Loss of Fnip1 alters kidney developmental transcriptional program and synergizes with TSC1 loss to promote mTORC1 activation and renal cyst formation

<div><p>Birt-Hogg-Dube’ Syndrome (BHDS) is a rare genetic disorder in humans characterized by skin hamartomas, lung cysts, pneumothorax, and increased risk of renal tumors. BHDS is caused by mutations in the <i>BHD</i> gene, which encodes for Folliculin, a cytoplasmic adapter protein that binds to Folliculin interacting proteins-1 and -2 (Fnip1, Fnip2) as well as the master energy sensor AMP kinase (AMPK). Whereas kidney-specific deletion of the <i>Bhd</i> gene in mice is known to result in polycystic kidney disease (PKD) and renal cell carcinoma, the roles of Fnip1 in renal cell development and function are unclear. In this study, we utilized mice with constitutive deletion of the <i>Fnip1</i> gene to show that the loss of <i>Fnip1</i> is sufficient to result in renal cyst formation, which was characterized by decreased AMPK activation, increased mTOR activation, and metabolic hyperactivation. Using RNAseq, we found that <i>Fnip1</i> disruption resulted in many cellular and molecular changes previously implicated in the development of PKD in humans, including alterations in the expression of ion and amino acid transporters, increased cell adhesion, and increased inflammation. Loss of <i>Fnip1</i> synergized with <i>Tsc1</i> loss to hyperactivate mTOR, increase Erk activation, and greatly accelerate the development of PKD. Our results collectively define roles for Fnip1 in regulating kidney development and function, and provide a model for how loss of Fnip1 contributes to PKD and perhaps renal cell carcinoma.</p></div

Conditional disruption of <i>Tsc1</i> results in polycystic kidney disease.

<p><i>Tsc1</i>^<i>fl/fl</i> mice were bred to <i>Mb1Cre</i>⁺ mice. (A) Representative gross pictures of <i>Tsc1</i>^<i>fl/fl</i> <i>Mb1Cre</i>⁺ and WT kidneys showing that loss of <i>Tsc1</i> results in kidney enlargement in 20-week old mice. (B) Representative histology images of <i>Tsc1</i>^<i>fl/fl</i> <i>Mb1Cre</i>⁺ kidneys showing development of polycystic kidney disease at 20 weeks of age. (C) Kidney-to-brain ratio graph showing ratios at 5, 10, and 20 week old <i>Tsc1</i>^<i>fl/fl</i> <i>Mb1Cre</i>⁺ and WT mice showing a gradual increase in kidney-to-brain weight ratio in <i>Tsc1</i>^<i>fl/fl</i> <i>Mb1Cre</i>⁺ kidneys. (n = 3 each genotype, p-values are shown) (D) Survival graph of <i>Tsc1</i>^<i>fl/fl</i> <i>Mb1Cre</i>⁺ (solid black line), <i>Tsc1</i>^<i>fl/+</i> <i>Mb1Cre</i>+ (sold blue line), and rapamycin treated <i>Tsc1</i>^<i>fl/fl</i> <i>Mb1Cre</i>⁺ (dotted red line) showing that polycystic kidney disease is fatal, but that disease induction can be blocked by inhibiting mTOR with Rapamycin. Rapamycin diet was started at weaning and continued until euthanasia. (E) Immunoblots of proteins isolated from whole kidney lysates from 12-week old mice showing increased p-S6R and p-4EBP indicative of increased mTORC1 activation, and increased p-Erk in <i>Tsc1</i>^<i>fl/fl</i> <i>Mb1Cre</i>⁺ relative to WT kidney tissue.</p

Loss of Fnip1 synergizes with loss of TSC1 resulting in accelerated PKD and mTOR activation.

<p>(A) Kidney-to-brain ratio graph comparing double null (<i>Tsc1</i>^<i>fl/fl</i><i>Mb1</i>Cre⁺<i>Fnip1</i>^<i>-/-</i>), <i>Fnip1</i>^<i>-/-</i> (<i>Fnip1</i>^<i>-/-</i><i>Tsc1</i>^<i>fl/+</i> <i>Mb1</i>Cre-), <i>Tsc1</i> null (<i>Fnip1</i>^<i>-/+</i> <i>Tsc1</i>^<i>fl/fl</i><i>Mb1</i>Cre⁺), and heterozygous (<i>Fnip1</i>^<i>-/+</i> <i>Tsc1</i>^<i>fl/+</i> <i>Mb1Cre</i>^-) mice at 5 weeks of age, showing an increase in kidney size of double null mice at an early age. (B) Representative histology image of <i>Tsc1</i>^<i>fl/fl</i> <i>Mb1</i>Cre⁺<i>Fnip1</i>^<i>-/-</i> double null mice showing the development of severe polycystic kidney disease at 5 weeks of age. (C) Kaplan Meier survival graph of <i>Tsc1</i>^<i>fl/fl</i><i>Mb1</i>Cre⁺<i>Fnip1</i>^<i>-/-</i> double null and <i>Tsc1</i>^<i>fl/+</i><i>Mb1</i>Cre⁺<i>Fnip1</i>^<i>-/-</i> mice. Double-null mice develop PKD very early in life, indicating that loss of <i>Fnip1</i> in <i>Tsc1</i> null mice greatly accelerates the onset of PKD relative to <i>Tsc1</i> loss alone. (D) Immunoblots of proteins isolated from whole kidney lysates derived from 5 week-old wildtype, <i>Tsc1</i>^<i>-/-</i>, and <i>Tsc1</i>^<i>-/-</i><i>Fnip1</i>^<i>-/-</i> blots showing increased p-Erk, p-S6R, and p-4E-BP in <i>Tsc1</i>^<i>-/-</i> <i>Fnip1</i>^<i>-/-</i> kidney tissue compared to <i>Tsc1</i>^<i>-/-</i> and WT mice indicating hyperactivation of the mTORC1 and Erk pathways. p-AMPK is decreased in double null mice relative to <i>Tsc1</i>^<i>-/-</i> and WT mice, suggesting decreased AMPK activation. (n = 3 each genotype, p-values are shown).</p

Loss of Fnip1 results in increased kidney size and cyst formation.

<p>(A) Increased kidney-to-brain weight ratio in <i>Fnip1</i>^<i>-/-</i> versus wildtype mice. n = 15 mice per genotype. Shown are means +/- SEM. (B) Representative hematoxylin and eosin stained histology sections of <i>Fnip1</i>^<i>-/-</i> and wildtype kidneys. <i>Fnip1</i>^<i>-/-</i> mice develop cysts and microcysts in the renal cortex. (C) Graph showing cyst counts, as determined by a blinded counter, of 12–18 week-old <i>Fnip1</i>^<i>-/-</i> and WT mice (n = 6 per group). *p-values are shown.</p

Differences in gene expression between <i>Fnip1</i>^<i>-/-</i> versus wildtype kidney tissue.

<p>RNAseq was performed on kidney cortical tissue from <i>Fnip1</i>^<i>-/-</i> (n = 3) and wildtype (n = 3) mice (11–18 weeks of age) using Illumina HiSeq 2500. (A) Volcano plot generated using EdgeR program showing genes significantly increased (red) and decreased (green) in <i>Fnip1</i>^<i>-/-</i> kidney versus WT kidney tissue. Gene ontology analyses derived from Bioconductor Open Source Software for Bioinformatics showing: (B) significantly downregulated genes grouped by expression levels and gene function in <i>Fnip1</i>^<i>-/-</i> compared to WT renal tissue, and (C) significantly upregulated expression of genes grouped by expression levels and gene function in <i>Fnip1</i>^<i>-/-</i> compared to WT renal tissue. The majority of significantly decreased genes were involved in ion transport and metabolism, whereas the majority of significantly increased genes were involved in cell adhesion and immune responses.</p

Increased mTOR and decreased AMPK activation in <i>Fnip1</i> null kidney tissue and renal tubular epithelial cells.

<p>(A) Representative immunoblots showing decreased AMPK activation (pAMPKThr172) and increased mTORC1 activation (p-S6R, p-4EBP1) in total renal tissue from <i>Fnip1</i>^<i>-/-</i> and WT mice. Statistical quantitation is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197973#pone.0197973.s001" target="_blank">S1 Fig</a>. n = 3 mice per genotype (each lane). (B) Representative immunohistochemistry images showing increased p-S6R activation around cystic tubules in <i>Fnip1</i>^<i>-/-</i> mice. Representative of 3 mice per genotype. (C) Real-time quantitative PCR showing no significant changes in expression of <i>Flcn (Bhd)</i> and <i>Fnip2</i> mRNA in <i>Fnip1</i>^<i>-/-</i> renal tissue versus WT (n = 3/genotype, * = p = 0.03). Mice used were between 12–18 weeks of age.</p

Increased immune cell infiltration in <i>Fnip1</i>^<i>-/-</i> null kidney tissue.

<p>(A) Flow cytometric analyses of immune cells isolated from <i>Fnip1</i>^<i>-/-</i> and wildtype kidney tissue showing increases in the representation of CD11c and GR1 labeled cells and decreases in B220 labeled cells (n = 3 of each genotype, p-values are shown). (B) Representative immunohistochemistry images showing increased representation of F4/80⁺ myeloid cells and (C) increased representation of CD3⁺ cells localized around renal cysts of <i>Fnip1</i>^<i>-/-</i> mice (arrows). Micrographs are representative of 3 mice per group (~12 weeks of age).</p

Real-time Quantitiative PCR analyses on kidney tissue from <i>Fnip1</i>^<i>-/-</i> versus wildtype mice.

<p>RNA was purified from <i>Fnip1</i>^<i>-/-</i> (n = 3) compared to WT (n = 3) kidney cortical tissue (mice were 11–18 weeks of age). Quantitative PCR was performed on cDNA derived from these tissues. Bar graphs show fold- differences in gene expression in <i>Fnip1</i>^<i>-/-</i> compared to WT mice (where WT is set to 1). (A) Genes encoding many of the solute carrier (Slc) family of transporter proteins are decreased in <i>Fnip1</i>^<i>-/-</i> compared to WT kidney tissue. (B) Altered expression of genes encoding acyl-CoA regulatory molecules and fatty acids regulatory molecules. (C) Expression of S100A genes including A14, 6, 8, and 9 are increased in <i>Fnip1</i>^<i>-/-</i> compared to WT kidney tissue. (D) Increased expression of TLR1 and 13 genes in <i>Fnip1</i>^<i>-/-</i> compared to WT kidney tissue. (n = 3 of each genotype, * = p<0.01).</p

<i>Fnip1</i>^<i>-/-</i> kidney tubular epithelial cells exhibit increased metabolism.

<p>(A) Seahorse analyses showing increased maximal extracellular acidification rate (ECAR), a measure of glycolysis, and (B) increased maximal oxidative phosphorylation (OCR) of cultured purified <i>Fnip1</i>^<i>-/-</i> (n = 3 mice) versus WT (n = 3 mice) renal tubular epithelial cells. Cells were stimulated with oligomycin (2.5 μM) at 30 minutes, FCCP (0.5 μM) at 50 minutes, and rotenone (2 μM) and antimycin A (2 μM) at 75 minutes. p-values are shown. (C and D) Total kidneys from <i>Fnip1</i>^<i>-/-</i> (n = 4) and WT (n = 3) mice (20–32 weeks old) were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine metabolite levels. Data were analyzed using MetaboAnalyst 3.0. Variable importance in projection (VIP) scores of metabolite changes in <i>Fnip1</i>^<i>-/-</i> versus wildtype renal tissue showing changes in nitrogenous waste products, amino acid metabolites, glycolysis, and TCA cycle metabolites. Metabolites that were significantly different and either increased (red) or decreased (green) are shown. (D) Whisker plots showing selected metabolites either significantly increased or decreased in <i>Fnip1</i>^<i>-/-</i> (red) versus <i>Fnip1</i>^<i>+/-</i>(green) kidney tissue. p-values are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197973#pone.0197973.s005" target="_blank">S1 Table</a>.</p