Perirenal fat β₃-adrenergic signaling alleviates renal fibrosis via regulating Bcat2/BCAA-TNFα pathway in diabetic kidney disease

Figures

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  Fig. 1. PRF is increased in DKD and associates with renal dysfunction. A KEGG over-representation analysis of the top differentially expressed gene (DEG)-related pathways (two-sided Fisher’s exact test with Benjamini–Hochberg FDR correction). B Heatmap of fibrosis-related genes in kidneys from controls and DKD patients (GEO: GSE166239). C Gross PRF appearance in db/m and db/db mice. D 7 T T1-weighted MRI pseudocolor maps and PRF-area quantification (n = 8 mice per group). E Representative H&E staining of PRF. F, G PRF triglyceride (TG) and total cholesterol (TCH) content (n = 8 mice per group). H–J Two-tailed Pearson correlations between PRF area and blood glucose, serum creatinine and UACR in individual mice (n = 8 mice per group). Data are mean ± SEM (D, F, G). Statistics: two-tailed unpaired Student’s t-test for (D, F, G); two-tailed Pearson correlation for (H–J). Exact P values are shown in the figure. Source data are provided as a Source Data file.

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  Fig. 2. Removal of PRF alleviates renal pathological injury and fibrosis in db/db mice. A Experimental timeline; mice underwent sham surgery or PRF removal and were sacrificed at week 24. [Created in BioRender. Hu, H. (2026) https://BioRender.com/cs6kgre]. B–D Body weight, fasting blood glucose, and urinary albumin-to-creatinine ratio (UACR) over time (n = 8 mice per group). E–G Representative H&E, PAS, and Masson staining of kidneys with quantification of tubular-interstitial damage score, mesangial matrix expansion, and fibrosis area (n = 8 mice per group). H Immunofluorescence for FN1 (red), αSMA (green), LTL (gray/white), and DAPI (blue) in kidney sections with fluorescence quantification of αSMA and FN1 (n = 8 mice per group). I Western blotting of isolated tubules showing αSMA and FN1 with densitometry (n = 8 mice per group). J–L Succinate dehydrogenase (SDH) histochemistry, dihydroethidium (DHE) staining, and Oil Red O (ORO) staining with quantification of SDH-positive area, DHE-positive area, and ORO-positive area (n = 8 mice per group). M Immunofluorescence of αSMA and FN1 in primary TECs with quantification (n = 8 biologically independent experiments). N Oxygen-consumption-rate…

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  Fig. 3. ADRB3 expression is downregulated in PRF in diabetes and correlates with renal function. A Volcano plot of DEGs in kidneys from controls and individuals with obesity (n = 10 samples per group; GEO: GSE2508; two-sided DESeq2 Wald test with Benjamini–Hochberg FDR correction). B KEGG over-representation analysis of DEG-associated pathways (two-sided Fisher exact test with Benjamini–Hochberg FDR correction). C Heatmap of lipid metabolism genes in kidneys from controls and DKD patients (n = 10 samples per group; GEO: GSE166239). D–F qPCR of Fabp1, Ppara, and Adrb3 mRNA in PRF from db/m and db/db mice (n = 8 mice per group). G, H Immunoblotting and densitometry of FABP1, PPARA, and ADRB3 in PRF (n = 8 mice per group). I, J ADRB3 immunofluorescence and immunohistochemistry in PRF with quantification (n = 8 mice per group). K–M Two-tailed Pearson correlations between PRF Adrb3 expression and blood glucose, serum creatinine and UACR (n = 8 mice per group). N Schematic of PRF single-cell RNA-seq. [Created in BioRender. Hu, H. (2026) https://BioRender.com/cs6kgre]. O–S PRF single-cell analyses showing cluster correlations, major cell types, adipocyte subclusters, UCP1 expression and ADRB3…

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  Fig. 4. Brown adipocyte-specific deletion of ADRB3 enlarges perirenal fat and increases TNFα secretion. A Experimental scheme for generating ADRB3BKO (UCP1-Cre;Adrb3fl/fl) mice and study timeline. [Created in BioRender. Hu, H. (2026) https://BioRender.com/cs6kgre]. B qPCR of Adrb3 in major organs showing selective loss in PRF (n = 8 mice per group). C Western blotting and densitometry of ADRB3 in PRF from ADRB3fl/fl (ctrl) and ADRB3BKO mice (n = 8 mice per group). D, E Body weight and fasting blood glucose in the indicated groups (n = 8 mice per group). F 7 T T1-weighted MRI pseudocolor maps with ROI encompassing both kidneys and quantification of PRF area (n = 8 mice per group). G, H Biochemical lipid content of PRF: triglycerides (TG) and total cholesterol (TCH) (n = 8 mice per group). I H&E staining of PRF and adipocyte size quantification (n = 8 mice per group). J Schematic of primary PRF adipocyte isolation. [Created in BioRender. Hu, H. (2026) https://BioRender.com/cs6kgre]. K Oil Red O (ORO) staining of primary adipocytes and quantification (n = 8 biologically independent experiments). L Seahorse traces of oxygen consumption rate (OCR) in primary adipocytes (n = 3 biologically…

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  Fig. 5. Brown adipocyte–specific loss of ADRB3 aggravates renal injury and fibrosis. A Representative kidney sections from ADRB3fl/fl and ADRB3BKO mice under saline or STZ/HFD showing Masson trichrome (fibrosis) and immunofluorescence for FN1 (red), αSMA (green), LTL (white) and DAPI (blue) (n = 8 mice per group). B Quantification of fibrosis area (Masson) and fluorescence intensities of αSMA and FN1 (n = 8 mice per group). C Western blotting of isolated renal tubules with densitometry for αSMA and FN1 (n = 8 mice per group). D–F qPCR of Il1b, Il6, and Tnf mRNA in PRF (n = 8 mice per group). G–I PRF cytokine content for IL-1β, IL-6, and TNF-α (n = 8 mice per group). J Seahorse traces of oxygen-consumption rate (OCR) in primary TECs (n = 3 biologically independent experiments). K Cellular ATP in TECs (n = 8 biologically independent experiments). L–M TFAM immunofluorescence in TECs and quantification (n = 8 biologically independent experiments). N Oil Red O (ORO) staining of TECs with quantification of ORO-positive area (n = 8 biologically independent experiments). O Immunofluorescence of FN1 (red) and αSMA (green) in TECs with quantification (n = 8 biologically independent experiments).…

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  Fig. 6. Activation of ADRB3 in PRF attenuates renal pathological injury and fibrosis in DKD. A Schematic of BRL37344 (BRL, 2.5 mg/kg, i.p., 2 weeks) treatment in saline- or STZ/HFD-treated mice. [Created in BioRender. Hu, H. (2026) https://BioRender.com/cs6kgre]. B qPCR showing Adrb3 mRNA in PRF (n = 8 mice per group). C Western blotting and densitometry of ADRB3 in PRF (n = 8 mice per group). D Representative H&E images of PRF with adipocyte-size quantification (n = 8 mice per group). E TNF-α content in PRF (n = 8 mice per group). F–I Body weight, fasting blood glucose, urinary albumin-to-creatinine ratio (UACR) and blood urea nitrogen (BUN) (n = 8 mice per group). J, K Kidney histology: representative H&E, PAS, and Masson staining with quantification of tubular–interstitial damage score, mesangial matrix expansion and fibrosis area (n = 8 mice per group). L Kidney neutral-lipid deposition assessed by Oil Red O (ORO) with area quantification (n = 8 mice per group). M Kidney immunofluorescence for αSMA (green), FN1 (red), and DAPI (blue) with fluorescence quantification (n = 8 mice per group). Data are mean ± SEM (B–M). Two-tailed two-way ANOVA with Sidak’s multiple-comparisons test…

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  Fig. 7. Knockout of ADRB3 in PRF rewires branched-chain amino-acid (BCAA) metabolism and promotes TNF-α secretion. A Schematic of primary PRF adipocyte isolation and RNA-seq. [Created in BioRender. Hu, H. (2026) https://BioRender.com/cs6kgre]. B Rank plot of DEGs in primary adipocytes from ADRB3fl/fl and ADRB3BKO DKD mice (two-sided DESeq2 Wald test with Benjamini–Hochberg FDR correction). C KEGG over-representation of DEGs (two-sided Fisher exact test with Benjamini–Hochberg FDR correction). D GSEA showing downregulated VALINE_LEUCINE_AND_ISOLEUCINE_DEGRADATION in ADRB3BKO adipocytes. E Targeted metabolomics of PRF BCAAs (Leu, Ile, Val) in primary adipocytes (n = 8 biologically independent experiments). F TNF-α levels in adipocyte culture medium (n = 8 biologically independent experiments). G OCR traces (n = 3 biologically independent experiments). H Heatmap of BCAA-degradation genes. I Volcano plot highlighting Bcat2 (two-sided DESeq2 Wald test with Benjamini–Hochberg FDR correction). J BCAT2 immunofluorescence in PRF (n = 8 biologically independent experiments). K TNF-α after BCAT2 overexpression (n = 8 biologically independent experiments). L OCR traces (n = 3 biologically…

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  Fig. 8. CREB1 upregulates METTL3-mediated m6A to suppress BCAT2 translation in PRF adipocytes. A qPCR of Bcat2 mRNA in ADRB3fl/fl and ADRB3BKO mice under saline or STZ/HFD (n = 8 biologically independent experiments). B Transcription-factor prediction identifies CREB1 as a candidate Bcat2 promoter regulator. C, D CREB1 overexpression decreases Bcat2 mRNA and protein (n = 8 biologically independent experiments). E GSEA indicates RNA-degradation pathway activation in ADRB3BKO. F Global m6A immunostaining in PRF with quantification (n = 8 biologically independent experiments). G qPCR of m6A regulators in primary adipocytes (n = 3 biologically independent experiments). H Structural model of CREB1 bound to the METTL3 promoter. I ChIP-PCR of CREB1 enrichment at the METTL3 promoter (n = 3 biologically independent experiments). J–M Luciferase assays testing METTL3 promoter truncations and CREB1-site mutation (n = 3 biologically independent experiments). N, O m6A-RIP-qPCR (n = 3 biologically independent experiments). P Polysome profiling of Bcat2 (n = 3 biologically independent experiments). Q OPP-labeling/puromycylation of nascent BCAT2 (n = 3 biologically independent experiments). R Working…