Regulation and Pathological Role of Bid in Ischemic Acute Kidney Injury

Abstract

Bid, a BH3-only member of the Bcl-2 family proteins, is most abundantly expressed in the kidneys. Recent research has shown Bid activation in renal tubular cells in vitro following ATP-depletion and hypoxic injury, and also in vivo during renal ischemia-reperfusion in rats and mice. Importantly, Bid-deficient mice are resistant to ischemic kidney injury. Targeting Bid may therefore offer a new strategy for the treatment of acute renal failure associated with ischemia-reperfusion.

Keywords:

• acute kidney injury
• Bid
• apoptosis
• Bcl-2 family protein
• ischemia

INTRODUCTION

Acute renal failure (ARF) is characterized by kidney tissue injury and rapid loss of renal function.[1–3] Clinically, there are two main causes of ARF: renal ischemia and nephrotoxicity. Renal ischemia is a condition of decreased blood supply to kidney tissues, which occurs as a consequence of cardiac failure, vasoconstriction, dehydration, septic shock, vessel-clamping during surgical operation, and kidney transplantation. Nephrotoxicity is due to kidney tissue exposure to toxins, which include environmental chemicals such as mercury, cadmium, and chloroform, as well as some of the commonly used clinical drugs such as the aminoglycoside class of antibiotics and cancer therapy agents including cisplatin and ifosfamide. A recent analysis indicates that more than 30% of ARF is related to renal ischemia. Even with the extensive use of dialysis and the improved medical care in the recent years, the mortality of ARF is still around 30% in hospital and rises to around 50% within six months.[4]

The development of ARF is a multifactorial process involving several inter-related mechanisms; nevertheless, it is ultimately precipitated by reversible and irreversible injury of renal tubular cells, particularly proximal tubules at the S3 segment.[5] Under the pathological condition, massive necrosis in proximal tubules occurs acutely. On the other hand, work during the last decade has provided compelling evidence for the involvement of tubular cell apoptosis in ARF.[2],[6],[7] Mechanistically, two major apoptotic pathways have been recognized: the intrinsic pathway mediated by mitochondria, and the extrinsic pathway initiated via death receptors.[8],[9] In the intrinsic pathway, cell stress induces Bax/Bak activation and the subsequent release of apoptotic factors (e.g. cytochrome c) from mitochondria. The released cytochrome c binds to Apaf-1 to recruit caspase 9 to form apoptosome in the cytosol, leading to further activation of the executive caspases and the development of apoptosis. In the extrinsic pathway, death receptors are activated following ligand binding to induce caspase 8 activation, which in turn proteolytically activate downstream executive caspases. Of note, these two apoptotic pathways are not mutually exclusive; instead, they can be well connected. For example, Bid, a Bcl-2 family protein, can be proteolytically activated by caspase-8 in the death receptor pathway and then triggers Bax/Bak activation to activate the mitochondrial pathway to amplify the apoptotic cascade.[10],[11] Recent studies including those from our laboratory have suggested an important role for Bcl-2 family proteins, including Bid, in ischemic renal injury.[12–17]

BCL-2 FAMILY PROTEINS

Bcl-2 family proteins are critical regulators of apoptosis, which is characterized by the conserved Bcl-2 homology (BH) domains.[18–21] According to their structural homology, Bcl-2 family protein can be separated into three groups (see Figure 1). Anti-apoptotic Bcl-2 proteins, such as Bcl-2, Bcl-XL and Mcl-1, contain four BH domains (BH1-4). Overexpression of anti-apoptotic proteins can greatly improve the survival of cells under various apoptotic stress. Pro-apoptotic multi-domain Bcl-2 proteins, such as Bax and Bak, do not have the BH4 domain. The activation of Bax/Bak is a critical step in mitochondrial outer membrane permeabilization in the intrinsic pathway of apoptosis.[22] In addition, there is a unique subclass of Bcl-2 family proteins that contain only the BH-3 domain. These proteins are important regulators of both the anti-apoptotic and the multi-domain pro-apoptotic Bcl-2 proteins. Good examples of the BH3-only Bcl-2 proteins include Bid, Bim, Bad, and Puma. It is generally believed that the balance between the anti-apoptotic and pro-apoptotic proteins determine the fate of a cell under stress, to live or to die.

Figure 1. Structure of Bcl-2 family proteins. Abbreviations: BH  =  Bcl-2 homology domain, TM  =  transmembrane domain.

BID

Bid was discovered in 1996 by interactive cloning using Bcl-2 and Bax probes.[23] The protein of 195 amino acids was termed BH3 interacting domain death agonist, or Bid, which antagonized the protective action of Bcl-2 and promoted cell death.[23] Bid contains eight α helices with four functional domains: the BH3 domain, the protease susceptible loop, the mitochondrial targeting motif, and the central hydrophobic region (see Figure 2). The BH3 domain appears to be the decisive structure for the function of Bid. Mutation or deletion of this domain abolishes its death-promoting activity. The BH3 domain is also required for Bid interaction with other Bcl-2 family proteins, including Bax.[23] The protease-sensitive loop of 35 amino acids is located just before the BH3 domain (see Figure 2). This loop is cleaved during Bid activation, releasing the BH3-containing carboxyl portion of ∼15kD, called truncated Bid. The mitochondrial targeting domain is normally buried in full-length Bid, but after cleavage at the protease-sensitive loop, the mitochondrial targeting domain is exposed, leading to the translocation to mitochondria.

Figure 2. Major functional domains in Bid.

Truncated Bid, or tBid, is the active form of Bid. Several proteases have been shown to proteolytically cleave or process Bid. Typically, Bid can be cleaved by caspase 8 at different Asp sites to generate tBid of 15kD (p15), 13kD (p13) or 11kD (p11).[10],[24],[25] In addition, calpains and other caspases have been reported to cleave Bid under certain experimental conditions.[25] tBid is the active form of Bid, which accumulates in mitochondria to interact with multi-BH domain proteins such as Bax and also induce the change of mitochondrial structure, leading to mitochondrial disruption and cell death. In addition to proteolytic activation, Bid is also regulated by other post-translational modifications such as N-myristoylation and phosphorylation.[26–28]

The mechanism whereby Bid induces mitochondrial injury is still not clear and is being actively investigated. Bid may directly interact with Bax/Bak to promote pore formation in the outer membrane of mitochondria, resulting in the release of apoptogenic factors.[29–31] Alternatively, Bid may interact with and neutralize Bcl-2/Bcl-XL and, as a result, to free Bax/Bak to form mitochondria permeabilizing pores. On the other hand, Bid may also alter the mitochondrial structure to facilitate the molecular release. In this regard, tBid may bind cardiolipin at the inner and outer membrane contact sites in mitochondria to induce the remodeling of mitochondrial cristae.[32–34] Recent work further suggests that Bid widens the cristae junctions, disrupts oligomerization of OPA1 (a protein involved in mitochondrial morphological regulation), and facilitates cytochrome c release.[35]

A powerful tool for the study of the involvement of Bid in different kinds of diseases is the Bid-deficient mouse model.[36] Using this model, Bid has been shown to be critical in death receptor related hepatocyte or liver injury.[36] In addition, Bid deficiency was reported to protect against ischemic neuronal cell death.[37] Under these conditions, cytochrome c release was significantly delayed in Bid-deficient mice. More relevant to our current discussion, Bid was shown to be cleaved by calpains during heart ischemia-reperfusion,[38] although definitive evidence for a role of Bid has not been demonstrated by using Bid-deficient models.

BCL-2 FAMILY PROTEINS IN ISCHEMIC KIDNEY INJURY

The first evidence for Bcl-2 family protein regulation during renal ischemia-reperfusion was demonstrated by Basile et al.[39] In this study, Bcl-2 and Bax were shown to be induced during renal ischemia-reperfusion of the rat. Subsequent studies by Gobe et al. further indicated that the pattern of Bcl-2 family protein expression was somewhat different in distal and proximal tubules. In proximal tubules, Bax was markedly induced after ischemia, while Bcl-XL was moderately increased and Bcl-2 remained. In distal tubules, there was a marked induction of Bcl-2 and a moderate increase in Bcl-XL and Bax.[40],[41] Using a rat model, we showed that following renal ischemia, both Bax and Bak were induced rapidly, Bcl-2 was induced later, and Bcl-XL was induced first and then decreased after 48 hours of reperfusion. In a mouse model with a much more severe injury, all of the tested Bcl-2 family proteins decreased at the beginning and then increased (Wei and Dong, unpublished). Thus, the expression of Bcl-2 proteins may depend on the severity of ischemic injury. Recently, Chiang-Ting et al. showed that the overexpression of Bcl-2 in the kidneys inhibited apoptosis during renal ischemia-reperfusion,[42] providing further support for the involvement of Bcl-2 family proteins in the development of renal pathology.

Using cultured renal tubular cells, we showed that Bcl-2 family proteins regulate the integrity of mitochondria following hypoxic or ATP depletion-induced injury. Upon injury, Bax translocates from cytosol to mitochondria and form oligomer pores, resulting in the release of cytochrome c.[13] The overexpression of Bcl-2 can suppress Bax oligomerization and prevent cytochrome c release and subsequent caspase activation and apoptosis.[13],[43] Similarly, the up-regulation of Bcl-2 pharmacologically by minocycline also ameliorates renal tubular cell death.[14] Moreover, tubular cells selected by repeated episodes of hypoxia express higher levels of Bcl-XL, which promotes cell survival by preserving mitochondrial integrity under a variety of stresses.[12] Together these results have established a critical role for the mitochondria-mediated intrinsic pathway of apoptosis in tubular cell injury and death during renal ischemia-reperfusion. By regulating mitochondria, Bcl-2 family proteins play an important role in the fate determination of the stressed cells.

BID IN ISCHEMIC ACUTE KIDNEY INJURY

When Bid was cloned in 1996, it was shown to be most abundantly expressed in the kidneys.[23] However, a role for Bid in renal patho-physiology has not been demonstrated until very recently. The first evidence for the involvement of Bid in renal injury by ischemia-reperfusion was shown in 2004.[15] In that study, we examined Bid activation in cultured kidney proximal tubular cells following ATP depletion, a condition that mimics renal ischemia in vivo. We showed that tubular cells underwent massive apoptosis during the recovery period following ATP-depletion. This was accompanied by Bid cleavage, releasing tBid of 15 and 13 kDa. Bid cleavage was suppressed in cells overexpressing Bcl-2. It was also suppressed by peptide inhibitors of caspases, particularly inhibitors of caspase-9. Similarly, Bid cleavage was also induced following hypoxic incubation of the cells. Of note, cleaved Bid translocated to mitochondria, suggesting a role for Bid in the development of mitochondrial defects under the experimental condition.[15] In 2006, we further examined Bid activation and its pathological role in renal ischemia-reperfusion using in vivo mouse models.[16] It was shown that in C57BL/6 mice, Bid was proteolytically processed into active forms of tBid during renal ischemia-reperfusion, which subsequently targeted mitochondria. The cleavage of Bid was accompanied by the development of tissue damage and severe renal failure. Importantly, the same ischemic insult induced significantly less severe acute kidney injury and renal failure in Bid-deficient mice. Histologically, Bid deficiency attenuated tubular disruption, tubular cell apoptosis, and caspase-3 activation. Compared with wild-type, animal death following renal ischemia was also delayed in Bid-deficient mice.[16] Collectively, these two studies suggest a role for Bid in ischemic renal injury and renal failure.

MECHANISMS OF BID REGULATION AND INVOLVEMENT IN ISCHEMIC RENAL INJURY

Despite the demonstrated role of Bid in ischemic renal failure, the mechanism underlying Bid activation under the pathological condition is elusive. Bid is proteolytically processed into active forms during both renal ischemia and the reperfusion periods,[16] suggesting that different activation mechanisms might be involved during these two periods of time. Fas and its associated death receptor pathway have been reported to participate in ischemic renal injury.[44] Thus, it is possible that caspase-8, after being activated via Fas pathway, may cleave and activate Bid. In addition, Bid could also be activated by calpains.[38] During renal ischemia, there is a marked increase of intracellular calcium, which induces calpain activation.[45],[46] However, it is still unclear if calpains are involved in cell injury and death, recovery of the injury, or both.[46],[47] Furthermore, Bid activation is regulated by N-myristoylation and phosphorylation. For example, JNK has been shown to phosphorylate Bid and regulate its activation.[26] JNK is activated during kidney ischemia-reperfusion.[48] However it is not clear when and where JNK is activated, and whether JNK phosphorylates Bid under this pathological condition. These possible regulations are summarized in Figure 3.

Figure 3. Bid activation and involvement in apoptosis. Bid is proteolytically processed into active forms of tBid via caspase-8, calpain, or other proteases. The activation may be regulated by JNK-mediated phosphorylation. tBid translocates to mitochondria, where it can directly interact with and activate Bax/Bak, or indirectly activate Bax/Bak by antagonizing Bcl-2/Bcl-XL. Activated Bax/Bak form porous defects in the outer membrane of mitochondria, leading to the release of apoptogenic factors.

As described in previous studies, Bid deficiency not only diminishes apoptosis but also ameliorates tubular cell necrosis. These observations suggest that Bid may act at different levels to regulate cell death/survival. First, tBid would directly interact with Bax and lead to Bax activation to permeabilizing outer mitochondrial membrane, resulting in cytochrome c release, caspase activation, and apoptosis. Second, tBid may interact with Bcl-2 and Bcl-XL to neutralize their anti-apoptotic activity and, as a result, to free Bax and Bak to injury porous defects in mitochondrial membrane. Finally, tBid may target the mitochondrial membrane to induce changes of cristae structure, facilitating the release of apoptogenic factors (see Figure 3). Our latest work has suggested an important role for mitochondrial morphological regulation in mitochondrial injury during apoptosis.[49] Is Bid involved in this regulation? Further investigation should test these possibilities to gain insights into the mechanisms of Bid involvement in mitochondrial injury and tubular cell apoptosis during renal ischemia-reperfusion.

CONCLUSIONS AND PERSPECTIVES

A pathological role for Bid has been demonstrated during ischemic renal injury and renal failure. However, it is unclear how Bid is activated and, after being activated, how tBid promotes tubular cell death. Regardless of the underlying mechanisms, targeting Bid may offer a new strategy for treatment of ischemic renal failure. By molecular modeling, Becattini et al have designed and synthesized chemicals that can block the translocation of tBid to mitochondria.[50],[51] Notably, the chemicals can inhibit neuronal apoptosis at low micromolar concentrations. Whether these inhibitors are effective in blocking ischemic renal injury remains to be studied. In addition, the upstream regulators of Bid activation also deserve investigation, which may provide additional therapeutic targets. The up-regulation of anti-apoptotic Bcl-2 proteins by genetic or pharmacological methods also holds great therapeutic potential. On the other hand, it is important to point out that Bid is an important, but not the sole, pathogenic factor in ischemic renal injury; thus, Bid-deficiency cannot completely block tubular cell apoptosis. This observation is consistent with the view that ischemic injury is a multifactorial process. The identification of new molecular targets and combinatorial therapy may lead to promising approaches for the treatment of ischemic renal failure.

ACKNOWLEDGMENTS

The work was supported in part by grants from National Institutes of Health and Department of Veterans Affairs of the United States of America.