Am J Physiol Renal Physiol 297: F244–F256, 2009.
First published March 11, 2009; doi:10.1152/ajprenal.00033.2009.
Review
Autophagy: molecular machinery, regulation, and implications
for renal pathophysiology
Sudharsan Periyasamy-Thandavan, Man Jiang, Patricia Schoenlein, and Zheng Dong
Department of Cellular Biology and Anatomy, Medical College of Georgia and Charlie Norwood Department of Veterans
Affairs Medical Center, Augusta, Georgia
Submitted 22 January 2009; accepted in final form 5 March 2009
apoptosis; Atg; cytoprotection; mammalian target of rapamycin; phosphatidylinositol 3-kinase
DURING NORMAL CELL GROWTH and development, a well-controlled balance is needed between the biosynthesis and catabolism of macromolecules. This balance is preserved by diverse
regulatory mechanisms that respond to the environmental circumstances and provoke specific signals into systematic
growth and developmental responses. In this continuous process, there is an invariable production of compounds that can
be toxic to the cells. It has been recognized that appropriate
degradation of irreversibly damaged proteins is vital to cellular
viability and function (121). Often, damaged proteins develop
abnormal intermolecular interactions to form aggregates that,
when trapped inside other cellular components, are cytotoxic.
In eukaryotic cells, the major systems responsible for protein
degradation are the ubiquitin-proteasome pathway and the
lysosomal system. The proteasomal pathway is restricted to the
cytosol and nucleus and mainly involved in the degradation of
short-lived proteins. In contrast, the lysosomal system comprises vesicular organelles with various hydrolases including
proteases for substance degradation. It is generally believed
that lysosomes are responsible for the degradation of longlived proteins. The major channel that delivers cytoplasmic
constituents including proteins to lysosomes for degradation is
autophagy (55).
Address for reprint requests and other correspondence: Z. Dong, Dept. of
Cellular Biology and Anatomy, Medical College of Georgia, 1459 Laney
Walker Blvd., Augusta, GA 30912 (e-mail: [email protected]).
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An Evolving View of Autophagy
Autophagy, a term derived from the Greek, means self
(auto)-eating (phagy). In a cell, three types of autophagy have
been recognized: macroautophagy, microautophagy, and chaperone-mediated autophagy (Fig. 1). Macroautophagy starts
with the de novo formation of a cup-shaped isolation double
membrane (also called phagophore or preautophagosome) that
engulfs a portion of cytoplasm. The isolation membrane then
encloses to form a mature vesicle, i.e., autophagosome, that
subsequently fuses with a lysosome, leading to the degradation
of intra-autophagosomal components by lysosomal hydrolases.
In contrast, microautophagy involves the engulfment of cytoplasm instantly at the lysosomal membrane by invagination,
protrusion, and separation. Chaperone-mediated autophagy, on
the other hand, is a process of direct transport of unfolded
proteins via the lysosomal chaperonin hsc70 and LAMP-2A, a
lysosomal membrane receptor. Of these three types, macroautophagy is most prevalent and most frequently referred to as
“autophagy,” the focus of our discussion in this review.
For many years, autophagy was considered to be primarily
involved in cellular architectural changes that prevail during
differentiation and development. However, in the late 1960s,
autophagy was found to be enhanced in various organs including newborn kidney (29). This and other related observations
led to the belief that breakdown of cytoplasmic components by
cellular autophagy could be functionally related to gluconeogenesis (106). Sybers et al. (125) further proposed that cellular
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Periyasamy-Thandavan S, Jiang M, Schoenlein P, Dong Z. Autophagy:
molecular machinery, regulation, and implications for renal pathophysiology.
Am J Physiol Renal Physiol 297: F244 –F256, 2009. First published March 11,
2009; doi:10.1152/ajprenal.00033.2009.—Autophagy is a cellular process of “selfeating.” During autophagy, a portion of cytoplasm is enveloped in double membrane-bound structures called autophagosomes, which undergo maturation and
fusion with lysosomes for degradation. At the core of the molecular machinery of
autophagy is a specific family of genes or proteins called Atg. Originally identified
in yeast, Atg orthologs are now being discovered in mammalian cells and have been
shown to play critical roles in autophagy. Traditionally, autophagy is recognized as
a cellular response to nutrient deprivation or starvation whereby cells digest
cytoplasmic organelles and macromolecules to recycle nutrients for self-support.
However, studies during the last few years have indicated that autophagy is a
general cellular response to stress. Interestingly, depending on experimental conditions, especially stress levels, autophagy can directly induce cell death or act as
a mechanism of cell survival. In this review, we discuss the molecular machinery,
regulation, and function of autophagy. In addition, we analyze the recent findings
of autophagy in renal systems and its possible role in renal pathophysiology.
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AUTOPHAGY AND RENAL PATHOPHYSIOLOGY
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injury stimulates the endoplasmic reticulum (ER) to enclose
the damaged components, permitting localized lysosomal digestion without causing injury to the entire cell. Later, autophagy was shown to be particularly selective in removing
cytoplasmic components from the cytoplasm and to play an
important role in intracellular turnover (107).
Autophagy is constitutive in all eukaryotic cell types containing
a lysosomal compartment. This basal autophagy contributes to
long-lived protein degradation as well as organelle turnover and
occurs in parallel with the proteasomal pathway to control the
quality or health of the cytoplasm. Autophagy can also be
induced as a cellular reaction to various physiological challenges including nutrient starvation, cell growth control, antiaging, intracellular protein and organelle turnover, development, eradication of microorganisms, cell death, tumor suppression, and inherent immunity (80). While a role for
autophagy in cell survival is well recognized, cell death from
excessive cellular consumption or autophagy has also been
suggested (9, 30). To complicate these circumstances further,
cytotoxic events generally induce autophagy, making it hard to
conclude whether autophagy is a death mechanism or a failed
attempt at cellular defense (79). Although autophagy may
engulf cytoplasm nonselectively, a selective or specific sequestration of organelles has been observed and proposed to play a
role in certain diseases including cancer, cardiomyopathy,
renal disorders, neurodegenerative disorders, and viral, bacterial, and parasitic infections (17, 23, 68, 81, 90, 94, 98, 145).
Core Molecular Machinery of Autophagy
The voyage into the understanding of the molecular mechanism of autophagy began with the identification of the autoAJP-Renal Physiol • VOL
phagy-related (Atg) genes in yeast (95–97). To date, over 30
Atg genes have been cloned. Notably, mammalian Atg orthologs have been identified, showing high conservation with
yeast genes (54). Atgs constitute the core molecular machinery
of autophagy, which is involved in the induction of the isolation membrane, engulfment of cytoplasm, formation of the
autophagic vesicles, and fusion with lysosome. A summary of
Atg proteins and their functions in autophagy is provided in
Table 1.
The formation of autophagosomes is reliant upon several
Atgs that are part of two novel conjugation systems required
for initiation and elongation of the isolation membrane (96)
(Fig. 2). The first conjugating event involves the conjugation of
a single lipid molecule, phosphatidylethanolamine, to the
COOH terminus of Atg8/LC3. In this conjugation event, Atg7
and Atg3 act as E1- and E2-homologous enzymes, respectively, in the ubiquitin pathway. For this conjugation to occur,
Atg8/LC3 is initially cleaved by Atg4, a cysteine protease (41).
The cleaved/activated Atg8/LC3 (LC3-I) is then activated by
Atg7 and further transferred to Atg3. Atg3 conjugates Atg8/
LC3 to phosphatidylethanolamine to form LC3-II, a lipidated
form associated with autophagic membranes or vesicles (53)
(Fig. 2A). Of note, redistribution of LC3 from the cytosol to
autophagic vesicles and formation of LC3-II are commonly
used as a marker for autophagy (83). The second conjugation
event of autophagy involves the covalent attachment of the
COOH-terminal glycine of Atg12 to Atg5 through an internal
lysine residue (84). In this conjugation, Atg7 and Atg10 act as
E1- and E2-homologous enzymes, respectively. Atg7 hydrolyzes ATP and activates Atg12 through the formation of a
thioester bond. The activated Atg12 is then transferred to Atg5
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Fig. 1. The 3 forms of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Macroautophagy starts with the de novo formation
of a cup-shaped isolation double membrane that engulfs a portion of cytoplasm. Microautophagy involves the engulfment of cytoplasm instantly at the lysosomal
membrane by invagination, protrusion, and separation. Chaperone-mediated autophagy is a process of direct transport of unfolded proteins via the lysosomal
chaperonin hsc70 and LAMP-2A. All forms of autophagy subsequently lead to the degradation of intra-autophagosomal components by lysosomal hydrolases.
PE, phosphatidylethanolamine.
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Table 1. Atg proteins in autophagy
Gene
Atg1
Atg2
Atg3
Atg4
Atg5
Atg7
Atg8
Atg9
Atg10
Atg11
Atg12
Atg13
Atg14
Atg15
Atg16
Atg17
Atg18
Atg19
Atg20
Atg21
Atg22
Cellular Localization
Protein serine/threonine kinase forming complex
with Atg13p and Atg17 during PAS
formation
Peripheral membrane protein required for
vesicle formation and cycling with Atg9
between preautophagosomal structure and
mitochondria
E2-like enzyme involved in Atg8-PE
conjugation
Conserved cysteine protease for Atg8 cleavage
and processing and attachment of
autophagosomes to microtubules
Conserved protein undergoing conjugation with
Atg12, leading to oligomerization with
Atg16p to form a complex involved in Atg8
lipidation
Subunit of PI3-kinase complexes I and II that
are essential for autophagy
Mediating conjugation of Atg12p with Atg5p
and Atg8p with PE
Conserved protein that undergoes COOHterminal conjugation to PE; Atg8p-PE is a
component of autophagosome vesicles
Transmembrane protein involved in formation
of autophagic vesicles; cycles between
preautophagosomal structure and other
cytosolic punctate structures; forms complex
with Atg23p and Atg27p
Conserved E2-like conjugating enzyme that
mediates formation of Atg12p-Atg5p
conjugate, a critical step in autophagy
Adapter protein that directs receptor-bound
cargo and recruits other proteins to
preautophagosomal structure
Conserved protein that undergoes conjugation
with Atg5p, leading to oligomerization with
Atg16p and formation of a complex involved
in Atg8p lipidation
Regulatory subunit of Atg1 signaling complex;
stimulates Atg1 kinase activity; required for
vesicle formation during autophagy and
involved in Atg9p, Atg23p, and Atg27p
cycling
Targets PI3-kinase complex I (with Vps34p,
Vps15p, and Vps30p) and other proteins to
preautophagosomal structure
Lipase required for intravacuolar lysis of
autophagosomes; targeted to intravacuolar
vesicles during autophagy via multivesicular
body pathway
Conserved protein that interacts with Atg12Atg5 conjugates to form Atg12-Atg5-Atg16
multimers in preautophagosomal structure
Scaffold protein responsible for
preautophagosomal structure organization;
forming Atg1-Atg13-Atg17 complex;
stimulates Atg1 kinase activity
Phosphoinositide binding protein required for
vesicle formation in autophagy
Receptor protein for cytoplasm-to-vacuole
targeting pathway
Contains a Phox homology domain that binds
PI3P
Similar to Atg18
Vacuolar integral membrane protein required for
efflux of amino acids during autophagic body
breakdown; null mutation causes a gradual
loss of viability during starvation
Reference
Other Names
Entrez Gene ID
Molecular Mass, kDa
Cytoplasm
75
AUT3, APG1
852695
10
Cytoplasm
137
SPO72, APG2
855479
17
Cytoplasm
140
AUT1, APG3
855741
35
Cytoplasm
62
AUT2, APG4
855498
55
Cytoplasm, autophagic vacuole
39
APG5
855954
33
Membrane fraction
20
855983
63
856576
71
Cytosol and membrane fraction
(mainly mitochondria)
Cytosolic to autophagic
vacuolar membrane
142
VPS30, Beclin1,
APG6
CVT2, APG7
128
APG8, LC3
852200
LC3 I: 18; LC3 II: 16
Preautophagosomal membrane
structure
114
APG9, AUT9
851406
115
Unclear
111
APG10
850684
20
Preautophagosomal membrane
structure
85
CVT9
856162
135
Membrane fraction
39
APG12
852518
21
114
APG13
856315
83
Extrinsic to membrane
Preautophagosomal membrane
structure
92
APG14, CVT12
852425
40
Vacuolar lumen
31
AUT5, CVT17
850432
58
Preautophagosomal membrane
structure
74
SAP18, APG16
855194
17
Preautophagosomal membrane
structure
45
APG17
851142
49
Cytosol
58
AUT10, CVT18
850577
55
Cytosol, preautophagosomal
membrane structure
Endosome, preautophagosomal
membrane structure
Cytosol
Integral to vacuolar membrane
21
CVT19
854072
48
147
CVT20
851445
72
122
144
HSV1
AUT4
856004
850319
55
59
Continued
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Atg6
Function in Autophagy
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Table 1.—Continued
Gene
Function in Autophagy
Atg23 Cycles between PAS and non-PAS locations;
forms a complex with Atg9p and Atg27p
Atg24 Contains PI-binding domain and forms
complexes with Atg20
Atg27 Type I membrane protein involved in autophagy
and involved in membrane delivery to
preautophagosomal structure; binds PI3P
Atg29 Autophagy-specific protein that is required for
recruitment of other Atg proteins (mainly
Atg17) to preautophagosomal structure
Cellular Localization
Reference
Other Names
Entrez Gene ID
Molecular Mass, kDa
Preautophagosomal membrane
structure
Cytosol, preautophagosomal
membrane structure
Membrane, preautophagosomal
membrane structure
134
CVT23
851154
51
5
CVT
853416
49
146
ETF1
853261
30
49
NA
855937
25
Cytosol, preautophagosomal
membrane structure
PE, phosphatidylethanolamine; PAS, preautophagosome; PI, phosphatidylinositol; PI3-kinase, phosphatidylinositol 3-kinase; PI3P, phosphatidylinositol
3-phosphate.
autophagic units (50). Therefore, the PI3-kinase complex not
only controls the nucleation of autophagic vesicles but also
guides the conjugation events. Notably, beclin-1 in this complex has several interacting partners including Bcl-2, UVRAG,
Ambra, and Bif-1 or Endophilin B1 (35, 103, 126). These
molecular interactions are critical to the regulation of autophagy.
The second kinase complex of autophagy is centered on
Atg1 and Atg13 (75). In the classical model of autophagy
induced by nutrient starvation, Atg13 is one of the downstream
effectors of target of rapamycin (TOR)2 kinase (48). In the
presence of nutrients including growth factors, TOR2 kinase is
active and phosphorylates Atg13, preventing its association
with Atg1, a serine/threonine protein kinase. Under this condition, Atg1 contributes to the formation of lysosomal hydrolase carrier vesicles, but not autophagosomes (3). During
starvation, TOR2 kinase is inhibited, resulting in the activation
of phosphatases and partial dephosphorylation of Atg13. Dephosphorylated Atg13 associates tightly with and activates
Atg1, leading to the recruitment of Atg17 and the formation of
the second kinase complex of autophagy. The Atg1-Atg13Atg17 complex participates in elongation of the isolation
membrane and progression toward a complete autophagosome
structure (51) (Fig. 3B).
Once the autophagosome is fused at each side, it gets ported
and blended with a neighboring lysosome, forming an autolysosome. In yeast, components of the SNARE machinery are
required for autophagosome porting and blending with the
lysosome (28). The inner membrane of the autophagosomes
and the sequestered components of the cytoplasm are then
degraded by lysosomal hydrolases. Once the macromolecules
are degraded inside the lysosome, the monomeric units are
exported to the cytosol for recycling (55).
Regulation of Autophagy
Fig. 2. Two essential ubiquitin-like conjugation systems in autophagy. A: the
first conjugating system includes Atg8, -4, -7, and -3 and PE. This system
conjugates a single PE molecule to the COOH terminus of Atg8/LC3. B: the
second conjugation system includes Atg12, -7, -10, -5, and 16. This conjugation leads to the covalent attachment of the COOH-terminal glycine of Atg12
to Atg5 through an internal lysine residue.
AJP-Renal Physiol • VOL
The complex molecular machinery of autophagy suggests
that its regulation can be extremely complicated and may
involve multiple signaling inputs. Indeed, many of the major
signaling pathways have been shown to regulate autophagy
under various conditions. Of note, these signaling pathways
may cross talk and regulate at different levels in the autophagic
cascade, including induction and expansion of the isolation
membrane, enclosure of the isolation membrane to form autophagosome, and fusion with lysosome.
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via Atg10. The Atg5-Atg12 complex associates noncovalently
to coiled-coil protein Atg16, which oligomerizes to form large
units that are required for targeting the Atg8/LC3 into the
limiting membrane and elongation of the sequestering membrane (61) (Fig. 2B). As these two conjugation events of
autophagy are interdependent, Atg8/LC3 conjugation depends
on the Atg5-Atg12-Atg16 complex activity. Consequently,
LC3 conjugation or LC3-II formation is not detectable in
Atg5-null cells and animals (133).
Besides the conjugation events, autophagy is also regulated
by two unique kinase complexes (Fig. 3). The first kinase
complex includes an autophagy-specific phosphatidylinositol
3-kinase (PI3-kinase), Vps34 (homolog to human class III
PI3-kinase). The involvement of PI3-kinases in autophagy was
initially suggested by the observation that autophagosome formation was inhibited by pharmacological inhibitors of PI3-kinases
including 3-methyladenine, wortmannin, and LY-294002 (14,
118). As a PI3-kinase, Vps34 phosphorylates phosphatidylinositol (PI) to produce phosphatidylinositol 3-phosphate (PI3P), a
docking lipid that promotes protein complex formation, membrane enclosing, and the consequent sequestration of cytoplasmic components in autophagic vacuoles (82, 105) (Fig. 3A).
Importantly, Vps34 is associated with and probably regulated
by Vps15 and Vps30/Atg6/beclin-1, which plays an important
role in focusing the two conjugation events to the emerging
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TOR kinase. TOR kinase is a principal regulator of autophagy that shuts off autophagy in cells growing in the presence of
nutrients including amino acids and growth factors. Being
autophagy inhibitory, TOR activity prevents the formation of
Atg complexes including the Atg1-Atg13-Atg17 serine/threonine protein kinase complex and the Vps34-Atg6-Vps15 lipid
kinase complex (51, 82, 124). It also interferes with the two
ubiquitin-like conjugation systems of autophagy. As a result,
induction and expansion of the isolation membrane is abrogated. Conversely, TOR is inactivated when a cell is nutrient
deprived, unleashing the autophagic cascade. Therefore, inhibition of TOR has been suggested to be obligatory for the
induction of autophagy (2). As a result, TOR may provide a
central platform for autophagic signaling pathways and their
integration (Fig. 4), although it remains to be determined
whether TOR inhibition is a universal mechanism for autophagy under other types of cellular stress.
Class I PI3-kinase/Akt. Class I PI3-kinase/Akt regulates
autophagy mainly through the modulation of TOR activity.
Briefly, PI3-kinase phosphorylates phosphatidylinositol (4,5)P2 to
Fig. 4. Major regulatory mechanisms of autophagy. The major signaling pathways of autophagy regulation under conditions of nutrient (amino acids, growth
factors) deprivation, endoplasmic reticulum (ER) stress, energetic stress or depletion (see detailed discussion in text) are depicted. The pathways may cross talk
and regulate autophagy at different levels, including induction and expansion of the isolation membrane, enclosure of the isolation membrane to form
autophagosome, and fusion with lysosome. PI3K, PI3-kinase; AMPK, AMP-dependent protein kinase; TSC, tuberous sclerosis complex; eIF-2␣, eukaryotic
initiation factor-2␣; InR, insulin receptor; RAS, rat sarcoma (a protein encoded by rat sarcoma virus oncogene).
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Fig. 3. Two essential kinase systems in autophagy. A: class III phosphatidylinositol 3-kinase
(PI3-kinase) complex. Vps34, a class III PI3kinase, phosphorylates phosphatidylinositol (PI)
to produce phosphatidylinositol 3-phosphate
(PI3P), a docking lipid that promotes protein
complex formation, membrane enclosing, and
the consequent sequestration of cytoplasmic
components in autophagic vacuoles. Vps34
forms complexes with and is regulated by beclin-1 and Vps15. Beclin-1 further interacts with
and is regulated by Bcl-2, Ambra, and UVRAG.
B: serine/threonine protein kinase complex. Regulation of this complex is centered on the interaction between Atg13 and Atg1, a serine/threonine protein kinase. In the presence of nutrients
and growth factors, target of rapamycin (TOR)
kinase is active and phosphorylates Atg13, preventing its association with Atg1. During starvation,
TOR is not active, resulting in the activation of
phosphatases and partial dephosphorylation of
Atg13. Dephosphorylated Atg13 associates tightly
with and activates Atg1, leading to the recruitment
of Atg17 and the formation of the autophagic
serine/threonine kinase complex.
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of AMPK in autophagy in metabolically stressed mammalian
cells. A major mechanism for AMPK to induce autophagy is
suppression of TOR signaling. AMPK can directly inhibit TOR
via phosphorylation. In addition, AMPK can activate TSC,
leading to the inhibition of TOR.
Eukaryotic initiation factor-2␣. In response to cellular
stress, eukaryotic initiation factor-2␣ (eIF-2␣) is phosphorylated and regulated by a family of evolutionarily conserved
serine/threonine kinases. In 2002, Talloczy et al. (127)
showed that eIF-2␣ phosphorylation by GCN2 (the yeast
eIF-2␣ kinase) is essential for starvation-induced autophagy
in yeast. A role for eIF-2␣ phosphorylation was also shown
to be important to autophagy during viral infection in
murine embryonic fibroblasts. More recently, eIF-2␣ has
been implicated in autophagy under ER stress. For example,
Kouroku et al. (57) demonstrated that ER stress-associated
autophagy can be prevented by eIF-2␣ mutations that block
its phosphorylation. ER stress-associated autophagy is also
attenuated by dominant-negative inhibition of protein kinase
regulated by RNA (PKR)-like ER kinase (PERK), the protein kinase responsible for eIF-2␣ phosphorylation (57). It
was suggested that eIF-2␣ phosphorylation/activation may
lead to selective translation of specific transcription factors,
resulting in the expression of Atg12 and formation of the
Atg5-Atg12-Atg16 complex that is critical to autophagy
induction (57).
Mitogen-activated protein kinases. Mitogen-activated protein kinases (MAPKs), including ERK1/2, p38, and JNK, are a
family of serine/threonine protein kinases involved in a wide
range of cellular responses. In various models of autophagy,
MAPK activation has been documented (26, 115). During
nutrient starvation both ERK1 and ERK2 are activated, and
inhibition of ERK1/2 abrogates starvation-induced autophagy
(105). Consistently, amino acids have been shown to suppress
autophagy by inhibiting ERK1/2 in human colon cancer cells
(102). A role for JNK in autophagy induction has also been
suggested. For example, the recent work by Wei et al. (138a)
demonstrated a critical role for JNK-mediated phosphorylation
of Bcl-2 in autophagy during cellular starvation. They showed
that JNK1, but not JNK2, is activated during starvation stress
and phosphorylates Bcl-2 at multiple sites, leading to its
dissociation from beclin-1 and autophagy induction. Similar
observations were shown by Pattingre et al. (101) in ceramideinduced autophagy. JNK signaling may also directly induce
beclin-1 transcription in response to ceramide treatment (64).
For autophagy regulation by p38, vom Dahl and colleagues
(136) conducted a series of studies showing that p38 plays a
key role in cell swelling-induced autophagy. It is important to
recognize that the role and regulation of individual MAPKs in
autophagy are very complicated and can vary from one experimental model to another.
It is important to note that the current knowledge of autophagy
regulation is mainly derived from research characterizing the
autophagic response to nutrient deprivation or starvation.
Whether the regulatory mechanisms are central to autophagy
under other stress conditions remains to be determined. It is
likely that specific stimuli may trigger unique signaling pathways that are then integrated with the core regulatory mechanisms to induce autophagy.
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produce phosphatidylinositol (3,4,5)P3, which activates phosphoinositide-dependent kinase-1 (PDK-1). PDK-1 can phosphorylate and activate Akt. Akt, also called protein kinase B,
can further phosphorylate and inactivate tuberous sclerosis
complex (TSC). TSC is a GTPase-activating protein complex,
and one of its downstream targets is Rheb. When TSC is
inactivated by Akt, the GTPase activity of Rheb is blocked,
leading to a GTP-bound form of Rheb, which is the major
activator of TOR (42). Therefore, by activating TOR, class I
PI3-kinase/Akt suppresses autophagy. Importantly, class I PI3kinase /Akt is the critical link between growth factor signaling
and TOR. Growth factors such as insulin-like growth factor
bind to their tyrosine kinase receptors, leading to the activation
of PI3-kinase/Akt and TOR, resulting in the inhibition of
autophagy (Fig. 4).
Amino acid signaling. It has been recognized for decades
that amino acids, the major end products of autophagic degradation, are also crucial regulators or inhibitors of autophagy
(89). This provides a feedback mechanism to control the level
of autophagy to maintain cellular homeostasis. Of note, not all
but only a few amino acids are involved in autophagy regulation (76, 88, 119). Alanine, leucine, glutamine, and phenylalanine seem particularly effective (12, 87, 119). These amino
acids have their own recognition sites at the cell surface and are
believed to initiate signal transduction to suppress autophagy
(46). A major mechanism for amino acid-mediated inhibition
of autophagy is TOR activation. In 1995, Blommaart et al. (15)
showed that in isolated rat hepatocytes the inhibitory effects of
amino acids on autophagy are prevented by rapamycin, the
TOR inhibitor. A role for TOR in amino acid-mediated autophagy inhibition was also demonstrated later in yeast by Noda
and Ohsumi (91). The mechanism whereby amino acids activate TOR remains unclear, but it seems to be independent of
class I PI3-kinase/Akt.
Bcl-2 family proteins. The interaction between Bcl-2 and
beclin-1 suggested that autophagy could be subjected to regulation by Bcl-2 family proteins (67). In 2005, Pattingre et al.
(103) further demonstrated the inhibitory effects of Bcl-2 on
autophagy in both yeast and mammalian cells. The inhibitory
effects of Bcl-2 were shown to depend on its physical interaction with beclin-1 (103). The antiautophagy activities have
now been shown not only for Bcl-2 but also for its homologs
including Bcl-XL, Bcl-w, and Mcl-1 (32, 70, 104). Binding and
sequestration of beclin-1 by these proteins is expected to
disturb the formation of the class III PI3-kinase (Vps34-Vps15beclin-1) complex that is critical to the induction of autophagy,
and as a result autophagy is inhibited. Conversely, BH3-only
proteins can bind Bcl-2/Bcl-XL to allow beclin-1 to induce
autophagy (70, 151). It is noteworthy that, in addition to
beclin-1, Bcl-2 family proteins may also regulate autophagy by
other mechanisms including Ca2⫹ homeostasis (40).
AMP-dependent protein kinase. AMP-dependent protein kinase (AMPK) is sensitive to the cytosolic AMP-to-ATP ratio
and responds to metabolic stresses affecting this ratio, such as
glucose deprivation, hypoxia, ischemia, heat shock, or oxidative stress (47). Not surprisingly, AMPK is involved in autophagy induction under these conditions. In yeast, autophagy
induction during metabolic stress requires AMPK (138). However, the involvement of AMPK in mammalian autophagy is
controversial. Nevertheless, recent studies by Meijer and Codogno (77) and Meley et al. (78) have further clarified the role
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Autophagy in Cell Survival
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Autophagic Cell Death
While a cytoprotective role for autophagy has been demonstrated in a variety of experimental models, autophagy has also
been suggested to induce or participate in cell death under
certain circumstances. As a matter of fact, cell death resulting
from autophagy has been classified as type II cell death, in
parallel to apoptotic (type I) and necrotic (type III) cell death
(18, 36). Autophagic cell death was originally suggested by
observations from apoptosis-defective or -blocked cells. A
notable example was shown by Shimizu et al. (120) using Bax
and Bak double-knockout (Bax⫺/⫺ Bak⫺/⫺) mouse embryonic
fibroblasts. In these cells, apoptosis is blocked because of the
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A role for autophagy in cell survival was initially recognized
in experimental models of nutrient depletion or starvation. This
is not surprising, as autophagy breaks down the engulfed
cytoplasmic constituents including proteins and organelles,
resulting in the production and recycling of nutrients to sustain
the intermediary metabolisms and biosynthetic pathways that
are essential to cell viability (117). This is clearly shown by
autophagic degradation of proteins, resulting in the generation
of amino acids. Under normal circumstances, cells maintain
amino acid levels by new synthesis or proteasomal degradation
of short-lived proteins rather than autophagy. However, during
an extended period of starvation, de novo synthesis and proteasomal degradation does not occur because of the lack of
substrates and energy and the vital amino acids are produced
by autophagy (79). In fact, immediately after birth neonates use
autophagy as an important source of oxidizable substrates
during sudden interruptions of maternal nutrient supply. Kuma
et al. (60) demonstrated that autophagy is upregulated during
early neonatal periods in the liver, heart, lung, diaphragm,
pancreas, and muscle. The autophagic degradation of proteins
in the liver produces amino acids, which are converted to
glucose and utilized by brain and erythrocytes as a metabolic
energy source. It is also understood that autophagy generates
amino acids to supply the tricarboxylic cycle as well as
substrates for new protein synthesis that are necessary for cell
survival during starvation (69, 79). In autophagy-deficient
cells, insufficient generation of starvation-induced proteins
such as argininosuccinate synthetase, heat shock protein, and
Ape1 were documented, leading to cell death in response to
starvation (132). Consistently, during the neonatal period Atg5
knockout mice are depleted of amino acids and show declined
cardiac ATP production, which is accompanied by myocardial
injury and animal death (60). Autophagy is also important to
the maintenance of cellular energy homeostasis and bioenergetics during growth factor deprivation. For example, in BAXBAK knockout mice, autophagy can maintain cell survival
during IL-3 deprivation for a remarkable period of several
weeks (69). Conversely, inhibition of autophagy by silencing
autophagy genes or pharmacological inhibitors ultimately
leads to cell death, further confirming a critical role for autophagy in cell viability (16).
In addition to the well-documented role of autophagy in cell
survival during nutrient and growth factor deprivation, autophagy has been shown to be protective against cell death
during stress by many recent studies. Ferraro and Cecconi (34)
showed that autophagy can postpone the beginning of the
apoptotic program or slow down apoptosis, and, when it fails
to do so, apoptosis takes over the fate of the cell. In this and
other related studies, pharmacological inhibitors of autophagy
such as 3-methyladenine, bafilomycin, monesin, and chloroquine increase apoptosis in various cell types. Of note, while
3-methyladenine blocks the formation of autophagosomes,
bafilomycin, monesin, and chloroquine inhibit the fusion of
autophagosome with lysosome or lysosomal degradative activity (19, 141). As a result, treatment with bafilomycin, monesin,
or chloroquine increases autophagic vacuoles, which can be
shown by LC3-GFP punctate accumulation in the cytoplasm
and also by increased LC3-II signal in immunoblot analysis.
The death-promoting effects of pharmacological inhibitors of
autophagy suggest that autophagy is cytoprotective in a variety
of cell injury models. Moreover, the protective role of autophagy has been supported by studies using Atg-deficient cells or
animals and also by studies using small interfering RNA
(siRNA) to knock down critical Atg genes. With these approaches, inhibition of autophagy accelerates starvation-induced apoptosis as well as mitochondrial membrane potential
loss and release of cytochrome c (16). In camptothecin-treated
cells, autophagy interrupts apoptosis and its inhibition triggers
apoptosis by mitochondrial depolarization and caspase 9 activation (1). More recently, Apel et al. (7) showed that irradiation
induces autophagy and autophagy-related genes in therapy-resistant cancer cells. Importantly, siRNA knockdown of the induced Atgs can sensitize the cells to radiotherapy. Autophagy
is also induced in normal kidney cells during nephrotoxicity
(100, 104, 143). As discussed below, blockade of autophagy
can exacerbate cell death in these experimental models, further
supporting a survival role for autophagy in malignant as well as
normal cells.
In vivo in mice, loss of Atg7 in the liver results in obvious
alterations in a series of stress-related proteins, followed by the
occurrence of ER and oxidative stresses and tissue injury (73).
Consistently, Kim et al. (52) showed that hepatocytes lose both
Atg7 and Atg6/beclin-1 by calpain-2 activation during anoxiareoxygenation or liver ischemia-reperfusion, leading to inhibition of autophagy. This autophagy blockade is associated with
the onset of mitochondrial permeability transition and ultimately cell death by necrosis and apoptosis (52). In the heart,
ischemia induces autophagy through an AMPK-dependent
mechanism, whereas subsequent reperfusion stimulates autophagy through an Atg6/beclin-1-dependent, AMPK-independent mechanism. Interestingly, it was concluded that autophagy may be protective during ischemia; however, it may be
detrimental during reperfusion (72).
Together, these in vitro and in vivo studies indicate that
autophagy may well be a cellular response to stress that,
depending on the experimental conditions, can be an important
protective mechanism for cell survival. Obviously, autophagy
can maintain the viability of starved cells by temporally digesting portions of cytoplasm to supply vital substrates and
energy. However, it is much less clear how autophagy protects
against cell death under other stress conditions. In this regard,
it has been suggested that autophagy may protect by eliminating damaged and potentially dangerous organelles, such as
leaky mitochondria (37). Nevertheless, this possibility remains
to be carefully tested and evaluated, and may not be the sole or
even major mechanism for cell survival via autophagy.
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AUTOPHAGY AND RENAL PATHOPHYSIOLOGY
AJP-Renal Physiol • VOL
when autophagy is defective, cancer cell growth is unchecked
by autophagic cell death, resulting in tumorigenesis. However,
this straightforward explanation has been seriously challenged
recently (71). Instead, the emerging idea is that autophagy is
cytoprotective in tumors. By limiting tumor cell necrosis and
inflammation, autophagy contributes to the maintenance of
genomic stability (71). In addition, autophagy may directly
inhibit cell growth in tumors by degrading organelles or proteins that are essential to cell cycle regulation such as cyclin E
and Rb (56, 66).
Autophagic Survival and Autophagic Cell Death: Where Is
the Connection?
It is clear from studies during the last few years that
autophagy is not just a response to starvation but a more
general response to cell stress. Moreover, it has been recognized that autophagy occurs early during cell stress and, when
the stress persists, cells die (Fig. 5). As discussed above,
autophagy may be a mechanism for cell survival but may also
induce cell death. Autophagy and its signaling seem to be
integrated with that of cell death. Clearly, many signaling
pathways activated by cell stress also play roles in the regulation of both autophagy and cell death. These include TOR,
PI3-kinase, Akt, and MAPK, to name a few (see Regulation of
Autophagy above). Even at the level of core machineries,
autophagy and cell death share key molecules, which may
determine the fate of the cells, to live or to die.
Atg6/beclin-1. Atg6 is a key autophagy protein involved in
the formation of PI3-kinase complex that controls the nucleation of autophagic vesicles. Interestingly, beclin-1, the mammalian ortholog of Atg6, was originally identified by yeast
two-hybrid screening as a protein that interacts with Bcl-2 (67),
a well-recognized apoptosis regulator that antagonizes apoptosis by interacting and neutralizing proapoptotic molecules
(148). Later studies have pinpointed the molecular interaction
via a Bcl-2 homology 3 (BH3) domain in beclin-1 (70, 93). The
beclin-1/Bcl-2 interaction suggests that these two molecules
may provide a cross talk channel between autophagy and
apoptosis. Indeed, by sequestering Bcl-2, beclin-1 has been
suggested to induce apoptosis or sensitize cells to apoptosis
(93). On the other hand, Bcl-2 and its homolog Bcl-XL can
suppress autophagy by sequestering beclin-1 (63). It is note-
Fig. 5. Roles of autophagy in cell death and survival. At the early stage of cell
stress, autophagy is induced and is cytoprotective. When cell stress is too
severe at late stage, excessive autophagy may trigger cell injury and death.
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loss of Bax and Bak, two proapoptotic Bcl-2 family proteins
that are essential to mitochondrial outer membrane permeabilization, and consequent release of apoptogenic factors including cytochrome c. When these cells are injured by classical
apoptotic insults, they do not die by apoptosis like wild-type
cells. However, in response to apoptotic insults, the Bax⫺/⫺
Bak⫺/⫺ cells do not show long-term viability or clonogenic
ability, either. Instead, they undergo massive autophagy followed by ultimate cell death. Remarkably, autophagy seems to
contribute to the delayed nonapoptotic cell death, because the
latter is abrogated when autophagy is blocked (120). Consistently, Yu et al. (150) showed that when the progression of
apoptosis is blocked by peptide caspase inhibitors, cells die
with autophagic features. Importantly, the cell death can be
prevented by autophagy inhibitors and siRNA knockdown of
autophagy genes including Atg6/beclin-1. More recently, lipopolysaccharide-treated U937 monocytoid cells were shown
to undergo autophagic cell death that was Atg6/beclin-1 dependent (139). Neuronal cells are predominantly vulnerable to
developing autophagic stress in aging, because of an increased
requirement for autophagic clearance of damaged constituents,
and decreased degradative and biosynthetic reserves (135). In
pancreatic cancer cells, Ozpolat et al. (99) found that protein
kinase C (PKC)-␦ constitutively suppresses autophagy through
the induction of tissue transglutaminase (TG)2 and inhibition
of PKC-␦/TG2 signaling leads to significant autophagy and
Atg6/beclin-1-dependent cell death. In the case of cancer
therapy, Aoki et al. (6) suggested that curcumin can restrain the
growth of malignant gliomas in vitro and in vivo through the
induction of autophagy and autophagic cell death.
It is unclear how autophagy triggers cell death. However,
several possible mechanisms have been proposed. The first
possibility can be called the autophagic stress hypothesis,
which emphasizes that excessive autophagy provokes an imbalance between autophagic stimulation and the cellular capability to complete autophagic degradation and restore essential
cellular components. This scenario culminates in a specific
form of stress, i.e., autophagic stress, followed by cell death
(24, 25). The second mechanism may be called the “wrong
eating” hypothesis, which indicates that autophagy may mistakenly digest vital organelles or proteins that are essential to
cell viability. Finally, the third mechanism is the “atrophy”
hypothesis, which emphasizes that excessive autophagy may
deplete the cells of too much cytoplasm, resulting in atrophic
cell death. In this case, cells die as a result of starvation and
self-digestion, and whether autophagy per se induces cell death
can be questioned.
It remains controversial as to whether autophagic cell death
is pathophysiologically relevant or just an observation from
artificial experimental models (59). This question was raised
when autophagic cell death was first revealed in studies using
apoptosis-defective models. Currently, the most hotly debated
issue regarding autophagic cell death is probably in the area of
tumorigenesis. There is a consensus that knockout of key
autophagic genes (and therefore suppression of autophagy) is
associated with tumor development, suggesting that autophagy
is a bona fide mechanism of tumor suppression. For example,
Atg6/beclin-1 knockout mice have a clearly increased frequency in spontaneous lymphoma and mammary neoplasia
(113). The tumor-suppressing effects of autophagy seem
readily explainable by the activation of autophagic cell death:
F251
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F252
AUTOPHAGY AND RENAL PATHOPHYSIOLOGY
Regulation of Autophagy in Renal Pathophysiology
The research on autophagy in renal physiology has been
scarce but has spun over three decades. In 1975, Pfeifer and
Scheller (109) suggested an important role for cellular autophagy in the turnover of cytoplasmic constituents in rat kidneys.
Interestingly, autophagy was shown to occur in a diurnal
fashion, minimum during the night and maximum during the
day. After that, Pfeifer along with Guder (108) studied the
stimulation of autophagy by parathyroid hormone and cAMP
in isolated tubular fragments from the rat kidney cortex. These
studies showed that the increase in the number of autophagic
vacuoles is proportional to the production of ammonia and
AJP-Renal Physiol • VOL
glucose, suggesting a role for autophagy in gluconeogenesis in
kidneys. Further studies by Pfeifer and Warmuth-Metz (110)
demonstrated the inhibitory effects of insulin on autophagy in
renal proximal tubular cells. Inhibition of autophagy was also
demonstrated in renal proximal tubules during uninephrectomy
and diabetes, providing an anticatabolic mechanism for renal
hypertrophy under these experimental conditions (11, 44). In
1983, Berkenstam et al. (13) isolated autophagic vacuoles from
kidney cortex, demonstrating enriched lysosomal enzymes and
high proteolytic activity of the organelles. In addition to
proximal tubules, autophagy has been implicated in the physiology of other segments or cells of the kidneys. In the thick
ascending limb, functional unloading resulted in an increased
sequestration of cytoplasmic components into autophagic
vacuoles within a short time interval (10). Autophagy has also
been identified in podocytes during cellular differentiation and
recovery from damage caused by experimental puromycin
aminonucleoside-induced nephrosis (8). Interestingly, electron
microscopy revealed two types of autophagy in podocytes in
renal biopsy specimens. While type I autophagy fails to transform into autophagosomes, type II forms complete autophagic
vacuoles and apparently plays a significant role in the clearance
of proteins and lipids (116).
The role of autophagy in renal pathology was not recognized
until very recently. In 2007, Chien et al. (23) demonstrated the
induction of autophagic genes (beclin-1 and LC3) during renal
ischemia-reperfusion in rats, suggesting the first evidence of
autophagy in this pathological condition. Although the role of
autophagy was not determined, beclin-1 and LC3 expression
was detected along with apoptosis in injured renal tubules.
Interestingly, both autophagy and apoptosis were suppressed
by the expression of Bcl-XL and Bcl-2 (23). In 2008, Suzuki
et al. (123) further demonstrated the formation of autophagosome-like structures in renal tubular cells during ischemiareperfusion in mice and, notably, in transplanted kidneys in
humans. In cultured renal tubular HK2 cells, autophagy inhibitors could suppress H2O2-induced cell death, suggesting that
autophagy might contribute to cell death under the condition
(123). Very recently, Gozuacik et al. (38) showed autophagy
in renal tubular cells following tunicamycin-induced ER
stress in mice. In vitro results from cell cultures further
suggested that autophagy may serve as a second cell-killing
mechanism that acts in concert with apoptosis during ER
stress (38).
In contrast, a protective role of autophagy has been suggested in other settings of renal pathology. In a mouse model
of cisplatin-induced nephrotoxicity, our recent work (104)
demonstrated a time-dependent increase of autophagic vacuoles by electron microscopy in renal tubular cells, accompanied by LC3-II accumulation. In cultured renal proximal tubular cells, cisplatin induced autophagy within hours, before
apoptosis. Importantly, inhibition of autophagy by pharmacological inhibitors and beclin-1 knockdown increased apoptosis
during cisplatin treatment, suggesting a protective role for
autophagy in cisplatin-induced renal injury and nephrotoxicity
(104). Similarly, Yang et al. (143) demonstrated cisplatininduced autophagy in cultured renal tubular cells and suggested
a cytoprotective or prosurvival role for autophagy in this
experimental model. Autophagy was also shown in renal tubular cells during cyclosporine A-induced nephrotoxicity. Notably, under this condition autophagy was induced by ER stress
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worthy that Bcl-2 and Bcl-XL may also have additional mechanisms to antagonize autophagy. For example, Bcl-2 expressed
in the ER can attenuate autophagy by blocking Ca2⫹ rise in the
ER and agonist-induced Ca2⫹ fluxes (40). Thus the beclin-1Bcl-2/Bcl-XL axis may be one of the key rheostats that govern
the cellular response to stress: autophagy or apoptosis.
Atg5. Atg5 is another protein at the crossroad of autophagy
and apoptosis. During autophagy, Atg5 forms a protein conjugation complex with Atg12 and Atg16, which is essential for
elongation of the isolation membrane. However, Yousefi et al.
(149) showed that Atg5 is proteolytically cleaved by calpains,
releasing a 24-kDa fragment. Notably, the truncated Atg5 then
translocates into mitochondria and binds Bcl-XL, leading to the
release of cytochrome c, caspase activation, and apoptosis
(149). In addition, Atg5 may trigger cell death by physically
interacting with Fas Associated via Death Domain (FADD) by
its COOH terminus (112). These observations, although requiring confirmation by studies in other model systems, suggest
that Atg5 can be a critical switch between autophagy and cell
death.
p53. The tumor suppressor p53 has a well-recognized role in
apoptosis induction. It may induce apoptosis indirectly via
transcription of proapoptotic genes or directly by activating
mitochondrial permeabilization molecules including Bax (43,
131). Interestingly, recent studies have also suggested a role for
p53 in autophagy regulation. However, it remains controversial
as to whether p53 signaling stimulates or inhibits autophagy. In
2005, Feng et al. (33) showed that transient activation of p53
by cellular stress can attenuate TOR signaling, leading to
autophagy. Crighton et al. (27) further identified damageregulated autophagy modulator (DRAM) as a transcriptional
target of p53 that, upon induction, activates autophagy. In line
with these observations, the involvement of p53 in autophagy
has been confirmed in other experimental models (4, 22, 43,
104, 129). In contrast, Tasdemir et al. (130) showed recently
that autophagy is dramatically induced when p53 is deleted or
inhibited. In this study, p53 was shown to be degraded during
autophagy and maintenance of p53 levels prevented autophagy. Morselli et al. (86) further suggested that cytosolic, and
not nuclear, p53 inhibits autophagy. While these findings
require further studies to confirm, they suggest that physiological levels of p53 may actually repress autophagy. Thus,
depending on the experimental conditions, p53 may have both
pro- and antiautophagy effects. Despite the recognition of the
roles played by p53 in both autophagy and apoptosis, little is
known about the mechanism that governs the dual roles and the
potential switch between them.
Review
AUTOPHAGY AND RENAL PATHOPHYSIOLOGY
and apparently acted as a protective mechanism against cell
death (100).
12.
Concluding Remarks
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
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