THE RNA-BINDING PROTEIN RBM3 PRESERVES AUTOPHAGIC FLUX AND MITIGATES RENAL INJURY IN ACUTE KIDNEY INJURY

Acute kidney injury (AKI) is a serious condition associated with high mortality and risk of progression to chronic kidney disease. However, information on factors that exert protective effects in AKI remains uncompletely understood. RNA-binding Motif Protein 3 (RBM3), a cold-inducible RNA-binding protein, regulates mRNA stability, splicing, and translation and has been implicated in cytoprotection. This study aimed to elucidate the protective mechanisms of RBM3 in AKI, particularly its role in maintaining autophagy flux and lysosomal function.

To investigate the role of RBM3 in AKI, ischemia-reperfusion (IR)-induced AKI was established in 8-week-old male C57BL/6 mice by clamping both renal arteries for 40 minutes, followed by reperfusion. RBM3 knockout mice were used to assess the impact of RBM3 deficiency on renal injury. 

In vitro, ischemic stress was modeled by exposing human kidney proximal tubular (HK-2) cells to 500 µM CoCl2 under serum-free DMEM medium for 24 hours under standard cell culture conditions (37°C, 5% CO2). RBM3 levels were modulated by siRNA-mediated silencing and mild hypothermia (33 °C) induced overexpression.

Molecular and pathological analyses began with microarray profiling, which confirmed the upregulated expression of RBM3 in the AKI model. Renal function was further evaluated by measuring blood urea nitrogen and serum creatinine levels, while protein expression was assessed using Western blotting. Tissue morphology was examined with Hematoxylin and Eosin and Periodic Acid–Schiff staining. To investigate autophagic flux and lysosomal function, immunohistochemistry and an autophagy flux assay kit were employed. Additionally, immunofluorescence co-staining for LAMP2 and LC3B was performed to evaluate autolysosome formation.

RBM3 knockout mice exhibited significantly aggravated renal injury compared with wild-type AKI controls. In both in vivo and in vitro models, RBM3 deficiency impaired autophagic flux, as evidenced by LC3B and LAMP2 immunofluorescence, p62 accumulation, and autophagy flux assays. Mechanistically, the loss of RBM3 diminished lysosomal degradative capacity, thereby disrupting autophagic progression. To compensate for the incomplete autophagy caused by RBM3 deficiency, excessive autophagic activity was induced, accompanied by mTORC1 suppression and activation of lysosomal factors including TFEB, LAMP2, and cathepsins B/D. This dysregulated autophagy ultimately resulted in exacerbated tubular injury, increased apoptosis, and worsened renal dysfunction in both RBM3 knockout mice and RBM3-silenced HK-2 cells. Conversely, mild hypothermia–induced RBM3 overexpression in HK-2 cells enhanced autophagic activity and preserved cellular integrity.

These findings demonstrate that RBM3 serves a crucial protective role in maintaining renal homeostasis during AKI. RBM3 deficiency leads to impaired autophagy completion, lysosomal dysfunction, and aggravated tubular injury. Conversely, RBM3 restoration mitigates renal damage and activates autophagic flux by sustaining lysosomal degradative capacity and preventing excessive compensatory activation of autophagy. These results suggest that RBM3 may serve as a potential therapeutic target for preserving autophagic flux and attenuating renal injury in AKI.

This research was supported by National Research Foundation of Korea (NRF) grant funded by the Korea government (MIST) (RS-2023-00212470)