Age sensitizes the kidney to heme protein-induced acute kidney injury

Abstract

Age increases the risk for ischemic acute kidney injury (AKI). We questioned whether a similar age-dependent injury occurs following exposure to hemoglobin, a known nephrotoxin. Old mice (~16 mo old), but not young mice (~6 mo old), when administered hemoglobin, exhibited marked elevation in blood urea nitrogen (BUN) and serum creatinine, and acute tubular necrosis with prominent tubular cast formation. The aged kidney exhibited induction of heme oxygenase-1 (HO-1) and other genes/proteins that may protect against heme-mediated renal injury, including ferritin, ferroportin, haptoglobin, and hemopexin. Old mice did not evince induction of HO-2 mRNA by hemoglobin, whereas a modest induction of HO-2 mRNA was observed in young mice. To determine the functional significance of HO-2 in heme protein-induced AKI, we administered hemoglobin to relatively young HO-2(+/+) and HO-2(-/-) mice: HO-2(-/-) mice, compared with HO-2(+/+) mice, exhibited greater renal dysfunction and histologic injury when administered hemoglobin. In addition to failing to elicit a protective system such as HO-2 in response to hemoglobin, old mice exhibited an exaggerated maladaptive response typified by markedly greater induction of the nephrotoxic cytokine IL-6 (130-fold increase vs. 10-fold increase in mRNA in young mice). We conclude that aged mice, unlike relatively younger mice, are exquisitely sensitive to the nephrotoxicity of hemoglobin, an effect attended by a failure to induce HO-2 mRNA and a fulminant upregulation of IL-6. Age thus markedly augments the sensitivity of the kidney to heme proteins, and HO-2 confers resistance to such insults.

Fig. 1.

Renal function in old (16 mo) and young (6 mo) mice 1 day after the intravenous administration of hemoglobin. Measurements of serum creatinine (A) and blood urea nitrogen (BUN; B) were performed at 1 day after the administration of saline or hemoglobin (Hgb). *P < 0.05 vs. similarly treated young mice; n = 4 and n = 5 for young and old saline-treated mice, and n = 6 and n = 11 for young and old Hgb-treated mice, respectively.

Fig. 2.

Histological appearance of the kidney in old mice 1 day after the intravenous administration of hemoglobin. Representative low power (×200) and high power (×400) views of saline-treated mice (A and C) and Hgb-treated mice (B and D) are displayed; in old mice, hemoglobin induces vacuolization, necrosis, and apoptosis of tubular epithelial cells, intratubular casts, and tubular dilatation.

Fig. 3.

Renal heme oxygenase-1 (HO-1) and HO-2 expression in old mice 1 day after the intravenous administration of hemoglobin. HO-1 (A) and HO-2 (B) mRNA expression in the kidneys of saline-treated mice and Hgb-treated mice was assessed by quantitative real-time RT-PCR and normalized to 18S rRNA expression. *P < 0.05 vs. saline-treated mice; n = 5 and n = 11 for saline-treated and Hgb-treated mice, respectively.

Fig. 4.

Renal ferritin content and ferroportin expression in old mice 1 day after the intravenous administration of hemoglobin. A: ferritin content in kidney lysates from saline-treated mice and Hgb-treated mice was assayed by ELISA and normalized to protein content. B: ferroportin mRNA expression was assessed by quantitative real-time RT-PCR and normalized to 18S rRNA expression. *P < 0.05 vs. saline-treated mice; n = 5 and n = 11 for saline-treated mice and Hgb-treated mice, respectively.

Fig. 5.

Renal haptoglobin (Hp) and CD163 expression in old mice 1 day after the intravenous administration of hemoglobin. Hp (A) and CD163 (B) mRNA expression in the kidneys of saline-treated mice and Hgb-treated mice was assessed by quantitative real-time RT-PCR and normalized to 18S rRNA expression. *P < 0.05 vs. saline-treated mice; n = 5 and n = 11 for saline-treated mice and Hgb-treated mice, respectively.

Fig. 6.

Renal hemopexin (Hpx) and CD91 expression in old mice 1 day after the intravenous administration of hemoglobin. Hpx (A) and CD91 (B) mRNA expression in the kidneys of saline-treated mice and Hgb-treated mice was assessed by quantitative real-time RT-PCR and normalized to 18S rRNA expression. *P < 0.05 vs. saline-treated mice; n = 5 and n = 11 for saline-treated mice and Hgb-treated mice, respectively. Renal Hpx protein expression (C) was also assessed in these mice by Western analysis, which employed β-actin as a housekeeping protein.

Fig. 7.

Renal megalin and cubilin expression in old mice 1 day after the intravenous administration of hemoglobin. Megalin (A) and cubilin (B) mRNA expression in the kidneys of saline-treated mice and Hgb-treated mice was assessed by quantitative real-time RT-PCR and normalized to 18S rRNA expression. *P < 0.05 vs. saline-treated mice; n = 5 and n = 11 for saline-treated mice and Hgb-treated mice, respectively.

Fig. 8.

Renal expression of proinflammatory genes in old mice 1 day after the intravenous administration of hemoglobin. TNF-α (A), monocyte chemoattractant protein-1 (MCP-1; B), and IL-6 (C) mRNA expression in the kidneys of saline-treated and Hgb-treated mice was assessed by quantitative real-time RT-PCR and normalized to 18S rRNA expression. *P < 0.05 vs. saline-treated mice; n = 5 and n = 11 for saline-treated mice and Hgb-treated mice, respectively.

Fig. 9.

Renal expression of profibrotic genes in old mice 1 day after the intravenous administration of hemoglobin. Transforming growth factor (TGF)-β1 (A), Smad3 (B), collagen I (Col I; C), and collagen III (Col III; D) mRNA expression in the kidneys of saline-treated mice and Hgb-treated mice was assessed by quantitative real-time RT-PCR and normalized to 18S rRNA expression. *P < 0.05 vs. saline-treated mice; n = 5 and n = 11 for saline-treated mice and Hgb-treated mice, respectively.

Fig. 10.

Serum creatinine concentration and BUN measurements in HO-2^+/+ and HO-2^−/− mice prior to and after the intravenous administration of hemoglobin. Serum creatinine (A) and BUN measurements (B) were performed at baseline and on days 1 and 2 after the intravenous administration of hemoglobin in HO-2^+/+ and HO-2^−/− mice. *P < 0.05 vs. HO-2^+/+ mice on that day; n = 6–8 in each group at baseline, and n = 13 and n = 15 for HO-2^+/+ and HO-2^−/− mice, respectively, on days 1 and 2.

Fig. 11.

Histological appearance of the kidney 2 days after the intravenous administration of hemoglobin in HO-2^+/+ and HO-2^−/− mice. Representative low power (×200) and high power (×400) views of HO-2^+/+ (A and C) and HO-2^−/− (B and D) mice are displayed; in HO-2^−/− mice, hemoglobin induces necrosis of tubular epithelial cells, intratubular casts, and tubular dilatation.