Figure 3.

KIM-1–deficient mice exhibit increased renal Gα12 activation and tissue damage after ischemia-reperfusion injury. A: Wild-type (Kim-1^+/+) and Kim-1–deficient (Kim-1^−/−) mice underwent unilateral renal pedicle clamping for 35 minutes, followed by 24 hours of reperfusion. Active Gα12 was measured in renal cortical lysates obtained from clamped and contralateral kidneys by GST-TPR pull-down assay, followed by SDS-PAGE and Western blot analysis for active and total Gα12, mKim-1, and GAPDH. B: The amount of active pY419 compared with total Src was measured by Western blot analysis in renal cortical lysates of mice treated as in A. Samples were analyzed by SDS-PAGE and Western blot analysis for indicated antibodies. Densitometric analysis of the ratio of active Gα12 to total Gα12 (or pY419-Src to c-Src) is shown as a representation of experiments. C–I: Independent groups of Kim-1^+/+ and Kim-1^−/− mice underwent sham surgery or bilateral renal pedicle clamping for 30 minutes (C–E) or 25 minutes (F–I), followed by 24, 48, or 168 hours of reperfusion. C and F: Kidney function was determined by plasma creatinine. D and G: Quantification of tubular damage in whole kidneys after 48 hours of reperfusion. E and H: Percentage of survival after reperfusion. I: Representative periodic acid-Schiff–stained kidney sections after 48 hours of reperfusion (Sham represents Kim-1^−/−). Arrows indicate areas with tubular injury. Data are expressed as means ± SEM. n = 3 per group in three independent experiments (A and B); n = 5 to 11 per group (C–E); n = 7 to 12 per group (F–I); n = 9 per group (D and G). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. Scale bar = 0.10 mm. Original magnification, ×200. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Gα12, G protein α-12; GST-TPR, glutathione S-transferase–fused tetratricopeptide repeat domain of Ser/Thr protein phosphatase type 5; KIM-1, kidney injury molecule-1; mKim-1, mouse kidney injury molecule-1; pY419, phosphorylated Src.