Severity of renal ischemia-reperfusion injury in wild type and AMPK-β1^−/− mice assessed by serum urea and creatinine at 24 hours post IR.

<p>Serum urea (A) and creatinine (B) were measured 24 hours post renal IR for 18 or 20 minutes in WT and AMPK-β1^−/− mice (n = 6–13 per group). Results are shown as mean ± SD. * P<0.001, ** P<0.01. No significant differences between WT and AMPK-β1^−/− mice were observed.</p

AMPK activation by acute renal ischemia in wild type, AMPK-α1^−/− and AMPK-β1^−/− mice.

<p>Lysates (1 mg protein) from control (□ no ischemia) and ischemic (▪ 10 min ischemia) kidneys of WT (C57Bl/6), AMPK-α1^−/− and AMPK-β1^−/− mice were immunoprecipitated with antibodies specific for the α1 and α2 AMPK catalytic subunits (n = 6–8 per group). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029887#s3" target="_blank">Results</a> are shown as mean ± SD. In WT mice AMPK activity was increased by acute renal ischaemia (P<0.001). In AMPK-β1^−/− mice, AMPK activity after acute renal ischaemia was reduced compared to WT for both AMPK-α1 (P<0.001) and AMPK-α2 (P<0.001) isoforms. In AMPK-α1^−/− mice there was no activation of AMPK-α1 by acute renal ischemia, whereas AMPK-α2 was activated by acute renal ischemia (P<0.01) by an amount not different to WT. * P<0.001, ** P<0.01.</p

Histological injury 24 hours after renal ischemia-reperfusion injury in wild type and AMPK-β1^−/− mice.

<p>IRI was performed for 20 minutes and the severity of injury was assessed by histology. A. Histological appearance at 24 hours following 20 mins IRI in WT and β1^−/− mice. Images shown are from the region of the corticomedullary junction (CMJ). 200× magnification. The severity of histological injury at 24-hours was quantified in cortex (B), CMJ (C) and medulla (D) as described in methods. No differences were seen between WT and β1^−/− mice. n = 7 for WT and 6 for β1^−/−.</p

AMPK activation and phosphorylation after acute renal ischemia in MIF^−/− mice.

<p>Lysates (1 mg protein) from control (□ no ischemia) and ischemic (▪ 10 min ischemia) kidneys of WT (C57Bl/6) and MIF^−/− mice were immunoprecipitated with antibodies specific for the α1 (A) and α2 (B) AMPK catalytic subunits (n = 10–12 per group for α1 and n = 6 for α2). AMPK activity was measured by ADR-1 peptide activity assay. Results are shown as mean ± SD. α1-AMPK and α2-AMPK activity were increased several fold after 10 minutes ischemia in WT and MIF^−/− mice (* P<0.001). There were no differences in α1-AMPK or α2-AMPK activity between WT and MIF^−/− under either control or ischemic conditions. C. Lysates (1 mg protein) from control (C) and ischemic (Isc) kidneys of WT and MIF^−/− mice were immunoprecipitated with antibodies specific for the α1 AMPK catalytic subunit and analyzed by Western blotting using antibodies against pAMPKαThr¹⁷², pAMPKαSer⁴⁸⁵ and α1AMPK (blot shown representative of 4 experiments). D. In addition, whole kidney lysates (50 µg) from the same experiment were separated by SDS-PAGE and analyzed by Western blot for AMPK phosphorylation at αThr¹⁷² and αSer⁴⁸⁵, with blotting for β-actin as a loading control (blot shown representative of 4 experiments). E. Kidney lysates from this experiment were blotted for inhibitory phosphorylation of ACC at Ser⁷⁹ and expression of total ACC1 (blot shown representative of 4 experiments). F. Heart and kidney lysate was blotted for expression of the MIF receptor CD74.</p

Renal expression of activated AMPK is markedly reduced in AMPK-β1^−/− mice.

<p>A and B. Lysate from WT and AMPK-β1^−/− kidneys were analyzed by Western blot for the activated form of AMPK, phosphorylated at Thr¹⁷² of the catalytic (α) subunit. The membrane was stripped and reblotted to determine expression of the catalytic (α1 and α2) (A) and scaffolding (β1 and β2) (B) subunits of AMPK. Expression of β-actin was determined to confirm even sample loading. C. Expression of activated (phospho-αThr¹⁷²) in kidneys from WT and AMPK-β1^−/− mice was quantified by densitometry using arbitrary units (n = 6 per group). Densitometry for phospho-αThr¹⁷² was corrected for β-actin. * P<0.001 by unpaired t test.</p