Pulse mTOR inhibitor treatment effectively controls cyst growth but leads
to severe parenchymal and glomerular hypertrophy in rat polycystic kidney
Ming Wu,1Alexandre Arcaro,2Zsuzsanna Varga,3Alexander Vogetseder,4Michel Le Hir,4
Rudolf P. Wu ¨thrich,1,5and Andreas L. Serra1,5
1Zurich Center for Integrative Human Physiology (ZIHP),2Department of Oncology, University Children’s Hospital,
3Institute of Surgical Pathology, University Hospital,4Anatomical Institute, University of Zurich-Irchel, and5Division
of Nephrology, University Hospital, Zurich, Switzerland
Submitted 27 July 2009; accepted in final form 19 September 2009
Wu M, Arcaro A, Varga Z, Vogetseder A, Hir ML, Wu ¨thrich
RP, Serra AL. Pulse mTOR inhibitor treatment effectively controls
cyst growth but leads to severe parenchymal and glomerular hyper-
trophy in rat polycystic kidney disease. Am J Physiol Renal Physiol
297: F1597–F1605, 2009. First published September 23, 2009;
doi:10.1152/ajprenal.00430.2009.—The efficacy of mammalian target
of rapamycin (mTOR) inhibitors is currently tested in patients af-
fected by autosomal dominant polycystic kidney disease. Treatment
with mTOR inhibitors has been associated with numerous side effects.
However, the renal-specific effect of mTOR inhibitor treatment ces-
sation in polycystic kidney disease is currently unknown. Therefore,
we compared pulse and continuous everolimus treatment in Han:
SPRD rats. Four-week-old male heterozygous polycystic and wild-
type rats were administered everolimus or vehicle by gavage feeding
for 5 wk, followed by 7 wk without treatment, or continuously for 12
wk. Cessation of everolimus did not result in the appearance of renal
cysts up to 7 wk postwithdrawal despite the reemergence of S6 kinase
activity coupled with an overall increase in cell proliferation. Pulse
everolimus treatment resulted in striking noncystic renal parenchymal
enlargement and glomerular hypertrophy that was not associated with
compromised kidney function. Both treatment regimens ameliorated
kidney function, preserved the glomerular-tubular connection, and
reduced proteinuria. Pulse treatment at an early age delays cyst
development but leads to striking glomerular and parenchymal hyper-
trophy. Our data might have an impact when long-term treatment
using mTOR inhibitors in patients with autosomal dominant polycys-
tic kidney disease is being considered.
Han:SPRD; S6K; 4E-BP1
AUTOSOMAL DOMINANT POLYCYSTIC kidney disease (ADPKD) is
the most common hereditary kidney disease, resulting in pro-
gressive renal failure and end-stage renal disease in adulthood
(31). The continuous growth of cysts is associated with in-
creased tubular epithelial cell (TEC) proliferation, leading to
progressive cystic kidney enlargement and a loss of renal
function (8). Currently, specific treatments for human ADPKD
other than supportive care do not exist.
The mammalian target of rapamycin (mTOR) is a key
controller of cell growth and proliferation (11). The pathway
has two branches, which start with the mTOR complexes
known as mTORC1, (mTOR, Raptor) and mTORC2 (mTOR,
Rictor) (23). The former is inhibited by sirolimus (rapamycin)
and its analog everolimus, while the latter is not. The direct
downstream targets of mTORC1, ribosomal protein S6 kinase
(S6K) and 4E-BP1, in turn tightly regulate the downstream
translational initiation machinery to control cell growth and
The mTOR pathway is inactive in the healthy adult kidney.
In this situation, tuberin, an upstream inhibitor of mTOR, is
attached to the cytoplasmic tail of the polycystin complex (25).
Mutations of the polycystin complex (which are the cause of
ADPKD) direct tuberin to the cytoplasmic compartment, re-
sulting in a continuously activated mTOR pathway. The spe-
cific inhibition of this pathway with mTOR inhibitors can
retard cyst growth and ameliorates kidney function loss in
various murine models of PKD (10, 25, 27, 28, 32, 33). Based
on these promising preclinical studies, the efficacy of rapamy-
cin and everolimus, immunosuppressants approved for kidney
transplantation, are currently examined in clinical trials for
ADPKD (15, 24). Treatment with mTOR inhibitors has been
associated with numerous side effects; therefore, short-term or
pulse treatment may offer an alternative treatment regimen to
increase treatment tolerability. Some ADPKD patients cur-
rently enrolled in clinical trials testing the efficacy of mTOR
inhibitor treatment have been withdrawn from the study drug
due to adverse events. However, the renal-specific effect of
such an mTOR inhibitor withdrawal has so far never been
examined. The purpose of the present investigation was there-
fore to study the effect of pulse compared with continuous
everolimus treatment in Han:SPRD rats, a rodent model for
PKD. Here, we show for the first time that the interruption of
the mTOR treatment did not result in the appearance of renal
cysts up to 7 wk postwithdrawal. However, cessation of
everolimus treatment resulted in striking glomerular and non-
cystic renal hypertrophy, which was not associated with com-
promised kidney function. Pulse compared with continuous
everolimus application differentially inhibited the mTOR path-
way regulators AKT and GSK and the mTOR effectors rpS6
and 4E-BP1, pointing to a potential distinct biochemical role of
mTORC1 and mTORC2 in the development and progression of
MATERIALS AND METHODS
Animals. The Han:SPRD rat colony was established in our animal
facility from a litter which was obtained from the Rat Resource and
Research Center (Columbia, MO). Heterozygous cystic (Cy/?) and
wild-type normal (?/?) rats were used in this study. Only male rats
were used since cysts develop more rapidly in male compared with
female rats (12). The regulatory commission for animal studies, a
Address for reprint requests and other correspondence: A. L. Serra, Div. of
Nephrology, Univ. Hospital, Ra ¨mistrasse 100, 8091 Zu ¨rich, Switzerland (e-mail:
Am J Physiol Renal Physiol 297: F1597–F1605, 2009.
First published September 23, 2009; doi:10.1152/ajprenal.00430.2009.
0363-6127/09 $8.00 Copyright © 2009 the American Physiological Society http://www.ajprenal.orgF1597
local government agency, approved the study protocol. Rats had free
access to tap water and a standard rat diet.
Study drug. Everolimus microemulsion (20 mg/g) and vehicle
microemulsion were kindly supplied by Novartis Pharma (Basel,
Switzerland) and stored at ?20°C. Everolimus and vehicle were
diluted with tap water before oral administration.
Experimental protocol. Male Cy/? and ?/? rats were weaned and
then treated at 4 wk of age with 3 mg?kg?1?day?1everolimus (Cy/?:
n ? 9; ?/?: n ? 5) or vehicle [Cy/?: n ? 11; ?/?: n ? 8; vehicle
treatment (VT)] by gavage at a volume of 2 ml?kg?1?day?1for 12 wk
throughout (continuous treatment). In a different schedule, everolimus
was discontinued after 5 wk, followed by 7 wk without treatment
(pulse treatment; Cy/?: n ? 5; ?/?: n ? 6). The dose of everolimus
or vehicle was adjusted daily according to the body weight of the rats.
All animals were killed at week 16.
Blood chemistries. Tail blood was obtained from rats at week 4, 8,
12, and 16. Blood urea nitrogen (BUN) and creatinine were deter-
mined in plasma, whereas everolimus trough levels were measured in
whole blood. All samples were stored at ?20°C before measurement.
BUN was analyzed by kinetic color testing and creatinine by the
isotope dilution mass spectrometry traceable modified Jaffe ´ method.
Everolimus blood levels were analyzed by HPLC-mass spectroscopy.
Determination of urinary protein excretion. Twenty-four-hour
urine samples were collected at week 16 in metabolic cages for
selected rats (n ? ?4/group) and centrifuged. Urinary albumin was
measured by a turbidimetric method (Roche Diagnostic, Basel, Swit-
zerland). Albuminuria was expressed as urinary albumin-to-creatinine
ratios (mg/mmol). All measurements of blood and urine samples were
performed by the Institute for Clinical Chemistry, University Hospi-
tal, Zurich, Switzerland.
Urine was also analyzed by nonreducing SDS-PAGE. Twenty-
microliter urine samples were boiled for 5 min at 95°C and resolved
in denaturing 7.5 and 15% polyacrylamide gels. Electrophoresis was
followed by staining of the gels with Coomassie brilliant blue R250
for 30 min. The gels were destained and washed in double distilled
water and dried for imaging.
Tissue processing. At the age of 16 wk, all rats were killed and
kidneys were excised, decapsulated, and weighed. For histological
examination, one of the kidneys from each animal was sliced perpen-
dicularly to the long axis at ?2-mm intervals. Slices from the
midportion of the kidneys were fixed in 4% buffered formalin and
submitted to subsequent paraffin embedding. Up to 65 serial sections
of 3-?m thickness per paraffin block were cut. Every second and third
consecutive slide was stained with regular hematoxylin and eosin
(H&E) and periodic acid-Schiff (PAS) stain.
Morphometric measurements. The investigators of the morphomet-
ric measurements were blinded to experimental groups, and repeated
measurements were performed without knowledge of each other’s
Determination of cyst volume density. Cyst volume density was
assessed on PAS-stained sections by morphometry, using the method
of point counting (16). Four to six micrographs were obtained for one
section per rat for morphometry as described before (28). The pixel
size of the micrographs was 1,300 ? 1,030 (1.34 ?m/pixel). An
orthogonal grid with line spacing of 150 pixels was used.
Glomerular volume measurement. Mean glomerular volume was
determined from the mean glomerular capillary tuft area (AG) by light
microscopy of PAS sections. The areas were determined by light
microscopy and analyzed by dedicated software (Analysis 3.0, Soft
Imaging System, Mu ¨nster, Germany) as the average area of 50
glomerular profiles (the capillary tuft omitting the proximal tubular
tissue and Bowman’s capsule) for each animal. Glomerular volume
was calculated using the formula GV ? ?/k ? (AG)3/2, where GV is
glomerular volume, ? ? 1.38, which is the shape coefficient for
spheres (the idealized shape of glomeruli), k ? 1.1, which is a size
distribution coefficient, and AGis the glomerular capillary tuft area.
Assessment of glomerular-tubular connection. Serial sections of
everolimus-treated and nontreated heterozygous polycystic (Cy/?)
and wild-type normal (?/?) rat kidneys of 2-?m thickness were cut
and stained with PAS. On the sections, glomeruli were counted and
each glomerulus was examined for the presence of a urinary pole. This
procedure was continued until 50 urinary poles were detected. If a
section did not contain 50 urinary poles, another section of that kidney
was evaluated. This section was at least 50 ?m from the previous one
to ensure that the same glomerulus was not evaluated twice. Sections
were independently evaluated by two investigators, and the results
were averaged. The number of glomeruli to obtain 50 urinary poles
was used as a measure of glomerular-tubular connection: i.e., the
higher the numbers of counted glomeruli, the more atubular glomeruli
exist in a kidney.
Antibodies for immunostaining and western blotting. The following
antibodies were used: rabbit anti-human polyclonal antibody recog-
nizing Ki-67 antigen (clone MIB 1; Dako Diagnostic, Zug, Switzer-
land); rabbit anti-mTOR (7C10); mouse anti-phospho-Akt (Ser473);
rabbit anti-phospho-GSK-3?/? (Ser21/9); rabbit anti-phospho-4E-
BP1 (Thr37/46); rabbit anti-phospho-S6 ribosomal protein (Ser235/
236); rabbit anti-phospho-S6 ribosomal protein (Ser240/244, Cell
Signaling Technology, Beverly, MA); rabbit anti-actin (Sigma, St.
Louis, MO); rabbit anti-RPS6 antibody (Bethyl Laboratories, Mont-
gomery, TX); and rabbit anti-Akt1/2/3 (H-136, Santa Cruz Biotech-
nology, Santa Cruz, CA). Goat anti-rabbit horseradish peroxidase
(HRP)-conjugated antibody (Dunn Labortechnik, Asbach, Germany)
and sheep anti-mouse HRP-conjugated antibody (GE Healthcare,
Buckinghamshire, UK) were used as the secondary antibodies.
Immunohistochemical detection of proliferation. Immunohisto-
chemistry was performed on paraffin sections of formalin-fixed tis-
sues using the Ventana Benchmark automated staining system (Ven-
tana Medical Systems, Tucson, AZ). For antigen retrieval, the slides
were heated with Ventana cell conditioner one (mild protocol). Anti-
Ki-67 (dilution 1:20) antibody was detected with a Ventana iVIEW
DAB detection kit, yielding a brown reaction product. Slides were
counterstained with hematoxylin. Micrographs were made randomly
using the ?200 magnification of the light microscope. The proximal
tubule was identified by the brush border, detectable in background
fluorescence, and was controlled with PAS stain on a corresponding
serial section. Nuclei of proximal epithelial cells positive for Ki-67
were counted on 100 consecutive tubules. A tubule with fewer than
five cells was not included for assessment. The proliferation index of
TECs was calculated as the percentage of positively stained Ki-67
cells to total cells. The staining was scored independently by two
Protein extraction and western blot analysis. Snap frozen kidney
tissue was homogenized by an automated homogenizer (Precellys 24;
Stretton Scientific, Stretton, UK) in freshly made tissue protein
extraction reagent (T-PER, Pierce Bioscience, Rockford, IL) contain-
ing 1 mM PMSF, 0.5 M EDTA, Halt Protease Inhibitor cocktail, and
Halt Phosphatase Inhibitor. Homogenates were centrifuged twice at
4°C at 13,000 g for 15 min, and supernatants were stored at ?80°C.
Sixty-microgram protein samples and a reducing loading buffer
were mixed and boiled for 5 min at 95°C. Samples were loaded on a
7.5% SDS-PAGE gel. After migration, the proteins of the gels were
transferred onto a 0.2-?m polyvinylidene difluoride membrane (Bio-
Rad, Hercules, CA). The blots were blocked by drying for 15–30 min
at room temperature and were then incubated overnight at 4°C in a 5%
BSA/PBST buffer containing primary antibodies against actin (dilu-
tion 1:20,000), mTOR (1:1,000), RPS6 (1:5,000), p240/244 S6 (1:
3,000), p235/236 S6 (1:1,000), Akt1/2/3 (1:1,000), p21/9 GSK-3?/?
(1:1,000), or p37/464E-BP1 (1:1,000). Secondary antibodies against
rabbit (1:10,000) or mouse (1:10,000) were applied for 45 min at
room temperature after 3? wash with PBST. After 4? wash with
PBST, the membranes were incubated for 5 min using a chemilumi-
nescence substrate kit (ChemiGlow; Alpha Innotech, San Leandro,
CA). Blots were visualized with the Chemi-Doc XRS system (Bio-
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