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Chronic kidney disease (CKD), which is associated with high morbidity, remains a worldwide health concern, while effective therapies remain limited. Hydroxychloroquine (HCQ), which mainly targets toll-like receptor-7 (TLR-7) and TLR-9, is associated with a lower risk of incident CKD. Taking into account that TLR-9 is involved in the development of renal fibrosis and serves as a potential therapy target for CKD, we investigated whether HCQ could attenuate CKD via TLR-9 signal pathway. The effects of HCQ on renal tubulointerstitial fibrosis were further explored using a mouse model of renal tubulointerstitial fibrosis after ischemia/reperfusion injury. Bone marrow-derived macrophages were isolated to explore the effects of HCQ in vitro. Judicious use of HCQ efficiently inhibited the activation of macrophages and MAPK signaling pathways, thereby attenuating renal fibrosis in vivo. In an in vitro model, results showed that HCQ promoted apoptosis of macrophages and inhibited activation of macrophages, especially M2 macrophages, in a dose-dependent manner. Because TLR-7 is not involved in the development of CKD post-injury, a TLR-9 knockout mouse was used to explore the mechanisms of HCQ. The effects of HCQ on renal fibrosis and macrophages decreased after depletion of TLR-9 in vivo and in vitro. Taken together, this study indicated that proper use of HCQ could be a new strategy for anti-fibrotic therapy and that TLR-9 could be a potential therapeutic target for CKD following acute kidney injury.

Toll-like receptor-9 (TLR-9) is a potent proinflammatory receptor that mediates renal injury. However, the reported effects of TLR-9 are contradictory. Here, using a traditional mouse AKI→CKD transition model, the roles of TLR-9 during the transition from acute kidney injury (AKI) to chronic kidney disease (CKD) were further explored. Using a TLR-9–/– mouse, the effects and mechanisms of TLR-9 were investigated. Loss of TLR-9 elicited no obvious effects as regards renal function or histology during AKI in the early phases (24–48 h), while TLR-9 KO attenuated renal fibrosis (as shown using fibronectin and collagen III) and epithelial-to-mesenchymal transition (EMT) [E-cadherin (E-Cad) and α-smooth muscle actin (α-SMA)] on the long-term after AKI through the inhibition of macrophages infiltration, especially M2 macrophages. The roles of TLR-9 on macrophages were also explored using Raw264.7 macrophage cell line, and results indicated that the inhibition of TLR-9 on Raw 264.7 macrophages decreased the induction of M2 type macrophage in a dose-dependent manner. The roles of TLR-9 on renal tubular epithelial (RTE) cells were also explored. Conversely, TLR-9 depletion did not contribute to the improvement of fibrosis and EMT in vitro. Therefore, TLR-9 plays a critical role in the AKI→CKD transition. Attenuation of CKD post-AKI in the TLR-9 KO group mainly relies on the effects of TLR-9 on macrophages. These results also suggest that TLR-9 could be a therapeutic target for CKD.