Progressive loss and dysfunction of islet β-cells has not yet been solved in the treatment of diabetes. Regenerating protein (Reg) has been identified as a trophic factor which is demonstrated to be associated with pancreatic tissue regeneration. We previously produced recombinant Reg3α protein (rReg3α) and proved that it protects against acute pancreatitis in mice. Whether rReg3α protects islet β-cells in diabetes has been elusive. In the present study, rReg3α stimulated MIN6 cell proliferation and resisted STZ-caused cell death. The protective effect of rReg3α was also found in mouse primary islets. In BALB/c mice, rReg3α administration largely alleviated STZ-induced diabetes by the preservation of β-cell mass. The protective mechanism could be attributed to Akt/Bcl-2/-xL activation and GRP78 upregulation. Scattered insulin-expressing cells and clusters with small size, low insulin density, and exocrine distribution were observed and considered to be neogenic. In isolated acinar cells with wheat germ agglutinin (WGA) labeling, rReg3α treatment generated insulin-producing cells through Stat3/Ngn3 signaling, but these cells were not fully functional in response to glucose stimulation. Our results demonstrated that rReg3α resists STZ-induced β-cell death and promotes β-cell regeneration. rReg3α could serve as a potential drug for β-cell maintenance in anti-diabetic treatment.

Key Points: Growth arrest and DNA damage–inducible 45 α (GADD45 α ) is crucial for maintaining kidney health by enhancing six-transmembrane epithelial antigen of the prostate 4 expression through promoter demethylation. In diabetic nephropathy, lower GADD45 α levels hinder R-loop formation, leading to decreased six-transmembrane epithelial antigen of the prostate 4 demethylation and expression. Targeting the GADD45 α -R-loop pathway holds promise for innovative diabetic nephropathy treatment strategies. Background: Diabetic nephropathy is a primary cause of kidney failure. Persistent hyperglycemia causes metabolic perturbations epigenetically dysregulating gene expression in kidney cells, thereby leading to diabetic nephropathy pathogenesis. On analyzing the Gene Expression Omnibus database by using machine learning algorithms, our preliminary results demonstrated that growth arrest and DNA damage–inducible 45 α (GADD45 α ) might serve as key regulators in diabetic nephropathy. Furthermore, emerging evidence has shown that R-loops, the three-stranded DNA–RNA structures, are crucial to gene expression during diabetic nephropathy. Therefore, this study aimed to investigate the role of GADD45 α in diabetic nephropathy by modulating epigenetic alterations through interaction with R-loops. Methods: A diabetic mouse model was established by injecting streptozotocin intraperitoneally into mice. Kidney histology and biochemical markers were analyzed in wild-type, GADD45 α knockout, and renal tubule–specific GADD45 α -overexpressing mice. The GADD45 α lentivirus was used to induce the overexpression of GADD45 α in human kidney-2 (a proximal tubular epithelial cell line) cells, while high-glucose treatment was applied to verify the mechanisms in vitro . Results: GADD45 α expression was reduced in kidneys of diabetic nephropathy, correlating with kidney dysfunction. GADD45 α knockout worsened kidney injuries, while overexpression mitigated them. Mechanistically, GADD45 α interacted with R-loops on the six-transmembrane epithelial antigen of the prostate 4 ( STEAP4 ) promoter, recruiting ten eleven translocation 1 to activate STEAP4 transcription. Deficiency in the GADD45 α -R-loop pathway exacerbated mitochondrial injury, disrupted lipid metabolism, and increased oxidative stress in diabetic nephropathy. Conclusions: Deficiency of GADD45 α exacerbates diabetic nephropathy by interacting with R-loops and inhibiting STEAP4 promoter demethylation. Targeting the GADD45 α -R-loop pathway offers therapeutic potential against diabetic nephropathy.

Microinflammation is a key driver of chronic kidney disease (CKD) progression, with interleukin‐1β (IL‐1β) playing a pivotal role. However, current anti‐IL‐1β antibody therapies face critical limitations, such as systemic side effects and substantial production costs, which hinder their therapeutic efficacy and clinical translation for CKD intervention. To address this, we developed Bacteroides fragilis ‐derived outer membrane vesicles (OMVs) encapsulating anti‐IL‐1β single‐chain variable fragment (scFv) and conjugated with kidney‐targeting peptides (KKEEE) ₃ K. Engineered OMV‐(KKEEE) ₃ K‐scFv's safety was evaluated in vitro (24, 48 h) and in vivo (2 months). Its anti‐inflammatory efficacy was assessed in a high glucose‐induced model in vitro, and in various kidney disease mouse models (streptozotocin‐induced diabetic nephropathy, lupus nephritis, unilateral ureteral obstruction) in vivo. OMV‐(KKEEE) ₃ K‐scFv showed high stability, precisely delivered scFv to proximal renal tubules, had excellent safety, reduced the expression of inflammatory cytokines including tumour necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6) and monocyte chemoattractant protein‐1 (MCP‐1), decreased cell infiltration, and alleviated renal injury. In conclusion, engineered OMVs effectively deliver anti‐IL‐1β scFv, mitigating local inflammation and CKD‐related renal damage.