Podocytes: Gaining a foothold

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Available online at www.sciencedirect.com

www.elsevier.com/locate/yexcr

Review Article

Podocytes: Gaining a foothold Puneet Garga , Lawrence B. Holzmanb, c,⁎ a

Division of Nephrology, University of Michigan, Ann Arbor, MI, USA Renal-Electrolyte and Hypertension Division, University of Pennsylvania, Philadelphia, PA, USA c Department of Veterans Affairs, Philadelphia, PA, USA b

A R T I C L E I N F O R M A T I O N

A B S T R A C T

Article Chronology:

In an attempt to understand the basis of glomerular disease, significant progress has been made in

Received 24 October 2011

understanding the mechanisms that determine podocyte development and the maintenance of

Accepted 24 February 2012

podocyte health. This review examines recent advances in this area focusing on the podocyte

Available online 7 March 2012

intercellular junction, actin cytoskeletal dynamics, and determinants of podocyte cell polarity, autophagy and mTOR biology. Published by Elsevier Inc.

Keywords: Kidney Podocyte Intercellular junction Cytoskeleton Glomerulus

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Podocyte development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Podocyte polarity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Nephrin and its associated complex in podocyte intercellular junction formation . MOTOR-ing podocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Crosstalk between podocyte, basement membrane and endothelial cells . . . . . . . . . Podocyte maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of mTOR in podocyte homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . Autophagy and podocyte homeostasis . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author at: Renal-Electrolyte and Hypertension Division, Hill Pavilion, 380 S. University Avenue, Philadelphia, PA 19104, USA. Fax: +1 215 898 1830. E-mail address: [email protected] (L.B. Holzman). 0014-4827/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.yexcr.2012.02.030

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Introduction The human kidney contains approximately a million individual filtering units called glomeruli composed of specialized capillaries that are surrounded by a basement membrane and glomerular epithelial cells or podocytes. These capillary tufts are structurally supported by modified smooth muscle-like cells called mesangial cells. Though all glomerular components are important for proper glomerular development and function, the podocyte has recently been the focus of numerous investigations. Over the past decade, identification of human diseases with podocyte-specific gene mutations and observations from animal models and cell culture studies have led to intense scientific interest in the role of podocytes in glomerular diseases (Table 2). Although investigation of the mechanisms of glomerular disease during this period has focused largely on this cell, there is growing evidence to support the importance of the inter-dependence between all components of the glomerulus. Study of podocyte biology can be broadly divided into investigation of mechanisms and physiology that define podocyte development, the maintenance of mature podocyte health, and the response of podocytes to injury. Podocyte development involves the metamorphosis of a cuboidal epithelial cell sitting on the glomerular basement membrane into a cell with octopus-like primary, secondary, and tertiary (or foot) processes that interdigitate to form unique intercellular junctions called the slit diaphragm. Podocyte maintenance in maturity requires cell signalingdependent structural changes to maintain filter integrity and to maintain glomerular health. In most glomerular disease states the podocyte undergoes dramatic morphological change best seen by scanning electron microscopy involving spreading and shortening of its finger-like tertiary processes, a process termed “foot process effacement”. While the morphological appearance of podocytes found in many glomerular diseases might be indistinguishable, it is likely that the underlying molecular disease mechanisms that cause these changes in morphology are distinct. Understanding the unique molecular mechanisms that are responsible for these differences will ultimately aid us in designing specific therapies for individual disease conditions. In the following discussion, we will review recent progress in understanding mechanisms inherent in podocyte development and in the maintenance of podocyte health.

Podocyte development Podocyte precursor cells are polarized cuboidal epithelial cells that undergo differentiation with emergence of octopus-like processes that ultimately extend to surround glomerular capillaries. These processes possess a highly organized three-dimensional structure with three levels of branching. Primary processes arise from the podocyte cell body and divide further to form secondary processes. Both the primary and secondary processes are primarily microtubule and intermediate filament-based structures. Additional processes arising from secondary processes form finger-like actin-based tertiary or foot processes that interdigitate in the plane of the basement membrane. The molecular mechanisms that determine the metamorphosis from podocyte precursors to differentiated podocytes remain poorly understood.

Podocyte polarity Cellular polarity or asymmetric distribution of cellular constituents including membrane proteins and organelles is a fundamental property that determines cell structure and function. Cell plasma membranes are polarized in the axis perpendicular to its basement membrane into apical and basolateral domains demarcated by a cell–cell junction. Morphological examination of early podocyte development revealed migration of the podocyte cell–cell junction from an apico-lateral to basolateral position prior to emergence of nascent primary processes that extend along the glomerular basement membrane [1,2]. It is remarkable that beyond the emergence of these initial podocyte processes, studies have not been performed that image the morphogenesis of secondary and tertiary podocyte processes. Therefore, it is not surprising that we have only a rudimentary understanding of the molecular mechanisms that provide the cues that initiate podocyte differentiation and ultimately define podocyte architecture. Evolutionarily conserved protein complexes that conspire to specify polarity in a variety of other polarized cell types also appear to play a role in establishing polarity in podocytes [3,4]. Among the established polarity complexes known, the Par3– Par6–aPKC complex is necessary for establishing and maintaining polarity in polarized epithelia and neurons. In polarized epithelial cells, the par3/par6/aPKC complex facilitates asymmetric targeting of proteins that maintain the functional differences between the apical and basolateral domain [5,6]. In podocyte precursors, the par3/par6/aPKC polarity complex is initially targeted to the apicolaterally-placed adherens junction. With cellular differentiation, this polarity complex moves with this cell junction toward the basement membrane before apparently taking up a position with the forming tertiary process intercellular junction. While mice engineered to obtain podocyte-specific deletion of aPKC lambda/iota (activated protein kinase C) exhibited normal podocyte development at birth [4], these animals ultimately exhibited abnormal podocyte morphology, proteinuria and glomerulosclerosis post-gestation, suggesting that there is an ongoing role of par3/par6/aPKC complex proteins in maintenance of podocyte morphology in maturity. The molecular mechanisms that define podocyte process patterning are of interest because determinates of these patterns might be disordered in podocyte disease or might be employed following podocyte injury to restore normal morphology. In addition to apicolateral polarity podocytes also exhibit polarity in the plane of the tissue defined by the unique pattern assumed by their interdigitating processes. To obtain this unique patterning, these cells appear to employ molecular mechanisms similar to those used in axonal path finding or in patterning of other complex tissues. For example, Nephrin and Neph1 are cell adhesion molecules of the Ig superfamily that are specifically targeted to podocyte intercellular junctions. When deleted in human inherited disease or in mouse genetic mutant models, absence of Nephrin or Neph1 results in a developmental phenotype in which podocyte tertiary process formation and podocyte intercellular junction is dramatically disrupted, suggesting that these proteins are required for normal podocyte patterning [7,8]. The hypothesis that Nephrin and Neph1 participate in tissue patterning is supported by evidence of the function of their homologs hibris and roughest in developing Drosophila compound eyes [9,10]. The fly eye is a complex tissue assembled from

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approximately 750 individual units, the ommatidia. Ommatidia are separated from each other by interommatidial cells (IOC). During development, IOCs are sorted from multiple rows into a single layer of cells. During this process, local contacts between primary pigment cells and IOCs are essential in determining where the IOC will be located in the adult eye or whether it is dispensable and is eliminated by apoptosis [9]. This process is precisely regulated by interactions between Nephrin homolog Hibris which is expressed on primary pigment cells and Neph1 homolog Roughest on juxtaposed IOCs since in Hibris and Roughest mutant fly eyes IOCs fail to move into their proper niche and form aberrant junctions [9]. Additional studies in Caenorhabiditis elegans suggest an important role of nephrin family proteins in tissue patterning by specifying cell junction formation. The nephrin and neph1 homologs syg2 and syg-1 appear to provide “guidepost” signals that specify the unique spatial relationship between an extending neuron and formation of a nascent synapse with its target cell [11,12]. Syg-1 and -2 mutants exhibit fewer synapses with their natural targets and form aberrant synapses with incorrect target cells presumably because the extending neurite fails to recognize its target cell for synapse formation [11–13]. Unlike hibris and roughest that are expressed uniquely on Drosophila primary pigment cells or IOCs, mammalian nephrin and neph1 are both expressed on homologous neighboring podocytes [14]. While the consequences of this distribution of nephrin and neph1 are not presently understood, there are instances in nature where transmembrane proteins and their ligands interact simultaneously in both cis and trans fashion with important functional consequences [15]. For example, Eph/ephrin interactions result in complex bidirectional signaling events that are induced uniquely when these interactions occur in cis or in trans and that determine unique tissue patterning events [16]. Similarly, interaction in trans of the receptor Notch with its membrane bound ligand delta results in nuclear signaling, while cis interaction results in attenuation of Notch signal transduction by endocytosis [17,18].

Role of Nephrin and its associated complex in podocyte intercellular junction formation Investigation of the signaling mechanisms by which nephrin and neph1 influence podocyte process patterning and cell junction formation revealed their ability to assemble a protein complex that can regulate actin cytoskeletal dynamics [19–22]. Early studies showed that the nephrin cytoplasmic domain is a target of Src family kinase Fyn [23–25]. Mice where Src kinases fyn and yes were deleted simultaneously developed proteinuria and foot process developmental abnormalities [23] similar to the developmental phenotype observed in nephrin null mice [26]. This suggested that nephrin phosphorylation is an early event necessary for podocyte foot process development. Similarly, podocyte injury models using puromycin aminonucleoside and protamine sulfate also show an increase in nephrin tyrosine phosphorylation [22,27] suggesting a related role of nephrin mediated signaling in podocyte injury/repair. A growing body of evidence suggests that once tyrosine phosphorylated, nephrin assembles a large protein complex that collaborates to regulate actin dynamics and by doing so, participates in regulating podocyte morphogenesis or response to injury (See Table 1). In artificial systems, induction of nephrin tyrosine phosphorylation results in actin filament nucleation and

Table 1 – Mouse nephrin tyrosine residues with their motifs and known interacting partners. Tyrosine number Motif Phosphorylation (mouse nephrin) Y1128

YEES

Inconclusive

Y1153

YYSM Yes

Y1154 Y1172 Y1191 Y1198 Y1208 Y1216 Y1225 Y1232

YYSM YRQA YDEV YGPP YDEV YDLR YEDP YDQV

Yes Inconclusive Yes Yes Yes Inconclusive Yes Inconclusive

Adaptor protein Direct PI3 kinase p85 subunit PI3 kinase p85 subunit

Indirect Cofilin

Cofilin

Nck Nck

PLC γ

Nck

elongation [20,21] in an Nck1/2-dependent fashion. The concept that activated nephrin assembles an Nck-dependent actin polymerization complex to influence podocyte process patterning is strengthened by the observed abnormality of podocyte foot process development following simultaneous deletion of Nck1 and 2 in a podocyte-specific fashion [21]. Whether the podocyte phenotype observed in this model is truly due specifically to the Nephrin–Nck interaction or to Nck function in another cell compartment requires further investigation. Nephrin's ability to regulate actin filament polymerization is augmented by Neph1 [19], recapitulating mechanisms employed in nature by pathogens to recruit an actin polymerization complex in host cells [28–30]. Nephrin also recruits other actin associated proteins like nWASp, Arp2/3 and cofilin which not only help in nucleating actin filaments but also are known to generate and maintain a branched actin network [31–33]. Nephrin's ability to recruit pI3 kinase at the membrane generates phosphoinositol moieties that play an important role in localizing signaling proteins including actin regulatory proteins to the inner wall of the membrane following an external stimulus.

MOTOR-ing podocytes Because kinesin and other non-muscle myosin motor proteins play well recognized roles in cellular function, survival and morphogenesis, it is not surprising that an expanding body of work has demonstrated that a variety of motor proteins play a critical role in podocyte development and function. Nevertheless, it might not have been anticipated that mutations in genes encoding these proteins are associated with human disease. For about a decade it has been known that missense mutations in non-muscle myosin heavy chain myh9 are associated with rare forms of FSGS associated with macrothrombocytopenia in Epstein's or Fetchner's syndrome. More recently, genome-wide association studies linked non-coding single-nucleotide polymorphisms (SNPs) at the nonmuscle myosin heavy chain myh9 locus to a dramatically increased risk of chronic kidney disease among patients of west African descent, particularly glomerular diseases such as HIV associated nephropathy and FSGS associated with hypertension [34,35]. Further, it has been suggested that myh9 serves as a

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Table 2 – List of human genetic mutations and deletion or transgenic genetic mouse models that result in proteinuria and/or foot process developmental abnormalities. Gene/protein Podocytes NPHS1/Neprhin NPHS2/podocin PLC epsilon 1 Nck Fyn Fyn/yes combined Kirrel/Neph1 TRPC6 Combined CD2ap/Fyn ACTN4/alpha-actinin-4 Notch1 Transgenic NFAT transgenic uPAR Focal adhesion kinase COQ2/coenzyme Q10 HGF/C-met

Human/animal

Disease/phenotype

Human/mouse Human Human/zebra fish Mouse Mouse Mouse Mouse Human Mouse Human/mouse Mouse Mouse Mouse Mouse Human Mouse

FP effacement/proteinuria FP effacement/proteinuria Diffuse mesangial sclerosis (DMS) FP formation defect Subtle changes in FP FP effacement/proteinuria FP effacement/proteinuria FSGS FSGS FSGS FSGS FSGS NA NA FSGS Normal

aPKC lambda/iota PDSS2/Prenly Diphosphate Synthase Subunit 2 Synaptopodin Cfl1/Cofilin 1

Mouse Human/mouse

FSGS/FP effacement/proteinuria FP effacement/proteinuria

Mouse Mouse/zebra fish

Normal FP effacement/proteinuria

Sidekick transgene INF2 ATG5 PI3Kc II EP4 receptor transgenic

Mouse Human Mouse Mouse Mouse

FSGS FSGS Glomerulosclerosis/proteinuria DMS/FP effacement/proteinuria Proteinuria

AT1 transgenic Beta catenin Podocalyxin FAT1

Mouse Mouse Transgenic mouse Mouse

GLEPP1 VEGF heterozygous deletion VEGF transgenic Myo1E Myo1E ArhGap24

Response to injury

Age of onset

NA NA NA NA NA NA NA NA NA NA NA NA Protected Protected NA Proteinuria following adriamycin NA NA

At birth Late Early At birth Early Early Early NA Late Late NA Early NA NA Late

NA Late

FP effacement NA FP formation defect FP effacement/proteinuria

Slow Recovery Abnormal foot process morphology NA NA NA NA 5/6th Nephrectomy required NA Protected NA NA

Broadening of FP/no proteinuria Endotheliosis/proteinuria Collapsing glomeruolopathy FP effacemet/proteinuria Focal segmental glomerulosclerosis NA

NA NA NA NA NA Late

C-mip transgenic Tsc1/transgenic mTORC1 mTORC1 (Raptor) mTORC2 (Rictor) Myh9

Mouse Mouse Mouse Mouse Human Human focal segmental glomerulosclerosis Mouse Mouse Mouse Mouse Mouse

Proteinuria/normal FP Diabetic glomerulosclerosis Glomerulosclersosis/proteinuria Normal/develop Proteinuria on injury Normal

NA NA NA BSA overload Proteinuria following adriamycin

8 weeks Early 8 weeks NA NA

Glomerular basement membrane Lamb2/laminin β2

Human/mouse

DMS/FSGS (Pierson syndrome)

NA

Beta 1 Integrin Integrin alpha 3 subunit

Mouse Mouse

NA NA

Integrin linked kinase

Mouse

Proteinuria Disorganized GBM/FP formation defect/proteinuria GBM alteration/FSGS

Perinatal lethality At birth Early

NA

Early

Tubules Cubulin Megalin

Mouse Mouse

Normal/minimal proteinuria LMW proteinuria

NA NA

NA Early

Transcription factors WT1

Human

Denys Drash and Frasier Syndrome

NA

Early

Late Late

Late Late Late Late NA Early NA NA Perinatal lethality NA Early Early Late Late

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Table 2 (continued) Gene/protein

Human/animal

Transcription factors PTIP Mouse LMX1B/LIM homeobox Human transcription factor SMARCAL1 Human

Disease/phenotype

Response to injury

Age of onset

Proteinuria Nail Patella syndrome

NA NA

Late Early

FSGS/steroid resistant NS

NA

Late

susceptibility locus, predisposing podocytes to a second disease initiating hit since deletion of myh9 from typically glomerular injury resistant C57B/6 mice predisposes these mice to acute podocyte injury [36]. However, the significance of these findings has been challenged by identification of non-synonymous mutations in the ApoL1 gene immediately neighboring the myh9 genetic locus which when homozygous are linked to markedly increased susceptibility to FSGS in subjects of African descent with a probability far exceeding that found for SNPs within the myh9 locus [35]. While the identified missense mutations in ApoL1 also confer resistance to human African trypanosomiasis caused by Tyrpanosoma brucei rhodesiense and explain its evolutionary selection [37], a causal relationship between these mutations and kidney disease susceptibility has not yet been identified. In addition to work on myh9, recent investigations have demonstrated additional roles for non-muscle myosin proteins in podocytes. The actin-based motor protein myo1c appears to be necessary for transporting neph1 to the podocyte intercellular junction and might be anticipated to be necessary in podocyte development or response to injury [38]. In addition, mutations were identified in non-muscle myosin 1e (Myo1E) in two families with focal segmental glomerulosclerosis [39] and a missense variant within the Myo1E gene in patients with steroid resistant nephrotic syndrome [39]. The mechanism by which these mutations affect glomerular structure and function is yet to be identified. Myo1E is a membrane-associated class I myosin with motor head domain that binds F-actin and ATP. In cell culture, inhibition of Myo1E function results in defects in endocytosis [40] and deletion of Myo1e in mice resulted in podocyte foot process spreading and proteinuria [41].

Crosstalk between podocyte, basement membrane and endothelial cells The glomerular basement membrane (GBM) is produced and maintained by both podocytes and glomerular endothelial cells and abnormalities of the glomerular basement membrane can alter the function of both podocytes and endothelial cells. Podocytes adhere to the GBM via adhesion proteins that include integrins. There are abundant data pointing to the importance of integrin-related signaling in determining podocyte behavior. Integrins are heterodimeric transmembrane receptors composed of an α and a β subunit. In podocytes, α3- and β1-integrin are most abundant. Mice deleted of α3- or β1-integrin develop nephrotic syndrome and foot process developmental defects [42–44]. Integrin-linked kinase (ILK) associates with the cytoplasmic domain of β-integrin [45] and contributes to integrin-mediated signal transduction. Mice deleted of ILK in a podocyte-specific manner exhibit foot process effacement, basement membrane abnormalities, proteinuria and progressive glomerulosclerosis [46].

Integrin heterodimer cluster, are linked to the actin cytoskeleton, and signal to focal adhesion protein complexes at the basal aspect of cells. Regulation of the assembly and disassembly of focal adhesions plays a vital role in movement of cells on their underlying basement membrane. Focal adhesion kinase (FAK) a cytoplasmic receptor tyrosine kinase links the actin cytoskeleton via talin [47] and serves as a signaling platform or scaffold that participates in regulating cellular adhesion, motility and spreading [48,49]. In breast cancer cells, inhibition of FAK results decreased mobility and the ability to metastasize in vivo [50,51]. In podocytes, deletion or inhibition of FAK [52] blocks foot process effacement and proteinuria in injury models suggesting that disruption or turnover of podocyte-GBM adhesions is required for movement of podocytes over the GBM in disease states. Best exemplified by the paracrine effects on glomerular endothelial cells of VEGF expressed by podocytes, podocytes also influence and are influenced by neighboring cells in the glomerulus. In the absence of podocyte generated VEGF, migration of endothelial cells into the forming glomerulus [53,54] during glomerular development is impaired. Post-gestation, both deficiency and excess VEGF in the glomerulus has deleterious effect on the filter. Excess VEGF results in a proliferative phenotype with basement membrane thickening, mesangial expansion and an inflammatory infiltrate [53] whereas deficiency of VEGF results in endothelial cell swelling and formation of microthrombi recapitulating thrombotic microangiopathic disease. The importance of maintaining a glomerular VEGF homeostatic set point has been emphasized by observations both in human diseases and in therapeutic interventions that affect VEGF signaling. Inhibition of VEGF by bevacizumab to treat diseases such as cancer that require pathological angiogenesis has resulted in proteinuria and in renal thrombotic microangiopathy in a minority of patients [55]. In murine models, administration of VEGF-blocking antibodies or expression of sFlt (soluble fms-like tyrosine kinase), the soluble VEGF receptor, caused a syndrome similar to pre-eclampsia characterized by endotheliosis, proteinuria and hypertension [56]. sFLT competitively inhibits VEGF for its binding with the VEGFR [56,57] and has been identified as a major mediator of pre-eclampsia.

Podocyte maintenance Podocytes are terminally differentiated cells that are not thought to undergo renewal [58,59]. Consequently, it has been suggested that segmental glomerular scarring is proportional to the extent of permanent podocyte loss incurred during injury [60–63]. Short of loss-induced segmental sclerosis, some have speculated that podocytes must remain motile to maintain podocyte health in the face of a hostile environment or to maintain coverage of

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the GBM following neighboring podocyte loss [62]. In support of this hypothesis, human mutations in actin-associated proteins, including podocin, α-actinin 4 [64] and INF2 [65] result in late manifestation of proteinuria and glomerulosclerosis. Rho family small GTPases are known regulators of cytoskeletal dynamics and play an important role in intercellular junction maintenance [66,67]. Recently, the GTPase activating protein Arhgap24 was associated with familial focal segmental glomerulosclerosis [68]. In the glomerulus Arhgap24 is enriched in podocytes where it co-localizes with the slit diaphragm marker synaptopodin [68]. Arhgap24 inactivates Rac1 and appears to disrupt the equilibrium between RhoA and Rac1 signaling [68]. Several additional studies indicate that decreased RhoA and increased Rac1 activity is harmful to the mature podocyte and is associated with proteinuria and foot process effacement [69,70]. For example, mice deleted of the GDP dissociation inhibitor Rho GDIα exhibit normal podocyte development yet develop a delayed FSGS phenotype [71]. These observations may indicate that proper balance between RhoA and Rac1 signaling is important for maintaining podocyte junction stability and add evidence supporting the importance of cytoskeletal dynamics in maintaining podocyte health.

Role of mTOR in podocyte homeostasis Target of rapamycin (TOR) is a central regulator of cellular growth and metabolism that integrates information about the cell's environment including nutrients, access to growth factors and cellular energy status [72,73]. TOR is conserved from yeast to mammals and exists in two distinct multi-protein complexes namely, TOR complex-1 (TORC1) and TORC2. The two complexes contain shared components including mTOR (mammalian TOR), and complex-specific components that include the protein raptor found in TORC1 and rictor found in TORC2. Rapamycin sensitive mTORC1 regulates diverse cellular processes like protein synthesis, ribosome biogenesis and autophagy whereas the rapamycin insensitive mTORC2 complex is involved in regulation of actin cytoskeleton dynamics. Two recent studies demonstrated that altering podocyte mTOR pathway activity above or below a homeostatic set point results in proteinuria and glomerulosclerosis producing histology reminiscent of diabetic nephropathy [74]. Podocytes exhibit low mTOR activity at maturity. Nevertheless, Godel et al. reported proteinuria and glomerulosclerosis when mTORC1 complex activity was interrupted by deleting raptor, a phenotype that was made more severe by disrupting both mTORC1 and mTORC2 activity simultaneously [75]. Inoki et al. reported that podocyte morphology was also disrupted by activating mTORC1 activity by deleting TSC1, an upstream inhibitor of mTOR [74]. Intriguingly, treatment of animals with mTOR inhibitor rapamycin in this mTOR activation model resulted in formation of new podocyte processes equivalent to re-initiation of the developmental program [74]. These studies suggest that healthy podocytes require tight regulation of mTOR activity and emphasize the vital role that mTOR activity plays in both development and maintenance of podocyte structure and function.

Autophagy and podocyte homeostasis Autophagy is the process by which cells degrade its own components through the lysosomal machinery. Autophagy is an important mechanism by which cells are able to reallocate nutrition and energy to essential cellular processes. It is also an important

part of cellular development and growth, helping in maintaining a balance between synthesis, degradation and recycling of cellular components. Typically, terminally differentiated cells like neurons exhibit low basal rates of autophagy [76,77], while hepatocytes that turn over rapidly demonstrate high rates of autophagy [78,79]. Podocytes appear to be unusual since they exhibit a high basal rate of autophagy even as terminally differentiated cells. Indeed, routine transmission electron micrographs of healthy podocytes reveal numerous vesicles in the cytoplasm resembling autophaghic vacuoles [80] and the examination of the GFP-LC3 (atg8) transgenic mouse model reveal that podocytes contain autophagosomes under basal conditions [80]. It has been hypothesized that the terminally differentiated podocyte requires autophagy to sustain its terminally differentiated state (reviewed in [81]). Supporting this hypothesis, a recent report identified TORautophagy spatial coupling compartment (TASCC) in podocytes, where (auto) lysosome and mTOR accumulate [82]. TASCC represent a sub-cellular compartment where both the synthetic and degradation machinery of the cells couple to improve the cell's regenerative capacity. In podocyte deletion of Atg5, an important component of the autophagic phagosomes, results in early aging of the podocytes [80]. Together, these studies indicate that podocytes might employ autophagy to sustain longevity in the context of a hostile environment.

Conclusions Accumulating evidence indicates that maintenance of the unique morphology of podocytes and long term maintenance of podocyte health is essential for its function in supporting the glomerular filter. Molecular events that occur during podocyte development are recapitulated during podocyte injury and subsequent recovery. For these reasons, the continued study of podocyte development and the mechanisms that support podocyte homeostasis should lead to a better understanding of the mechanisms underlying various glomerular disease processes and might provide potential therapeutic targets useful for preventing podocyte loss and progression to end stage kidney disease.

Acknowledgments This work was supported by grants to L.B.H. from the NIDDK (DK080751) and the Department of Veterans Affairs, and to P.G. from the NIDDK (K08 DK081403).

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