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Transgenic expression of nonclassically secreted FGF suppresses kidney repair.

Kirov A, Duarte M, Guay J, Karolak M, Yan C, Oxburgh L, Prudovsky I - PLoS ONE (2012)

Bottom Line: The effects of nonclassical FGF export in vivo are not sufficiently studied.This was manifested by a strong decrease of postischemic kidney size and weight, whereas the undamaged contralateral kidney exhibited an enhanced compensatory size increase.We suggest that nonclassical export may be used as a target for the treatment of pathologies involving signal peptide-less FGFs.

View Article: PubMed Central - PubMed

Affiliation: Maine Medical Center Research Institute, Maine Medical Center, Scarborough, Maine, United States of America.

ABSTRACT
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.

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Transgenic FGF1 expression and release in FGF1/Tek mice.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.
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pone-0036485-g001: Transgenic FGF1 expression and release in FGF1/Tek mice.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.

Mentions: To assess the effect of EC-derived FGF1 on postischemic kidney repair, we produced transgenic mice with EC-specific expression of human FGF1. C-terminal HA tag, which does not interfere with FGF1 release [24], was used for immunohistochemical FGF1 detection. To ensure cell type specific FGF1 expression, we chose a Tet-based system. FGF1 was cloned in the pTRE-Tight plasmid (Clontech) and the resultant construct was injected into fertilized mouse oocytes. We produced several independent lines of FGF1 transgenic FVB mice. By immunoblotting or immunohistochemistry, none were found to express detectable amounts of FGF1:HA in kidney, muscle, or liver (data not shown). To assess the potential of rtTA-dependent FGF1 expression in transgenic mice, we isolated fibroblasts from their tails and transiently transfected them with rtTA. Immunofluorescence analysis demonstrated that doxycycline treatment induced FGF1:HA expression in cultured mouse fibroblasts derived from transgenic mice (data not shown). We bred pTRE-Tight/FGF1 transgenic mice with Tg(Tek-rtTA, TRE-lacZ)1425Tpr/J transgenic mice expressing rtTA under the EC-specific Tek promoter. Double transgenic FGF1/Tek mice were bred onto the FVB background. Immunoperoxidase staining demonstrated endothelium-specific expression of FGF1:HA in kidneys without doxycycline treatment (Figure 1A). Western immunoblot analysis confirmed the expression of FGF1 in the kidneys of the FGF1/Tek mice dramatically exceeding endogenous FGF1 level (Figure 1B). Furthermore, FGF1 was detected by ELISA in the vasculature of FGF1/Tek mice untreated with doxycycline but not in the control mice (Figure 1C). FGF1/Tek mice did not display any visible pathology and had normal fertility. The kidneys of FGF1/Tek animals were indistinguishable from those of control animals by their macroscopic ad microscopic morphology. The unexpected stable uninduced EC-specific expression of FGF1 in FGF1/Tek mice simplified our experiments by eliminating the need for doxycycline stimulation.


Transgenic expression of nonclassically secreted FGF suppresses kidney repair.

Kirov A, Duarte M, Guay J, Karolak M, Yan C, Oxburgh L, Prudovsky I - PLoS ONE (2012)

Transgenic FGF1 expression and release in FGF1/Tek mice.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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351418&req=5

pone-0036485-g001: Transgenic FGF1 expression and release in FGF1/Tek mice.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.
Mentions: To assess the effect of EC-derived FGF1 on postischemic kidney repair, we produced transgenic mice with EC-specific expression of human FGF1. C-terminal HA tag, which does not interfere with FGF1 release [24], was used for immunohistochemical FGF1 detection. To ensure cell type specific FGF1 expression, we chose a Tet-based system. FGF1 was cloned in the pTRE-Tight plasmid (Clontech) and the resultant construct was injected into fertilized mouse oocytes. We produced several independent lines of FGF1 transgenic FVB mice. By immunoblotting or immunohistochemistry, none were found to express detectable amounts of FGF1:HA in kidney, muscle, or liver (data not shown). To assess the potential of rtTA-dependent FGF1 expression in transgenic mice, we isolated fibroblasts from their tails and transiently transfected them with rtTA. Immunofluorescence analysis demonstrated that doxycycline treatment induced FGF1:HA expression in cultured mouse fibroblasts derived from transgenic mice (data not shown). We bred pTRE-Tight/FGF1 transgenic mice with Tg(Tek-rtTA, TRE-lacZ)1425Tpr/J transgenic mice expressing rtTA under the EC-specific Tek promoter. Double transgenic FGF1/Tek mice were bred onto the FVB background. Immunoperoxidase staining demonstrated endothelium-specific expression of FGF1:HA in kidneys without doxycycline treatment (Figure 1A). Western immunoblot analysis confirmed the expression of FGF1 in the kidneys of the FGF1/Tek mice dramatically exceeding endogenous FGF1 level (Figure 1B). Furthermore, FGF1 was detected by ELISA in the vasculature of FGF1/Tek mice untreated with doxycycline but not in the control mice (Figure 1C). FGF1/Tek mice did not display any visible pathology and had normal fertility. The kidneys of FGF1/Tek animals were indistinguishable from those of control animals by their macroscopic ad microscopic morphology. The unexpected stable uninduced EC-specific expression of FGF1 in FGF1/Tek mice simplified our experiments by eliminating the need for doxycycline stimulation.

Bottom Line: The effects of nonclassical FGF export in vivo are not sufficiently studied.This was manifested by a strong decrease of postischemic kidney size and weight, whereas the undamaged contralateral kidney exhibited an enhanced compensatory size increase.We suggest that nonclassical export may be used as a target for the treatment of pathologies involving signal peptide-less FGFs.

View Article: PubMed Central - PubMed

Affiliation: Maine Medical Center Research Institute, Maine Medical Center, Scarborough, Maine, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus