Fig 2: BTK knockout alleviates kidney inflammation in diabetic mice. (A) TNF-α, IL-1β and MCP-1 levels of in mouse serum were detected using ELISA kits. (B) TNF-α, IL-1β, MCP-1 and IL-6 mRNA expression levels in mouse kidneys were detected by RT-qPCR. (C) TNF-α, IL-1β and MCP-1 immunohistochemistry analysis of protein expression was conducted in mouse kidneys (scale bar, 50 µm). (D) Protein expression levels of TNF-α, IL-1β and MCP-1 were detected by western blot analysis (*P<0.05 vs. WT group; #P<0.05 vs. STZ group). WT, wild-type; BTK, Bruton's tyrosine kinase; STZ, Streptozotocin; MCP-1, monocyte chemoattractant protein-1; RT-qPCR, reverse transcription-quantitative PCR.

Fig 3: BTK is notably activated in renal macrophages in the kidneys of patients with DN. (A) Immunohistochemistry of BTK expression in the glomerulus and tubulointerstitium (scale bar, 50 µm; *P<0.05 vs. control group). (B) CD68 was co-stained with phosphorylated-BTK and BTK in the kidney (scale bar, 20 µm). BTK, Bruton's tyrosine kinase; DN, diabetic nephropathy.

Fig 4: Irreversible targeting of BTK by SY-1530. (A) The compounds were incubated with BTK for 2 h, after which HTRF assays were performed with dilution of the mixture. The reactions were stopped at different time points. Data are shown as the mean ± SEM. (B) The content of the compounds was measured with LC-MS/MS after incubation with BTK for 4 h. Data are shown as the mean ± SEM (n = 3). Statistical analysis was performed with Student’s t-test (***P < 0.001). (C) Chemical structure of the Bodipy-FL fluorophore attached to the BTK inhibitor SY-1530 (FL-SY-1530). (D) DOHH2 cells were treated with various concentrations of FL-SY-1530 for 1 h, after which cells were lysed and analyzed by SDS-PAGE with the indicated antibodies and fluorescent gel scanning with a Typhoon imaging system. (E) Chemical structure of the labeled BTK inhibitor ibrutinib (FL-ibrutinib). (F) HEK293 cells expressing wild-type or C481A mutant BTK were treated with FL-SY-1530 or FL-ibrutinib for 1 h, and the cells were then lysed and analyzed. (G) Jurkat, Pfeiffer, and DOHH2 cells were treated with SY-1530 or ibrutinib for 1.5 h before incubation with FL-SY-1530 (FL). Cells were lysed and analyzed by Western blot and fluorescent gel scanning.

Fig 5: SY-1530 is a highly selective and non-competitive BTK inhibitor. (A–D) The inhibitory effect of SY-1530 or ibrutinib on the activity of BTK or ITK was measured with HTRF assays. Different concentrations of SY-1530 or ibrutinib (gradient dilution of compounds with DMSO) were used, and IC50 was calculated with GraFit6.0. (E and F) HTRF assays were performed with different concentrations of SY-1530 (3, 1, 0.3, and 0 nM) and ATP (0–30 μM) to detect the activity of BTK. The enzyme kinetic parameters (Km, Vmax, and Ki) were calculated (E), and the Lineweaver-Burk plot was obtained according to the Michaelis-Menten equation (F).