Kidney-targeted Birt-Hogg-Dube gene inactivation in a mouse model: Erk1/2 and Akt-mTOR activation, cell hyperproliferation, and polycystic kidneys.
BACKGROUND
Patients with Birt-Hogg-Dubé (BHD) syndrome harbor germline mutations in the BHD tumor suppressor gene that are associated with an increased risk for kidney cancer. BHD encodes folliculin, a protein that may interact with the energy- and nutrient-sensing 5'-AMP-activated protein kinase-mammalian target of rapamycin (AMPK-mTOR) signaling pathways.


METHODS
We used recombineering methods to generate mice with a conditional BHD allele and introduced the cadherin 16 (KSP)-Cre transgene to target BHD inactivation to the kidney. Kidney cell proliferation was measured by BrdU incorporation and phospho-histone H3 staining. Kidney weight data were analyzed with Wilcoxon's rank-sum, Student's t, and Welch's t tests. Hematoxylin and eosin staining and immunoblot analysis and immunohistochemistry of cell cycle and signaling proteins were performed on mouse kidney cells and tissues. BHD knockout mice and kidney cells isolated from BHD knockout and control mice were treated with the mTOR inhibitor rapamycin. Mouse survival was evaluated by Kaplan-Meier analyses. All statistical tests were two-sided.


RESULTS
BHD knockout mice developed enlarged polycystic kidneys and died from renal failure by 3 weeks of age. Targeted BHD knockout led to the activation of Raf-extracellular signal-regulated protein kinase (Erk)1/2 and Akt-mTOR pathways in the kidneys and increased expression of cell cycle proteins and cell proliferation. Rapamycin-treated BHD knockout mice had smaller kidneys than buffer-treated BHD knockout mice had (n = 4-6 mice per group, relative kidney/body weight ratios, mean = 4.64% vs 12.2%, difference = 7.6%, 95% confidence interval = 5.2% to 10.0%; P < .001) and longer median survival time (n = 4-5 mice per group, 41.5 vs 23 days; P = .0065 ).


CONCLUSIONS
Homozygous loss of BHD may initiate renal tumorigenesis in the mouse. The conditional BHD knockout mouse may be a useful research model for dissecting multistep kidney carcinogenesis, and rapamycin may be considered as a potential treatment for Birt-Hogg-Dubé syndrome.
Baba
Masaya
M
Urologic Oncology Branch, National Cancer Institute-Frederick, Frederick, MD 21702, USA.
Furihata
Mutsuo
M
Hong
Seung-Beom
SB
Tessarollo
Lino
L
Haines
Diana C
DC
Southon
Eileen
E
Patel
Vishal
V
Igarashi
Peter
P
Alvord
W Gregory
WG
Leighty
Robert
R
Yao
Masahiro
M
Bernardo
Marcelino
M
Ileva
Lilia
L
Choyke
Peter
P
Warren
Michelle B
MB
Zbar
Berton
B
Linehan
W Marston
WM
Schmidt
Laura S
LS
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D015139:Blotting, Southern; D049109:Cell Proliferation / Q000187:drug effects; D004195:Disease Models, Animal; D004970:Estrone / Q000235:genetics; D005455:Fluorescent Antibody Technique; D020868:Gene Silencing; D005838:Genotype; D018095:Germ-Line Mutation; D015151:Immunoblotting; D007150:Immunohistochemistry; D053208:Kaplan-Meier Estimate; D007668:Kidney / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D007680:Kidney Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout; D019950:Mitogen-Activated Protein Kinase 1 / Q000187:drug effects / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000187:drug effects / Q000378:metabolism*; D007690:Polycystic Kidney Diseases / Q000150:complications / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D011494:Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D011897:Random Allocation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology; D013577:Syndrome; D058570:TOR Serine-Threonine Kinases; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D000903:Antibiotics, Antineoplastic; C526060:Bhd protein, mouse; D011518:Proto-Oncogene Proteins; D025521:Tumor Suppressor Proteins; D004970:Estrone; D011494:Protein Kinases; C546843:mTOR protein, mouse; D058570:TOR Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020123:Sirolimus