Proteostasis in endoplasmic reticulum--new mechanisms in kidney disease.

REVIEWS Proteostasis in endoplasmic reticulum —new mechanisms in kidney disease Reiko Inagi, Yu Ishimoto and Masaomi Nangaku Abstract | Cells use an exquisite network of mechanisms to maintain the integrity and functionality of their protein components. In the endoplasmic reticulum (ER), these networks of protein homeostasis—referred to as proteostasis—regulate protein synthesis, folding and degradation via the unfolded protein response (UPR) pathway. The UPR pathway has two components: the adaptive UPR pathway, which predominantly maintains the ER function or ER proteostasis, and the apoptotic UPR pathway, which eliminates dysfunctional cells that have been subject to long-term or severe ER stress. Dysregulation of the UPR pathway often occurs in glomerular or tubulointerstitial cells under a pathogenic microenvironment, such as oxidative stress, glycative stress or hypoxia. A defective UPR is highly deleterious to renal cell function and viability and is thereby implicated in the pathophysiology of various kidney diseases. Accumulating evidence provides a link between the UPR pathway and mitochondrial structure and function, indicating the important role of ER proteostasis in the maintenance of mitochondrial homeostasis. Restoration of normal proteostasis, therefore, holds promise in protecting the kidney from pathogenic stresses as well as ageing. This Review is focused on the role of the ER stress and UPR pathway in the maintenance of ER proteostasis, and highlights the involvement of the derangement of ER proteostasis and ER stress in various pathogenic stress signals in the kidney. Inagi, R. et al. Nat. Rev. Nephrol. advance online publication 22 April 2014; doi:10.1038/nrneph.2014.67

Introduction

Division of Chronic Kidney Disease Pathophysiology (R.I.), and Division of Nephrology and Endocrinology (Y.I., M.N.), The University of Tokyo Graduate School of Medicine, 7‑3‑1 Hongo, Bunkyo-ku, Tokyo 113‑8655, Japan. Correspondence to: R.I. [email protected]

Protein homeostasis, or proteostasis, is conducted via sophisticated networks of mechanisms that act to main tain the quality of proteins and the evolutionary diver sity of the biological functions of proteins.1,2 Proteostasis, therefore, exerts substantial influence on cell homeostasis or, in other words, on cell fate. Proteostasis networks in eukaryotes are composed of intricate linkages between cytosolic proteostasis and organelle proteostasis, includ ing the endoplasmic reticulum (ER) and mitochondria. These networks act together to reduce proteotoxic stress. The cytosolic proteostasis networks include the protein folding system with heat shock proteins and the machin ery for degradation of unfavourable (that is, misfolded, aggregated or dysfunctional) proteins with the ubiquitin– proteasome system or autophagy–proteasome pathway. As for the organelles, proteostasis in the ER is carefully orchestrated by mechanisms for protein synthesis, folding, trafficking and degradation—all of which are subject to control by the unfolded protein response (UPR) pathway. Proteostasis in the mitochondria regulates the synth esis and folding of mitochondrial proteins to optimize mitochondrial homeostasis via the mitochondrial UPR.3 The ER and mitochondrial UPR pathways have important roles in sensing the accumulation of unfolded proteins in the ER and mitochondria, respectively, and transmit signals out of the organelle to the nucleus, where genes encoding the chaperones or molecules related to Competing interests The authors declare no competing interests.

the unfolded protein degradation system are upregu lated.3 The two UPR pathways are conceptually similar, but are conducted by organelle-specific UPR molecules. Both pathways are rapidly activated and crosstalk with each other when cells respond to extrinsic stimuli, such as temperature change, hypoxia, oxidative stress and metabolic disorder.3 This rapid activation and crosstalk mechanism indicates a physiological effect of proteostasis networks on the maintenance of protein function and the determination of cell fate. When the force of proteostasis fails to protect cells from pathogenic stimuli, or the proteostasis machinery is deranged by a stressful microenvironment, cell func tion and viability are decreased. Insufficient proteo stasis has been suggested to have a pathophysiological role in various diseases, such as Parkinson disease and Alzheimer disease. 4,5 In particular, renal pathogenic factors that include proteinuria, uraemic toxins and metabolic derangement act in association with renal cell dysfunction to disrupt proteostasis.6 Importantly, these phenomena are often associated with the activation of intracellular stress signals—for example, ER stress, oxidative stress or hypoxia—indicating a close link between proteostasis and stress.6 Thus, the maintenance of normal proteostasis is important for the protection of organs against adverse environments. In this Review, we focus on the role of stress and the UPR pathway in the maintenance of ER proteostasis, and on the involvement disrupted ER proteostasis has in various pathogenic stress signals in the kidney.

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REVIEWS Key points ■■ Endoplasmic reticulum (ER) proteostasis is regulated by the adaptive unfolded protein response (UPR) pathway, which determines cell fate and maintains cell structure and function ■■ Stress (hypoxia, inflammation and oxidative and glycative stress) disturbs ER proteostasis; cells subjected to long-term or severe ER stress are cleared via the apoptotic UPR pathway ■■ In kidney cells, including glomerular cells, tubular cells and interstitial cells, derangement of ER proteostasis leads to development and progression of kidney diseases ■■ Optimizing ER proteostasis using pharmacological approaches, such as small-molecule UPR modulators, might prove beneficial to prevent and treat kidney diseases

ER

Cytoplasm ER stress

UPR

P ATF6

P

eIF2α P

Golgi apparatus

Ca2+

P

P XBP1

Cleavage

ER stress

IRE1

PERK

Mfn2

TRAF2

UPRmt

ASK1 JNK

Mitochondrial fusion/fission

Cyt c P

Mitochondria Cyt c

Adaptive UPR genes ATF6(p50)

ATF4

XBP1s

Cellular maintenance

Apoptotic UPR genes CHOP Apoptosis

Nucleus

Figure 1 | UPR pathway in ER proteostasis. Under ER stress, the UPR modulators PERK and IRE1 are activated by dimerization followed by phosphorylation, and ATF6 is cleaved in the Golgi apparatus, inducing the activation of UPR transcription factors ATF6 (p50), ATF4 and XBP1s. These transcription factors mainly upregulate the adaptive UPR pathway and normalize ER function via the ATF6, PERK–eIF-2α– ATF4 or IRE1–XBP1 pathways. Under long-term ER stress, the adaptive UPR pathway fails to rescue the cells, and the apoptotic UPR pathway, namely the PERK–eIF-2α–ATF4–CHOP or IRE1–TRAF2–ASK1–JNK pathway, is induced. The interaction of ER and mitochondria (via Mfn2) also has an important role in the maintenance of mitochondrial fusion and fission, optimization of the UPR mt and induction of Ca2+–Cyt c‑dependent apoptosis signalling. The balance between the adaptive and apoptotic UPR pathways is closely associated with glomerular and tubulointerstitial cell functions, and disruption of this balance can result in kidney disease. Abbreviations: ASK1, apoptosis signal-regulating kinase 1; ATF, activating transcription factor; CHOP, C/EBP-homologous protein; Cyt c, cytochrome c; eIF-2α, eukaryotic translation initiation factor 2α; ER, endoplasmic reticulum; IRE1, inositol-requiring protein 1; JNK, c‑Jun N‑terminal kinase; Mfn, mitofusin; P, phosphate; PERK, pancreatic eIF-2α; TRAF2, tumour necrosis factor receptorassociated factor 2; UPR, unfolded protein response; UPRmt, mitochondrial UPR; XBP1, X‑box binding protein 1; XBP1s, spliced form of XBP1.

Role of the UPR pathway The UPR pathway controls the core proteostasis net works in the ER. The pathway is a stress signal that is triggered by conditions associated with ER dysfunction, namely ER stress, which includes physiological stress associated with organ development 7 and pathogenic stress associated with the development and progres sion of various diseases, including kidney disease.6 The UPR pathway is the predominant adaptive response to

ER stress conditions. Once the adaptive UPR pathway is overcome by severe and/or long-term ER stress, it can no longer maintain ER proteostasis and the UPR-mediated proapoptotic signal becomes dominant, thereby inducing apoptotic cell death of the damaged cells. Although the mechanisms that convert an adaptive UPR to an apop totic UPR remain unclear, the UPR is a double-edged sword; the balance between activation of the adap tive and apoptotic pathways is an important factor in deciding cell fate. The adaptive UPR pathway is regulated by three major ER‑resident transducers—inositol-requiring protein 1 (IRE1), pancreatic eukaryotic translation initiation factor 2α (eIF-2α) kinase (PERK) and activating tran scription factor 6 (ATF6)—that are known as ER stress sensors (Figure 1).6,8,9 These transducers are i nactive under normal conditions via binding with the ER chaper one GRP78 (78 kDa glucose-regulated protein), which is a key sensor of the UPR pathway. When ER function is decreased under pathogenic conditions, unfolded proteins accumulate in the ER lumen. This accumula tion causes GRP78 to dissociate from the transducers and bind to the unfolded proteins to refold them. The transducers are thereby activated by dimerization or t ranslocation, and induce the nuclear translocation of UPR transcription factors, such as X‑box-binding protein 1 (XBP1) and ATF4. 6,8,9 Activated ATF6 is cleaved in the Golgi apparatus to form ATF p50, which acts as a transcription factor. Together, the UPR pro cesses produce a rapid intensification and acceleration of ER proteostasis, involving attenuated translation of a wide range of proteins through the PERK–eIF-2α axis, enhanced protein folding via an increase in ER chaperone expression through the IRE1–XBP1 and PERK–ATF4 axes, and degradation of unfolded and/or misfolded proteins by ER‑associated protein degradation (ERAD) through the IRE1–XBP1 and ATF6 axes (Figure 1). As described above, GRP78 binding to the UPR transducers is involved in UPR activation. Interestingly, several reports have demonstrated that IRE1 activation (oligomerization and phosphorylation) might also occur by direct binding of unfolded proteins. For example, IRE1 mutants that do not bind to GRP78 were shown to activate the IRE1–XBP1 axis, indicating the existence of a GRP78-independent UPR activation pathway.8,9 This phenomenon is supported by the finding that the yeast IRE1 luminal domain contains a site positioned for peptide binding.10 The apoptotic UPR pathway is induced when the adaptive UPR pathway cannot rectify deranged cell homeostasis (Figure 1). Long-term or excessive activa tion of IRE1, PERK or ATF6 causes apoptosis, or inhibits the antiapoptotic signal via activation of CHOP, caspase signalling or the TRAF2–ASK1–JNK axis.6 In parallel, Ca2+ leakage from the ER caused by stress enhances mito chondrial Ca2+ uptake and induces cytochrome c (Cyt c) release, thereby activating Cyt c‑dependent apoptosis signalling.11 Together, these processes trigger the apop totic UPR pathway. These findings reveal a correlation between decreased ER proteostasis and apoptosis.

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REVIEWS a

ER chaperones Ac 3'

Unfolded protein < Mature protein

NH3

+

Folding

NH3

+

Ribosome 5' mRNA

b

Synthesis

Ac P

Post-translational modification

ER Cytoplasm

?

ER stress

ER chaperones

P Unfolded protein > Mature protein Hypoxia, NH3 metabolic Folding + disorder NH3

3'

Ac

+

ERAD

Ribosome 5' mRNA

Me

P

Post-translational modification

Synthesis

Oxidative stress

Ac Acetylation P Phosphorylation Me Methylation

Malfolded protein ER stress

Aggregates

Degradation

Glycative stress

Ubiquitination ? Other post-translational modification

Autophagy

Proteasome

Figure 2 | Proteostasis networks in the ER. a | Under normal conditions, newly synthesized (unfolded) proteins in the ER are folded by the action of ER chaperones and post-translationally modified via acetylation, phosphorylation, methylation or glycosylation into mature and functional proteins. The balance between protein synthesis and protein folding is regulated by the UPR pathway to maintain the quality of proteins and, subsequently, cell homeostasis. b | Under conditions of stress—such as hypoxia, metabolic disorder (for example, hyperglycaemia and dyslipidaemia) and oxidative and glycative stress—imbalance between synthesis and folding of proteins results in accumulation of unfolded or malfolded proteins, a process termed ER stress. These abnormal proteins are transferred to the cytoplasm, degraded by the ERAD pathway, ubiquitinated and subsequently degraded by the proteasome. In addition to the ERAD pathway, autophagy also has an important role in the elimination of these abnormal proteins. The defective UPR pathway in the degradation system exacerbates loss of cell function and cell death. Abbreviations: ER, endoplasmic reticulum; ERAD, ER‑associated protein degradation; UPR, unfolded protein response.

Proteostasis and stress signals Accumulating evidence demonstrates that derangement of ER proteostasis by pathogenic UPR activation is an important contributor to the development or progression, or both, of various diseases, including those of the kidney (Figure 2).6,12–18 The microenvironmental stresses associ ated with alteration of ER proteostasis in kidney disease include hypoxia, oxidative stress and ER stress. The signals induced by these stresses—namely, the hypoxiainducible factor (HIF), nuclear factor e rythroid 2‑related factor 2 (Nrf2) and UPR pathways—intricately cross talk with each other and reflect the manifestation of kidney disease.6

Hypoxia and oxidative stress Hypoxia is induced by an imbalance in the supply and demand of oxygen. Chronic hypoxia is a common patho genetic factor in kidney disease,19 and triggers other stresses, including oxidative stress and ER stress. For example, hypoxia-induced aberrant oxygen metabolism exacerbates the oxidative stress state. In parallel, energy loss owing to aberrant oxygen metabolism decreases ER function, inducing ER stress.19 This evidence suggests a vicious cycle of hypoxia, oxidative stress and ER stress. The likely roles of both hypoxia and oxidative stress as major causal factors of ER stress suggest that they also affect ER proteostasis. Interestingly, reports have demonstrated that renal pathogenic factors that exacerbate renal hypoxia, such as uraemic toxins or metabolic disorder (for example, high glucose or hyperinsulinaemia), cause renal epigenetic changes related to the development and progression of chronic kidney disease (CKD).20–22 The major posttranslational modifications of proteins that act to control proteostasis are acetylation and methylation. A genomewide analysis study involving chromat in immuno precipitation with deep sequencing of the binding sites of HIF‑1α (a transcription factor of HIF pathway) revealed that, under hypoxic conditions, acetylation of lysine 27 of histone 3 covers the HIF‑1α binding sites and that HIF‑1α enhances expression of glucose transporter 3 (GLUT3; encoded by SLC2A3) by interaction with lysine (K)-specific demethylase 3A (KDM3A).23 This finding demonstrates that epigenetic changes in HIF‑1α chromatin conformational structure and histone modi fications under hypoxic conditions induces downstream target gene expression in a ssociation with a change in proteostasis. Epigenetic changes in microRNA (miRNA) expression could also modulate response to oxidative and ER stress. In one study, expression of miR‑205, an miRNA expressed in renal tubular cells, was found to be downregulated under conditions of stress.24 This downregulation was associated with a reduction in antioxidant enzyme expression via changes in the HIF and UPR pathways, by regulation of transcription factors HIF‑1α and ATF4.24 Consistently, expression of other miRNAs has been shown to be regulated by hypoxia, which leads to activation of the adaptive UPR pathway.25,26 Altogether, these results indicate that hypoxia-induced regulation of miRNA expression is involved in UPR pathway activation and, accordingly, in ER proteostasis.25,26 Glycative stress Glycation, which is a nonenzymatic post-translational modification of proteins with carbohydrates (sugars), is known to modify protein function. Glycated proteins with loss of function strongly contribute to the develop ment or progression of various conditions, includ ing CKD with type 2 diabetes mellitus.27 In particular, because glycation is substantially enhanced under oxi dative stress, the phenotypes of oxidative-stress-related diseases or ageing often reflect the pathogenic effects of glycated proteins.28,29 Reports have demonstrated that



REVIEWS glycative stress induces ER stress, which in turn leads to defective ER proteostasis associated with cell damage.30,31 Murine podocytes exposed to glycated albumin show UPR activation, specifically UPR-related apoptosis, and this process is ameliorated by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA).32 Glycative stress is known to be involved in accelerated ageing and decreased longevity.33,34 One study demon strated that under conditions of stress, the receptor for advanced glycation (or glycosylation) end products (RAGE) prematurely promotes a senescence pheno type (via activation of p21 signalling) in murine tubular cells by UPR induction, which indicates that glycative stress triggers ER stress, thereby reducing life span.35 In another study, diet-derived AGE disrupt the protea some activity of proteostasis networks in Drosophila and reduce the life span of flies.36 These findings suggest that glycative stress contributes to the derangement of proteostasis networks.

Inflammation A common outcome of inflammation is the alteration of proteostasis through the demanding protein synthesis of inflammatory cytokines. To avoid this unfavourable situation, the UPR pathway regulates the inflammatory response. For example, the UPR pathway suppresses activation of nuclear factor (NF)-κB, a transcription factor in the inflammatory response. 37 A previous report demonstrated that the cytokine-triggered NF‑κB activation in rat mesangial cells was blunted by the UPR pathway via PERK-mediated and IRE1-mediated preferential induction of C/EBP‑β.38 Interestingly, meta bolic stress via glucose deprivation activates the UPR pathway in cultured human renal cortical tubular cells, which subsequently activates NF‑κB via IRE1 and pro motes the transcription of proinflammatory cytokines and chemokines.39 Furthermore, acute ischaemia acti vates ER stress and inflammation in rat kidneys and human kidney transplant biopsy samples.39 These find ings emphasize the link between inflammation and the UPR pathway, but also indicate that the effect of the UPR pathway on inflammatory responses might differ by cell type or stress condition. Autophagy Autophagy is an important proteostasis system in the cytoplasm.40 In autophagy, misfolded and/or aggregated proteins in the cytosol, many of which threaten cell homeostasis, are first captured and then degraded via lysosomes (Figure 2). In addition to its role in protein degradation, the autophagy system also engulfs damaged ER and mitochondria (through a mechanism called mitophagy) to maintain the structural and functional homeostasis of cells, indicating a close link between autophagy and ER proteostasis. In the kidney, autophagy has an important role in the control of glomerular and tubular cell function.41,42 ER stress induces autophagy in renal proximal tubular cells;43 insufficient autophagy observed in obesity exacerbates proteinuria-induced tubulointerstitial injury.44 Autophagy induced by acute

kidney injury consists of at least two independent path ways, involving the p53–sestrin‑2 and HIF-1α–BNIP3 axes, which might be activated by different types of stress to protect the renal tubules during acute injury.45

Proteostasis and organelle crosstalk The dynamics of mitochondrial structure, which are regu lated by the combined actions of mitochondrial fission and fusion, are controlled by the interaction between the ER and mitochondria.46,47 This control mechanism sug gests that ER proteostasis is essential to sustain the struc ture and function of the mitochondria, or in other words, to maintain mitochondrial homeostasis. In a new study, scientists showed that apoptotic signals are transferred from mitochondria to the ER and back to the mitochondria, and noted that this mechanism serves as a platform for the activation of procaspase‑8, the pre cursor of the apoptotic protein caspase 8.48 Moreover, the ER and mitochondria are tightly bound via mitofusin‑2 (the first direct ER–mitochondria tether discovered), which is localized on both the ER membrane and outer mitochondrial membrane, and regulates the shape of both organelles (Figure 1).49 Thus, the fact that ER stress influences the morphology and bioenergetic activity of the mitochondrial population is not surprising. In a new study, PERK has been shown to be a key regulator of mitochondrial morphology and function.50 Furthermore, ER stress induces mitochondrial stress, which results in the loss of mitochondrial membrane potential, fragmentation of the mitochondrial network and subsequent mitophagy. 51 ER stress and mito chondrial dysfunction are causative factors in insulin resistance52 and type 2 diabetes mellitus, 52,53 indicat ing that CKD with diabetes might also progress with the crosstalk of these organelles. Although no direct evidence has been found for a pathophysiological role of proteostatic crosstalk between ER and mitochon dria in kidney disease, unravelling the effect of ER and mitochondrial proteostasis will enable better under standing of the molecular mechanisms involved in the development and progression of CKD.

ER proteostasis in kidney disease Various renal pathogenic factors induce stress signals that derange ER proteostasis in glomerular and tubulo interstitial cells and lead to alterations in the structure and function of kidney cells (Figure 2). In a rat model of glomerulonephritis, we showed that precondition ing with ER stress ameliorated glomerular damage by damping the excessive UPR activation caused by disease induction.54 Furthermore, the pathogenesis of ER stress closely contributes to cardiorenal syndrome.12 Cardiac hypertrophy is associated with long-term or severe ER stress caused by increased protein synthesis, thereby leading to apoptosis of cardiac myocytes and resulting in chronic heart failure.12 In the following section, we summarize the mechanisms through which the derange ment of ER proteostasis affects the fate of kidney cells and results in the development and progression of kidney disease.



REVIEWS Podocytes The podocyte slit diaphragm has an important role in maintaining glomerular filtration. Mutation of slit dia phragm components, such as nephrin, α‑actinin‑4 and CD2-associated protein (CD2AP), is a major causal factor of a number of congenital nephrotic syndromes, which have similar manifestations to those of ‘conformational diseases’.55–57 These manifestations are characterized by inadequate protein folding, which results in loss of func tion by way of a decrease in the production of mature and functional proteins. Thus, congenital nephrotic syndrome with mutation of slit diaphragm components is associated with the accumulation of mutated and misfolded proteins in the ER and disruption of the slit diaphragm structure, leading to proteinuria. 55–57 The accumulation of misfolded proteins in the ER strongly triggers the UPR pathway to maintain ER proteostasis. Mutation of the glomerular basement membrane (GBM) component laminin subunit β2 also leads to nephrotic syndrome, which is caused by defective secretion of laminin from podocytes to the GBM and is accompanied by podocyte ER stress.58 These various findings highlight the possibility that UPR augmentation therapy might be used to enhance ER proteostasis as a new therapeutic approach to congenital nephrotic syndrome. Previously, the only cause of defective ER proteostasis in podocytes was thought to be protein-folding muta tion, which leads to congenital nephrotic syndrome development.55–57 Importantly, however, current evidence demonstrates that a decrease in ER proteostasis activ ity is a common occurrence under various pathogenic microenvironments, contributing to the progression of various glomerular diseases.59–61 Abnormal protein accumulation associated with ER stress in the ER of podocytes produces structural and functional damage in the cells, which in turn leads to severe proteinuria. This phenomenon was first identified in transgenic rats that overexpressed a transgene-encoded protein that is easily aggregated under conditions of excessive expres sion in podocytes.62 Accumulating evidence has since revealed that various other pathogenetic factors that induce podocyte damage also lead to UPR activation. For example, in one study, complement component C5b‑9 membrane attack complex shows pathogenic UPR activation in podocytes in a passive Heymann nephri tis model in rats.41,63 In another study, calcium entry via transient receptor protein 6 (TRPC6) or downregulation of CD2AP by albumin overload (a mimic of protein uria) induces UPR-mediated apoptosis in podocytes.57,64 Moreover, activation of rapamycin-sensitive protein kinase complex TORC1, which contributes to multiple cellular processes associated with proteostasis, also trig gers UPR activation in podocytes, leading to nephrotic syndrome65 and diabetic nephropathy.66 ER stress also contributes to podocyte injury caused by increased expression of monocyte chemoattractant protein 1 (MCP‑1), which has a central role in the inflammation associated with diabetic nephropathy.67 Taken together, these findings demonstrate that not only are mutations in slit-diaphragm-related protein

folding associated with ER stress, but renal patho gens that induce podocyte injury are as well. These data illustrate a pathophysiological effect of the UPR pathway—namely, the alteration of ER proteostasis—on podocyte homeostasis. Further studies will reveal the relative effect of pathogenesis of the UPR pathway in podocytopathy, and might give way to new therapeu tic approaches that target ER stress in podocyte-related kidney disease.

Tubular cells The structural and functional properties of tubular epi thelial cells are closely dependent on the degree of ER proteostasis activity. The UPR pathway in these cells is physiologically active, enabling them to adapt to multi ple and diverse stress conditions. However, when a cell undergoes long-term or severe stress, the adaptive UPR pathway is unable to optimize homeostasis; instead, the cell is eliminated by induction of the apoptotic UPR pathway. 6 Thus, an imbalance in the UPR pathway induces tubular apoptosis, which—in association with the deterioration of ER proteostasis—leads to the progression of CKD. In particular, proteinuria,68 hyperglycaemia,69 uraemic toxins,70 nephrotoxins (that include cisplatin71) and oxidative stress72 are major pathogens that induce either cell apoptosis or a decrease in the repair ability of tubular cells, both of which are mediated by the UPR pathway and accelerate the progression of kidney disease. Other studies have shown that berberine, an antioxidant, attenuates hypoxia and/or reoxygenation-induced renal proximal cell injury by inhibiting ER stress73 and that an epoxyeicosatrienoic acid analogue with antioxidative and antiapoptotic activities protects the tubular cells from cisplatin-induced apoptosis via a decrease in activity of the apoptotic UPR pathway.71 Of note, tubular UPR state changes with ageing, whereby the adaptive UPR pathway with GRP78 expression is substantially suppressed in aged tubular cells exposed to proteinuria, whereas the pro apoptotic UPR with CHOP overexpression is enhanced.74 These changes indicate a link between ER proteostasis and tubular ageing, as well as tubular-damage-related kidney diseases. Interstitial cells Pericytes are characteristic interstitial cells that are known for their role as effecter cells in tubulointerstitial fibrosis. 75 Pericytes differentiate to myofibroblasts (pericyte–myofibroblast transition), leading to fibrosis under pathogenetic conditions, such as obstruction of the kidney.76,77 In parallel, other reports have demon strated that the pericytes are not the major source of myofibroblasts, but that local resident fibroblasts are.78 These discrepancies might be attributable to differences in techniques or in the state of progression of fibro sis. Nevertheless, phenotypic change from pericyte or fibroblast to myofibroblast is closely associated with the alteration of proteostasis. Activation of the UPR pathway has been demonstrated in a unilateral ureteral obstruction fibrosis rat model.79 In this model, an imbalance in UPR activation, causing



REVIEWS

Hypoxia Oxygen tension

Proteostasis Protein synthesis, folding and degradation Adaptive UPR pathway Cell-fate determination Cell maintenance of development, function and structure

Glycative stress Unfavourable protein modification

Oxidative stress ROS

Proteostasis

the function of EPO-producing cells.83 Cell fate mapping research revealed that the number of EPO-producing cells varies with changes in microenvironment (includ ing hypoxia),81 and suggested that ER proteostasis is con trolled by a phenotypic change at a functional level in EPO-producing cells.

ER stress

Targeting proteostasis in kidney disease Maladaptive UPR system

Ageing of kidney and kidney disease progression (CKD)

Figure 3 | ER proteostasis in kidney disease. Proteostasis in the ER is regulated by protein synthesis, folding and degradation through the UPR pathway. The UPR pathway determines cell fate (left side). Specifically, the adaptive UPR pathway maintains cell development, structure and function. By contrast, ER dysfunction or ER stress (right side), which is associated with a decrease in proteostasis in kidney cells, occurs under various conditions, such as low oxygen tension (hypoxia), increased production of ROS by oxidative stress and loss of protein function by glycative stress. These conditions induce overwhelming activation of adaptive and/ or apoptotic UPR pathways, and often lead to an imbalance in the UPR networks. Such maladaptive UPR system leads to kidney ageing as well as kidney disease. Abbreviations: CKD, chronic kidney disease; ER, endoplasmic reticulum; ROS, reactive oxygen species; UPR, unfolded protein response.

predominant activation of the apoptotic over the adap tive UPR pathway, leads to renal cell apoptosis medi ated by the apoptotic UPR and subsequent fibrosis, and is attenuated via the optimization of UPR activation by candesartan, an angiotensin receptor blocker.79 In a new study, the ER‑resident protein ERP57 (58 kDa glucoseregulated protein), which modulates the folding of newly synthesized glycoproteins via disulfide isomerase activ ity, has been reported to exacerbate the accumulation of extracellular matrix and progression of fibrosis in renal cells under ER stress conditions.80

Erythropoietin-producing cells Renal erythropoietin (EPO)-producing cells are a second characteristic type of interstitial cell. EPO is essential to erythropoiesis and its transcription is strictly regulated by the HIF pathway;81 renal hypoxia strongly enhances EPO production as an adaptive response to low oxygen tension. Renal anaemia associated with CKD was previ ously thought to result from the loss of EPO-producing cells. However, a growing consensus considers that the EPO-producing cells suffer a decrease in EPO produc tion activity under pathogenic conditions owing to derangement of the oxygen-sensing machinery by the HIF pathway.81 A previous report stated that the HIF pathway was downregulated by NF‑κB, which resulted in a decrease in EPO transcription.82 We have since demonstrated that ER proteostasis contributes to the derangement of hypoxia-induced EPO production.83 When the kidney is exposed to ER stress or excess UPR activation, EPO production is sup pressed by ATF4, which acts by binding to a putative ATF4-binding site adjacent to the HIF binding site in the 3' enhancer region of the EPO gene.83 This mecha nism suggests that a change in ER proteostasis influences

Until now, the idea of manipulating proteostasis was considered viable only for conformational diseases, such as Alzheimer disease or prion disease.13,14 For example, small-molecule UPR modulators, such as chemical chaperones, have been developed to optimize the proteo stasis of neuronal cells by increasing protein folding capacity and decreasing proteotoxicity, thereby suppress ing UPR-mediated apoptosis.84,85 Of note, several studies have demonstrated that certain chemical chaperones are also beneficial in preventing type 2 diabetes mellitus in experimental disease models in rats,86 suggesting a new therapeutic avenue involving the use of proteostasis regulators in various diseases associated with imbal anced proteostasis or ER stress. Indeed, other studies have also revealed that these chemical chaperones have therapeutic efficacy in cardiovascular disease,15,16 cancer 17 and kidney disease,6 as well as to type 2 diabetes melli tus.18 Proteostasis networks weaken with ageing, but are strengthened by cell signalling that controls l ongevity and ‘youthfulness’.87 Oxidative or glycative stress induce the stress signals that influence premature ageing in associ ation with defective proteostasis (Figure 3); thus, the small molecules inhibiting these stresses also act to regu late proteostasis.18,88 These findings are consistent with the idea that the maintenance of proteostasis is closely linked to healthy ageing. The finding that an imbalance in ER proteostasis, or pathogenic UPR activation, contributes to structural and functional damage of various renal cells has sparked investigations into the possibility of protein-folding- augmentation therapy as a new approach to kidney disease.89 In this regard, we hereby emphasize that not only the augmentation of protein folding but also the regulation of protein synthesis, 90,91 degradation, 92,93 aggregation94,95 and post-translational modification96 are promising strategies in rescuing defective proteostasis networks and subsequent cell homeostasis. The following section focuses primarily on the mechanisms targeted by the small molecules that act as pharmacological agents to regulate proteostasis in renal disease (Table 1).

Protein folding augmentation Some small molecules—such as 4‑PBA, TUDCA and BIX—enhance the protein folding capacity and rescue kidney cells from pathogenic microenvironmental stress. 4‑PBA and TUDCA are chemical chaperones that pri marily enhance ER homeostasis and have been shown to restore leptin resistance and glucose homeostasis in mice with obesity and type 2 diabetes mellitus, respectively.97,98 4‑PBA also ameliorates the development and progression of diabetic nephropathy in rats.99 Moreover, 4‑PBA treat ment increases the secretion rate of complement factor H



REVIEWS Table 1 | Regulators of proteostasis by type Agent

Mechanism of action

References

4-PBA

Enhances protein folding

15, 16, 97–101

TUDCA


97, 98, 102

Migalastat

Selectively prevents mutant GLA from misfolding

105–107

ORP150


125

Selectively upregulates immunoglobulin binding protein

104

Bortezomib

Inhibits chymotrypsin-like sites and peptidylglutamyl peptide hydrolysing sites of proteasome

120–122

Carfilzomib

Inhibits chymotrypsin-like sites of proteasome and activates eIF-2α–ATF4 axis

123

Oprozomib

Inhibits chymotrypsin-like sites of proteasome and activates eIF-2α–ATF4 axis

123

Chaperone

Chaperone inducer BIX Protease inhibitor

Protein translation attenuator Salubrinal

Inhibits cellular eIF-2α phosphatases

83, 109–111

Guanabenz

Inhibits stress-induced eIF-2α phosphatases

112, 113

Activates IRE1–XBP1 axis

114–116

Other Quercetin

Abbreviations: 4‑PBA, chaperone 4‑phenylbutyrate; ATF4, activation transcription factor 4; BIX, binding immunoglobulin protein inducer X; eIF-2α, eukaryotic translation initiation factor 2α; GLA, α‑galactosidase A; IRE1, inositol-requiring protein 1; ORP150, 150 kDa oxygen-regulated protein; TUDCA, tauroursodeoxycholate; XBP1, X‑box binding protein 1.

(CFH), a regulatory protein of the complement system, in fibroblasts of patients with CFH deficiency, which—in association with the accumulation of mutant CFH in the ER—causes kidney disease such as atypical haemolytic uraemic syndrome.100 Both TUDCA and 4‑PBA effec tively ameliorate cardiac, liver and lung fibrosis by nor malization of UPR activation.15,16,101,102 These findings lead us to speculate that these small molecules might have similar effects on chemical chaperones in renal fibrosis as well. BIX is a small-molecule proteostasis regulator that induces the expression of GRP78. BIX was identified using high-throughput screening with a GRP78 reporter assay system.103 Activation of the ATF6 axis selectively induces BIX-associated GRP78 production, and a previ ous report showed a protective effect of BIX on kidney injury following ischaemia–reperfusion injury via the regulation of ER stress.104 In patients with Fabry disease, a progressive X‑linked inherited disorder of glycosphingolipid metabo lism that results from deficient or absent lysosomal α‑galactosidase A (GLA) activity, migalastat, an orally bioavailable molecule, binds to and stabilizes mutant GLA in the ER, preventing it from misfolding and degrading, leading to effective restoration of enzyme trafficking to lysosomes.105 Two phase 2 clinical studies have explored the safety of and pharmacodynamic responses to migalastat, demonstrating that migalastat is a candidate pharmacological chaperone and a new

ge notype-specific treatment for Fabry disease.106,107 Further evaluation of the pharmacological effect of chemical chaperones in m ultiple kidney diseases is necessary.

Modulation of UPR pathway Salubrinal (an eIF-2α inhibitor) and guanabenz (an α2-adrenergic receptor agonist) are compounds that target proteostasis by modulating the PERK–eIF-2α axis, which attenuates protein translation.108–113 The specific PERK inhibitor GSK2606414, which targets the modulation of UPR pathway, effectively prevents protein-folding-related diseases associated with defec tive proteostasis, such as neurodegeneration and clinical disease in prion-infected mice.108 Accumulating evi dence demonstrates that salubrinal also attenuates the multiple phenotypic changes associated with ER stress in podocytes, tubules and renal EPO-producing cells. For example, salubrinal ameliorates podocyte damage by hyperglycaemia,109 tubular and endothelial injuries by nephrotoxin110,111 and endoplasmic-reticulum-stressinduced derangement of EPO production in renal EPO-producing cells, all via the regulation of UPR acti vation.83 These findings revealed that activation of the PERK–eIF-2α axis by salubrinal substantially restores ER proteostasis, maintaining cellular functions in different cell types. Guanabenz is used as an antihypertension drug 112 and selectively modulates the stress-responsive PERK axis, but not the PERK axis in the absence of stress.113 Although the proteostatic effect of guanabenz in the kidney remains unclear, we speculate that selective PERK axis modulation might be more useful than the broader effect of salubrinal. Quercetin is a plant-derived flavonoid that acts as an antioxidant. 114 Notably, quercetin activates the IRE1–XBP1 axis and protects the cell from ER stress.115 Although quercetin exerts a protective effect against renal ischaemia–reperfusion injury via the inhibition of oxidative stress,116 its inhibitory effects on the UPR pathway have not been revealed. Further investigation is necessary to investigate whether the renoprotec tive effect of quercetin can be atttributed to regulation of proteostasis. Modulation of protein degradation The clearance of misfolded proteins generated in the ER is regulated by ERAD and the ubiquitin–proteasome pathway. Ubiquitin modification also influences podo cyte differentiation and injury.117 Ubiquitin C‑terminal hydrolase isozyme L1 (UCHL1) is a key modulator of ubiquitin modification, and normal kidneys express no UCHL1 and little ubiquitin; however, a subset of human glomerulopathies associated with podocyte foot process effacement (for example, membranous nephropathy and focal segmental glomerulosclerosis) display de novo expression of UCHL1 in podocytes.117 On this basis, therapeutic approaches that target protein degradation might also be useful. Proteasome inhibitors, such as bortezomib, that induce ER stress and UPR-mediated apoptosis in tumour cells, have been developed for



REVIEWS cancer therapy.118,119 Interestingly, findings have shown that proteasome inhibitors also have other effects on nor malization of proteostasis via the regulation of protein degradation state. For example, bortezomib ameliorates the antibody-mediated rejection of kidney transplanta tion;120,121 a combination of bortezomib and chaperones rescues the mutation of angiotensin-converting enzyme (ACE) that is associated with impaired trafficking to the cell surface.122 Additionally, the second-generation pro teasome inhibitors carfilzomib and oprozomib activate the UPR pathway, particularly the adaptive UPR axis, via eIF-2α–ATF4 to promote cell survival.123 Considering these findings together, proteasome inhibitors might act not only to induce apoptotic cell death but also to maintain proteostasis via the modulation of protein degradation state for cell survival. Further investigation will clarify the potential use of proteasome inhibitors as proteostasis modulators in renal disease.

Regulation by post-translational acetylation Modulation of protein acetylation might be beneficial in rebalancing proteostasis in cells with defective proteo stasis. In this regard, sirtuin 1 (SIRT1), a histone deacety lase, has been shown to attenuate UPR activation in mice with type 2 diabetes mellitus and restores insulin resist ance.124 Moreover, SIRT1 ameliorates palmitate-induced UPR activation and insulin resistance in liver cells via the induction of 150 kDa oxygen-regulated protein (ORP150), an ER chaperone.125 In another study, SIRT1 deacetylates IRE1-generated active XBP1 (XBP1s), and acts as a transcription factor of the IRE1 axis, thereby inhibiting its transcriptional activity.126 These findings suggest that epigenetic modulators (including SIRT1 modulators) might be beneficial in the modulation of UPR or proteostasis state. 1.

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Conclusions Proteostasis networks have important roles in the quality control and maintenance of the functional diversity of proteins. Proteostasis in cells is modulated by various microenvironmental conditions and determines cell fate. In the kidney, accumulating evidence emphasizes that the UPR pathway is altered by stress conditions, such as hypoxia, and oxidative and glycative stress, which modulate ER proteostasis through the interaction of UPR with other stress signals. Thus, deranged ER proteo stasis in kidney cells strongly contributes to the develop ment or progression of kidney disease. Normalization of ER proteostasis might, therefore, be effective for cell maintenance, and protect against kidney pathologies. A growing consensus considers that optimizing ER proteostasis using pharmacological approaches, such as small-molecule UPR modulators, is a promising approach to preventing or slowing kidney disease. In particular, given that proteostasis is closely linked to cellular senescence phenotypes and multiple age-related diseases, including CKD, UPR modulators might protect kidney cells against the functional dysregulation, which occurs with ageing or via age-related sensitivity to renal pathogens. Review criteria A search for original articles published between 2007 and 2013 focusing on endoplasmic reticulum proteostasis was performed in MEDLINE and PubMed. The search terms used were “proteostasis”, “endoplasmic reticulum”, “unfolded protein response”, “hypoxia”, “glycation”, “oxidative stress”, “stress signalling”, and “kidney disease”, alone and in combination. All articles identified were Englishlanguage, full-text papers. The reference lists of identified articles were also searched for further relevant papers.

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