Mechanisms of mTORi and Evidence of Improved Renal Function after the Change from
CNIs to mTORi
Abstract
The use of immunosuppressive drugs calcineurin inhibitors (CNIs) have led to
remarkable increases in organ transplant success, however they have high nephrotoxicity leading
to kidney impairment. Mammalian target of rapamycin inhibitors (mTORi) are a newer class of
immunosuppressants that are believed to have less harmful side effects. It is suggested that the
mechanism of immunosuppression using mTORi involves the dissociation of raptor and TOR at
the FRB domain. To function in this way, the FKBP12/rapamycin complex is used to disrupt the
coupling between mTOR with its complex member, raptor, and decreases phosphorylation of its
substrate, p70S6k. In liver transplant recipients with chronic kidney disease, it has been found
that the gradual withdrawal of CNIs and the introduction of everolimus, an mTORi, leads to
improvement of renal function tests. Despite the improvement of renal function not being
constant, there was no immunological transplant rejections in any of the patients. With
continued research, mTORi have the potential to replace CNIs as the first line of immunotherapy
following an organ transplant.
Introduction
Renal dysfunction is a common side effect of organ transplantation.1 Common indicators
of renal failure are decreased glomerular filtration rates (GFR) and increased serum creatinine
levels.2 There are multiple factors which contribute to the onset and severity of renal
dysfunction post-transplantation, including renal disease before transplantation, diabetes
mellitus, hypertension, and nephrotoxicity of drug therapy.1,2 Although they are not the only
cause, the use of calcineurin inhibitors (CNIs) as an immunosuppressive treatment to prevent
transplant rejection are believed to be a significant contributing factor to nephrotoxicity leading
to increased renal dysfunction.3 While the use of CNIs have led to remarkable increases in graft
survival, especially in liver transplants, their adverse effects have led researchers to begin the
search for new immunosuppressive therapies with fewer side effects.2,3
A new class of drugs called mammalian target of rapamycin inhibitors (mTORi) are
possible candidates for immunotherapy in organ transplants with less adverse effects compared
to CNIs.3 mTORi affect the immune system by inhibiting mTOR causing an increase in
production of pro-inflammatory cytokines, such as IL-12 and IL-1β, and a decrease in production
of anti-inflammatory cytokines, such as IL-10.3 MHC antigen presentation by APCs is also
increased as well as regulation of type 1 interferon production, expression of chemokine
receptors and costimulatory molecules.3 While an inflammatory response is not desirable in
organ transplants, the cytokines are needed to induce an adaptive immunity response.3,4 Instead
of CD4+ T cells differentiating into one of the subsets of T helper cells, differentiation into T
regulatory cells is increased.3,4 Regulatory T cells therefore function to suppress an immune
response by decreasing the differentiation of effector T cells.
mTOR is a Serine/Threonine-specific protein kinase that plays a crucial role in a nutrientsensitive signaling pathway to regulate cell growth in response to mitogens, amino acid and
energy sufficiency. Rapamycin, the immunosuppressive agent, acts to inhibit mTOR by
complexing with FKBP12 and binding to the receptor at the FRB domain. It has been
established that the mTOR polypeptide functions in a complex with two other proteins, raptor
and GβL. Raptor serves as a scaffold for the union of mTOR with its substrates p70S6k and 4EBP1. Raptor does not alter the catalytic activity of mTOR, rather it binds p70S6k and 4E-BP1
and the association of raptor with mTOR increases mTOR phosphorylation of p70S6k in vitro.
The ability of the FKBP12/rapamycin complex to promote the dissociation of the scaffold
protein raptor from mTOR and thereby disrupt the coupling between mTOR with its substrates is
a major component of the mechanism by which rapamycin inhibits TOR signaling in vivo.
However, the molecular mechanisms by which the binding of the FKBP12/rapamycin complex
to the mTOR FRB domain interferes with mTOR signaling remain incompletely understood.5
For this project, we seek to explore the mechanism of action of the class of
immunosuppressive drugs, mTORi, and the evidence showing that they are a better alternative to
CNIs in treatment after organ transplantation. Our first paper, Dissociation of raptor from mTOR
is a mechanism of rapamycin-induced inhibition of mTOR function by Oshiro et. al. investigates
the mechanism of mTORi. We chose Figure 1 which tests the effect of rapamycin on the
association between raptor and mTOR, and Figure 4 (Figure 2) which tests the effects of
FKBP12/rapamycin complex on mTOR-catalysed phosphorylation of p70S6k.5 Our second
paper, Improvement of Renal Function After the Switch from a Calcineurin Inhibitor to
Everolimus in Liver Transplant Recipients with Chronic Renal Dysfunction by Castroagudin et.
al. explores the effects on renal function of switching from CNIs to an mTORi, everolimus, as
immunosuppressive treatment after liver transplantation.6 Figure 1 (Figure 3) shows the effects
on serum creatinine levels and Figure 3 (Figure 4) shows the effects on GFR.6
Results
For Figure 1, they were testing the effect of rapamycin on the association between mTOR
and its complex proteins raptor and GβL. Because raptor and GβL form a complex with mTOR,
if rapamycin interferes with either of these associations then we expect to see a decrease in the
amount of raptor or GβL attached to the mTOR immunoprecipitates. Withdrawal of serum and
amino acids (lanes 2 and 3) had no significant effect on the binding of either raptor or GβL
compared to those cells receiving both serum and amino acids (lane 4). When rapamycin is
present, however, association of raptor is decreased and GβL is unaffected (lane 5). Panels 4 and
5 show blotting of the whole cell lysates as a control for equal amounts of raptor and GβL
present prior to immunoprecipitation of mTOR.5
In 1B, they tested the ability of rapamycin treatment to affect the association of
endogenous raptor with wild-type, rapamycin-resistant, or kinase-inactive mTOR. In both the
wild-type and kinase-inactive cells raptor/mTOR association was decreased after treatment with
rapamycin (lanes 3-4, 7-8). However, the rapamycin-resistant cells, which lack the ability to
bind the FKBP12/rapamycin complex, showed no change in the association of raptor with
mTOR after treating with rapamycin. The third panel shows blotting of the whole cell lysates
again as a control for equal amounts of raptor present prior to immunoprecipitation.5
Figure 1. Effects of rapamycin on the association between mTOR and raptor. (A) HEK293 cells were
treated with rapamycin (lane 5), withdrawal of serum (lanes 2,3), or withdrawal of amino acids (lane 2)
and then tested for the association of mTOR with both raptor and GβL. The cells were lysed and
immunoprecipitated for mTOR, analysed using SDS-PAGE, and then immunoblotted for mTOR, raptor,
and GβL. Panels 4 and 5 show whole cell lysates immunoblotted for raptor and GβL as a control to show
equal loading. The results are representative of three reproducible experiments. (B) The ability of
rapamycin treatment to affect the association of endogenous raptor with wild-type (WT), rapamycinresistant (ST), or kinase-inactive (NK) mTOR was tested. Each cell type was either not treated or treated
with 200 nM rapamycin for 30 minutes and then immunoprecipitated for FLAG which was tagged onto the
mTOR. The immunoprecipitates were then analysed using SDS-PAGE and blotted for FLAG-mTOR and
raptor. Whole cell lysates were immunoblotted for raptor as a control to show equal loading.The results
are representative of three reproducible experiments.5
In Figure 2, researchers questioned whether rapamycin would limit mTOR’s
phosphorylation of p70S6k-F28A, in an environment with excess FKBP12, which forms a
complex with rapamycin required for binding to the mTOR. This was done in order to test the
inhibitory effect of rapamycin, in excess FKBP12, upon binding TOR. The kinase activity by
mTOR on p70S6k-F28A (lanes 3-8) was about 25% of the kinase activity by mTOR on the wildtype (lanes 10-15).5 By observing the effects of rapamycin, it was seen that, when in high
amounts, rapamycin limited the phosphorylation of p70S6k-WT an additional 75-80% (lane 1516). Rapamycin had seemingly no effect on mTOR’s phosphorylation of p70S6k-F28A (lane 38).5
Figure 2. In vitro effects of FKBP12/rapamycin complex on mTOR‐ catalysed phosphorylation of p70S6k.
First, the immunoprecipitated mTOR complex was incubated in 5 µM of FKBP12 and in varying
concentrations of rapamycin (0, 10, 30, 100, 300, or 1000 nM). Following 90 minutes of incubation, the
complexes were tested for their ability to phosphorylate both mutated and wild type p70S6k. The mTOR
complex was then incubated by itself, and tested for its phosphorylation ability. Normal mouse
immunoglobulin was used to provide control immunoprecipitation (lanes 1 and 9). The mTOR/raptor
complexes were washed and subjected to the mTOR kinase assay using a GST tag, using p70S6k‐ F28A
(lanes 1–8) or p70S6k‐ WT (lanes 9–16) as substrate. Next, these samples were separated by SDS‐
PAGE, transferred on to a membrane, and analysed by autoradiography. mTOR‐ catalysed
phosphorylation of GST‐ p70S6k is shown in the upper panel. The bar graph represents the phosphorous
integrated into GST‐ p70S6k, which was then read and quantified. These results are representative of
three reproducible experiments.5
In Figure 3, they were monitoring the improvement of serum creatinine levels in patients
after gradually switching their immunosuppressive treatment from CNIs to the mTORi,
everolimus. These patients had previously received a liver transplant and were exhibiting
symptoms of chronic kidney disease. The average basal serum creatinine levels in the 21
patients was 1.79 ± 0.39 mg/dL with a range of 1.50-2.90 mg/dL. The values at follow-up after
30, 90, 180, 360 days were 1.68 ± 0.40, 1.67 ± 0.34, 1.70 ± 0.41, and 1.57 ± 0.30 mg/dL
respectively. The serum creatinine values decrease after the first 30 days of treatment with
everolimus, remain fairly stable for the next 150 days, and then decrease during the last 180
days.6
Figure 3. Evolution of serum creatinine values (mean ± standard deviation) in a cohort of everolimustreated liver transplant recipients with chronic renal dysfunction. The basal levels were measured prior to
starting treatment with everolimus, and then measured at follow-up after beginning treatment at 30, 90,
180, and 360 days.6
For Figure 4, they were monitoring the improvement of glomerular filtration rate (GFR)
in the same patients. The average basal GFR was 42.14 ± 8.71 mL/minute/m2. The values at
follow-up after 30, 90, 180, 360 days were 45.81 ± 11.29, 46.11 ±10.12, 46.13 ± 11.75, and
49.79 ± 10.33 mL/minute/m2 respectively. According to these values, 18 patients were classified
as stage 3 CKD and 3 as stage 4 CKD. The GFR values increase after the first 30 days of
treatment with everolimus, remain fairly stable for the next 150 days, and then increase during
the last 180 days. By the end of the study, 4 patients were classified as stage 2 CKD, 14 as stage
3 CKD, and 3 as stage 4 CKD.6
Figure 4. Evolution of the glomerular filtration rate (GFR) values (mean ± standard deviation) in a cohort
of everolimus-treated liver transplant recipients with chronic renal dysfunction. The basal levels were
measured prior to starting treatment with everolimus, and then measured at follow-up after beginning
treatment at 30, 90, 180, and 360 days.6
Conclusions
From Figure 1A it can be concluded that rapamycin decreases the association of raptor
with mTOR, but the association of GβL with mTOR is unaffected. Figure 1B suggests that in
order for rapamycin to inhibit the association of raptor with mTOR, mTOR must be able to bind
the FKBP12/rapamycin complex and inhibition is independent of the catalytic activity of mTOR.
This is interesting because, even though it is believed that the raptor/mTOR interaction occurs at
multiple sites along the length of raptor, the binding of FKBP12/rapamycin to the mTOR FRB is
enough to cause dissociation of raptor from mTOR.5
Figure 2 suggests that the mechanism of immunosuppression using mTORi will involve
the dissociation of raptor and TOR at the FRB domain. In a complex with FKBP12, Rapamycin
will promote the dissociation of TOR and raptor, which serves as a substrate for TOR, and is
suggested to decrease the phosphorylation of p70S6k. The ability of FKBP12/rapamycin
complex to disrupt the coupling between mTOR with its substrates is the major component of
the mechanism by which rapamycin inhibits TOR signaling. This figure is interesting because it
is evident that functioning p70S6k is required for rapamycin mTOR inhibition and that patients
who have a mutated p70S6k will not benefit from rapamycin treatment.5
From Figures 3 and 4 they conclude that the gradual withdrawal of CNIs and the
introduction of everolimus, an mTORi, in liver transplant recipients with chronic kidney disease
leads to improvement of renal function tests. Improvement of serum creatinine values to normal
was seen in 30% to 50% of patients, however the improvement of renal function was not
constant which could be a result of other underlying factors that contribute to chronic kidney
disease. Nevertheless, no immunological transplant rejection was seen in any patients and other
adverse effects were low, making conversion from CNIs to treatment with everolimus successful
in over 95% of patients (one patient had to discontinue treatment because of myelotoxicity).
Everolimus is considered safe and useful as a rescue immunosuppresive agent in chronic kidney
disease after liver transplantation. The success of this study in comparison to previous studies is
likely due to the gradual withdrawal of CNIs. The earlier the conversion to everolimus can be
introduced after the onset of renal dysfunction the more likely the treatment will be successful.6
The Oshiro et. al. paper sheds light on the mechanism of mTORi, while the Castroagudın
et. al. study represents a comparison of CNI and mTORi treatments in liver transplant recipients.
mTORi have the potential to replace CNIs as immunotherapy after organ transplants because it is
evident that they are less toxic to the kidneys. Further research is necessary to gain more
knowledge of the mechanism of mTORi and the long term effects of their use.
Future Directions
It would be beneficial to further investigate the mechanism of action of mTORi
immunosuppressive drugs. Better understanding the mechanism through which these drugs work
to suppress the immune system can help us to understand why mTORi treatment may be more
favorable than CNIs, why they cause less renal impairment, and other side effects that may cause
them to be unfavorable in some patients. The more we can understand, the greater the likelihood
of their use in treatment after organ transplantation will be more successful. It would be
advantageous to continue to study the effects of rapamycin on mTOR and other proteins
associated with it, as well as the cytokines, transcription factors, and other molecules that mTOR
regulates.
To further study the comparative effects of CNI and mTORi treatments and gather more
conclusive evidence, a higher number of participants in the study would be necessary and follow up after
treatment should be longer to monitor the long term effects. A shorter amount of time between the
transplant and participation in the study would also be favorable. It would be interesting to study the
effects of starting mTORi treatment sooner or right after organ transplantation instead of waiting for renal
dysfunction to worsen before switching to mTORi. The more studies that can be done for longer periods
of time, the better we can understand the long term effects of using mTORi as an immunosuppressive
treatment after organ transplants and to assess whether or not improvements in renal function will
continue long term and outweigh any other possible side effects that could develop.
References
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2. Pawarode A, Fine DM, and Thuluvath PJ. 2003. Independent Risk Factors and Natural
History of Renal Dysfunction in Liver Transplant Recipients. Liver Transpl. 9(7): 741-7.
3. Kawahara T, Asthana S, and Kneteman NM. 2011. m-TOR inhibitors: What role in liver
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