Autophagy in kidney disease and aging - CyberLeninka

review

www.kidney-international.org

Autophagy in kidney disease and aging: lessons from rodent models 1,2

Olivia Lenoir , Pierre-Louis Tharaux

1,2,3,4*

and Tobias B. Huber

OPEN

4,5,6,7*

1

Paris Cardiovascular Research Centre—PARCC, Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France; Université Paris Descartes, Sorbonne Paris Cité, Paris, France; 3Nephrology Division, Georges Pompidou European Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France; 4FRIAS, Freiburg Institute for Advanced Studies and Center for Biological System Analysis— ZBSA, Freiburg, Germany; 5Department of Medicine IV, Faculty of Medicine, University of Freiburg, Germany; 6BIOSS Center for Biological Signalling Studies, Albert-Ludwigs-University Freiburg, Freiburg, Germany; and 7Center for Systems Biology (ZBSA), Albert-LudwigsUniversity, Freiburg, Germany 2

Autophagy is a highly regulated lysosomal protein degradation pathway that removes protein aggregates and damaged or excess organelles to maintain intracellular homeostasis and cell integrity. Dysregulation of autophagy is involved in the pathogenesis of a variety of metabolic and age-related diseases. Growing evidence suggests that autophagy is implicated in cell injury during renal diseases, both in the tubulointerstitial compartment and in glomeruli. Nevertheless, the impact of autophagy on renal disease progression and aging is still not fully understood. This review summarizes the recent advances in understanding the role of autophagy for kidney disease and aging. Kidney International (2016) j.kint.2016.04.014

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http://dx.doi.org/10.1016/

KEYWORDS: acute kidney injury; aging; autophagy; endothelium; glomerulus; kidney; kidney transplantation; mTOR; podocyte; polycystic kidney disease Copyright ª 2016, International Society of Nephrology. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

A

utophagy (“self-eating” in Greek) is a highly regulated lysosomal protein degradation pathway that removes protein aggregates and damaged organelles to maintain intracellular homeostasis and cell integrity.1–3 This process was first described in 1957 by Sam Clark Jr.,4 but the term autophagy was coined by Christian de Duve only in the late 1950s.5 The autophagy process is well conserved in the evolution from yeast to mammals.6,7 Characterization of the molecular regulators of autophagy was first described in the 1990s with the identification of autophagy-related genes (ATGs) in autophagy-defective yeast cells,8,9 Then mammal orthologs and the molecular machinery were identified.10 Three types of autophagy have been described to date as follows: macroautophagy, the most studied form and the focus of this review; microautophagy; and chaperonemediated autophagy. All differ in their mechanisms and functions11: microautophagy involves the engulfment of small cytoplasmic cargos within lysosomal membrane invaginations12 and chaperone-mediated autophagy involves the heat shock cognate protein 70–mediated recruitment of KFERQ motif–bearing proteins to the lysosome,13,14 whereas macroautophagy is the most prevalent and probably less selective type and is referred to in this review as autophagy. In this review, we describe the pathway of autophagy and highlight its role in renal physiology, renal aging, and kidney diseases. We will also discuss the potential implication of manipulating autophagy as a potential novel renoprotective therapeutic strategy. AUTOPHAGY Functions of autophagy

Correspondence: Pierre-Louis Tharaux, INSERM Paris Cardiovascular Research Centre, 56, rue Leblanc, 75015 Paris, France. E-mail: pierre-louis. [email protected] or Tobias B. Huber, University Hospital Freiburg, Nephrology, Breisacherstrasse 66, Freiburg, BW 79106, Germany. E-mail: [email protected] *These authors contributed equally to this work. Received 10 October 2015; revised 17 April 2016; accepted 20 April 2016 Kidney International (2016) -, -–-

Autophagy has been demonstrated to be essential for a number of fundamental biological activities,15 such as the maintenance of cellular homeostasis and cellular stress response, particularly in postmitotic cells. Autophagy participates in recycling of organelles such as mitochondria (mitophagy),16,17 endoplasmic reticulum (ER), and peroxisomes7,18–22; the clearance of polyubiquitinated proteins aggregates23; and lipid degradation (lipophagy).24 Dysregulation of autophagy is involved in the pathogenesis of a variety of metabolic and age-related diseases.25–31 The major role of 1

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autophagy is to provide metabolic precursors for survival in stress conditions and to serve as a quality control by clearing misfolded proteins and others cellular debris. Interestingly, genetic evidence (i.e., polymorphisms in ATGs) indicates that autophagy is implicated in several immune diseases associated with kidney dysfunction, such as systemic lupus erythematosus (autophagy related 5 gene [ATG5])32 and rheumatoid arthritis (the PR domain 1 gene [PRDM1]-ATG5 intergenic region).33 Furthermore, activation of autophagy at the wholebody level extends the life span of various model organisms, including mice.28 There is a growing amount of evidence that dysregulation of the autophagic pathway is implicated in the pathogenesis of kidney aging and in several renal diseases such as acute kidney injury (AKI), polycystic kidney disease (PKD), diabetic nephropathy (DN), obstructive nephropathy, focal and segmental glomerulosclerosis (FSGS), and potentially other kidney diseases.34–37 Molecular mechanisms of autophagy

Autophagy proceeds through at least five steps. Autophagosomes are initiated by expansion and sealing of a small vesicle made of a double-membrane structure called phagophore or isolation membrane. The origin of the autophagosome

O Lenoir et al.: Autophagy in kidney disease and aging

membrane may involve different sources, such as lipid droplets, mitochondria, Golgi, ER, plasma membrane, and recycling endosomes.38,39 The phagophore is generated at a specialized site known as the phagophore assembly site or preautophagosomal structure. Sixteen ATG proteins comprise the conserved core ATG machinery that catalyzes formation of the phagophore and its expansion into autophagosomes in all eukaryotes40 (Figure 1). The metabolites generated in the lysosomes/vacuoles are subsequently transported in the cytoplasm and recycled. Macroautophagy has recently been shown to achieve some selectivity for lipid droplets, ribosomes, pathogens, surplus reticulum, peroxisomes, enzymes, and proteins aggregates in processes that are called lipophagy, ribophagy, xenophagy, reticulophagy, pexophagy, zymophagy, and aggrephagy, respectively (reviewed and discussed in Birgisdottir et al.,41 Johansen and Lamark,42 Johansen and Lamark,43 Mochida et al.,44 Khaminets et al.,45 and Khaminets et al.46). Selectivity of autophagy is controlled by autophagy receptors that physically associate with the autophagy compartment by interacting simultaneously with cargo and Atg8- or microtubule-associated protein 1A/1B–light chain 3 (LC3)/g-aminobutyric acid receptor–associated protein–like

Figure 1 | Autophagy pathway. The autophagic pathway responds to signals from the environment. Nutrients status controls autophagy through the mTOR signaling pathway. The class I PI3K-AKT can also activate the mTORC1 complex in response to insulin and other growth factors, acting as a negative regulator of autophagy. Activation of AMPK in response to low energy status (i) inhibits the mTORC1 complex and (ii) activates the ULK1 complex through ULK1 and ATG13 phosphorylation, thereby acting as a positive regulator of autophagy in response to energy depletion. The BECN1/class III PI3K complex, which is inactivated by Bcl2, and the class I PI3K-AKT complex also regulate autophagy. Upon activation, the Beclin1-VPS34-ATG14L-P150 complex will generate PI3P, which promotes autophagosomal membrane nucleation in coordination with the ULK1-ATG13-ATG101-FIP200 complex. The ATG5-ATG12-ATG16L complex and the LC3-phosphatidylethanolamine system will play roles in cargo recruitment, membrane elongation, and autophagosome maturation. Lysosome fusion to autophagosome to form autolysosome is mediated through SNARE proteins, and lysosomal hydrolases degrade proteins, lipids, and nucleic acids. AKT, protein kinase B; AMPK, adenosine monophosphate–activated protein kinase; ATG, autophagy-related gene; BCL2, B-cell lymphoma 2; BCLXL, B-cell lymphoma–extra large; DEPTOR, DEP domain-containing mTOR-interacting protein; FIP200, FAK family kinase-interacting protein of 200 kDa; LC3, microtubule-associated protein 1 light chain 3; MLST8, target of rapamycin complex subunit LST8; mTOR, mammalian target of rapamycin; mTORC1, mTOR complex 1; PI3K, phosphatidylinositol-3-kinase; PI3P, phosphatidylinositol-3-phosphate; PRAS40, proline-rich v-akt murine thymoma viral oncogene homolog 1 substrate 40 kDa; Raptor, regulatory-associated protein of mTOR; SNARE, soluble NSF attachment protein receptor; ULK1, Unc51-like kinase 1. 2

Kidney International (2016) -, -–-


proteins on autophagosomes.47 The best-characterized cargo receptors are probably sequestosome 1 (SQSTM1)/p62 and neighbor of breast cancer 1 gene (BRCA1) 1, which can sequester aggregated proteins and form the SQSTM1/p62 bodies. One of the prevailing mechanisms to regulate and often stimulate selective autophagy pathways includes phosphorylation of both cargo signals and autophagy receptors. For example, the phosphate and tensin homolog–induced putative kinase protein 1 and ubiquitin (UB) ligase Parkin act cooperatively in sensing mitochondrial functional state and directing damaged mitochondria for disposal via the autophagy pathway (mitophagy). Putative kinase protein 1 phosphorylates UB to activate the UB ligase Parkin, which builds UB chains on mitochondrial outer membrane proteins, where they act to recruit autophagy receptors such as NDP52 and optineurin.48,49 The fast-evolving field of UB-dependent and independent signals in selective autophagy has been reviewed recently.46 Monitoring autophagy

The accumulation of autophagosomes can be used to monitor autophagy. It is important to keep in mind that accumulation of autophagosomes can be caused by an increased autophagic flux or can be due to the inhibition of autophagosomelysosome fusion or inhibition of lysosomal enzyme function.50,51 The formation and maturation of autophagosomes depends on several genes, including microtubule-associated protein 1 light chain 3 (MAP1LC3A/B), beclin 1 gene (BECN1), and ATGs.1,26,52,53 Microtubule-associated protein 1 light chain 3A/B is a soluble protein that belongs to the UBrelated system. The cytoplasmic LC3 is conjugated to phosphatidylethanolamine to produce LC3–PE (or LC3-II), which is subsequently incorporated into to the double-membrane structure of the autophagosome. Therefore, detecting the intracellular level of LC3-II and conversion of cytoplasmic LC3 into LC3-II by immunofluorescence and Western blot analysis can be used as marker for autophagy. However, accumulation of LC3-positive vacuoles or LC3II protein could be due to impaired autophagosome maturation or an acceleration of autophagic flux, which can be further differentiated by inhibition of autolysosomal degradation with bafilomycin A1 or chloroquine. Additionally, the lysosomal turnover of p62, which interacts with LC3-II, can also be used to monitor the autophagic flux.54 The use of an monomeric red fluorescent protein–green fluorescent protein tandem fluorescent-tagged LC3 has also been proposed for visualizing the autophagic flux.55 We refer readers to landmark articles and methodological guidelines.50,51,56,57 Regulation of autophagy

Autophagy is regulated by the major nutrient-sensing pathways, including the mammalian target of rapamycin complex 1 (mTORC1), adenosine monophosphate–activated protein kinase (AMPK), and oxidized nicotinamide adenine dinucleotide–dependent histone deacetylase (sirtuin 1 [SIRT1]).1,58–62 The most powerful autophagy inducer is Kidney International (2016) -, -–-

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starvation by induction of low levels of amino acids, glucose, adenosine triphosphate, and growth factors. The activated unc-51 like autophagy activating kinase 1 (ULK1) complex (ULK1-ATG13-ATG101–FAK family kinase-interacting protein of 200 kDa) is recruited to the ER,52 where it activates the class III phosphoinositide 3–kinase complex (beclin 1 [BECN1]-ATG14L-Vps34-p150) by phosphorylating BECN1.63 The complex generates phosphatidylinositol 3– phosphate, which recruits effectors to initiate autophagosome formation.64,65 The lipid kinase activity of this phosphoinositide 3–kinase complex I is enhanced upon dimerization provoked by nuclear receptor binding factor 2 binding to Atg14L.66 Membrane elongation, autophagosome maturation, and cargo recruitment depend on two conjugation complexes: the ATG5-ATG12-ATG16L1 complex and the LC3phosphatidylethanolamine complex.40 LC3 and SQSTM1 have primordial roles in cargo selection. LC3 is also essential for autophagosomal membrane closure.67 Nutrition-related regulation of autophagy. Most of the studies on autophagy regulation focus on the role of mTORC1 complex. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase whose activity is associated with nutrient levels and redox status. The function of TORC1 as a repressor of autophagy is conserved in mammals and mTORC1 inhibits autophagy in many mammalian cell types.68 It has been shown that mTORC1 interacts with the ULK1 complex and inhibits autophagy induction through phosphorylation of ULK1 and ATG13. Upon starvation, inhibitory phosphorylation of the serine/threonine-protein kinase ULK1 at Ser757 is removed, thereby allowing autophagy induction.58,62,69 The functions of mTORC1 in glomerular physiology have been well documented.70–72 In situations of low energy, cellular adenosine monophosphate levels rise, leading to the activation of AMPK.73 AMPK is ubiquitously expressed and plays a major role in cellular energy homeostasis by sensing levels of intracellular calcium and adenosine triphosphate/adenosine monophosphate ratio. AMPK is strongly expressed in several renal cells including podocytes, mesangial cells, glomerular endothelial cells and tubular cells. AMPK is activated through phosphorylation on Ser72 by the calcium calmodulindependent protein kinase kinase and the tumor suppressor protein serine/threonine kinase 11.74,75 AMPK is an autophagy inducer by phosphorylating ULK1 on Ser317 and Ser777.62 Once activated, AMPK phosphorylates tuberous sclerosis complex 2,76 which forms a complex with tuberous sclerosis complex 1 protein, which acts as a guanosine triphosphatase–activating protein toward Ras homolog enriched in brain.77–79 Additionally, tuberous sclerosis complex 2 phosphorylation by AMPK is a primer for further phosphorylation by glycogen synthase kinase 3 beta, which leads to Ras homolog enriched in brain–dependent inhibition of mTORC1.80 AMPK was also shown to directly phosphorylate raptor.81 However, the relative contribution of raptor phosphorylation to overall mTORC1 regulation by AMPK and the context in which raptor is implicated in mTORC1 3

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regulation are not well understood. The link between autophagy and AMPK regulation in kidney cells is only incompletely understood.34 The oxidized nicotinamide adenine dinucleotide–dependent histone deacetylase (SIRT1) also regulates autophagy through different targets and effectors.82,83 Sirtuins are a family of histone and nonhistone deacetylases that are regulated by nicotinamide adenine dinucleotide as a cofactor linking their activity to the metabolic status of the cell.84 SIRT1 has been demonstrated to be an inducer of autophagy by directly and indirectly acting on components of the autophagy machinery. SIRT1 coimmunoprecipitates with ATG5, ATG7, and ATG8/LC3 and can deacetylate these proteins in a nicotinamide adenine dinucleotide–dependent manner in vitro.61 Through deacetylation of transcription factors that in turn activate autophagy genes, SIRT1 can also indirectly modulate autophagy. This is the case for members of the forkhead box O family.85,86 Interestingly, SIRT1mediated forkhead box O1 activation was shown to increase expression of Rab7, a small guanosine triphosphatase that mediates late autophagosome-lysosome fusion. Furthermore, forkhead box O3 deacetylation by SIRT1 can upregulate multiple autophagy-related genes such as unc-51 like autophagy activating kinase 2 gene (ULK2), BECN1, phosphatidylinositol 3–kinase catalytic subunit type 3 gene (VPS34), B-cell lymphoma 2 (BCL2)/adenovirus E1B 19kDa interacting protein 3 gene (Bnip3) and BCL2/adenovirus E1B 19kDa interacting protein 3 gene-like (Bnip3L), autophagy related 12 gene (Atg12), autophagy related 4B gene (Atg4B), LC3, and gaminobutyric acid receptor gene (GABAR)–amino acid permase like gene (APL1).87 Recent evidence demonstrated SIRT1 involvement in renal pathophysiology88–93 and the aging kidney (see later). Stress-induced regulation of autophagy (oxidative stress, ER stress, hypoxia). In addition to nutrient starvation, intracel-

lular stress effectors can also modulate autophagy levels. Most of the following evidence has been collected from nonrenal cellular models. For example, reactive oxygen species can influence LC3 production through activation of c-Jun Nterminal protein kinase 194 and activation of protein kinase RNA-like endoplasmic reticulum kinase.95 In the absence of clearly deciphered interaction between ER stress and autophagy in the kidney, we will refer to a recent review of the literature demonstrating that ER stress serves dual roles by favoring both induction and inhibition of autophagy.96 Modulation by hypoxia: interplay with oxidative stress and proapoptotic and metabolic pathways. Evidence has accu-

mulated that hypoxia plays a significant role in the pathogenesis and progression of chronic renal disease.97 Depending on the severity and duration of the variations in oxygen tension, hypoxia has been demonstrated to regulate autophagy pathways differently. For instance, chronic and moderate hypoxia induces autophagy through hypoxiainducible factor-1 alpha and PKCd–c-Jun N-terminal protein kinase 1–implicated pathways.98 Conversely, rapid and 4


severe oxygen fluctuation could induce autophagy via hypoxia-inducible factor–independent pathways. Hypoxia can also activate autophagy through induction of BCL2/adenovirus E15 19kDa interacting protein 3 (BNIP3),99 which can disrupt the inhibitory interaction between BECN1 and BCL2.100,98,101–103 Under conditions of hypoxia, BNIP3 transcription triggers mitochondrial dysfunction, formation of autophagosomes, and promotion of the apoptosis process in neonatal cardiac myocytes.104 By causing mitochondrial dysfunction, BNIP3 or BCL2/ adenovirus E1B 19kDa interacting protein 3-like may increase production of reactive oxygen species, which can activate autophagy.105 Competition by BNIP3 or BCL2/adenovirus E1B 19kDa interacting protein 3-like for binding to BCL2 (or a related protein) could also liberate BECN1 from BCL2 complexes and activate autophagy.106 In fact, this interplay between proapoptotic pathways and proautophagy pathways may lead to misinterpretation of the role of autophagy. For example, overexpression of Bcl2 homolgy domain 3–only proteins at some stages of kidney diseases may increase autophagy by freeing BECN1 from BCL2, but the net result may still be proapoptotic.107,108 Finally, BNIP3 binds to and inhibits Ras homolog enriched in brain,109 which is an upstream activator of mTOR. Therefore, BNIP3 may activate autophagy by repressing mTOR. Oxygen is also an essential regulator of cellular metabolism affecting protein translation, autophagy, and cell growth through mTOR regulation. In response to hypoxia, regulation of mTORC1 occurs through a conserved pathway that is independent of energy signaling pathways.110,111 Under conditions of oxygen deprivation, mTORC1 dissociates from the ULK1 to phosphorylate ATG13/FAK family kinase-interacting protein of 200 kDa complex and, thus, initiates autophagy in cells.79 Regulation of mTORC1 and autophagy by hypoxia, however, seems to be highly cell type–dependent and context dependent. Another level of chronic hypoxia-dependent regulation is activation of tuberous sclerosis complex 2 protein by the reduction of adenosine triphosphate depletion and upregulation of AMPK phosphorylation.112 Overall, both laboratory evidence and clinical evidence highlight hypoxia as an important inducer of autophagy. Future studies will have to elucidate whether the steep oxygen gradient in the kidney is a physiological modulator of autophagy. AUTOPHAGY IN AGING AND DISEASED KIDNEYS

To date, the roles of autophagy in the diseased kidney have been studied mainly in proximal tubular cells and podocytes. The availability of mouse models genetically targeting the autophagy machinery has facilitated our understanding of the role of autophagy in kidney physiology and pathology (Tables 1, 2, and 3). Autophagy in glomerular aging and diseases

Podocytes have been identified as the most fragile cell types of the glomerulus, and a decrease in the number of podocytes Kidney International (2016) -, -–-

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Table 1 | Effects on kidneys of global genetic targeting of autophagy genes

Name

Renal disease Tissue model

Atg3/, Atg5/, Atg7/, Atg9/, Atg16L1/ Beclin1/

Global

N/A

Global

N/A

FIP200/

Global

N/A

Ambra1gt/gt

Global

N/A

Ulk1/

Global None

Atg4C/ Map1lc3/ Gabarap/

Global None Global None Global None

Sirt1þ/-

Global

Map1lc3-GFP

Global None

UUO

IRI CAG-RFP-EGFP-Map1lc3 Global

IRI

Phenotype/renal phenotype

Comments

References

Neonatal lethal

No kidney abnormalities in Atg5-/- pups

113–117

Early embryonic lethal (E7.5) with defects in proamniotic canal closure Embryonic lethal (E13.5–E16.5) because of defective heart and liver development Embryonic lethal (wE14) with defects in neural tube development Viable. Increased reticulocyte number with Kidney not studied delayed mitochondrial clearance Viable. Increased tumorigenesis Kidney not studied None Normal renal histology in young adults None Gabarap(-/-) mice have reduced urinary excretion of P(i), higher Na(þ)-dependent (32)P(i) uptake in BBM vesicles, and increased expression of NaPi-IIa in renal BBM compared with Gabarap(þ/þ) mice. The expression of Na(þ)/H(þ) exchanger regulatory factor 1, an important scaffold for the apical expression of NaPi-IIa, is also increased in Gabarap(-/-) mice Increased oxidative stress, apoptosis and Lower autophagy activity and decreased BNIP3 fibrosis after UUO expression None Demonstrate high level of autophagy in podocytes at basal state None Demonstrate that autophagic activity in the tubule system is low at basal state and increased after IRI None Demonstrate that autophagic activity in the proximal tubule system is increased after IRI

118, 119

120

121

122

123 124 125, 126

88

56, 141

192

127

Atg3, autophagy related 3 gene; Atg4C, autophagy related 4C gene; Atg5, autophagy related 5 gene; Atg7, autophagy related 7 gene; Atg9, autophagy related 9 gene; Atg16L1, autophagy related 16-like 1 gene; BBM, brush border membrane; EGFP, enhanced green fluorescent protein; FIP200, FAK family kinase-interacting protein of 200 kDa gene; GABARAP, g-aminobutyric acid receptor–associated pro; GFP, green fluorescent protein; IRI, ischemia-reperfusion injury; LC3, microtubule-associated protein 1A/1B–light chain 3 gene; LC3B, microtubule-associated protein 1A/1B–light chain 3B gene; N/A, not applicable; RFP, red fluorescent protein; SIRT1, sirtuin 1 gene; ULK1, unc-51 like autophagy activating kinase 1; UUO, unilateral ureteral obstruction.

and/or in their function is directly correlated with disease progression in several renal diseases, such as DN, IgA nephropathy, HIV-associated nephropathies, or FSGS.132–134 Glomerular endothelial cells are also altered in several renal diseases (i.e., primary hypertension, DN, and other chronic kidney diseases). Mechanisms of podocyte and glomerular endothelial cell injuries are incompletely understood, but ER stress, mitochondrial damage, and oxidative stress are implicated in most types of glomerulosclerosis.135–138 Autophagy and glomerular maintenance during aging. Aging induces the accumulation of damaged organelles and mitochondria as well as protein aggregates that are physiological causes of organ dysfunction. The kidney is particularly susceptible to age-related renal damage such as tubular atrophy, interstitial fibrosis, and glomerulosclerosis. Elderly individuals are particularly sensitive to ischemia and toxic stress and show high rates of end-stage renal disease and chronic kidney disease.139,140 In glomeruli, podocytes are terminally differentiated postmitotic cells. Therefore, their capacities for regeneration are limited and they require efficient cellular mechanisms to “clean” themselves from protein aggregates and altered organelles that will accumulate throughout a lifetime. Recent evidence suggests that autophagy might be this cleaning mechanism allowing podocyte maintenance during aging. Kidney International (2016) -, -–-

Unlike other renal and nonrenal cell types, podocytes have a high level of basal autophagy, suggesting that autophagy has important functions in this cell type.56,141 Furthermore, podocyte-specific deletion of Atg5 results in proteinuria, loss of podocytes, and aging-related glomerulosclerosis, thus demonstrating the primordial function of autophagy for podocyte maintenance.141 Importantly, Atg5 knockout podocytes showed the following typical age-related alteration: lipofuscin accumulation, an increase in oxidized proteins, and UB-positive and SQSTM1-positive protein aggregates.141 Concomitant with such results, podocyte-specific Vps34 deletion leads to spontaneous development of glomerulosclerosis, although defective autophagy was not primarily responsible for the severe phenotype observed in Vps34-deficient podocytes.142–144 Finally, pharmacological inhibition of autophagy induces podocyte apoptosis by activating the apoptotic pathway of ER stress.145 Autophagy in FSGS. FSGS is a glomerular disease associated with podocytopathy that is associated with a poor prognosis and evolves easily into end-stage renal disease.146 Several pieces of evidence suggest that autophagy is implicated in podocytopathy during FSGS.141,147–150 In an initial study it was demonstrated that podocyte-specific deficiency of Atg5 (Nphs2.Cre Atg5lox/lox mice) sensitizes mice for the 5

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Table 2 | Effects of kidney cell–specific deletion of autophagy genes Name fl/fl

Nphs2-cre Atg5

Nphs2-cre Vps34fl/fl

Tissue

Cre-driven expression

Renal disease model

Podocyte

Nphs2

None


Age-dependent late-onset glomerulosclerosis. Podocytes loss associated with ubiquinated protein aggregates. LPS-, PAN-, doxorubicin- Increased sensitivity to the development (Adriamycin-), and of proteinuria bortezomib-induced proteinuria STZ-induced DN Increased sensitivity to the development of podocyte injury and apoptosis HFD-induced proteinuria HFD-fed podo-Atg5/ mice develop albuminuria with severe podocyte injury None Massive proteinuria and early lethality. Podocyte degeneration and early-onset glomerulosclerosis

Podocyte

Nphs2

Cdh5-cre Atg5fl/fl

Endothelial

Cdh5

STZ-induced DN

Six2-cre Atg5fl/fl

Podocytes, PEC, tubules

Six2

None

Six2-cre Atg7fl/fl

Podocytes, PEC, tubules

Six2

None

KAP-cre Atg5fl/fl

Proximal tubule

KAP

Cisplatin and IRI FFA-albumin overload

PEPCK-cre Atg7fl/fl Pax8.rtTA;tetO.Cre Atg5fl/fl

Proximal tubule Proximal, distal tubules l and collecting duct

PEPCK Pax8

Cisplatin and IRI None

IRI Ksp-cre Atg5fl/fl

Distal tubule

Ksp

Comments

None

Increased sensitivity to the development of DN Tubulointerstitial damage and FSGS by 4 mo. Lethality by 5 mo FSGS by 4 mo with moderate albuminuria. Less tubular injury than for the Six2-cre Atg5fl/fl mice. Survive until 1 yr Increased sensitivity to AKI Increased sensitivity to the development of tubular lesions Increased sensitivity to AKI Few aberrant ultrastructure in tubular cells 5 mo after autophagy blockade Severe tubular injury. P62 and oxidative injury markers accumulation Normal renal function. P62 accumulation and increased oxidative stress markers

References 141

141

167

178

Abrogated autophagic flux is not causative of the podocyte phenotype

142, 144

167

150

150

202, 230 162

201 192

192

192

AKI, acute kidney injury; Atg5, autophagy related 5 gene; Cdh5, cadherin 5 gene; DN, diabetic nephropathy; FFA, free fatty acid; FSGS, focal and segmental glomerulosclerosis; HFD, high-fat diet; IRI, ischemia-reperfusion injury; KAP, kidney androgen regulated protein gene; Ksp, cadherin 16, KSP-cadherin gene; LPS, lipopolysaccharide; Nphs2, nephrosis 2, idiopathic, steroid-resistant (Podocin) gene; PAN, puromycin aminonucleoside nephrosis; Pax8, paired box 8 gene; PEPCK, phosphoenolpyruvate carboxykinase 2, mitochondrial gene; Six2, SIX2 homeobox 2 gene; STZ, streptozotocin; Vps34, phosphatidylinositol 3– kinase gene.

development of several renal diseases, including FSGS as a general theme of podocyte biology and glomerular disease.141 Podocyte injury was characterized by ER stress, accumulation of oxidized proteins, and altered mitochondria.141 These results could also be confirmed using Six2.Cre Atg5lox/lox mice or Six2.Cre Atg7lox/lox mice.150 The more dramatic phenotype caused by deletion of autophagy throughout the nephron than that reported in models of autophagy deletion in tubular or podocyte compartments solely unravels a strong effect of autophagy on nephron development or integration of physiological cross talk between the distinct parts of the nephron. Autophagy was also found to have protective properties in puromycin-aminonucleoside–treated human podocytes.151 A decreased number of autophagic vacuoles was described in podocytes from patients with FSGS.149 Finally, pharmacological induction of autophagy delays the progression of 6

FSGS. Indeed, Wu et al. found that rapamycin could reduce podocyte injury by inducing autophagy.152 Concomitantly, inhibition of autophagy by 3-methyladenine treatment sensitizes rats to the development of puromycin-aminonucleoside–induced FSGS, whereas rapamycin partially protects podocytes from puromycin-aminonucleoside–induced nephropathy.149 Autophagy in other nondiabetic glomerular diseases. A limited amount of studies brought direct evidence for autophagy in other models of glomerulopathies, principally antibody-mediated glomerular diseases. A major role for B-cell autophagy was shown in a mouse model of lupus nephritis, the spontaneous mutant mouse Y-linked autoimmune accelerator with duplication of the Toll-like receptor 7 (Tlr7) gene. Autophagy in CD19þ cells mediates Toll-like receptor 7–dependent autoimmunity and Kidney International (2016) -, -–-

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Table 3 | Effects of kidney cell–specific deletion of potential modulators of autophagy Name

Tissue fl/fl

Cre-driven expression Renal disease model

Nphs2-cre Tsc1

Podocytes

Nphs2

Nphs2-cre Raptorfl/fl

Podocytes

Nphs2

Nphs2.rtTA;tetO Raptorfl/fl

Podocytes

Nphs2

Nphs2-cre mTorfl/fl

Podocytes

Nphs2

Ksp-cre Raptorfl/fl

Distal tubule

Ksp

Pax8.rtTA;tetO.Cre Raptorfl/fl

Pax8

Nphs2-cre Sirt1fl/fl

Proximal, distal, and collecting duct tubules Podocytes

Nphs2

Nphs2-cre Sirt1fl/fl

Podocytes

Nphs2

Proximal tubule

Npt2

Sirt1Tg (Npt2)

g-GT-cre Sirt1fl/fl

Proximal tubule

g-GT

None


Comments

DN-like phenotype: glomerular No link with autophagy membrane thickening, established mesangial expansion, podocyte loss, and proteinuria None Increased proteinuria by 4 wk of No link with autophagy age. established Podocyte degeneration and progressive glomerulosclerosis by 12 mo None Glomerular synechiae No link with autophagy established None Massive proteinuria by 4 wk mTOR inhibition of age. disrupts the Podocyte degeneration and autophagic pathway in early-onset glomerulosclerosis podocytes in vitro None Defective concentrating No link with autophagy mechanisms established and polyuria IRI More severe cell sloughing, cell No link with autophagy flattening, established and distal tubular cast formation db/db DN Increased urinary proteins No link with autophagy established NTS-induced RPGN, Increased sensitivity to podocyte No link with autophagy PS-induced podocyte injury established injury cisplatin-induced AKI Less oxidative stress and No link with autophagy decreased established cisplatin-induced renal injuries db/db DN and 5/6 Proximal tubule-specific Sirt1 No link with autophagy Nphx overexpression alleviates established diabetic albuminuria in db/db mice but not in 5/6th nephrectomized STZ-induced DN Increased sensitivity to DN No link with autophagy established

References 72

71

71

128

129

130

131

89

89

90

AKI, acute kidney injury; DN, diabetic nephropathy; IRI, ischemia-reperfusion injury; Ksp, cadherin 16, KSP-cadherin gene; mTOR, mammalian target of rapamycin; Nphs2, nephrosis 2, idiopathic, steroid-resistant (Podocin) gene; Nphx, nephrectomy; NTS, nephrotoxic serum; Pax8, paired box 8 gene; PS, protamine sulfate; RPGN, rapid and progressive glomerulonephritis; Sirt1, sirtuin 1; STZ, streptozotocin; Tsc1, tuberous sclerosis 1 gene.

inflammation because lupus nephritis development in Tlr7 transgenic (Tg) mice with B-cell–specific ablation of autophagy (Cd19-Cre Atg5lox/lox) was markedly alleviated compared with mice without B-cell–specific autophagy deficiency.153 By contrast, the role of autophagy in other cell types is protective in models of antibody-mediated nephritis, such as lupus nephritis and nephrotoxic serum nephritis. Amino acid metabolism triggers interferon-g–mediated induction of indoleamine 2,3-dioxygenase 1 enzyme activity with subsequent activation of a stress response dependent on the eIF2alpha kinase general control nonderepressible 2. Consistent with a role in prevention of apoptotic cell driven autoreactivity, myeloid deletion of general control nonderepressible 2 in lupus-prone mice resulted in increased immune cell activation, humoral autoimmunity, and renal disease.154 Interestingly, the indoleamine 2,3-dioxygenase– general control nonderepressible 2 pathway in podocytes and other cells was suggested to be a critical negative feedback Kidney International (2016) -, -–-

cascade that limits inflammatory renal injuries by inducing autophagy in a model of nephrotoxic serum nephritis.155 Increasing kidney indoleamine 2,3-dioxygenase 1 activity or treating mice with a general control nonderepressible 2 agonist induced autophagy and protected mice from nephritic kidney damage. Interestingly, kidneys from patients with antibody-driven nephropathy showed increased indoleamine 2,3-dioxygenase 1 abundance and stress gene expression, suggesting interplay between autophagy and interferong–mediated protective feedback cascade.155 Autophagy in DN. DN is the leading cause of end-stage renal disease in industrialized countries. Progressive loss of podocytes and microvascular alterations appear to most closely correlate with the functional renal decline in DN.156–158 Progressive podocyte injury characterized by cell detachment, hypertrophy, and foot process effacement plays a central role in the development of DN in both type 1 and type 2 diabetes mellitus.132,158–160 Over the past decade, several studies have provided indirect and direct evidence that 7

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autophagy is implicated in the pathogenesis of DN.34,161,162 First, autophagy can be induced by high glucose levels in various cell types, partly through hyperglycemia-mediated production of reactive oxygen species, and it has protective effects in vitro.163–165 In podocytes, high glucose levels lead to podocyte apoptosis in vitro that is mediated through caspase-3 activation.166 High glucose concentration also induces autophagy.165,167,168 However, prolonged exposure to hyperglycemia results in depressed podocyte autophagy.169 This secondary downregulation of autophagy may play a role in disease progression and may be linked to podocyte hypertrophy through the upregulation of the cyclindependent kinase inhibitor p27/Kip1170 and activation of mTOR.71,72 Furthermore, activation of mTOR was associated with an accelerated glomerular injury in DN.71,152 Although rapamycin, resveratrol, and caloric restriction treatments affect many pathways besides the autophagy pathway, several studies have shown that they have positive effects on inflammation, tubular injury, glomerulosclerosis, and podocyte injury in rodent models of DN.93,171–177 Taurineconjugated ursodeoxycholic acid also restores high-glucose– suppressed autophagy and podocin expression in podocyte culture, and it attenuates albuminuria in diabetic mice.168 Direct evidence for the protective role of autophagy during DN is coming from recent work demonstrating that podocyte Atg5 deficiency increased high-glucose–induced apoptosis. Moreover, specific podocyte autophagy deficiency (in Nphs2.Cre Atg5lox/lox mice) resulted in accelerated diabetesinduced podocytopathy as shown in models of type I167 and type II DN.178 Thus, chronic alteration of podocyte autophagy may contribute to DN progression, and fluctuations of autophagy in the diabetic podocyte over time could be an interesting paradigm for stage-specific interventions. Histone deacetylase 4 may participate to suppression of podocyte autophagy.179 Expression level of histone deacetylase 4 was shown to negatively correlate with estimated glomerular filtration rate in individual patients with DN and was upregulated in kidney tissues of rats with streptozotocininduced diabetes as well as in diabetic db/db mice. A pattern of histone deacetylase 4 expression was prominent in cultured podocytes upon incubation with a high concentration of glucose and in podocytes from individuals with DN or FSGS. Histone deacetylase 4 inhibition sustained the high constitutive autophagy in podocytes. Alteration of proteostasis and autophagy in the tubular compartment also takes place in diabetic kidneys and in cells exposed to a high concentration of glucose or advanced glycation end products. An early work investigating the activity of chaperone-mediated autophagy in rat kidney cortical lysates upon induction of diabetes revealed that reduced chaperone-mediated autophagy contributes to accumulation of specific proteins in diabetic-induced renal hypertrophy.180 The implication of such changes in DN progression is unknown. A recent work demonstrated that ubiquitinated protein bodies were delivered to the autophagy machinery but 8


not degraded by the impaired lysosomes of HK-2 cells exposed to advanced glycation end product–bovine serum albumin.181 Again, this study suggests that the autophagylysosome pathway is disrupted by advanced glycation end products in tubular epithelial cells during the development of DN, which results in the accumulation of abnormal proteins. Progressive and irreversible microvascular damage and loss are also observed in the diabetic kidney, and a decrease in the function and density of intrarenal microvessels has been reported in several studies.182–185 Interestingly, the treatment of diabetic mice with resveratrol, a well-known inducer of autophagy, protects from intraglomerular capillary rarefaction, suggesting that resveratrol attenuates DN by modulating angiogenesis.186 Moreover, autophagy protects from high glucose–induced senescence in cord human venous endothelial cells.187 Finally, endothelial-specific deletion of Atg5 (Cdh5.Cre Atg5lox/lox mice) leads to increased diabetes-induced renal endothelial injury, as shown by interstitial and glomerular capillary rarefactions and glomerular endothelial cells’ loss of fenestrations.167 Interestingly, epidermal growth factor receptor activation detected in diabetic glomeruli was associated with ER stress, decreased autophagy, and accelerated diabetic glomerulopathy.169 Therefore, pharmacologic epidermal growth factor receptor kinase inhibition may have therapeutic implications in DN.169 In summary, autophagy acts as a protective mechanism on both cellular layers of the glomerular filtration barrier in DN. Autophagy in the tubulointerstitial compartment Autophagy in AKI. Most cases of AKI are caused by renal

ischemia-reperfusion, sepsis, xenobiotic agents, drugs, and metals, which are risk factors for development of chronic kidney diseases.188,189 Autophagy is now generally accepted as a renoprotective cellular response in AKI of various causes.36,190–203 An in-depth review of the relationship between autophagy and AKI has been published in this journal.204 Autophagy in renal fibrosis. Unilateral ureteral obstruction (UUO) in rodents has been established as a classic model of progressive renal fibrosis.205 Autophagy markers were increased in obstructed renal tubules in a UUO model. Indeed, BECN1 and LC3-II are overexpressed during the time course of renal injury and accompany tubular atrophy and tubular cell death.77,206,207 Nevertheless, the positive or negative impact of autophagy induction in UUO is not clear. The autophagy inhibitor 3-methyladenine enhances tubular cell apoptosis and tubulointerstitial fibrosis in the obstructed kidney in rats after ureter obstruction, suggesting that autophagy induced by UUO has a renoprotective role in the obstructed kidney.77 The protective function of autophagy in tubules during ureteral obstruction was also demonstrated recently by the use of genetically modified animals deficient for the autophagy pathway.207 In renal tubular epithelial cells, autophagy regulates expression of transforming growth factor beta (TGF-b) by promoting autophagic degradation of Kidney International (2016) -, -–-


mature TGF-b. Deletion of LC3B (in LC3-/- mice) or Beclin1 haploinsufficiency (in Bcn1þ/- mice) resulted in increased levels of mature TGFbeta and was associated with more severe fibrosis and renal tubular epithelial cell apoptosis in the obstructed kidney after UUO. Interestingly, TGF-b itself stimulates autophagy.207 On the other hand, it was found that under the stimulus of persistent UUO, the induction of autophagy in the proximal tubules contributes to cell death. Indeed, the authors found that mitochondrial structure changes during UUO and lipid peroxidation product, reduced nicotinamide adenine dinucleotide phosphate oxidase 4, and reduced nicotinamide adenine dinucleotide phosphate oxidase activity are increased in the obstructed renal cortex, whereas mitochondrial injury leads to autophagy and apoptosis through the BECN1 pathway and Bcl2 interaction. Therefore, during UUO, oxidative stress that leads to mitochondrial damage could drive autophagy-dependent cell death and apoptosis and could be a mechanism of tubular atrophy.208 The sequestration of damaged lysosomes by autophagy is another emerging role for autophagy in epithelial maintenance. This involves complex process of selective lysosome recognition, engulfment, and degradation or so-called lysophagy.209 This proved to be the case in tubular kidney cells, in which under conditions of lysosomal damage, loss of autophagy caused inhibition of lysosomal biogenesis in vitro and deterioration of AKI in vivo.210 LC3-associated phagocytosis in tubular cells. Although several studies suggested that cells from proximal tubules play a significant role in removing apoptotic cells and debris via phagocytosis or heterophagy, recent evidence suggests that proximal tubular cells also exert noncanonical autophagy in cell corpse clearance, termed LC3-associated phagocytosis. In this clearance pathway, LC3 is targeted to the phagosome in an ATG5-, ATG7-, and BECN1-dependent manner; however, this process does not classically involve the engagement of upstream autophagy proteins such as ULK1.211–213 A recent study found that kidney injury molecule-1 (KIM-1)/T-cell Ig and mucin domain 1, a phosphatidylserine phagocytosis and scavenger receptor expressed by kidney proximal tubule epithelial cells, is the dominant apoptotic cell phagocytosis receptor in these cells.214 Originally, upon ligand extracellular recognition, binding and phosphorylation of KIM-1 cytosolic domain interacted with the phosphoinositide 3–kinase pathway and stimulated LC3 lipidation and autophagy induction with progressive appearance of KIM-1-and LC3-positive organelles characteristic of autophagy. Unlike standard LC3-associated phagocytosis described in professional phagocytic cells, KIM1 expression induced phosphorylation and activation of ULK1/ATG1, which is essential for autophagosome formation. These results also suggest that KIM-1 phagosomes are degraded by autophagy. In summary, this cascade described in kidney proximal epithelial cells represents a novel mechanism by which autophagosomes generated in an ULK1-dependent manner target phagosomes for degradation, which has potential pathophysiological implications as kidney injury in mice Kidney International (2016) -, -–-

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carrying a nonfunctional mutant KIM-1 results in a worsening of AKI with the accumulation of unphagocytosed apoptotic cells and increased inflammation.214,215 Beyond the case of AKI, we hypothesize that interplay between KIM-1–mediated phagocytosis, tubular autophagy, and inflammation may play a role in resolution of inflammation, scarring processes, and progression of chronic nephropathies. Autophagy and metabolic acidosis. Metabolic acidosis induces autophagy in proximal tubular cells in vitro and in vivo. Kidneys of acid-loaded proximal tubule–specific autophagydeficient mice (Atg5lox/lox:KAP-Cre mice) exhibited an increased number of SQSTM1/p62 and UB-positive aggregates in the kidney proximal tubular cells compared with in the vehicle-treated Atg5lox/lox:KAP-Cre mice. Furthermore, acid-loaded Atg5lox/lox:KAP-Cre mice showed significantly more severe acidosis than did the acid-loaded controls, with impaired ammoniagenesis indicating a protective role for ATG5 on ammoniagenesis. This was linked to deficiency in proper mitochondrial functions, including ammoniagenesis in Atg5lox/lox:KAP-Cre animals, as basal mitochondrial oxygen consumption rate, the adenosine triphosphate–linked oxygen consumption rate, and maximum respiratory function were significantly more reduced by acid loading in ATG5-deficient proximal tubular cells than in ATG5-competent proximal tubular cells.216 Autophagy PKDs. Several pieces of evidence suggest that autophagy could play roles in PKDs in cyst formation and growth. Indeed, electron microscopy, immunofluorescence, and immunoblot demonstrated LC3-II and BECN1 overexpression and increased number of autophagosomes in polycystic kidneys, thus suggesting autophagic flux dysregulation in PKD.217 Moreover, PKD is a cilia-related disease and autophagy was recently shown to be implicated in ciliogenesis,218 whereas primary cilia are required for the activation of autophagy.219 Therefore, the close relationship between ciliogenesis and autophagy constitutes an exciting method of investigation in the field of cilia-related disease such as PKD. Finally, autophagy modulators have been implicated in PKD progression. The PtdIns3K–v-akt murine thymoma viral oncogene homolog 1–tuberous sclerosis complex–mTOR pathway is activated in PKD, and rapamycin has shown proof of therapeutic effects on animal models of PKD220–226 but not in patients.227,228 Interestingly, a low dose of rapamycin results in protection from PKD without effects on apoptosis,225 whereas a higher dose of rapamycin increases apoptosis of cyst-lining epithelial cells in PKD.226 Moreover, it was shown recently that metformin, a known AMPK inducer, slows cyst formation.229 Even if the authors did not establish any link between AMPK activation and autophagy induction, this study suggests that autophagy induction, mediated by metformin, could be renoprotective in PKD. Further studies on the link between autophagy and cyst formation in PKD are required to clarify the functions of autophagy. Autophagy and aging. The renal tubulointerstitial compartment is not spared from aging-induced damage. Tubular atrophy, apoptosis, and fibrosis are associated with a decline 9

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in the filtration function in the aged kidney. Experimental evidence suggests that autophagy is protective in proximal tubules cells during aging. Indeed, mice with a specific autophagy deficiency in renal proximal tubular cells (Atg5lox/lox;KSP.Cre and Atg5lox/lox; Pax8.rtTA;TetO.Cre mice) have increased accumulation of damaged mitochondria, ubiquitinated proteins, and SQSTM1-positive aggregates, which are associated with tubular apoptosis and tubulointerstitial fibrosis.192,230 These studies showed that autophagy deficiency in renal tubular cells leads to premature aging. Consequently, autophagy activation in the aging kidney would prevent tubular lesions development and protect from age-related renal dysfunction. Calorie restriction is a major inducer of autophagy and has been correlated with increased life span in eukaryotes.231,232 Interestingly, calorie restriction protects from age-related kidney damage233,234 and autophagy underlies calorie restriction–mediated renal protection.233 It was recently demonstrated that SIRT1 mediates forkhead box O3 deacetylation in response to calorie restriction and leads to restoration of autophagy in aged kidneys.233,235 Resveratrol is an antioxidant that occurs naturally in many plant parts and products, such as grapes, berries, red wine, and peanut skins. Resveratrol activates SIRT1, and previous reports have shown that resveratrol can ameliorate several types of renal injury, such as DN, ischemia-reperfusion injury, and UUO among others, in animal models through its antioxidant effect or SIRT1 activation. Therefore, resveratrol may be a promising treatment for preventing age-related renal injury.236 PUTATIVE THERAPEUTIC TARGETS AND CLINICAL IMPLICATIONS

Inducing autophagy could be a promising therapeutic strategy for the treatment of renal diseases. Nevertheless, all the cytoprotective effects of autophagy were found in rodent models of renal diseases, and so far, proof is lacking that these findings can be transferred to human diseases. To date, the only autophagy inducers commonly used in human medicine are mTOR inhibitors (sirolimus and everolimus), which are widely used as immunosuppressive agents. However, it is now well established that in humans these antiproliferative mTOR inhibitors have adverse effects on compensatory renal epithelial cell hypertrophy and recovery from ischemia following renal transplantation.237–239 Moreover, sirolimus is associated with numerous side effects, including diabetes mellitus, systemic inflammation, or edema among others.237 Thus, the use of mTOR inhibitors as autophagy inducers appears to not be a feasible strategy in many cases. Resveratrol supplementation is currently being tested in several clinical trials, essentially for safety of use, for weight loss therapy, and in elderly individuals, and it seems to be safe at the doses tested.240–243 In summary, to date there is a lack of reliable biomarkers and clear interventional strategies targeting autophagy in renal diseases. Thus, a specific approach that enables pathway-specific and kidney-selective regulation of autophagy would be required. Recently, a Tat-Beclin fusion protein has 10


been found to selectively induce autophagy without side effects on other signaling pathways, offering novel perspectives for potential treatment strategies.244 CONCLUSION

Because autophagy is a dynamic process, instant pictures from cells or tissues cannot bring robust information regarding induction or inhibition of this homeostatic phenomenon. Therefore, information gained from animal models has been precious. The development of a conditional knockout mice enabling targeting of autophagy in rodent models of renal diseases has significantly deepened our knowledge of the role of autophagy in these conditions. Most of the direct studies of autophagy systems in vivo have used loss-of-function approaches rather than specific gain-offunction approaches, which may be seen as a limitation. Meanwhile, most of these studies are in accord regarding a protective function of autophagy in the kidney, highlighting autophagy as a novel and very promising target for renal diseases therapy. However, further studies are required to elucidate the precise and cell-specific programs regulating autophagy in renal diseases. There is also a pressing need for robust biomarkers that accurately assess the clinical utility of modulating autophagy in various disease contexts. Investigations of kidney disease–dependent triggers, sensors, and adaptors that tailor the autophagy machinery might drive biomarker discovery to achieve target specificity. Moreover, selective autophagy modulators and (cell-)specific delivery routes remain to be elucidated to ultimately hopefully offer novel treatment options for our kidney patients. DISCLOSURE

All the authors declared no competing interests. ACKNOWLEDGMENTS

This study was supported by INSERM, Joint Transnational Call 2011 for “Integrated Research on Genomics and Pathophysiology of the Metabolic Syndrome and the Diseases arising from It” from l’Agence Nationale de la Recherche (ANR) of France (PLT); the LefoulonDelalande Foundation; and the Francophone Diabetes Society (to OL). This study was further supported by the German Research Foundation (DFG): CRC 1140 (to TBH) and CRC 992 (to TBH), Heisenberg program (to TBH) and HU 1016/8-1; by the European Research Council-ERC grant 616891 (to TBH) and grant 311255 (to PLT); by the BMBF-Joint transnational grant 01KU1215 (to TBH) and STOP-FSGS 01GM1518C (to TBH); by the Else-Kröner Fresenius Stiftung-NAKSYS; and by the Excellence Initiative of the German Federal and State Governments (GSC-4, Spemann Graduate School and EXC294, BIOSS II to TBH). REFERENCES 1. Boya P, Reggiori F, Codogno P. Emerging regulation and functions of autophagy. Nat Cell Biol. 2013;15:713–720. 2. Choi AM, Ryter SW, Levine B. Autophagy in human health and disease. N Engl J Med. 2013;368:1845–1846. 3. Glick D, Barth S, Macleod KF. Autophagy: cellular and molecular mechanisms. J Pathol. 2010;221:3–12. 4. Clark SL Jr. Cellular differentiation in the kidneys of newborn mice studies with the electron microscope. J Biophys Biochem Cytol. 1957;3: 349–362. 5. De Duve C. The lysosome. Sci Am. 1963;208:64–72. Kidney International (2016) -, -–-


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