114 (1µM) and negative control i-body (P<0.001, n=4). However, AD-114 did not change the phosphorylation of NF-κB (p65).

Conclusion

Blocking CXCR4 using the i-body AD-114 is a promising therapeutic strategy to prevent the development of CKD.

Funding

  • Government Support - Non-U.S.