Fig 2: Assessment of standard kidney morphology in female rats on P39. A, B: Glomerular number and glomerular area were analyzed on H&E-stained slices. Representative H&E stains for each group are shown in (G). C: Podocyte number was assessed by WT-1 positive cells per glomerular area. D: Collagen deposition was analyzed using Sirius Red staining (positive staining per cortex-medulla area). E: CD68-positive cells per cortex-medulla area indicate the number of infiltrating monocytes and macrophages, and (F) Ki67-positive cells per cortex-medulla area indicate proliferating cells. Values are shown as mean ± SD, no significant differences (P < 0.05; Bonferroni-adjusted Mann-Whitney test) were observed in the presented analyses.

Fig 3: 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 4: Representative micrographs of immunohistochemical staining for nephrin (a1-4), WT-1 (b1-4), beclin-1 (c1-4) and TUNEL (d1-4). Graphical representation of the results of the evaluation of immunohistochemical staining (a5, b5, c5 and d5, respectively). *P < 0,001 vs the other groups; #P < 0,001 vs C group; ** P < 0,05 vs C and D groups; *** P < 0,05 vs C

Fig 5: Confirmation of podocyte expression by immunostaining.Frozen human kidney sections (5 µm) were double stained for the foot process marker NPHS2 or podocyte marker WT1 together with ARMH4 or WIPF3. A: ARMH4 shows perinuclear staining in cells that are outlined by NPHS2-positive contours, and ARMH4 is present in most of the cells that are positive for WT1. This is highlighted in the insets in the merged images to the right. B: WIPF3 immunoreactivity is observed in the cytosol of cells positive for NPHS2 or WT1. The upper inset in the merged images shows a cell body encircled by podocin that is also positive for WIPF3 and localized in close vicinity to podocyte foot processes. Bottom rows in A and B show negative control images for the primary antibodies used. Scale bars: 50 µm. C: DAB staining (in brown) of 2 µm thick paraffin-embedded kidney sections shows ARMH4 and WIPF3 immunoreactivity in glomerular podocytes. At higher magnification, ARMH4 shows an excentric perinuclear pattern, whereas WIPF3 seems to be distributed through the podocyte cytosol and along the glomerular basal membrane. Scale bars: 100 µm.