Inhibition of lysyl oxidase‑like 2 ameliorates folic acid‑induced renal tubulointerstitial fibrosis

Tubulointerstitial fibrosis is characterized by accumulation of the extracellular matrix in the interstitium. Lysyl oxidase-like 2 (LOXL2), a member of the lysyl oxidase family, is known for promoting cancer metastasis, invasion and stromal fibrosis in various organs. Our previous study demonstrated expression of LOXL2 in kidney podocytes and tubular epithelial cells, and the association between elevated LOXL2 and tubulointerstitial fibrosis. The present study evaluated the effect of LOXL2 inhibition using an inhibitory monoclonal antibody (AB0023) on tubulointerstitial fibrosis in a folic acid-induced tubulointerstitial fibrosis mouse model. The association of LOXL2 with epithelial-mesenchymal transformation-related molecules was also evaluated in vitro using HK-2 cells. The present data demonstrated that AB0023 prevented the progression of tubulointerstitial fibrosis significantly, as determined by trichrome and picro-sirius red staining, as well as the total collagen assay. The mean expression of phosphorylated Smad2 and Smad4 was lower in the AB0023-treated group although it was not statistically significant. Following transforming growth factor-β (TGF-β) challenge, LOXL2-deficient HK-2 cells exhibited significantly lower expression of the mesenchymal markers vimentin and fibronectin than control HK-2 cells. In conclusion, LOXL2 inhibition ameliorates renal fibrosis through the TGF-β/Smad signalling pathway.


Introduction 27
As tubulointerstitial fibrosis is a common endpoint in renal disease with no effective treatment other 28 than dialysis, the need to understand the molecules and mechanisms involved is increasingly urgent. 29 Histologically, tubulointerstitial fibrosis is an accumulation of the extracellular matrix (ECM) in the 30 interstitium. ECM-producing cells are primarily activated fibroblasts [1]. Various cells such as pericytes, 31 endothelial cells, residual fibroblasts, and tubular epithelial cells are known to be the origin of 3 32 fibroblasts [2]. 33 The epithelial-mesenchymal transformation (EMT) has been studied in cancer and benign 34 fibrotic diseases [3]. Once acute injury is imposed on the kidney, various chemokines and growth factors 35 cause inflammation, which in turn leads to the secretion of transforming growth factor-β (TGF-β) via 36 release of active TGF-β from latent TGF-β-binding protein via protease cleavage [4]. TGF-β is the 37 primary molecule responsible for EMT [5,6], and the canonical and non-canonical pathways are the 38 downstream pathways of 7]. The hallmark of EMT is loss of epithelial phenotypes and 39 acquisition of mesenchymal phenotypes with activation of profibrotic genes to produce the ECM, 40 including fibronectin and types I and III collagen [3,5,8].

41
Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family, originally known as a 42 copper-dependent amine oxidase, that is involved in cross-linking collagen and elastin of the ECM [9]. 43 Studies have also demonstrated additional functions for LOXL2 independent of its catalytic activity, 44 such as organ development [10], tumour invasion [11], and EMT [12,13]. 45 In our previous study, we found that LOXL2 is expressed in kidney podocytes and tubular 46 epithelial cells, and its expression is increased in the folic acid-induced murine fibrosis model [14]. In 47 this study, we evaluated the effect and therapeutic role of LOXL2 inhibitor AB0023 on the progression 48 of tubulointerstitial fibrosis in mice. In order to evaluate the contribution of LOXL2 in EMT, an in vitro 49 study using immortalized human proximal tubular epithelial cells (HK-2 cells) was also performed. Male CD1 mice at 8 weeks of age (Orient Bio, Inc., Seongnam, South Korea) were used in this study.

55
The animals were housed in a facility maintained at 20°C and 12-h alternating light/dark cycles with 4 56 free access to rodent chow and water. Tubulointerstitial fibrosis was induced by intraperitoneal injection 57 of folic acid (240 μg/g body weight) [15,16]. The folic acid solution was prepared by dissolving folic 58 acid powder (Sigma-Aldrich) in 0.3 M NaHCO 3 . Control CD1 mice were injected intraperitoneally with 59 the same volume of vehicle (NaHCO 3 ). Urinary excretion of neutrophil gelatinase-associated lipocalin 60 (NGAL) was measured immediately before injection and at 3 days after injection using a Mouse    control IgG in the CD1 mouse model. 84 The red arrow indicates AB0023 or control IgG intraperitoneal injection (15 mg/kg). AB0023 or control 85 IgG was injected 4 and 1 day before folic acid injection, and twice weekly until 4 weeks after folic acid 86 injection. Urinary neutrophil gelatinase-associated lipocalin (NGAL) was measured 3 days after folic 87 acid injection to ensure successful induction of renal fibrosis. Germany) for 1 hour at room temperature to visualize collagen before washing with 0.5% glacial acid.    The amount of fibrosis measured by trichrome (Fig 2A) and picro-sirius red staining (Fig 2B) decreased 175 in mice treated with AB0023, compared to the control IgG-treated group (Fig 2C and D). Quantitative 176 measurement of fibrosis by total collagen analysis also showed that fibrosis decreased in mice treated 177 with AB0023 (Fig 2E). Smad signaling pathway molecules, including p-Smad3, p-Smad2, and Smad4 exhibited no significant 188 difference with LOXL2 inhibition (Fig. 3). However, the amounts of p-Smad2 and Smad4 tended to 189 decrease in the AB0023-treated group relative to the control group.  is the first study demonstrating that LOXL2 inhibition leads to attenuated fibrosis in the murine kidney 233 injry model..

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In addition to fibrosis, TGF-β is involved in various biological activities, such as cell 235 proliferation, apoptosis, differentiation, autophagy, and the immune response [21]. Thus, it is critical to 236 investigate therapeutic strategies related to the downstream pathways of TGF-β due to the possible 237 adverse effects of directly targeting this cytokine [22].

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EMT is a major mechanism that contributes to renal fibrosis in response to multiple molecules, here. However, cell types and experimental methods used in this study differ from those reported 266 previously [12,13,26]. Although E-cadhrin and ZO-1 level remained insignificant, it can be inferred 267 that LOXL2 might be related to EMT pathway based on significant change of vimentin. Further studies 268 are warranted to elucidate the mechanisms underlying the LOXL2-EMT-related pathway.

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In conclusion, inhibition of LOXL2 ameliorates renal fibrosis. LOXL2 is associated with TGF-270 β-mediated tubulointerstitial fibrosis and EMT. Improved understanding of the role of LOXL2 in the 271 kidney may illuminate the pathophysiology of tubulointerstitial fibrosis and glomerulosclerosis, and 272 potentially lead to the discovery of novel therapeutic targets for treating these conditions.