The indispensability of heme oxygenase-1 in protecting against acute heme protein-induced toxicity in vivo

Abstract

Heme oxygenase (HO) is the rate limiting enzyme in the degradation of heme, and its isozyme, HO-1, may protect against tissue injury. One posited mechanism is the degradation of heme released from destabilized heme proteins. We demonstrate that HO-1 is a critical protectant against acute heme protein-induced toxicity in vivo. In the glycerol model of heme protein toxicity-one characterized by myolysis, hemolysis, and kidney damage-HO-1 is rapidly induced in the kidney of HO-1 +/+ mice as the latter sustain mild, reversible renal insufficiency without mortality. In stark contrast, after this insult, HO-1 -/- mice exhibit fulminant, irreversible renal failure and 100% mortality; HO-1 -/- mice do not express HO-1, and evince an eightfold increment in kidney heme content as compared to HO-1 +/+ mice. We also demonstrate directly the critical dependency on HO-1 in protecting against a specific heme protein, namely, hemoglobin: doses of hemoglobin which exert no nephrotoxicity or mortality in HO-1 +/+ mice, however, precipitate rapidly developing, acute renal failure and marked mortality in HO-1 -/- mice. We conclude that the induction of HO-1 is an indispensable response in protecting against acute heme protein toxicity in vivo.

Figure 1.

Effect of hypertonic glycerol (50%), 7.5 and 10 ml/kg, in dehydrated C57BL/6 mice on HO-1 mRNA expression, determined 6 hours after glycerol. The individual and mean standardized densitometric readings are provided below the Northern analysis.

Figure 2.

Alterations in plasma creatinine in HO-1 +/+ mice (open columns) and HO-1 −/− mice (solid columns) after glycerol. Basal plasma creatinine was determined and mice were then dehydrated overnight. Hypertonic glycerol (50%) was injected (7.5 ml/kg) after which plasma creatinine was measured on sequential days. Each group consisted of eight mice before glycerol. *, P < 0.05 between HO-1 +/+ and HO-1 −/− mice on the same day.

Figure 3.

Photomicrographs of kidney sections 2 days after administration of glycerol to HO-1 +/+ and HO-1 −/− mice. HO-1 −/− mice subjected to glycerol (right panel) demonstrate diffuse and extensive tubular epithelial cell necrosis and tubular cast formation; such changes are much less prominent in HO-1 +/+ mice subjected to glycerol (left panel). Original magnification, ×200.

Figure 4.

a: Alterations in plasma lactate dehydrogenase (LDH) in HO-1 +/+ (open column) and HO-1 −/− mice (solid column). Mice were subjected to glycerol as described in Figure 2 ▶ , and plasma LDH was measured sequentially. *, P < 0.05 between HO-1 +/+ and HO-1 −/− mice on the same day. b: Percent survival in HO-1 +/+ (open square) and HO-1 −/− mice (solid square) subjected to glycerol-induced heme protein injury. Mice were subjected to glycerol as described in Figure 2 ▶ , and these survival data are derived from studies summarized in Figure 2 ▶ . Each group consisted of eight mice before the administration of glycerol.

Figure 5.

a: Northern analysis for HO-1 expression in kidneys in HO-1 +/+ and HO-1 −/− mice, before (basal) and 6 hours after glycerol (7.5 ml/kg). Each lane represents RNA extracted from a single kidney of an individual mouse, and in each group, three mice were studied. b: Western analysis for HO-1 expression in kidneys of HO-1 +/+ and HO-1 −/− mice, before (basal) and 6 hours after the administration of hypertonic glycerol (7.5 ml/kg). Each lane represents a single kidney of an individual mouse, and in each group, two mice were studied. c: Increment in kidney heme content above basal values in whole kidney homogenates in HO-1 +/+ and HO-1 −/− mice 6 hours after the administration of hypertonic glycerol (7.5 ml/kg). Each group consisted of eight mice. *, P < 0.05 between HO-1 +/+ and HO-1 −/− mice.

Figure 6.

Immunoperoxidase staining for HO-1 in HO-1 +/+ mice, before and 6 hours after glycerol (7.5 ml/kg). In the basal state the kidneys of HO-1 +/+ mice do not express HO-1 (left panel). In response to glycerol prominent induction of HO-1 was observed in the proximal tubules of HO-1 +/+ mice (right panel). There was no evidence of HO-1 staining in HO-1 −/− mice either in the basal state or 6 hours after glycerol (data not shown). Original magnification, ×400.

Figure 7.

a: Alterations in kidney function as assessed by daily plasma creatinine in HO-1 +/+ mice (open column) and HO-1 −/− mice (solid column) after infusions of mouse hemoglobin. Each group consisted of eight mice before the administration of hemoglobin. *, P < 0.05 between HO-1 +/+ and HO-1 −/− mice on the same day. b: Percent survival in HO-1 +/+ (open square) and HO-1 −/− mice (solid square) after infusions of mouse hemoglobin as described in a. Each group consisted of eight mice before the administration of hemoglobin.

Figure 8.

Alteration in kidney function in younger mice (age, 7 to 10 weeks) as assessed by plasma creatinine in HO-1 +/+ mice (open column) and HO-1 −/− mice (solid column) after infusion of mouse hemoglobin (n = 5 and n = 6 in the HO-1 +/+ and HO-1 −/− group, respectively). *, P < 0.05 between HO-1 +/+ and HO-1 −/− mice on the same day.

Figure 9.

Photomicrographs of histological sections from the kidney in HO-1 +/+ and HO-1 −/− mice 1 day after infusion of mouse hemoglobin as described in Figure 8 ▶ . HO-1 −/− mice subjected to hemoglobin (right panel) demonstrated diffuse and extensive tubular epithelial cell necrosis; histologically, the kidney in the HO-1 +/+ mice appeared normal after hemoglobin (left panel).