Q CAO1, C HUANG1, H YI1, S STANGENBERG1, M FOLEY2,3, X-M CHEN1, CA POLLOCK1
1Kolling Institute of Medical Research, Royal North Shore Hospital, Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia; 2AdAlta Pty. Ltd., 15/2 Park Dr., Bundoora, Victoria, Australia; 3Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia
Aim: To define the role of i-body AD-114 in renal fibrosis.
Background: Fibrosis is the final common pathway of various types of chronic kidney disease (CKD). CXCR4 has been demonstrated to be a central player in the development of tissue fibrosis. i-body AD-114, which binds CXCR4 with high specificity and affinity, 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) for 48 hours, and 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 TGFβ1 (2ng/ml) in the absence or presence of AD-114 (1mM or 3mM) for 48 hours. The supernatant and cell lysis protein were collected and fibronectin (FN) and collagen 4 (Col 4) were measured by Western-blotting.
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 compared to a lower concentration of AD-114 (1µM) and negative control i-body (P<0.001, n=4).
Conclusions: Blocking CXCR4 using the i-body AD-114 is a promising therapeutic strategy to prevent the development of CKD.