Nephrol Dial Transplant (2014) 29: iv33–iv44
doi: 10.1093/ndt/gfu231
Full Review
Protein trafficking defects in inherited kidney diseases
Céline Schaeffer, Anna Creatore and Luca Rampoldi
Molecular Genetics of Renal Disorders Unit, Division of Genetics and Cell Biology, Dulbecco Telethon Institute c/o IRCCS San Raffaele
Scientific Institute, Milan, Italy
Correspondence and offprint requests to: Luca Rampoldi; E-mail: [email protected]
A B S T R AC T
The nephron, the basic structural and functional unit of the
kidney, is lined by different, highly differentiated polarized
epithelial cells. Their concerted action modifies the composition of the glomerular ultrafiltrate through reabsorption and
secretion of essential solutes to finally produce urine. The
highly specialized properties of the different epithelial cell
types of the nephron are remarkable and rely on the regulated
delivery of specific proteins to their final subcellular localization. Hence, mutations affecting sorting of individual proteins
or inactivating the epithelial trafficking machinery have severe
functional consequences causing disease. The presence of
mutations leading to protein trafficking defect is indeed a mechanism of pathogenesis seen in an increasing number of disorders, including about one-third of monogenic diseases affecting
the kidney. In this review, we focus on representative diseases to
discuss different molecular mechanisms that primarily lead to
defective protein transport, such as endoplasmic reticulum retention, mistargeting, defective endocytosis or degradation,
eventually resulting in epithelial cell and kidney dysfunction.
For each disease, we discuss the type of reported mutations,
their molecular and cellular consequences and possible strategies for therapeutic intervention. Particular emphasis is given
to new and prospective therapies aimed at rescuing the trafficking defect at the basis of these conformational diseases.
Keywords: inherited kidney disease, misfolded protein, molecular and pharmacological chaperone, protein homeostasis,
protein trafficking
INTRODUCTION
The nephron, the functional unit within the kidney, contains
several types of highly differentiated polarized epithelial cells
© The Author 2014. Published by Oxford University Press
on behalf of ERA-EDTA. All rights reserved.
that ensure barrier function and vectorial transport of ions,
proteins and other molecules across the apical and basolateral
plasma membranes. The sequential action of the different epithelial cell types lining the nephron, each with specific transport properties, modifies the composition of the primary
ultrafiltrate through reabsorption and secretion of essential
solutes, to eventually produce urine. The accurate delivery of
specific proteins, such as channels or transporters, on the appropriate membrane domain ensures the complex and highly
specialized functions of renal epithelia. In this context, mutations affecting intracellular sorting of individual proteins or
inactivating the epithelial trafficking machinery can have
severe functional consequences causing disease. To date, mutations in ∼120 genes are known to be responsible for monogenic diseases affecting the kidney [1]. About one-third of
them directly interfere with protein transport, affecting one or
more segments of the nephron (Table 1). With few exceptions,
protein products encoded by such genes enter the secretory
pathway to reach their final destination. The first step of this
journey occurs in the co-translational translocation in the
endoplasmic reticulum (ER) where a quality control system
ensures that only properly folded proteins can move forward.
From the ER, proteins reach the Golgi where they undergo
different modifications, the most common being the ones on
N-linked glycans. At this stage, complex processes regulate anterograde and retrograde transport with the final goal of reaching the trans-Golgi network, where proteins are dispatched
towards apical, basolateral or endolysosomal compartments
(Figure 1a). Mutations in secretory proteins can affect their
trafficking at any of these steps (Figure 1b–d). In this review,
we ideally move along the secretory pathway and discuss examples of inherited kidney diseases characterized by different
pathogenetic mechanisms. Storage disorders due to the accumulation of substrates of defective lysosomal enzymes are not
specifically addressed and have recently been the focus of
iv33
Table 1. List of Mendelian renal diseases associated with defective protein trafficking
Diseasea
Gene
Alport syndrome
COL4A3, A4, Collagen α-3(IV), α-4(IV),
A5
α-5(IV) chains
COL4A1
Collagen α-1(IV) chain
Angiopathy, hereditary, with nephropathy,
aneurysms and muscle cramps (HANAC)
Focal segmental glomerulosclerosis 1
Focal segmental glomerulosclerosis 2
Focal segmental glomerulosclerosis 5
Focal segmental glomerulosclerosis 6
Nephrotic syndrome type 1 (steroid-resistant,
Finnish type)
Nephrotic syndrome type 2 (steroid-resistant)
INF2
MYO1E
NPHS1
α-Actinin-4
Short transient receptor
potential channel 6
Inverted formin 2
Unconventional myosin Ie
Nephrin
NPHS2
Podocin
Pierson syndrome
LAMB2
Laminin subunit β2
Cystinosis
Cystinuria, type A
CTNS
SLC3A1
Dent’s disease 1
Dent’s disease 2
CLCN5
OCRL
Hypophosphataemic rickets with
hypercalciuria
Hypophosphataemic rickets
SLC34A3
Cystinosin
Neutral and basic amino acid
transport protein rBAT
H+/Cl− exchange transporter 5
Inositol polyphosphate 5phosphatase OCRL-1
Na+/Pi-co-transporter 2C
ACTN4
TRPC6
PHEX
Mutation effectb
Nephron
segment
Reduced secretion, ER retention shown
for α-3
Intracellular retention; reduced
secretion
Aggregation; increased affinity for actin
Increased expression/activity at the PM
Glomerulus
Glomerulus
Glomerulus
Altered subcellular localization
Intracellular retention
ER retention; defective function
Glomerulus
Glomerulus
Glomerulus
ER and small intracellular vesicles
retention; inclusion bodies in cytoplasm
ER retention; defective basement
membrane
Mistargeting to PM; defective function
ER retention; defective association with
B(0,+)-type amino acid transporter 1
ER retention; defective activity
Defective function
Glomerulus
Defective function
PT
ER retention; mistargeting to AM
PT
Intracellular retention; defective
function
ER retention; defective function
PT
ER retention; defective function
TAL
ER retention; defective function
TAL
ER retention; mistargeting to AM;
defective interaction with ClC-K
channels
ER and Golgi retention; mislocalization
to lysosome; defective function
Intracellular retention; defective
function
Intracellular retention
TAL, DCT
SLC4A4
Renal glucosuria
SLC5A2
Renal glucosuria (glucose/galactose
malabsorption)
Bartter syndrome, antenatal, type 1
SLC5A1
Na+/glucose co-transporter 1
SLC12A1
Bartter syndrome, antenatal, type 2
KCNJ1
Bartter syndrome, type 4
BSND
Na+–K+–2Cl− co-transporter
(NKCC2)
ATP-sensitive inward rectifier
K+ channel 1 (ROMK)
Barttin
Hypomagnesaemia type 3
CLDN16
Claudin-16
Hypomagnesaemia type 5
CLDN19
Claudin-19
Hypomagnesaemia type 2
FXYD2
Uromodulin-associated kidney disease
Gitelman syndrome
UMOD
SLC12A3
Distal renal tubular acidosis
SLC4A1
Liddle syndrome
SCNN1B,
SCNN1G
AVPR2
AQP2
Na+/K+ transporting ATPase
subunit γ
Uromodulin
ER retention
Na+–Cl− co-transporter (NCC) ER retention; intracellular retention;
defective function
Anion exchanger 1
ER or Golgi retention; mistargeting
to AM
Epithelial Na+ channel subunit Increased expression at the AM
β, subunit γ
Vasopressin V2 receptor
ER retention; defective function
Aquaporin-2
ER and Golgi retention; mislocalization
to late endosomes, lysosome, BM
Cystic fibrosis transmembrane ER retention; defective function
conductance regulator
α-Galactosidase A
ER retention; defective function
Fabry disease
GLA
PT
PT
PT (secondary)
PT
Proximal renal tubular acidosis
CFTR
PT
PT
Altered processing
Intracellular retention
OCRL
Cystic fibrosis
Glomerulus
PT
Lowe syndrome
Nephrogenic diabetes insipidus
Glomerulus
ER retention; increased degradation;
defective activity
ER retention; defective activity
Phosphate-regulating neutral
endopeptidase
Fibroblast growth factor 23
Dentin matrix acidic
phosphoprotein
Inositol polyphosphate 5phosphatase OCRL-1
Electrogenic Naþ HCO3 co-transporter 1
Na+/glucose co-transporter 2
FGF23
DMP1
FULL REVIEW
Protein product
PT (secondary)
PT
TAL
TAL
DCT
TAL
DCT
CD
CD
CD
CD
Secondary/PT
Secondary/
multiple
Continued
iv34
C. Schaeffer et al.
Table 1. Continued
Diseasea
Gene
Protein product
Mutation effectb
Nephron
segment
Haemolytic uraemic syndrome, atypical
CFH
CD46
Complement factor H
Membrane cofactor protein
Intracellular retention; defective
function
ER retention; defective function
Hyperoxaluria, primary, type I
AGXT
Alanine:glyoxylate
aminotransferase
Mistargeting to mitochondria;
aggregation
Secondary
(glomerulus)
Secondary
(glomerulus)
Secondary
PM, plasma membrane; AM, apical plasma membrane; BM, basolateral plasma membrane; ER, endoplasmic reticulum; PT, proximal tubule; TAL, thick ascending limb; DCT, distal
convoluted tubule; CD, collecting duct; Multiple, disease primarily affecting more than one nephron segments; Secondary, disease affecting the kidney via secondary effect.
a
Disease nomenclature according to the Online Mendelian Inheritance in Man (OMIM) database.
b
Loss of transcript/protein product is not indicated.
other reviews (as in [2]). For each disease, we discuss the type
of reported mutations, their functional consequences and
present and prospective strategies for therapeutic intervention.
ER RETENTION
Nephrogenic Diabetes Insipidus
NDI is a water balance disease characterized by the incapacity to concentrate urine resulting in severe polyuria and
polydipsia. This may lead to dehydration, electrolyte imbalance (hypernatremia) and growth and mental retardation. Persistent polyuria can also result in urologic complications, from
slight dilation of the urinary tract to severe hydronephrosis.
The conventional therapy consists of adequate fluid supply to
compensate for water loss and low sodium diet combined with
thiazide and amiloride treatment. The most common forms in
clinic are the acquired ones (mainly secondary to drug treatment, electrolyte imbalance and urinary tract obstruction) [3].
Congenital NDI is caused by mutations in the AVPR2 or
AQP2 genes, responsible for X-linked (90%) and autosomal
(10%; recessive and dominant) forms of the disease, respectively. These two genes are essential for the control of water
balance mediated by the peptide hormone arginine vasopressin (AVP). AVP binds to vasopressin receptor type 2 (AVPR2),
a G-protein-coupled receptor (GPCR) localized at the basolateral membrane of the principal cells of the kidney collecting
duct. Upon activation by AVP, AVPR2 activates adenylate cyclases triggering a signalling cascade eventually resulting in activation of protein kinase A and phosphorylation of aquaporin
2 (AQP2) in the endosomal compartment. This leads to fusion
of endosomal vesicles containing AQP2 with the apical plasma
membrane where AQP2 allows water reabsorption. Once water
balance is restored, AVP level decreases and AQP2 is removed
from the plasma membrane by ubiquitylation-mediated endocytosis [4].
Protein trafficking defects in kidney diseases
iv35
FULL REVIEW
Retention in the ER is a mechanism shared by a wide range of
conformational diseases and it is essentially due to mutations
affecting protein folding (Figure 1b).
In some cases, misfolding mutations have a main loss-offunction effect, since the mutated protein is effectively degraded via ER-associated degradation (ERAD), as for example
in nephrogenic diabetes insipidus (NDI).
The X-linked form of congenital NDI is caused by lossof-function mutations in the AVPR2 gene [5, 6], resulting in
insensitivity of principal cells of the collecting duct to AVP.
Mutations can be divided into four classes on the basis of their
molecular mechanism of action [7]. Class 1 mutations (usually
frameshift or nonsense) affect synthesis or processing of the
mRNA (i.e. promoter alterations, aberrant splicing) and lead
to absent (mRNA decay) or truncated protein. Class 2 mutations (missense, in-frame insertion/deletion) are the most
common and cause misfolding of the protein, its retention in
the ER and degradation. Class 3 mutations (missense and inframe insertion/deletion) do not affect plasma membrane localization, but interfere with G protein or AVP binding. Class
4 mutations lead to protein missorting from the plasma membrane to different cellular compartments [8]. Some mutations
can present features of different classes, as they can affect different stages of protein maturation and function. Class 2
AVPR2 mutations represent >50% of the reported mutations.
Although ER retention and degradation are common features
of all Class 2 mutants, the amount of retention differs between
mutants, likely reflecting their degree of misfolding [9]. Some
mutants are indeed partially trafficked to the plasma membrane
where they can be functional or defective in AVP binding or
G-protein coupling.
Partial trafficking to the basolateral membrane of functional protein could explain why some patients respond to high
doses of the vasopressin synthetic analogue desmopressin
[10]. Thus, a possible therapeutic strategy for mutants retaining functional activity is to induce their escape from ER retention and ERAD and their insertion at the plasma membrane.
While the effect of non-selective chemical chaperones, as glycerol or dimethyl sulfoxide (DMSO), has been modest [11],
interesting results were obtained with receptor antagonists
( peptides or pharmacological chaperones). These molecules
specifically bind to AVPR2 and aid the folding of mutated
protein and its exit from the ER [12]. One such molecule
(SR49059) had some beneficial effects in patients [13]. However, further clinical studies have been limited by the difficulty
in identifying effective low-affinity ligands, able to rescue
AVPR2 trafficking and to be efficiently displaced by AVP, and
by the high concentrations required, raising significant safety
issues. An alternative and more valid approach seems to be the
use of non-peptide agonists [as OPC51, VA(9990)88 and VA
(9990)89] that do not rescue trafficking of the receptor, but
can directly activate intrinsically functional AVPR2 mutants
F I G U R E 1 : (a) Simplified representation of membrane trafficking pathways in kidney epithelial cells. Proteins entering the secretory pathway
FULL REVIEW
are synthesized in the ER, trafficked first to the ER-Golgi intermediate compartment (ERGIC) and then to the cis-Golgi. Retrograde transport
can retrieve cargoes from the Golgi to the ER. In the Golgi, proteins proceed towards the trans-Golgi network from where they are dispatched
towards the apical or basolateral plasma membranes, secretory granules or the endolysosomal compartment. Plasma membrane proteins can
be internalized by endocytosis and either cycle back to the membrane or are degraded in the lysosomes. (b) Mutations of proteins entering the
secretory pathway can induce retention in the ER or in the Golgi leading to loss-of-function (as, for example, some mutants in NDI, dRTA
and Dent’s disease) and gain-of-function (as in UAKD) effects. (c) Mutations affecting sorting signals in the protein sequence can lead to
mislocalization in a different organelle (as seen in PH1) or to loss or reversal of polarized trafficking (as seen in dRTA). (d) Some mutations can
affect protein turnover at the plasma membrane due to defective internalization and degradation, as seen in Liddle syndrome. Mutations in
proteins involved in endosomal function cause defective endocytosis, leading to accumulation of proteins and lipids at the plasma membrane
and lack of internalization of extracellular components (as seen in Dent’s disease).
within the ER. This leads to GPCR activation and cyclic AMP
increase, eventually resulting in increased AQP2 delivery to
the plasma membrane [14]. Likely, this effect is due to ERactivated AVPR2 signalling to pre-formed receptor G-protein–
adenylate cyclase complexes at the ER membrane, as observed
for the activation of the ER-localized GPR30 by oestrogen [15]
or for the β2-adrenergic receptor signalosome [16]. Further
studies will be needed to assess the clinical applicability of this interesting class of molecules. A third strategy that could be beneficial for different AVPR2 mutants is to bypass AVPR2 function
and induce AQP2 localization at the apical membrane, as recently demonstrated by treatment with agonists of E-prostanoid
receptors EP2 and EP4 in rodent models [17, 18].
AQP2 is the gene mutated in the autosomal dominant and
recessive forms of NDI [19]. In the dominant form, representing 10% of autosomal NDI cases, mutations are found in the
carboxy-terminal tail of the protein that is essential for proper
trafficking. These mutations introduce missorting signals leading
to mislocalization of AQP2 in the Golgi, late endosomes, lysosomes or basolateral membrane [20]. They can also affect apical
membrane insertion by interfering with AVP-induced phosphorylation of AQP2 at serine 256 [21, 22]. These mutations are
dominant since mutant protein can interact with wild-type
AQP2 and affect its localization [23]. In the recessive form, the
vast majority of mutations are missense changes in the region
forming the aquaporin pore and they lead to protein misfolding,
ER retention and proteasomal degradation. A different mechanism of recessive inheritance was discovered in two families, in
which patients were compound heterozygotes for either A190T
or R187C mutations, along with mutation P262L. While A190T
iv36
and R187C AQP2 mutants are retained in the ER, the P262L
isoform is retained in intracellular vesicles only in the absence of
wild-type protein [24].
In the dominant forms, strategies aimed at increasing cyclic
AMP and hence AQP2 phosphorylation could be effective, as
successfully demonstrated by treatment with Rolipram, a
phosphodiesterase 4 inhibitor, in a murine model of autosomal
dominant NDI [25]. As for AVPR2 Class 2 mutants, modulating
protein folding could be envisaged as a potential treatment for
recessive AQP2 mutations. An Hsp90 inhibitor (17-AAG) has
already been shown to partially rescue AQP2 trafficking in a
conditional murine model of NDI [26]. Despite the interesting
results in preclinical models, the applicability of molecules inhibiting phosphodiesterase 4 or Hsp90 in the therapy of NDI
needs further studies addressing safety issues, given the abundance and composite biological roles of such proteins.
In some conformational diseases, ER-retained mutant protein
is not effectively degraded and its accumulation exerts a gain-oftoxic-function effect. This mechanism is proved by the different
phenotype of knock-out (KO) models and of models of expression of the mutant protein. An example is uromodulin-associated
kidney disease (UAKD) that is caused by mutations of the
UMOD gene encoding uromodulin (or Tamm–Horsfall protein)
[27]. Studies in cellular models demonstrated that UMOD mutations lead to mutant protein retention and aggregation in the ER,
likely due to protein misfolding [28]. This was also observed in
patient renal biopsies and in the kidneys of mice expressing
mutant uromodulin [29, 30]. These models recapitulate most of
the features of the human disorder. Indeed, ER retention of
C. Schaeffer et al.
mutant uromodulin leads to progressive structural and functional damage of the thick ascending limb (TAL) of Henle’s loop, the
nephron segment where uromodulin is expressed, to interstitial
fibrosis and inflammation. On the contrary, Umod KO mice do
not show any sign of tubulo-interstitial disease [31], demonstrating the gain-of-function effect of uromodulin mutations. Accumulation of mutant protein in the ER can impact on several
cellular processes, ranging from protein homeostasis, redox
balance and chronic induction of different cell stress pathways.
Among these, the unfolded protein response (UPR) has been extensively studied. UPR is a complex adaptive pathway that counteracts protein folding overload in the ER by decreasing protein
translation and by increasing the protein folding and degradation
machinery. Such an adaptive pathway can turn into maladaptive
when chronically induced, as in the case of accumulating misfolded mutant protein, and can eventually lead to apoptosis. Induction of the UPR has been detected in conformational renal
diseases, as in nephrotic syndrome type 1 with nephrin mutations [32] and in Alport syndrome and Pierson syndrome, disorders of the glomerular basement membrane due to mutations in
collagen IV [33] and laminin β2 [34], respectively. The pathogenic role of UPR and the effect of its modulation in renal disorders
are still unclear and deserve further investigation.
M I S TA R G E T I N G
Primary hyperoxaluria type I
Mistargeting is among the causes of primary hyperoxaluria
type I (PH1). PH1 is a rare autosomal recessive disease caused
by mutations in the AGXT gene coding for the liver-specific
peroxisomal enzyme alanine-glyoxylate aminotransferase (AGT)
[35]. Deficiency in the activity of AGT causes accumulation of
glyoxylate and consequently overproduction of the metabolicend product oxalate. This leads to progressive deposition of insoluble calcium oxalate in the kidney and urinary tract. Calcium
oxalate deposition eventually spreads throughout the body due
to chronic kidney failure. Reported AGXT mutations are missense (50%), nonsense (13%), major or minor insertions or
deletions (25%) or affect splicing (12%) [36]. These mutations
can have different consequences altering protein function (loss
of catalytic activity or co-factor binding), structure (increased
degradation, reduced dimerization and aggregation) or subcellular localization (aberrant mitochondrial localization) [36]. The
AGXT gene has a number of polymorphic variants in linkage
disequilibrium forming ‘major’ and ‘minor’ (present in 15–20%
of Caucasians) haplotypes. Main differences in the minor allele
include an imperfect duplication in intron 1 and two coding
non-synonymous polymorphisms (P11L and I340M). Interestingly, the frequency of AGXT minor allele raises to 46% in PH1
Protein trafficking defects in kidney diseases
In epithelial cells, mistargeting mutations can affect polarized
protein trafficking to the apical or basolateral membranes, causing
rerouting of the mutant protein to the incorrect membrane
domain (Figure 1c), as for example in distal renal tubular acidosis
(dRTA, also named Type 1 renal acidosis or classic RTA).
Distal renal tubular acidosis
dRTA is characterized by the inability to properly acidify
the urine due to impaired secretion of hydrogen ions (H+) by
iv37
FULL REVIEW
Protein targeting is the process by which proteins reach their
final destination. It is controlled by information contained in the
protein sequence and its dysregulation often leads to disease
(Figure 1c). Mutations have a loss-of-function effect, due to the
absence of the protein function in the physiological cell compartment. This can be associated with a gain-of-function component,
due to the aberrant localization of functional mutant protein.
patients [36]. The substitution of a proline with a leucine at position 11 creates an N-terminal mitochondrial targeting sequence
(MTS). This MTS is normally ineffective, as dimerization of the
protein masks the binding motif for the mitochondrial import
receptor TOM20 and it keeps AGT in a conformation that is incompatible with mitochondrial import. However, in conditions
that affect protein dimerization, as for some AGT mutations, the
MTS becomes accessible and AGT can be imported to the mitochondria [37]. This synergistic mechanism was first described
for the most common mutation G170R [35], present in ∼30% of
PH1 cases. It was recently confirmed for three other mutants
(I244T, F152I and G41R), showing different extent of protein
mitochondrial re-localization [38]. These mutations do not completely abolish dimerization and binding to the peroxisomal
import protein Pex5p and retain some catalytic activity, suggesting that they do not fully compromise protein folding and dimerization [38, 39]. Likely, they decrease the dimerization
efficiency, and this is sufficient to allow the interaction of mutant
protein MTS with TOM20. Delayed or defective folding is likely
at the bases of the effect of other AGT mutations that are mainly
associated with protein aggregation (in the cytosol or in peroxysomes) or with proteasome-mediated degradation [40]. Also in
this case, AGT mutations show a synergistic effect with the
minor allele and mutant proteins partially retain folding, transport and catalytic properties [39].
Patient treatment envisages high fluid intake and alkalinization of the urine to reduce urinary Ca-oxalate saturation. Once
renal function is lost, PH1 patients require liver and kidney
transplantation. Administration of pyridoxine (vitamin B6)
was proposed as a conservative PH1-specific measure, and
about one-third of patients, mainly carrying G170R and F152I
mutations, have shown to respond best [41]. A rational
explanation for the therapeutic effect of this drug has been
provided by a recent study in cell models stably expressing wildtype or mutant AGT. The authors proposed that treatment with
pyridoxine, a metabolic precursor of pyridoxal phosphate
(PLP), leads to increased concentration of PLP, an AGT cofactor. Increased intracellular PLP is able to act as a prosthetic
group (increasing catalytic activity) and as a chemical chaperone (increasing expression and peroxisomal import) [42].
These promising findings demonstrate that AGT is a ‘druggable
target’. The development of pharmacological compounds able
to synergize with pyridoxine would thus seem an interesting
strategy to ameliorate protein folding and stability, decrease aggregation and redirect mutant protein to peroxysomes. Potential
targets are likely to be identified through further characterization of the complex molecular machinery that regulates AGT
homeostasis, as the chaperones Hsp70, Hsp60 and Hsp90 [40].
FULL REVIEW
α-intercalated cells of the connecting tubules and cortical collecting ducts. This leads to chronic metabolic acidosis, which
is responsible for growth retardation, bone disease and kidney
stones [43]. Associated features include polyuria, nephrolithiasis, hypercalciuria and hypocitraturia. Alkali therapy allows
the correction of biochemical abnormalities and also improves
growth in children, kidney stones and bone abnormalities.
The disease can be acquired, mainly attributed to immune disorders or drug-induced kidney damage, or can be hereditary.
Hereditary forms are due to mutations in proteins involved in
acid–base transport in the intercalated cells, i.e. the anion exchanger 1 (AE1), the H+-ATPase and the cytosolic carbonic
anhydrase II (CA II). Mutations in the H+-ATPase and CA II
are responsible for recessive forms of dRTA, due in both cases
to loss-of-function mutations that lead to defective activity of
the enzymes [44, 45]. Mutations (missense, small deletions
and insertions) in AE1 lead to both autosomal dominant and
recessive forms of dRTA [46, 47]. AE1, a chloride-bicarbonate
exchanger, is encoded by the SLC4A1 gene. It is expressed at
the plasma membrane of erythrocytes and, as a shorter
isoform lacking the first N-terminal residues, at the basolateral
membrane of alpha-intercalated cells [48]. In vitro studies performed in polarized Madin-Darby Canine Kidney (MDCK)
cells have shown that mutations responsible for dominant
dRTA can have two distinct mechanisms of action. A first
group leads to ER retention and degradation of the mutant
protein [49]. Interestingly, for one such mutation (S613F),
AE1 intracellular retention was confirmed in patient kidney
biopsy [50]. These mutant isoforms are partly trafficked to the
plasma membrane and retain some activity in Xenopus oocytes,
excluding a major effect on protein folding, suggesting the
possibility of functional rescue [46, 51]. Another group of mutations is delivered to the membrane but with a reversal or loss
of polarity. Indeed, mutant protein can be found either on
both the basolateral and apical membrane or on the apical
membrane only of polarized MDCK cells. Defective polarized
trafficking of AE1 mutants is likely due to the disruption of
basolateral targeting signals, as in the case of R901X mutation
deleting the C-terminal signal sequence 904YDVE907 [52], and
possibly to the introduction of apical targeting motifs, as reported for mutation M909T [53]. Mistargeted AE1 mutants
were shown to be functional. Thus, their apical mislocalization
could lead to the secretion of HCO
3 into the collecting duct
lumen resulting in highly alkaline urine. Mutations responsible for the recessive form of dRTA can lead to the production
of partially functional or non-functional protein. Mutant isoforms are partly trafficked to the basolateral membrane (e.g.
S773P), or retained in the Golgi compartment (G701D) or in
the ER (S667F) of polarized MDCK cells [49, 54]. Dominant
or recessive inheritance of AE1 mutations is determined by
the interaction of mutant isoforms with co-expressed wildtype protein. Dominant mutants heterodimerize with the
wild-type isoform and exert a negative effect on its trafficking
[49]. On the contrary, trafficking of recessive mutants can actually be rescued by the presence of wild-type protein, thus explaining the lack of phenotype in heterozygous individuals
[49, 54]. Interestingly, most of the dRTA-associated dominant
mutations do not alter the trafficking and function of AE1 in
iv38
erythrocytes and are not associated with haematological abnormalities. This suggests that these cells have a less stringent
quality control system or express specific chaperones. One
such molecule could be glycophorin A (GPA), an erythroidspecific AE1-binding protein. Indeed, co-expression of GPA
increases membrane localization of mutant AE1 kidney isoforms [47, 54] and its deficiency alters AE1 structure [55].
As trafficking defect of mutant AE1 is at the basis of dRTA
pathogenesis, several studies have been performed to find molecules able to rescue the delivery of active AE1 to the membrane. ER-retained dominant mutants (R589H and R901X)
can be partially rescued to the membrane through inhibition
of their interaction with the ER chaperone calnexin (by treatment with castanospermine) or with Hsc70 (by treatment with
MAL3). Trafficking rescue is also observed after treatment with
two small molecules (C3 and C4) that were already shown to increase folding efficiency of the ΔF508 CFTR mutant through a
yet unclear mechanism [56]. Such treatments were not effective
on the Golgi-retained mutant G701D. The trafficking of this
mutant, and not the one of the ER-retained ones (R589H and
C479W), could be rescued by low temperature and by treatment
with chemical chaperones (DMSO and glycerol) and pharmacological agents (VX-809, another ΔF508 CFTR corrector). This
effect could be explained by an increase in overall protein levels,
possibly overcoming the cellular quality control systems. However,
only DMSO was also able to restore mutant AE1 function [57].
These studies in cellular models lay the foundations for developing
new therapeutic strategies in dRTA by rescuing mutant AE1 trafficking and function.
DEFECTIVE ENDOCYTOSIS
Endocytosis is a fundamental cellular process that involves internalization of extracellular molecules, plasma membrane
proteins and lipids. The principal components of the mammalian endocytic pathways are early and late endosomes and lysosomes (Figure 1a). These membrane compartments allow
the internalization of molecules from the plasma membrane,
their recycling to the surface and their intracellular sorting or
degradation. Defective endocytosis has been linked to several
diseases (Figure 1d), such as Dent’s disease.
Dent’s disease
Dent’s disease is an X-linked recessive disorder that is characterized by dysfunction of the proximal tubule (renal Fanconi
syndrome), resulting in low-molecular-weight proteinuria
(LMWP), hypercalciuria, nephrocalcinosis, nephrolithiasis and
progressive renal failure. The disease is genetically heterogeneous, being caused by mutations in the CLCN5 gene in 60% of
patients (Dent’s disease 1) [58] and in the OCRL1 gene, also responsible for Lowe syndrome, in 15% of patients (Dent’s disease
2) [59]. Genes mutated in the remaining 25% of patients are still
to be identified. The disease is essentially due to defective endocytosis in the proximal tubule. This process is of pivotal importance for reabsorbance of >80% of the proteins, as albumin and
low-molecular-weight proteins (including vitamin-binding proteins and hormones), that are filtered by the glomeruli.
C. Schaeffer et al.
Protein trafficking defects in kidney diseases
with no induction of the UPR [73, 74]. Class 2 mutations
(∼20%) are mainly localized at the dimer interface. Mutant
proteins are only partially retained in the ER, can acquire
complex glycosylation and are present in the endosomes and
at the plasma membrane. However, while the distribution of
Class 2 mutants in early endosomes is normal, their plasma
membrane levels are reduced due to delayed protein maturation, reduced protein stability and increased degradation.
Class 3 mutations (20%) do not affect protein folding and stability, acquire complex glycosylation and are present at the
plasma membrane and in the endosomes at levels comparable
with the wild-type protein. However, they have reduced or no
ion permeation. It is of note that these studies, providing important insights into the molecular mechanisms of ClC-5 mutations, were mostly conducted in non-polarized immortalized cell
lines and in Xenopus oocytes. Since other studies already underlined the different behaviour of mutant membrane proteins in
non-polarized versus polarized cell models (e.g. AE1 missorting
mutations [52, 75]), additional analysis in differentiated and polarized primary proximal tubule cells [76] may be needed to
further characterize the trafficking effect of ClC-5 mutations.
Current treatment for Dent’s disease patients is only supportive and mainly involves increased water intake to decrease
the risk of nephrolithiasis. However, no specific therapy aimed
at correcting the molecular defect due to ClC-5 deficiency is
available. In principle, one would expect that molecules restoring folding and stability of mutant ClC-5 could be effective in
rescuing Class 1 and 2 mutants. In vitro studies have already
shown that the use of the histone deacetylase (HDAC) inhibitor sodium butyrate (PBA) or low temperature conditions is
not able to rescue the trafficking of Class 1 ClC-5 mutants, as
opposed to what has been observed for misfolded ΔF508
CFTR [74]. These results suggest that ClC-5 mutations lead to
severe protein misfolding, and that the development of
pharmacological treatment able to restore proper folding will
be the next challenge in the field. Molecules able to rescue
ClC-5 mutations could be tested in vivo in the clinically relevant mouse models [62, 63], in vitro in murine primary proximal tubule cells [76] and possibly in immortalized proximal
tubule epithelial cells from Dent’s disease patients [77].
OCRL1 is a lipid phosphatase controlling the pool of phosphatidyl-inositol 4,5 biphosphate (PIP2), an intracellular messenger regulating membrane trafficking and cytoskeleton
structure and function. OCRL1 is localized in the Golgi and
early endosomes and may play a role in protein trafficking
between these two compartments [78]. OCRL1 mutations in
Dent’s disease 2 are mainly clustered in the 50 half region of the
gene and hit the pleckstrin homology (exons 2–5) or the PIP
5-phosphatase (exons 9–15) domains. Nonsense, frameshift and
splicing mutations, predicted to lead to premature termination,
are localized in OCRL1 exons 2–7, whereas missense mutations
are mainly clustered in exons 9–15 [79]. Different models have
been proposed to explain the relatively mild phenotype of Dent’s
disease 2 compared with Lowe syndrome, in which extra-renal
symptoms affecting brain and eye are present. Dent’s missense
mutations in the phosphatase domain might lead to a milder reduction in protein activity since they map to the domain surface.
On the contrary, Lowe syndrome missense changes tend to
iv39
FULL REVIEW
CLCN5 encodes the chloride/proton (2Cl−/H+) exchanger
ClC-5, functioning as a homodimer and expressed in renal
and intestinal epithelia. In the kidney, ClC-5 is predominantly
expressed in the proximal tubule and to a lesser extent in the
TAL and in the α-intercalated cells of the collecting duct [60].
In the proximal tubule, ClC-5 is mainly found in subapical endosomes and a small fraction at the apical plasma membrane.
Reported mutations in CLCN5 are nonsense (36%), missense
(33%), frameshift (19%) or in-frame (2%) deletions or insertions, intragenic or complete deletions (3%) or affect splicing
(7%) [61]. In the majority of the cases, they are predicted to
lead to loss of protein function. This is confirmed in vivo by
ClC-5 KO mice that faithfully recapitulate the disease features
[62, 63]. It has been proposed that ClC-5 activity contributes
to endosomal acidification, a condition indispensable for
progression along the endocytic pathway, ligand–receptor
dissociation, ligand processing and receptor degradation or
recycling. As a chloride transporter, ClC-5 could provide
the shunt conductance allowing the neutralization of H+
pumped into endosomes/vesicles by the v-type H+-ATPase for
acidification [64]. However, this hypothesis has been challenged. Indeed, a knock-in mouse model carrying a ClC-5 mutation (E211A) that converts the exchanger in an uncoupled
Cl− channel shows normal endosomal acidification. Still, it
presents defective endocytosis and the same renal phenotype
as ClC-5 KO mice and as patients with Dent’s disease. These
results indicate that functional endocytosis requires not only a
low endosomal pH but most importantly the endosomal accumulation of Cl− , and that this could be at the bases of the
pathogenesis of Dent’s disease [65]. Moreover, ClC-5 deficiency has been associated with more general defects of
protein trafficking in proximal tubule cells. Indeed, the levels
of cubilin and megalin, the two multiligand receptors implicated in endocytosis, are reduced at the apical brush border of
proximal tubules in ClC-5 KO mice. This contributes to
LMWP and possibly to indirect downstream effects such as
hyperphosphaturia and hypercalciuria, due to increased
urinary levels of parathyroid hormone and vitamin D-binding
protein [66, 67]. Defective exocytosis of Na+-H+ exchanger
isoform 3 (NHE3) and altered polarity of H+-ATPase have also
been reported in proximal tubules of ClC-5 KO mice [68] and
in kidney biopsies of Dent’s disease patients [69], respectively.
The mechanism by which the absence of ClC-5 leads to vesicular trafficking defect is still unknown. It could be correlated
with the loss of key interactions of ClC-5 with components
of the intracellular trafficking machinery such as cofilin, an
actin-depolymerizing protein, the Na+-H+ exchange regulatory
factor-2, Nedd-4, and the kinesin KIF3B [70].
In vitro studies of different missense ClC-5 mutations
(∼20% of the reported ones) have led to their classification
into three main classes, according to the different functional
and cell biological consequences [71]. About 60% of studied
mutations belong to Class 1. They are mostly located at the
interface between monomers and are predicted to affect the
homodimer formation [72]. Few mutations of the same class
are found away from the dimer interface and are thought to
affect protein structure. Class 1 mutations lead to ER retention
followed by proteasomal degradation of the mutant protein,
cluster in the domain core and are predicted to have a destabilizing effect [78]. Based on bioinformatics analysis, Shrimpton
et al. [80] proposed that truncating mutations could be rescued
in brain and eye tissues by the presence of an alternative OCRL1
isoform starting from exon 8.
D E F E C T I V E D E G R A D AT I O N
Vesicular trafficking can modulate the function of plasma membrane proteins, since the rate of insertion and internalization
modifies their amount at the plasma membrane (Figure 1a).
Several studies have shown that ubiquitylation is an important
signal for internalization and sorting of plasma membrane proteins. Its defects are associated with diseases (Figure 1d) such as
Liddle syndrome and pseudohypoaldosteronism type 2 (PHAII).
FULL REVIEW
Liddle syndrome
Liddle syndrome is a rare autosomal dominant disease
characterized by early onset salt-sensitive hypertension associated with low plasma renin activity, hypokalaemia and metabolic alkalosis. The disease is due to mutations (premature
termination, frameshift or missense) in SCNN1B and SCNN1G
genes coding for the β and γ subunit of the epithelial sodium
channel (ENaC), respectively [81, 82]. ENaC is present in many
epithelial cells of the body. In the kidney, it is found at the
apical membrane of collecting duct principal cells. Mutations
are responsible for high ENaC activity resulting in excessive
sodium reabsorption in the distal nephron [83]. This increased
activity is independent of the action of aldosterone, as patients
have low levels of aldosterone secretion. Consistently, they are
not responsive to aldosterone antagonists, but are responsive to
low salt diet combined with treatment with ENaC inhibitors
such as amiloride or triamterene [84]. With treatment, the prognosis is generally good. An early diagnosis is thus important to
avoid cardiovascular and renal complications due to long-term
undetected hypertension. Mutations are mainly located in the
cytoplasmic C-terminal region and lead to its deletion or
mutate a proline or tyrosine residue within a short proline-rich
sequence called the PY motif (Pro-Pro-x-Tyr). This motif is
highly conserved in all the ENaC subunits and acts as a binding
site for NEDD4-2, an E3 ubiquitin ligase [85]. The WW
domains of NEDD4-2 interact with the ENaC PY motif to
induce its ubiquitylation. This process is required for ENaC
endocytosis and degradation, thus reducing the number of
active channels at the membrane. Mutations hence exert a gain-
F I G U R E 2 : Effect of the treatment with pharmacological chaperones or proteostasis regulators to counteract mutations affecting protein
folding. In the absence of treatment, misfolded proteins are degraded or form intracellular aggregates. This not only leads to the absence of
functional protein in its final compartment, but can also have a broader impact on other proteins sharing common proteostasis pathways.
Pharmacological chaperones bind to their specific targets and stabilize them in a native or semi-native conformation rescuing protein trafficking
and reducing degradation or aggregation. Proteostasis regulators, by acting on pathways controlling protein homeostasis as protein synthesis,
folding, trafficking, aggregation and degradation (green shading), can increase the pool of proteins available for folding attempts and for
pharmacological chaperones. The synergistic action of the two treatments can further increase the amount of functional protein in its
physiological subcellular compartment. In some cases (not shown), functional rescue can also be obtained without restoring protein native
localization, as discussed in the text for AVPR2 mutants.
iv40
C. Schaeffer et al.
of-function effect by inhibiting the binding of NEDD4-2 and
consequently leading to an increased amount of active channel
at the apical membrane [83]. To date, only two mutations
(R563Q and N530S in the β and γ subunits, respectively) have
been reported outside of the C-terminus in an extracellular loop
of the ENaC subunit [86, 87]. It is also likely that these mutations lead to increased ENaC activity, although through a different mechanism. Mutation N530S was indeed shown to increase
the channel open probability [87]. In order to counteract ENaC
over-activation, effective treatments could be designed by developing more specific blockers. Since ENaC is activated through different proteolytic cleavages, the use of specific protease inhibitors
could also be envisaged as an alternative therapeutic option [88].
P R O S P E C T I V E T H E R A P E U T I C S T R AT E G I E S
Identification of the genes responsible for monogenic diseases
and the functional study of mutations and their consequences
on the protein product are essential steps to understand
disease pathophysiology, improve diagnosis and develop possible therapeutic strategies. In a growing number of genetic
diseases associated with dysfunctional protein trafficking mutations affect protein folding, leading to gain- and/or loss-offunction effects. Often misfolded proteins retain their intrinsic
activity, but their mutation results in disease because of
protein mislocalization. For conformational diseases, the ideal
therapeutic approach would be to identify small molecules
that can bind to and stabilize the native state of mutant proteins, thereby increasing the level of folded protein while
decreasing the amount of misfolded ones (Figure 2). Three
different types of small molecules have been tested to this
purpose: chemical chaperones, pharmacological chaperones
and proteostasis regulators. Chemical chaperones [e.g. DMSO,
trimethylamine N-oxide (TMAO), glycerol] are small molecules that do not directly bind to misfolded proteins, but
stabilize them by reducing free movement and increasing hydration. Although chemical chaperones have been shown to be
Protein trafficking defects in kidney diseases
iv41
FULL REVIEW
Pseudohypoaldosteronism type 2
The importance of protein ubiquitylation in the regulation
of renal Na+ transport has been extended by recent studies on
the genetic bases of PHAII [89]. PHAII is due to increased activity of Na+-Cl− co-transporter (NCC) at the apical membrane of the distal convoluted tubule leading to hypertension,
reduced distal Na+ delivery and consequent hyperkalaemia.
Mutations were found in the two main regulators of NCC, the
with-no-lysine kinases WNK1 and WNK4 [90]. Recently, mutations in the genes encoding the Kelch-like protein 3
(KLHL3)/Cullin 3 (CUL3) ubiquitin protein–ligase complex
have been identified in PHAII patients [91, 92]. Such mutations result in defective NCC ubiquitylation and degradation.
Interestingly, renal tubule-specific ablation of Nedd4L in the
mouse results in a phenotype resembling PHAII, with increased expression and phosphorylation of NCC. This suggests
that NEDD4-2 also plays a role in the regulation of NCC activity [93]. Targeting NEDD4-2 activity might thus be a possible
strategy to treat hypertension in these diseases.
effective in rescuing transport and function of some misfolded
proteins, as for example TMAO for ΔF508 CFTR [94], their
pharmacological application seems limited by the lack of specificity and the high concentration required. This can be circumvented by the use of pharmacological chaperones, small
molecules that specifically bind to a misfolded protein and can
induce conformational stabilization. Their mode of action is
still not well understood. These molecules could either bind
and stabilize the native or native-like state of the target protein
or they could bind to non-native folding intermediates,
serving as a scaffold for subsequent folding attempts. Regardless of the molecular mechanism, the final effect is to increase
the cellular availability of functional protein. Pharmacological
chaperones bind with high affinity to their targets and they
can thus be effective at low concentration. Their binding affinity should be taken into account when selecting new molecules, as binding at or near natural ligand-binding sites would
require displacement of the chaperone to rescue the antagonistic effect and protein function. Due to their specificity, the use
of pharmacological chaperones might fail to provide a comprehensive treatment for an individual conformational disease,
as the variety of mutations could limit the application of a
given molecule to a subset of mutations. However, the development of new powerful high-content screening methods could
overcome this limitation [95]. Although only two pharmacological chaperones are currently approved for clinical use in
conformational disorders (i.e. sapropterin dihydrochloride for
phenylketonuria and tafamidis for transthyretin-related hereditary amyloidosis), several molecules are under preclinical
and clinical trials [96]. Increasing interest is also given to modulators of protein homeostasis (or proteostasis). The maintenance of proteostasis is crucial for cellular and organismal
health and is achieved by an integrated network of several
hundred proteins. As our knowledge of these complex mechanisms steadily grows, we can identify targets and molecules
to tune these processes, with the rationale that tipping the
balance between protein synthesis, folding and degradation
may have therapeutic relevance. Proteostasis modulators
indeed act by increasing protein folding and/or enhancing
protein degradation. Modulators of stress responses, as the
heat shock response or the UPR, or of proteasomal or lysosomal/autophagic degradation pathways have already proved
to be effective in cellular and animal models. This is the case
of celastrol and guanabenz (UPR activators) in lysosomal
storage diseases and in Type I diabetes, respectively, and of
PBA (HDAC inhibitor) in cystic fibrosis [97]. Finally, interesting studies in cellular models of Gaucher and Tay-Sachs diseases demonstrated that combining more than one strategy, as
acting on a stress response signalling pathway and using a specific pharmacological chaperone, could be the best approach
to treat conformational diseases in the future [98].
Small molecules modulating protein homeostasis are being
discovered at an impressive rate thanks to the development of
improved screening strategies (high-throughput platforms and
new compound libraries) and to the increasing knowledge of
physiological and disease-associated mechanisms modulating
protein folding and trafficking. The expansion of this relatively
young class of pharmaceutical molecules may, in the near
future, improve current treatment or even offer treatment for
yet incurable conformational diseases.
AC K N O W L E D G E M E N T S
We are grateful to Eelco Van Anken for critical reading of the
manuscript. Work in our laboratory is supported by TelethonItaly (TCR08006) and the Italian Ministry of Health (grant
RF-2010-2319394).
C O N F L I C T O F I N T E R E S T S TAT E M E N T
None declared. The authors declare that the results presented
in this paper have not been published previously in whole
or part.
FULL REVIEW
REFERENCES
1. Hildebrandt F. Genetic kidney diseases. Lancet 2010; 375: 1287–1295
2. Parenti G, Pignata C, Vajro P et al. New strategies for the treatment of
lysosomal storage diseases (review). Int J Mol Med 2013; 31: 11–20
3. Moeller HB, Rittig S, Fenton RA. Nephrogenic diabetes insipidus: essential
insights into the molecular background and potential therapies for treatment. Endocr Rev 2013; 34: 278–301
4. Kamsteeg EJ, Hendriks G, Boone M et al. Short-chain ubiquitination
mediates the regulated endocytosis of the aquaporin-2 water channel.
Proc Natl Acad Sci USA 2006; 103: 18344–18349
5. Rosenthal W, Seibold A, Antaramian A et al. Molecular identification of
the gene responsible for congenital nephrogenic diabetes insipidus.
Nature 1992; 359: 233–235
6. Pan Y, Metzenberg A, Das S et al. Mutations in the V2 vasopressin receptor gene are associated with X-linked nephrogenic diabetes insipidus. Nat
Genet 1992. 2: 103–106
7. Wesche D, Deen PM, Knoers NV. Congenital nephrogenic diabetes insipidus: the current state of affairs. Pediatr Nephrol 2012. 27: 2183–2204
8. Barak LS, Oakley RH, Laporte SA et al. Constitutive arrestin-mediated
desensitization of a human vasopressin receptor mutant associated with
nephrogenic diabetes insipidus. Proc Natl Acad Sci USA 2001; 98: 93–98
9. Hermosilla R, Oueslati M, Donalies U et al. Disease-causing V(2) vasopressin receptors are retained in different compartments of the early secretory pathway. Traffic 2004; 5: 993–1005
10. Postina R, Ufer E, Pfeiffer R et al. Misfolded vasopressin V2 receptors
caused by extracellular point mutations entail congential nephrogenic diabetes insipidus. Mol Cell Endocrinol 2000; 164(1–2): 31–39
11. Robben JH, Sze M, Knoers NV et al. Rescue of vasopressin V2 receptor
mutants by chemical chaperones: specificity and mechanism. Mol Biol
Cell 2006; 17: 379–386
12. Los EL, Deen PM, Robben JH. Potential of nonpeptide (ant)agonists to
rescue vasopressin V2 receptor mutants for the treatment of X-linked nephrogenic diabetes insipidus. J Neuroendocrinol 2010; 22: 393–399
13. Bernier V, Morello JP, Zarruk A et al. Pharmacologic chaperones as a potential treatment for X-linked nephrogenic diabetes insipidus. J Am Soc
Nephrol 2006; 17: 232–243
14. Robben JH, Kortenoeven ML, Sze M et al. Intracellular activation of vasopressin V2 receptor mutants in nephrogenic diabetes insipidus by nonpeptide agonists. Proc Natl Acad Sci USA 2009; 106: 12195–12200
15. Revankar CM, Cimino DF, Sklar LA et al. A transmembrane intracellular
estrogen receptor mediates rapid cell signaling. Science 2005; 307:
1625–1630
16. Dupre DJ, Baragli A, Rebois RV et al. Signalling complexes associated with
adenylyl cyclase II are assembled during their biosynthesis. Cell Signal
2007; 19: 481–489
iv42
17. Olesen ET, Rutzler MR, Moeller HB et al. Vasopressin-independent targeting of aquaporin-2 by selective E-prostanoid receptor agonists alleviates
nephrogenic diabetes insipidus. Proc Natl Acad Sci USA 2011; 108:
12949–12954
18. Li JH, Chou CL, Li B et al. A selective EP4 PGE2 receptor agonist alleviates
disease in a new mouse model of X-linked nephrogenic diabetes insipidus.
J Clin Invest 2009; 119: 3115–3126
19. Van Lieburg AF, Verdijk MA, Knoers VV et al. Patients with autosomal
nephrogenic diabetes insipidus homozygous for mutations in the aquaporin 2 water-channel gene. Am J Hum Genet 1994; 55: 648–652
20. Robben JH, Knoers NV, Deen PM. Cell biological aspects of the vasopressin type-2 receptor and aquaporin 2 water channel in nephrogenic diabetes insipidus. Am J Physiol Renal Physiol 2006; 291: F257–F270
21. De Mattia F, Savelkoul PJ, Kamsteeg EJ et al. Lack of arginine vasopressininduced phosphorylation of aquaporin-2 mutant AQP2-R254L explains
dominant nephrogenic diabetes insipidus. J Am Soc Nephrol 2005; 16:
2872–2880
22. Savelkoul PJ, De Mattia F, Li Y et al. p.R254Q mutation in the aquaporin2 water channel causing dominant nephrogenic diabetes insipidus is due
to a lack of arginine vasopressin-induced phosphorylation. Hum Mutat
2009; 30: E891–E903
23. Kamsteeg EJ, Wormhoudt TA, Rijss JP et al. An impaired routing of wildtype aquaporin-2 after tetramerization with an aquaporin-2 mutant explains
dominant nephrogenic diabetes insipidus. EMBO J 1999; 18: 2394–2400
24. De Mattia F, Savelkoul PJ, Bichet DG et al. A novel mechanism in recessive
nephrogenic diabetes insipidus: wild-type aquaporin-2 rescues the apical
membrane expression of intracellularly retained AQP2-P262L. Hum Mol
Genet 2004; 13: 3045–3056
25. Sohara E, Rai T, Yang SS et al. Pathogenesis and treatment of autosomaldominant nephrogenic diabetes insipidus caused by an aquaporin 2 mutation. Proc Natl Acad Sci USA 2006; 103: 14217–14222
26. Yang B, Zhao D, Verkman AS. Hsp90 inhibitor partially corrects nephrogenic diabetes insipidus in a conditional knock-in mouse model of aquaporin-2 mutation. FASEB J 2009; 23: 503–512
27. Hart TC, Gorry MC, Hart PS et al. Mutations of the UMOD gene are
responsible for medullary cystic kidney disease 2 and familial juvenile
hyperuricaemic nephropathy. J Med Genet 2002; 39: 882–892
28. Rampoldi L, Scolari F, Amoroso A et al. The rediscovery of uromodulin
(Tamm-Horsfall protein): from tubulointerstitial nephropathy to chronic
kidney disease. Kidney Int 2011; 80: 338–347
29. Bernascone I, Janas S, Ikehata M et al. A transgenic mouse model for uromodulin-associated kidney diseases shows specific tubulo-interstitial
damage, urinary concentrating defect and renal failure. Hum Mol Genet
2010; 19: 2998–3010
30. Kemter E, Prueckl P, Sklenak S et al. Type of uromodulin mutation and
allelic status influence onset and severity of uromodulin-associated kidney
disease in mice. Hum Mol Genet 2013; 22: 4148–4163
31. Raffi H, Bates JM, Laszik Z et al. Tamm-Horsfall protein knockout mice do
not develop medullary cystic kidney disease. Kidney Int 2006; 69: 1914–1915
32. Drozdova T, Papillon J, Cybulsky AV. Nephrin missense mutations: induction of endoplasmic reticulum stress and cell surface rescue by reduction
in chaperone interactions. Physiol Rep 2013; 1: e00086
33. Pieri M, Stefanou C, Zaravinos A et al. Evidence for activation of the unfolded protein response in collagen IV nephropathies. J Am Soc Nephrol
2014; 25: 260–275
34. Chen YM, Zhou Y, Go G et al. Laminin beta2 gene missense mutation
produces endoplasmic reticulum stress in podocytes. J Am Soc Nephrol
2013; 24: 1223–1233
35. Purdue PE, Takada Y, Danpure CJ. Identification of mutations associated
with peroxisome-to-mitochondrion mistargeting of alanine/glyoxylate
aminotransferase in primary hyperoxaluria type 1. J Cell Biol 1990; 111
(6 Pt 1): 2341–2351
36. Williams EL, Acquaviva C, Amoroso A et al. Primary hyperoxaluria type
1: update and additional mutation analysis of the AGXT gene. Hum
Mutat 2009; 30: 910–917
37. Leiper JM, Oatey PB, Danpure CJ. Inhibition of alanine:glyoxylate
aminotransferase 1 dimerization is a prerequisite for its peroxisome-tomitochondrion mistargeting in primary hyperoxaluria type 1. J Cell Biol
1996; 135: 939–951
C. Schaeffer et al.
Protein trafficking defects in kidney diseases
61. Devuyst O, Thakker RV. Dent’s disease. Orphanet J Rare Dis 2010; 5: 28
62. Piwon N, Gunther W, Schwake M et al. ClC-5 Cl−-channel disruption
impairs endocytosis in a mouse model for Dent’s disease. Nature 2000; 408:
369–373
63. Wang SS, Devuyst O, Courtoy PJ et al. Mice lacking renal chloride
channel, CLC-5, are a model for Dent’s disease, a nephrolithiasis disorder
associated with defective receptor-mediated endocytosis. Hum Mol Genet
2000; 9: 2937–2945
64. Gunther W, Luchow A, Cluzeaud F et al. ClC-5, the chloride
channel mutated in Dent’s disease, colocalizes with the proton pump in
endocytotically active kidney cells. Proc Natl Acad Sci USA 1998. 95:
8075–8080
65. Novarino G, Weinert S, Rickheit G et al. Endosomal chloride-proton exchange rather than chloride conductance is crucial for renal endocytosis.
Science 2010; 328: 1398–1401
66. Christensen EI, Devuyst O, Dom G et al. Loss of chloride channel ClC-5
impairs endocytosis by defective trafficking of megalin and cubilin in
kidney proximal tubules. Proc Natl Acad Sci USA 2003; 100: 8472–8477
67. Claverie-Martin F, Ramos-Trujillo E, Garcia-Nieto V. Dent’s disease: clinical features and molecular basis. Pediatr Nephrol 2011; 26: 693–704
68. Lin Z, Jin S, Duan X et al. Chloride channel (Clc)-5 is necessary for exocytic trafficking of Na+/H+ exchanger 3 (NHE3). J Biol Chem 2011; 286:
22833–22845
69. Moulin P, Igarashi T, Van Der Smissen P et al. Altered polarity and
expression of H+-ATPase without ultrastructural changes in kidneys of
Dent’s disease patients. Kidney Int 2003; 63: 1285–1295
70. Lippiat JD, Smith AJ. The CLC-5 2Cl(−)/H(+) exchange transporter in
endosomal function and Dent’s disease. Front Physiol 2012; 3: 449
71. Lourdel S, Grand T, Burgos J et al. ClC-5 mutations associated with Dent’s
disease: a major role of the dimer interface. Pflugers Arch 2012; 463:
247–256
72. Wu F, Roche P, Christie PT et al. Modeling study of human renal chloride
channel (hCLC-5) mutations suggests a structural-functional relationship.
Kidney Int 2003; 63: 1426–1432
73. Grand T, Mordasini D, L’hoste S et al. Novel CLCN5 mutations in patients
with Dent’s disease result in altered ion currents or impaired exchanger
processing. Kidney Int 2009; 76: 999–1005
74. D’antonio C, Molinski S, Ahmadi S et al. Conformational defects underlie
proteasomal degradation of Dent’s disease-causing mutants of ClC-5.
Biochem J 2013; 452: 391–400
75. Quilty JA, Li J, Reithmeier RA. Impaired trafficking of distal renal tubular
acidosis mutants of the human kidney anion exchanger kAE1. Am J
Physiol Renal Physiol 2002; 282: F810–F820
76. Terryn S, Jouret F, Vandenabeele F et al. A primary culture of mouse
proximal tubular cells, established on collagen-coated membranes. Am J
Physiol Renal Physiol 2007; 293: F476–F485
77. Gorvin CM, Wilmer MJ, Piret SE et al. Receptor-mediated endocytosis
and endosomal acidification is impaired in proximal tubule epithelial
cells of Dent disease patients. Proc Natl Acad Sci USA 2013; 110:
7014–7019
78. Pirruccello M, De Camilli P. Inositol 5-phosphatases: insights from the
Lowe syndrome protein OCRL. Trends Biochem Sci 2012; 37: 134–143
79. Hichri H, Rendu J, Monnier N et al. From Lowe syndrome to Dent
disease: correlations between mutations of the OCRL1 gene and clinical
and biochemical phenotypes. Hum Mutat 2011; 32: 379–388
80. Shrimpton AE, Hoopes RR, Jr, Knohl SJ et al. OCRL1 mutations in Dent
2 patients suggest a mechanism for phenotypic variability. Nephron
Physiol 2009; 112: p27–p36
81. Shimkets RA, Warnock DG, Bositis CM et al. Liddle’s syndrome: heritable
human hypertension caused by mutations in the beta subunit of the
epithelial sodium channel. Cell 1994; 79: 407–414
82. Hansson JH, Nelson-Williams C, Suzuki H et al. Hypertension caused by
a truncated epithelial sodium channel gamma subunit: genetic heterogeneity of Liddle syndrome. Nat Genet 1995; 11: 76–82
83. Snyder PM, Price MP, Mcdonald FJ et al. Mechanism by which Liddle’s
syndrome mutations increase activity of a human epithelial Na+ channel.
Cell 1995; 83: 969–978
84. Warnock DG. Liddle syndrome: an autosomal dominant form of human
hypertension. Kidney Int 1998; 53: 18–24
iv43
FULL REVIEW
38. Fargue S, Lewin J, Rumsby G et al. Four of the most common mutations
in primary hyperoxaluria type 1 unmask the cryptic mitochondrial targeting sequence of alanine:glyoxylate aminotransferase encoded by the polymorphic minor allele. J Biol Chem 2013; 288: 2475–2484
39. Mesa-Torres N, Fabelo-Rosa I, Riverol D et al. The role of protein denaturation energetics and molecular chaperones in the aggregation and
mistargeting of mutants causing primary hyperoxaluria type I. PLoS ONE
2013; 8: e71963
40. Pey AL, Albert A, Salido E. Protein homeostasis defects of alanine-glyoxylate
aminotransferase: new therapeutic strategies in primary hyperoxaluria type
I. Biomed Res Int 2013; 2013: 687658
41. Hoppe B, Langman CB. A United States survey on diagnosis, treatment, and
outcome of primary hyperoxaluria. Pediatr Nephrol 2003; 18: 986–991
42. Fargue S, Rumsby G, Danpure CJ. Multiple mechanisms of action of pyridoxine in primary hyperoxaluria type 1. Biochim Biophys Acta 2013; 1832:
1776–1783
43. Alper SL. Familial renal tubular acidosis. J Nephrol 2010; 23(Suppl 16):
S57–S76
44. Karet FE, Finberg KE, Nelson RD et al. Mutations in the gene encoding
B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural
deafness. Nat Genet 1999; 21: 84–90
45. Sly WS, Whyte MP, Sundaram V et al. Carbonic anhydrase II deficiency
in 12 families with the autosomal recessive syndrome of osteopetrosis with
renal tubular acidosis and cerebral calcification. N Engl J Med 1985; 313:
139–145
46. Bruce LJ, Cope DL, Jones GK et al. Familial distal renal tubular acidosis is
associated with mutations in the red cell anion exchanger (Band 3, AE1)
gene. J Clin Invest 1997; 100: 1693–1707
47. Tanphaichitr VS, Sumboonnanonda A, Ideguchi H et al. Novel AE1 mutations in recessive distal renal tubular acidosis. Loss-of-function is rescued
by glycophorin A. J Clin Invest 1998; 102: 2173–2179
48. Kollert-Jons A, Wagner S, Hubner S et al. Anion exchanger 1 in human
kidney and oncocytoma differs from erythroid AE1 in its NH2 terminus.
Am J Physiol 1993; 265(6 Pt 2): F813–F821
49. Cordat E, Kittanakom S, Yenchitsomanus PT et al. Dominant and recessive distal renal tubular acidosis mutations of kidney anion exchanger 1
induce distinct trafficking defects in MDCK cells. Traffic 2006; 7: 117–128
50. Walsh S, Turner CM, Toye A et al. Immunohistochemical comparison of
a case of inherited distal renal tubular acidosis (with a unique AE1 mutation) with an acquired case secondary to autoimmune disease. Nephrol
Dial Transplant 2007; 22: 807–812
51. Jarolim P, Shayakul C, Prabakaran D et al. Autosomal dominant distal
renal tubular acidosis is associated in three families with heterozygosity
for the R589H mutation in the AE1 (band 3) Cl−/HCO−
3 exchanger. J Biol
Chem 1998; 273: 6380–6388
52. Devonald MA, Smith AN, Poon JP et al. Non-polarized targeting of AE1
causes autosomal dominant distal renal tubular acidosis. Nat Genet 2003;
33: 125–127
53. Fry AC, Su Y, Yiu V et al. Mutation conferring apical-targeting motif on
AE1 exchanger causes autosomal dominant distal RTA. J Am Soc Nephrol
2012; 23: 1238–1249
54. Toye AM, Williamson RC, Khanfar M et al. Band 3 Courcouronnes
(Ser667Phe): a trafficking mutant differentially rescued by wild-type band
3 and glycophorin A. Blood 2008; 111: 5380–5389
55. Bruce LJ, Pan RJ, Cope DL et al. Altered structure and anion transport
properties of band 3 (AE1, SLC4A1) in human red cells lacking glycophorin A. J Biol Chem 2004; 279: 2414–2420
56. Patterson ST, Reithmeier RA. Cell surface rescue of kidney anion exchanger 1 mutants by disruption of chaperone interactions. J Biol Chem 2010;
285: 33423–33434
57. Chu CY, King JC, Berrini M et al. Functional rescue of a kidney anion exchanger 1 trafficking mutant in renal epithelial cells. PLoS ONE 2013; 8: e57062
58. Lloyd SE, Pearce SH, Fisher SE et al. A common molecular basis for three
inherited kidney stone diseases. Nature 1996; 379: 445–449
59. Hoopes RR, Jr, Shrimpton AE, Knohl SJ et al. Dent disease with mutations
in OCRL1. Am J Hum Genet 2005; 76: 260–267
60. Devuyst O, Christie PT, Courtoy PJ et al. Intra-renal and subcellular distribution of the human chloride channel, CLC-5, reveals a pathophysiological basis for Dent’s disease. Hum Mol Genet 1999; 8: 247–257
93. Ronzaud C, Loffing-Cueni D, Hausel P et al. Renal tubular NEDD4-2 deficiency causes NCC-mediated salt-dependent hypertension. J Clin Invest
2013; 123: 657–665
94. Fischer H, Fukuda N, Barbry P et al. Partial restoration of defective chloride conductance in DeltaF508 CF mice by trimethylamine oxide. Am J
Physiol Lung Cell Mol Physiol 2001; 281: L52–L57
95. Naik S, Zhang N, Gao P et al. On the design of broad based screening assays
to identify potential pharmacological chaperones of protein misfolding diseases. Curr Top Med Chem 2012; 12: 2504–2522
96. Muntau AC, Leandro J, Staudigl M et al. Innovative strategies to
treat protein misfolding in inborn errors of metabolism: pharmacological chaperones and proteostasis regulators. J Inherit Metab Dis
2014; doi: 10.1007/s10545-014-9701-z [epub ahead of print]
97. Lindquist SL, Kelly JW. Chemical and biological approaches for adapting
proteostasis to ameliorate protein misfolding and aggregation diseases:
progress and prognosis. Cold Spring Harb Perspect Biol 2011; 3:
a004507
98. Mu TW, Ong DS, Wang YJ et al. Chemical and biological approaches
synergize to ameliorate protein-folding diseases. Cell 2008; 134: 769–781
Received for publication: 1.2.2014; Accepted in revised form: 27.5.2014
FULL REVIEW
85. Staub O, Dho S, Henry P et al. WW domains of Nedd4 bind to the
proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle’s
syndrome. EMBO J 1996; 15: 2371–2380
86. Jones ES, Owen EP, Davidson JS et al. The R563Q mutation of the epithelial sodium channel beta-subunit is associated with hypertension. Cardiovasc J Afr 2011; 22: 241–244
87. Hiltunen TP, Hannila-Handelberg T, Petajaniemi N et al. Liddle’s syndrome
associated with a point mutation in the extracellular domain of the epithelial
sodium channel gamma subunit. J Hypertens 2002; 20: 2383–2390
88. Rotin D, Schild L. ENaC and its regulatory proteins as drug targets for
blood pressure control. Curr Drug Targets 2008; 9: 709–716
89. Ronzaud C, Staub O. Ubiquitylation and control of renal Na+ balance and
blood pressure. Physiology (Bethesda) 2014; 29: 16–26
90. Wilson FH, Disse-Nicodeme S, Choate KA et al. Human hypertension
caused by mutations in WNK kinases. Science 2001; 293: 1107–1112
91. Louis-Dit-Picard H, Barc J, Trujillano D et al. KLHL3 mutations cause familial hyperkalemic hypertension by impairing ion transport in the distal
nephron. Nat Genet 2012; 44: 456–460, S1–3
92. Boyden LM, Choi M, Choate KA et al. Mutations in Kelch-like 3 and
Cullin 3 cause hypertension and electrolyte abnormalities. Nature 2012;
482: 98–102
iv44
C. Schaeffer et al.