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Pflügers Archiv - European Journal of Physiology

, Volume 466, Issue 8, pp 1591–1604 | Cite as

Polycystin-1 but not polycystin-2 deficiency causes upregulation of the mTOR pathway and can be synergistically targeted with rapamycin and metformin

  • Djalila MekahliEmail author
  • Jean-Paul Decuypere
  • Eva Sammels
  • Kirsten Welkenhuyzen
  • Joost Schoeber
  • Marie-Pierre Audrezet
  • Anniek Corvelyn
  • Georges Dechênes
  • Albert C. M. Ong
  • Martijn J. Wilmer
  • Lambertus van den Heuvel
  • Geert Bultynck
  • Jan B. Parys
  • Ludwig Missiaen
  • Elena Levtchenko
  • Humbert De Smedt
Molecular and cellular mechanisms of disease

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is caused by loss-of-function mutations in either PKD1 or PKD2 genes, which encode polycystin-1 (TRPP1) and polycystin-2 (TRPP2), respectively. Increased activity of the mammalian target of rapamycin (mTOR) pathway has been shown in PKD1 mutants but is less documented for PKD2 mutants. Clinical trials using mTOR inhibitors were disappointing, while the AMP-activated kinase (AMPK) activator, metformin is not yet tested in patients. Here, we studied the mTOR activity and its upstream pathways in several human and mouse renal cell models with either siRNA or stable knockdown and with overexpression of TRPP2. Our data reveal for the first time differences between TRPP1 and TRPP2 deficiency. In contrast to TRPP1 deficiency, TRPP2-deficient cells did neither display excessive activation of the mTOR-kinase complex nor inhibition of AMPK activity, while ERK1/2 and Akt activity were similarly affected among TRPP1- and TRPP2-deficient cells. Furthermore, cell proliferation was more pronounced in TRPP1 than in TRPP2-deficient cells. Interestingly, combining low concentrations of rapamycin and metformin was more effective for inhibiting mTOR complex 1 activity in TRPP1-deficient cells than either drug alone. Our results demonstrate a synergistic effect of a combination of low concentrations of drugs suppressing the increased mTOR activity in TRPP1-deficient cells. This novel insight can be exploited in future clinical trials to optimize the efficiency and avoiding side effects of drugs in the treatment of ADPKD patients with PKD1 mutations. Furthermore, as TRPP2 deficiency by itself did not affect mTOR signaling, this may underlie the differences in phenotype, and genetic testing has to be considered for selecting patients for the ongoing trials.

Keywords

ADPKD mTOR Rapamycin Metformin 

List of abbreviations

ADPKD

Autosomal dominant polycystic kidney disease

AMPK

AMP-activated protein kinase

[Ca2+]c

Free Ca2+ concentration in the cytosol

CaMKKβ

Ca2+/calmodulin-dependent protein kinase kinase-β

CD13

Aminopeptidase N

ciPTEC

Conditionally immortalized proximal-tubule epithelial cell

ECL

Enhanced-chemiluminescence

ESRD

End-stage renal disease

FACS

Fluorescence-activated cell sorting

FITC

Fluoresceinisothiocyanate

KD

Knockdown

mTOR

Mammalian target of rapamycin

TRPP1

Polycystin-1

TRPP2

Polycystin-2

P-AMPK

Phosphorylated AMP-activated protein kinase

PKD1

Gene for TRPP1

PKD2

Gene for TRPP2

P-S6Rp

Phosphorylated S6 ribosomal protein

P-S6Rp/Tot S6Rp

Phosphorylated S6 ribosomal protein/total S6 ribosomal protein

PVDF

Polyvinylidene fluoride

S6Rp

S6 ribosomal protein

siRNA

Small interfering RNA

STO609

7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid-acetic acid

TBS

Tris-buffered saline

Notes

Acknowledgments

This work was supported by grant G0A/09/012 of the Concerted Actions Program of the Research Council of the KU Leuven, grant G.0B13.13 from the Research Foundation Flanders and Clinical PhD fellowship of the Research Foundation Flanders (1700613N0). The authors are grateful for the excellent technical assistance by Tomas Luyten. The authors thank Marina Crabbé, Anja Florizoone, Sandra Van Aerschot, and Inge Bongaers for their help with the cell cultures; Dr. Kathleen Claes, Dr. Bert Bammens, and Dr. Björn Meijers from the University Hospital of Leuven, Belgium for including patients; and Dr. Y. Cai and Dr. S. Somlo, Yale University (New Haven, CT) for sending the TRPP2−/− and TRPP2+/− renal proximal-tubule epithelial cells.

Competing interests

The authors declare that there are no competing interests.

Supplementary material

424_2013_1394_MOESM1_ESM.tif (77 kb)
ESM Fig. 1 Representative analysis of a ciPTEC line (10.064) from an ADPKD patient by flow cytometry. Cells were examined for the presence of the proximal tubular marker CD13 (aminopeptidase N) by flow cytometry. Readings were initially made in unstained cells, then in CD13-FITC-treated cells. (TIFF 77 kb)
424_2013_1394_Fig7_ESM.jpg (36 kb)
High-resolution image

(JPEG 35 kb)

424_2013_1394_MOESM2_ESM.tif (135 kb)
ESM Fig. 2 Representative analysis by RT-PCR for Aquaporin-1 (Aq1) and P-glycoprotein (Pgp) expression in different ciPTEC lines from ADPKD patients as compared with control ciPTEC (positive control). (TIFF 135 kb)
424_2013_1394_Fig8_ESM.jpg (43 kb)
High-resolution image

(JPEG 42 kb)

424_2013_1394_MOESM3_ESM.tif (62 kb)
ESM Fig. 3 mTORC1 activity after siRNA TRPP1-KD and siRNA TRPP2-KD in ciPTEC control cells (34.8). The siRNA data are compared with an siRNA scrambled control. a Representative immunoblot of TRPP1 and TRPP2 expression in siRNA TRPP1-KD, siRNA TRPP2-KD and siRNA scrambled controls (n = 4). b Representative immunoblot of mTORC1 activity assessed by the ratio P-S6Rp/Tot S6Rp (n = 4). The lower panels show the quantification of the data presented as means ± SEM. The values of the siRNA scrambled control cells were set at 100 %. The means were compared using a Student's t test for paired data: *p < 0.05. NS not significant. (TIFF 61 kb)
424_2013_1394_Fig9_ESM.jpg (47 kb)
High-resolution image

(JPEG 47 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Djalila Mekahli
    • 1
    • 2
    Email author
  • Jean-Paul Decuypere
    • 1
  • Eva Sammels
    • 1
  • Kirsten Welkenhuyzen
    • 1
  • Joost Schoeber
    • 3
  • Marie-Pierre Audrezet
    • 4
  • Anniek Corvelyn
    • 5
  • Georges Dechênes
    • 6
  • Albert C. M. Ong
    • 7
  • Martijn J. Wilmer
    • 8
  • Lambertus van den Heuvel
    • 3
    • 8
  • Geert Bultynck
    • 1
  • Jan B. Parys
    • 1
  • Ludwig Missiaen
    • 1
  • Elena Levtchenko
    • 2
    • 3
  • Humbert De Smedt
    • 1
  1. 1.Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular MedicineKU LeuvenLeuvenBelgium
  2. 2.Department of Pediatric NephrologyUniversity Hospital of LeuvenLeuvenBelgium
  3. 3.Laboratory of PediatricsKU LeuvenLeuvenBelgium
  4. 4.Laboratoire de Génétique MoléculaireCHU - INSERM U613BrestFrance
  5. 5.Laboratory for Molecular Diagnosis, Center for Human GeneticsKU LeuvenLeuvenBelgium
  6. 6.Department of Pediatric NephrologyHôpital Robert-DebréParisFrance
  7. 7.Academic Unit of NephrologyUniversity of SheffieldSheffieldUK
  8. 8.Radboud University Nijmegen Medical CentreNijmegenThe Netherlands

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