Fig 2: DPEP1 and leukocyte recruitment during kidney IRI.(A) Kidney IVM in Dpep1+/+ and Dpep1−/− mice at 2 hours following renal IRI. Labels: leukocytes (CD11b, red), tubules (autofluorescence, green), and capillaries (QTracker, blue). Scale bars, 100 μm. (B) Quantification of stationary CD11b+ cells/field in the kidney (Dpep1+/+ versus Dpep1−/−, **P = 0.003, n = 5 to 6 per group, Student’s t test). (C) Kidney IVM in LysMgfp/gfp mice 2 hours following IRI. Mice were pretreated with vehicle (phosphate-buffered saline), scrambled (Scr) LSALT peptide, or LSALT peptide. Labels: leukocytes (LysM-GFP, bright green) and capillaries (QTracker, blue). Scale bars, 50 μm. (D) Quantification of stationary GFP+ leukocytes/field in the kidneys of mice 2 hours after IRI treatment with vehicle, LSALT peptide, or scrambled LSALT peptide (LSALT peptide versus scrambled peptide, *P = 0.03, n = 3 to 5). (E) Kidney IVM and (F) quantification of stationary GFP+ leukocytes in LysMgfp/gfp mice 2 hours after IRI treatment with increasing doses of LSALT peptide. Labels: macrophages (F4/80, red), leukocytes (LysM-GFP, yellow), tubules (autofluorescence, dark green), and capillaries (QTracker, blue) [LSALT peptide 0 μg/kg versus 0.1 μg/kg, ***P = 0.0002; 1 μg/kg, ***P = 0.0001; 10 μg/kg, ***P = 0.0003; 100 μg/kg, ***P = 0.0001; n = 3 to 5 per group, analysis of variance (ANOVA) with Dunnett’s post hoc test].

Fig 3: DPEP1-mediated leukocyte recruitment and tubular injury.(A) Immunoblotting for LRP2, GPX4, ACSL4, and DPEP1 expression in whole-kidney tissue from Dpep1−/− and Dpep1+/+ mice 48 hours after renal IRI or sham operation. (B) Kidney IVM in LysMgfp/gfp mice at 90 min following LPS administration with or without cilastatin or LSALT peptide treatment. Scale bars, 100 μm. (C) Stationary GFP+ leukocytes/field in the kidney were quantified (versus LPS: cilastatin, *P = 0.01; LSALT, **P = 0.002; n = 4 to 5 per group, ANOVA with Dunnett’s post hoc test). (D) Kidney IVM in Dpep1+/+ and Dpep1−/− mice at 90 min following LPS administration. Labels: leukocytes (CD11b, red), capillaries (QTracker, blue), and tubules (autofluorescence, yellow-green). Scale bars, 100 μm. (E) Kidney IVM with SYTOX Red staining in LysMgfp/gfp mice 2 hours following LPS administration. Non-LPS (NT)–treated mice are shown as a control. Labels: leukocytes (LysM-GFP, bright green/yellow), tubules (autofluorescence, dark green), capillaries (QTracker, blue), and necrotic cells (SYTOX, red). Scale bars, 100 μm.

Fig 4: DPEP1-mediated neutrophil and inflammatory monocyte recruitment to the injured kidney.Flow cytometry of neutrophil (A) and inflammatory monocyte (B) recruitment to the kidneys of Dpep1+/+ and Dpep1−/− at 24 hours (circles) and 48 hours (squares) after IRI. Contralateral (Cntrl) kidneys are used as controls. [Neutrophils: Dpep1+/+ (24 hours) Cntrl versus IRI, **P = 0.005; IRI (24 hours) Dpep1+/+ versus Dpep1−/−, **P = 0.008; Dpep1+/+ (48 hours) Cntrl versus IRI, **P = 0.006; IRI (48 hours) Dpep1+/+ versus Dpep1−/−, ***P = 0.001; n = 5 per group, Student’s t test.] [Monocytes: Dpep1+/+ (24 hours) Cntrl versus IRI, ***P = 0.001; IRI (24 hours) Dpep1+/+ versus Dpep1−/−, **P = 0.004; Dpep1+/+ (48 hours) Cntrl versus IRI, **P = 0.004; IRI (48 hours) Dpep1+/+ versus Dpep1−/−, ns = not significant; n = 5 per group, Student’s t test.] Flow cytometry of neutrophil (C) and inflammatory monocyte (D) recruitment to the kidneys of LSALT peptide–treated mice at 24 hours (circles) and 48 hours (squares) after IRI. Contralateral (Cntrl) kidneys are used as controls. [Neutrophils: Cntrl versus IRI (24 hours), **P = 0.005; IRI (24 hours) no treatment versus LSALT, **P = 0.01; Cntrl versus IRI (48 hours), *P = 0.048; IRI (48 hours) no treatment versus LSALT, *P = 0.04; n = 6 to 8 per group, Student’s t test.] [Monocytes: Cntrl versus IRI (24 hours), ***P = 0.0003; IRI (24 hours) no treatment versus LSALT, **P = 0.005; Cntrl versus IRI (48 hours), **P = 0.002; IRI (48 hours) no treatment versus LSALT, *P = 0.047; n = 5 to 8 per group, Student’s t test.]

Fig 5: DPEP1 expression in the kidney.(A) DPEP1 immunohistochemistry in normal human kidney and allograft biopsies with clinical ATN/acute tubular necrosis (ATN). Arrows denote peritubular capillaries. Pt1 and Pt2 denote biopsies from 2 individual patients. Scale bars, 50 μm. Right panels are magnification of hatched boxes. (B and C) Immunoblot probing for DPEP1 in kidney lysates from mice treated with renal IRI and lipopolysaccharide (LPS) at 8 hours. Sham and untreated (NT) mice are controls. (D) DPEP1 immunohistochemistry in wild-type (WT) mouse kidneys following IRI or LPS treatment at 8 hours. Kidney from untreated Dpep1−/− mouse is used as a control. Arrows denote peritubular capillaries. Scale bars, 50 μm. (E) Kidney IVM in mice following sham operation, LPS administration, and IRI (8 hours). Tubules are visualized using autofluorescence and DPEP1 expression by fluorescent-labeled LSALT peptide. Arrows denote peritubular capillaries. Labels: tubules (green) and DPEP1 (red). Scale bars, 100 μm. Bottom panels are magnification of hatched boxes. (F) Representative flow cytometry for DPEP1 expression in cells isolated from the kidneys of untreated (NT), LPS-, or IRI-treated mice (8 hours). Cells sorted using LTL (proximal tubular epithelial cells), CD31 (endothelial cells), and CD45 (leukocytes).