Heme oxygenase-2 protects against ischemic acute kidney injury: influence of age and sex

Abstract

Heme oxygenase (HO) activity is exhibited by inducible (HO-1) and constitutive (HO-2) proteins. HO-1 protects against ischemic and nephrotoxic acute kidney injury (AKI). We have previously demonstrated that HO-2 protects against heme protein-induced AKI. The present study examined whether HO-2 is protective in ischemic AKI. Renal ischemia was imposed on young and aged HO-2^+/+ and HO-2^-/- mice. On days 1 and 2 after renal ischemia, there were no significant differences in renal function between young male HO-2^+/+ and HO-2^-/- mice, between young female HO-2^+/+ and HO-2^-/- mice, or between aged female HO-2^+/+ and HO-2^-/- mice. However, in aged male mice, HO-2 deficiency worsened renal function on days 1 and 2 after ischemic AKI, and, on day 2 after ischemia, such deficiency augmented upregulation of injury-related genes and worsened histological injury. Renal HO activity was markedly decreased in unstressed aged male HO-2^-/- mice and remained so after ischemia, despite exaggerated HO-1 induction in HO-2^-/- mice after ischemia. Such exacerbation of deficiency of HO-2 protein and HO activity may reflect phosphorylated STAT3, as activation of this proinflammatory transcription factor was accentuated early after ischemia in aged male HO-2^-/- mice. This exacerbation may not reflect impaired induction of nephroprotectant genes, since the induction of HO-1, sirtuin 1, and β-catenin was accentuated in aged male HO-2^-/- mice after ischemia. We conclude that aged male mice are hypersensitive to ischemic AKI and that HO-2 mitigates such sensitivity. We speculate that this protective effect of HO-2 may be mediated, at least in part, by suppression of phosphorylated STAT3-dependent signaling.

Keywords: acute kidney injury; age; biology of sex differences; heme oxygenase-2; phospho-STAT3.

Fig. 1.

Renal filtration markers in aged male mice with targeted disruption of the heme oxygenase (HO)-2 gene (HO-2^−/− mice) and littermate control (HO-2^+/+) mice after acute renal ischemia [ischemia-reperfusion (IR)]. Plasma levels of creatinine (A) and blood urea nitrogen (BUN; B) were measured in aged male HO-2^+/+ and HO-2^−/− mice on days 1 and 2 after 15 min of IR. Values are means ± SE; n = 11 HO-2^+/+ and 12 HO-2^−/− mice. *P < 0.05 vs. HO-2^+/+ mice on the same day.

Fig. 2.

Renal expression of acute inflammation-related genes [chemokine (C-C motif) ligand 2 (CCL2), IL-6, matrix metalloproteinase (MMP)-9, and plasminogen activator inhibitor 1 (PAI-1)] in aged male mice with targeted disruption of the heme oxygenase (HO)-2 gene (HO-2^−/− mice) and littermate control (HO-2^+/+) mice on day 2 after acute (15 min) renal ischemia (IR). mRNA expression of CCL2 (A), IL-6 (B), MMP-9 (C), and PAI-1 (D) was determined by quantitative real-time RT-PCR. Values are means ± SE; n = 4 mice subjected to sham operation and n = 11 HO-2^+/+ and 12 HO-2^−/− mice subjected to IR. *P < 0.05 vs. HO-2^+/+ mice subjected to IR.

Fig. 3.

Renal expression of chronic inflammation-related genes [transforming growth factor (TGF)-β₁, collagen type I (Col I), collagen type III (Col III), and collagen type IV (Col IV)] in aged male mice with targeted disruption of the heme oxygenase (HO)-2 gene (HO-2^−/− mice) and littermate control (HO-2^+/+) mice on day 2 after acute (15 min) renal ischemia (IR). mRNA expression of TGF-β₁ (A), Col I (B), Col III (C), and Col IV (D) was determined by quantitative real-time RT-PCR. Values are means ± SE; n = 4 mice subjected to sham surgery and n = 11 HO-2^+/+ and 12 HO-2^−/− mice subjected to IR. *P < 0.05 vs. HO-2^+/+ mice subjected to IR.

Fig. 4.

Renal expression of injury-related genes [platelet-derived growth factor-A (PDGFA), matrix metalloproteinase (MMP)-2, p21, and p16] in aged male mice with targeted disruption of the heme oxygenase (HO)-2 gene (HO-2^−/− mice) and littermate control (HO-2^+/+) mice on day 2 after acute renal ischemia (IR). mRNA expression of PDGFA (A), MMP-2 (B), p21 (C), and p16 (D) was determined by quantitative real-time RT-PCR. Values are means ± SE; n = 4 mice subjected to sham surgery and n = 11 HO-2^+/+ and 12 HO-2^−/− mice subjected to IR. *P < 0.05 vs. HO-2^+/+ mice subjected to IR.

Fig. 5.

Histological appearance of the kidney in aged male mice with targeted disruption of the heme oxygenase (HO)-2 gene (HO-2^−/− mice; B and D) and littermate control (HO-2^+/+) mice (A and C) on day 2 after acute (15 min) renal ischemia (IR). Lower-power ( ×100; A and B) and higher-power ( ×200; C and D) views are shown. Aged male HO-2^−/− mice displayed more severe renal injury than aged male HO-2^+/+ mice in response to IR, with a greater percentage of tubules exhibiting necrosis and with greater extension of necrosis into the cortex. No histological injury was observed in kidneys of aged male HO-2^+/+ and HO-2^−/− mice subjected to sham IR (not shown). Tissue sections were stained with hematoxylin and eosin. Scale bars = 250 μm (A and B) and 100 μm (C and D).

Fig. 6.

Semiquantitative assessment of necrosis in the kidney on day 2 after acute renal ischemia (IR) in aged male mice with targeted disruption of the heme oxygenase (HO)-2 gene (HO-2^−/− mice) and littermate control (HO-2^+/+) mice. A: percentage of tubules exhibiting tubular epithelial cell necrosis. B: extension of cortical necrosis, determined as the mean percentage of the distance from the corticomedullary junction to the surface of the kidney. Values are means ± SE; n = 11 HO-2^+/+ and 12 HO-2^−/− mice subjected to IR. *P < 0.05 vs. HO-2^+/+ mice subjected to IR.

Fig. 7.

Basal expression of heme oxygenase (HO)-1 mRNA (A) and HO activity (B) in unstressed kidneys of aged male mice with targeted disruption of the HO-2 gene (HO-2^−/− mice) and littermate control (HO-2^+/+) mice. HO-1 mRNA was determined by quantitative real-time RT-PCR (A), and microsomal HO activity was measured in the intact unstressed kidney (B). Values are means ± SE; n = 8 for each group. *P < 0.05 vs. HO-2^+/+ mice.

Fig. 8.

Renal microsomal heme oxygenase (HO) activity (A) and renal HO-1 mRNA expression (B) in aged male mice with targeted disruption of the HO-2 gene (HO-2^−/− mice) and littermate control (HO-2^+/+) mice 6 h after acute renal ischemia (IR). Values are means ± SE; n = 4 mice subjected to sham surgery and n = 9 mice subjected to IR in A and n = 4 mice subjected to sham surgery and n = 7 HO-2^+/+ and 8 HO-2^−/− mice subjected to IR in B. *P < 0.05 vs. HO-2^+/+ mice subjected to IR; †P < 0.05 vs. HO-2^+/+ mice subjected to sham surgery.

Fig. 9.

Renal phosphorylated (p-)STAT3 expression in aged male mice with targeted disruption of the heme oxygenase (HO)-2 gene (HO-2^−/− mice) and littermate control (HO-2^+/+) mice 6 h after acute renal ischemia (IR). Renal expression of p-STAT3 protein was determined by Western blot analysis. Values are means ± SE; n = 4 mice subjected to sham surgery and n = 7 HO-2^+/+ and 8 HO-2^−/− mice subjected to IR. *P < 0.05 vs. HO-2^+/+ mice subjected to IR.

Fig. 10.

Renal expression of β-catenin and sirtuin (SIRT)1 in aged male mice with targeted disruption of the heme oxygenase (HO)-2 gene (HO-2^−/− mice) and littermate control (HO-2^+/+) mice 6 h after acute renal ischemia (IR). Renal expression of β-catenin and SIRT1 protein was determined by Western blot analysis. Values are means ± SE; n = 4 mice subjected to sham surgery and n = 7 HO-2^+/+ and 8 HO-2^−/− mice subjected to IR. *P < 0.05 vs. HO-2^+/+ mice subjected to IR.