Autophagy: Emerging Therapeutic Target for Diabetic Nephropathy Shinji Kume, MD, PhD,a Kosuke Yamahara, MD,a Mako Yasuda, MD,a Hiroshi Maegawa, MD, PhD,a and Daisuke Koya, MD, PhDb Summary: Autophagy is a major catabolic pathway by which mammalian cells degrade and recycle macromolecules and organelles. It plays a critical role in removing protein aggregates, as well as damaged or excess organelles, to maintain intracellular homeostasis and to keep cells healthy. The accumulation of damaged proteins and organelles induced by hyperglycemia and other metabolic alterations is strongly associated with the development of diabetic nephropathy. Autophagy is up-regulated under conditions of calorie restriction and environmental stress, such as oxidative stress and hypoxia in proximal tubular cells, and occurs even under normal conditions in podocytes. These findings have led to our hypothesis that autophagy is involved in the pathogenesis of diabetic nephropathy, a hypothesis increasingly supported by experimental evidence. To date, however, the exact role of autophagy in diabetic nephropathy has not been fully revealed. This article therefore reviews recent findings and provides perspectives on the involvement of autophagy in diabetic nephropathy. Semin Nephrol 34:9-16 C 2014 Elsevier Inc. All rights reserved. Keywords: Autophagy, diabetic nephropathy, mTORC1, AMPK, proteinuria
D
iabetic nephropathy is a leading cause of endstage renal diseases worldwide. Proteinuria is a sign of glomerular lesions in diabetic nephropathy and causes tubulointerstitial lesions that lead to renal dysfunction.1,2 Reducing proteinuria therefore is considered a principal therapeutic target to improve renal outcomes in patients with diabetic nephropathy (Fig. 1). The pathogenesis of diabetic nephropathy involves altered intracellular metabolism related to hyperglycemia, including the activation of protein kinase C, the accumulation of advanced glycation end-products, increased flux of the polyol pathway, and oxidative stress (Fig. 2).3–8 Moreover, hemodynamic changes such as systemic and glomerular hypertension are involved in diabetic nephropathy (Fig. 2).9,10 Clinical evidence has shown that intensive control of glycemia and blood pressure could successfully reduce or abrogate proteinuria, improving renal prognosis in patients with diabetic nephropathy.10–13 Unfortunately, however, some patients develop treatment-resistant proteinuria, resulting in end-stage renal disease. Additional therapeutic options are needed to further reduce proteinuria and/or to protect proximal tubular cells from proteinuria-related toxicity. a
Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga, Japan. b Diabetes and Endocrinology, Kanazawa Medical University, Uchinada, Ishikawa, Japan. Address reprint requests to Daisuke Koya, MD, PhD, Diabetes and Endocrinology, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan. E-mail: [email protected] Financial support: Supported in part by the Japan Society for Promotion of Sciences (JSPS) KAKENHI grants 25713033 (S.K.), 25670414, and 25282028 (D.K.). Conflict of interest statement: none. 0270-9295/ - see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.semnephrol.2013.11.003
Seminars in Nephrology, Vol 34, No 1, January 2014, pp 9–16
Among several types of proteinuric kidney diseases, the renal prognosis of patients with diabetic nephropathy is particularly poor, suggesting that the diabetic condition makes various renal cells vulnerable to damage. Cells have evolved several mechanisms to cope with stress and to maintain cellular homeostasis, such as anti-oxidative stress response6 and endoplasmic reticulum (ER) stress.14 Recent studies have focused on autophagy as a stress-responsive mechanism.15 Autophagy is an intracellular catabolic process in which proteins and organelles are degraded via lysosomes to maintain intracellular homeostasis under starvation conditions and some types of stress, including hypoxia and ER stress (Fig. 3).15 The autophagy process is composed of several steps. Autophagosome formation is regulated by some autophagy-related (Atg) proteins. The initiation of starvation-induced autophagy is regulated by the phosphorylation of Atg1 (unc-51–like kinase [Ulk1]) via intracellular nutrient-sensing signals such as mammalian target of rapamycin and adenosine monophosphate (AMP)-activated protein kinase (AMPK).16 The conjugation of microtubule-associated protein 1 light chain 3 (LC3I), the mammalian homolog of Atg8, to phosphatidylethanolamine by Atg7 and Atg3 to form LC3II is a critical step in autophagosome formation, with LC3II remaining on the lysosomal membrane even after the lysosome fuses with the autophagosome.16 Moreover, this LC3 conjugation reaction is regulated positively by the Atg12–Atg5–Atg16 complex.16 Accumulating evidence has shown that autophagy activity decreases with obesity in some metabolic organs, a decrease associated with the pathogenesis of obesity-related metabolic diseases.17,18 In addition, the functional roles of autophagy in the kidney have been investigated intensely.19 For example, autophagy has been reported to play renoprotective roles during both 9
10
Figure 1. Typical histologic changes and clinical course in diabetic nephropathy. (A) A renal biopsy specimen was obtained from a patient with type 2 diabetes and overt proteinuria. This section showed glomerular sclerosis with severe tubulointerstitial lesions. The patient provided written informed consent for inclusion in this study, and the protocol was approved by the ethics committee/institutional review board of Shiga University of Medical Science. (B) Pathway by which proteinuria resulting from glomerular damage leads to subsequent tubulointerstitial lesions, which strongly are associated with a decrease in renal function. Proteinuria therefore may be reduced by ameliorating glomerular lesions, and protecting proximal tubular cells from proteinuria may be a new therapeutic target in diabetic nephropathy.
normal aging and acute kidney injury.20–25 These findings can lead us to hypothesize that diabetic kidneys are deficient in autophagic activity, making kidney cells vulnerable to diabetes-associated damage.26 This, in turn, can lead to treatment-resistant proteinuria and a progressive decrease in renal function in patients with diabetic nephropathy. Thus, regulation of the autophagy system may become a new therapeutic option to protect kidneys against diabetes-associated damage (Fig. 2).26 In this review, we summarize experimental findings regarding autophagy in diabetic nephropathy and discuss therapeutic possibilities.
S. Kume et al.
vesicles (autophagosomes).16 Autophagosomes can originate from several sources, including the ER, mitochondria, and plasma membrane. Four major steps are involved in the formation of autophagosomes: initiation, nucleation, elongation, and closure (Fig. 3),16 each of which involves autophagy-related proteins. Autophagy is initiated by the Ulk1 complex, the mammalian ortholog of the yeast autophagy-related (Atg)1 complex. The Ulk1 complex is composed of the Ulk1 Ser/Thr protein kinase, Atg13, and FIP200, the mammalian homolog of the yeast Atg17 protein. Ulk1derived phosphorylation of Atg13 and FIP200 is essential for triggering autophagy. Phagophore nucleation is dependent on beclin 1 (Atg6 in yeast), an hVps34 or class III phosphatidylinositol 3-kinase complex, which consists of hVps34, hVps15, beclin 1, and Atg14.30,31 Autophagosome elongation/ closure involves two dependent ubiquitin-like conjugation systems: Atg12 and LC3, the mammalian ortholog of yeast Atg8. The Atg12–Atg5 conjugate, which forms the Atg12–Atg5–Atg16 complex, contributes to the stimulation and localization of the LC3 conjugation reaction. The cytosolic isoform of LC3 is conjugated to phosphatidylethanolamine through two consecutive ubiquitination-like reactions catalyzed by the E1-like enzyme Atg7 and the E2-like enzyme Atg3, forming LC3-phosphatidylethanolamine conjugate (LC3-II). Thus, LC3-II formation is recognized as a marker of the presence of autophagosomes in cell and animal experiments.16,32 After formation, the autophagosomes merge with the lysosomal compartment to form autolysosomes. The protein p62, also known as sequestosome 1, localizes to autophagosomes by
AUTOPHAGY The term autophagy is derived from Greek, and means self-eating. Autophagy, which is highly conserved from yeast to mammals, is a bulk degradation process that is involved in the clearance of long-lived proteins and organelles.16,27 Autophagy has two major roles in cells: to recycle intracellular energy resources in response to conditions of nutrient depletion28,29 and to remove cytotoxic proteins and organelles under stressful conditions,15 thus maintaining cellular homeostasis. Several types of autophagy have been recognized in cells, including macroautophagy, microautophagy, and chaperone-mediated autophagy, all of which differ in mechanisms and functions. Of these three types, macroautophagy is the most prevalent and hereafter is referred to as autophagy. This review focuses on the mechanisms and functions of autophagy in diabetic nephropathy. During autophagy, de novo isolation membranes (phagophores) elongate and fuse while engulfing a portion of the cytoplasm within double-membraned
Figure 2. Systemic metabolic and hemodynamic changes in diabetes mellitus leading to intracellular metabolic alterations strongly associated with the development of renal damage, including fibrosis, inflammation, apoptosis, and epithelial mesenchymal transition (EMT). Strict control of blood glucose and blood pressure (BP) is essential to inhibit the progression of diabetic complications. Although several therapeutic targets have been identified and new agents have been developed, diabetic nephropathy remains a problem. Autophagy may be involved in the pathogenesis of diabetic nephropathy and therefore may be a target for its treatment. AGE, advanced glycation end product; AR, aldose reductase; RAS, renin-angiotensin system.
Autophagy and diabetic nephropathy
11
Figure 3. Steps of the autophagic pathways. Four steps have been identified: initiation, nucleation, elongation, and expansion/closure. Autophagy is initiated by the nucleation of an isolated membrane, or phagophore. The phagophore elongates and closes on itself to form an autophagosome. The fusion of an autophagosome with a lysosome forms an autolysosome, in which the acid hydrolases in the lysosome, such as cathepsin B and cathepsin L, breaking down the inner membrane and cytoplasmic contents. Initiation of autophagy is regulated positively by ULK1 and beclin-1, which is regulated positively by AMPK and suppressed by mTORC1.
interacting with LC3 and consistently is degraded by the autophagy-lysosome system.33 The accumulation of p62 has been observed in autophagy-deficient cells.33
PODOCYTE AUTOPHAGY IN DIABETIC NEPHROPATHY Proteinuria is a major and primary clinical aspect of diabetic nephropathy and is caused by disruption of the glomerular filtration barrier. Glomerular epithelial cells, also called podocytes, are predominantly responsible for maintaining the glomerular filtration barrier.34 Podocytes are highly specialized, terminally differentiated, and unable to proliferate. Podocyte loss caused by apoptosis and podocyte dysfunction result in proteinuria in patients with diabetic nephropathy.35 Thus, maintaining podocyte cell homeostasis is regarded as a therapeutic target in diabetic nephropathy. The autophagy-lysosomal degradation pathway is likely to play an essential role in maintaining podocyte function. Interestingly, podocytes show active autophagy even under nonstress conditions, suggesting that podocytes require a basal level of autophagy to maintain cellular homeostasis.32 Podocyte-specific autophagy-deficient mice, resulting from Atg5 gene deletion, were found to have glomerular lesions accompanied by podocyte loss and increased albuminuria with advancing age.20 Furthermore, the impairment of lysosomal function in podocytes by deletion of the mammalian target of rapamycin (mTOR), prorenin receptor, and mVps34 genes caused severe glomerular sclerosis, massive proteinuria, and higher mortality.36–38 Because autophagy involves degradation by lysosomes, autophagosomal degradation apparently was disturbed in the podocytes of these mouse models. These results also support the idea that the autophagy-lysosomal degradation pathway is essential for maintaining podocyte cell homeostasis.
Despite this knowledge of the physiological role of podocyte autophagy, the role of autophagy in diabetic nephropathy still is unclear. Autophagy may be involved in the pathogenesis of diabetic glomerular lesions. Autophagic activity in podocytes of streptozotocininduced diabetic mice was found to decrease with the duration of diabetes.39 Cultured podocytes exposed to high concentrations of glucose also showed lower autophagic activity, along with decreased levels of expression of autophagy-related proteins, such as beclin-1 and the Atg5–Atg12 complex.39 Transfection of beclin-1 small interfering RNA into cultured podocytes decreased podocin expression and subsequent albumin leakage.39 These findings suggest that hyperglycemia reduces autophagy activity, leading to alterations in podocyte function to maintain the glomerular filtration barrier. Although many of these results support the hypothesis that autophagy is involved in the pathogenesis of diabetic nephropathy, a study using mice deficient in podocyte-specific autophagy, by deleting Atg genes, is required to confirm this hypothesis.
mTOR Complex 1 in Diabetic Podocytes mTOR is an evolutionarily conserved serine/threonine kinase that forms two functional complexes, mTOR complex 1 (mTORC1) and mTORC2.40 mTORC1 is a rapamycin-sensitive protein kinase complex involved in the regulation of a wide array of cellular processes, including cell growth, proliferation, and apoptosis.41–43 mTORC1 activity is modulated by, for example, growth factors, stress, energy status, and amino acids. Studies therefore have assessed the role of mTORC1 in the pathogenesis of diabetic nephropathy.41,44,45 Interestingly, several recent studies have shown that mTORC1 signaling is highly activated in podocytes of diabetic kidneys in human beings and animals, and that both pharmacologic and genetic inactivation of mTORC1 ameliorated podocyte dysfunction in diabetic nephropathy.46–50 mTORC1 therefore may be a
12
therapeutic target in patients with diabetic nephropathy, especially because diabetes-induced hyperactivation of the mTORC1 signal is involved in the pathogenesis of diabetic nephropathy. However, the detailed downstream molecular mechanism by which mTORC1 induces podocyte dysfunction has not been determined. In addition to being involved in cell growth, proliferation, and apoptosis, mTORC1 is involved in autophagy.51–53 After its activation by certain amino acids, insulin, and high glucose conditions, mTORC1 phosphorylates Ulk1 protein, which is critical in initiating autophagosome formation, thereby inactivating Ulk1 function and suppressing autophagosome formation. mTORC1 thus senses a hypernutrient state and inhibits autophagy.51,53 Interestingly, rapamycin, a specific mTORC1 inhibitor, restores autophagic activity in cultured podocytes exposed to high glucose conditions, suggesting that mTORC1 may be involved in suppressing autophagy in podocytes under diabetic conditions.39 It remains unclear, however, whether the renoprotective effects of mTORC1 inhibition are owing solely to the restoration of autophagy activity. Although mTORC1 inhibition successfully ameliorates glomerular lesions in diabetic nephropathy, it has serious adverse effects on podocytes under nondiabetic conditions.44,45 Patients treated with rapamycin are at risk for the development of proteinuria.44 Moreover, deletion of genes associated with activation of mTORC1 led to severe glomerular damage and proteinuria, as well as higher mortality.46,47 mTORC1 is essential for regulating cellular homeostasis and growth by regulating protein translation. Thus, mTORC1 function is both critical for maintaining podocyte function and is involved in the pathogenesis of diabetic nephropathy. These findings suggest that rapamycin now is unacceptable for patients with diabetic nephropathy. If mTORC1-dependent autophagy deficiency is critical for podocyte dysfunction in diabetic nephropathy, specific agents that inhibit mTORC1-dependent autophagy suppression should be safer for patients with diabetic nephropathy. Further studies are needed to clarify the details of the relationship between mTORC1 signaling and autophagy in podocytes under diabetic conditions, perhaps leading to the development of new therapeutic agents to treat diabetic nephropathy.
AUTOPHAGY IN PROXIMAL TUBULAR CELLS Among the several types of glomerular diseases, the renal prognosis of patients with diabetic nephropathy is extremely poor, although the underlying mechanisms remain unclear. Because the severity of proteinuriainduced tubulointerstitial lesions is correlated strongly with renal outcome,1,2,54,55 diabetic conditions may exacerbate proteinuria-induced tubulointerstitial lesions.9
S. Kume et al.
Thus, identifying the molecular mechanisms underlying the obesity-mediated vulnerability of proximal tubular cells may lead to new therapies that improve renal outcomes in obese and type 2 diabetes patients with persistent proteinuria. The autophagy-mediated stress resistance machinery in proximal tubular cells therefore may be considered a new therapeutic target in the treatment of diabetic nephropathy. The physiological role of autophagy in proximal tubular cells differs from its role in podocytes. Autophagy activity is very low in proximal tubular cells under basal conditions, but higher rates of autophagy are required by cells under stress conditions. Acute kidney injury caused by some nephrotoxic agents and ischemia is becoming a serious health problem in clinical settings. Several recent animal studies have shown that autophagy in proximal tubular cells is up-regulated during acute kidney injury caused by ischemic reperfusion and cisplatin, a nephrotoxic anticancer drug.21,23–25,56,57 Mice deficient in proximal tubular cell–specific autophagy, generated by deleting the Atg5 and Atg7 genes, showed progressive renal damage, suggesting that activation of autophagy during acute kidney injury is renoprotective.23–25,56 These mice also showed premature renal aging, suggesting that a low but sufficient level of basal autophagy is needed to maintain cellular homeostasis even in proximal tubular cells, or that autophagy induction is needed to cope with age-related extracellular and intracellular stresses, such as hypoxia and ER stress. Proteinuria filtered from the glomeruli is a nephrotoxic stress in many proteinuric kidney diseases including diabetic nephropathy,1,2,54 An increased flux of protein into the urinary lumen from the glomeruli was found to activate autophagy in proximal tubular cells, reabsorbing the protein. Atg5 knockout mice, deficient in proximal tubular cell–specific autophagy, developed severe proteinuria-induced tubulointerstitial lesions, along with enhanced proximal tubular cell apoptosis, similar to results obtained in animal models of acute kidney injury (Fig. 4).58 Collectively, proximal tubular cells can induce autophagy to cope with both acute and chronic nephrotoxic stresses. Studies also have assessed the effects of obesity and diabetes on renoprotective autophagy in proximal tubular cells exposed to nephrotoxicity.58 Autophagy activity was shown to be significantly suppressed in the kidneys of streptozotocin-induced diabetic mice, high-fat-diet– induced obese mice, and Wistar fatty rats, leading to the accumulation of damaged molecules and organelles, including p62 protein and damaged mitochondria, which should be degraded via the autophagy-lysosomal pathway. Interestingly, autophagy insufficiency was confirmed in renal biopsy specimens obtained from patients with obesity and/or type 2 diabetes mellitus. The proximal tubular cells of patients with type
Autophagy and diabetic nephropathy
Figure 4. Involvement of autophagy in the development of diabetic nephropathy. Podocyte homeostasis is maintained by basal autophagy activity. Autophagy in proximal tubular cells can be induced in response to various cellular stresses including proteinuria, hypoxia, and ER stress. Obesity or diabetes suppresses stress-inducible autophagy in proximal tubular cells, leading to severe proximal tubular cell damage. The involvement of podocyte autophagy in the pathogenesis of diabetic nephropathy still is undetermined.
2 diabetes showed the accumulation of p62 protein, which can be removed by the autophagy machinery, suggesting that a deficiency in autophagy also occurs in human beings with obesity and/or type 2 diabetes.58 Studies also have investigated the mechanism involved in autophagy-deficient proximal tubular cells of obese animals and human beings. This mechanism apparently involves the hyperactivation of mTORC1 signaling in proximal tubular cells.58 Histologic analysis showed that the proximal tubular cells of obese type 2 diabetic mice and human beings were intensely positive for phosphorylated S6 protein, a marker of mTORC1 activation, and strongly associated with an obesity-related deficiency in autophagy.58 These findings indicate that autophagy deficiency and the pathogenesis of diabetic nephropathy are closely associated. Obesity-mediated autophagy deficiency likely is involved in the pathogenesis of vulnerability of proximal tubular cells in diabetic kidneys. Restoration of autophagy activity therefore may be a new therapeutic strategy for diabetic patients with overt proteinuria (Fig. 4).
THERAPEUTIC STRATEGIES TARGETING THE ACTIVATION OF AUTOPHAGY Autophagy developed during evolution and was designed to be activated under nutrient-depleted conditions to overcome long-term periods of starvation. For the past several decades, many investigators have tried to identify the calorie restriction–mediated antiaging effects in eukaryotes. Activation of autophagy has been found to be essential for calorie restriction– mediated life span elongation and anti-aging effects in lower species, and perhaps even in mammals.59,60 Autophagy in proximal tubular cells is activated by short-term starvation, suggesting that these cells possess a mechanism by which autophagy is induced in
13
response to energy depletion. Calorie restrictions have a renoprotective action against several kinds of renal injury.22,61 A calorie restriction regimen, which activates autophagy, therefore should become a potent therapeutic strategy to prevent diabetic nephropathy. Calorie restriction improves renal damage in type 2 diabetic Wistar fatty rats, as well as restores autophagy activity in their proximal tubular cells.62 Although mTORC1 inhibition can activate autophagy, excessive mTORC1 inhibition leads to podocyte dysfunction, indicating that mTORC1 inhibition is still under debate for treating patients with diabetic nephropathy. AMPK is another nutrient-sensing kinase that positively regulates autophagy. AMPK is activated under conditions of energy depletion and likely is suppressed in diabetic nephropathy.63 AMPK plays a central role in the integration of several stress stimuli and is a positive regulator of autophagy in response to conditions of nutrient depletion.64,65 AMPK monitors the energy status of the cell by sensing its AMP/adenosine triphosphate ratio. Several upstream kinases, including liver kinase B1, calcium/ calmodulin kinase kinase β, and transforming growth factor-β–activated kinase-1, can activate AMPK by phosphorylating a threonine residue on its catalytic α subunit.65 AMPK can cross-talk with mTORC1 signaling during multiple steps of autophagy regulation. AMPK induces autophagy by inhibiting mTORC1 activity via phosphorylation of its regulatory-associated proteins. Recent studies have shown that AMPK-dependent phosphorylation of Ulk1 induces autophagy.66,67 A balance between mTORC1 and AMPK likely directly regulates Ulk1 activity and subsequent autophagy initiation.53 Thus, AMPK-mediated induction of autophagy may be involved in its renoprotective mechanism. AMPK activation may be linked to autophagy for maintaining renal homeostasis in diabetic kidneys. AMPK is inactivated by dephosphorylation in the glomeruli and tubules of both type 1 and type 2 diabetic animal models, with this inactivation reversed by agents such as metformin and resveratrol, along with the attenuation of diabetic glomerular and tubular injury.63,68–72 Decreases in AMPK activity may be involved in the pathogenesis of diabetic nephropathy by reducing autophagy, suggesting that AMPK activation may be a target for restoring autophagy activity, even in diabetic kidneys.
CONTRADICTORY ROLES OF AUTOPHAGY IN THE DEVELOPMENT OF DIABETES MELLITUS In addition to diabetic nephropathy, autophagy deficiency is associated with the pathogenesis of cardiac complications. Mice deficient in cardiac myocyte–specific autophagy, resulting from the deletion of the Atg5 gene, develop severe cardiac hypertrophy and cardiac dysfunction (Fig. 5).73 These findings suggest that the deficiency in autophagy contributes to the progression of
14
the disease state. However, a more recent report showed that autophagy has contradictory roles in the development of obesity and insulin resistance in individuals with type 2 diabetes mellitus (Fig. 5). The primary triggers of type 2 diabetes are obesity and insulin resistance. Pancreatic β cells proliferate to compensate for insulin resistance and maintain blood glucose level.74,75 However, β cells have a limited capacity to proliferate and β-cell apoptosis occurs during the latter phase of diabetes, leading to insufficient insulin secretion and severe hyperglycemia. Autophagy can have an impact on each step in the development of diabetes mellitus. Glucotoxicity, free fatty acid–related lipotoxicity, and subsequent ER stress strongly contribute to β-cell apoptosis during the latter phase of diabetes development.75 A deficiency in pancreatic β-cell–specific autophagy, resulting from the deletion of the Atg7 gene, enhances high-fat-diet– induced β-cell apoptosis and subsequent insufficient βcell mass, leading to glucose intolerance (Fig. 5).76,77 This result indicates that inductive autophagy plays a critical role in the adaptive responses of β cells to insulin resistance induced by a high-fat diet.76 Thus, autophagy in pancreatic β cells plays a protective and antidiabetic role, similar to its roles in diabetic complications. In contrast, liver-, skeletal muscle–, and adipose tissue–specific autophagy-deficient mice are highly resistant to high-fat-diet–induced obesity and subsequent insulin resistance (Fig. 5).78,79 Moreover, autophagy is
S. Kume et al.
essential for lipid storage in species such as Caenorhabditis elegans.80 Because autophagy originally was activated under conditions of nutrient depletion, these findings may indicate that autophagy also is used to store sources of energy to cope with long-term starvation. Thus, autophagy has two distinct physiological roles: storing lipids under starvation conditions and an antistress function. The autophagy machinery may have evolved to encompass these two functions at the same time because organisms are exposed to long-term starvation during the course of evolution. In the present age of hypernutrition, it is likely that autophagy should be suppressed to prevent obesity and insulin resistance, whereas a higher level of autophagy is required to protect organs once patients become insulin resistant and diabetic. Thus, the relationships among autophagy, diabetes mellitus, and complications of the latter are now becoming more complex. Studies on the roles of autophagy in the pathogenesis of both diabetes and diabetic complications are needed to determine whether regulation of autophagy is an effective therapy for patients with diabetic nephropathy.
CONCLUDING COMMENTS In recent decades, many investigators have attempted to identify the molecular mechanisms involved in the initiation and progression of diabetic nephropathy and to develop new therapeutic strategies. However, the incidence of end-stage renal disease resulting from diabetic nephropathy continues to increase worldwide. There is an urgent need to identify additional new therapeutic targets for the prevention of diabetes and its complications, especially diabetic nephropathy. We have provided a perspective on the involvement of autophagy in the pathogenesis of diabetic nephropathy and whether it can be a new therapeutic target to treat this condition. To date, however, few studies have focused on autophagy in diabetic nephropathy, although studies over the next decade are expected to do so. Moreover, future studies may provide a clearer perspective as to whether addressing autophagy should be considered a novel therapeutic target in patients with diabetic nephropathy.
REFERENCES
Figure 5. Effects of autophagy deficiency on diabetes and diabetes-related organ dysfunction, with lessons learned from tissue-specific autophagy-deficient mice. Autophagy deficiency in insulin-sensitive organs inhibits the development of diet-induced obesity and insulin resistance. In contrast, autophagy deficiency in organs damaged by diabetes exacerbates tissue damage and dysfunction. BAT, brown adipose tissue; LV, left ventricular.
1. Abbate M, Zoja C, Remuzzi G. How does proteinuria cause progressive renal damage? J Am Soc Nephrol. 2006;17: 2974-2984. 2. Burton C, Harris KP. The role of proteinuria in the progression of chronic renal failure. Am J Kidney Dis. 1996;27:765-75. 3. Brownlee M. The pathobiology of diabetic complications: a unifying mechanism. Diabetes. 2005;54:1615-25. 4. Dunlop M. Aldose reductase and the role of the polyol pathway in diabetic nephropathy. Kidney Int Suppl. 2000;77:S3-12. 5. Forbes JM, Thallas V, Thomas MC, Founds HW, Burns WC, Jerums G, et al. The breakdown of preexisting advanced
Autophagy and diabetic nephropathy
6.
7.
8.
9.
10.
11.
12.
13.
14.
15. 16. 17.
18.
19.
20.
21.
22.
23.
24.
glycation end products is associated with reduced renal fibrosis in experimental diabetes. FASEB J. 2003;17:1762-4. Ha H, Hwang IA, Park JH, Lee HB. Role of reactive oxygen species in the pathogenesis of diabetic nephropathy. Diabetes Res Clin Pract. 2008;82 (Suppl 1):S42-5. Hwang I, Lee J, Huh JY, Park J, Lee HB, Ho YS, et al. Catalase deficiency accelerates diabetic renal injury through peroxisomal dysfunction. Diabetes. 2012;61:728-38. Koya D, King GL. Protein kinase C activation and the development of diabetic complications. Diabetes. 1998;47: 859-866. Mallamaci F, Ruggenenti P, Perna A, Leonardis D, Tripepi R, Tripepi G, et al. ACE inhibition is renoprotective among obese patients with proteinuria. J Am Soc Nephrol. 2011;22: 1122-8. Brenner BM, Cooper ME, de Zeeuw D, Keane WF, Mitch WE, Parving HH, et al. Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy. N Engl J Med. 2001;345:861-9. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulindependent diabetes mellitus. N Engl J Med. 1993;329:977-86. UK Prospective Diabetes Study (UKPDS) Group. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet. 1998;352: 837-853. Ohkubo Y, Kishikawa H, Araki E, Miyata T, Isami S, Motoyoshi S, et al. Intensive insulin therapy prevents the progression of diabetic microvascular complications in Japanese patients with non-insulin-dependent diabetes mellitus: a randomized prospective 6-year study. Diabetes Res Clin Pract. 1995;28:103-17. Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol. 2007;8:519-29. Kroemer G, Marino G, Levine B. Autophagy and the integrated stress response. Mol Cell. 2010;40:280-93. Mizushima N, Komatsu M. Autophagy. Renovation of cells and tissues. Cell. 2011;147:728-41. Singh R, Kaushik S, Wang Y, Xiang Y, Novak I, Komatsu M, et al. Autophagy regulates lipid metabolism. Nature. 2009; 458:1131-5. Yoshizaki T, Kusunoki C, Kondo M, Yasuda M, Kume S, Morino K, et al. Autophagy regulates inflammation in adipocytes. Biochem Biophys Res Commun. 2012;417:352-7. Huber TB, Edelstein CL, Hartleben B, Inoki K, Dong Z, Koya D, et al. Emerging role of autophagy in kidney function, diseases and aging. Autophagy. 2012;8:1009-31. Hartleben B, Godel M, Meyer-Schwesinger C, Liu S, Ulrich T, Kobler S, et al. Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice. J Clin Invest. 2010;120:1084-96. Jiang M, Liu K, Luo J, Dong Z. Autophagy is a renoprotective mechanism during in vitro hypoxia and in vivo ischemiareperfusion injury. Am J Pathol. 2010;176:1181-92. Kume S, Uzu T, Horiike K, Chin-Kanasaki M, Isshiki K, Araki S, et al. Calorie restriction enhances cell adaptation to hypoxia through Sirt1-dependent mitochondrial autophagy in mouse aged kidney. J Clin Invest. 2010;120:1043-55. Liu S, Hartleben B, Kretz O, Wiech T, Igarashi P, Mizushima N, et al. Autophagy plays a critical role in kidney tubule maintenance, aging and ischemia-reperfusion injury. Autophagy. 2012;8:826-37. Periyasamy-Thandavan S, Jiang M, Wei Q, Smith R, Yin XM, Dong Z. Autophagy is cytoprotective during cisplatin
15
25.
26. 27. 28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41. 42. 43. 44.
45.
injury of renal proximal tubular cells. Kidney Int. 2008; 74:631-40. Takahashi A, Kimura T, Takabatake Y, Namba T, Kaimori J, Kitamura H, et al. Autophagy guards against cisplatin-induced acute kidney injury. Am J Pathol. 2012;180:517-25. Kume S, Thomas MC, Koya D. Nutrient sensing, autophagy, and diabetic nephropathy. Diabetes. 2012;61:23-9. Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell. 2008;132:27-42. Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, Tanida I, et al. Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J Cell Biol. 2005;169:425-34. Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T, et al. The role of autophagy during the early neonatal starvation period. Nature. 2004;432:1032-6. Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature. 1999;402:672-6. Pattingre S, Espert L, Biard-Piechaczyk M, Codogno P. Regulation of macroautophagy by mTOR and Beclin 1 complexes. Biochimie. 2008;90:313-23. Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell. 2004;15:1101-11. Komatsu M, Waguri S, Koike M, Sou YS, Ueno T, Hara T, et al. Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell. 2007; 131:1149-63. Kawachi H, Miyauchi N, Suzuki K, Han GD, Orikasa M, Shimizu F. Role of podocyte slit diaphragm as a filtration barrier. Nephrology (Carlton). 2006;11:274-81. Pagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers BD, Rennke HG, et al. Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Invest. 1997; 99:342-8. Chen J, Chen MX, Fogo AB, Harris RC, Chen JK. mVps34 deletion in podocytes causes glomerulosclerosis by disrupting intracellular vesicle trafficking. J Am Soc Nephrol. 2013;24: 198-207. Cina DP, Onay T, Paltoo A, Li C, Maezawa Y, De Arteaga J, et al. Inhibition of MTOR disrupts autophagic flux in podocytes. J Am Soc Nephrol. 2012;23:412-20. Oshima Y, Kinouchi K, Ichihara A, Sakoda M, Kurauchi-Mito A, Bokuda K, et al. Prorenin receptor is essential for normal podocyte structure and function. J Am Soc Nephrol. 2011; 22:2203-12. Fang L, Zhou Y, Cao H, Wen P, Jiang L, He W, et al. Autophagy attenuates diabetic glomerular damage through protection of hyperglycemia-induced podocyte injury. PLoS One. 2013;8:e60546. Jacinto E, Loewith R, Schmidt A, Lin S, Ruegg MA, Hall A, et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol. 2004; 6:1122-8. Inoki K, Corradetti MN, Guan KL. Dysregulation of the TSCmTOR pathway in human disease. Nat Genet. 2005;37:19-24. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012;149:274-93. Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell. 2006;124:471-84. Huber TB, Walz G, Kuehn EW. mTOR and rapamycin in the kidney: signaling and therapeutic implications beyond immunosuppression. Kidney Int. 2011;79:502-11. Lieberthal W, Levine JS. The role of the mammalian target of rapamycin (mTOR) in renal disease. J Am Soc Nephrol. 2009;20:2493-502.
16 46. Godel M, Hartleben B, Herbach N, Liu S, Zschiedrich S, Lu S, et al. Role of mTOR in podocyte function and diabetic nephropathy in humans and mice. J Clin Invest. 2011; 121:2197-209. 47. Inoki K, Mori H, Wang J, Suzuki T, Hong S, Yoshida S, et al. mTORC1 activation in podocytes is a critical step in the development of diabetic nephropathy in mice. J Clin Invest. 2011;121:2181-96. 48. Lloberas N, Cruzado JM, Franquesa M, Herrero-Fresneda I, Torras J, Alperovich G, et al. Mammalian target of rapamycin pathway blockade slows progression of diabetic kidney disease in rats. J Am Soc Nephrol. 2006;17:1395-404. 49. Mori H, Inoki K, Masutani K, Wakabayashi Y, Komai K, Nakagawa R, et al. The mTOR pathway is highly activated in diabetic nephropathy and rapamycin has a strong therapeutic potential. Biochem Biophys Res Commun. 2009; 384:471-5. 50. Sakaguchi M, Isono M, Isshiki K, Sugimoto T, Koya D, Kashiwagi A. Inhibition of mTOR signaling with rapamycin attenuates renal hypertrophy in the early diabetic mice. Biochem Biophys Res Commun. 2006;340:296-301. 51. Hosokawa N, Hara T, Kaizuka T, Kishi C, Takamura A, Miura Y, et al. Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol Biol Cell. 2009;20:1981-91. 52. Moscat J, Diaz-Meco MT. Feedback on fat: p62-mTORC1autophagy connections. Cell. 2011;147:724-7. 53. Kim J, Kundu M, Viollet B, Guan KL. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol. 2011;13:132-41. 54. Nath KA. Tubulointerstitial changes as a major determinant in the progression of renal damage. Am J Kidney Dis. 1992; 20:1-17. 55. Risdon RA, Sloper JC, De Wardener HE. Relationship between renal function and histological changes found in renal-biopsy specimens from patients with persistent glomerular nephritis. Lancet. 1968;2:363-6. 56. Kimura T, Takabatake Y, Takahashi A, Kaimori JY, Matsui I, Namba T, et al. Autophagy protects the proximal tubule from degeneration and acute ischemic injury. J Am Soc Nephrol. 2011;22:902-13. 57. Yang C, Kaushal V, Shah SV, Kaushal GP. Autophagy is associated with apoptosis in cisplatin injury to renal tubular epithelial cells. Am J Physiol Renal Physiol. 2008;294: F777-F787. 58. Yamahara K, Kume S, Koya D, Tanaka Y, Morita Y, ChinKanasaki M, et al. Obesity-mediated autophagy insufficiency exacerbates proteinuria-induced tubulointerstitial lesions. J Am Soc Nephrol. 2013;24:1769-81. 59. Colman RJ, Anderson RM, Johnson SC, Kastman EK, Kosmatka KJ, Beasley TM, et al. Caloric restriction delays disease onset and mortality in rhesus monkeys. Science. 2009; 325:201-4. 60. Fontana L, Partridge L, Longo VD. Extending healthy life span–from yeast to humans. Science. 2010;328:321-6. 61. Cherry Engelman RW, Wang BY, Kinjoh K, El-Badri NS, Good RA. Calorie restriction delays the crescentic glomerulonephritis of SCG/Kj mice. Proc Soc Exp Biol Med. 1998; 218:218-22. 62. Kitada M, Takeda A, Nagai T, Ito H, Kanasaki K, Koya D. Dietary restriction ameliorates diabetic nephropathy through anti-inflammatory effects and regulation of the autophagy via restoration of Sirt1 in diabetic Wistar fatty (fa/fa) rats: a model of type 2 diabetes. Exp Diabetes Res. 2011;2011:908185.
S. Kume et al. 63. Kume S, Uzu T, Araki S, Sugimoto T, Isshiki K, ChinKanasaki M, et al. Role of altered renal lipid metabolism in the development of renal injury induced by a high-fat diet. J Am Soc Nephrol. 2007;18:2715-23. 64. Steinberg GR, Kemp BE. AMPK in health and disease. Physiol Rev. 2009;89:1025-78. 65. Shackelford DB, Shaw RJ. The LKB1-AMPK pathway: metabolism and growth control in tumour suppression. Nat Rev Cancer. 2009;9:563-75. 66. Alers S, Loffler AS, Wesselborg S, Stork B. Role of AMPKmTOR-Ulk1/2 in the regulation of autophagy: cross talk, shortcuts, and feedbacks. Mol Cell Biol. 2012;32:2-11. 67. Lee JW, Park S, Takahashi Y, Wang HG. The association of AMPK with ULK1 regulates autophagy. PLoS One. 2010;5: e15394. 68. Chang CC, Chang CY, Wu YT, Huang JP, Yen TH, Hung LM. Resveratrol retards progression of diabetic nephropathy through modulations of oxidative stress, proinflammatory cytokines, and AMP-activated protein kinase. J Biomed Sci. 2011;18:47. 69. Takiyama Y, Harumi T, Watanabe J, Fujita Y, Honjo J, Shimizu N, et al. Tubular injury in a rat model of type 2 diabetes is prevented by metformin: a possible role of HIF1alpha expression and oxygen metabolism. Diabetes. 2011; 60:981-92. 70. Ding DF, You N, Wu XM, Xu JR, Hu AP, Ye XL, et al. Resveratrol attenuates renal hypertrophy in early-stage diabetes by activating AMPK. Am J Nephrol. 2010;31:363-74. 71. Lee MJ, Feliers D, Mariappan MM, Sataranatarajan K, Mahimainathan L, Musi N, et al. A role for AMP-activated protein kinase in diabetes-induced renal hypertrophy. Am J Physiol Renal Physiol. 2007;292:F617-27. 72. Sharma K, Ramachandrarao S, Qiu G, Usui HK, Zhu Y, Dunn SR, et al. Adiponectin regulates albuminuria and podocyte function in mice. J Clin Invest. 2008;118:1645-56. 73. Nakai A, Yamaguchi O, Takeda T, Higuchi Y, Hikoso S, Taniike M, et al. The role of autophagy in cardiomyocytes in the basal state and in response to hemodynamic stress. Nat Med. 2007;13:619-24. 74. Weyer C, Bogardus C, Mott DM, Pratley RE. The natural history of insulin secretory dysfunction and insulin resistance in the pathogenesis of type 2 diabetes mellitus. J Clin Invest. 1999;104:787-94. 75. Kasuga M. Insulin resistance and pancreatic beta cell failure. J Clin Invest. 2006;116:1756-60. 76. Ebato C, Uchida T, Arakawa M, Komatsu M, Ueno T, Komiya K, et al. Autophagy is important in islet homeostasis and compensatory increase of beta cell mass in response to high-fat diet. Cell Metab. 2008;8:325-32. 77. Jung HS, Chung KW, Won Kim J, Kim J, Komatsu M, Tanaka K, et al. Loss of autophagy diminishes pancreatic beta cell mass and function with resultant hyperglycemia. Cell Metab. 2008;8:318-24. 78. Kim KH, Jeong YT, Oh H, Kim SH, Cho JM, Kim YN, et al. Autophagy deficiency leads to protection from obesity and insulin resistance by inducing Fgf21 as a mitokine. Nat Med. 2013;19:83-92. 79. Singh R, Xiang Y, Wang Y, Baikati K, Cuervo AM, Luu YK, et al. Autophagy regulates adipose mass and differentiation in mice. J Clin Invest. 2009;119:3329-39. 80. Lapierre LR, Silvestrini MJ, Nunez L, Ames K, Wong S, Le TT, et al. Autophagy genes are required for normal lipid levels in C. elegans. Autophagy. 2013;9:278-86.
D
iabetic nephropathy is a leading cause of endstage renal diseases worldwide. Proteinuria is a sign of glomerular lesions in diabetic nephropathy and causes tubulointerstitial lesions that lead to renal dysfunction.1,2 Reducing proteinuria therefore is considered a principal therapeutic target to improve renal outcomes in patients with diabetic nephropathy (Fig. 1). The pathogenesis of diabetic nephropathy involves altered intracellular metabolism related to hyperglycemia, including the activation of protein kinase C, the accumulation of advanced glycation end-products, increased flux of the polyol pathway, and oxidative stress (Fig. 2).3–8 Moreover, hemodynamic changes such as systemic and glomerular hypertension are involved in diabetic nephropathy (Fig. 2).9,10 Clinical evidence has shown that intensive control of glycemia and blood pressure could successfully reduce or abrogate proteinuria, improving renal prognosis in patients with diabetic nephropathy.10–13 Unfortunately, however, some patients develop treatment-resistant proteinuria, resulting in end-stage renal disease. Additional therapeutic options are needed to further reduce proteinuria and/or to protect proximal tubular cells from proteinuria-related toxicity. a
Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga, Japan. b Diabetes and Endocrinology, Kanazawa Medical University, Uchinada, Ishikawa, Japan. Address reprint requests to Daisuke Koya, MD, PhD, Diabetes and Endocrinology, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan. E-mail: [email protected] Financial support: Supported in part by the Japan Society for Promotion of Sciences (JSPS) KAKENHI grants 25713033 (S.K.), 25670414, and 25282028 (D.K.). Conflict of interest statement: none. 0270-9295/ - see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.semnephrol.2013.11.003
Seminars in Nephrology, Vol 34, No 1, January 2014, pp 9–16
Among several types of proteinuric kidney diseases, the renal prognosis of patients with diabetic nephropathy is particularly poor, suggesting that the diabetic condition makes various renal cells vulnerable to damage. Cells have evolved several mechanisms to cope with stress and to maintain cellular homeostasis, such as anti-oxidative stress response6 and endoplasmic reticulum (ER) stress.14 Recent studies have focused on autophagy as a stress-responsive mechanism.15 Autophagy is an intracellular catabolic process in which proteins and organelles are degraded via lysosomes to maintain intracellular homeostasis under starvation conditions and some types of stress, including hypoxia and ER stress (Fig. 3).15 The autophagy process is composed of several steps. Autophagosome formation is regulated by some autophagy-related (Atg) proteins. The initiation of starvation-induced autophagy is regulated by the phosphorylation of Atg1 (unc-51–like kinase [Ulk1]) via intracellular nutrient-sensing signals such as mammalian target of rapamycin and adenosine monophosphate (AMP)-activated protein kinase (AMPK).16 The conjugation of microtubule-associated protein 1 light chain 3 (LC3I), the mammalian homolog of Atg8, to phosphatidylethanolamine by Atg7 and Atg3 to form LC3II is a critical step in autophagosome formation, with LC3II remaining on the lysosomal membrane even after the lysosome fuses with the autophagosome.16 Moreover, this LC3 conjugation reaction is regulated positively by the Atg12–Atg5–Atg16 complex.16 Accumulating evidence has shown that autophagy activity decreases with obesity in some metabolic organs, a decrease associated with the pathogenesis of obesity-related metabolic diseases.17,18 In addition, the functional roles of autophagy in the kidney have been investigated intensely.19 For example, autophagy has been reported to play renoprotective roles during both 9
10
Figure 1. Typical histologic changes and clinical course in diabetic nephropathy. (A) A renal biopsy specimen was obtained from a patient with type 2 diabetes and overt proteinuria. This section showed glomerular sclerosis with severe tubulointerstitial lesions. The patient provided written informed consent for inclusion in this study, and the protocol was approved by the ethics committee/institutional review board of Shiga University of Medical Science. (B) Pathway by which proteinuria resulting from glomerular damage leads to subsequent tubulointerstitial lesions, which strongly are associated with a decrease in renal function. Proteinuria therefore may be reduced by ameliorating glomerular lesions, and protecting proximal tubular cells from proteinuria may be a new therapeutic target in diabetic nephropathy.
normal aging and acute kidney injury.20–25 These findings can lead us to hypothesize that diabetic kidneys are deficient in autophagic activity, making kidney cells vulnerable to diabetes-associated damage.26 This, in turn, can lead to treatment-resistant proteinuria and a progressive decrease in renal function in patients with diabetic nephropathy. Thus, regulation of the autophagy system may become a new therapeutic option to protect kidneys against diabetes-associated damage (Fig. 2).26 In this review, we summarize experimental findings regarding autophagy in diabetic nephropathy and discuss therapeutic possibilities.
S. Kume et al.
vesicles (autophagosomes).16 Autophagosomes can originate from several sources, including the ER, mitochondria, and plasma membrane. Four major steps are involved in the formation of autophagosomes: initiation, nucleation, elongation, and closure (Fig. 3),16 each of which involves autophagy-related proteins. Autophagy is initiated by the Ulk1 complex, the mammalian ortholog of the yeast autophagy-related (Atg)1 complex. The Ulk1 complex is composed of the Ulk1 Ser/Thr protein kinase, Atg13, and FIP200, the mammalian homolog of the yeast Atg17 protein. Ulk1derived phosphorylation of Atg13 and FIP200 is essential for triggering autophagy. Phagophore nucleation is dependent on beclin 1 (Atg6 in yeast), an hVps34 or class III phosphatidylinositol 3-kinase complex, which consists of hVps34, hVps15, beclin 1, and Atg14.30,31 Autophagosome elongation/ closure involves two dependent ubiquitin-like conjugation systems: Atg12 and LC3, the mammalian ortholog of yeast Atg8. The Atg12–Atg5 conjugate, which forms the Atg12–Atg5–Atg16 complex, contributes to the stimulation and localization of the LC3 conjugation reaction. The cytosolic isoform of LC3 is conjugated to phosphatidylethanolamine through two consecutive ubiquitination-like reactions catalyzed by the E1-like enzyme Atg7 and the E2-like enzyme Atg3, forming LC3-phosphatidylethanolamine conjugate (LC3-II). Thus, LC3-II formation is recognized as a marker of the presence of autophagosomes in cell and animal experiments.16,32 After formation, the autophagosomes merge with the lysosomal compartment to form autolysosomes. The protein p62, also known as sequestosome 1, localizes to autophagosomes by
AUTOPHAGY The term autophagy is derived from Greek, and means self-eating. Autophagy, which is highly conserved from yeast to mammals, is a bulk degradation process that is involved in the clearance of long-lived proteins and organelles.16,27 Autophagy has two major roles in cells: to recycle intracellular energy resources in response to conditions of nutrient depletion28,29 and to remove cytotoxic proteins and organelles under stressful conditions,15 thus maintaining cellular homeostasis. Several types of autophagy have been recognized in cells, including macroautophagy, microautophagy, and chaperone-mediated autophagy, all of which differ in mechanisms and functions. Of these three types, macroautophagy is the most prevalent and hereafter is referred to as autophagy. This review focuses on the mechanisms and functions of autophagy in diabetic nephropathy. During autophagy, de novo isolation membranes (phagophores) elongate and fuse while engulfing a portion of the cytoplasm within double-membraned
Figure 2. Systemic metabolic and hemodynamic changes in diabetes mellitus leading to intracellular metabolic alterations strongly associated with the development of renal damage, including fibrosis, inflammation, apoptosis, and epithelial mesenchymal transition (EMT). Strict control of blood glucose and blood pressure (BP) is essential to inhibit the progression of diabetic complications. Although several therapeutic targets have been identified and new agents have been developed, diabetic nephropathy remains a problem. Autophagy may be involved in the pathogenesis of diabetic nephropathy and therefore may be a target for its treatment. AGE, advanced glycation end product; AR, aldose reductase; RAS, renin-angiotensin system.
Autophagy and diabetic nephropathy
11
Figure 3. Steps of the autophagic pathways. Four steps have been identified: initiation, nucleation, elongation, and expansion/closure. Autophagy is initiated by the nucleation of an isolated membrane, or phagophore. The phagophore elongates and closes on itself to form an autophagosome. The fusion of an autophagosome with a lysosome forms an autolysosome, in which the acid hydrolases in the lysosome, such as cathepsin B and cathepsin L, breaking down the inner membrane and cytoplasmic contents. Initiation of autophagy is regulated positively by ULK1 and beclin-1, which is regulated positively by AMPK and suppressed by mTORC1.
interacting with LC3 and consistently is degraded by the autophagy-lysosome system.33 The accumulation of p62 has been observed in autophagy-deficient cells.33
PODOCYTE AUTOPHAGY IN DIABETIC NEPHROPATHY Proteinuria is a major and primary clinical aspect of diabetic nephropathy and is caused by disruption of the glomerular filtration barrier. Glomerular epithelial cells, also called podocytes, are predominantly responsible for maintaining the glomerular filtration barrier.34 Podocytes are highly specialized, terminally differentiated, and unable to proliferate. Podocyte loss caused by apoptosis and podocyte dysfunction result in proteinuria in patients with diabetic nephropathy.35 Thus, maintaining podocyte cell homeostasis is regarded as a therapeutic target in diabetic nephropathy. The autophagy-lysosomal degradation pathway is likely to play an essential role in maintaining podocyte function. Interestingly, podocytes show active autophagy even under nonstress conditions, suggesting that podocytes require a basal level of autophagy to maintain cellular homeostasis.32 Podocyte-specific autophagy-deficient mice, resulting from Atg5 gene deletion, were found to have glomerular lesions accompanied by podocyte loss and increased albuminuria with advancing age.20 Furthermore, the impairment of lysosomal function in podocytes by deletion of the mammalian target of rapamycin (mTOR), prorenin receptor, and mVps34 genes caused severe glomerular sclerosis, massive proteinuria, and higher mortality.36–38 Because autophagy involves degradation by lysosomes, autophagosomal degradation apparently was disturbed in the podocytes of these mouse models. These results also support the idea that the autophagy-lysosomal degradation pathway is essential for maintaining podocyte cell homeostasis.
Despite this knowledge of the physiological role of podocyte autophagy, the role of autophagy in diabetic nephropathy still is unclear. Autophagy may be involved in the pathogenesis of diabetic glomerular lesions. Autophagic activity in podocytes of streptozotocininduced diabetic mice was found to decrease with the duration of diabetes.39 Cultured podocytes exposed to high concentrations of glucose also showed lower autophagic activity, along with decreased levels of expression of autophagy-related proteins, such as beclin-1 and the Atg5–Atg12 complex.39 Transfection of beclin-1 small interfering RNA into cultured podocytes decreased podocin expression and subsequent albumin leakage.39 These findings suggest that hyperglycemia reduces autophagy activity, leading to alterations in podocyte function to maintain the glomerular filtration barrier. Although many of these results support the hypothesis that autophagy is involved in the pathogenesis of diabetic nephropathy, a study using mice deficient in podocyte-specific autophagy, by deleting Atg genes, is required to confirm this hypothesis.
mTOR Complex 1 in Diabetic Podocytes mTOR is an evolutionarily conserved serine/threonine kinase that forms two functional complexes, mTOR complex 1 (mTORC1) and mTORC2.40 mTORC1 is a rapamycin-sensitive protein kinase complex involved in the regulation of a wide array of cellular processes, including cell growth, proliferation, and apoptosis.41–43 mTORC1 activity is modulated by, for example, growth factors, stress, energy status, and amino acids. Studies therefore have assessed the role of mTORC1 in the pathogenesis of diabetic nephropathy.41,44,45 Interestingly, several recent studies have shown that mTORC1 signaling is highly activated in podocytes of diabetic kidneys in human beings and animals, and that both pharmacologic and genetic inactivation of mTORC1 ameliorated podocyte dysfunction in diabetic nephropathy.46–50 mTORC1 therefore may be a
12
therapeutic target in patients with diabetic nephropathy, especially because diabetes-induced hyperactivation of the mTORC1 signal is involved in the pathogenesis of diabetic nephropathy. However, the detailed downstream molecular mechanism by which mTORC1 induces podocyte dysfunction has not been determined. In addition to being involved in cell growth, proliferation, and apoptosis, mTORC1 is involved in autophagy.51–53 After its activation by certain amino acids, insulin, and high glucose conditions, mTORC1 phosphorylates Ulk1 protein, which is critical in initiating autophagosome formation, thereby inactivating Ulk1 function and suppressing autophagosome formation. mTORC1 thus senses a hypernutrient state and inhibits autophagy.51,53 Interestingly, rapamycin, a specific mTORC1 inhibitor, restores autophagic activity in cultured podocytes exposed to high glucose conditions, suggesting that mTORC1 may be involved in suppressing autophagy in podocytes under diabetic conditions.39 It remains unclear, however, whether the renoprotective effects of mTORC1 inhibition are owing solely to the restoration of autophagy activity. Although mTORC1 inhibition successfully ameliorates glomerular lesions in diabetic nephropathy, it has serious adverse effects on podocytes under nondiabetic conditions.44,45 Patients treated with rapamycin are at risk for the development of proteinuria.44 Moreover, deletion of genes associated with activation of mTORC1 led to severe glomerular damage and proteinuria, as well as higher mortality.46,47 mTORC1 is essential for regulating cellular homeostasis and growth by regulating protein translation. Thus, mTORC1 function is both critical for maintaining podocyte function and is involved in the pathogenesis of diabetic nephropathy. These findings suggest that rapamycin now is unacceptable for patients with diabetic nephropathy. If mTORC1-dependent autophagy deficiency is critical for podocyte dysfunction in diabetic nephropathy, specific agents that inhibit mTORC1-dependent autophagy suppression should be safer for patients with diabetic nephropathy. Further studies are needed to clarify the details of the relationship between mTORC1 signaling and autophagy in podocytes under diabetic conditions, perhaps leading to the development of new therapeutic agents to treat diabetic nephropathy.
AUTOPHAGY IN PROXIMAL TUBULAR CELLS Among the several types of glomerular diseases, the renal prognosis of patients with diabetic nephropathy is extremely poor, although the underlying mechanisms remain unclear. Because the severity of proteinuriainduced tubulointerstitial lesions is correlated strongly with renal outcome,1,2,54,55 diabetic conditions may exacerbate proteinuria-induced tubulointerstitial lesions.9
S. Kume et al.
Thus, identifying the molecular mechanisms underlying the obesity-mediated vulnerability of proximal tubular cells may lead to new therapies that improve renal outcomes in obese and type 2 diabetes patients with persistent proteinuria. The autophagy-mediated stress resistance machinery in proximal tubular cells therefore may be considered a new therapeutic target in the treatment of diabetic nephropathy. The physiological role of autophagy in proximal tubular cells differs from its role in podocytes. Autophagy activity is very low in proximal tubular cells under basal conditions, but higher rates of autophagy are required by cells under stress conditions. Acute kidney injury caused by some nephrotoxic agents and ischemia is becoming a serious health problem in clinical settings. Several recent animal studies have shown that autophagy in proximal tubular cells is up-regulated during acute kidney injury caused by ischemic reperfusion and cisplatin, a nephrotoxic anticancer drug.21,23–25,56,57 Mice deficient in proximal tubular cell–specific autophagy, generated by deleting the Atg5 and Atg7 genes, showed progressive renal damage, suggesting that activation of autophagy during acute kidney injury is renoprotective.23–25,56 These mice also showed premature renal aging, suggesting that a low but sufficient level of basal autophagy is needed to maintain cellular homeostasis even in proximal tubular cells, or that autophagy induction is needed to cope with age-related extracellular and intracellular stresses, such as hypoxia and ER stress. Proteinuria filtered from the glomeruli is a nephrotoxic stress in many proteinuric kidney diseases including diabetic nephropathy,1,2,54 An increased flux of protein into the urinary lumen from the glomeruli was found to activate autophagy in proximal tubular cells, reabsorbing the protein. Atg5 knockout mice, deficient in proximal tubular cell–specific autophagy, developed severe proteinuria-induced tubulointerstitial lesions, along with enhanced proximal tubular cell apoptosis, similar to results obtained in animal models of acute kidney injury (Fig. 4).58 Collectively, proximal tubular cells can induce autophagy to cope with both acute and chronic nephrotoxic stresses. Studies also have assessed the effects of obesity and diabetes on renoprotective autophagy in proximal tubular cells exposed to nephrotoxicity.58 Autophagy activity was shown to be significantly suppressed in the kidneys of streptozotocin-induced diabetic mice, high-fat-diet– induced obese mice, and Wistar fatty rats, leading to the accumulation of damaged molecules and organelles, including p62 protein and damaged mitochondria, which should be degraded via the autophagy-lysosomal pathway. Interestingly, autophagy insufficiency was confirmed in renal biopsy specimens obtained from patients with obesity and/or type 2 diabetes mellitus. The proximal tubular cells of patients with type
Autophagy and diabetic nephropathy
Figure 4. Involvement of autophagy in the development of diabetic nephropathy. Podocyte homeostasis is maintained by basal autophagy activity. Autophagy in proximal tubular cells can be induced in response to various cellular stresses including proteinuria, hypoxia, and ER stress. Obesity or diabetes suppresses stress-inducible autophagy in proximal tubular cells, leading to severe proximal tubular cell damage. The involvement of podocyte autophagy in the pathogenesis of diabetic nephropathy still is undetermined.
2 diabetes showed the accumulation of p62 protein, which can be removed by the autophagy machinery, suggesting that a deficiency in autophagy also occurs in human beings with obesity and/or type 2 diabetes.58 Studies also have investigated the mechanism involved in autophagy-deficient proximal tubular cells of obese animals and human beings. This mechanism apparently involves the hyperactivation of mTORC1 signaling in proximal tubular cells.58 Histologic analysis showed that the proximal tubular cells of obese type 2 diabetic mice and human beings were intensely positive for phosphorylated S6 protein, a marker of mTORC1 activation, and strongly associated with an obesity-related deficiency in autophagy.58 These findings indicate that autophagy deficiency and the pathogenesis of diabetic nephropathy are closely associated. Obesity-mediated autophagy deficiency likely is involved in the pathogenesis of vulnerability of proximal tubular cells in diabetic kidneys. Restoration of autophagy activity therefore may be a new therapeutic strategy for diabetic patients with overt proteinuria (Fig. 4).
THERAPEUTIC STRATEGIES TARGETING THE ACTIVATION OF AUTOPHAGY Autophagy developed during evolution and was designed to be activated under nutrient-depleted conditions to overcome long-term periods of starvation. For the past several decades, many investigators have tried to identify the calorie restriction–mediated antiaging effects in eukaryotes. Activation of autophagy has been found to be essential for calorie restriction– mediated life span elongation and anti-aging effects in lower species, and perhaps even in mammals.59,60 Autophagy in proximal tubular cells is activated by short-term starvation, suggesting that these cells possess a mechanism by which autophagy is induced in
13
response to energy depletion. Calorie restrictions have a renoprotective action against several kinds of renal injury.22,61 A calorie restriction regimen, which activates autophagy, therefore should become a potent therapeutic strategy to prevent diabetic nephropathy. Calorie restriction improves renal damage in type 2 diabetic Wistar fatty rats, as well as restores autophagy activity in their proximal tubular cells.62 Although mTORC1 inhibition can activate autophagy, excessive mTORC1 inhibition leads to podocyte dysfunction, indicating that mTORC1 inhibition is still under debate for treating patients with diabetic nephropathy. AMPK is another nutrient-sensing kinase that positively regulates autophagy. AMPK is activated under conditions of energy depletion and likely is suppressed in diabetic nephropathy.63 AMPK plays a central role in the integration of several stress stimuli and is a positive regulator of autophagy in response to conditions of nutrient depletion.64,65 AMPK monitors the energy status of the cell by sensing its AMP/adenosine triphosphate ratio. Several upstream kinases, including liver kinase B1, calcium/ calmodulin kinase kinase β, and transforming growth factor-β–activated kinase-1, can activate AMPK by phosphorylating a threonine residue on its catalytic α subunit.65 AMPK can cross-talk with mTORC1 signaling during multiple steps of autophagy regulation. AMPK induces autophagy by inhibiting mTORC1 activity via phosphorylation of its regulatory-associated proteins. Recent studies have shown that AMPK-dependent phosphorylation of Ulk1 induces autophagy.66,67 A balance between mTORC1 and AMPK likely directly regulates Ulk1 activity and subsequent autophagy initiation.53 Thus, AMPK-mediated induction of autophagy may be involved in its renoprotective mechanism. AMPK activation may be linked to autophagy for maintaining renal homeostasis in diabetic kidneys. AMPK is inactivated by dephosphorylation in the glomeruli and tubules of both type 1 and type 2 diabetic animal models, with this inactivation reversed by agents such as metformin and resveratrol, along with the attenuation of diabetic glomerular and tubular injury.63,68–72 Decreases in AMPK activity may be involved in the pathogenesis of diabetic nephropathy by reducing autophagy, suggesting that AMPK activation may be a target for restoring autophagy activity, even in diabetic kidneys.
CONTRADICTORY ROLES OF AUTOPHAGY IN THE DEVELOPMENT OF DIABETES MELLITUS In addition to diabetic nephropathy, autophagy deficiency is associated with the pathogenesis of cardiac complications. Mice deficient in cardiac myocyte–specific autophagy, resulting from the deletion of the Atg5 gene, develop severe cardiac hypertrophy and cardiac dysfunction (Fig. 5).73 These findings suggest that the deficiency in autophagy contributes to the progression of
14
the disease state. However, a more recent report showed that autophagy has contradictory roles in the development of obesity and insulin resistance in individuals with type 2 diabetes mellitus (Fig. 5). The primary triggers of type 2 diabetes are obesity and insulin resistance. Pancreatic β cells proliferate to compensate for insulin resistance and maintain blood glucose level.74,75 However, β cells have a limited capacity to proliferate and β-cell apoptosis occurs during the latter phase of diabetes, leading to insufficient insulin secretion and severe hyperglycemia. Autophagy can have an impact on each step in the development of diabetes mellitus. Glucotoxicity, free fatty acid–related lipotoxicity, and subsequent ER stress strongly contribute to β-cell apoptosis during the latter phase of diabetes development.75 A deficiency in pancreatic β-cell–specific autophagy, resulting from the deletion of the Atg7 gene, enhances high-fat-diet– induced β-cell apoptosis and subsequent insufficient βcell mass, leading to glucose intolerance (Fig. 5).76,77 This result indicates that inductive autophagy plays a critical role in the adaptive responses of β cells to insulin resistance induced by a high-fat diet.76 Thus, autophagy in pancreatic β cells plays a protective and antidiabetic role, similar to its roles in diabetic complications. In contrast, liver-, skeletal muscle–, and adipose tissue–specific autophagy-deficient mice are highly resistant to high-fat-diet–induced obesity and subsequent insulin resistance (Fig. 5).78,79 Moreover, autophagy is
S. Kume et al.
essential for lipid storage in species such as Caenorhabditis elegans.80 Because autophagy originally was activated under conditions of nutrient depletion, these findings may indicate that autophagy also is used to store sources of energy to cope with long-term starvation. Thus, autophagy has two distinct physiological roles: storing lipids under starvation conditions and an antistress function. The autophagy machinery may have evolved to encompass these two functions at the same time because organisms are exposed to long-term starvation during the course of evolution. In the present age of hypernutrition, it is likely that autophagy should be suppressed to prevent obesity and insulin resistance, whereas a higher level of autophagy is required to protect organs once patients become insulin resistant and diabetic. Thus, the relationships among autophagy, diabetes mellitus, and complications of the latter are now becoming more complex. Studies on the roles of autophagy in the pathogenesis of both diabetes and diabetic complications are needed to determine whether regulation of autophagy is an effective therapy for patients with diabetic nephropathy.
CONCLUDING COMMENTS In recent decades, many investigators have attempted to identify the molecular mechanisms involved in the initiation and progression of diabetic nephropathy and to develop new therapeutic strategies. However, the incidence of end-stage renal disease resulting from diabetic nephropathy continues to increase worldwide. There is an urgent need to identify additional new therapeutic targets for the prevention of diabetes and its complications, especially diabetic nephropathy. We have provided a perspective on the involvement of autophagy in the pathogenesis of diabetic nephropathy and whether it can be a new therapeutic target to treat this condition. To date, however, few studies have focused on autophagy in diabetic nephropathy, although studies over the next decade are expected to do so. Moreover, future studies may provide a clearer perspective as to whether addressing autophagy should be considered a novel therapeutic target in patients with diabetic nephropathy.
REFERENCES
Figure 5. Effects of autophagy deficiency on diabetes and diabetes-related organ dysfunction, with lessons learned from tissue-specific autophagy-deficient mice. Autophagy deficiency in insulin-sensitive organs inhibits the development of diet-induced obesity and insulin resistance. In contrast, autophagy deficiency in organs damaged by diabetes exacerbates tissue damage and dysfunction. BAT, brown adipose tissue; LV, left ventricular.
1. Abbate M, Zoja C, Remuzzi G. How does proteinuria cause progressive renal damage? J Am Soc Nephrol. 2006;17: 2974-2984. 2. Burton C, Harris KP. The role of proteinuria in the progression of chronic renal failure. Am J Kidney Dis. 1996;27:765-75. 3. Brownlee M. The pathobiology of diabetic complications: a unifying mechanism. Diabetes. 2005;54:1615-25. 4. Dunlop M. Aldose reductase and the role of the polyol pathway in diabetic nephropathy. Kidney Int Suppl. 2000;77:S3-12. 5. Forbes JM, Thallas V, Thomas MC, Founds HW, Burns WC, Jerums G, et al. The breakdown of preexisting advanced
Autophagy and diabetic nephropathy
6.
7.
8.
9.
10.
11.
12.
13.
14.
15. 16. 17.
18.
19.
20.
21.
22.
23.
24.
glycation end products is associated with reduced renal fibrosis in experimental diabetes. FASEB J. 2003;17:1762-4. Ha H, Hwang IA, Park JH, Lee HB. Role of reactive oxygen species in the pathogenesis of diabetic nephropathy. Diabetes Res Clin Pract. 2008;82 (Suppl 1):S42-5. Hwang I, Lee J, Huh JY, Park J, Lee HB, Ho YS, et al. Catalase deficiency accelerates diabetic renal injury through peroxisomal dysfunction. Diabetes. 2012;61:728-38. Koya D, King GL. Protein kinase C activation and the development of diabetic complications. Diabetes. 1998;47: 859-866. Mallamaci F, Ruggenenti P, Perna A, Leonardis D, Tripepi R, Tripepi G, et al. ACE inhibition is renoprotective among obese patients with proteinuria. J Am Soc Nephrol. 2011;22: 1122-8. Brenner BM, Cooper ME, de Zeeuw D, Keane WF, Mitch WE, Parving HH, et al. Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy. N Engl J Med. 2001;345:861-9. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulindependent diabetes mellitus. N Engl J Med. 1993;329:977-86. UK Prospective Diabetes Study (UKPDS) Group. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet. 1998;352: 837-853. Ohkubo Y, Kishikawa H, Araki E, Miyata T, Isami S, Motoyoshi S, et al. Intensive insulin therapy prevents the progression of diabetic microvascular complications in Japanese patients with non-insulin-dependent diabetes mellitus: a randomized prospective 6-year study. Diabetes Res Clin Pract. 1995;28:103-17. Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol. 2007;8:519-29. Kroemer G, Marino G, Levine B. Autophagy and the integrated stress response. Mol Cell. 2010;40:280-93. Mizushima N, Komatsu M. Autophagy. Renovation of cells and tissues. Cell. 2011;147:728-41. Singh R, Kaushik S, Wang Y, Xiang Y, Novak I, Komatsu M, et al. Autophagy regulates lipid metabolism. Nature. 2009; 458:1131-5. Yoshizaki T, Kusunoki C, Kondo M, Yasuda M, Kume S, Morino K, et al. Autophagy regulates inflammation in adipocytes. Biochem Biophys Res Commun. 2012;417:352-7. Huber TB, Edelstein CL, Hartleben B, Inoki K, Dong Z, Koya D, et al. Emerging role of autophagy in kidney function, diseases and aging. Autophagy. 2012;8:1009-31. Hartleben B, Godel M, Meyer-Schwesinger C, Liu S, Ulrich T, Kobler S, et al. Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice. J Clin Invest. 2010;120:1084-96. Jiang M, Liu K, Luo J, Dong Z. Autophagy is a renoprotective mechanism during in vitro hypoxia and in vivo ischemiareperfusion injury. Am J Pathol. 2010;176:1181-92. Kume S, Uzu T, Horiike K, Chin-Kanasaki M, Isshiki K, Araki S, et al. Calorie restriction enhances cell adaptation to hypoxia through Sirt1-dependent mitochondrial autophagy in mouse aged kidney. J Clin Invest. 2010;120:1043-55. Liu S, Hartleben B, Kretz O, Wiech T, Igarashi P, Mizushima N, et al. Autophagy plays a critical role in kidney tubule maintenance, aging and ischemia-reperfusion injury. Autophagy. 2012;8:826-37. Periyasamy-Thandavan S, Jiang M, Wei Q, Smith R, Yin XM, Dong Z. Autophagy is cytoprotective during cisplatin
15
25.
26. 27. 28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41. 42. 43. 44.
45.
injury of renal proximal tubular cells. Kidney Int. 2008; 74:631-40. Takahashi A, Kimura T, Takabatake Y, Namba T, Kaimori J, Kitamura H, et al. Autophagy guards against cisplatin-induced acute kidney injury. Am J Pathol. 2012;180:517-25. Kume S, Thomas MC, Koya D. Nutrient sensing, autophagy, and diabetic nephropathy. Diabetes. 2012;61:23-9. Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell. 2008;132:27-42. Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, Tanida I, et al. Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J Cell Biol. 2005;169:425-34. Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T, et al. The role of autophagy during the early neonatal starvation period. Nature. 2004;432:1032-6. Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature. 1999;402:672-6. Pattingre S, Espert L, Biard-Piechaczyk M, Codogno P. Regulation of macroautophagy by mTOR and Beclin 1 complexes. Biochimie. 2008;90:313-23. Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell. 2004;15:1101-11. Komatsu M, Waguri S, Koike M, Sou YS, Ueno T, Hara T, et al. Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell. 2007; 131:1149-63. Kawachi H, Miyauchi N, Suzuki K, Han GD, Orikasa M, Shimizu F. Role of podocyte slit diaphragm as a filtration barrier. Nephrology (Carlton). 2006;11:274-81. Pagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers BD, Rennke HG, et al. Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Invest. 1997; 99:342-8. Chen J, Chen MX, Fogo AB, Harris RC, Chen JK. mVps34 deletion in podocytes causes glomerulosclerosis by disrupting intracellular vesicle trafficking. J Am Soc Nephrol. 2013;24: 198-207. Cina DP, Onay T, Paltoo A, Li C, Maezawa Y, De Arteaga J, et al. Inhibition of MTOR disrupts autophagic flux in podocytes. J Am Soc Nephrol. 2012;23:412-20. Oshima Y, Kinouchi K, Ichihara A, Sakoda M, Kurauchi-Mito A, Bokuda K, et al. Prorenin receptor is essential for normal podocyte structure and function. J Am Soc Nephrol. 2011; 22:2203-12. Fang L, Zhou Y, Cao H, Wen P, Jiang L, He W, et al. Autophagy attenuates diabetic glomerular damage through protection of hyperglycemia-induced podocyte injury. PLoS One. 2013;8:e60546. Jacinto E, Loewith R, Schmidt A, Lin S, Ruegg MA, Hall A, et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol. 2004; 6:1122-8. Inoki K, Corradetti MN, Guan KL. Dysregulation of the TSCmTOR pathway in human disease. Nat Genet. 2005;37:19-24. Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012;149:274-93. Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell. 2006;124:471-84. Huber TB, Walz G, Kuehn EW. mTOR and rapamycin in the kidney: signaling and therapeutic implications beyond immunosuppression. Kidney Int. 2011;79:502-11. Lieberthal W, Levine JS. The role of the mammalian target of rapamycin (mTOR) in renal disease. J Am Soc Nephrol. 2009;20:2493-502.
16 46. Godel M, Hartleben B, Herbach N, Liu S, Zschiedrich S, Lu S, et al. Role of mTOR in podocyte function and diabetic nephropathy in humans and mice. J Clin Invest. 2011; 121:2197-209. 47. Inoki K, Mori H, Wang J, Suzuki T, Hong S, Yoshida S, et al. mTORC1 activation in podocytes is a critical step in the development of diabetic nephropathy in mice. J Clin Invest. 2011;121:2181-96. 48. Lloberas N, Cruzado JM, Franquesa M, Herrero-Fresneda I, Torras J, Alperovich G, et al. Mammalian target of rapamycin pathway blockade slows progression of diabetic kidney disease in rats. J Am Soc Nephrol. 2006;17:1395-404. 49. Mori H, Inoki K, Masutani K, Wakabayashi Y, Komai K, Nakagawa R, et al. The mTOR pathway is highly activated in diabetic nephropathy and rapamycin has a strong therapeutic potential. Biochem Biophys Res Commun. 2009; 384:471-5. 50. Sakaguchi M, Isono M, Isshiki K, Sugimoto T, Koya D, Kashiwagi A. Inhibition of mTOR signaling with rapamycin attenuates renal hypertrophy in the early diabetic mice. Biochem Biophys Res Commun. 2006;340:296-301. 51. Hosokawa N, Hara T, Kaizuka T, Kishi C, Takamura A, Miura Y, et al. Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol Biol Cell. 2009;20:1981-91. 52. Moscat J, Diaz-Meco MT. Feedback on fat: p62-mTORC1autophagy connections. Cell. 2011;147:724-7. 53. Kim J, Kundu M, Viollet B, Guan KL. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol. 2011;13:132-41. 54. Nath KA. Tubulointerstitial changes as a major determinant in the progression of renal damage. Am J Kidney Dis. 1992; 20:1-17. 55. Risdon RA, Sloper JC, De Wardener HE. Relationship between renal function and histological changes found in renal-biopsy specimens from patients with persistent glomerular nephritis. Lancet. 1968;2:363-6. 56. Kimura T, Takabatake Y, Takahashi A, Kaimori JY, Matsui I, Namba T, et al. Autophagy protects the proximal tubule from degeneration and acute ischemic injury. J Am Soc Nephrol. 2011;22:902-13. 57. Yang C, Kaushal V, Shah SV, Kaushal GP. Autophagy is associated with apoptosis in cisplatin injury to renal tubular epithelial cells. Am J Physiol Renal Physiol. 2008;294: F777-F787. 58. Yamahara K, Kume S, Koya D, Tanaka Y, Morita Y, ChinKanasaki M, et al. Obesity-mediated autophagy insufficiency exacerbates proteinuria-induced tubulointerstitial lesions. J Am Soc Nephrol. 2013;24:1769-81. 59. Colman RJ, Anderson RM, Johnson SC, Kastman EK, Kosmatka KJ, Beasley TM, et al. Caloric restriction delays disease onset and mortality in rhesus monkeys. Science. 2009; 325:201-4. 60. Fontana L, Partridge L, Longo VD. Extending healthy life span–from yeast to humans. Science. 2010;328:321-6. 61. Cherry Engelman RW, Wang BY, Kinjoh K, El-Badri NS, Good RA. Calorie restriction delays the crescentic glomerulonephritis of SCG/Kj mice. Proc Soc Exp Biol Med. 1998; 218:218-22. 62. Kitada M, Takeda A, Nagai T, Ito H, Kanasaki K, Koya D. Dietary restriction ameliorates diabetic nephropathy through anti-inflammatory effects and regulation of the autophagy via restoration of Sirt1 in diabetic Wistar fatty (fa/fa) rats: a model of type 2 diabetes. Exp Diabetes Res. 2011;2011:908185.
S. Kume et al. 63. Kume S, Uzu T, Araki S, Sugimoto T, Isshiki K, ChinKanasaki M, et al. Role of altered renal lipid metabolism in the development of renal injury induced by a high-fat diet. J Am Soc Nephrol. 2007;18:2715-23. 64. Steinberg GR, Kemp BE. AMPK in health and disease. Physiol Rev. 2009;89:1025-78. 65. Shackelford DB, Shaw RJ. The LKB1-AMPK pathway: metabolism and growth control in tumour suppression. Nat Rev Cancer. 2009;9:563-75. 66. Alers S, Loffler AS, Wesselborg S, Stork B. Role of AMPKmTOR-Ulk1/2 in the regulation of autophagy: cross talk, shortcuts, and feedbacks. Mol Cell Biol. 2012;32:2-11. 67. Lee JW, Park S, Takahashi Y, Wang HG. The association of AMPK with ULK1 regulates autophagy. PLoS One. 2010;5: e15394. 68. Chang CC, Chang CY, Wu YT, Huang JP, Yen TH, Hung LM. Resveratrol retards progression of diabetic nephropathy through modulations of oxidative stress, proinflammatory cytokines, and AMP-activated protein kinase. J Biomed Sci. 2011;18:47. 69. Takiyama Y, Harumi T, Watanabe J, Fujita Y, Honjo J, Shimizu N, et al. Tubular injury in a rat model of type 2 diabetes is prevented by metformin: a possible role of HIF1alpha expression and oxygen metabolism. Diabetes. 2011; 60:981-92. 70. Ding DF, You N, Wu XM, Xu JR, Hu AP, Ye XL, et al. Resveratrol attenuates renal hypertrophy in early-stage diabetes by activating AMPK. Am J Nephrol. 2010;31:363-74. 71. Lee MJ, Feliers D, Mariappan MM, Sataranatarajan K, Mahimainathan L, Musi N, et al. A role for AMP-activated protein kinase in diabetes-induced renal hypertrophy. Am J Physiol Renal Physiol. 2007;292:F617-27. 72. Sharma K, Ramachandrarao S, Qiu G, Usui HK, Zhu Y, Dunn SR, et al. Adiponectin regulates albuminuria and podocyte function in mice. J Clin Invest. 2008;118:1645-56. 73. Nakai A, Yamaguchi O, Takeda T, Higuchi Y, Hikoso S, Taniike M, et al. The role of autophagy in cardiomyocytes in the basal state and in response to hemodynamic stress. Nat Med. 2007;13:619-24. 74. Weyer C, Bogardus C, Mott DM, Pratley RE. The natural history of insulin secretory dysfunction and insulin resistance in the pathogenesis of type 2 diabetes mellitus. J Clin Invest. 1999;104:787-94. 75. Kasuga M. Insulin resistance and pancreatic beta cell failure. J Clin Invest. 2006;116:1756-60. 76. Ebato C, Uchida T, Arakawa M, Komatsu M, Ueno T, Komiya K, et al. Autophagy is important in islet homeostasis and compensatory increase of beta cell mass in response to high-fat diet. Cell Metab. 2008;8:325-32. 77. Jung HS, Chung KW, Won Kim J, Kim J, Komatsu M, Tanaka K, et al. Loss of autophagy diminishes pancreatic beta cell mass and function with resultant hyperglycemia. Cell Metab. 2008;8:318-24. 78. Kim KH, Jeong YT, Oh H, Kim SH, Cho JM, Kim YN, et al. Autophagy deficiency leads to protection from obesity and insulin resistance by inducing Fgf21 as a mitokine. Nat Med. 2013;19:83-92. 79. Singh R, Xiang Y, Wang Y, Baikati K, Cuervo AM, Luu YK, et al. Autophagy regulates adipose mass and differentiation in mice. J Clin Invest. 2009;119:3329-39. 80. Lapierre LR, Silvestrini MJ, Nunez L, Ames K, Wong S, Le TT, et al. Autophagy genes are required for normal lipid levels in C. elegans. Autophagy. 2013;9:278-86.
