Transgenic Expression of Nonclassically Secreted FGF Suppresses Kidney Repair

FGF1 is a signal peptide-less nonclassically released growth factor that is involved in angiogenesis, tissue repair, inflammation, and carcinogenesis. The effects of nonclassical FGF export in vivo are not sufficiently studied. We produced transgenic mice expressing FGF1 in endothelial cells (EC), which allowed the detection of FGF1 export to the vasculature, and studied the efficiency of postischemic kidney repair in these animals. Although FGF1 transgenic mice had a normal phenotype with unperturbed kidney structure, they showed a severely inhibited kidney repair after unilateral ischemia/reperfusion. This was manifested by a strong decrease of postischemic kidney size and weight, whereas the undamaged contralateral kidney exhibited an enhanced compensatory size increase. In addition, the postischemic kidneys of transgenic mice were characterized by hyperplasia of interstitial cells, paucity of epithelial tubular structures, increase of the areas occupied by connective tissue, and neutrophil and macrophage infiltration. The continuous treatment of transgenic mice with the cell membrane stabilizer, taurine, inhibited nonclassical FGF1 export and significantly rescued postischemic kidney repair. It was also found that similar to EC, the transgenic expression of FGF1 in monocytes and macrophages suppresses kidney repair. We suggest that nonclassical export may be used as a target for the treatment of pathologies involving signal peptide-less FGFs

AHNAK2 participates in the stress-induced nonclassical FGF1 secretion pathway.

FGF1 is a nonclassically released growth factor that regulates carcinogenesis, angiogenesis, and inflammation. In vitro and in vivo, FGF1 export is stimulated by cell stress. Upon stress, FGF1 is transported to the plasma membrane where it localizes prior to transmembrane translocation. To determine which proteins participate in the submembrane localization of FGF1 and its export, we used immunoprecipitation mass spectrometry to identify novel proteins that associate with FGF1 during heat shock. The heat shock-dependent association of FGF1 with the large protein AHNAK2 was observed. Heat shock induced the translocation of FGF1 and AHNAK2 to the cytoskeletal fraction. In heat-shocked cells, FGF1 and the C-terminal fragment of AHNAK2 colocalized with F-actin in the vicinity of the cell membrane. Depletion of AHNAK2 resulted in a drastic decrease of stress-induced FGF1 export but did not affect spontaneous FGF2 export and FGF1 release induced by the inhibition of Notch signaling. Thus, AHNAK2 is an important element of the FGF1 nonclassical export pathway

Massive infiltration of neutrophils and macrophages in the postischemic kidneys of FGF1/Tek mice.

<p>Twenty-one days after ischemia, paraffin sections of the postischemic and contralateral kidneys of FGF1/Tek and control FVB mice were stained using the immunoperoxidase method for a neutrophil marker or F4/80, a macrophage marker. Hematoxylin counterstaining. Bar –40 μ.</p

Effect of taurine treatment on FGF1 release and postischemic kidney weight in FGF1/Tek mice.

<p>FGF1/Tek mice were fed with water containing taurine (10 mg/ml) or taurine-free water from 2 days before to day 21 after ischemia/reperfusion, when they were sacrificed. FGF1 content in the vasculature was determined by the ELISA method.</p

Transgenic FGF1 expression and release in FGF1/Tek mice.

<p>A. FGF1 is expressed in kidney EC. Immunoperoxidase staining was used to detect transgenic FGF1 (anti-HA antibodies) and EC (anti-PECAM antibodies) in the paraffin sections of kidneys obtained from FGF1/Tek mice. Preparations were counterstained with hematoxylin. Bar –30 µ. B. Lysates of kidney tissue obtained from FGF1/Tek and control FVB mice were resolved by SDS-PAGE and immunoblotted using rabbit anti-FGF1 antibodies and mouse monoclonal anti-ß-actin antibodies (loading control). C. Transgenically expressed FGF1 is released into the vasculature of FGF1/Tek mice. Seven male FGF1/Tek mice and 7 control WT FVB males were sacrificed; their vasculatures were perfused with cold heparinized PBS and the content of FGF1 (ng/ml blood) was determined using an FGF1 ELISA kit from R&D.</p

Decrease of organ size and loss of tubular structures in the postischemic kidneys of FGF1/c-fms transgenic mice.

<p>A. Induction of FGF1/HA expression in the peritoneal macrophages of FGF1/c-fms transgenic mice. Forty-eight hours before being sacrificed, the animals were intraperitoneally injected with 0.2 ml PBS containing 10 µg/ml doxycycline (right) or with doxycycline-free PBS (left). Macrophages were obtained by flushing the peritoneal cavity with PBS, plated on coverslips in DMEM with 10% FBS, fixed 12 h after plating and stained using anti-HA antibodies (green) and TOPRO3 (red). Confocal images are presented. Bar –20µ. B. FGF1 release in the vasculature of FGF1/c-fms mice. The animals were intraperitoneally injected with 0.2 ml PBS containing 10 µg/ml doxycycline (right) or with doxycycline-free PBS (left). Forty-eight hours later, the animals were sacrificed. Their vasculatures were perfused with cold heparinized PBS, and the content of FGF1(ng/ml blood) was determined using an FGF1 ELISA kit. C. Contralateral (top) and postischemic (bottom) kidneys of an FGF1/c-fms mouse, 21 days after ischemia/reperfusion, during which period the animal was receiving water with doxycycline (660 mg/l). D. Sharp decrease of postischemic kidneys weight in FGF1/c-fms mice in comparison with control FVB animals (WT). Means and SEM are presented. Both types of mice received doxycycline in water throughout the experiment. E. Loss of tubular structures in the postischemic kidney of an FGF1/c-fms mouse. Representative hematoxylin and eosin stained paraffin sections of postischemic and contralateral kidneys of FGF1/c-fms and wild WT mice. Bar –80 µ.F. Postischemic/contralateral % ratio (mean and SEM) of kidney tubule density in FGF1/c-fms and WT mice. Numbers of epithelial tubular structures in ten ×10 objective fields were counted in postischemic and contralateral kidneys of six FGF1/c-fms and nine WT mice.</p

Early postischemic response in FGF1/Tek and control animals.

<p>A. Expression of the kidney injury marker 1, Havcr1, in the postischemic kidneys of FGF1/Tek and control WT FVB mice one day after ischemia. qRT-PCR results normalized to ß-actin expression. Kidneys of five FGF1/Tek and five WT mice were studied. Mean and SEM are presented. B. PAS staining of the paraffin sections of postischemic and contralateral kidneys of control WT FVB and FGF1/Tek mice. One day after ischemia, hematoxylin counterstaining. Bar –40 μ C. Increase of the percentage of tubules with enlarged lumen (lumen occupies more than ¼ of tubule section) in postischemic kidneys comparatively to contralateral organs. Kidneys of four FGF1/Tek and four WT mice were studied. Mean and SEM of fold increase are presented. D. Glomeruli diameters in postischemic and contralateral kidneys. Kidneys of four FGF1/Tek and four WT mice were studied. Mean and SEM are presented.</p

Taurine inhibits FGF1 release in FGF1/Tek mice and rescues the postischemic kidney recovery.

<p>FGF1/Tek mice were fed with water containing taurine (10 mg/ml) or taurine-free water from 2 days before to day 21 after ischemia/reperfusion, when they were sacrificed. FGF1 content in the vasculature was determined by the ELISA method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036485#pone-0036485-t001" target="_blank">Table 1</a>). Four taurine-treated mice with inhibited FGF1 export and six untreated mice were studied. A. Weights of contralateral and postischemic kidneys. Means and SEM are presented. B. Representative hematoxylin/eosin stained paraffin sections of the postischemic and contralateral kidneys of a taurine-treated mouse and a control mouse. Bar −120µ. C. Postischemic/contralateral % ratio (mean and SEM) of kidney tubule density in taurine-treated and untreated mice. Numbers of epithelial tubular structures in ten ×10 objective fields were counted in postischemic and contralateral kidneys of four FGF1/Tek mice with taurine-inhibited FGF1 release and six FGF1/Tek mice untreated with taurine. D. Paraffin sections of the postischemic and contralateral kidneys of taurine-treated and untreated mice were stained using the immunoperoxidase method for a neutrophil marker or F4/80, a macrophage marker. Hematoxylin counterstaining. Bar –40 µ.</p

Resistance to visceral obesity is associated with increased locomotion in mice expressing an endothelial cell-specific fibroblast growth factor 1 transgene.

Overdevelopment of visceral adipose is positively correlated with the etiology of obesity-associated pathologies including cardiovascular disease and insulin resistance. However, identification of genetic, molecular, and physiological factors regulating adipose development and function in response to nutritional stress is incomplete. Fibroblast Growth Factor 1 (FGF1) is a cytokine expressed and released by both adipocytes and endothelial cells under hypoxia, thermal, and oxidative stress. Expression of Fibroblast Growth Factor 1 (FGF1) in adipose is required for normal depot development and remodeling. Loss of FGF1 leads to deleterious changes in adipose morphology, metabolism, and insulin resistance. Conversely, diabetic and obese mice injected with recombinant FGF1 display improvements in insulin sensitivity and a reduction in adiposity. We report in this novel, in vivo study that transgenic mice expressing an endothelial-specific FGF1 transgene (FGF1-Tek) are resistant to high-fat diet-induced abdominal adipose accretion and are more glucose-tolerant than wild-type control animals. Metabolic chamber analyses indicate that suppression of the development of visceral adiposity and insulin resistance was not associated with alterations in appetite or resting metabolic rate in the FGF1-Tek strain. Instead, FGF1-Tek mice display increased locomotor activity that likely promotes the utilization of dietary fatty acids before they can accumulate in adipose and liver. This study provides insight into the impact that genetic differences dictating the production of FGF1 has on the risk for developing obesity-related metabolic disease in response to nutritional stress