ATP7A-fibulin-4 complex delivers copper in the Golgi to activate LOX in renal fibrosis
Wenqian Zhou, Yan Zheng, Yuqing Liu, Jing Liu, Yiguo Liu, Yangyang Niu, Ying Yu, Xiaoqin Zhang, Yingying Zhang, Chen Yu
Wenqian Zhou, Yan Zheng, Yuqing Liu, Jing Liu, Yiguo Liu, Yangyang Niu, Ying Yu, Xiaoqin Zhang, Yingying Zhang, Chen Yu
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Research Article Cell biology Nephrology

ATP7A-fibulin-4 complex delivers copper in the Golgi to activate LOX in renal fibrosis

Abstract

Lysyl oxidase (LOX) is a copper-dependent monoamine oxidase whose primary function is the covalent cross-linking of collagen and elastin in the extracellular matrix (ECM). However, the regulation of LOX activity in renal fibrosis is not well understood. Here, our study showed that (a) LOX expression and ECM cross-linking were markedly increased in fibrotic kidneys. Reduction of copper levels in the Golgi apparatus by treatment with the copper chelator tetrathiomolybdate or by specific knockdown of copper transporter 1 (CTR1) decreased LOX activity and ameliorated renal fibrosis. (b) Overexpression of ATP7A caused an elevation of copper ions within the Golgi apparatus, resulting in increased LOX activity and enhanced ECM crosslinking, thereby promoting the progression of renal fibrosis. Knockdown of ATP7A showed the opposite result. (c) FBLN4 was essential for the ATP7A-mediated transfer of copper to LOX and formed a ternary complex of ATP7A-FBLN4-LOX. Our research revealed that high ATP7A expression induced copper overload in the Golgi apparatuses. FBLN4 then assisted ATP7A in transporting this excess copper to LOX, resulting in LOX overactivation. This, in turn, catalyzed the cross-linking of ECM components, thereby accelerating renal fibrosis.

Authors

Wenqian Zhou, Yan Zheng, Yuqing Liu, Jing Liu, Yiguo Liu, Yangyang Niu, Ying Yu, Xiaoqin Zhang, Yingying Zhang, Chen Yu

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Figure 8

Disruption of FBLN4-mediated copper transfer from ATP7A to LOX suppresses renal fibrosis.

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Disruption of FBLN4-mediated copper transfer from ATP7A to LOX suppresse...
(A) Representative images of immunohistochemical staining with FBLN4, H&E, and Masson’s trichrome in kidney sections from patients with and without renal fibrosis (n = 5). Original magnification, ×200. Scale bar: 100 μm. (B) Representative images of immunohistochemical staining with FBLN4, H&E, and Masson’s trichrome in kidney subjected to IR injury (n = 5). Original magnification, ×200. Scale bar: 100 μm. (C) Western immunoblots of FBLN4 in kidney tissues among different groups (n = 5). All mice were randomly divided into 4 groups (n = 5/each group): sham-operated mice treated with AAV9-Con (shNC + sham), sham-operated mice treated with AAV9-shFBLN4 (shFBLN4 + sham), IR mice treated with AAV9-Con (shNC + IR), and IR mice treated with AAV9- shFB LN4 (shFBLN4 + IR) (D–F). (D) LOX activity in kidney tissues was determined using the Fluorometric Lysyl Oxidase Assay Kit. (E) Western immunoblots analysis of FBLN4, aLOX, collagen I, and elastin expression in mouse kidneys among different groups. (F) Representative images of H&E and Masson’s trichrome staining of kidney sections. Original magnification, ×200. Scale bar: 100 μm. (G) LOX activity as determined using the Fluorometric Lysyl Oxidase Assay Kit in the culture medium of NRK-52E cells after Fbln4 knockdown (n = 3). (H) Western immunoblots of FBLN4 and aLOX in NRK-52E cells among different groups (n = 3). (I and J) LOX activity was determined in the culture medium of NRK-52E cells treated with CuSO4 after downregulation of Fbln4 alone and downregulation of both Atp7a and Fbln4 (n = 3). Data are shown as the mean ± SEM. Statistics used included 1-way ANOVA. **P < 0.01, ***P < 0.001.