Activation of Trpv4 reduces the hyperproliferative phenotype of cystic cholangiocytes from an animal model of ARPKD.
OBJECTIVE
In polycystic liver diseases, cyst formation involves cholangiocyte hyperproliferation. In polycystic kidney (PCK) rats, an animal model of autosomal-recessive polycystic kidney disease (ARPKD), decreased intracellular calcium [Ca(2+)](i) in cholangiocytes is associated with hyperproliferation. We recently showed transient receptor potential vanilloid 4 (Trpv4), a calcium-entry channel, is expressed in normal cholangiocytes and its activation leads to [Ca(2+)](i) increase. Thus, we hypothesized that pharmacologic activation of Trpv4 might reverse the hyperproliferative phenotype of PCK cholangiocytes.


METHODS
Trpv4 expression was examined in liver of normal and PCK rats, normal human beings, and patients with autosomal-dominant polycystic kidney disease or ARPKD. Trpv4 activation effect on cell proliferation and cyst formation was assessed in cholangiocytes derived from normal and PCK rats. The in vivo effects of Trpv4 activation on kidney and liver cysts was analyzed in PCK rats.


RESULTS
Trpv4 was overexpressed both at messenger RNA (8-fold) and protein (3-fold) levels in PCK cholangiocytes. Confocal and immunogold electron microscopy supported Trpv4 overexpression in the livers of PCK rats and ARPKD or autosomal-dominant polycystic kidney disease patients. Trpv4 activation in PCK cholangiocytes increased [Ca(2+)](i) by 30%, inhibiting cell proliferation by approximately 25%-50% and cyst growth in 3-dimensional culture (3-fold). Trpv4-small interfering RNA silencing blocked effects of Trpv4 activators by 70%. Trpv4 activation was associated with Akt phosphorylation and beta-Raf and Erk1/2 inhibition. In vivo, Trpv4 activation induced a significant decrease in renal cystic area and a nonsignificant decrease in liver cysts.


CONCLUSIONS
Taken together, our in vitro and in vivo data suggest that increasing intracellular calcium by Trpv4 activation may represent a potential therapeutic approach in PKD.
Gradilone
Sergio A
SA
Miles and Shirley Fiterman Center for Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota, USA.
Masyuk
Tatyana V
TV
Huang
Bing Q
BQ
Banales
Jesus M
JM
Lehmann
Guillermo L
GL
Radtke
Brynn N
BN
Stroope
Angela
A
Masyuk
Anatoliy I
AI
Splinter
Patrick L
PL
LaRusso
Nicholas F
NF
D000818:Animals; D001652:Bile Ducts / Q000166:cytology*; D002118:Calcium / Q000378:metabolism; D049109:Cell Proliferation; D004195:Disease Models, Animal; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D010641:Phenotype; D010703:Phorbol Esters / Q000494:pharmacology; D017044:Polycystic Kidney, Autosomal Recessive / Q000473:pathology / Q000628:therapy*; D048493:Proto-Oncogene Proteins B-raf / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D013449:Sulfonamides / Q000494:pharmacology; D050916:TRPV Cation Channels / Q000502:physiology*
C530602:N-(1-((4-(2-(((2,4-dichlorophenyl)sulfonyl)amino)-3-hydroxypropanoyl)-1-piperazinyl)carbonyl)-3-methylbutyl)-1-benzothiophene-2-carboxamide; D010703:Phorbol Esters; D013449:Sulfonamides; D050916:TRPV Cation Channels; C416986:TRPV4 protein, human; C486577:Trpv4 protein, rat; C008876:phorbol-12,13-didecanoate; D048493:Proto-Oncogene Proteins B-raf; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D007930:Leucine; D002118:Calcium
J Biol Chem. 2004 Sep 24;279(39):40419-30
15263001
J Biol Chem. 2008 Mar 7;283(10):6272-80
18174177
Hepatology. 2004 Oct;40(4):774-82
15382167
J Am Soc Nephrol. 2008 Mar;19(3):455-68
18235088
Proc Natl Acad Sci U S A. 2007 Nov 27;104(48):19138-43
18024594
Biochem Soc Trans. 2007 Feb;35(Pt 1):91-5
17233610
J Pharmacol Exp Ther. 2008 Aug;326(2):443-52
18499744
Mol Cell Biol. 2007 Apr;27(8):3241-52
17283055
J Physiol. 2005 Aug 15;567(Pt 1):53-8
15961428
J Cell Biol. 2008 Aug 11;182(3):437-47
18695040
Biochem Biophys Res Commun. 2005 Dec 16;338(2):880-9
16243292
Nat Genet. 2002 Mar;30(3):259-69
11919560
Nat Clin Pract Nephrol. 2006 Jan;2(1):40-55; quiz 55
16932388
Nat Cell Biol. 2000 Oct;2(10):695-702
11025659
J Pharmacol Exp Ther. 2008 Aug;326(2):432-42
18499743
Gastroenterology. 2008 Sep;135(3):937-46, 946.e1-2
18565335
Nature. 2003 Jul 24;424(6947):434-8
12879072
Circ Res. 2005 Oct 28;97(9):908-15
16179585
J Cell Sci. 2005 Jun 1;118(Pt 11):2435-40
15923656
J Biol Chem. 2008 Nov 14;283(46):31559-66
18782757
Am J Hum Genet. 2000 Dec;67(6):1598-604
11047756
J Biol Chem. 2000 Sep 1;275(35):27354-9
10869359
Kidney Int. 2004 Sep;66(3):964-73
15327388
Annu Rev Physiol. 2006;68:619-47
16460286
Medicine (Baltimore). 2006 Jan;85(1):1-21
16523049
Circ Res. 2005 Apr 29;96(8):873-80
15790956
J Am Soc Nephrol. 2006 Jan;17(1):178-87
16319189
Am J Hum Genet. 2003 Mar;72(3):691-703
12529853
Gastroenterology. 2003 Nov;125(5):1303-10
14598246
Proc Natl Acad Sci U S A. 2007 Mar 13;104(11):4389-94
17360534
Kidney Int. 2000 Apr;57(4):1460-71
10760082
Am J Med Genet. 1998 Mar 5;76(2):137-44
9511976
Am J Physiol Gastrointest Liver Physiol. 2008 Oct;295(4):G725-34
18687752
World J Gastroenterol. 2006 Jun 14;12(22):3466-70
16773703
FEBS Lett. 2000 Nov 24;485(2-3):127-34
11094154
Cell. 2000 Oct 27;103(3):525-35
11081638
Hepatology. 2009 Jan;49(1):160-74
19065671
Am J Hum Genet. 2002 May;70(5):1305-17
11898128
Am J Pathol. 2004 Nov;165(5):1719-30
15509540
Am J Pathol. 2008 Dec;173(6):1637-46
18988797
Lab Invest. 2006 Sep;86(9):940-50
16783394
J Biol Chem. 2002 Apr 19;277(16):13569-77
11827975
Hepatology. 2004 Oct;40(4):836-46
15382115
Curr Opin Nephrol Hypertens. 2006 Jul;15(4):456-63
16775462
Hepatology. 2006 May;43(5):906-8
16628666
Kidney Int. 2003 Jun;63(6):1983-94
12753285
Semin Nephrol. 2001 Mar;21(2):107-23
11245774
Am J Physiol Renal Physiol. 2008 Apr;294(4):F890-9
18256315
J Neurosci. 2006 Apr 5;26(14):3864-74
16597741
J Am Soc Nephrol. 2009 Jul;20(7):1527-32
19423689
Nephrol Dial Transplant. 2009 Feb;24(2):526-34
18826972
Pediatr Res. 2002 Dec;52(6):830-1
12438655
Gastroenterology. 2007 Mar;132(3):1104-16
17383431
Pediatr Transplant. 2005 Oct;9(5):634-9
16176423
Nat Genet. 2004 Jun;36(6):575-7
15133510