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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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Decrease of organ size and loss of tubular structures in the postischemic kidneys of FGF1/c-fms transgenic mice.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.
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pone-0036485-g007: Decrease of organ size and loss of tubular structures in the postischemic kidneys of FGF1/c-fms transgenic mice.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.

Mentions: Like EC [37], macrophages and monocytes present a source of nonclassically secreted FGF1 and FGF2 in the organism [10]. To assess the effect of macrophage-derived FGF1 on postischemic kidney repair, we produced mice with monocyte/macrophage specific overexpression of FGF1. pTRE-Tight/FGF1 transgenic mice were crossed with rtTA/c-fms transgenic animals [30] that had been bred on an FVB background. Peritoneal macrophages that were obtained from the bi-transgenic FGF1/c-fms mice pretreated with doxycycline expressed FGF1:HA, while it was undetectable in macrophages obtained form the animals that did not receive doxycycline (Figure 7A). ELISA analysis demonstrated that doxycycline injection resulted in the appearance of FGF1 in the vasculature of FGF1/c-fms mice (Figure 7B). Thus, unlike FGF1/Tek mice, the cell type-specific expression of FGF1 in FGF1/c-fms animal depended on doxycycline stimulation. However, to equalize the mice for potential side effects of long-term doxycycline treatment, we choose to treat experimental (FGF1/c-fms) and control WT animals with doxycycline instead of comparing doxycycline-treated and untreated FGF1/c-fms mice. In unilateral kidney ischemia experiments, FGF1/c-fms and control WT two-month-old males were fed with doxycycline-containing water (660 mg/l), beginning 48 h before and up to day 21 after surgery. We found that like FGF1/Tek mice, FGF1/c-fms animals displayed a significant decrease of the weight of postischemic kidneys (Figure 7C,D) comparatively to WT animals. In addition, when compared to control mice the postischemic kidneys of FGF1/c-fms mice were characterized by enhanced hyperplasia of interstitial cells and significantly stronger loss of tubular structures (Figure 7E,F). Thus, similar to EC-derived, monocyte/macrophage-derived FGF1 can attenuate the postischemic kidney recovery.


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)

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

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getmorefigures.php?uid=PMC3351418&req=5

pone-0036485-g007: Decrease of organ size and loss of tubular structures in the postischemic kidneys of FGF1/c-fms transgenic mice.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.
Mentions: Like EC [37], macrophages and monocytes present a source of nonclassically secreted FGF1 and FGF2 in the organism [10]. To assess the effect of macrophage-derived FGF1 on postischemic kidney repair, we produced mice with monocyte/macrophage specific overexpression of FGF1. pTRE-Tight/FGF1 transgenic mice were crossed with rtTA/c-fms transgenic animals [30] that had been bred on an FVB background. Peritoneal macrophages that were obtained from the bi-transgenic FGF1/c-fms mice pretreated with doxycycline expressed FGF1:HA, while it was undetectable in macrophages obtained form the animals that did not receive doxycycline (Figure 7A). ELISA analysis demonstrated that doxycycline injection resulted in the appearance of FGF1 in the vasculature of FGF1/c-fms mice (Figure 7B). Thus, unlike FGF1/Tek mice, the cell type-specific expression of FGF1 in FGF1/c-fms animal depended on doxycycline stimulation. However, to equalize the mice for potential side effects of long-term doxycycline treatment, we choose to treat experimental (FGF1/c-fms) and control WT animals with doxycycline instead of comparing doxycycline-treated and untreated FGF1/c-fms mice. In unilateral kidney ischemia experiments, FGF1/c-fms and control WT two-month-old males were fed with doxycycline-containing water (660 mg/l), beginning 48 h before and up to day 21 after surgery. We found that like FGF1/Tek mice, FGF1/c-fms animals displayed a significant decrease of the weight of postischemic kidneys (Figure 7C,D) comparatively to WT animals. In addition, when compared to control mice the postischemic kidneys of FGF1/c-fms mice were characterized by enhanced hyperplasia of interstitial cells and significantly stronger loss of tubular structures (Figure 7E,F). Thus, similar to EC-derived, monocyte/macrophage-derived FGF1 can attenuate the postischemic kidney recovery.

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