Deficiency of FLCN in mouse kidney led to development of polycystic kidneys and renal neoplasia

Abstract

The Birt-Hogg-Dubé (BHD) disease is a genetic cancer syndrome. The responsible gene, BHD, has been identified by positional cloning and thought to be a novel tumor suppressor gene. BHD mutations cause many types of diseases including renal cell carcinomas, fibrofolliculomas, spontaneous pneumothorax, lung cysts, and colonic polyps/cancers. By combining Gateway Technology with the Ksp-Cre gene knockout system, we have developed a kidney-specific BHD knockout mouse model. BHD(flox/flox)/Ksp-Cre mice developed enlarged kidneys characterized by polycystic kidneys, hyperplasia, and cystic renal cell carcinoma. The affected BHD(flox/flox)/Ksp-Cre mice died of renal failure at approximate three weeks of age, having blood urea nitrogen levels over tenfold higher than those of BHD (flox/+)/Ksp-Cre and wild-type littermate controls. We further demonstrated that these phenotypes were caused by inactivation of BHD and subsequent activation of the mTOR pathway. Application of rapamycin, which inhibits mTOR activity, to the affected mice led to extended survival and inhibited further progression of cystogenesis. These results provide a correlation of kidney-targeted gene inactivation with renal carcinoma, and they suggest that the BHD product FLCN, functioning as a cyst and tumor suppressor, like other hamartoma syndrome-related proteins such as PTEN, LKB1, and TSC1/2, is a component of the mTOR pathway, constituting a novel FLCN-mTOR signaling branch that regulates cell growth/proliferation.

Figure 1. Targeting strategy and generation of BHD conditional knockout mice.

(A) Construction of the BHD gene targeting vector using a combination of the Gateway and loxp systems. A 3.5-kb 5′ homology arm containing exon 2 and a 3.0-kb 3′ arm carrying exons 5 and 6 were integrated into the pDONR P4-P1R and pDONR P2R-P through a BP (attB and attP) reaction to generate the BHD-5′ and BHD-3′ homology entry clones, respectively. A 1.3-kb fragment of genomic DNA bearing exons 3 and 4 of the BHD gene was inserted to the modified pDONR vector pENTR3C-loxPMCS-loxP-FRT-neo-FRT between the SalI and NotI sites to generate a BHD-exon3-4-pENTR3C entry clone. The three entry clones, in combination with the modified destination vector, were incubated to create a BHD-pDESTR4R3 targeting construct through BP recombination reaction. (B) Positive-targeting ES clones were selected by long-range PCR and confirmed by Southern blot analysis. (C) PCR genotyping of mouse offspring using tail DNA. (D, E) Knockout mice and normal controls were validated by Southern blot analysis.

Figure 2. Phenotypes of BHD^flox/flox/Ksp-Cre mice.

(A) BHD^flox/flox/Ksp-Cre mice developed polycystic kidney and died at age of three weeks. The kidneys from heterozygotes were phenotypically normal, similar to the ones from wild-type mice. (B) The organs from the affected mice were normal except for the kidneys (10 days old). There was not much size difference between the normal control kidneys and the affected kidneys at birth. However, the difference became apparent after the age of 5 days. At age of 10 days, the polycystic kidneys were approximate 15 times larger than those from normal controls. (C) Biochemical analysis revealed that the conditional knockout mice died of kidney failure due to high level of blood urea nitrogen (BUN). The BUN level dramatically elevated 15 days after birth. Most of the mice died at the age of 21 days. (D) The mRNA level of BHD in the kidneys of BHD^flox/flox/Ksp-Cre mice is significantly lower than that in wild-type kidneys. The heterozygote also shows lower mRNA expression relative to the wild type. m = month; d = day. f/f = flox/flox; f/+ = flox/+.

Figure 3. Inactivation of FLCN in BHD^flox/flox/Ksp-Cre mice led to polycystic disease, hyperplasia, cystic renal cell RCC.

(A,B) Deficiency of FLCN resulted in polycystic disease. (C, D) No FLCN expression was detected in cysts (enlarged tubules). However, FLCN was still expressed in relatively normal tubules where BHD was not deleted or completely eliminated by Ksp-Cre due to no or low Ksp-Cre expression in some proximal tubules. (E, F) Most of the relative normal tubules were proximal tubules stained by proximal tubule-specific marker lotus tetragonolobus lectin (LTL). Many of the proximal tubules remained relatively normal, though some proximal tubules were also enlarged (indicated by arrow. (G, H) Hyperplasia (indicated by arrow) was frequently observed in the cysts. (I, J) Cystic renal cell carcinomas were also one of the important consequences of kidney-targeted BHD gene inactivation in BHD^flox/flox/Ksp-Cre mice, which is morphologically distinct from regular cysts showed in A and B. (K, L) FLCN is predominately expressed in proximal tubules, which was demonstrated by the proximal-specific marker LTL (M). FLCN expression is quite weak in distal tubules, which was marked by the distal-tubule-specific marker, Na-Cl-cotransporter (TSC) (N). Scale bar = 50 µm.

Figure 4. mTOR signaling pathway was activated in the cystic cells, cystic RCC cells.

(A) Cystic RCC was stained by hematoxylin and eosin (H&E). (B) No FLCN expression was detected in cystic RCC, indicating deletion of the BHD gene. Phosphorylated mTOR (C) and phosphorylated S6 (D) staining was observed in the corresponding FLCN-deficient cells. Scale bar = 50 µm.