Proximal tubule-targeted heme oxygenase-1 in cisplatin-induced acute kidney injury

Abstract

Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that catalyzes the breakdown of heme to biliverdin, carbon monoxide, and iron. The beneficial effects of HO-1 expression are not merely due to degradation of the pro-oxidant heme but are also credited to the by-products that have potent, protective effects, including antioxidant, anti-inflammatory, and prosurvival properties. This is well reflected in the preclinical animal models of injury in both renal and nonrenal settings. However, excessive accumulation of the by-products can be deleterious and lead to mitochondrial toxicity and oxidative stress. Therefore, use of the HO system in alleviating injury merits a targeted approach. Based on the higher susceptibility of the proximal tubule segment of the nephron to injury, we generated transgenic mice using cre-lox technology to enable manipulation of HO-1 (deletion or overexpression) in a cell-specific manner. We demonstrate the validity and feasibility of these mice by breeding them with proximal tubule-specific Cre transgenic mice. Similar to previous reports using chemical modulators and global transgenic mice, we demonstrate that whereas deletion of HO-1, specifically in the proximal tubules, aggravates structural and functional damage during cisplatin nephrotoxicity, selective overexpression of HO-1 in proximal tubules is protective. At the cellular level, cleaved caspase-3 expression, a marker of apoptosis, and p38 signaling were modulated by HO-1. Use of these transgenic mice will aid in the evaluation of the effects of cell-specific HO-1 expression in response to injury and assist in the generation of targeted approaches that will enhance recovery with reduced, unwarranted adverse effects.

Keywords: acute kidney injury; cisplatin; heme oxygenase-1; p38.

Fig. 1.

Characterization of proximal tubule-specific heme oxygenase-1-overexpressing mice (ROSA-HO-1). A: schematic of the floxed and ROSA-HO-1 alleles. Before cre-mediated deletion (ROSA-Flox-HO-1; HO-1^+/+), the expression of the human HO-1 gene is prevented by the stop codon cassette flanked by LoxP sites (triangles). After cre-mediated recombination (ROSA-HO-1) at the LoxP sites, the stop codon is deleted, allowing the expression of HO-1. Total RNA was isolated from the kidneys of HO-1^+/+ and ROSA-HO-1 mice and analyzed for the expression of mouse (B) and human (C) HO-1. Data are expressed following normalization with GAPDH. D: HO-1 and HO-2 expression in the indicated tissues was determined by Western analysis. Anti-GAPDH antibody was used as a loading control. E: localization of HO-1 protein was determined by staining with lotus lectin (proximal tubule marker) and HO-1 on serial sections of kidneys from HO-1^+/+ and ROSA-HO-1 mice under basal conditions. PEPCK, phosphoenolpyruvate carboxykinase.

Fig. 2.

Proximal tubule-specific HO-1 overexpression protects against cisplatin nephrotoxicity. A: body weight loss following cisplatin administration (20 mg/kg body wt; intraperitoneal single dose) is expressed as a percentage of the original weight. Data are expressed as means ± SE. B: serum creatinine levels were measured and expressed as milligrams/deciliter day 3 after cisplatin administration. Data are expressed as means ± SE; *P < 0.05. C: representative periodic acid-Schiff (PAS) staining of kidney sections from HO-1^+/+ and ROSA-HO-1 mice at day 3 following cisplatin administration. D: whole-kidney protein lysates from HO-1^+/+ and ROSA-HO-1 mice were analyzed for the expression of cleaved caspase-3 (CC3) and HO-1. Anti-GAPDH antibody was used as a loading control. E: whole-kidney protein lysates from saline- and cisplatin-administered HO-1^+/+ and ROSA-HO-1 mice were analyzed for the expression of organic cation transporters 1 and 2 (OCT1 and -2) and HO-1. Anti-GAPDH antibody was used as a loading control. F: serum creatinine levels were measured and expressed as milligrams/deciliter at day 5 after cisplatin administration. Data are expressed as means ± SE; *P < 0.05. G: representative PAS of kidney sections from HO-1^+/+ and ROSA-HO-1 mice at day 3 following cisplatin administration. H: scoring of structural damage was performed by counting the number of casts and necrotic tubules in the cortex and outer medulla. Data are represented as means ± SE; *P < 0.05.

Fig. 3.

Characterization of proximal tubule-specific HO-1 deletion mice (HO-1^PT−/−). A: schematic of the floxed vector, PEPCK Cre, and HO-1^PT−/− alleles. Following PEPCK Cre-mediated recombination, exons 3 and 4 (E3 and E4) are deleted, leading to loss of HO-1 expression in the proximal tubules. Total RNA was isolated from the indicated organs of HO-1^+/+ and HO-1^PT−/− mice and analyzed for the expression of HO-1 (B) and HO-2 (C). Data are normalized to GAPDH and expressed as fold change compared with HO-1^+/+ mice; *P < 0.05. D: protein lysates from the indicated organs were analyzed for HO-1 expression by Western analysis. Anti-GAPDH antibody was used as a loading control. E: deletion of HO-1 specifically in the proximal tubules of HO-1^PT−/− mice was confirmed by staining with lotus lectin (proximal tubule marker) and HO-1 on serial sections of kidneys from HO-1^+/+ and HO-1^PT−/− mice following 24 h of renal ischemia reperfusion.

Fig. 4.

HO-1^PT−/− exacerbates cisplatin nephrotoxicity. A: loss of body weight following cisplatin administration was calculated and expressed as a percentage of the original weight. Data are expressed as means ± SE. B: serum creatinine levels were measured and expressed as milligrams/deciliter at day 3 after cisplatin administration. Data are expressed as means ± SE; *P < 0.05. C: representative PAS-stained sections of the cortex and outer medulla from HO-1^+/+ and HO-1^PT−/− mice at day 3 following cisplatin administration. D: scoring of structural damage was performed by counting the number of casts and necrotic tubules in the cortex and outer medulla. Data are represented as means ± SE; *P < 0.05. E: whole-kidney lysates of cisplatin-administered HO-1^+/+ and HO-1^PT−/− mice were analyzed for the expression of HO-1, phospho-p38 (pP38), p38, and CC3 by Western analysis. Membranes were stripped and reprobed for GAPDH to demonstrate equal loading. F: densitometric analysis of HO-1 and CC3 was performed and normalized to GAPDH expression. Phospho-p38 expression was normalized to total p38 expression. Data are represented as means ± SE; *P < 0.05. G and H: whole-kidney protein lysates from saline- and cisplatin-administered HO-1^+/+ and HO-1^PT−/− mice were analyzed for the expression of OCT1 and -2 and HO-1. Anti-GAPDH antibody was used as a loading control.