Met activation is required for early cytoprotection after ischemic kidney injury

Abstract

Renal proximal tubule epithelial cells express high levels of the hepatocyte growth factor receptor Met, and both the receptor and ligand are upregulated after ischemic injury. Activation of the Met receptor after hepatocyte growth factor stimulation in vitro promotes activities involved in kidney repair, including cell survival, migration, and proliferation. However, characterizing the in vivo role of these signaling events in proximal tubule responses to kidney injury has been difficult because global Met knockout results in embryonic lethality due to placental and liver abnormalities. Here, we used γGT-Cre to knockout Met receptor expression selectively in the proximal tubules of mice (γGT-Cre;Met(fl/fl)). The kidneys of these mice developed normally, but exhibited increased initial tubular injury, tubular cell apoptosis, and serum creatinine after ischemia/reperfusion compared with γGT-Cre;Met(+/+) kidneys. These changes in γGT-Cre;Met(fl/fl) mice correlated with a selective reduction in PI3K/Akt activation in response to injury and subsequent decreases in inhibitory phosphorylation of the proapoptotic factor Bad and activating phosphorylation of the ribosomal regulatory protein p70-S6 kinase. Moreover, tubular cell proliferation after ischemia/reperfusion was delayed in γGT-Cre;Met(fl/fl) mice. In conclusion, this study identifies Met-dependent phosphoinositide 3-kinase activation in proximal tubules as a critical determinant of initial tubular cell survival and reparative proliferation after ischemic injury.

Figure 1.

Activation of Met, Akt, and Erk in wild-type mice after renal ischemia. (A) Western blot analysis of Met receptor phosphorylation at the activation site in wild-type mice at 6 hours, and 1, 2, and 3 days after I/R compared with sham operation. The graph shows quantification of pMet normalized to β-actin from four separate mice. (B) Kidney whole cell lysates of wild-type mice at the indicated times after I/R injury immunoblotted with pAkt and pErk (p42/44). The graph shows quantification from four separate mice as in A. (C) Lysates as in B immunoblotted and quantified for pErk. *P<0.05 versus sham; **P<0.01 versus sham; ***P<0.001 versus sham. n=4–6 per group.

Figure 2.

Renal proximal tubule–specific deletion of the Met receptor. (A) Illustration of LoxP containing the Met allele with the location of genotyping primers. (B) PCR of genomic DNA using primers A–C. The 406 bp band occurs with inclusion of the LoxP sites, whereas the 313 bp band is the wild-type sequence. γGT-Cre recombinase is seen in all three groups. (C) Western blot of lysates from the renal cortex and medulla (left panel) in γGT-Cre;Met^fl/fl and γGT-Cre;Met^+/+ mice with mouse proximal tubule (MPT) cells as a positive control. Western blot analysis of primary tubular epithelial cells isolated from renal cortex (right panel). β-Actin is used as a loading control. (D) Immunofluorescence of the renal cortex with α-Met (green) and α-megalin (red, proximal tubule marker). Note the absence of basolateral staining for the Met receptor in the proximal tubules of γGT-Cre;Met^fl/fl mice. DAPI (blue) is included in the merged image to identify nuclei.

Figure 3.

γGT-Cre;Met^fl/fl mice exhibit worse tubule injury after I/R. (A) Serum creatinine values are obtained on the indicated days in both groups. *P<0.05; ****P<0.0001 versus γGT-Cre;Met^+/+ by two-way ANOVA. n=3–7 per group at each time point. (B) Renal histology 48 hours after injury reveals greater loss of brush border, nuclear drop out, and cast formation in γGT-Cre;Metf^l/fl mice. (C) Tubular injury quantified in a single-blinded fashion. *P<0.05 versus γGT-Cre;Met^+/+. n=5.

Figure 4.

Decreased antiapoptotic PI3K/Akt signaling in γGT-Cre;Met^fl/fl mice after I/R. (A) Representative outer medullary kidney sections from γGT-Cre;Met^fl/fl and γGT-Cre;Met^+/+ mice with TUNEL-positive staining for apoptotic cells (TUNEL-positive nuclei green stain) and α-megalin (red stain) for identification of proximal tubules. DAPI is used for counterstaining. (B) Percentage of apoptotic proximal tubular cells quantified as seen in A. ***P<0.001 versus γGT-Cre;Met^+/+. n=4 per group. (C) Kidney lysates prepared from sham-operated mice, at indicated times after I/R surgery in γGT-Cre;Met^+/+ and γGT-Cre;Met^fl/fl mice and immunoblotted with pAkt antibody with β-actin as a loading control. (D) Quantification of pAkt as in C. ****P<0.0001 versus γGT-Cre;Met^+/+ by two-way ANOVA. n=6 per group. (E) Western blot analysis of kidney lysates from γGT-Cre;Met^+/+ and γGT-Cre;Met^fl/fl mice obtained at day 1 after injury and immunoblotted with pBad. (F) Quantification of pBad as in E normalized to β-actin. *P<0.05 versus γGT-Cre;Met^+/+. n=5 per group.

Figure 5.

Met receptor inactivation leads to a reduction in early p70S6K phosphorylation and delayed tubular proliferation. (A) Kidney lysates prepared from sham-operated mice at 6 hours and 1 day after I/R surgery in γGT-Cre;Met^+/+ and γGT-Cre;Met^fl/fl mice. Lysates are immunoblotted with phosphorylated p70S6K antibody using β-actin as a loading control. (B) Quantification of phosphorylated p70S6K as in A. **P<0.01; ***P<0.001 versus γGT-Cre;Met^+/+ by two-way ANOVA. n=5 per group. (C) Immunostaining of the renal outer medulla of γGT-Cre;Met^fl/fl and γGT-Cre;Met^+/+ mice with Ki-67 (nuclei stained brown) to assess tubular cell proliferation. (D) Quantification of proliferation as in C. ***P<0.001 versus γGT-Cre;Met^+/+. n=4 per group. (E) Whole kidney lysates of γGT-Cre;Met^+/+ and γGT-Cre;Met^fl/fl mice probed with pErk (42/44) antibody after sham operation or I/R surgery at the indicated times with β-actin as the loading control. (F) Quantification of Erk activation as in E. ***P<0.001 versus γGT-Cre;Met^+/+, by two-way ANOVA. n=6 in each group. (G) Quantification of tubular cell proliferation on day 7 after I/R injury. **P<0.01 versus γGT-Cre;Met^+/+. n=4 per group.