Figure 4. IRI-induced ADAM17 pathway components are reduced in ADAM17ex/ex mice.

The expression levels of ADAM17 and its pathway components were examined at different time points after ischemic injury in whole kidneys. (A) qPCR analysis of ADAM17 mRNA expression levels in ex/ex or WT/WT mice, expressed as fold over respective sham-injured mice (n = 3–5). (B) Western blot analysis of ADAM17 protein levels in ex/ex or WT/WT mice subjected to sham or IRI (day 5 after ischemia) (left: sample blots; right: quantification; GAPDH was used as loading control; n = 5). (C–H) qPCR analysis of EGFR ligands, EGFR, TNFα, and TNFR1/2 mRNA expression levels in ex/ex or WT/WT mice, expressed as fold over respective sham-injured mice (time points as indicated; n = 3–6). (I) Soluble ectodomains of the ADAM17 substrates TNFα, TGFα, and AREG were measured in whole kidney lysates of WT/WT or ex/ex mice at day 2 after ischemia by ELISA (ADAM10 substrate cMET is used as control; n = 4). (J) Time-course of EGFR phosphorylation (Y1068) as measured by Western blot in whole kidney lysates of WT/WT or ex/ex mice (n = 5). (K) Representative images of EGFR phosphorylation (Y1173) examined in kidney cortex by immunostaining in WT/WT or ex/ex mice at day 2 after injury (left: representative images; right: quantification; n = 3; scale bar: 50 μm). *P < 0.05; **P < 0.01; ***P < 0.001 as determined by an unpaired 2-tailed Student’s t test. ADAM17; a disintegrin and metalloprotease 17; EGFR, epidermal growth factor receptor; IRI, ischemia reperfusion injury; NS, nonspecific band; TNFα, tumor necrosis factor α; TNFR, tumor necrosis factor receptor; AREG, amphiregulin; ADAM10, a disintegrin and metalloprotease 10; cMet, met proto-oncogene; WB, Western blot.