Fig 2: Reduction of Tim-3 ameliorated podocyte injury and proteinuria in DN mice. UACR (A) and the ratio of kidney weight to body weight (KBWR) (B) in Tim-3 KD mice at 16 weeks of age after STZ injection. (C) Representative photomicrographs of TUNEL staining for apoptotic cells in the different groups. Bars = 50 µm. (D) Representative western blot analysis showing the relative protein levels of Tim-3 and Nephrin in renal tissue of the different groups. (E) Representative photomicrographs and quantifications of PAS staining and IHC staining of Nephrin and WT-1 in renal tissue of the different groups. Bars = 10 µm. (F) Representative transmission electron micrographs and quantifications of glomerular basement membrane (GBM) thickness and numbers of podocyte foot processes in the glomeruli of different groupss. Bars = 2 µm; 1 µm. UACR (G) and KBWR (H) levels from WT and Tim-3 KO mice at 12 weeks of age after STZ injection. (I) Representative photomicrographs of TUNEL staining for apoptotic cells in the different groups. Bars = 50 µm. (J) Representative photomicrographs and quantifications of PAS staining and IHC staining of Nephrin and WT-1 in renal tissue of the different groups. Bars = 10 µm *P < 0.05, **P < 0.01, ***P < 0.001. Tim-3 KD mice, shTim-3-lv knockdown diabetic mice (n = 6); Tim-3 KO mice, Tim-3 talen-target knockout diabetic mice (n = 3).

Fig 3: Enhanced Tim-3 expression in macrophage leading to severe podocyte injury with HG treatment. (A) Western blot analysis showing the relative protein level of Tim-3 treated with various stimulants in PMs and BMs. (B–E) Macrophage from either WT mice or Tim-3 KO mice were cultured with HG or NG medium and the culture medium was collected as macrophages conditioned medium (CM). Mouse podocyte (MPC) were stimulated with macrophage CM. Western blot (B) and RT-PCR (C) analysis showing the relative protein level of Nephrin and the relative mRNA levels of Podocin and CD2AP genes expressions. The podocyte cytoskeleton and its migration ability were assessed by immunofluorescence confocal (D), scratch assay (E) and transwell assay (F). *P < 0.05, **P < 0.01, ***P < 0.001 vs normal glucose (n = 3). Data are expressed as means ± SEM, Student's t-test was employed for comparisons between two groups. PM, Peritoneal macrophage, BM, bone marrow-derived macrophage, HG, high glucose medium (20 and 40 mmol/L), AGE, advanced glycation end product (50, 100, 200 µg/ml), NG, normal glucose (11 mmol/L), MA, mannitol (40 mmol/L), BSA, bovine serum albumin (200 µg/ml).

Fig 4: Adoptive transfer of Tim-3-positive macrophages aggravated podocyte dysfunction. (A) Experimental design for the BMT of diabetic mice. Transferred Tim-3-positive or deficient BMs back to Tim-3 KD mice. (B–C) UACR and KBWR in each group at 12 weeks of age after STZ injection. (D) Representative photomicrographs and quantifications of TUNEL staining for apoptotic renal cells in BMT mice. Bars = 50 µm. (E) Western blot analysis showing the relative protein levels of Nephrin and WT-1 in renal tissue of BMT mice. (F) Representative photomicrographs and quantifications of PAS staining and IHC staining of Nephrin and WT-1 in renal tissue in the different groups. Bars = 10 µm *P < 0.05, **P < 0.01 between such two BMT groups. BMT, bone marrow derived macrophage transfer.

Fig 5: Silencing of PVT1 or the overexpression of FOXA1 inhibited the apoptosis and damage of podocytes in DN in vivo.a The expression of PVT1 and FOXA1 in mice with DN in vivo. b FOXA1 promoter recruitment in vivo detected by ChIP assay, n = 10. c The degree of glomerulosclerosis and renal tubule atrophy in response to the treatment of sh-PVT1 and/or GSK126 assessed by periodic acid-Schiff (PAS) staining (the original magnification is ×400). d Podocyte apoptosis in response to the treatment of sh-PVT1 and/or GSK126 in vivo. e Immunofluorescence staining displaying the expression of podocyte marker Nephrin and FOXA1 in vivo (the original magnification is ×400). f The protein level of FOXA1 in vivo detected by western blot analysis; *p < 0.05 vs. the sh-NC group. The measurement data are expressed as the mean ± standard deviation, and the comparisons among multiple groups were analyzed by one-way analysis of variance (ANOVA). The experiment was repeated three times. DN, diabetic nephropathy; PVT1, plasmacytoma variant translocation 1; IgG, immunoglobulin G; FOXA1, forkhead box A1; NC, negative control; DAPI, 4',6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSK126, EZH2 inhibitor