Heme Oxygenase-1 Inhibits Renal Tubular Macroautophagy in Acute Kidney Injury

Autophagy is a tightly regulated, programmed mechanism to eliminate damaged organelles and proteins from a cell to maintain homeostasis. Cisplatin, a chemotherapeutic agent, accumulates in the proximal tubules of the kidney and causes dose-dependent nephrotoxicity, which may involve autophagy. In the kidney, cisplatin induces the protective antioxidant heme oxygenase-1 (HO-1). In this study, we examined the relationship between autophagy and HO-1 during cisplatin-mediated acute kidney injury (AKI). In wild-type primary proximal tubule cells (PTC), we observed a time-dependent increase in autophagy after cisplatin. In HO-1^−/− PTC, however, we observed significantly higher levels of basal autophagy, impaired progression of autophagy, and increased apoptosis after cisplatin. Restoring HO-1 expression in these cells reversed the autophagic response and inhibited apoptosis after treatment with cisplatin. In vivo, although both wild-type and HO-1–deficient mice exhibited autophagosomes in the proximal tubules of the kidney in response to cisplatin, HO-1–deficient mice had significantly more autophagosomes, even in saline-treated animals. In addition, ecdysone-induced overexpression of HO-1 in cells led to a delay in autophagy progression, generated significantly lower levels of reactive oxygen species, and protected against cisplatin cytotoxicity. These findings demonstrsate that HO-1 inhibits autophagy, suggesting that the heme oxygenase system may contain therapeutic targets for AKI.Oxidative stress plays a major role in the pathogenesis of cisplatin-induced nephrotoxicity.^1,2 In response to injury, the kidney is able to elicit adaptive and protective mechanisms to limit further damage. One such mechanism is the rapid and robust induction of heme oxygenase-1 (HO-1).^3–5 Heme oxygenase is the rate-limiting enzyme in the degradation of heme to iron, carbon monoxide, and biliverdin.^3,6,7 Studies have shown that HO-1 mRNA is induced in the kidney as early as 3 to 6 hours in animal models of both ischemia/reperfusion and nephrotoxin-induced acute kidney injury (AKI).^3,8 Such induction occurs predominantly in the proximal tubule segment of the nephron,^3,8 which coincides with the location of maximal cisplatin accumulation and oxidative stress.^9,10 Chemical inhibition of HO enzyme activity in rats³ and genetically deficient HO-1 mice¹¹ treated with cisplatin have significantly worse kidney function and tubular injury, suggesting a protective role for HO-1 expression in cisplatin-induced renal tubular cell death, specifically necrosis and apoptosis.Recent evidence indicates that autophagy, a type II programmed cell death, is induced during cisplatin injury in proximal tubular epithelial cells (PTC) and is a protective response.^12–15 Autophagy, a physiologically regulated and evolutionarily conserved process, refers to an intracellular degradation system in which cytoplasmic components, such as damaged organelles, long-lived proteins, protein aggregates, and other macromolecules, are directed to the lysosome.^16–19 Autophagy (also referred to as macroautophagy) begins with the formation of an initiation membrane (vesicle nucleation) that sequesters cytoplasmic components as it expands (vesicle elongation); finally, the edges fuse to form a double-membraned vesicle called autophagosome. This vesicle fuses with the lysosome to form an autolysosome where the sequestered components are degraded by the acidic lysosomal enzymes.^17,20 At least 31 Atg (Autophagy) genes have been identified in yeast and their mammalian orthologs have also been recently characterized.^21–24 Expression of the mammalian orthologs of Atg5, Atg6 (beclin 1), Atg7, and Atg8 (LC3, microtubule-associated protein 1 light chain 3) are used as markers to detect autophagy in mammalian cells.^13,25,26 Both Atg5 and beclin play an important role in autophagosome initiation and vesicle nucleation. Vesicle elongation requires several autophagy proteins such as Atg7 and Atg4. These proteins conjugate the lipid phosphatidylethanolamine to LC3 to form membrane-associated LC3-II. LC3-II is one of the autophagy proteins that specifically interacts only with the autophagic vesicles and remains associated until vesicle breakdown. All of the other proteins associate with the vesicle at different stages of maturation and have alternate functions in the cell. Therefore, LC3-II is a valuable marker to assess the presence of autophagosomes in cells.Several in vitro and in vivo studies suggest that autophagy can induce cell survival or death depending on the stress or the cellular environment.^{12,13,18,22,27} Under normal physiologic conditions, cells use autophagy to maintain homeostasis. If insufficient autophagy occurs, long-lived proteins and damaged organelles accumulate and cell death occurs. Even under certain pathologic conditions, autophagy is induced and is cytoprotective. However, if autophagy is prolonged or unregulated, it can lead to cell death. This suggests that autophagy may act as a cytoprotective mechanism but converges into apoptotic pathways during severe stress. Therefore, it is important to understand how autophagy is modulated as both insufficient and excessive autophagy have deleterious effects.Because both autophagy and HO-1 are induced during cisplatin injury, the purpose of this study was to evaluate whether HO-1 expression modulated autophagy in PTC and protected them from cisplatin-induced cell death. We studied the effects of HO-1 deficiency using HO-1 knockout (HO-1^−/−) mice on the progression of autophagy during cisplatin injury. Also, PTC cultures generated from HO-1^+/+ (heme oxygenase-1 wild-type) and HO-1^−/− mice were analyzed for cisplatin-mediated autophagy and cell death. Furthermore, HO-1^−/− mice that specifically express only the human HO-1 gene (HBAC mice, human HO-1 overexpressing bacterial artificial chromosome mice) were generated and PTC isolated from these mice were used to study the effects of restoring HO-1 expression on autophagy progression during cisplatin injury. Also, ecdysone inducible HO-1 overexpressing renal epithelial cells were generated and analyzed for cisplatin-mediated autophagy.