University of Zurich - UZH€¦ · Serra, A L Serra, A L. Mammalian Target of Rapamycin (mTOR) ... more than 10’000 patients in Switzerland. ADPKD is caused by mutations in the

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Mammalian Target of Rapamycin (mTOR)-mediated Effect onDisease Progression in Autosomal Dominant Polycystic Kidney

Disease (ADPKD)

Serra, A L

Serra, A L. Mammalian Target of Rapamycin (mTOR)-mediated Effect on Disease Progression in AutosomalDominant Polycystic Kidney Disease (ADPKD). 2009, University of Zurich, Faculty of Medicine.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:University of Zurich, Faculty of Medicine, 2009.

Serra, A L. Mammalian Target of Rapamycin (mTOR)-mediated Effect on Disease Progression in AutosomalDominant Polycystic Kidney Disease (ADPKD). 2009, University of Zurich, Faculty of Medicine.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:University of Zurich, Faculty of Medicine, 2009.

Habilitationsschrift

Mammalian Target of Rapamycin (mTOR)-mediated Effect on Disease Progression in Autosomal Dominant Polycystic Kidney Disease (ADPKD)

Zur Erreichung der Venia Legendi der Universität Zürich

Andreas L. Serra

9. März 2008

Serra, AL. Mammalian Target of Rapamycin (mTOR)-mediated Effect on Disease Progression in Autosomal Dominant Polycystic Kidney Disease (ADPKD) Background My research interest is focused on autosomal dominant polycystic kidney disease (ADPKD). ADPKD is the most common hereditary cause of end-stage renal disease (ESRD), affecting more than 4.5 Mio worldwide and more than 10’000 patients in Switzerland. ADPKD is caused by mutations in the polycystic kidney disease 1 gene (PKD1) or in the polycystic kidney disease gene 2 (PKD2). The protein products of these two genes, polycystin-1 (PC-1) and polycystin-2 (PC-2) are expressed on renal tubular epithelial cells (TECs), specifically by the cilia of these cells. The polycystin complexes on cilia function as sensors of the extracellular environment and elicit intracellular responses through phosphorylation pathways. The disease characterized by the progressive development of innumerable cysts in both kidneys, which distort the normal kidney architecture and leads to a loss of renal function. Currently there is no therapy available. As a physician-scientist I work at the interface between the research laboratory and patient care. My hope is that such a translational research and medicine change the outcome of this serious disease. Basic science research The basic research science group is located at the University Zurich –Irchel within the Physiological Institute and the Zurich Center for Integrative Human Physiology (ZIHP). Group members are Ming Wu and Shagun Raina, both PhD students and Claudia Wuhrmann, technical assistant. Former member is Patricia Wahl, PhD. Sirolimus treatment slows rodent polycystic kidney disease progression In ADPKD renal cysts are lined by a single layer of cells originating from the epithelia of the nephrons. These tubular epithelial cells (TECs) have an increased proliferation rate. The mammalian target of Rapamycin (mTOR) is a key intermediary kinase in multiple mitogenic signalling pathways and plays a central role in modulating cellular proliferation, cell growth processes and angiogenesis. Activated mTOR directly phosphorylates p70S6 protein kinase (S6K) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1) which promotes cell growth through an increase in translation. Rapamycin (sirolimus; Rapamune®) and its analogue everolimus (Certican®) are macrocyclic lactones that bind to FKBP-12, thereby inhibiting specifically mTOR kinase activity. Both drugs are clinically used as immunosuppressive agents and exhibit well-known anti-proliferative effects. Several rodent models for ADPKD exist, whereas the Han:SPRD Cy/+ rat has emerged as an excellent ADPKD model which closely resembles the human disease. Heterozygous male Han:SPRD rats with the Pkdr1 gene mutation (Cy/+) develop a slowly progressing polycystic kidney disease (PKD). To test our hypothesis that the inhibition of the mTOR pathway will slow polycystic kidney disease progression, we examined the effect of sirolimus on cyst growth and renal function loss in the Han:SPRD rat model [1]. The expression and phosphorylation of the downstream effector of the mTOR pathway, S6K was also analyzed. The oral administration of sirolimus through the drinking water for 3 months reduced indeed cyst volume and the steady increase in BUN and creatinine in male Cy/+ rats. A relatively low sirolimus blood level of 0.5 to 1.9 µg/l effectively reduced 2-kidney/total body weight (2K/TBW) and cyst volume density (CVD) by 36% and 18%, respectively. Western blot analysis from wild type Han:SPRD rat and treated and untreated Cy/+ rat kidneys for total amount and phosphorylated S6K showed that the mTOR pathway is not expressed in normal adult kidneys but in untreated Cy/+. Sirolimus effectively reduced phosphorylation at Thr389 and Thr421/Ser424 of S6K in Cy/+ kidneys and that correlated well with the histomorphometric findings. Our findings pointed to an important role of the mTOR pathway in the pathogenesis of cyst growth. Akt/mTOR/S6K pathway is upregulated in polycystic kidney disease Because the Akt kinase is an upstream regulator of mTOR, we hypothesized that the activity of Akt could be enhanced in the kidneys of Han:SPRD rats. The primary pathway by which growth factors and cytokines activate mTOR and its downstream targets is via the PI3K/Akt pathway. Growth factors stimulate phosphatidylinositol 3-kinase (PI3K) activating Akt via phosphorylation on two residues (Ser473 and Thr308) which in turn activates mTOR. Renal cyst fluid from PKD mice and rodent models contain epidermal growth factor (EGF)-like peptides in mitogenic concentration and treatment with EGFR tyrosine kinase inhibitor attenuated disease progression in Han:SPRD rats. We therefore analyzed the Akt expression on RNA and protein level as well in the tissue by immunohistochemistry of polycystic rat kidneys [2]. Western blotting and enzyme-linked immunosorbent assay showed a significant increase in phosphorylated Akt in Cy/+ compared to wild type kidneys. Immunohistochemistry revealed that phosphorylated Akt is highly expressed in mitotic cells, beginning with the onset of the prophase. Staining by bromo-deoxyuridine (BrdU) and cell cycle marker Ki67 demonstrated a 6 to 9-fold higher rate of proliferation of TECs lining cysts compared to normal tubules. Our study showed that Akt is instrumental in mitosis in TECs of normal and cystic tubules. Furthermore, by examining the distribution of pS6K(Thr421/Ser424) with immunofluorescence, we could show that S6K was also highly phosphorylated throughout mitosis and because the phosphorylation of S6K on the residue 421 and 424 is sensitive to mTOR inhibition, it is likely that the Akt/mTOR/S6K pathway plays a role TECs proliferation.

2

Serra, AL. Mammalian Target of Rapamycin (mTOR)-mediated Effect on Disease Progression in Autosomal Dominant Polycystic Kidney Disease (ADPKD) Inhibition of cystogenesis is a class effect of mTOR inhibitors Sirolimus slows polycystic kidney disease progression and to determine whether this is a class effect of all mTOR inhibitors we analyzed the effect of everolimus on cyst growth and renal function in Cy/+ [3]. Furthermore by applying 3 mg/kg/day everolimus by gavage feeding, we aimed to reach whole blood everolimus through levels between 5- 10 ng/ml, a drug level known to prevent organ rejection in rat models of renal allograft transplantation. Comparable to rapamycin, everolimus treatment for 5 weeks significantly inhibited cyst growth and 2-kidney weights in Cy/+ rats (Figure 1). Everolimus trough levels between 5 and 7 µg/l slowed renal functional deterioration and reduced the proliferation of TECs in Cy/+ rats. In summary, an everolimus dosage, clinically applied to prevent organ rejection, effectively inhibits cyst growth and the inhibitory effect of everolimus on cytogenesis most likely represents a class effect of mTOR inhibitors. Figure 1: Representative paraffin-embedded kidney sections of Han:SPRD rats showing the effect of everolimus treatment on kidney seize and cyst number (A) Wild type (+/+) kidneys after treatment with vehicle, (B) heterozygous (Cy/+) with vehicle and (C) heterozygous (Cy/+) with everolimus treatment showing a marked reduction of kidney size and reduced number of cysts.

B C
A
Sirolimus reduces extracellular remodTo obtain a full understanding of the thdevelop human therapies it is importanto cyst formation, reorganization of tuthe renal interstitium are characteristicremodelled. Matrix metalloproteinasesdependent endopeptidases which can denzymes in the turnover of the ECM. Aand animal models of PKD. The two mantiproliferative effects on renal cysticdegradation. In collaboration with the arrays from 4 months old male Han:SPmonths. The analysis of the microarrayremodelling, including the metalloprotupregulated in Cy/+ rats and that this wthen validated by real time PCR and bconfirmed that the mRNA and proteinheterozygous Cy/+ animals compared MMP-2 and MMP-14 levels near to thclose to cysts, as revealed by immunohmatrix remodelling in the process of cassociated with reduced extracellular r Conclusion Our studies provide a broader understainhibitors to treat human ADPKD. Basfirst clinical trial, the so called SUISSEmTOR inhibitor in young patients with

elling in kidneys of Cy/+ erapeutic potential of mTOR inhibitors it to precisely unravel the mechanisms of

bular basement membranes (TBM) and ths of PKD, suggesting that the extracellula (MMPs) are a large family of secreted aegrade a wide spectrum of matrix substrabnormalities in the expression of MMPsTOR inhibitors rapamycin and everolim

epithelia, but they may have additional eFunctional Genomics Center Zürich (FGRD rats and +/+ control animals which w data was then focused on molecules wheinases. The array data revealed that MMas reversed by the treatment with rapam

y measuring protein expression and MMP levels of MMP-2 and MMP-14 were marto wild-type +/+. Furthermore the treatmose of the wild-type. Notably, MMP-2 wistochemistry. Overall these data point to

ytogenesis and that the mTOR-mediated emodelling [4].

nding of the mechanisms of action and thed on these promising results we designe ADPKD Study to examine the effective early manifestations of ADPKD.

n ADPKD and to optimally action of these drugs. In addition e development of fibrosis within r matrix (ECM) is intensely

nd membrane-bound zinc-tes, and therefore represent key have been described for human us not only display ffects on matrix synthesis and

CZ) we produced kidney gene ere treated with rapamycin for 3

ich are relevant in matrix P-2 and MMP-14 were highly ycin. These microarray data were activity. These analyses kedly increased in kidneys of

ent with rapamycin decreased as predominantly overexpressed wards an important role of

effect on cystogensis in Cy/+ is

e therapeutic potential of mTOR d and initiated the world wide ness and safety of a specific

3

Serra, AL. Mammalian Target of Rapamycin (mTOR)-mediated Effect on Disease Progression in Autosomal Dominant Polycystic Kidney Disease (ADPKD) Clinical research In human ADPKD progressive cyst growth generally precedes the development or renal insufficiency. Compensatory mechanisms (hyperfiltration) maintain renal function virtually normal for decades despite continuous cyst growth. By the time renal function starts to decline, the kidneys are usually grossly enlarged with little normal renal parenchyma recognisable on imaging studies. Data from the consortium for radiologic imaging studies of polycystic kidney disease (CRISP) and others have shown that the rate of kidney volume growth is a predictor of renal functional decline and therefore kidney volume is used as surrogate marker of disease progression especially in clinical intervention trials for ADPKD. Typically patients develop end-stage renal disease (ESRD) by the age of 40 to 50 years, necessitating renal replacement therapy (RRT) and/or kidney transplantation. Apart from blood pressure control and symptomatic treatment of cyst bleedings and infections there is no curative therapy for this disease. The SUISSE ADPKD [NCT00346918] study seeks to determine if sirolimus halts kidney volume growth in patients with ADPKD early in the disease course [5]. Study participation is restricted to young patients with preserved kidney function because we hypothesize that any effective treatment of ADPKD needs to be started early in the course of the disease to have an impact on long term kidney function. The study is a single center randomised controlled open label trial. The clinical study was designed and implanted and shall be reported in accordance with Guidelines for Good Clinical Practice (GCP) and with the ethical principles laid down in the Declaration of Helsinki. During the study an external field monitor visits the study center regularly to check the completeness of the patient records, the accuracy of entries on the CRFs, the adherence to the protocol and to GCP and to ensure that the study drug is being stored, dispensed, and accounted according to specifications. The primary objective of the SUISSE ADPKD study is to assess the effectiveness of sirolimus to retard kidney volume growth and to prevent the loss of renal function. The secondary objectives are to follow renal function and blood pressure and to monitor for the occurrence of proteinuria. In addition, cardiovascular function as assessed by echocardiography, ambulatory blood pressure measurements and electrocardiogram will be monitored. Safety and tolerability of sirolimus treatment in ADPKD patients will also be assessed. Patients with ADPKD and kidney volume growth that can be documented within 6 months are randomized to treatment with sirolimus 2 mg/day for 18 months or standard treatment (Figure Appendix). The study will involve 100 ADPKD-patients aged 18-40 years with a creatinine clearance >70 ml/min. Kidney volumes will be measured by MRI without contrast media at study month 0 and 6. Patients with documented volume progression are randomized at a 1:1 ratio to sirolimus or standard treatment. Adherence to the prescribed study drug will be assessed by using the Medication Event Monitoring V Track-Cap System (MEMS®, Aardex, Ltd., Zug, Switzerland) during the complete treatment period. Principal statistical analysis will be undertaken using an intention-to-treat approach. A secondary on-treatment analysis will also be performed. The annual percent change in kidney volume will be determined by regressing the log transformed total kidney volume at month 6, 12 and month 24 against time for each patient through the least squares method. To account for possible baseline imbalances, a secondary analysis will be performed in which comparison of treatment groups for all endpoints will be adjusted for predefined covariates using multiple regressions (ANCOVA). Recruitment has started in May 2006 and finished December 2007. The study will be completed by December 2009. Our trial is the first clinical study addressing this question. If sirolimus treatment can reduce or stop volume growth in patients with maintained kidney function and prior documented kidney volume progression, these ADPKD patients could benefit the most from a treatment with an anti-proliferative agent like sirolimus. Further information is available here: www.ADPKD.ch. Similar studies using specific mTOR inhibitors to halt disease progression have been announced by others (Mayo Clinic, USA; Certican® trial, Novartis, Germany). We anticipate that sirolimus might be an effective therapeutic option for ADPKD patients that are prone to progress to end-stage renal disease. Outlook To obtain a full understanding of the therapeutic potential of mTOR inhibitors in ADPKD and to optimally develop human therapies it is important to precisely unravel the mechanisms of action of these drugs, both in vitro and in vivo. Our ongoing basic research project supported by the SNF has the goal to characterize the mechanisms of the antiproliferative effect of rapamycin and everolimus on TECs in vitro and in vivo. The two mTOR inhibitors rapamycin and everolimus may not only display antiproliferative effects on renal cystic epithelia, but they have additional effects on matrix synthesis and degradation and on angiogenesis. Neo-angiogenesis around cysts is a crucial process in PKD, and mTOR inhibitors might act in a beneficial way by a combined effect on tubular epithelial cell (TEC) proliferation, pericystic matrix turnover and angiogenesis. The results of these studies will provide a broader understanding of the mechanisms of action and the therapeutic potential of mTOR inhibitors to treat human ADPKD. We anticipate that mTOR inhibitors might be very effective drugs, due to their unique mechanism of action that targets cystic epithelia, matrix production and pericystic capillaries. Together with our proof-of-concept clinical trial that is currently recruiting patients we should be able to definitively prove the therapeutic potential of mTOR inhibitors for patients with ADPKD.

4

Serra, AL. Mammalian Target of Rapamycin (mTOR)-mediated Effect on Disease Progression in Autosomal Dominant Polycystic Kidney Disease (ADPKD) References 1. Wahl PR, Serra AL, Le Hir M, Molle KD, Hall MN, Wuthrich RP: Inhibition of

mTOR with sirolimus slows disease progression in Han:SPRD rats with autosomal dominant polycystic kidney disease (ADPKD). Nephrol Dial Transplant 2006, 21(3):598-604.

2. Wahl PR, Le Hir M, Vogetseder A, Arcaro A, Starke A, Waeckerle-Men Y, Serra AL, Wuthrich RP: Mitotic activation of Akt signalling pathway in Han:SPRD rats with polycystic kidney disease. Nephrology (Carlton) 2007, 12(4):357-363.

3. Wu M, Wahl PR, Le Hir M, Wackerle-Men Y, Wuthrich RP, Serra AL: Everolimus retards cyst growth and preserves kidney function in a rodent model for polycystic kidney disease. Kidney Blood Press Res 2007, 30(4):253-259.

4. Berthier CC, Wahl PR, Hir ML, Marti HP, Wagner U, Rehrauer H, Wuthrich RP, Serra AL: Sirolimus ameliorates the enhanced expression of metalloproteinases in a rat model of autosomal dominant polycystic kidney disease. Nephrol Dial Transplant 2008, 23(3):880-889.

5. Serra AL, Kistler AD, Poster D, Struker M, Wuthrich RP, Weishaupt D, Tschirch F: Clinical proof-of-concept trial to assess the therapeutic effect of sirolimus in patients with autosomal dominant polycystic kidney disease: SUISSE ADPKD study. BMC Nephrol 2007, 8(1):13.

Appendix Flow chart of SUISSE ADPKD study

Screening/Enrollment

Kidney Volumetry

Kidney Volumetry

Progression

Randomisation

YESYES

Rapamune 2 mg/d Standard therapy

Re-VolumetryNONO

Kidney Volumetry

Kidney Volumetry

Progression

Exclusion

YESYES

month 0

month 6

month 12

month 24

Screening period

Interventional period

NONO

Clinical visits (5) Clinical visits (1)

Clinical visits (1) Clinical visits (1)

Kidney Volumetrymonth 18

5

Nephrol Dial Transplant (2006) 21: 598–604

doi:10.1093/ndt/gfi181

Advance Access publication 11 October 2005

Original Article

Inhibition of mTOR with sirolimus slows disease progression

in Han:SPRD rats with autosomal dominant polycystic

kidney disease (ADPKD)

Patricia R. Wahl1, Andreas L. Serra1,2, Michel Le Hir3, Klaus D. Molle4,Michael N. Hall4 and Rudolf P. Wuthrich1,2

1Physiological Institute, 3Anatomical Institute, University Zurich-Irchel, 2Division of Nephrology,University Hospital, Zurich and 4Division of Biochemistry, Biozentrum, University of Basel, Switzerland

Abstract

Background. Autosomal dominant polycystic kidneydisease (ADPKD) is characterized by dysregulatedtubular epithelial cell growth, resulting in the forma-tion of multiple renal cysts and progressive renalfailure. To date, there is no effective treatment forADPKD. The mammalian target of rapamycin(mTOR) is an atypical protein kinase and a centralcontroller of cell growth and proliferation. Weexamined the effect of the mTOR inhibitor sirolimus(rapamycin) on renal functional loss and cyst progres-sion in the Han:SPRD rat model of ADPKD.Methods. Five-week-old male heterozygous cystic(Cy/þ) and wild-type normal (þ/þ) rats were adminis-tered sirolimus (2mg/kg/day) orally through thedrinking water for 3 months. The renal function wasmonitored throughout the treatment phase, and ratswere sacrificed thereafter. Kidneys were analysedhistomorphometrically, and for the expression andphosphorylation of S6K, a well-characterized target ofmTOR in the regulation of cell growth.Results. The steady increase in BUN and creatininein Cy/þ rats was reduced by 39 and 34%, respectivelywith sirolimus after 3 months treatment. Kidneyweight and 2-kidney/total body weight (2K/TBW)ratios were reduced by 34 and 26% in sirolimus-treatedCy/þ rats. Cyst volume density was also reduced by18%. Of importance, Cy/þ rats displayed enhancedlevels of total and phosphorylated S6K. Sirolimuseffectively reduced total and phosphorylated levelsof S6K.Conclusion. We conclude that oral sirolimus markedlydelays the loss of renal function and retards cystdevelopment in Han:SPRD rats with ADPKD.

Our data also suggest that activation of the S6Ksignalling pathway plays an important role in thepathogenesis of PKD. Sirolimus could be a useful drugto retard progressive renal failure in patients withADPKD.

Keywords: ADPKD; Han:SPRD rats; mTOR;rapamycin; sirolimus; S6K

Introduction

Autosomal-dominant polycystic kidney disease(ADPKD) is the most common form of renal cysticdisease, affecting all ethnic groups worldwide, with anincidence of 1 in 500 to 1 in 1000. The disease accountsfor up to 10% of all patients requiring renal replace-ment therapy. Affected individuals usually presentin the 3rd and 4th decade of life, progressing to end-stage renal disease (ESRD) within 5–10 years after thedevelopment of renal insufficiency [1].

The disease is characterized by the progressivedevelopment of fluid-filled cysts in the kidney. Therenal cysts originate from the epithelia of the nephronsand are lined by a single layer of cells that have ahigher rate of cellular growth and proliferation.Various abnormalities in gene expression, fluid secre-tion, apoptosis and extracellular matrix productionhave been described in ADPKD [2]. PKD1 and PKD2encode for the proteins polycystin-1 and polycystin-2,and various mutations in these genes are linked to theoccurrence of ADPKD [3–6]. How defects in thesegenes lead to the massive cyst growth in patients withADPKD is being investigated [7].

Currently, there is no specific treatment for PKDother than supportive care and blood pressure control.Usually dialytic treatment or renal transplantation

Correspondence and offprint requests to: Prof. Dr Rudolf P.Wuthrich, Renal Division, University Hospital, Ramistrasse 100,8091 Zurich, Switzerland. Email: [email protected]

� The Author [2005]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.For Permissions, please email: [email protected]

becomes necessary when the disease has progressedto end-stage renal failure. There is significant interestin searching for specific drugs which decrease or haltthe progression of cyst development, thus slowing renalfunctional deterioration and preventing the develop-ment of ESRD [8–10].

The Han:SPRD rat is a well-known model ofADPKD, although the gene defect has not beenidentified yet [11]. Recently, the drug rapamycin(sirolimus) – an inhibitor of the mammalian target ofrapamycin (mTOR) – was found to significantly inhibitcyst growth in the Han:SPRD rat [12]. The proteinkinase mTOR has emerged as a major effector of cellgrowth and proliferation via the regulation of proteinsynthesis. mTOR controls protein synthesis througha number of downstream targets, some of which arephosphorylated directly by mTOR, while others arephosphorylated indirectly, and could be dysregulatedin ADPKD.

The mTOR inhibitor sirolimus is an immunosup-pressant drug with antiproliferative and growthinhibiting effects [13,14]. It is used clinically inrecipients of renal allografts to prevent transplantrejection. It is also used in coated stents to preventcoronary artery re-stenosis after angioplasty [15]. Veryrecently, sirolimus has shown clinical effectiveness inkidney transplant recipients with Kaposi’s sarcoma[16]. We hypothesized that mTOR and its downstreamcell growth regulatory proteins are dysregulated inPKD, and that sirolimus could be an effective oral drugto inhibit cyst progression. We therefore used theHan:SPRD rat model of ADPKD [11] to test the effectof oral sirolimus (Rapamune�) on loss of renal functionand cyst progression. We also examined the activityof the mTOR downstream target S6K (p70S6K) [17] inthese ADPKD rats and tested the effect of sirolimus onthis effector protein.

Subjects and methods

Animals

The study was conducted in heterozygous (Cy/þ) and nor-mal littermate control (þ/þ) Han:SPRD rats. Only male ratswere used since the disease progresses faster in male comparedwith female rats. A colony of Han:SPRD rats was establishedin our animal care facility from breeding pairs that wereobtained from the Rat Resource & Research Center(Columbia, MO, USA). The study protocol was approvedby the regulatory commission for animal studies, a localgovernment agency. Rats had free access to tap water andstandard rat diet.

Study drug

Rapamune oral solution (1mg/ml) was kindly provided byWyeth-AHP (Schweiz) AG, Switzerland.

Experimental protocol

Male Cy/þ and þ/þ rats were weaned and were then treatedat 5 weeks of age (n¼ 12) with 2mg/kg/day sirolimus.

Additional male Cy/þ andþ/þ rats (n¼ 16) were not treated,and they served as controls. The drug was administered in theform of Rapamune� oral solution (Wyeth) in the drinkingwater for 3 months. Drinking bottles were protected fromlight with aluminum foil and were changed every 2 days.The concentration of sirolimus (Rapamune� oral stocksolution 1mg/ml) was adjusted according to rat bodyweight. Blood was drawn from the tail veins every month.After 3 months treatment, rats were anaesthetized withisoflurane, and kidneys were removed, decapsulated andweighed. Slices of each kidney were preserved in 4%paraformaldehyde and embedded in paraffin for histologicalexaminations. Additional slices were snap frozen in liquidnitrogen and used for protein extraction.

Blood chemistry

Blood was collected in lithium-heparin tubes and centrifugedat 4�C. Plasma was stored at �20�C, and was analysed laterfor blood urea nitrogen (BUN) and creatinine using kineticcolor test and the modified Jaffe method, respectively. Bloodfor sirolimus levels was collected under the same conditions,but using EDTA tubes and analysed using HPLC-massspectroscopy. All samples were analysed at the Institutefor Clinical Chemistry, University Hospital (Zurich,Switzerland).

Histology

Mid-transversal slices of the kidney, about 2mm-thick, werefixed by immersion in phosphate-buffered 4% paraformalde-hyde, dehydrated in alcohol series and embedded in paraffin.Three micrometer thick sections were stained with theperiodic acid-Schiff (PAS) technique. The cyst volume density(CVD) was evaluated in these sections by morphometry,using the technique of point counting [18].

To avoid subjective sampling biases, micrographs formorphometry were taken according to an invariable protocol.At the magnification used (Zeiss Plan-Neofluar 10�/0.30), thediameter of the field of view was close to the thickness of therenal cortex in healthy rats. The first micrograph was taken asthe field was positioned on one extremity of the horseshoe-shaped cortex. For further sampling, the microscope stagewas moved clockwise by steps of one field diameter, whilethe surface of the kidney remained at the limit of the field.A micrograph was made at every second step. A total of 4–6micrographs was obtained for one section. One section wasevaluated per rat. The pixel size of the micrographs was1300� 1030 (1.34 mm/pixel). The cyst volume density of cystswas evaluated by point counting. An orthogonal grid witha line spacing of 150 pixels was used. The person responsiblefor morphologic documentation and for morphometry wasblinded to the experimental groups.

Analysis of S6K activity

The amount of total and phosphorylated S6K was assessedby immunoblotting, using specific antibodies. Samples ofkidney tissue were snap-frozen in liquid nitrogen and stored at�80�C. On the day of immunoblotting, ice-cold lysis bufferwas prepared. The lysis buffer consisted of 50mM Tris base,pH 7.4; 150mM NaCl; 1% deoxycholate; 1% SDS; and 1%Triton X-100. Immediately before use, the lysis buffer was

Sirolimus inhibits PKD progression 599

supplemented with 1mM PMSF, 10 mg/ml aprotinin,10mg/ml leupeptin, and protease and phosphatase inhibitorcocktail. Each sample was homogenized in this buffer witha Dounce homogenizer. The protein concentration of eachsample was measured.

SDS-PAGE sample buffer was added to the extracts.Extracts were heated to 90�C for 5min for denaturation.A total of 30 mg protein was loaded per lane for standardSDS-PAGE (7.5%), and Western analysis. The followingantibodies were used for detection: sc-230 (St CruzBiotechnology, Inc., Santa Cruz, CA, USA; detects S6K),#9205 and #9204 (Cell Signaling Technology, Inc., Beverly,MA, USA; detect S6K phosphorylated on Thr389 andThr 421/Ser424, respectively), and MAB 1501 (Chemicon,Temecula, CA, USA; detects actin). For signal detection,horseradish peroxidase-conjugated secondary antibodiesand SuperSignal Femto/Pico ECL kits were used (PierceBiotechnology, Rockford, IL, USA).

Statistical analysis

Data are presented as means±SD and were analysed byone-way ANOVA with post-test according to Newman–Keuls. A P-value of <0.05 was considered significant.

Results

Judging by physical appearance, the treatment withsirolimus was well tolerated. None of the 12 sirolimus-treated rats died, and all 16 control rats survived.Sirolimus blood levels were obtained in all treatedanimals, and were between 0.5 and 1.9 mg/l. Figure 1shows that body weight increased steadily in untreatedand sirolimus treated Cy/þ and þ/þ rats. Treatmentwith sirolimus did not significantly change body weightafter 3 months of treatment (�3.3% in þ/þ and �11%in Cy/þ rats).

Renal functional loss is inhibited with sirolimus

Figure 2 demonstrates that the BUN and creatininelevels remained within normal limits in þ/þ rats butincreased progressively in untreated Cy/þ rats. BUNand creatinine levels also increased in sirolimus-treatedCy/þ rats, but to a lesser degree. After 3 months oftreatment, the average BUN level was 39% less insirolimus-treated Cy/þ rats (n¼ 7) compared withuntreated Cy/þ rats (n¼ 9) (45.6±4.2 vs 75.3±16.9mg/dl; P<0.001), and creatinine levels were 34%less (0.89±0.13 vs 1.34±0.24mg/dl; P<0.001). Thisdemonstrates that sirolimus effectively inhibited theloss of renal function in male Han:SPRD rats.

Renal weight and cyst volume densityare reduced with sirolimus

Figure 3 shows that the increase in kidney weight waseffectively reduced in sirolimus-treated Cy/þ comparedwith untreated Cy/þ rats. Treatment with sirolimusresulted in a 34% lower kidney weight in Cy/þ rats.Sirolimus treatment did not have a significant effecton kidney weight in wild-type þ/þ rats (Figure 3A).We also determined the 2K/TBW ratios. Figure 3Bdemonstrates that sirolimus reduced the 2K/TBW ratioby 26% in Cy/þ rats.

Histological examination of the Han:SPRD kidneysrevealed a significant reduction in cyst size in sirolimus-treated Cy/þ rats compared with untreated Cy/þanimals (Figure 4). A more detailed microscopicalexamination revealed the presence of tubular cysts inthe cortex, and to a lesser extent in the outer medullaof untreated Cy/þ kidneys. The inner medulla wasnot affected. Cystic expansion of Bowman’s capsulewas observed in a few glomeruli. Mononuclear cellinfiltrates were observed focally in the peritubularinterstitium. This histopathological pattern was not

1 2 3 40

150

300

450 Cy/+ no treatment

Cy/+ sirolimus

+/+ no treatment

+/+ sirolimus

Age (months)

Wei

gh

t (g

ram

s)

Fig. 1. Weight gain (g) in the four animal groups at 1, 2, 3 and 4 months of age. Treatment with sirolimus was started at 5 weeks and didnot significantly influence weight gain (ANOVA).

600 P. R. Wahl et al.

affected qualitatively by sirolimus treatment in Cy/þkidneys. Untreated and sirolimus-treated wild-typeþ/þ kidneys showed no difference in histology (datanot shown).

The most significant effect of sirolimus on kidneyhistology was a significant reduction of the cyst volumedensity in the cortex of Cy/þ rats. Figure 5 demon-strates that cyst volume density was 18% less insirolimus-treated Cyþ/rats (P<0.001). Since the refer-ence volume for evaluation of volume density was thetotal cortical volume and since sirolimus treatmentconsiderably reduced the increase of kidney weightin Cy/þ rats, the effect of sirolimus on the cyst volumedensity certainly underestimates its effect on theabsolute volume of cysts.

Total and phosphorylated levels of S6Kare enhanced in ADPKD rats

mTOR controls protein synthesis and cell growth byphosphorylating and thereby activating the 70 kDaribosomal S6 kinase (S6K) [19]. Thus far it has notbeen tested whether mTOR-S6K signalling is altered inADPKD, and whether the mTOR inhibitor sirolimusaffects ADPKD. We therefore examined S6K expres-sion and phosphorylation in untreated and sirolimus-treated Cy/þ rats. Figure 6 shows that total levels ofS6K are markedly increased in the kidneys of 4-month-old Cy/þ Han:SPRD rats compared with wild-type þ/þ rats. The level of expression of S6K waseffectively reduced in sirolimus-treated Cy/þ rats.

A

1 2 3 40

25

50

75

100 Cy/+ no treatment

Cy/+ sirolimus

+/+ no treatment

+/+ sirolimus

Age (months)

BU

N (

mg

/dl)

B

1 2 3 40.0

0.3

0.6

0.9

1.2

1.5

1.8 Cy/+ no treatment

Cy/+ sirolimus

+/+ no treatment

+/+ sirolimus

Age (months)

Ser

um

cre

atin

ine

(mg

/dl)

∆= −39%

∆ = −34%

Fig. 2. Blood urea nitrogen (BUN) (A) and serum creatinine (B) levels were measured monthly in all rats. The rise in BUN and creatininewas significantly reduced by sirolimus in Cy/þ rats.


We then measured the activity of S6K by analysingthe mTOR-dependent phosphorylation of S6K atthreonine 389 and threonine 421/serine 424 [20–23].Figure 5 shows that the phosphorylated forms of S6Kwere undetectable in normal þ/þ kidneys, but mark-edly enhanced in Cy/þ kidneys. Sirolimus effectivelyreduced phosphorylation at Thr389 and Thr421/Ser424of S6K in Cy/þ kidneys. Overall, there was an excellentcorrelation between the histomorphometric findingsand the degree of S6K phosphorylation.

Discussion

The massive and spontaneous development of cystsresults in progressive deterioration of renal functionin male Han:SPRD rats. This leads to symptomaticrenal failure and premature death in these animals.Here, we demonstrate that sirolimus, when given orallyfor 3 months, significantly improves renal function inmale Han:SPRD rats. Sirolimus treatment effectively

Fig. 4. Paraffin sections of kidneys of a wild-type, sirolimus-treated(A), of a Cy/þ untreated (B) and of a Cy/þ sirolimus-treated rat(C). Sirolimus reduced the kidney size, cyst volume and cyst densitymarkedly in Cy/þ rats. PAS staining. Bar length: 1.5mm.

A

0

1

2

3

4

5

6

7

sirolimus − −+ +

− −+ +

*

**

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t (g

ram

s)

B

0.0

0.6

1.2

1.8

sirolimus

*

**

2K/T

BW

t %

+/+

Cy/+

+/ +

Cy/+

Fig. 3. The 2-kidney weights (2KWt) (A) and 2-kidney/total bodyweight (2K/TBWt) ratios (B) were determined after 3 months oftreatment with sirolimus. Both parameters were significantlyreduced by sirolimus in Cy/þ rats. *P<0.001 (Cy/þ untreated vsþ/þ untreated); **P<0.001 (Cy/þ untreated vs Cy/þ sirolimus-treated).

0

10

20

30*

sirolimus −+− +

CV

D (

%)

+/+

Cy /+

Fig. 5. Cyst volume density (CVD) was massive in Cy/þ rats andwas significantly decreased (�18%) in sirolimus-treated Cy/þ rats.*P<0.001 (Cy/þ untreated vs Cy/þ sirolimus-treated).


controlled the massive increase in kidney weight inCy/þ rats and significantly reduced the cyst volumedensity. Our data confirm and extend a very recentreport by Tao et al. [12]. They found that rapamycin,when injected intraperitoneally for 5 weeks, improvedrenal function and slowed cyst progression in the samerat model of ADPKD.

We applied sirolimus by using the Rapamune� oralsolution. This is the galenic form which is approvedto prevent graft rejection in renal transplant patients.Using a 2mg/kg/day dose resulted in relatively lowblood levels in our animals (between 0.5 and 1.9 mg/l).This indicates that the drug was absorbed poorly.Despite these low levels, the therapeutic effect wasimpressive. Tao et al. used a different preparation ofsirolimus at a 10-fold lower daily dose intraperitoneally[12]. They did not measure blood levels, but judgingby the significantly reduced body weight (�22%) intheir rapamycin-treated rats it is likely that blood levelswere higher in their animals. The 3-month treatmentwas well tolerated in our rats with no significant effecton body weight.

In the study by Tao et al., treatment with sirolimusdid not completely prevent progression of cystic disease[12]. This was the case in our study also. However,both studies show that the treatment with sirolimusmarkedly retarded renal functional loss. In our studywe treated animals for an extended period of 3 monthsand found a continued inhibitory effect on renalfunctional loss and cystic expansion. More studies areneeded to determine whether mortality is reduced inHan:SPRD rats by prolonging treatment with sirolimusor other mTOR inhibitors.

Sirolimus is known to inhibit its molecular targetmTOR. As it was highly effective in Han:SPRDrats, we hypothesize that the mTOR pathway isdysregulated in the kidneys of these rats. mTOR

regulates a vast array of cellular functions, andpromotes cell growth and proliferation [19]. A well-characterized function of mTOR in mammalian cells isthe upregulation of protein synthesis via S6K, adirect substrate of mTOR. Phosphorylation of S6Kby mTOR enhances translation of certain mRNAs,which play an important role in translation regulation,increasing thereby the overall translation capacity ofcells [24].

We describe here for the first time that total amountsof S6K and activity of S6K (determined as phosphor-ylation of S6K) are enhanced in the kidneys ofHan:SPRD rats with ADPKD and that this can bereversed by the application of sirolimus. In an acuterenal failure model, it has been shown that activatedS6K is increased in rat kidneys, suggesting that thissignalling pathway is activated in other types of renalinjury [17]. The mechanisms which lead to the enhancedexpression and phosphorylation of S6K in cystickidneys of Han:SPRD need to be investigated further.Our findings point to an important role of the mTORpathway in Han:SPRD. We hypothesize that therecould be an interaction between molecules which aredefective in Han:SPRD and the mTOR pathway inthe pathogenesis of cyst growth. The nature and theimportance of these interactions need to be investigatedin further studies.

In summary, we demonstrate that oral sirolimusmarkedly inhibits the rapid deterioration of renalfunction by inhibiting cyst growth in Han:SPRD ratswith ADPKD. We speculate that mTOR and itsdownstream effector S6K play a critical role in thepathogenesis of ADPKD. Further studies are inprogress to elucidate the mechanisms of the alteredexpression and phosphorylation of these proteins,and to examine the effect of mTOR inhibition inother animal models of cystic kidney disease.

actin

sirolimus − − + + − − + +

1 2 3 4 5 6 7 8

Cy /++/+

P-Thr421/Ser424-S6K

P-Thr389-S6K

total S6K

Fig. 6. SDS-PAGE and Western blot analysis from kidney protein extracts for total amounts of S6K, phosphorylated S6K (Thr389, andThr421/Ser424) and actin. Two representative animals were analyzed from each group. The amount of total and phosphorylated S6K wasmarkedly increased in Cy/þ kidneys (lanes 5 and 6), and this was effectively suppressed by sirolimus (lanes 7 and 8).


Acknowledgements. This study was supported by an unrestrictedresearch grant from Wyeth-AHP (Schweiz) AG, Switzerland(R.P.W.) and by a grant from the Swiss National Foundation(M.N.H.). Wyeth also provided Rapamune� free of charge.

Conflict of interest statement. All authors listed declare to have hadno involvements that might raise the question of bias in the workor in the conclusions, implications, or opinions stated.

References

1. Wilson PD. Polycystic kidney disease. N Engl J Med 2004; 350:151–164

2. Igarashi P, Somlo S. Genetics and pathogenesis ofpolycystic kidney disease. J Am Soc Nephrol 2002; 13:2384–2398

3. Harris PC. Molecular basis of polycystic kidney disease: PKD1,PKD2 and PKHD1. Curr Opin Nephrol Hypertens 2002; 11:309–314

4. The European Polycystic Kidney Disease Consortium.The polycystic kidney disease 1 gene encodes a 14 kb transcriptand lies within a duplicated region on chromosome 16.Cell 1994; 77: 881–894

5. The International Polycystic Kidney Disease Consortium.Polycystic kidney disease: the complete structure of the PKD1gene and its protein. Cell 1995; 81: 289–298

6. Mochizuki T, Wu G, Hayashi T et al. PKD2, a gene forpolycystic kidney disease that encodes an integral membraneprotein. Science 1996; 272: 1339–1342

7. Delmas P. Polycystins: from mechanosensation to generegulation. Cell 2004; 118: 145–148

8. Torres VE, Sweeney WE Jr, Wang X et al. Epidermal growthfactor receptor tyrosine kinase inhibition is not protective inPCK rats. Kidney Int 2004; 66: 1766–1773

9. Torres VE. Therapies to slow polycystic kidney disease.Nephron Exp Nephrol 2004; 98: e1–e7

10. Wang X, Gattone V 2nd, Harris PC, Torres VE. Effectivenessof vasopressin V2 receptor antagonists OPC-31260 and OPC-41061 on polycystic kidney disease development in the PCKrat. J Am Soc Nephrol 2005; 16: 846–851

11. Gretz N, Kranzlin B, Pey R et al. Rat models of autosomaldominant polycystic kidney disease. Nephrol Dial Transplant1996; 11 [Suppl 6]: 46–51

12. Tao Y, Kim J, Schrier RW, Edelstein CL. Rapamycinmarkedly slows disease progression in a rat model of polycystickidney disease. J Am Soc Nephrol 2005; 16: 46–51

13. Guba M, von Breitenbuch P, Steinbauer M et al. Rapamycininhibits primary and metastatic tumor growth by antiangio-genesis: involvement of vascular endothelial growth factor.Nat Med 2002; 8: 128–135

14. Schmelzle T, Hall MN. TOR, a central controller of cellgrowth. Cell 2000; 103: 253–262

15. Moses JW, Leon MB, Popma JJ et al. Sirolimus-elut stentsversus standard stents in patients with stenosis in a nativecoronary artery. N Engl J Med 2003; 349: 1315–1323

16. Stallone G, Schena A, Infante B et al. Sirolimus for Kaposi’ssarcoma in renal-transplant recipients. N Engl J Med 2005; 352:1317–1323

17. Lieberthal W, Fuhro R, Andry CC et al. Rapamycin impairsrecovery from acute renal failure: role of cell-cycle arrest andapoptosis of tubular cells. Am J Physiol Renal Physiol 2001;281: F693–F706

18. Mathieu O, Cruz-Orive LM, Hoppeler H, Weibel ER.Measuring error and sampling variation in stereology:comparison of the efficiency of various methods for planarimage analysis. J Microsc 1981; 121: 75–88

19. Inoki K, Corradetti MN, Guan KL. Dysregulation of theTSC-mTOR pathway in human disease. Nat Genet 2005; 37:19–24

20. Pearson RB, Dennis PB, Han JW et al. The principal target ofrapamycin-induced p70S6K inactivation is a novel phosphor-ylation site within a conserved hydrophobic domain. Embo J1995; 14: 5279–5287

21. Khaleghpour K, Pyronnet S, Gingras AC, Sonenberg N.Translational homeostasis: eukaryotic translation initiationfactor 4E control of 4E-binding protein 1 and p70 S6 kinaseactivities. Mol Cell Biol 1999; 19: 4302–4310

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23. Saitoh M, Pullen N, Brennan P, Cantrell D, Dennis PB,Thomas G. Regulation of an activated S6 kinase 1 variantreveals a novel mammalian target of rapamycin phosphoryla-tion site. J Biol Chem 2002; 277: 20104–20112

24. Hay N, Sonenberg N. Upstream and downstream of mTOR.Genes Dev 2004; 18: 1926–1945

Received for publication: 3.6.05Accepted in revised form: 6.9.05


Original Article

Mitotic activation of Akt signalling pathway in Han:SPRDrats with polycystic kidney disease

PATRICIA R WAHL,1 MICHEL LE HIR,2 ALEXANDER VOGETSEDER,2 ALEXANDRE ARCARO,3

ASTRID STARKE,1 YING WAECKERLE-MEN,1 ANDREAS L SERRA1,4 andRUDOLF P WUTHRICH1,4

1Physiological Institute and Zurich Center for Human Integrative Human Physiology and 2Anatomical Institute,University Zurich-Irchel, 3Division of Clinical Chemistry and Biochemistry, University Children’s Hospital, and

4Clinic for Nephrology, University Hospital, Zurich, Switzerland

SUMMARY:

Aim: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by an imbalance betweentubular epithelial cell proliferation and apoptosis. We have previously shown that the mammalian target ofrapamycin (mTOR) signalling pathway is aberrantly activated in the cystic kidneys of Han:SPRD rats withADPKD. Because the Akt kinase is an upstream regulator of mTOR, we hypothesized that the activity of Akt couldbe enhanced in the kidneys of Han:SPRD rats.Methods: Reverse transcription-polymerase chain reaction, western blot, enzyme-linked immunosorbent assayand immunohistochemistry were used to analyse Akt expression in rat polycystic kidneys.Results: Wild-type (+/+) and heterozygous (Cy/+) Han:SPRD rats showed constitutive expression of Akt-1, -2and -3 mRNA by reverse transcription-polymerase chain reaction analysis with no significant difference betweenCy/+ and +/+ kidneys. Western blotting and enzyme-linked immunosorbent assay showed a significant increasein phosphorylated Akt in Cy/+ compared with +/+ kidneys. The pattern of immunoreactivity for phosphorylated Aktin kidney sections was the same in +/+ and in Cy/+ rats, with very low levels in interphase cells, but extremelybright signals in mitotic cells, beginning with the onset of the prophase. The in vivo incorporation of bromo-deoxyuridine revealed approximately a ninefold higher rate of proliferation in Cy/+ cyst epithelia compared withnormal tubule epithelia in +/+ rats, while the expression of the cell cycle marker Ki67 revealed approximately asixfold higher rate of proliferation. In summary, enhanced phosphorylation of Akt can be demonstrated in Cy/+kidneys which correlates with a markedly elevated proliferation rate of epithelial cells in cysts. Mitotic but notresting cells display strong phosphorylation of Akt.Conclusion: Because Akt is a proximal target of mTOR, its inhibition with specific antagonists could be usefulto prevent or halt cystogenesis in ADPKD.

KEY WORDS: ADPKD, Akt kinase, cell proliferation, Han:SPRD rat, mitosis, mTOR.

Autosomal dominant polycystic kidney disease (ADPKD) isa common monogenetic disorder affecting as many as 1 in800 people in the general population.1,2 The disease is char-acterized by the formation of multiple fluid-filled renal cyststhat expand over time and destroy the architecture of thekidney. About 8–10% of all cases of chronic renal failure are

due to ADPKD, and around 50% of ADPKD patients willdevelop end-stage renal disease by the time they are 60 yearsof age.1,2

Renal cyst formation is due to enhanced proliferation oftubular epithelial cells.3 Microdissection of cystic kidneyshas revealed that cystic enlargement is due to an increase inthe number of epithelial cells lining the cyst and not to thestretching of the cyst epithelium.4,5 Additionally, culturedepithelial cells from ADPKD cysts display an enhancedproliferation rate, and genes associated with increasedproliferation are overexpressed in cystic epithelium.6

The mammalian target of rapamycin (mTOR), a serine/threonine kinase, plays an important role in the transla-tional control of gene expression during cell growth, and for

Correspondence: Dr Rudolf P Wuthrich, Clinic for Nephrology,University Hospital, Ramistrasse 100, 8091 Zurich, Switzerland.Email: [email protected]

Accepted for publication 18 April 2007.

© 2007 The AuthorsJournal compilation © 2007 Asian Pacific Society of Nephrology

NEPHROLOGY 2007; 12, 357–363 doi:10.1111/j.1440-1797.2007.00811.x

mailto:[email protected]

the overall rate of protein synthesis in response to nutrientsand other environmental factors.7 The mTOR kinase is alsoa key intermediary in multiple mitogenic signalling path-ways. Thus, mTOR plays a central role in regulating cellproliferation, such as angiogenesis in normal tissues, andneoplastic cell growth. An important pathway by whichgrowth factors and cytokines activate mTOR and its down-stream targets is represented by the phosphoinositide3-kinase (PI3K)/Akt pathway.8

There is evidence that signals upstream from mTOR areactivated in ADPKD. The serine/threonine protein kinaseAkt, a downstream effector of PI3K, has emerged as a criti-cal mediator of mTOR activity.8 The mTOR kinase pos-sesses two adjacent phosphorylation sites (Thr 2446 and Ser2448), and it is known that Ser 2448 is directly phosphory-lated by Akt.9–11 Recently, it has been shown that Akt isalso linked to mTOR via tuberin (TSC2) which is directlyphosphorylated by Akt.12,13

We have previously shown that mTOR is involved in theprogression of cyst development in the Han:SPRD model ofADPKD.14 The purpose of the present investigation was todetermine whether Akt kinase, an upstream regulator ofmTOR, is also activated in the kidney of Han:SPRD rats, awell-characterized rodent model of ADPKD. Here we showthat phosphorylated Akt (p-Akt) is markedly increased inheterozygous (Cy/+) Han:SPRD rats in comparison withwild-type (+/+) rats, particularly in cells undergoing mitosis,correlating with an increased proliferation of epithelial cellsin cysts.

METHODS

Reagents

Unless otherwise stated, all reagents used were from Sigma (St. Louis,MO, USA).

Animals

The study was conducted in heterozygous (Cy/+) and normal littermatecontrol (+/+) Han:SPRD rats. Only male rats were used because thedisease progresses faster in male compared with female rats. A colony ofHan:SPRD rats was established in our animal care facility from breed-ing pairs that were obtained from the Rat Resource & Research Center(Columbia, MO, USA). The study protocol was approved by the regu-latory commission for animal studies, a local government agency.Unless otherwise stated, the rats had free access to tap water andstandard rat diet. For immunohistochemistry 8-week-old rats wereinjected with 0.3 mL of bromo-deoxyuridine (BrdU) subcutaneously at10 am on day 0 because this is the stage of cyst progression at whichproliferation is at its peak. From that time on, the drinking solutionconsisted of 1 mg/mL BrdU – a synthetic analogue of thymidine – in tapwater up to sacrifice 4 days later between 9 am and 11 am. Whenapplied in vivo, BrdU is incorporated in the DNA of cycling cells,allowing detection by specific antibodies. In addition, 16-week-old ratswere sacrificed and kidneys were removed for mRNA and proteinextraction. This later time point was chosen to detect more significantchanges in RNA and protein levels, because previous results using genearray analyses (data not shown) have revealed that expression changeswere generally more apparent in older animals. After decapsulation,

kidney slices were prepared and were stored in RNAlater (Qiagen,Basel, Switzerland), an RNA stabilization solution for tissue, or weresnap frozen in liquid nitrogen for protein extraction. All samples werestored at -80°C.

RNA extraction and RT-PCR for Akt

Kidney tissue was mechanically disrupted with 0.1 mm diameterzirconia-silica beads in a Fast Prep FP120 instrument (Qbiogene,Heidelberg, Germany). RNA isolation from this lysate was performedwith a Qiagen RNeasy mini kit (Qiagen, Basel, Switzerland), accordingto the instructions of the supplier. All RNA samples were quantified bymeasurement of the OD at 260 nm and were then analysed for Aktexpression by semiquantitative reverse transcription-polymerase chainreaction (RT-PCR), using a Qiagen OneStep PCR kit (Qiagen, Basel,Switzerland). Varying numbers of cycles (25–35) were performed toensure that the PCR analysis was in the linear phase of amplification.The primer sequences for rat Akt-1, -2 and -3 and the housekeepinggene glyceraldehyde-3-phosphate dehydrogenase were determinedby using Primer 3 software (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). RT-PCR products were resolved on 1%agarose gels, and were stained with ethidium bromide and thenphotographed with ultraviolet light.

Protein extraction

To prepare protein extracts, frozen kidney tissue samples were homog-enized, using a Dounce homogenizer in T-PER extraction reagent(Pierce, Rockford, IL, USA). Immediately before use, the lysis bufferwas supplemented with 1 mM PMSF, Halt™ protease and Halt™ phos-phatase inhibitor cocktail (Pierce, Rockford, IL, USA). After centrifu-gation at 10 000 ¥ g for 5 min, supernatants were adjusted for proteinconcentration, and were used for western blotting and enzyme-linkedimmunosorbent assay (ELISA).

Western blot

Frozen cell lysates were defrosted and equal amounts of protein(0.335 mg) diluted to 0.5 mL using lysis buffer (20 mM HEPES(pH 7.4), 150 mM NaCl, 10% (w/v) glycerol, 1% (w/v) Triton X-100,2 mM EDTA, 10 mM sodium fluoride, 1 mM phenylmethylsul-phonyl fluoride, 5 mM benzamidine, 1 mM Na-tosyl-L-lysinechloromethyl ketone, 20 mM leupeptin, 18 mM pepstatin, 10 mMb-glycerophosphate, 2 mM sodium orthovanadate). Immunoprecipita-tion was performed using polyclonal rabbit anti-Akt1/2 antibody(Santa Cruz Biotechnology, Santa Cruz, CA, USA) and proteinA-sepharose CL-4B (GE Healthcare Bio-Sciences Corp., Piscataway,NJ, USA). Samples were incubated for 3 h at 4°C with constantmixing. The immunoprecipitates were then washed three times with1 mL of lysis buffer and boiled in SDS-PAGE sample buffer. Thesamples were analysed by SDS-PAGE (10%, reducing) and westernblotting, using polyclonal rabbit antiphospho-Akt antibody (Ser473,Cell Signalling Technology, Danvers, MA, USA). Blots were devel-oped using enhanced chemiluminescence (ECL kit, GE HealthcareBio-Sciences Corp., Piscataway, NJ, USA).

ELISA

Expression levels of total and p-Akt (phosphorylation sites Ser473 andThr308) were determined by sensitive and specific ELISA (Sigma, St.Louis, MO, USA). A monoclonal capture antibody specific for Akt

PR Wahl et al.358


http://www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi

http://www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi

(independent of phosphorylation) was coated onto a 96-well plate.Standard dilutions and equal concentrations of samples were incubatedfor 2 h at RT allowing Akt antigen to bind to the capture antibody.After washing, a detection antibody specific for either total Akt, p-Akt(Ser473), or p-Akt (Thr308) was added and incubated for 1 h at RT.After washing, an anti-rabbit IgG-HRP was added. The reaction wasvisualized by tetramethylbenzidine (TMB) substrate, followed by theaddition of stop solution. The optical density was measured in a multi-well plate reader at 450 nm and is directly proportional to the concen-tration of Akt/PKB in the sample. The values of p-Akt (Ser473) andp-Akt (Thr308) were normalized for total Akt content.

Immunohistochemistry

Fixation and tissue treatment

The rats were anaesthetized by an intraperitoneal injection of pento-barbital (100 mg/kg body weight) and fixed by vascular perfusion.15 Thefixative contained 3% paraformaldehyde, 0.01% glutardialdehyde and0.5% picric acid, dissolved in a 3:2 mixture of 0.1 M cacodylate buffer(pH 7.4, added with sucrose, final osmolality 300 mosmol) and 4%hydroxyl ethyl starch (Fresenius Kabi, Bad Homburg, Germany) in0.9% NaCl. The kidneys were fixed for 5 min, and then rinsed byvascular perfusion with 0.1 M cacodylate buffer for 5 min.

Immunofluorescence staining

2 mm slices of fixed kidney were frozen in liquid propane which wascooled down to the temperature of liquid nitrogen, and were cut into4 mm cryostat sections. Sections were thawed on slides and microwavedfor 10 min in 0.01 M citrate buffer at pH 6.0. After pretreatment for 1 hin 5% normal goat serum in phosphate-buffered saline, the sectionswere incubated overnight in a humidified chamber at 4°C with theprimary antibodies, diluted in phosphate-buffered saline supplementedwith 1% bovine serum albumin. p-Akt (rabbit anti-p-Akt (Thr308)),p-S6 kinase (rabbit anti-p-S6K(Thr421/Ser424)) (both Cell SignallingTechnology, Danvers, MA, USA), BrdU (mouse anti-BrdU clone B44,BD Biosciences Pharmingen, San Diego, CA, USA) and the prolifera-tion marker Ki67 (rabbit anti-Ki67, Novocastra Laboratories Ltd.,Newcastle, UK) were analysed on cryostat sections. Binding sites of theprimary antibodies were revealed with Cy3-conjugated goat-anti-rabbitIgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).For nuclear staining, 4′,6-diamidino-2-phenylindole (DAPI; Sigma, St.Louis, MO, USA) was added to the working dilution of the secondaryantibodies.

Quantitative evaluation of nuclear labelling

Using the nuclear DNA-labelling of DAPI, the outer stripe and cortexwere located and micrographs were made randomly, using the 40¥magnification of a laser scanning microscope (CLSM SP2, Leica, Man-nheim, Germany). The surface area of tissue evaluated for each rat was3.5 mm2. The proximal tubule was identified by the brush border,detectable in background fluorescence. Nuclei of proximal epithelialcells positive for BrdU, and/or Ki67 were counted in all proximaltubules in the micrographs. DAPI fluorescence was used for assessmentof the total number of nuclei in the proximal tubules. Because of thehigh number of DAPI-positive cells, these were evaluated in a uniformrandom systematic sample (10% sampling fraction) of the area of themicrograph. Those parameters were evaluated separately in cystictubules and normal tubules.

Statistical analysis

Unless otherwise stated, data are presented as means 1 SEM and wereanalysed by Student’s t-test for unpaired samples. A P-value of <0.05was considered significant. Statistical analysis of the data was performedwith the GraphPad Prism software (GraphPad Software Inc. San Diego,CA, USA).

RESULTS

Renal Akt mRNA expression in Cy/+ and +/+ rats

First, we examined the kidneys of Cy/+ and wild-type +/+rats for the presence of the three different Akt mRNAs,using specific primers for Akt-1, -2 and -3 and RT-PCRanalysis. Figure 1 shows that all three Akt mRNA tran-scripts were constitutively expressed. This RT-PCR analysisdid not reveal a distinct difference between healthy +/+kidneys and polycystic Cy/+ kidneys.

Upregulation of p-Akt in Cy/+ versus +/+ rats

We then examined whether an enhanced activity of Aktcould be found in Cy/+ kidneys, as revealed by phosphory-lation of Akt at Ser473. Total Akt was immunoprecipitatedfrom protein extracts of Cy/+ and +/+ kidneys, was separatedby SDS-PAGE and analysed by western blot using an

Fig. 1 Reverse transcription-polymerase chain reactionmRNA expression of the three Akt isoforms in 16-week-oldwild-type (+/+) and heterozygous (Cy/+) rats. Akt-1, -2 and -3mRNA transcript levels are all constitutively expressed,with no change between the two strains. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a house-keeping gene to ensure equal amounts of mRNA. Results arerepresentative of repeated experiments.

Akt in PKD 359


antibody which targets the Ser473 phosphorylation site.Figure 2 shows that p-Akt (Ser473) was only weakly detect-able in healthy 16-week-old rats. However, there was asignificant increase in the expression levels in age-matchedheterozygous (Cy/+) rats.

We confirmed the increase in Akt activity, using sensi-tive and specific ELISA for p-Akt, examining both theSer473 and the Thr308 phosphorylation sites. Figure 3Ashows that there was a significant increase in the activitylevel of p-Akt at Ser473 in heterozygous (Cy/+) kidneys, ascompared with untreated wild-type (+/+) kidneys (2.5-foldincrease; P 2 0.001). Figure 3B shows that there was also asignificant increase in levels of p-Akt at Thr308 in heterozy-gous (Cy/+) versus untreated wild-type (+/+) rats (1.3-foldincrease; P 2 0.05). Thus, Akt phosphorylation is enhancedat both sites in Cy/+ kidneys.

High levels of p-Akt and p-S6K in mitotic cells

We then examined the expression of p-Akt at Thr308 inkidneys of Cy/+ and +/+ rats by immunofluorescence stain-ing. Figure 4 shows that p-Akt was detected only in a smallfraction of tubular cells. Examination of the chromatin inthe blue channel (DAPI) revealed that all of those cellswere undergoing mitosis. Immunolabelling for p-Akt wasweak to moderate in the prophase and it was restricted tothe nucleus. In the metaphase and the anaphase, the fluo-rescence was very bright and it decreased sharply in thetelophase. A very similar pattern was observed for p-S6K, adownstream effector in the Akt pathway, with a weak signalin the interphase, a moderate-to-strong nuclear fluorescencein the prophase and very bright fluorescence in the meta-phase and the anaphase. Figure 5 shows a typical cyst with acell in metaphase that is brightly positive for p-S6K.

Increased cell proliferation in cysts of Cy/+Han:SPRD rats

In Figure 6, it can be seen that the increased proliferationrate in the Cy/+ rat is completely attributable to cyst epi-thelia, because in normal tubules of Cy/+ rats the incidence

of cells labelled with BrdU or Ki67 were not significantlydifferent from values in +/+ rats. At 8 weeks of age thepercentage of BrdU-positive tubular cells in the cortex was4.0% in +/+ rats, 5.3% in Cy/+ rats and reached 37.5% incyst epithelia. The corresponding numbers for Ki67 were1.2%, 1.0% and 7.3%.

DISCUSSION

Here we show that the phosphorylation of Akt kinase andhence its activity is markedly elevated in the kidney ofHan:SPRD rats with polycystic kidney disease. It has beenshown that Akt is activated by phospholipid binding andactivation loop phosphorylation at Thr308 and by phospho-rylation within the carboxy terminus at Ser473. Phospho-rylation of both sites is necessary for full activation.16

Enhanced phosphorylation of Akt was evident at Ser473and Thr308 in heterozygous Cy/+ compared with wild-type+/+ kidneys. The most striking observation was p-Akt in

Fig. 2 SDS-PAGE and western blot analysis of protein extractsfrom 16-week-old male Cy/+ and +/+ rat kidney tissue samples,testing for phosphorylated Akt (p-Akt) (Ser473). The amountof p-Akt was markedly increased in Cy/+ rat kidneys. Resultsare representative of several repeated experiments.

Fig. 3 Protein samples from 16-week-old wild-type (+/+) andheterozygous (Cy/+) male rats were analysed for (A) phospho-rylated Akt (p-Akt) (Ser473) or (B) p-Akt (Thr308), usingsensitive enzyme-linked immunosorbent assay. There was asignificant increase in both phosphorylated forms of Akt inheterozygous Cy/+ in comparison to wild-type (+/+) rats.*P 2 0.05, **P 2 0.001.

PR Wahl et al.360


mitotic cells, starting with the prophase. This suggests thatactivation of Akt is instrumental in mitosis in tubularepithelial cells. Because an enhanced proliferation rate ofepithelial cells is central in cyst formation, Akt could be anattractive target for treating ADPKD.

The detection of p-Akt in mitotic cells fits with previousstudies showing the requirement for Akt activation in G2/Mtransition in cell culture.6,17 Because we observed previouslythat phosphorylation of S6K, a downstream member of theAkt signalling cascade, is increased in ADPKD rats, we wereinterested in examining the distribution of p-S6K(Thr421/Ser424) by immunofluorescence. Like Akt, S6K was highly

phosphorylated throughout mitosis. Because the phospho-rylation of S6K on the residues 421 and 424 is sensitive tomTOR inhibition,18,19 it is likely that the Akt/mTOR/S6Kpathway plays a role in mitosis in tubular epithelial cells.

Others and we have recently shown that mTOR and itsdownstream target S6K are markedly activated in polycystickidney disease. Thus, we could show that S6K, a key regu-lator of cell growth and proliferation is aberrantly phospho-rylated in the Han:SPRD rat, a model of ADPKD.14 Taoet al. as well as our group could also show that rapamycintreatment of Han:SPRD rats markedly delayed cyst growthand renal functional deterioration in these animals.14,20 Shil-lingford et al. extended these findings to three mouse modelsof cystic diseases with different affected genes, as well as inhuman renal tissue samples from patients with ADPKD.21 Intheir landmark paper, they also provided evidence thattuberin (TSC2), a protein that directly inhibits mTOR,associated with polycystin 1, is the gene which is defectivein 85% of patients with ADPKD.21 They suggested thatmutations in polycystin 1 could lead to aberrant activationof mTOR, thus allowing phosphorylation of S6K, themaster switch of cell growth and proliferation.

The activity of the mTOR kinase may not be solelyregulated by tuberin. Renal cyst fluid from PKD mice androdent models contain epidermal growth factor (EGF)-likepeptides in mitogenic concentrations.22–24 EGF has also beenshown to induce proximal tubular cyst formation in murinemetanephric organ cultures.25 It is well established invarious systems that EGF receptor (EGFR) ligation leads toactivation of phosphatidylinositol 3-kinase (PI3K). Aktactivation is one of the major downstream events of PI3Ksignalling. Navé et al. showed that Akt, as part of the PI3K/

Fig. 4 Localization of phosphorylated Akt (p-Akt) (Thr308)by immunofluorescence. A normal proximal tubule (A, B) anda cyst (C) are shown in the kidney of a 2-month-old male Cy/+rat. (A) p-Akt positivity in a nucleus of a cell in prophase(arrowhead). The prophase nucleus, but not the neighbouringinterphase nuclei, is positive for p-Akt. (B) the pattern of DAPIfluorescence indicates that the cell shown in A is in theprophase (arrowhead). (C) Dual staining for p-Akt and DAPI.A cell in the metaphase in a cyst is positive for p-Akt (red). Theinsert shows the blue channel alone (chromatin staining withDAPI), demonstrating mitosis in the p-Akt-positive cell. Thegreen channel displays background fluorescence of the tissue.Bar: 10 mm in A and B, 100 mm in C.

Fig. 5 Localization of phosphorylated S6K (p-S6K) (Thr421/Ser424) by immunofluorescence. A cell in the metaphase in acyst is strongly positive for p-S6K (red), whereas interphasenuclei show only a faint signal. The insert shows the bluechannel alone (chromatin staining with DAPI), demonstratingmitosis in the p-S6K-positive cell. The green channel displaysbackground fluorescence of the tissue. Bar: 100 mm.

Akt in PKD 361


Akt pathway, directly targets mTOR, modulating the sig-nalling pathways involved in protein translation.10 Thus, itmight be possible that the PI3K/Akt kinase pathway acti-vates mTOR in a major way because of the enhanced EGFRactivation which is characteristic in ADPKD. In support ofthis hypothesis is an experimental study with an EGFRtyrosine kinase inhibitor which attenuated the developmentof PKD in Han:SPRD rats.26

In summary, we provide evidence that activation of Aktvia phosphorylation at Ser473 and Thr308 occurs in theearly phase of polycystic kidney disease in the Han:SPRDmodel of ADPKD. We hypothesize that mTOR activationby the PI3K/Akt pathway occurs in addition to thepolycystin-1-dependent regulation of mTOR via tuberin.Although we do not know precisely which signals activateAkt in ADPKD, it is tempting to speculate that Akt inhi-bition with selective antagonist could have a beneficialtherapeutic effect in ADPKD.

ACKNOWLEDGEMENT

This study was supported by an unrestricted research grantfrom Wyeth-AHP (Schweiz) AG, Switzerland (R.P.W.).

REFERENCES

1. Sutters M, Germino GG. Autosomal dominant polycystic kidneydisease: Molecular genetics and pathophysiology. J. Lab. Clin.Med. 2003; 141: 91–101.

2. Wilson PD. Polycystic kidney disease. N. Engl. J. Med. 2004; 350:151–64.

3. Comperat E, Ferlicot S, Camparo P et al. Expression of epidermalgrowth factor receptor and proliferative activity of cyst epitheliumin human renal cystic diseases. Urol. Int. 2006; 76: 269–73.

4. Yamaguchi T, Hempson SJ, Reif GA, Hedge AM, Wallace DP.Calcium restores a normal proliferation phenotype in human poly-cystic kidney disease epithelial cells. J. Am. Soc. Nephrol. 2006;17: 178–87.

5. Yamaguchi T, Wallace DP, Magenheimer BS, Hempson SJ,Grantham JJ, Calvet JP. Calcium restriction allows cAMP activa-tion of the B-Raf/ERK pathway, switching cells to a cAMP-dependent growth-stimulated phenotype. J. Biol. Chem. 2004;279: 40419–30.

6. Lee JE, Park MH, Park JH. The gene expression profile of cystepithelial cells in autosomal dominant polycystic kidney diseasepatients. J. Biochem. Mol. Biol. 2004; 37: 612–17.

7. Schmelzle T, Hall MN. TOR, a central controller of cell growth.Cell 2000; 103: 253–62.

8. Hay N, Sonenberg N. Upstream and downstream of mTOR. GenesDev. 2004; 18: 1926–45.

9. Scott PH, Brunn GJ, Kohn AD, Roth RA, Lawrence JC Jr.Evidence of insulin-stimulated phosphorylation and activation ofthe mammalian target of rapamycin mediated by a protein kinaseB signaling pathway. Proc. Natl. Acad. Sci. USA 1998; 95: 7772–7.

10. Navé BT, Ouwens M, Withers DJ, Alessi DR, Shepherd PR. Mam-malian target of rapamycin is a direct target for protein kinase B:Identification of a convergence point for opposing effects of insulinand amino-acid deficiency on protein translation. Biochem. J.1999; 344: 427–31.

11. Sekulic A, Hudson CC, Homme JL et al. A direct linkage betweenthe phosphoinositide 3-kinase-AKT signaling pathway and the

Fig. 6 Enhanced proliferation in cysts of 2-month-old male Cy/+ Han:SPRD rats. Proliferation is represented as the percentage ofnuclei of proximal tubular cells that are positive for BrdU (A) or for Ki67 (B). For evaluation of the total number of nuclei we usedDAPI, a fluorescent DNA-binding dye. *P 2 0.05, **P 2 0.001. No significant difference was seen in tubules from control (+/+) ratsas compared with normal tubules in Cy/+ rats. Panel C shows the distribution of BrdU as detected by immunofluorescence (green).The red channel displays background fluorescence of the tissue. The incidence of BrdU-positive nuclei is higher in the cysts than inthe neighbouring healthy tubules. Bar: 100 mm.

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mammalian target of rapamycin in mitogen-stimulated and trans-formed cells. Cancer Res. 2000; 60: 3504–13.

12. Inoki K, Li Y, Zhu T, Wu J, Guan KL. TSC2 is phosphorylated andinhibited by Akt and suppresses mTOR signalling. Nat. Cell Biol.2002; 4: 648–57.

13. Manning BD, Tee AR, Logsdon MN, Blenis J, Cantley LC. Iden-tification of the tuberous sclerosis complex-2 tumor suppressorgene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol. Cell 2002; 10: 151–62.

14. Wahl PR, Serra AL, Le Hir M, Molle KD, Hall MN, Wuthrich RP.Inhibition of mTOR with sirolimus slows disease progression inHan:SPRD rats with autosomal dominant polycystic kidneydisease (ADPKD). Nephrol. Dial. Transplant. 2006; 21: 598–604.

15. Dawson CA, Bongard RD, Rickaby DA, Linehan JH, Roerig DL.Effect of transit time on metabolism of a pulmonary endothelialenzyme substrate. Am. J. Physiol. 1989; 257: H853–65.

16. Tsurutani J, Fukuoka J, Tsurutani H et al. Evaluation of twophosphorylation sites improves the prognostic significance of Aktactivation in non-small-cell lung cancer tumors. J. Clin. Oncol.2006; 24: 306–14.

17. Dangi S, Cha H, Shapiro P. Requirement forphosphatidylinositol-3 kinase activity during progression throughS-phase and entry into mitosis. Cell. Signal. 2003; 15: 667–75.

18. Huang C, Li J, Ke Q et al. Ultraviolet-induced phosphorylation ofp70(S6K) at Thr (389) and Thr (421)/Ser (424) involves hydro-gen peroxide and mammalian target of rapamycin but not Akt andatypical protein kinase C. Cancer Res. 2002; 62: 5689–97.

19. Le XF, Hittelman WN, Liu J et al. Paclitaxel induces inactivationof p70, S6 kinase and phosphorylation of Thr421 and Ser424 viamultiple signaling pathways in mitosis. Oncogene 2003; 22: 484–97.

20. Tao Y, Kim J, Schrier RW, Edelstein CL. Rapamycin markedlyslows disease progression in a rat model of polycystic kidneydisease. J. Am. Soc. Nephrol. 2005; 16: 46–51.

21. Shillingford JM, Murcia NS, Larson CH et al. The mTORpathway is regulated by polycystin-1, and its inhibition reversesrenal cystogenesis in polycystic kidney disease. Proc. Natl. Acad.Sci. USA 2006; 103: 5466–71.

22. Munemura C, Uemasu J, Kawasaki H. Epidermal growth factorand endothelin in cyst fluid from autosomal dominant polycystickidney disease cases: Possible evidence of heterogeneity incystogenesis. Am. J. Kidney Dis. 1994; 24: 561–8.

23. Moskowitz DW, Bonar SL, Liu W, Sirgi CF, Marcus MD, ClaymanRV. Epidermal growth factor precursor is present in a variety ofhuman renal cyst fluids. J. Urol. 1995; 153: 578–83.

24. Lakshmanan J, Eysselein V. Hereditary error in epidermal growthfactor prohormone metabolism in a rat model of autosomal domi-nant polycystic kidney disease. Biochem. Biophys. Res. Commun.1993; 197: 1083–93.

25. Pugh JL, Sweeney WE Jr, Avner ED. Tyrosine kinase activity ofthe EGF receptor in murine metanephric organ culture. KidneyInt. 1995; 47: 774–8.

26. Torres VE, Sweeney WE Jr, Wang X et al. EGF receptor tyrosinekinase inhibition attenuates the development of PKD inHan:SPRD rats. Kidney Int. 2003; 64: 1573–9.

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Fax +41 61 306 12 34E-Mail [email protected]

Original Paper

Kidney Blood Press Res 2007;30:253–259 DOI: 10.1159/000104818

Everolimus Retards Cyst Growth and Preserves Kidney Function in a Rodent Model for Polycystic Kidney Disease

Ming Wu

a Patricia R. Wahl

a Michel Le Hir

b Ying Wäckerle-Men

a

Rudolf P. Wüthrich

a, c Andreas L. Serra

a, c

a Physiological Institute and b

Anatomical Institute, University Zürich Irchel, and c Clinic for Nephrology,

University Hospital, Zürich , Switzerland

Moderate dosage of everolimus inhibits cystogenesis in Han:SPRD rats. The inhibitory effect of everolimus appears to represent a class effect of mTOR inhibitors.

Copyright © 2007 S. Karger AG, Basel

Introduction

Autosomal dominant polycystic kidney disease(ADPKD) is one of the leading causes of end-stage kidney disease and occurs in 1–2 per 1,000 live births [1, 2] . The disease is characterized by aberrant proliferation of tubu-lar epithelial cells (TEC) forming multiple renal cysts [1] . Currently there is no specific treatment for ADPKD oth-er than supportive care, and dialytic treatment or renal transplantation when patients have reached end-stage re-nal disease. Because ADPKD progresses slowly, there is a window of opportunity to treat the disease by using ther-apeutical principles which retard cystic expansion, thus preventing the development of end-stage renal disease.

The mammalian target of rapamycin (mTOR), an atypical serine/threonine kinase, is a central controller of cell growth and proliferation [3] . The mTOR pathway is not activated in normal adult kidneys but is aberrantly activated in kidneys of different rodent models for poly-cystic kidney disease (PKD) as well as in cyst-lining cells in human ADPKD [4, 6] . Rapamycin (also termed siroli-

Key Words

Polycystic kidney disease � Everolimus � mTOR � Han:SPRD rat

Abstract

Background/Aims: Rapamycin inhibits cyst growth in poly-cystic kidney disease by targeting the mammalian target of rapamycin (mTOR). To determine if this is a class effect of the mTOR inhibitors, we examined the effect of everolimus,the analogue of rapamycin, on disease progression in the Han:SPRD rat model of polycystic kidney disease. Methods: Four-week-old male heterozygous cystic (Cy/+) and wild-type normal (+/+) Han:SPRD rats were administered everoli-mus or vehicle (3 mg/kg/day) by gavage for 5 weeks. Kidney function and whole-blood trough levels of everolimus were monitored. After treatment kidney weight and cyst volume density were assessed. Tubule epithelial cell proliferation was assessed by BrdU staining. Results: Everolimus trough levels between 5 and 7 � g/l were sufficient to significantly reduce kidney and cyst volume density by approximately 50 and 40%, respectively. The steady decrease of kidney func-tion in Cy/+ rats was reduced by 30% compared with vehicle-treated Cy/+ rats. Everolimus treatment markedly reduced the number of 5-bromo-2-deoxyuridine-labeled nuclei in cyst epithelia. Body weight gain and kidney function were impaired in everolimus-treated wild-type rats. Conclusion:

Received: April 19, 2007 Accepted: May 18, 2007 Published online: June 27, 2007

Andreas L. Serra, MD Clinic for Nephrology, University Hospital Rämistrasse 100 CH–8091 Zürich (Switzerland) Tel. +41 44 255 33 84, Fax +41 44 255 45 93, E-Mail [email protected]

© 2007 S. Karger AG, Basel1420–4096/07/0304–0253$23.50/0

Accessible online at:www.karger.com/kbr

JANA

copyright

Wu /Wahl /Le Hir /Wäckerle-Men /Wüthrich /Serra

Kidney Blood Press Res 2007;30:253–259254

mus) is a specific inhibitor of mTOR with strong anti-proliferative properties. Rapamycin is clinically used to prevent organ rejection in renal transplant patients. Fur-thermore, cardiac stents coated with rapamycin abolish intrastent neointimal proliferation. Recently we and oth-ers have shown that rapamycin when administered intra-peritoneally or through the drinking water successfully retards disease progression in different rodent models of PKD [5–7] . Relatively low blood level of rapamycin de-creased cyst volume density by approximately 20% in the Han:SPRD rat, a model for PKD [6] .

Everolimus [40-O-(2-hydroxyethyl)-rapamycin], also called RAD001, is a macrolide antibiotic that binds to the intracellular protein FKBP and thereby inhibits mTOR activity [8] . It is an orally active derivative of sirolimus (rapamycin), with a hydrophilic 2-hydroxyethyl chain at position 40 [9] . Compared to rapamycin, everolimus has a shorter half-life and a higher biological availability [10] . Everolimus is also used as an immunosuppressive drug in organ transplantation. Currently an ongoing clini-cal trial is testing whether everolimus (Certican � ; NCT00414440) whole-blood trough levels between 3 and 8 � g/l halt disease progression in human ADPKD. Sur-prisingly, it is not known if everolimus halts disease pro-gression in PKD animal models.

Han:SPRD rats represent one of the most commonly used rodent models of PKD, phenotypically resembling human ADPKD [11] . To determine whether the observed cyst inhibition is rapamycin-specific or if this is a class effect of the mTOR inhibitors, we examined the effect of everolimus on renal functional loss and cyst progression in the Han:SPRD rat model of ADPKD. To achieve con-sistent blood levels of everolimus, we employed daily ga-vage feeding in this study.

Materials and Methods

Animals This study was conducted in heterozygous cystic (Cy/+) and

wild-type normal (+/+) rats. Only male rats were used, since cysts develop more rapidly in male than female rats [12] . The Han:SPRD rat colony was established in our animal facility from a litter which was obtained from the Rat Resource and Research Center (Colum-bia, Mo., USA). The study protocol was approved by the regulatory commission for animal studies, a local government agency. Rats had free access to tap water and standard rat diet.

Study Drug Everolimus microemulsion (20 mg/g) and vehicle microemul-

sion were kindly supplied by Novartis (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 with

3 mg/kg/day everolimus (Cy/+, n = 8; +/+, n = 10) or vehicle (Cy/+, n = 4; +/+, n = 11) at 4 weeks of age. Everolimus was administered once daily by gavage feeding for 5 weeks (day 0 to day 35) at a vol-ume of 2 ml/day. The dose of everolimus administered was ad-justed daily according to the rat body weight.

Tail blood was obtained from rats on days 0, 17 and 36. Whole blood for everolimus levels and plasma for blood urea nitrogen (BUN) were stored at –20 ° C. Blood everolimus levels and BUN were analyzed later by kinetic color test and HPLC mass spectros-copy at the Institute for Clinical Chemistry, University Hospital (Zürich, Switzerland). Everolimus 6-hour levels were measured on day 17 and everolimus trough levels (24-hour level) on day 36. BUN concentrations were determined on days 0, 17 and 36.

For the assessment of cell proliferation 5-bromo-2-deoxyuri-dine (BrdU) (Sigma, St. Louis, Mo., USA) was injected subcutane-ously (20 mg/kg/day BrdU in 0.9% NaCl) in 3 rats per group on days 33, 34 and 35.

After the 5-week treatment the rats were anesthetized with iso-flurane, and kidneys were excised, decapsulated and weighed.

For histologic examinations, 3 rats per group were anesthe-tized by an intraperitoneal injection of pentobarbital (100 mg/kg body weight) and fixed by vascular perfusion [13] . The fixative contained 3% paraformaldehyde (PFA), 0.01% glutardialdehyde and 0.5% picric acid, dissolved in a 3: 2 mixture of 0.1 M cacodyl-ate buffer (pH 7.4, added with sucrose, final osmolality 300 mos-mol) and 4% hydroxyl ethyl starch (Fresenius Kabi, Bad Hom-burg, Germany) in 0.9% NaCl. The kidneys were fixed for 5 min and then rinsed by vascular perfusion with 0.1 M cacodylatebuffer for 5 min.

Histology Two- to three-millimeter-thick mid-transversal rat kidney

slices were dehydrated in ethanol series, embedded in paraffin and subjected to periodic acid-Schiff staining for cyst volume density determination.

Cyst volume density was assessed by morphometry, using the method of point counting [14] . For morphometry 4–6 micro-graphs were obtained for 1 section per rat as described before [6] . The pixel size of the micrographs was 1,300 ! 1,030 (1.34 � m/pixel). An orthogonal grid with a line spacing of 150 pixels was used. The reviewer who performed the morphometry was blinded to experimental groups.

Anti-BrdU clone 3D4 (BD Biosciences Pharmingen, San Die-go, Calif., USA) was mouse monoclonal. For the detection of BrdU, 2-mm-thick slices of perfusion-fixed kidney were frozen in liquid propane cooled down to –196 ° C by liquid nitrogen and cut into 4- � m-thick cryostat sections. The cryostat sections were mi-crowaved for 10 min in 0.01 M citrate buffer at pH 6.0. After pre-treatment in 5% normal goat serum in PBS, the cryostat sections were incubated overnight in a humidified chamber at 4 ° C with the primary antibody, diluted in PBS-1% BSA. Binding sites of the primary antibodies were revealed with a fluorescein-isothiocya-nate-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa., USA). For staining of brush border in the proximal tubule, rabbit-anti-mXT2 [targeting the 18 amino acid peptide antigen NH 2 -MAQ ASG MDP LVD IED ERC-COOH; affinity purified, dilution 1: 200] was applied and fol-lowed by the secondary antibody Alexa Fluor 594 donkey anti-

Everolimus in Rat Polycystic Kidney Disease

Kidney Blood Press Res 2007;30:253–259 255

rabbit IgG (1: 1,000; Molecular Probes, Portland, Oreg., USA) [15] . For control of unspecific binding of secondary antibodies we made control incubations by omitting the primary antibody. These control experiments were negative. The sections were placed on coverslips, using DAKO-Glycergel (Dakopatts), to which 2.5% 1,4-diazabicyclo[2,2,2]-octane (DABCO; Sigma, St. Louis, Mo., USA) was added as fading retardant. Sections were studied by epifluorescence with a Polyvar microscope (Reichert Jung, Vienna, Austria). The fluorescence for Alexa Fluor 594 and fluorescein isothiocyanate were recorded separately using U1 and B2 filter modules, respectively. Overlays of the 3 channels were performed with the Image-Pro software. Images were acquired with a charge-coupled device camera (Visicam 1280, Visitron Systems, Puching, Germany) and processed by Image-Pro and Photoshop software.

Statistical Analyses Statistical analyses were performed by one-way ANOVA with

the Newman-Keul post hoc test using GraphPad Prism 4 soft-ware. All data are expressed as means 8 SEM and p ! 0.05 was considered as statistically significant.

0

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30

35

Ev

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*

–25%

Fig. 1. Whole-blood everolimus levels in male Cy/+ and +/+ rats. Whole-blood 6- and 24-hour trough levels of everolimus were obtained in all everolimus-treated rats. The everolimus 6- and 24-hour trough levels were 27.7 8 3.1 and 5.8 8 0.4 � g/l in +/+ rats, and 20.7 8 2.4 and 7.1 8 1.2 � g/l in Cy/+ rats. * p ! 0.05 Cy/+ at 6 h versus +/+ at 6 h.

0

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–39%

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B

Fig. 2. Everolimus retards the increase in kidney weight in Cy/+ rats. A Two-kidney weight (2KWt) of Cy/+ rats was reduced by 53% after everolimus treatment. B Two-kidney/total body weight (2K/TBW) of Cy/+ rats was reduced by 39% after everolimus treatment. * p ! 0.001 Cy/+ with vehicle versus Cy/+ with evero-limus group.

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)

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Fig. 3. Everolimus retards body weight gain in rats. After 5 weeks of everolimus treatment (day 36) the body weight gain of +/+ and Cy/+ rats was significantly retarded by 24 and 23%, respectively (ANOVA). * p ! 0.001 +/+ or Cy/+ with vehicle versus +/+ or Cy/+ with everolimus group.



Results

Blood Level of Everolimus The everolimus 6-hour and 24-hour trough whole-

blood levels were 27.7 8 3.1 and 5.8 8 0.4 � g/l in +/+ rats, and 20.7 8 2.4 and 7.1 8 1.2 � g/l in Cy/+ rats ( fig. 1 ). The lower everolimus blood levels at 6 h in Cy/+ rats (25%, p ! 0.05) compared with +/+ rats suggest a differ-ence in absorption and/or metabolism. The difference of everolimus trough levels between +/+ and Cy/+ rats was not significant.

Everolimus Treatment Retarded the Increase of Kidney Weight The once a day administration of everolimus by ga-

vage feeding for 5 weeks reduced 2-kidney weight in Cy/+ Han:SPRD rats by 53% (p ! 0.001; fig. 2 A). Figure 2 B shows that everolimus reduced the 2-kidney/total body weight ratio by 39% (p ! 0.001) in Cy/+ rats. Everolimus treatment had no effect on the 2-kidney/total body weight of +/+ rats. Everolimus treatment lowered body weight gain in the same magnitude in +/+ (n = 10) and Cy/+ (n = 8) rats. The mean weight difference was –24% for+/+ (p ! 0.001) and –23% for Cy/+ rats (p ! 0.001) on day 36 compared to vehicle-treated rats ( fig. 3 ). These results

Fig. 4. Everolimus decreases the renal cyst growth in Cy/+ rats. Representative paraffin-embedded kidney sec-tions of wild-type/vehicle (+/+; A ), heterozygous/vehicle (Cy/+; B ), heterozygous/everolimus rat (Cy/+; C ). D Cyst volume density (CVD) of Cy/+ rat was reduced by 48% after everolimus treatment. * p ! 0.001 Cy/+ with ve-hicle versus Cy/+ with everolimus group. Bar scale: 1 mm.

0

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)

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–48%

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Everolimus

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*



show that everolimus treatment inhibited body weight and kidney weight gain in +/+ and Cy/+ rats. However, the effect on Cy/+ kidney weight was more pronounced than on +/+ kidney weight.

Everolimus Inhibited the Increase of Renal Cyst Size and the Number of Proliferating Epithelial Cells Histological examination was performed on Han:

SPRD rat kidneys by periodic acid-Schiff staining. Figure 4 B shows that renal cysts predominantly exist in the cortex and to a lesser extent in the outer medulla. No cysts were found in the inner medulla in Cy/+ rats at 9 weeks of age. The everolimus treatment did not change the histology pattern in Cy/+ rats but it significantly reduced cyst den-sity by 48% (p ! 0.001) in Cy/+ rats ( fig. 4 C, D).

We then examined the effect of everolimus treatment on cyst epithelial cell proliferation by BrdU application in everolimus- or vehicle-treated Cy/+ rats (n = 3) on days 33, 34 and 35. Figure 5 A demonstrates that BrdU pre-dominantly labeled cyst epithelial cells in vehicle-treated Cy/+ rats. Everolimus strikingly reduced the number of BrdU-labeled TEC in the cortex ( fig. 5 B) and in the outer medulla (not shown) of Cy/+ rats.

Renal Functional Deterioration Was Stabilized in Everolimus-Treated Cy/+ Rats The reduction of cyst growth and kidney weight by

everolimus was accompanied by a significant ameliora-tion of renal functional loss on day 36. The BUN level was 31% (p ! 0.001) lower in Cy/+ rats compared with vehicle-

Fig. 5. Proliferating cells are reduced after everolimus treatment in Cy/+ rats. BrdU (green fluorescence) is incorporated in prolif-erating cells. BrdU-positive cells were evaluated in vehicle-treated Cy/+ rats ( A ) and everolimus-treated Cy/+ rats ( B ). The brush border of the proximal tubule is shown by a red fluorescence with an antibody against XT-2. More normal tubule structures and fewer BrdU-positive cells are visible in everolimus-treated Cy/+ rats compared to vehicle-treated Cy/+ rats. Bar scale: 100 � m. Fig. 6. Everolimus retards loss of renal function in Cy/+ rats. BUN as a parameter of renal function was monitored on days 0, 17 and 36. The steady increase of BUN levels was reduced by 31% in Cy/+ rats after 5 weeks of everolimus treatment. * * p ! 0.001 Cy/+ with vehicle versus Cy/+ with everolimus group. * p ! 0.05 +/+ with everolimus versus +/+ with vehicle group.

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treated Cy/+ rats ( fig. 6 ). Everolimus treatment did not completely prevent the renal functional deterioration in Cy/+ rats, as BUN levels in everolimus-treated Cy/+ rats were 28% (p ! 0.01) higher than in vehicle-treated +/+ rats. Everolimus treatment also resulted in a 20% (p ! 0.05) increase of BUN level in +/+ rats.

Discussion

There is current experimental evidence indicating that mTOR is out of control in inherited PKD. Aberrant signals through mTOR have been shown in different ro-dent models of PKD and in human ADPKD [4, 5] . Here we show that mTOR inhibition with everolimus efficient-ly retarded the development of renal cysts and amelio-rated kidney function in Han:SPRD rats. These results extend previous studies with the mTOR inhibitor rapa-mycin, which also slowed the progression of PKD in the same rat model. Both mTOR inhibitors efficiently retard-ed disease progression, suggesting that the observed ef-fects represent a class effect of mTOR inhibition.

Rapamycin and its analogue everolimus are both clin-ically used as immunosuppressants and approximately similar whole-blood drug levels are effective in prevent-ing allograft rejection in rats and humans [16, 17] . In our previous study, relatively low rapamycin whole-blood levels (from 0.5 to 1.9 � g/l) led to reduced BUN values after 1 month (by –19%) and prevented cyst growth (by –18%) after 3 months of treatment. Compared to that, moderately higher everolimus whole-blood trough levels (between 5 and 7 � g/l) reduced BUN and cyst growth ap-proximately 3-fold more than rapamycin, pointing to a profound dose effect of mTOR inhibition on cystic kid-ney disease progression. The bioavailability of oral rapa-mycin is low but increases markedly when given intra-peritoneally. In the study of Tao et al. [5] the administra-tion of rapamycin (0.2 mg/kg/day) intraperitoneally for 5 weeks in Han:SPRD rats reduced BUN (by –34%) and cyst volume density by (–40%). The rapamycin blood lev-els were not reported in their study, but the treatment was comparably effective in halting disease progression as everolimus treatment. Notably, Tao et al. also stated a sig-nificant reduction of body weight gain. In contrast, low trough level of rapamycin did not cause a significant body weight loss in our previous study.

Interestingly, treatment for 5 weeks with everolimus also caused a slight deterioration of renal function in nor-mal +/+ rats, as shown by increasing BUN levels. It has been proposed that rapamycin or everolimus can pro-

mote glomerular lesions and impair TEC regeneration in certain conditions [18–20] . Like rapamycin, everolimus can also impair renal function [21] . In agreement with our study, it has been shown by others that 1 week of everolimus treatment by gavage feeding with a similar dosage (3 mg/kg/day) impaired renal function, in normal rats on a low-salt diet [21] . Furthermore, higher-dose (3 mg/kg/day) as opposed to a lower-dose (0.3 mg/kg/day) everolimus treatment worsened glomerular injury in a rat model of glomerulonephritis, suggesting again that everolimus could impair renal functions dose-de-pendently [22] . In our previous study the lower trough level of rapamycin did not increase BUN levels in +/+ rats. In future clinic studies using everolimus, the dosage needs to be adjusted carefully to balance the beneficial and adverse effects of everolimus in human ADPKD pa-tients.

BrdU staining revealed that the number of proliferat-ing renal epithelial cells was reduced by everolimus treat-ment. The same observation was made by Tao et al. [5] by using proliferating cell nuclear antigen staining in Han:SPRD rats. The effect of mTOR inhibitors on TEC is also demonstrated by the finding that rapamycin severe-ly impairs the recovery from delayed graft function in patients who received a renal transplant and were treated

with rapamycin [19, 20] . Rapamycin decreases the prolif-eration of TEC, and a hallmark of PKD is aberrant pro-liferation of renal epithelial cells [23] . Therefore, the ma-jor effect of mTOR inhibition in PKD could be the inhibi-tion of renal tubular cell proliferation. However, we conducted this study in the early stages of PKD. In later stages of PKD, the expansion of growing renal cysts could occupy and squeeze the space for normal nephrons. Oth-er pathophysiological events such as interstitial fibrosis, hypoxia and angiogenesis could then emerge. Therefore, it would be interesting to test if mTOR inhibitors could exert their beneficial effect beyond antiproliferative mechanisms in the later stages of PKD.

We conclude that the observed inhibitory effect on cyst progression with everolimus is a class effect of mTOR inhibitors. Everolimus could be a promising new drug to treat ADPKD patients.

Acknowledgments

This study was supported by an unrestricted research grant provided by Novartis Pharma (Switzerland) AG, Novartis Foun-dation (Basel, Switzerland) and Kamillo Eisner Foundation (Her-giswil, Switzerland). We thank Dr. Schuler (Novartis, Basel, Swit-zerland) for providing everolimus and vehicle emulsion.



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JANA

copyright

Nephrol Dial Transplant (2007) 0: 1–10doi: 10.1093/ndt/gfm697Advance Access publication xx xx xxxx

Original Article

Sirolimus ameliorates the enhanced expression of metalloproteinasesin a rat model of autosomal dominant polycystic kidney disease

Celine C. Berthier1,∗, Patricia R. Wahl1,∗, Michel Le Hir2, Hans-Peter Marti3, Ulrich Wagner4,Hubert Rehrauer4, Rudolf P. Wuthrich5 and Andreas L. Serra5

1Institute of Physiology and Center for Integrative Human Physiology, University of Zurich, Zurich, 2Institute of Anatomy,University of Zurich, Zurich, 3Clinic and Policlinic for Nephrology, Inselspital, Bern, 4Functional Genomics Center, University ofZurich, Zurich and 5Clinic for Nephrology, University Hospital, Zurich

AbstractBackground. Remodelling of matrix and tubular basementmembranes (TBM) is a characteristic of polycystic kid-ney disease. We hypothesized that matrix and TBM degra-dation by metalloproteinases (MMPs) could promote cystformation. We therefore investigated the renal expressionof MMPs in the Han:SPRD rat model of autosomal domi-nant polycystic kidney disease (ADPKD) and examined theeffect of sirolimus treatment on MMPs.Methods. 5-week-old male heterozygous (Cy/+) andwild-type normal (+/+) rats were treated with sirolimus(2 mg/kg/day) through drinking water for 3 months.Results. The mRNA and protein levels of MMP-2 andMMP-14 were markedly increased in the kidneys ofheterozygous Cy/+ animals compared to wild-type +/+as shown by RT-PCR and Western blot analyses forMMP-2 and MMP-14, and by zymography for MMP-2.Strong MMP-2 expression was detected by immunoperoxi-dase staining in cystic epithelial cells that also displayedan altered, thickened TBM. Tissue inhibitor ofmetalloproteinases-2 (TIMP-2) expression was not changedin Cy/+ kidneys. Sirolimus treatment leads to decreasedprotein expression of MMP-2 and MMP-14 in Cy/+,whereas MMP-2 and MMP-14 mRNA levels and TIMP-2protein levels were not affected by sirolimus.Conclusion. In summary, in kidneys of the Han:SPRD ratmodel of ADPKD, there is a marked upregulation of MMP-2 and MMP-14. Sirolimus treatment was associated with amarked improvement of MMP-2 and MMP-14 overexpres-sion, and this correlated also with less matrix and TBMalterations and milder cystic disease.

Keywords: matrix metalloproteinases (MMPs);polycystic kidney disease (PKD); sirolimus; tissueinhibitors of metalloproteinases (TIMPs)

Correspondence and offprint requests to: Andreas L. Serra, MD, Clinicfor Nephrology, University Hospital Zurich, Ramistrasse100–8091 Zurich,Switzerland. Tel: +41442553384; Fax: +41442554593; E-mail: [email protected]∗Both authors contributed equally.

Introduction

Autosomal dominant polycystic kidney disease (ADPKD)is a chronic progressive kidney disease, which is charac-terized by cystic enlargement of both kidneys. Affectedindividuals usually present in the third to fourth decadeof life hypertension, haematuria, polyuria, flank pain, andprogress to end-stage renal disease within 5–10 years afterthe development of renal insufficiency [1]. The presence ofnumerous fluid-filled cysts is the predominant pathologicalfeature of PKD. In addition to cyst formation, reorganisa-tion of tubular basement membranes (TBM) and the devel-opment of fibrosis within the renal interstitium are char-acteristics of PKD, suggesting that the extracellular matrix(ECM) is intensely remodelled [2]. Tubular epithelial cellsplay an important role in the synthesis and degradation ofECM components, which in turn play a regulatory role inepithelial cell differentiation and growth [3,4].

Matrix metalloproteinases (MMPs) are a large family ofsecreted and membrane-bound zinc-dependent endopepti-dases, which can degrade a wide spectrum of matrix sub-strates, and therefore represent key enzymes in the turnoverof the extracellular matrix [5–7]. Most MMPs are releasedas zymogens before being activated in the extracellularenvironment. However, MMP-14, a membrane-boundmetalloproteinase, is processed before its insertion intospecific plasma membrane domains. In addition to itsmatrix-degrading properties, MMP-14 mediates the acti-vation of pro-MMP-2 together with the tissue inhibitor ofmetalloproteinases TIMP-2, which acts as a cofactor. Acti-vation of pro-MMP-2 from this ternary complex is finallyaccomplished by a second, non-TIMP-2-bound MMP-14molecule [7,8]. Thus, net activation of MMP-2 dependson local MMP-14 and TIMP-2 levels, following the gen-eral concept that the interaction of a cell with extracellularmatrix is critically determined by the concentrations ofmetalloproteinases and their natural inhibitors.

The purpose of the present investigations was to exam-ine alterations in the expression of MMPs and TIMPs ina rodent model of PKD, namely the Han:SPRD rat, and

C© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.For Permissions, please e-mail: [email protected]

NDT Advance Access published November 27, 2007

2 C. C. Berthier et al.

to correlate these molecules with changes in matrix andTBM structure. In a previous work, we and others havedemonstrated that sirolimus treatment of Han:SPRD ratsmarkedly reduced cyst growth and improved renal his-tology [9,10]. Here, we investigated the renal expressionof MMP-2, MMP-14 and TIMP-2 in the Han:SPRD ratmodel of PKD, and examined the effect of sirolimus on theexpression of these molecules.

Subjects and methods

Animals

The study was conducted in heterozygous (Cy/+) and nor-mal littermate control (+/+) Han:SPRD rats. Only malerats were used since the disease progresses faster in malecompared with female rats. A colony of Han:SPRD ratswas established in our animal care facility from breedingpairs that were obtained from the Rat Resource & ResearchCenter (Columbia, MO, USA). The regulatory commissionfor animal studies, a local government agency, approvedthe study protocol. The rats had free access to tap waterand a standard rat diet. Rapamycin (Rapamune R© oral so-lution) was kindly provided by Wyeth-AHP (Schweiz) AG,Switzerland.

Reagents

The following antibodies from Calbiochem were usedfor western blot analyses: anti-MMP-2 (468–483) (Ab-3)human (mouse), anti-MMP-14 (Ab-4) human (mouse),anti-TIMP-2 (Ab-2) mouse mAb (67–4H11) and as sec-ondary antibody the peroxidase goat anti-mouse IgG.Anti-MMP-2 (Ab-3) clone A-Gel VC2 from Neomarkers(Fremont, CA, USA) was used for immunohistochemistry.

Experimental protocol

Male Cy/+ (n = 6) and +/+ (n = 4) rats were weanedand were then treated at 5 weeks of age (n = 10) with2 mg/kg/day sirolimus, as described previously [9]. Addi-tional male Cy/+ (n = 4) and +/+ rats (n = 3) were nottreated and served as controls. The drug was administeredin the form of a Rapamune R© oral solution in the drink-ing water for 3 months. The concentration of sirolimus(Rapamune R© oral stock solution 1 mg/ml) was adjustedaccording to rat body weight. After 3 months of treatment,the rats were anesthetized with isoflurane, the kidneys wereremoved, decapsulated and snap frozen in liquid nitrogen.Samples were stored at −80◦C.

RNA extraction

Frozen kidney tissues were homogenized in lysis buffer(RNeasy; Qiagen), using Lysing Matrix D tubes and aBIO101 FastPrep R© Instrument (QBiogene, Basel, Switzer-land). The total RNA isolation (including DNase treatment)was performed from the homogenates according to the man-ufacturer’s instructions (RNeasy; Qiagen). All aliquots ofsamples were stored at −70◦C until use. RNA concentrationwas measured by using a spectrophotometer, and the quality

was analyzed by Bioanalyzer 2100 electropherograms(Agilent, Waldbronn, Germany).

Genechip expression analysis

Affymetrix GeneChip R© rat genome 230 2.0 arrayswere used according to the manufacturer’s instructions(Affymetrix Inc, Santa Clara, CA). Briefly, 5 µg of totalRNA from Cy/+ and +/+ rat kidneys was used as startingmaterial to generate biotin-labeled cRNA samples, whichincludes cDNA synthesis using oligo-dT/T7 primers,followed by in vitro transcription (one-cycle labelingprotocol). Labeled cRNA samples (15 µg) were randomlyfragmented to 35–200 bp and hybridized on arrays for 16 h,and arrays were washed. The fluorescent intensity emittedby the labeled target was measured by an AffymetrixGeneChip Scanner 3000. Finally, the hybridization imageswere analyzed using Affymetrix GCOSTM 1.2software.

Reverse transcription and real-time taqman PCR analysesfor MMP-2, MMP-14 and TIMP-2

RT and Taqman PCR analyses for MMP-2, MMP-14 andTIMP-2 were performed as described previously [11].Total RNA was reverse-transcribed and PCR was carriedout using TaqMan Universal PCR Master Mix (AppliedBiosystems; containing AmpliTaq Gold DNA Polymerase),Gene Expression Assay Mix and cDNA. Real-time PCRanalyses were performed with the ABI PRISM 7500 Se-quence Detection System (Applied Biosystems, Rotkreuz,Switzerland), according to the instructions of AppliedBiosystems. The expression levels of the 18S subunit ofribosomal RNA (18S rRNA) were used as a housekeepinggene. Relative quantification of all targets was calculatedby the comparative cycle threshold method outlined by themanufacturer (User Bulletin No. 2; Applied Biosystems).For MMP-2, MMP-14, TIMP-2 and 18S, assays-on-demandgene expression products were used as described in themanufacturer’s protocol (Applied Biosystems).

Protein extraction

A fresh ice-cold lysis buffer was prepared for proteinextraction from frozen rat kidneys. The lysis buffer con-sisted of 50 mM Tris base, pH 7.4; 150 mM NaCl; 1%deoxycholate; 1% SDS; and 1% Triton X-100. Immedi-ately before use, the lysis buffer was supplemented with 1mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, anda protease and phosphatase inhibitor cocktail. Each sam-ple was homogenized using a Dounce homogenizer. Allaliquots of samples were stored at −70◦C until use. Proteinconcentrations were determined using the BCATM ProteinAssay reagent kit (Pierce, Lausanne, Switzerland).

Zymography for MMP-2

Activity of the gelatinase MMP-2 was analyzed by gelatinsubstrate zymography. Briefly, 20 µg of each sample wasloaded on a 7.5% SDS-PAGE gel containing 0.1% gelatin.After migration, gels were first incubated for 1 h in a mix-ture containing 2.5% Triton X-100 and 50 mM Tris/HCl

Effect of sirolimus on MMPs and TIMPs in ADPKD 3

pH 8.0, then overnight in 50 mM Tris/HCl pH 8.0, 5 mMCaCl2, 5 µM ZnCl2. After incubation, gels were stainedwith 0.4% Coomassie blue and destained with 10% aceticacid and 50% methanol. The white bands obtained on thegels corresponding to MMP-2 activity were analyzed bydensitometry (Scion Image Corporation) after scanning.The Precision Plus ProteinTM Standard (Biorad, Reinach,Switzerland) was used as molecular weight marker, and arecombinant MMP-2 protein from OncogeneTM was usedas positive control.

Western blot analysis for MMP-2, MMP-14 and TIMP-2

Equal amounts of protein samples and a reduced load-ing buffer were incubated for 5 min at 95◦C. Sampleswere loaded on a 7.5% SDS-PAGE gel containing 40%acrylamid and 10% SDS. After migration, the proteins ofthe gels were transferred onto a supported nitrocellulosemembrane (Biorad). The blots were blocked with 5% milkfor 1 h at room temperature and were then incubatedovernight at 4◦C in a blocking buffer containing monoclonalrat anti-α tubulin, or monoclonal mouse anti-MMP-2, anti-MMP-14 or anti-TIMP-2 (dilutions 1:2000, 1:100, 1:25,1:50). Subsequently, the blots were washed in a PBS-Tweensolution and were incubated with a goat anti-mouse HRP-conjugated secondary antibody (dilution 1:1000). Finally,the membranes were incubated for 5 min in an enhancedchemiluminescent (ECL+) reagent (Amersham PharmaciaBiotech), followed by the exposure to Hyperfilm (SuperRXFujifilm). The molecular weight of the bands of interestwas determined by the Precision Plus ProteinTM Standardand by recombinant MMP-2 and TIMP-2 proteins fromOncogeneTM as positive controls.

Immunohistochemical detection of MMP-2

Rat kidney slices of about 2 mm thickness were cutimmediately after the excision. The slices were fixed byimmersion for 2 h at 4◦C in 4% freshly de-polymerizedparaformaldehyde in phosphate-buffered saline (PBS), de-hydrated through an alcohol series and embedded in paraf-fin. For immunohistochemistry, 3 µm thick sections werecut and rehydrated through an alcohol series. They weremicrowaved for 10 min in a 0.01M sodium citrate buffer,pH 6.0. After blocking with 5% normal goat serum, the sec-tions were incubated overnight at 4◦C with the primary an-tibody. A mouse monoclonal antibody against MMP-2 wasdiluted 1:500. After three washes in PBS, the binding sitesof the primary antibody were revealed with the VectastainABC kit (Vector Laboratories, Burlingame, CA, USA) us-ing 3,3’-diaminobenzidine tetrahydrochloride as substrate,according to the instructions of the supplier. The sectionswere then washed in water and were counterstained with theperiodic acid-Schiff (PAS) reagent. The nuclei were stainedwith haematoxylin.

Statistics

All data are expressed as means ± SD and were considerednormally distributed. Data were analyzed by applying at-test using the GraphPad Prism software version 4.0 forwindows (San Diego, CA, USA). The analysis of Genechip

MAS5 normalized expression values was performed usingGeneSpring 6.1.1 (Agilent, CA, USA). A t-test was car-ried out between the group of samples belonging to therespective conditions, and p-values <0.05 were consideredsignificant.

Results

Affymetrix gene chip analysis

Microarray analysis revealed an upregulation of several in-teresting genes in the family of metalloproteinases in Cy/+kidneys, compared to wild-type +/+ kidneys. In particular,we found a significant upregulation of MMP-2 (3.9-fold,P < 0.05) and MMP-14 (3.3-fold P < 0.05) in the mi-croarrays (Figure 1A). Sirolimus treatment did not blockthis upregulation (data not shown). Real-time Taqman PCRanalysis of RNA from Cy/+ and control +/+ rat kidneyswas performed to determine steady-state mRNA changesfor MMP-2, MMP-14 and TIMP-2. Figure 1B shows a sig-nificant increase of MMP-2 mRNA (2.2-fold, P < 0.05),of MMP-14 mRNA (2.4-fold, P < 0.05) and an unchangedTIMP-2 mRNA level in Cy/+ rats compared with +/+rats, thereby validating the expression data obtained frommicroarray experiments.

Enhanced MMP-2 expression and activity in Cy/+ kidneysis reduced in sirolimus-treated rat kidneys

The expression of MMP-2 protein was then analyzed bywestern blot (Figure 2A), and quantified by densitome-try (Figure 2B). Both the pro-form (72 kD) and the activeform of MMP-2 (66 kD) were detected in wild-type rats, andthese forms were not altered by treatment with sirolimus.There was a significant increase in the expression levelsof the pro-form and the active form of MMP-2 in un-treated heterozygous (Cy/+) vs untreated wild-type (+/+)rats (5.08- and 3.17-fold increase, respectively; P < 0.05).Treatment with sirolimus effectively decreased the expres-sion of MMP-2 pro-form and active form by 3.08-fold and2.57-fold (P < 0.05). In Cy/+ rats, respectively, comparedto untreated Cy/+ rats (Figure 2B).

Analysis of MMP-2 activity with zymography was thenperformed to correlate the level of MMP-2 expressionwith its activity (Figure 3). A weak activity for the 72 kDpro-form of MMP-2 was detectable in +/+ rats; however,a significantly stronger activity was measured in Cy/+ ani-mals (6.65-fold increase of the pro-form activity, P < 0.01).Activity for the 66 kD active form could not be detectedin +/+ kidneys but was detectable in Cy/+ rats. Treatmentwith sirolimus was associated with a significant decrease inthe pro-MMP-2 activity (1.55-fold decrease; P < 0.01), andthe activity of the 66 kD active form was again undetectable.

Immunohistochemical staining in Cy/+ kidneys showspredominant tubular upregulation of MMP-2

In +/+ rats, immunohistochemistry revealed a moderateto strong expression of MMP-2 in interstitial cells, in theendothelia of peritubular capillaries and in the epithelium of


Fig. 1. (A) Microarray results of increased levels of MMP-2 and MMP-14 gene expression in Cy/+ heterozygous rats compared with +/+ wild-typerats. TIMP-2 mRNA levels were not modified. (B) Real-time TaqMan PCR analysis of MMP-2, MMP-14 and TIMP-2 gene expression in the same setof rats confirmed the original microarray data. The results are expressed as mean ± SD. ∗P < 0.05 compared to the +/+ group, n = 4 to 6 per group.

the capsule of Bowman (Figure 4A). Most of the convolutedpart of the proximal tubule showed a weak immunolabeling.MMP-2 was not detected in the late convoluted part, thestraight part of the proximal tubule, the whole distal tubuleand the collecting duct.

In histologically normal tubular profiles in Cy/+ rats,the expression of MMP-2 was similar to +/+ rats (Figure4B). In dilated tubules, there was a striking heterogeneity ofimmunolabeling (Figure 4C). The expression was similar to+/+ rats in epithelial cells that looked morphologically nor-mal. In contrast, MMP-2 staining in Cy/+ was moderate tovery strong where the epithelium displayed an abnormallydeveloped tubular basement membrane, as revealed byPAS staining (Figure 4D). The association between a highexpression of MMP-2 and a modified basement membrane

was found in the proximal tubule, in the capsule of Bowmanand in the distal tubule. MMP-2 was expressed in all largecysts, mostly at a high level (Figure 4E).

Cysts in Cy/+ sirolimus-treated rats displayed similarlevels of MMP-2 as cysts of placebo-treated rats. Ex-pression of MMP-2 was also focally observed in tubules,which displayed thickening of the basement membrane(Figure 4F).

Effect of sirolimus treatment on MMP-14 and TIMP-2expression in Cy/+ kidneys

In addition to its matrix-degrading properties, MMP-14mediates the activation of pro-MMP-2 together with the tis-sue inhibitor of metalloproteinases TIMP-2, which acts as a


Fig. 2. MMP-2 expression. (A) Western blot and (B) densitometry confirmed the upregulation of the pro- and active forms of MMP-2 in Cy/+ comparedto +/+. Treatment with sirolimus decreased these levels near to those of the wild-type. Two representative samples for each group are depicted. ∗P <

0.05 compared with the untreated or treated wild-type (+/+) rats, #P < 0.05 compared with the untreated heterozygous (Cy/+) rats, n = 4 per group.

cofactor. Since MMP-14 and TIMP-2 play decisive roles inMMP-2 activation, we hypothesized that levels of MMP-14and TIMP-2 would correlate with the increase of MMP-2in the Cy/+ kidneys of ADPKD rats. The expression ofMMP-14 was investigated by western blot analysis. Figure5 shows that MMP-14 was detected in two different forms,as reflected by the bands at 63 and 58 kD, correspondingto the MMP-14 proform and the active form, respectively.MMP-14 protein expression was stronger in Cy/+ com-pared to +/+ rat kidneys (Figure 5A). Densitometry (Fig-ure 5B) revealed a 4.55-fold and 6.35-fold increase in bothforms of MMP-14 in Cy/+ compared to +/+ rat kidneys(P < 0.05). Sirolimus was again associated with reducedMMP-14 protein levels (4.93-fold and 5.73-fold, respec-tively; P < 0.05) down to levels observed in +/+ controlrats.

TIMP-2 is involved in MMP-2 activation in conjunctionwith MMP-14, but can also inhibit activation of MMP-2[12]. Three different forms of TIMP-2 were found by west-ern blot analysis (Figure 6A). The active form of TIMP-2

at 27 kD and the bimolecular complex of TIMP-2/MMP-14at 70 kD were not significantly higher in Cy/+ rat kidneyscompared with +/+, and were not affected by treatmentwith sirolimus (Figure 6B). However, the dimeric complexof TIMP-2 at 54 kD increased approximately 1.59-fold(P < 0.05) when compared with the untreated +/+ rats,and tended to be reduced (1.45-fold; non significant) insirolimus-treated rats. Interestingly, of all three forms, theTIMP-2/MMP-14 complex was the least expressed; theTIMP-2 active form had the highest overall expression levelof the three forms.

Discussion

Others and we have shown that treatment with the mTORinhibitors sirolimus and everolimus delays the loss of renalfunction and retards cyst development in Han-SPRD rats[9,13–15]. Here, we show that treatment of Han:SPRD ratswith sirolimus was associated with a marked improvement


Fig. 3. (A) Zymography showed increased activities of pro- and active forms of MMP-2 (72 and 66 kDa respectively) in untreated heterozygous(Cy/+) rats compared with untreated wild-type (+/+) rats. MMP-2 activities were not modified by sirolimus treatment in +/+, but were decreasedin Cy/+ as determined by (B) densitometry. Ctrl = purified MMP-2 antigen (Calbiochem, Switzerland) as a reference for MMP-2 identification. Tworepresentative samples for each group are depicted. ∗∗P < 0.01 compared with the untreated or treated wild-type (+/+) rats, #P < 0.05 compared withthe untreated heterozygous (Cy/+) group, n = 4 per group.

of MMP-2 and MMP-14 overexpression, and this correlatedalso with less matrix and TBM alterations and milder cysticdisease.

By gene array analysis, we identified MMP-2 and MMP-14 as the two major MMPs, which are markedly enhancedin Han-SPRD rat kidneys with PKD. RT-PCR analysis,and the examination of MMP-2 and MMP-14 protein bywestern blot and zymography confirmed the upregulation.In agreement with our findings, the abnormalities in the ex-pression of MMPs have been described for human [16,17]and animal models of PKD [18–20]. In humans, increasedlevels of MMP-2 and -9 have been preferentially localizedin tubular epithelia forming renal cysts. In animal models,it has been demonstrated that cystic kidney tubules cansynthesize and secrete high levels of MMPs, in particularMMP-2 and MMP-14 [19,21,22]. In contrast to our find-ings, Schaefer et al. found a decrease of MMP-2 expressionand activity in Cy/+ rats in early stages of PKD. However,

this may be explained by several factors. In our experiments,we used whole kidney homogenate instead of isolatedproximal tubules because MMP-2 has been localized to theinterstitium and to the epithelial cells of all cyst-formingnephron segments [18]. Also, our rats were at a latter stageof disease progression. The expression of genes involved infibrosis and inflammation increase with age in Cy/+ ratsand further explain our finding of increased MMP-2 ex-pression [20]. MMP-2 expression was prominent in tubularepithelial cells lining cysts and with an altered basementmembrane. Those two features remained associated at thedifferent stages of cyst growth, indicating that cystogen-esis is associated with tubular induction of MMP-2. Theparallel increase of cyst growth and expression of genesinvolved in inflammation and fibrosis suggests that MMPsmay regulate collagen accumulation at sites where cystictransformation occurs, linking the progression of cystformation with ECM remodelling in PKD.


Fig. 4. Immunohistochemistry for MMP-2 in kidney sections. In wild-type (+/+), the segments S1 and S2 of the proximal (P) tubule display a weakimmunoreactivity; the segment S3 and the distal (D) tubule are negative (A). Patches of cells that produce excess amounts of basement membrane(arrows in B and C) represent the earliest histological alteration in (B) proximal and (C) distal tubules in Cy/+ kidneys. The cells in such patchesup-regulate MMP-2. Note the sharp transition in (D) between the normal and the altered epithelium with its thickened basement membrane and its highimmunoreactivity for MMP-2. (E) MMP-2 remains abundant in the large cysts with their thin epithelia. In Cy/+ sirolimus-treated rats, MMP-2 tubularexpression is detected in cysts and focally in tubules, which displayed thickening of the basement membrane (F).

Remodelling ECM has been hypothesized as a necessarystep for cyst expansion [21]. In a study which also used theHan:SPRD rat, treatment with the global MMP inhibitorbatimastat reduced cyst number and kidney weight [18].Treatment of cystic mice with WAR-1 and WTACE2, MMPinhibitors that share some degree of activity against MMP-9 and MMP-13 slowed disease progression [23], pointingagain to an important role of MMPs in cyst formationin PKD. Interestingly, transgenic expression of MMP-2 inproximal tubular epithelial leads to structural alterations inthe tubular basement membrane, fibrosis and renal failure[24].

Sirolimus is an immunosuppressant with potent antipro-liferative effects on non-lymphoid cells. We and others havepreviously shown that sirolimus effectively retards renal

cyst development in the Han:SPRD rat [9,10]. Shilling-ford et al. have confirmed the potent anticystic effects ofsirolimus in additional animal models of PKD [13]. Inhibi-tion of the mTOR signalling pathway with sirolimus mostlikely inhibits cell proliferation in PKD via control of ribo-somal translation. However, this may not be the sole mode ofaction. The reduced MMP-2 and MMP-14 protein expres-sion, which occurred in response to the sirolimus treatment,was not associated with reduced mRNA levels. Sirolimuscontrols gene expression at the translational level via in-hibition of downstream protein kinases that are requiredfor translation of mRNAs [25]. Effectors of the mTORpathway are upregulated in the kidneys of Cy/+ rats, andprotein expression of MMP-2 and MMP-14 is transducedat least in part by these effectors of the mTOR pathway


Fig. 5. MMP-14 protein expression. (A) Western blot analysis and (B) densitometry showed an increase of pro- and active forms of MMP-14 protein(63 and 58 kD respectively) in the kidneys of untreated heterozygous (Cy/+) rats compared with untreated wild-type (+/+) rats. Sirolimus treatmentdid not affect both MMP-14 forms in +/+ rats. MMP-14 protein levels decreased in kidneys of Cy/+ rats treated with sirolimus. Two representativesamples for each group are depicted. ∗P < 0.05 compared with the untreated wild-type (+/+), #P < 0.05 compared with the untreated heterozygous(Cy/+) group, n = 4 per group.

in vitro [2,26]. Thus, our results indicate that MMP-2/-14protein expression in response to sirolimus treatment ismainly due to translational regulation, because mRNA lev-els of neither protein show a decrease with sirolimus treat-ment. It is unlikely that the reduction of MMP-2 and MMP-14 in sirolimus-treated rats is mainly due to TIMP inhibitionbecause the active form of TIMP-2 remained unchanged.Increased TIMP-2 expression has been shown in Cy/+ ratsby in situ hybridization, but when TIMP-2 RNA expressionwas analysed in total kidney by an RNase protection assay,no pronounced effect was seen and the favourable effectof batimastat on cyst growth was different from TIMP-2expression [18]. In line with this result is our finding thatsirolimus treatment did not change TIMP-2 expression. In8-week-old Cy/+ rats, an increased expression of TIMP-2was found in proximal tubules [22]. We cannot excludethat in the early disease stage, locally expressed TIMP-2 is

associated with cyst development. Although in our studywe cannot prove a direct effect of sirolimus on MMP-2 andMMP-14 expression, it is plausible that part of the benefi-cial effects of sirolimus might be caused by inhibition ofMMPs.

Renal fibrosis is a hallmark of cystic disorder in hu-man ADPKD [27] and in Han:SPRD rats [28]. Fibrosisincreases with the progression of disease, indicating a linkbetween cystogenesis and fibrogenesis. It is therefore dif-ficult to deduce the effect of sirolimus on reduced cystgrowth from improved MMP activities, and it can eitherbe a consequence of the specific action of sirolimus or thereflection of the overall improvement of the severity of thedisease. A number of recent studies show that sirolimushas prominent therapeutic effects on various renal dis-eases where matrix production and fibrosis are enhanced.In a model for experimental membranous nephropathy, a


Fig. 6. TIMP-2 protein expression. (A) Western blot analyses and (B) densitometry revealed three forms of TIMP-2: the active form (TIMP-2 freeform), the dimer TIMP-2 and the complex TIMP-2/MMP-14 are depicted by three bands of 27 kD, 54 kD and 70 kD, respectively. Whereas the twocomplexes appeared to be strongly increased in heterozygous (Cy/+) kidneys, the free form was not differentially expressed compared to the wild-type(+/+). Sirolimus treatment decreased the TIMP-2 complexed forms in +/+ and Cy/+ rats, but did not affect the levels of TIMP-2 active form. Tworepresentative samples for each group are depicted. ∗P < 0.05 compared with the untreated wild-type (+/+) rats, n = 4 per group.

relatively low dose of sirolimus resulted in reducedinterstitial inflammation and fibrosis [29]. Treatmentwith sirolimus blunted compensatory renal hypertrophy;a similar effect has been observed in the rat modelfor hydronephrosis [30]. Sirolimus also blocked tubularepithelial-to-mesenchymal transition, a critical step in fi-brogenesis. These results support the hypothesis that thebeneficial effect of sirolimus is not only mediated by itsantiproliferative effect but by multiple mechanisms.

In conclusion, MMP-2, MMP-14 and TIMP-2 are dys-regulated in the Han:SPRD rat model of ADPKD. The ac-tivation of MMP-2 and MMP-14 is markedly suppressed invivo by the treatment of Han:SPRD rats with sirolimus, andthis is correlated with less matrix and TBM alterations andmilder cystic disease.

Acknowledgements. This project was funded in part by a research grantfrom the Swiss National Science Foundation to H.P.M. (NFP-46), and byfunds provided by the University of Zurich. We are particularly grateful toMarzanna Kunzli of the Functional Genomics Center of Zurich (FGCZ)for her technical support.

Conflict of interest statement. None declared.

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Received for publication: 1.5.07Accepted in revised form: 7.9.07

BioMed CentralBMC Nephrology

ss
Open AcceStudy protocolClinical proof-of-concept trial to assess the therapeutic effect of sirolimus in patients with autosomal dominant polycystic kidney disease: SUISSE ADPKD studyAndreas L Serra*1, Andreas D Kistler1, Diane Poster1, Marian Struker1, Rudolf P Wüthrich1, Dominik Weishaupt2 and Frank Tschirch2
Address: 1Clinic for Nephrology, University Hospital, CH-8091 Zürich, Switzerland and 2Institute of Diagnostic Radiology, University Hospital, CH-8091 Zürich, Switzerland

Email: Andreas L Serra* - [email protected]; Andreas D Kistler - [email protected]; Diane Poster - [email protected]; Marian Struker - [email protected]; Rudolf P Wüthrich - [email protected]; Dominik Weishaupt - [email protected]; Frank Tschirch - [email protected]

* Corresponding author

AbstractBackground: Currently there is no effective treatment available to retard cyst growth and toprevent the progression to end-stage renal failure in patients with autosomal dominant polycystickidney disease (ADPKD). Evidence has recently been obtained from animal experiments thatactivation of the mammalian target of rapamycin (mTOR) signaling pathway plays a crucial role incyst growth and renal volume expansion, and that the inhibition of mTOR with rapamycin(sirolimus) markedly slows cyst development and renal functional deterioration. Based on thesepromising results in animals we have designed and initiated the first randomized controlled trial(RCT) to examine the effectiveness, safety and tolerability of sirolimus to retard diseaseprogression in ADPKD.

Method/design: This single center, randomised controlled, open label trial assesses thetherapeutic effect, safety and tolerability of the mTOR inhibitor sirolimus (Rapamune®) in patientswith autosomal dominant polycystic kidney disease and preserved renal function. The primaryoutcome will be the inhibition of kidney volume growth measured by magnetic resonance imaging(MRI) volumetry. Secondary outcome parameters will be preservation of renal function, safety andtolerability of sirolimus.

Discussion: The results from this proof-of-concept RCT will for the first time show whethertreatment with sirolimus effectively retards cyst growth in patients with ADPKD.

Trial registration: NCT00346918

BackgroundAutosomal dominant polycystic kidney disease (ADPKD)is the most common hereditary cause of end-stage renaldisease (ESRD), affecting all ethnic groups worldwide,

with an incidence of 1 in 500 to 1 in 1000 [1]. ADPKD ischaracterized by the progressive development of innumer-able cysts in both kidneys, which distort the normal kid-ney architecture and leads to a loss of renal function. The

Published: 15 September 2007

BMC Nephrology 2007, 8:13 doi:10.1186/1471-2369-8-13

Received: 30 June 2007Accepted: 15 September 2007

This article is available from: http://www.biomedcentral.com/1471-2369/8/13

© 2007 Serra et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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development of renal failure is highly variable, but typi-cally patients develop ESRD by the age of 40 to 50 years,necessitating renal replacement therapy (RRT) and/or kid-ney transplantation [2]. Apart from blood pressure con-trol and symptomatic treatment of cyst bleedings andinfections there is no curative therapy for this disease [3].PKD1 and PKD2 encode the proteins polycystin-1 andpolycystin-2 which are expressed in the kidney and func-tion together to regulate growth and morphologic config-uration of renal epithelial cells [4]. Mutation in PKD1leads to a more severe phenotype of ADPKD than muta-tions in PKD2, with ESRD occurring on average 20 yearsearlier (53.4 versus 72.7 years) [5].

In ADPKD progressive cyst growth generally precedes thedevelopment of renal insufficiency. Compensatory mech-anisms (hyperfiltration) maintain renal function virtuallynormal for decades despite continuous cyst growth. By thetime renal function starts to decline, the kidneys are usu-ally grossly enlarged with little normal renal parenchymarecognisable on imaging studies. Data from the consor-tium for radiologic imaging studies of polycystic kidneydisease (CRISP) and others have shown that the rate ofkidney volume growth is a predictor of renal functionaldecline and therefore kidney volume is used as surrogatemarker of disease progression especially in clinical inter-vention trials for ADPKD [6,7]

Non-invasive radiologic methods are available to monitorthe growth rate of kidney volume. Renal ultrasound meas-urements are operator-dependent and not precisely repro-ducible. Unenhanced and contrast enhanced Computertomography (CT) scanning is reported to be an accuratemethod to determine kidney volume, but it involves ion-izing radiation and potentially nephrotoxic contrastmedium and is therefore not an ideal method in patientswith reduced kidney function needing repetitive measure-ments [8,9]. Due to its high soft tissue contrast and thelack of ionizing irradiation Magnetic resonance imaging(MRI) is also considered useful to monitor kidney volumechanges in ADPKD. The analysis of sequential MRI scanswas shown to be accurate to monitor rates of kidney vol-ume enlargement in ADPKD [7].

Sirolimus is an immunosuppressant that binds to FKBinding Protein-12 (FKBP-12) and inhibits the activationof the mTOR, a key regulatory kinase of growth and pro-liferation. Sirolimus is approved for the prevention ofgraft rejection following renal transplantation. Due to itsantiproliferative properties it is also used in coated stentsto prevent coronary artery restenosis after angioplasty[10]. Furthermore it has shown clinical effectiveness inkidney transplant recipients with Kaposi's sarcoma [11].

We have shown previously that the mTOR inhibitorsrapamycin and everolimus effectively reduce cyst growthand loss of renal function in an experimental animalmodel for PKD [12,13]. Additional studies have shownthat rapamycin is also effective in various mouse modelsof polycystic kidney disease, including dominant andrecessive forms [14]. Of interest, an analysis of ADPKDpatients which received a renal transplant, revealed thatcystic kidney volumes regressed under immunosuppres-sion with sirolimus [15]. Based on these promising resultswe have designed and initiated the first clinical trial toexamine the effectiveness and safety of sirolimus in youngpatients with early manifestations of ADPKD and intactrenal function.

Methods/DesignStudy aimThe primary objective of the SUISSE ADPKD study is toassess the effectiveness of sirolimus to retard kidney vol-ume growth and to prevent the loss of renal function inyoung patients with ADPKD and preserved renal function.Patients with ADPKD and kidney volume growth that canbe documented within 6 months are randomized to treat-ment with sirolimus 2 mg/day for 18 months (Figure 1) orstandard treatment. The secondary objectives are to followrenal function and blood pressure and to monitor for theoccurrence of proteinuria. Safety and tolerability ofsirolimus treatment in ADPKD patients will also beassessed.

Study design and settingThe study is a single center randomised controlled openlabel trial which is open to ADPKD patients with a posi-tive family history of ESRD due to ADPKD thereby select-ing mostly patients with mutations in the PKD1 gene. Thestudy will involve 100 ADPKD-patients aged 18–40 yearswith a creatinine clearance >70 ml/min. Kidney volumeswill be measured by MRI without contrast media at studymonth 0 and 6. Patients with documented volume pro-gression are randomized at a 1:1 ratio to sirolimus 2 mg/day or standard treatment for 18 months. Recruitment hasstarted in May 2006 and will last until December 2007.The study will be completed by December 2009.

Ethical considerationsEthical approval has been obtained form the local ethicscommittee of the University Hospital Zürich.

Study drug and dosingRapamune® (sirolimus) will be used at a fixed dose of 2mg daily to achieve sirolimus through levels between 4–10 µg/L. By using this rather low dose of sirolimus asmonotherapy we expect a low incidence of sirolimus-related adverse events. The dose will be reduced or with-held in case of a through level exceeding 10 µg/l, elevated



liver enzymes (> 2-fold above normal values), thrombo-penia (< 100'000/mm3), leukopenia (< 3'000/mm3) orserious sirolimus-associated toxicity.

Study drug adherenceAdherence to the prescribed study drug will be assessed byusing the Medication Event Monitoring V Track-Cap Sys-tem (MEMS®, Aardex, Ltd., Zug, Switzerland) during thecomplete treatment period. The MEMS® assesses the med-ication adherence reliably and sensitive as a period with alack of medication bottle opening documentation, repre-senting most likely an episode of non-adherence [16,17].The system monitors electronically the date and time ofthe medication bottle opening. We will measure 1) treat-ment adherence as the proportion of medication vial capsopened in a given month relative to the prescribed dosesfor that month, 2) dosing adherence as the percentage ofdays with correct dosing and 3) drug holidays, as thenumber of periods without drug intake that exceeded 48hours.

Identification of eligible patientsStudy participation is offered to all eligible patients withADPKD. ADPKD patients suffering from advanced renalfailure including dialysis patients and transplant recipi-ents treated at our clinic are informed about the study andscreening for ADPKD is offered to their relatives. All neph-rology clinics and dialysis units in Switzerland have beeninformed and information material (print and in theinternet [18]) for study participants and health profes-sionals has been provided. Potential study participants areinvited for a screening visit including medical history,physical examination, renal ultrasonography and bloodand urine analyse.

Male or female ADPKD patients aged 18 to 40 years witha creatinine clearance ≥ 70 ml/min are eligible for thestudy if they exhibit a kidney volume progression over theobservational pre-randomisation period of 6 months. Thediagnosis of ADPKD is based on ultrasonographic diag-nostic criteria in patients with a family history of poly-cystic kidney disease [19]. In patients with negative familyhistory, proof of a mutation in the PKD1 or PKD 2 genesis required for inclusion (sequencing analysis; Athena

Flow chart of SUISSE ADPKD studyFigure 1Flow chart of SUISSE ADPKD study.



Diagnostics, Inc., Worcester, MA, USA). Detailed studyinclusion and exclusion criteria are given in table 1 and 2.

Randomization and study blindingPatients are randomized at a one to one ratio to sirolimusor standard treatment alone. The randomisation list hasbeen generated by a biostatistics unit which is independ-ent of the study team using a permuted blocks design witha random block size of 4 and 6 to guarantee a balancedallocation. The randomisation codes are kept in sealedsequentially numbered opaque envelopes and are notopened until two MR scans within 6 months have shownan enlargement of the total kidney volume of ≥ 2 %.

Primary outcomeThe primary objective of the SUISSE ADPKD study is todetermine the effect of sirolimus treatment on kidney vol-ume enlargement in ADPKD patients with preserved renalfunction. Patients with documented kidney volumegrowth in the last six months will be randomized tosirolimus or standard treatment. Kidney volume will bemeasured 6 and 18 months after randomization and thepercent annual growth of the combined (left and right)kidney volume will be calculated.

Secondary outcomeAbsolute kidney volume growth from inclusion to month18 will be assessed as a secondary outcome. Other second-ary objectives are to compare blood pressure, renal func-tion and proteinuria in patients with sirolimus orstandard treatment and to assess safety and tolerability ofsirolimus treatment. New onset or progression of arterialhypertension might reflect disease progression of ADPKDor a potential adverse drug effect and will be assessedcomparing the change of blood pressure and the changeof antihypertensive drug dosage during follow up. Renalfunction will be assessed by using estimation equations(Cockcroft-Gault and cystatin C) as well as measured cre-atinine clearance determined by 24-hour urine collection.Proteinuria will also be measured by 24-hour urine collec-tion. The frequency and severity of all reported adverseevents will be recorded, including laboratory abnormali-

ties such as anemia, thrombocytopenia and hyperlipi-demia.

Magnetic resonance imagingAll individuals undergo MR imaging of the kidneys usinga 1.5 Tesla scanner. For signal reception in all examina-tions an 8-channel anteroposterior phased-array surfacecoil (torso array coil) is placed around the patient andcovers the entire kidneys. The imaging protocol includesunenhanced sequences only. In order to get an overviewof the extent of the cystic disease of the kidneys a coronalsingle shot fast spin echo (SSFSE) sequence is acquired inbreath hold technique. The MR imaging parameters ofthis sequence are as follows: repetition time (TR) msec/echo time (TE) msec 1349/90.1; field of view 48 × 48 cm;acquisition matrix 384 × 224; section thickness 4 mm; nointerslice gap. Based on this coronal sequence the transax-ial sequences are planned. The transaxial sequences con-sists of two breath hold T1-weighted fast spoiled gradientecho (FSPGR) sequences (TR msec/TE msec = 85/1.4)with two different slice thicknesses (3 and 4 mm, respec-tively). Other parameters of the T1-weighted gradient-echo pulse (GRE) sequences are: field of view 48 × 48 cm,matrix 256 × 160; no interslice gap. In addition a transax-ial T2-weighted fast spin echo (FSE) sequence with respi-ratory triggering is performed (TR msec/TE msec = 17143/102.8; field of view 48 × 48 cm, matrix 256 × 160, thick-ness 3 mm; no interslice gap). To measure the kidney vol-umes the transaxial breath hold T1-weighted FSPGRsequence with a slice thickness of 3 mm is primarily used.In case of a more advanced disease with large polycystickidneys and/or when respiratory artefacts are present thevolume measurements are either performed on thetransaxial breath hold T1-weighted FSPGR sequence witha slice thickness of 4 mm or on the transaxial T2-weightedFSE sequence. Once one of these sequences is chosen the

Table 2: Study exclusion criteria

• Female patient of childbearing potential who is unwilling to use effective means of contraception• Increased liver enzymes (2-fold above normal values)• Hypercholesterolemia (fasting cholesterol > 8 mmol/l) or hypertriglyceridaemia (> 5 mmol/l) not controlled by lipid lowering therapy• Granulocytopenia (white blood cell < 3,000/mm3) or thrombocytopenia (platelets < 100,000/mm3)• Infection with hepatitis B or C, HIV• History of malignancy• Mental illness that interferes with the patient ability to comply with the protocol• Drug or alcohol abuse within one year of baseline• Co-medication with strong inhibitor of CYP3A4 and or P-gp like voriconazole, ketoconazole, diltiazem, verapamil, erythromycin or with a strong CYP3A4 and or P-gp inductor like rifampicin• Known hypersensitivity to macrolides or Rapamune®

• Patients who are unwilling or unable to give informed consent

Table 1: Study inclusion criteria

• Age 18 to 40 years• GFR ≥ 70 ml/min (Cockcroft - Gault formula)• Diagnosis of ADPKD:

❍ Positive family history for ADPKD■ patients < 30 years: ≥ 2 cysts in either kidney■ patients ≥ 30 years: ≥ 2 cysts in each kidney

❍ Negative family history for ADPKD but sonographically cystic kidney disease: proof of a mutation in the PKD1 or PKD2 gene is required (Athena Diagnostics, Inc., Worcester, MA, USA)

• Documented kidney volume enlargement (MRI volumetry)• Signed informed consent



same sequence is used for volume measurements in thefollowing MR imaging sessions.

Renal volume measurementsTwo independent trained observers perform a manualsegmentation of both kidneys for each patient. To preventbias, the observers are blinded to all clinical and radiolog-ical data, their first measurements and the results of theother observer. The measurements are performed in ran-dom order. Blinding is performed with regard to patientsand the different time points when imaging has beenobtained. Manual segmentation is performed electroni-cally on an interactive workstation (Advantage WindowsWorkstation; GE Medical Systems Europe, Buc, France).Each kidney is assessed separately. On each section, theoutlines of the kidney are manually drawn by using thecomputer mouse. The vessels and the ureter in the area ofthe renal hilum are excluded from manual volumetricmarking. The volume corresponding to each outline isobtained by multiplying the area of the outline by the sec-tion thickness. The total volume of the kidney segments isobtained by summing the volume of each section. Themanufacturer's software automatically calculates the totalvolume after drawing the outlines of the kidney on all sec-tions. Mean values of the measurements performed by thetwo independent observers will be used for analysis. Incase of a large disagreement, both observers will repeatmeasurement.

Data collectionData will be collected into a web-based data base designedto capture all visit information including medical history,results form laboratory analysis and adverse events. Base-line data and ongoing data collection as outlined in table3 will be obtained.

Patient follow-up proceduresStudy duration for all patients will be 24 months in total,consisting of a 6 months pre-randomisation observa-tional period and 18 months follow up after randomisa-tion (Figure 1). Four main study visits including kidneyMRI and 24 h urine collection will take place at baseline,month 6, 12 and 24. Randomisation and inclusion in thestudy takes place after evaluation of kidney volumegrowth from baseline to month 6 within 2 weeks after thesecond MRI. Three additional visits including clinicalassessment and blood and urine chemistry will take placeat month 9, 15 and 18. Patients in the treatment arm willhave four extra visits at week 2, 4, month 1 and 2 after ran-domisation to allow for blood level monitoring andpotential dose adjustment of the study medication.Patients without volume progression during the pre-ran-domisation period will be followed by MRI for additional6 months and enrolled if kidney volume enlargement isdetectable.

Study withdrawalPatients will be censored and withdrawn from follow-upat their request. Patients will also be censored if they arenot randomized after the pre-randomization observationperiod.

Statistical analysisPrincipal analysis will be undertaken using an intention-to-treat approach. A secondary on-treatment analysis willalso be performed. The annual percent change in kidneyvolume will be determined by regressing the log trans-formed total kidney volume at month 6, 12 and month 24against time for each patient through the least squaresmethod. Mean annual decline of renal function will becalculated by regressing GFR or creatinine clearanceagainst time. All primary and secondary end point varia-bles will be compared using a two-sided α-level of 0.05.To account for possible baseline imbalances, a secondaryanalysis will be performed in which comparison of treat-ment groups for all endpoints will be adjusted for prede-fined covariates using multiple regressions. Thepredefined covariates are age, sex, presence of hyperten-sion, medication with angiotensin converting enzymeinhibitors or angiotensin receptor blockers, baseline totalkidney volume and percentage kidney growth during thepre-randomisation period.

Table 3: Baseline and ongoing data collection

Baseline data and follow-up data at month 6, 12 and 24

MRI kidney volumetryCreatinine clearance and estimated GFR (Cockcroft-Gault)Proteinuria (24-hour urine and spot urine)Physical examination and vital signsLaboratory tests

Haematology/BiochemistryLipid profileSirolimus trough level

Pregnancy test1

Serological testing for hepatitis B, C and human immunodeficiency virus (HIV)1

MEMS® checkAdverse events and concomitant therapy

Follow-up data every 3 months

Serum creatinineProteinuria (spot urine)Physical examination and vital signsLaboratory tests

Haematology/BiochemistryLipid profile

Sirolimus trough levelAdverse events and concomitant therapy

1Only at baseline and month 24



Sample size considerationsIn a large cohort of ADPKD patients, the mean annual kid-ney volume growth rate was 5.27% ± 3.92% (SD)[7].Because patients with lack of progression during the pre-randomisation period will be excluded from our study, weexpect to select for a higher progression rate in our studypopulation. Due to a shorter observation interval com-pared to the mentioned observational study, the standarddeviation might be higher. Presuming an annual kidneygrowth rate of 6% ± 4.75% (SD) in the control group, asample size of 40 patients per group will have 80% statis-tical power to detect a 50% relative reduction of kidneyvolume growth using a two-sided α-level of 0.05. Toaccount for a drop out rate of up to 20%, we plan to ran-domise a total of 100 patients.

DiscussionThe SUISSE ADPKD study seeks to determine if sirolimushalts kidney volume growth in patients with ADPKD earlyin the disease course. Study participation is restricted toyoung patients with preserved kidney function becauseany effective treatment of ADPKD needs to be started earlyin the course of the disease to have an impact on longterm kidney function. Our trial is the first clinical studyaddressing this question. If sirolimus treatment canreduce or stop volume growth in patients with main-tained kidney function and prior documented kidney vol-ume progression, these ADPKD patients could benefit themost from a treatment with an anti-proliferative agent likesirolimus. Similar studies using specific mTOR inhibitorsto halt disease progression have been announced by oth-ers (Mayo Clinic, USA; Certican® trial, Novartis, Ger-many). We anticipate that sirolimus might be an effectivetherapeutic option for ADPKD patients that are prone toprogress to end-stage renal disease.

Competing interestsThe author(s) declare that they have no competing inter-ests.

Authors' contributionsALS, ADK and RPW were responsible for identifying theresearch question and drafting the study protocol. Allauthors have contributed to the development of the pro-tocol and study design, as members of the study team. ALSand FT were responsible for drafting of this manuscriptand all authors provided comments and have read andapproved the final version.

AcknowledgementsThe SUISSE ADPKD trial is an investigator-initiated clinical trial. We thank Wyeth Europe for financial support and for providing the study drug Rapa-mune®. Wyeth Europe had no role in the design or conduct of the study, or in the writing and submission of the manuscript. We thank M. Sollberger for secretarial assistance.

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University of Zurich - UZH€¦ · Serra, A L Serra, A L. Mammalian Target of Rapamycin (mTOR) ... more than 10’000 patients in Switzerland. ADPKD is caused by mutations in the