Soluble receptor for advanced glycation end-products prevents unilateral ureteral obstruction-induced renal fibrosis

Abstract

Background

Accumulating evidence suggests that advanced glycation end-products (AGEs) and its receptor (RAGE) play an important role in extracellular matrix (ECM) accumulation, which is consistently observed in various renal diseases associated with progressive glomerulosclerosis and tubulointerstitial fibrosis. A recent study also demonstrated that high mobility group protein box1 (HMGB1), one of several ligands of RAGE, directly induced ECM accumulation in cultured renal tubular epithelial cells. Meanwhile, soluble RAGE (sRAGE) acts as a decoy. Therefore, blocking RAGE signaling using sRAGE is considered a potential therapeutic candidate for renal fibrosis, but it has never been explored. The aim of this study was to investigate the therapeutic effects of sRAGE on HMGB1-induced tubular epithelial cell injury in vitro and unilateral ureter obstruction (UUO)-induced renal tubulointerstitial fibrosis in vivo.

Methods

In vitro, NRK-52E cells were cultured in DMEM media with or without HMGB1 (10 μg/ml). To examine the effect of sRAGE on HMGB1-induced tubular cell injury, the cells were also incubated with sRAGE (1 μg/ml). In vivo, twenty-four Sprague-Dawley rats were divided into 4 groups, each of which consisted of 6 rats: sham operation with diluent (Control); sham operation with sRAGE (4 μg/kg); UUO operation with diluent; and UUO operation with sRAGE. The animals were treated intraperitoneally with either diluent or sRAGE at 1 hr before and every 48 hr after sham or UUO surgery. Rats were sacrificed at 10 days after operation, and the left kidneys were removed. The expression of fibrosis-related molecules and RAGE-dependent signaling cascades, including the mitogen-activated protein (MAP) kinases and NF-κB, were evaluated by Western blot analysis in cultured NRK-52E cells and the kidneys. Masson’s trichrome and immunohistochemical staining (IHC) for ED-1, fibronectin, and type I collagen were also performed with the kidneys.

Results

The protein expression of fibronectin, type I collagen, α-smooth muscle actin (α-SMA), and connective tissue growth factor were significantly increased in HMBG1-stimulated NRK-52E cells, and these increases were attenuated by sRAGE. The MAP kinase pathway and NF-κB were also activated in NRK-52E cells exposed to HMBG1, and sRAGE treatment significantly abrogated the activation of these pathways. Compared to sham-operated rats, RAGE and HMGB1 protein expression were significantly increased in the kidney of UUO rats. Renal expression of fibrosis-related molecules such as fibronectin, type I collagen, and α-SMA were also significantly increased in UUO rat compared to the control group. All these changes were significantly ameliorated by the administration of sRAGE. IHC and Masson’s trichrome staining also confirmed the anti-fibrotic effect of sRAGE in a UUO rat model. In addition, the activation of the MAP kinase pathway and NF-κB along with an increased number of infiltrated macrophages within the tubulointerstitium in the kidney of UUO rats was significantly attenuated by sRAGE treatment.

Conclusion

These findings suggest that RAGE plays a pivotal role in the pathogenesis of renal fibrosis and its inhibition by sRAGE may be a potential therapeutic approach to various kidney diseases associated with renal fibrosis.