Roles of mTOR complexes in the kidney: implications for renal
disease and transplantation
Daniel Fantus
1
, Natasha M. Rogers
1,2
, Florian Grahammer
3
, Tobias B. Huber
3,4,*
, and
Angus W. Thomson
1,*
1
Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh School of
Medicine, Pittsburgh, PA, USA
2
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine,
Pittsburgh, PA, USA
3
Department of Medicine IV, Medical Center-Faculty of Medicine, University of Freiburg, Freiburg
Germany
4
BIOSS Center for Biological Signalling Studies, University of Freiburg, Freiburg Germany
Abstract
The mTOR pathway has a central role in the regulation of cell metabolism, growth and
proliferation. Studies involving selective gene targeting of mTOR complexes (mTORC1 and
mTORC2) in renal cell populations and/or pharmacologic mTOR inhibition have revealed
important roles of mTOR in podocyte homeostasis and tubular transport. Important advances have
also been made in understanding the role of mTOR in renal injury, polycystic kidney disease and
glomerular diseases, including diabetic nephropathy. Novel insights into the roles of mTORC1 and
mTORC2 in regulation of immune cell homeostasis and function are helping to improve
understanding of the complex effects of mTOR targeting on immune responses, including those
that impact both
de novo
renal disease and renal allograft outcomes. Extensive experience in
clinical renal transplantation has resulted in successful conversion of patients from calcineurin
inhibitors to mTOR inhibitors at various times post-transplantation, with excellent long-term graft
function. Widespread use of this practice has, however, been limited owing to mTOR-inhibitor-
related toxicities. Unique attributes of mTOR inhibitors include reduced rates of squamous cell
carcinoma and cytomegalovirus infection compared to other regimens. As understanding of the
mechanisms by which mTORC1 and mTORC2 drive the pathogenesis of renal disease progresses,
clinical studies of mTOR pathway targeting will enable testing of evolving hypotheses.
*
Correspondence to: Tobias B. Huber tobias.huber@uniklinik-freiburg.de, Angus W. Thomson thomsonaw@upmc.edu.
Author contributions
D.F., N.M.R. and F.G. are co-first authors and T.B.H. and A.W.T are co-senior authors of this Review. D.F., N.M.R. and F.G.
contributed equally to writing the article. T.B.H. and A.W.T. researched literature for the article, provided substantial discussion of the
content and contributed equally to review and/or editing of the manuscript before and after submission.
Competing interests statement
The authors declare no competing interests.
HHS Public Access
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Nat Rev Nephrol
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Published in final edited form as:
Nat Rev Nephrol
. 2016 October ; 12(10): 587–609. doi:10.1038/nrneph.2016.108.
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Introduction
Since the discovery of rapamycin (also known as sirolimus more than 40 years ago,
1
advances in the understanding of its molecular mode of action as well as the functional
biology of its primary target — mTOR — have permeated many areas of medicine,
including cardiovascular disease, autoimmunity and cancer. mTOR is an evolutionarily-
conserved serine-threonine kinase that regulates cell growth, proliferation and metabolism.
Increasing evidence indicates that mTOR has an important role in the regulation of renal cell
homeostasis and autophagy. Moreover, this kinase has been implicated in the development
of glomerular disease, polycystic kidney disease (PKD), acute kidney injury (AKI) and
kidney transplant rejection.
The development of rapamycin and its analogues (known as rapalogstemsirolimus and
everolimus, has expanded the pharmacological armamentarium for treatment of renal
disease. Owing to its ability to potently inhibit T cell proliferation, rapamycin was initially
developed as an immunosuppressive agent in kidney transplantation.
2
Rapalogs have now
also been added to the immunosuppressive repertoire for glomerulonephritides (although not
a therapeutic mainstay for these conditions) and renal cell carcinoma. In this Review, we
discuss aspects of mTOR function and its inhibition in relation to renal physiology, kidney
disease including malignancy, and the role of mTOR complexes and their inhibitors in renal
transplantation.
mTOR complexes
mTOR operates in at least two distinct, multi-protein complexes: mTOR complex 1
(mTORC1) and mTOR complex 2 (mTORC2) (FIG. 1). Details of the structural
biochemistry of mTOR and role in cellular signalling have been reviewed in detail
elsewhere.
3–5
mTORC1 is often described as a ‘nutrient sensor’ as it can be activated by
amino acids and inhibited by severe oxidative stress and energy depletion. The primary roles
of mTOR are to facilitate cell growth and anabolism as well as to prevent autophagy.
Although mTORC1 was localized initially to the cytoplasm, this complex has since been
identified in association with endosomal compartments (outer mitochondrial membranes and
nuclei
6–8
) and has been shown to have a role in stress granule formation.
9
These findings
provide further evidence that mTOR is a ‘metabolic rheostat’ for eukaryotic cells.
Growth factors and cytokines can activate mTORC1 via upstream signalling through
phosphoinositide 3-kinase (PI3K). Generation of phosphatidylinositol (3,4,5) triphosphate
(PIP3) by PI3K activates 3-phosphoinositide-dependent protein kinase-1 (PDK1), which
enhances Akt (also known as protein kinase B) activity by phosphorylating the activation
loop at threonine 308. Interestingly, mTORC2 uniquely stabilizes Akt via phosphorylation
of the turn motif at serine 450, and further stimulates Akt kinase activity by phosphorylating
the hydrophobic motif at serine 473. The activity of tuberous sclerosis complex 1 (TSC1)
and TSC2, the major upstream inhibitors of mTORC1, is dampened directly by Akt-
dependent phosphorylation and provides a functional link between the PI3K–Akt signalling
axis and mTOR. TSC1 stabilizes and TSC2 inhibits the activity of the guanosine
triphosphate (GTP)-ase Rheb (RAS homolog enriched in brain), through its GTPase-
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activating protein activity. Rheb enhances the catalytic activity of mTORC1 when they are in
close proximity.
Tools to study mTORC2 are limited owing to a lack of mTORC2-specific inhibitors.
Substrates of mTORC2 include Akt, protein kinase Cα (PKCα) and serum and
glucocorticoid-induced kinase-1 (SGK-1). Furthermore, phosphorylation and cytoplasmic
sequestration of the forkhead box proteins 01 (FOXO1) and FOXO3 by Akt has been shown
to require mTORC2.
10
Consistent with these signalling cascades, mTORC2-orchestrated
processes include cell survival, protein synthesis, re-organization of the actin cytoskeleton
and sodium homeostasis. Although active mTORC2 physically associates with ribosomes in
mammalian cells, the intricacies of this pathway have not been completely characterized.
11
Importantly, crosstalk between the mTORC1 and mTORC2 pathways
12
adds an extra layer
of complexity in dissecting mTORC1 and mTORC2 biology. Thus, mTORC1 inhibits the
PI3K–mTORC2 pathway through a negative feedback mechanism.
mTOR inhibitors
Although rapamycin immediately inhibits mTORC1, its ability to destabilize and inhibit
mTORC2 requires prolonged exposure and is more sensitive to fluctuations in
concentrations of the immunophilin FK506 binding protein 12 (FKBP12), which binds
rapamycin and mediates its interaction with mTOR
13
The negligible effect of rapamycin on
mTORC2 function has been disputed, however, with evidence that this agent might inhibit
mTORC2 assembly and signalling.
14
The C-terminal part of Avo3, a subunit unique to
TORC2 in yeast, is located close to the FKBP12/rapamycin-binding domain of mTORC2
and removal of Avo3 from TORC2 confers sensitivity to rapamycin.
15
Although less is
known about how rapalogs affect mTORC1 and 2, inhibition of mTORC2 has been
demonstrated in acute myeloid leukemia cells exposed to everolimus and temsirolimus.
16
Novel dual inhibitors of TORC1 and TORC2 (TORKinibs) that compete for the adenosine
triphosphate (ATP)-binding site of mTOR were developed to limit activation of both mTOR
complexes
17
and provide broader clinical efficacy than the currently available rapalogs.
These molecules were first described to inhibit P13K,
17
a critical component upstream of the
Akt/mTOR pathway, and subsequently also found to inhibit mTOR, presumably owing to
sequence homology. Initial TORKinibs (mTOR and PI3K dual inhibitors) failed to
selectively inhibit either signalling moiety,
18
but newer molecular inhibitors (mTORC1 and
mTORC2 dual inhibitors) exhibit greater potency for mTOR.
19
These agents are being
investigated for the treatment of malignancy in phase I and II clinical trials
20
but have yet to
be tested in clinical organ transplantation.
mTOR in renal physiology
Given the ubiquitous expression of mTORC1 and mTORC2 in the kidney, elucidating the
roles of mTOR in renal physiology is a formidable undertaking. Nonetheless, pharmacologic
inhibition and targeted cell-specific genetic deletion have enabled analyses of mTOR
function in the rodent and human kidney (FIG 2). Studies of patients with TSC, a syndrome
with autosomal dominant inheritance characterized by upregulation of mTORC1, have also
increased understanding of the consequences of enhanced mTORC1 activity on renal
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physiology. Although much of the current understanding of mTOR function pertains to the
podocyte or tubular epithelial cell, renal endothelial and immune cells are emerging as
important players in the regulation of renal homeostasis and metabolism.
Podocytes
Glomerular podocytes are highly-differentiated, voluminous cells with numerous foot
processes that line the outer aspects of the glomerular basement membrane (GBM). The foot
processes interdigitate with each other and form the slit diaphragm, an intercellular junction
composed of several membrane proteins that regulate protein movement into the urinary
space.
21
Although glomerular diseases can be caused by disturbances in various barrier
components, podocyte loss and effacement is a critical event that, if not constrained, leads to
proteinuria, renal fibrosis and end-stage renal disease (ESRD).
Much of the understanding of podocyte mTORC1 and mTORC2 function stems from
genetic deletion studies. In mice, podocyte-specific embryonic knockout of mTORC1
(owing to deletion of the regulatory-associated protein of mTOR (Raptor) (Ed: Yes) resulted
in early albuminuria, later development of glomerulosclerosis, weight loss and increased
mortality.
22
Loss of podocyte mTORC1 in adult mice failed to induce similar deleterious
effects. Surprisingly, heterozygous deletion of Raptor in podocytes expression and podocyte
volume. Mice with podocyte-specific loss of mTORC2 (owing to deletion of rapamycin-
insensitive companion of mTOR (Rictor) did not exhibit significant phenotypic differences
compared to littermate controls, with the exception of transient albuminuria following
protein overload. However, combined deletion of both mTOR complexes from podocytes
precipitated an early (6 weeks of age) fulminant proteinuric phenotype, suggesting some
degree of interaction between mTORC1 and mTORC2 in the regulation of podocyte
development and homeostasis.
Excessive mTORC1 activity can also result in severe pathologic effects, including hallmarks
of diabetic nephropathy. In a murine study, podocyte-specific deletion of
Tsc1
led to
aberrant mTORC1 activation, enhanced pS6 expression, mesangial expansion, GBM
thickening, podocyte loss, podocyte foot-process effacement and proteinuria (that was
attenuated by rapamycin), as well as glomerular expression of transforming growth factor β1
(TGF-β1), type IV collagen and fibronectin.
23
The remaining podocytes of the transgenic
mice had a fibroblastic phenotype with markers of endoplasmic reticulum stress.
Immunofluorescence studies demonstrated altered distribution of podocyte nephrin and
synaptopodin that was corrected by rapamycin administration.
Tubular epithelial cells
Tubular epithelial cells (TECs) are bathed in urine via their apical membranes and have an
important role in maintaining salt and water balance, typically via their basolateral surfaces.
This role is accomplished largely via energy-dependent cellular transporters. Initial evidence
that mTOR might regulate tubular homeostasis came from repeated observations of
electrolyte disturbances in renal transplant recipients treated with rapamycin, although the
underlying cause of these disturbances is still disputed. The renal-related adverse effects of
rapamycin are typically hypokalaemia,
24
hypomagnesaemia and hypophosphataemia,
25, 26
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as well as decreased urinary calcium excretion.
27
Sirolimus decreases the expression of the
apical Na-K-2Cl co-transporter (NKCC2) in the thick ascending limb (TAL), resulting in
renal tubular wasting of sodium, magnesium and potassium.
26
The phosphaturic actions of
rapamycin might be linked to the induction of klotho, Rictor and mTORC2, which has been
shown
in vivo
and in an immortalized renal tubular cell line.
28
Post-transplant phosphaturia
is aggravated by administration of sirolimus, although this effect is reportedly not the result
of altered phosphate uptake via sodium–phosphate co-transporters in the brush border of the
proximal tubule.
29
Nevertheless, in
Xenopus
oocytes expressing sodium–phosphate
transporters, co-transfection with mTOR increased phosphate currents and this effect was
abrogated by exposure to rapamycin.
30
The past few years have seen several reports linking
mTORC1 to proximal tubular transport mechanisms. Work performed mainly in
Drosophila
melanogaster
showed that the proton pump V-ATPase responsible for intracellular
compartment acidification and apical amino acid uptake requires mTORC1, This interplay
seems to be regulated through the multi-ligand-binding receptor megalin.
31
Indeed, in
C57BL/6 mice, long-term rapamycin exposure reduced proximal tubular megalin expression
and caused concurrent low molecular weight proteinuria, consistent with renal proximal
TEC dysfunction.
Most recently, however, it has been demonstrated that mice deficient in proximal tubular
mTORC1 present with a Fanconi Syndrome-like phenotype consisting of phosphaturia,
glucosuria, low-molecular weight proteinuria, albuminuria and aminoaciduria.
32
Deep
proteomic and phosphoproteomic analysis shows that mTORC1 deficiency affects the
translation and phosphorylation of specific transport systems, its regulators as well as
proteins involved in endocytosis in a kinase-dependent manner. Interestingly, and in contrast
to the earlier study,
31
no alterations in the abundance of the two scavenger receptors cubilin
and megalin could be detected.
In mice, specific deletion of Raptor (mTORC1) in distal TECs did not cause significant
developmental defects, but these animals developed polyuria and hypercalciuria after
weaning as a result of a defect in TAL countercurrent multiplication
33
. Consistent with these
perturbations, transcriptional profiling identified reduced levels of NKCC2 in these mice.
Generation of mTORC1/2-deficient animals resulted in aggravated pathology and increased
mortality, suggesting that these complexes have complimentary roles.
Although the molecular pathways downstream of mTORC1 and mTORC2 that regulate ion
channels in the TAL (such as NKCC2) remain to be determined, significant advances have
occurred in the understanding of how mTOR regulates the aldosterone-sensitive epithelial
sodium channel (ENaC), a protein that is most abundant in the distal tubule. Serum and
glucocorticoid inducible kinase 1 (SGK1) acts as a central driver of this process by
increasing cell surface expression of ENaC via inhibition of the E3 ubiquitin-protein ligase
Nedd4–2
34
and mTORC2 has been found to activate SGK1 via phosphorylation of its
hydrophobic motif.
35
To investigate the physiological significance of the mTORC2–SGK1–ENaC axis
in vivo
,
researchers compared the effect on electrolyte homeostasis of targeting mTORC1 (using
rapamycin) with that of dual inhibition of mTORC1 and mTORC2 (using the TORKinibs
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