Figure 5.

Slit2 improves acute tubular necrosis and reduces neutrophil infiltration after renal IRI. (A–D) Representative hematoxylin and eosin-stained kidney sections from mice subjected to (A) sham surgery, (B) vehicle administration before IRI, (C) N-mSlit2 administration before IRI, and (D) N-mSlit2–RoboN administration before IRI examined using a Zeiss AxioSkop microscope equipped with a SPOT-RT Camera. Original magnification, ×20; ×2 in insets. (E) To quantify the degree of tubular injury, kidney sections were examined using a Zeiss AxioSkop microscope and scored by counting the percentage of tubules that displayed cell necrosis, loss of brush border, cast formation, and tubular dilation; they were scored as follows: 1<10%, 2=10%–25%, 3=26%–50%, 4=51%–75%, and 5>75%. Two to three fields from the outer medulla of each section were evaluated. Original magnification, ×200. (F) Experiments were performed as in Figure 4, and kidneys were collected after 18 hours. Kidneys were weighed, minced, and digested with collagenase type IA (10 µg/ml). The cell pellet was washed, nonspecific Fc binding was blocked with anti-mouse CD16/32 antibody (2.4G2), and cell suspensions were incubated with fluorophore-tagged anti-mouse CD45 antibody (30-F11) to determine total leukocyte cell numbers. Anti-CD45 antibody-labeled samples were further labeled with anti-mouse GR-1-FITC antibody (Ly6G) and anti-mouse CD11b-PE antibody to identify neutrophils. Appropriate fluorochrome-conjugated, isotype-matched, irrelevant monoclonal antibodies were used as negative controls. Flow cytometry data acquisition was performed on BD FACS Calibur, and data were analyzed using FlowJo software 6.4. The number of GR-1⁺CD11b⁺CD45⁺ neutrophils was normalized to kidney weight, indexed to the value for sham surgery kidneys, and log-transformed to generate a neutrophil infiltration score. Mean values ± SEM from experiments involving four to seven mice per experimental group. ATN, acute tubular necrosis. *P<0.05 versus sham-operated mice. ^†P<0.05 versus vehicle-treated IRI mice. ^‡P<0.05 versus N-mSlit2–treated IRI mice.