Autophagy in Glomerular Health and Disease Björn Hartleben, MD,*,† Nicola Wanner, PhD,*,‡,|| and Tobias B. Huber, MD*,¶

Summary: Glomerular filtration coupled to tubular reabsorption was the prerequisite for one of the most important milestones in evolution, when animals made their way from water onto land. To fulfill the enormous filtration task the filter is composed of the most sophisticated postmitotic epithelial cells—the podocytes, which have only a very limited ability to regenerate. Podocyte injury and loss owing to genetic, toxic, immunologic, or metabolic insults underlie the most common glomerular diseases. Thus, the understanding of the factors and mechanisms that help to maintain podocytes are of major clinical importance. Recently, autophagy emerged as a key mechanism to eliminate unwanted cytoplasmic materials, thereby preventing cellular damage and stress to safeguard long-lived podocytes. Here, we highlight the accumulating evidence suggesting that autophagy plays a critical role in the homeostasis of podocytes during glomerular disease and aging. Semin Nephrol 34:42-52 C 2014 Elsevier Inc. All rights reserved. Keywords: Podocyte, kidney, autophagy, mTOR, ATG5, LC3, RAPTOR, FSGS, diabetic nephropathy, glomerulonephritis, lysosomal storage diseases

U

ltrafiltration is executed by a multilayered filtration barrier that is composed of the fenestrated glomerular endothelium, the glomerular basement membrane, and the slit diaphragm, which bridges the filtration slits between the podocyte foot processes.1,2 These structures are maintained by four resident cell types: the endothelial cells of the glomerular capillaries; the mesangial cells, holding the glomerular capillary tuft from the inside; the podocytes, covering the capillary tuft from the outside with their primary processes and interdigitating foot processes; and the glomerular parietal epithelial cells, lining the bowman capsule.1,2 The highly specialized podocytes are the most vulnerable component of the filtration barrier. Podocyte injury is responsible for proteinuria, and loss of podocytes by cell death or detachment is a critical step for the progression of glomerular diseases and glomerular aging (Fig. 1).3–5 Transgenic mouse models of podocyte-selective *

Renal Division, University Hospital Freiburg, Freiburg, Germany. Institute of Pathology, University Hospital Hamburg-Eppendorf, Hamburg, Germany. ‡ Spemann Graduate School of Biology and Medicine, Albert Ludwigs University, Freiburg, Germany. || Faculty of Biology, Albert Ludwigs University, Freiburg, Germany. ¶ BIOSS Centre for Biological Signalling Studies, Albert Ludwigs University, Freiburg, Germany. Financial support: Supported by the Deutsche Forschungsgemeinschaft (T.B.H.); the Excellence Initiative of the German Federal and State Governments (EXC 294 to T.B.H.), GSC-4 Spemann Graduate School (N.W. and T.B.H.), the BMBF–Gerontosys II–NephAge (T.B.H.), and a Joint Transnational Grant (T.B.H.). Conflict of interest statement: none. Address reprint requests to Tobias B. Huber, MD, Renal Division, University Hospital Freiburg, Breisacher Str. 66, 79106 Freiburg, Germany. E-mail: [email protected] 0270-9295/ - see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.semnephrol.2013.11.007 †

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depletion showed that loss of more than 20% of podocytes is sufficient to cause glomerulosclerosis.6,7 Because podocytes are postmitotic cells with a very limited regenerative potential,8,9 mechanisms such as autophagy, which control the cellular homeostasis, are essential to maintain the podocyte compartment. Macroautophagy (hereafter referred to as autophagy) is the major cellular bulk degradation pathway. It serves as a quality control mechanism by inactivating misfolded proteins and nonfunctional organelles and supplies nutrients for survival. The process of autophagic degradation consists of several phases, the initiation of a double-membrane structure, called isolation membrane or phagophore, the elongation of this phagophore, the sequestration of the cargo, and the maturation to an autophagosome, which fuses with a lysosome to an autolysosome, where the cargo is degraded and nutrients are shuttled back to the cytoplasm for metabolic recycling (Fig. 2A).10,11 For a detailed description of the molecular autophagy machinery we refer the reader to recent excellent reviews by Mizushima et al,10 Yang and Klionsky,11 Ravikumar et al,12 and Choi et al.13 In brief, the initiation of autophagy and the nucleation of the isolation membrane depends on two protein complexes: the Unc-51-like kinase 1 (ULK1)-autophagy related 13 (ATG13)-FIP200-complex, which is activated by 50 -AMP activated protein kinase (AMPK) in response to energy depletion and negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1); and the beclin 1–interacting complex, comprising beclin 1, the class III phosphatidylinositol3-kinase VPS34 (vacuolar protein sorting 34), its regulatory subunit VPS15, ATG14L, and other interacting proteins. Activation of the beclin 1–interacting complex results in the generation of phosphatidylinositol3 (PI3) phosphate, which promotes autophagosomal membrane nucleation.13 The elongation of the membrane depends on two ubiquitin-like conjugation systems, the ATG5-ATG12 conjugation system and the Seminars in Nephrology, Vol 34, No 1, January 2014, pp 42–52

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Figure 1. Podocytes are a main target of glomerular diseases. (A) Podocytes are exposed to a wide range of cellular stress events, but have only a very limited regenerative potential. (B) Injury and loss of podocytes are the key to the progression of glomerular diseases and glomerular aging. MCD, minimal change disease; ROS, reactive oxygen species; RPGN, rapid progressive glomerulonephritis.

microtubule-associated protein 1 light chain 3 (LC3)conjugation system,12,13 where cytosolic LC3-I is converted to LC3-II by conjugation with phosphatidylethanolamine.14 LC3-II commonly serves as a marker for autophagosomes and can be visualized by immunofluorescence staining, appearing as dots in the cytosol, which represents autophagosomes.15 By using LC3 as a reporter system, a green fluorescent protein (GFP)-LC3 transgenic mouse model recently enabled the in vivo monitoring of autophagy.16 In addition, LC3-I and LC3II can be detected by Western blot analysis, in which the lipidation of LC3-I results in a higher electrophoretic mobility.14 Glomerular podocytes were identified as cells with high levels of basal autophagy (Fig. 2B).16–18 Subsequent studies recently showed a critical involvement and protective role of autophagy for glomerular maintenance, aging, and disease progression, which is summarized in this review. The understanding of the mechanisms by which autophagy can prevent glomerular disease progression may lead to the identification of new diagnostic and therapeutic approaches. In fact, agents directly acting on autophagy may offer novel opportunities for targeted therapies of glomerulopathies.

ROLE OF AUTOPHAGY FOR PODOCYTE MATURATION AND DIFFERENTIATION Podocytes are derived from the metanephric mesenchyme and undergo a series of complex transdifferentiation processes from mesenchymal cells to highly

differentiated epithelial cells. This differentiation process is accompanied by a stop in cell division,2 and, during the late capillary loop stage, an up-regulation of autophagy.18 The latter remains at a high basal level and becomes a characteristic hallmark of differentiated podocytes in vivo and in vitro.16–19 Constitutive knockout of Atg5, however, did not result in an obvious alteration of podocyte maturation,18 indicating that autophagy is not necessarily required for the differentiation process itself, but it is part of the podocyte’s phenotypic and metabolic shift to a postmitotic secretory cell.

ROLE OF AUTOPHAGY FOR PODOCYTE MAINTENANCE A functional block of the autophagy machinery in podocytes by deletion of Atg5 caused a slowly progressing cellular degeneration.18 Podocyte-specific conditional Atg5 knockout mice (Atg5 PcKO mice) developed aging-related albuminuria and late-onset glomerulosclerosis between 20 and 24 months of age.18 The phenotype phenocopied typical age-related alterations, such as the formation of ubiquitin- and SQSTM1/p62-positive protein aggregates, the accumulation of lipofuscin, the occurrence of damaged mitochondria, and the increase in the total load of oxidized proteins (Fig. 2C).18 The Calnexin signal was increased, and ultrastructural studies showed cytosolic vacuolization, apparently caused by expanded endoplasmic reticulum (ER) membranes. The compensatory

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Figure 2. Autophagy maintains podocyte homeostasis. (A) The autophagic process consists of the initiation of a double-membrane structure, called the isolation membrane or phagophore, the elongation of this phagophore, the sequestration of the cargo and the maturation to an autophagosome, the fusion with a lysosome to an autolysosome, the degradation of the cargo, and the release of its components to the cytoplasm. (B) In vivo analysis of the GFP-LC3 transgenic mouse shows high levels of basal autophagy in podocytes (nidogen serves as a basement membrane marker, arrows indicate autophagosomes in podocytes). Scale bars: 10 mm. (C) Autophagy deficiency in podocytes causes a slowly progressing cellular degeneration with typical agingrelated alterations. (D) Impaired podocyte autophagy increases the susceptibility to glomerular stress events.

Glomerular autophagy

up-regulation of glomerular proteasome activity observed in young Atg5 PcKO mice decreased with age, reflecting the total collapse of podocyte protein degradation machineries.18 Thus, the accumulation of misfolded proteins and damaged organelles in autophagy-deficient podocytes ultimately resulted in podocyte loss and the development of segmental and global glomerulosclerosis, highlighting the fundamental role of autophagy for the long-time maintenance of glomerular podocytes.

ROLE OF AUTOPHAGY FOR PODOCYTE AGING The decline of kidney function with age is a wellknown phenomenon, and a major part of the decline is associated with age-related glomerulosclerosis caused by podocyte depletion.20 Similarly to cells in other tissues, such as the brain, where, for instance, autophagy-related genes (such as ATG5 and ATG7) are transcriptionally down-regulated during normal aging,13,21–24 podocytes appear to display diminished autophagosome formation with age, which might contribute to age-related glomerulosclerosis. Podocytes of aging mice show a progressive accumulation of oxidized proteins and of SQSTM1/p62-positive protein aggregates,9 and preliminary data indicate an increased mTORC1 activity and a decrease of LC3-I to LC3-II conversion in glomeruli of old mice (Hartleben and Huber, unpublished data).

ROLE OF AUTOPHAGY FOR GLOMERULAR DISEASES Autophagy appears to be a quality-control mechanism and essential for the protein and organelle turnover of podocytes under physiological and pathophysiological conditions. Studies describe a boost in autophagic activity in podocytes during the recovery from puromycin aminonucleoside–nephritis and from passive Heymann nephritis in rats.17,25 Consistently, loss of autophagy in podocytes results in a markedly increased susceptibility to various models of glomerular disease.18 For instance, young Atg5 PcKO mice developed severe proteinuria and glomerulosclerosis in the Adriamycin (ADR)-model,18 whereas their control littermates were unaffected in agreement with the known phenomenon that C57BL/6J mice are resistant to this toxin. In Balb/c mice, the ADR-susceptibility gene has been identified as the protein kinase, DNAactivated, catalytic polypeptide, a component of the mitochondrial genome maintenance pathway.26 Sensitivity to ADR nephropathy is produced by a mutation in the protein kinase, DNA-activated, catalytic polypeptide gene, resulting in mitochondrial DNA depletion upon ADR treatment.26 In addition, acquired mutations of mitochondrial DNA have been described

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in focal segmental glomerulosclerosis (FSGS).27,28 The inability of Atg5 KO podocytes to cope with damaged mitochondria by degrading them via mitophagy might be the mechanism underlying glomerulosclerosis in ADR-injected young Atg5 PcKO mice on a C57BL/6J background. This possibility highlights the particular importance of autophagy as a key homeostatic mechanism for podocytes not only under physiological conditions but also under stress conditions (Fig. 2D). Focal Segmental Glomerulosclerosis A hallmark in the development of FSGS is the loss of podocytes, resulting in adhesion of the glomerular capillary tuft to the Bowman's capsule, proceeding to obliteration of the capillaries and eventual total nephron degeneration.29,30 Autophagy appears to be a general cellular stress surveillance factor for podocytes, and the functional block of autophagy in podocytes of Atg5 PcKO mice was associated with slowly progressing podocyte degeneration, loss of podocytes, and glomerulosclerosis.18 This finding prompts the question of whether genome-wide association studies can identify glomerular disease susceptibility loci in autophagosomal regulatory genes. Recently, two sequence variants in the apolipoprotein L1 gene (APOL1), termed G1 (a pair of nonsynonymous coding sequence single-nucleotide polymorphisms, leading to the replacement of two amino acids, S342G and I384M) and G2 (a 6–base pair in-frame deletion, resulting in the deletion of two amino acids, delta N388Y389), were shown to be associated with significantly increased rates of FSGS, hypertension-attributed endstage renal disease, and human immunodeficiency virus–associated nephropathy in African Americans.31–33 Individuals with two risk alleles have an estimated lifetime risk of developing FSGS of 4%,32 whereas APOL1 variants do not appear to increase the risk for developing diabetic nephropathy or IgA nephropathy.33,34 FSGS associated with two APOL1 risk alleles occurs at a younger age and shows a faster progression to end-stage renal disease compared with individuals with FSGS associated with only one or with no risk alleles.32 Three different ApoL1 protein isoforms exist as a result of alternative splicing; isoform a is a 398–amino acid polypeptide with an N-terminal pore-forming domain, followed by a membrane-addressing domain consisting of pHsensitive α-helices, a BH3 domain, and a C-terminal leucine zipper domain embedded in a serum resistanceassociated protein (SRA) interacting domain.35 Extracellular ApoL1 associates with high-density lipoprotein particles and is a trypanolytic factor, which is taken up by trypanosomes, targeted to the lysosomal membrane, forming anion channels, leading to membrane depolarization, an influx of chloride with swelling of the

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lysosome, and death of the parasite.36,37 The subspecies Trypanosoma brucei rhodesiense and gambiense are resistant to ApoL1 and cause African sleeping sickness, transmitted by tsetse flies. T brucei rhodesiense inhibits the trypanolytic activity of ApoL1 by an SRA that interacts with the C-terminus of ApoL1,36 a region containing the G1 and G2 sequence variants.31 Both G1 and G2 rescue the ability of ApoL1 to lyse T brucei rhodesiense, and it has been shown that SRA cannot bind the gene product of the G2 risk allele.31 This finding suggests a heterozygous advantage model: one copy of the G1 or G2 variants provides protection against T brucei rhodesiense, whereas two copies increase the risk for renal disease.31 In mammalian cells, intracellular ApoL1 is localized in the cytosol and in lysosomes.35 Its expression could be induced by interferon-γ and tumor necrosis factor-α and by p53 in p53-induced cell death.38,39 Further, ApoL1 was highly up-regulated in cultured normal human oral keratinocytes undergoing senescence.40 ApoL1 is a lipidbinding protein with a high affinity for the following: phosphatidic acid, a positive regulator of mTOR41; cardiolipin; and phosphatidylinositol phosphates, including PI3 phosphate, the product of VPS34, which is required for autophagosomal membrane nucleation.39 Overexpression of ApoL1 induces autophagy and autophagy-associated cell death in cell culture. The latter effect was abrogated by the PI3-kinase inhibitors 3-methyladenine and wortmannin and in Atg5 or Atg7 knockout embryonic fibroblasts.39 However, the molecular mechanisms by which cytosolic and/or lysosomal ApoL1 modulates autophagic activity are unknown and the manner in which the risk sequence variants alter its function is not solved yet. In normal kidneys, ApoL1 is expressed in podocytes, the proximal tubular epithelium, and endothelial cells of preglomerular arterial vessels42; the former two cell types have high levels of autophagy. In FSGS and in human immunodeficiency virus–associated nephropathy, however, ApoL1 is up-regulated in smooth muscle cells of the arterial wall, reflecting vascular wall remodeling, and its glomerular expression is reduced, regardless of whether the patients are homozygous, heterozygous, or null for the G2 risk variant.42 The function of ApoL1 in the kidney remains unknown, but its expression in podocytes and its ability to induce autophagy point toward the possibility that ApoL1 risk variants might induce glomerular disease by dysregulating autophagy. Membranous and IgA Glomerulopathies

In passive Heymann nephritis, an experimental rat model of membranous nephropathy, induction of ER stress by the complement C5b-9 membrane attack complex and up-regulation of autophagy could be

B. Hartleben, N. Wanner, and T.B. Huber

shown in podocytes in vivo.43,44 Similar observations were made in cultured podocytes incubated with the ER stress inducer tunicamycin.44 Furthermore, an increased LC3 signal was observed in podocytes in human biopsy samples of patients with membranous nephropathy,18 and autophagosomes were detected by electron microscopy in podocytes in IgA nephropathy.45 However, functional data resolving the role of glomerular autophagy in the course of glomerulonephritis are limited to date. A recent study described a negative correlation of mTORC1 and autophagy in the passive Heymann nephritis model with an initial activation of glomerular mTORC1 signaling and a corresponding decrease in autophagy, followed by an increase in autophagy during the recovery phase.25 In vitro studies showed that application of rapamycin reduced injury to puromycin aminonucleoside–treated murine podocytes by inhibiting mTOR-dependent ULK1 phosphorylation and autophagy suppression,25 raising the possibility that stimulation of autophagy by rapamycin also could work as a therapeutic tool in vivo. Diabetic Nephropathy Although the mTOR pathway has been studied in detail in diabetic nephropathy, the data about autophagy in this context are limited. Diabetic nephropathy is characterized by podocyte injury, glomerular basement thickening, mesangial expansion, and proteinuria. Activation of mTORC1 signaling in podocytes recently was characterized as a hallmark in mouse models of type 1 and 2 diabetes mellitus.46–48 In addition, patient biopsy samples of diabetic nephropathy displayed increased phosphorylation of the mTORC1 downstream target S6 and transcriptional up-regulation of mTORC1 target genes.46,47 Constant activation of mTORC1 signaling in podocyte-specific conditional Tsc1 knockout mice (Tsc1 PcKO) caused hypertrophy and dedifferentiation of podocytes with mislocalization and reduced expression of slit diaphragm proteins and ER stress; hypertrophy and dedifferentiation finally resulted in podocyte detachment and glomerulosclerosis, resembling the phenotype of diabetic nephropathy.47 Treatment with the chemical chaperone 4-phenyl butyric acid attenuated the mTORC1-induced ER stress and the loss of podocytes but had no effect on the S6 phosphorylation level and proteinuria.47 Furthermore, curtailing mTORC1 signaling by administration of rapamycin or by a podocyte-specific reduction of the Raptor copy number prevented progression of glomerular disease in mouse models of type 1 and type 2 diabetes mellitus as well as in Tsc1 PcKO mice, indicating that mTORC1 activation is a critical step in inducing diabetic nephropathy.46–48 However, the role of autophagy and its dependence

Glomerular autophagy

on mTORC1 signaling has not been studied in these mouse models. Recently, suppression of autophagy, detected as reduced expression levels of beclin 1, LC3, and ATG5-ATG12, has been described in podocytes in the streptozotocin mouse model of type 1 diabetes mellitus and in immortalized murine podocytes incubated with high glucose for 48 hours.49 These effects were accompanied by reduced expression of podocin and increased paracellular leakage across a podocyte monolayer in vitro, which could be rescued by administration of rapamycin.49 Prolonged incubation with high glucose (for up to 60 hours) resulted (after an initial increase) in a decreased phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which is a part of the adaptive unfolded protein response signaling and an upstream activator of autophagy. In contrast, the expression of proapoptotic Chop was increased, indicating a switch from a cytoprotective to a cytotoxic ER stress response. Administration of the chemical chaperone tauroursodeoxycholic acid partially reversed these effects in vitro as well as in vivo with restored autophagy and minor progression of diabetic nephropathy.49 A second study about the influence of glucose on immortalized murine podocytes focused on the short-term effects, showing that incubation with high glucose for 24 hours induced the generation of reactive oxygen species by podocytes.50 This oxidative stress resulted in an up-regulation of autophagy with an increase of beclin 1 expression, LC3-II levels, and autolysosomes, which could be diminished by the antioxidant N-acetylcysteine.50 These studies showed the time-dependence of the effect of high glucose on podocyte autophagic activity, with an obvious short-term up-regulation and a long-term down-regulation of autophagy, possibly owing to a switch in the unfolded protein response signaling. However, the interplay of mTOR signaling and autophagy remains to be elucidated further. In addition, studying the course of diabetic nephropathy in autophagy-deficient mouse models will be of great interest. Lysosomal Storage Diseases The class III phosphatidylinositol 3-kinase VPS34 catalyzes the phosphate transfer from adenosine triphosphate (ATP) to the D3 position of the inositol ring of phosphatidylinositols.51 As a part of the beclin 1–interacting complex, VPS34 promotes autophagosomal membrane nucleation. In addition, VPS34 regulates endosomal trafficking.51 Podocyte-specific knockout of Vps34 resulted in podocyte degeneration, severe proteinuria starting from the third week after birth, and the death of mice between 3 and 9 weeks of age.52,53 Podocytes showed a massive cytosolic vacuolization,

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which might have originated from lysosomes and endosomes.52,53 Conjugation of LC3-I with phosphatidylethanolamine still could occur in the VPS34deficient podocytes.52,53 However, accumulation of LC3-I, of LC3-II (showing only little colocalization with lysosomes), of SQSTM1/p62, and of vacant lysosomes indicated defects in early autophagosome formation as well as in autolysosome formation. Impaired autophagic flux, however, did not appear to be the sole cause of early podocyte degeneration because Atg5 PcKO mice showed a rather late-onset phenotype.18 Furthermore, double knockout of Vps34 and Atg5 in podocytes resulted in a phenotype similar to that caused by a single knockout of Vps34.53 These findings indicate that the early podocyte degeneration is caused only in part by an accumulation of aberrant autophagosomes and autolysosomes. Further in vivo and in vitro studies in VPS34-deficient podocytes as well as in VPS34 knockdown Garland cell nephrocytes, the podocyte-like cells of Drosophila melanogaster, showed that endosomal trafficking (with inhibited fluid phase uptake), receptor-mediated endocytosis, and maturation of early to late endosomes (with accumulation of Rab5) were blocked in VPS34deficient cells.53,54 Given the severe phenotype of the Vps34 podocyte-specific knockout, endocytosis, vesicle trafficking, and an efficient autophagosomallysosomal degradation system appear to be a fundamental part of podocyte physiology (Fig. 3A). Consistently, impairment of lysosomal function in mouse models as well as in human diseases resulted in podocyte dysfunction and disruption of the glomerular filtration barrier. The carboxy-terminal fragment of the prorenin receptor (ATPase, Hþ-transporting, lysosomal accessory protein 2, ATP6AP2) is associated with the vacuolar-type Hþ-ATPase,55 a proton pump consisting of a V1 domain, which hydrolyzes ATP, and a V0 pore domain, which translocates protons from the cytosol to the organelle lumen or to the extracellular space. Vacuolar-type Hþ-ATPases are responsible for intracellular vesicle acidification and are located in endosomes, lysosomes, the trans-Golgi network, and secretory vesicles.56 Knockdown of ATP6AP2 in human cultured podocytes resulted in both down-regulation of the V0 subunit and failure of vesicle acidification.57,58 Podocyte-specific conditional Atp6ap2 knockout mice (Atp6ap2 PcKO) developed severe proteinuria and renal failure and died between 2 and 4 weeks after birth.57,58 Knockout podocytes showed massive accumulation of intracellular vacuoles, including multivesicular bodies, dilated rough ER and lysosomes. Enrichment of LC3, SQSTM1/p62, and ubiquitin in knockout podocytes pointed toward impaired protein degradation by the autophagosomal-lysosomal system.57,58 In addition, the up-regulation of GRP78 illustrated the increased

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Figure 3. Proposed spatial regulation of mTORC1 signaling and autophagy in podocytes. (A) mTORC1 is recruited by the amino acid–rich environment of the TASCC to the (auto)lysosomal surface and is activated there, whereas autophagosomes are excluded, but move to the TASCC during maturation. Membrane components from autolysosomes are recycled for the formation of new lysosomes during mTOR-dependent ALR. VPS34 promotes autophagosomal membrane nucleation and regulates endosomal trafficking. (B) The spatial separation by the TASCC allows activation of both autophagy-dependent degradation of proteins and organelles and mTORC1dependent protein synthesis.

ER stress.57 Although the specific functional knockout of the autophagy machinery in Atg5 PcKO mice resulted in a delayed phenotype with podocyte degeneration between 1 and 2 years after birth,18 the phenotype of podocyte-specific Atp6ap2 knockout resembled the one observed in Vps34 PcKO mice with podocyte degeneration during the first weeks after birth.52,53 Aside from complete lysosomal failure, impairments in protein sorting, vesicle trafficking, receptor-mediated endocytosis, and mTOR activation might cause this accelerated phenotype.56–59 Several lysosomal storage diseases are associated with an accumulation of metabolic products in podocytes, leading to podocyte damage, proteinuria, and renal failure.60–64 A relevant genetic disease in this context is Fabry disease, which is caused by mutations of the X-chromosomal gene encoding for α-galactosidase A, resulting in the accumulation of glycosphingolipids, mostly globotriaosylceramide, in the lysosomes of podocytes, endothelial cells, and other cells. The clinical phenotype comprises impaired kidney function, systemic vasculopathy, and cardiomyopathy.65 The characteristic histologic hallmarks are lysosomal inclusion bodies, typically with a lamellated structure (the so-called myelin-like and zebra bodies), which are deposited mainly in podocytes in the kidney.66 Increased LC3, SQSTM1/p62, and ubiquitin signals in glomerular cells in kidney biopsy samples and in fibroblast und lymphoblast cell lines derived from patients with Fabry disease point toward impaired autophagic flux. Under starvation, Fabry fibroblasts show an accumulation of enlarged autophagic vacuoles in electron microscopy and defective autophagic lysosomal reformation (ALR).66,67 ALR depends on mTORC1 reactivation during starvation, which is impaired in Fabry fibroblasts, most likely owing to compromised autolysosomal degradative capability.67

Studies on an immortalized human podocyte α-galactosidase A knockdown cell line showed decreased mTOR activity and an increase in autophagosomes in these cells.68

PODOCYTE-SPECIFIC ASPECTS OF AUTOPHAGY REGULATION The master regulator of cellular metabolism, mTOR, exists in two different functional complexes: mTORC1 and mTORC2.69,70 mTORC1 comprises mTOR, the scaffolding protein regulatory associated protein of mTOR (RAPTOR), and several additional components. mTORC1 is activated by growth factor and nutrient signals and is inhibited directly by the rapamycinFKBP1A/FKBP12 complex.70 After amino acid stimulation, the heterodimeric Rag small guanosine triphosphatases recruit mTORC1 to the lysosomal membrane,71 where it is activated by the small guanosine triphosphatase Rheb, which itself is regulated negatively by the heterodimer tuberous sclerosis (TSC) 1-TSC2, a major upstream regulator of mTORC1.72,73 Activated mTORC1 phosphorylates several downstream targets; stimulates protein synthesis, lipid biogenesis, and cell growth; and inhibits autophagy induction. The second complex, mTORC2, is relatively rapamycininsensitive and consists of mTOR, rapamycin-insensitive companion of mTOR (RICTOR), and additional proteins. mTORC2 is activated by growth factors and regulates cytoskeletal organization and cell survival.70 mTORC1 signaling in podocytes appears to be required for postnatal growth, when podocytes have to adjust to an increasing glomerular size and volume.46,48 Because podocytes stop cell division in the capillary loop stage, extension of the glomerular surface must be accompanied by a potentially mTORC1-

Glomerular autophagy

dependent growth of every single podocyte that covers the glomerular capillaries. Podocyte-specific knockout of Raptor resulted in initiation of proteinuria between the second and fourth week after birth and smaller glomeruli and glomerulosclerosis during the first year.46 Furthermore, podocyte-specific expression of a mutant construct of the mTORC1 downstream target 4E-BP1, which regulates cap-dependent translation, caused proteinuria and glomerulosclerosis related to body weight and to glomerular enlargement in rats.74 These effects could be prevented by caloric restriction, diminishing the increase of body weight and glomerular size.74 Recently, a cellular compartment has been described in human diploid fibroblasts undergoing senescence; this compartment, termed TOR-autophagy spatial coupling compartment (TASCC), facilitates the dual activation of autophagy and mTOR-dependent protein synthesis and cell growth.75,76 The TASCC is located in close proximity to the trans-Golgi network and provides high levels of lysosomes, autolysosomes, and mTOR. mTORC1 is recruited to the (auto)lysosomal surface by the autolysosome-generated amino acid–rich environment and is activated by Rheb. This facilitates and stimulates synthesis of secretory proteins, which are required to retain the senescence phenotype, and reinforces lysosome biogenesis in the rough ER-Golgi apparatus. Autophagosomes are excluded from the TASCC but move to it during maturation.75,76 This spatial separation allows the autophagosome formation to remain unaffected by the mTORC1-dependent autophagy suppression. In vivo, a TASCC-like structure was detected in podocytes, which might enable postmitotic podocytes to activate both mTORC1-dependent protein synthesis and autophagy-dependent degradation of proteins and organelles (Fig. 3A and B).75,76 Notably, the synthesis of secreted factors, such as vascular endothelial growth factor, is required to maintain the filtration barrier.77 Podocyte-specific knockout of mTor caused a phenotype similar to the double knockout of Raptor and Rictor.46,78 Knockout mice developed severe proteinuria between the second and fourth week after birth, followed by podocyte degeneration, glomerulosclerotic destruction of the capillary tuft, and death of the mice between 8 and 12 weeks. mTor knockout podocytes displayed increased levels of LC3-II. In addition, it was proposed that deletion of mTOR results in the enhanced formation of autophagosomes as well as the accumulation of autolysosomes owing to failure of the mTOR-dependent so-called ALR in these podocytes.78 It recently was shown in normal rat kidney cells that during prolonged starvation, autophagy-dependent reactivation of mTOR is required to recycle lysosomal membrane components from autolysosomes and to form autolysosome-derived

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protolysosomes, which mature into new lysosomes.67 Knockdown of mTOR resulted in giant autolysosomes in these cells. In addition, fibroblasts derived from patients with lysosomal storage diseases, such as Fabry disease, showed impaired mTOR reactivation and defective lysosomal reformation under starvation.67 However, the specific functional deletion of mTORC1 by knockout of Raptor in podocytes resulted in a very distinct phenotype compared with that of a podocyte-specific complete mTor knockout or double knockout of both Raptor and Rictor.46,78 Raptor PcKO mice developed stable proteinuria and increasing glomerulosclerosis during the first year. Accumulation of LC3-II was not evident in these podocytes in vivo, and no giant autolysosomes were detected.46 These findings suggest that the phenotype of abrogated mTORC1 signaling in podocytes is not caused mainly by dysregulated autophagy.

SUMMARY AND OUTLOOK Recent research has highlighted the role of autophagy for the cellular homeostasis of podocytes in health and disease. Autophagy appears to be an important cytoprotective process that mediates protective effects in both glomerular maintenance and glomerular injury. Future work will have to elucidate further the interplay of glomerular mTOR signaling and autophagy in the course of aging, diabetic nephropathy, FSGS, glomerulonephritis, and lysosomal storage diseases. Moreover, the role of autophagy as a potential therapeutic target for the treatment of glomerulopathies needs to be evaluated. Recent studies have indicated that pharmacologic induction of autophagy enhances clearance of toxic proteins and diminishes the progression of neuronal degeneration in cell and animal models of neurodegenerative diseases such as Huntington disease and Parkinson disease.79–83 Next to mTOR-dependent induction of autophagy by rapamycin, several mTOR-independent autophagy enhancers such as lithium, rilmenidine, and trehalose have been identified and evaluated in neuronal disease models.84–87 It will be exciting to explore, if these already existing experimental treatment strategies for neurodegenerative diseases potentially can be transferred and adopted for the treatment of glomerular diseases.

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