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A zebrafish model of conditional targeted podocyte ablation and regeneration.

Huang J, McKee M, Huang HD, Xiang A, Davidson AJ, Lu HA - Kidney Int. (2013)

Bottom Line: Treatment of these transgenic zebrafish with metronidazole results in podocyte apoptosis, a loss of nephrin and podocin expression, foot process effacement, and a leaky glomerular filtration barrier.Following metronidazole washout, proliferating cells were detected in the glomeruli of recovering transgenic fish with a restoration of nitroreductase-GFP fluorescence, nephrin and podocin expression, a reestablishment of normal foot process architecture, and glomerular barrier function.Thus, our studies show that zebrafish podocytes are capable of regenerating following depletion, and establish the Tg(podocin:NTR-GFP) fish as a new model to study podocyte injury and repair.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems Biology, Program in Membrane Biology, Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.

ABSTRACT
Podocytes are specialized cells that contribute critically to the normal structure and function of the glomerular filtration barrier. Their depletion plays an important role in the pathogenesis of glomerulosclerosis. Here, we report generation of a genetic model of conditional podocyte ablation and regeneration in zebrafish using a bacterial nitroreductase strategy to convert a prodrug, metronidazole, into a cytotoxic metabolite. A transgenic zebrafish line was generated that expresses green fluorescence protein (GFP) and the nitroreductase fusion protein under the control of the podocin promoter Tg(podocin:nitroreductase-GFP). Treatment of these transgenic zebrafish with metronidazole results in podocyte apoptosis, a loss of nephrin and podocin expression, foot process effacement, and a leaky glomerular filtration barrier. Following metronidazole washout, proliferating cells were detected in the glomeruli of recovering transgenic fish with a restoration of nitroreductase-GFP fluorescence, nephrin and podocin expression, a reestablishment of normal foot process architecture, and glomerular barrier function. Thus, our studies show that zebrafish podocytes are capable of regenerating following depletion, and establish the Tg(podocin:NTR-GFP) fish as a new model to study podocyte injury and repair.

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Ultrastructural examination of podocytes in Mtz treated Tg(podocin:GFP) and Tg(podocin:NTR-GFP) fish larvaeA. Electron microscopy examination of Mtz treated Tg(podocin:GFP) larval fish shows normal podocyte morphology, intact foot processes and normal appearing glomerular basement membrane (a). Examination of Tg(podocin:NTR-GFP) fish treated for 12 hours with Mtz reveals the presence of foot process enfacement (b and c, indicated by arrows). Chromatin condensation and early nuclear fragmentation in podocytes is clearly seen at 12 hours post exposure to Mtz indicating podocyte apoptosis (c). A complete loss of foot process and significant podocyte destruction are observed in fish treated with Mtz for 72 hours (d), however, the morphology of neighboring endothelial cells appear grossly normal with intact intercellular junctional structures and glomerular basement membrane (b and d, arrowheads indicate intercellular junctional structures). 4 days after Mtz washout, foot process-like structures appear attached to the glomerular basement membrane in the glomerulus (arrows in e). 7 days after washout, near complete recovery of foot processes and slit diaphragms are found in the glomerulus (f). B. The change in podocyte ultrastructure in response to Mtz treatment and subsequent recovery after Mtz washout is categorized into mild, moderate, severe and denuded injuries based on established methods (25). C. Quantitation of damage of podocytes after Mtz treatment and podocyte recovery 4 day and 7 days after Mtz washout. Podo, podocyte. Endo, endothelial cell. BS, Bowman’s space. Cap, capillary space.
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Figure 3: Ultrastructural examination of podocytes in Mtz treated Tg(podocin:GFP) and Tg(podocin:NTR-GFP) fish larvaeA. Electron microscopy examination of Mtz treated Tg(podocin:GFP) larval fish shows normal podocyte morphology, intact foot processes and normal appearing glomerular basement membrane (a). Examination of Tg(podocin:NTR-GFP) fish treated for 12 hours with Mtz reveals the presence of foot process enfacement (b and c, indicated by arrows). Chromatin condensation and early nuclear fragmentation in podocytes is clearly seen at 12 hours post exposure to Mtz indicating podocyte apoptosis (c). A complete loss of foot process and significant podocyte destruction are observed in fish treated with Mtz for 72 hours (d), however, the morphology of neighboring endothelial cells appear grossly normal with intact intercellular junctional structures and glomerular basement membrane (b and d, arrowheads indicate intercellular junctional structures). 4 days after Mtz washout, foot process-like structures appear attached to the glomerular basement membrane in the glomerulus (arrows in e). 7 days after washout, near complete recovery of foot processes and slit diaphragms are found in the glomerulus (f). B. The change in podocyte ultrastructure in response to Mtz treatment and subsequent recovery after Mtz washout is categorized into mild, moderate, severe and denuded injuries based on established methods (25). C. Quantitation of damage of podocytes after Mtz treatment and podocyte recovery 4 day and 7 days after Mtz washout. Podo, podocyte. Endo, endothelial cell. BS, Bowman’s space. Cap, capillary space.

Mentions: We next determined the conditions under which Mtz will induce conditional ablation of podocytes. Wild type, Tg(podocin:GFP) and Tg(podocin:NTR-GFP) larval fish at 70 hpf were incubated with Mtz for 12–48 hours at concentrations ranging from 1–20 mM. Exposure to Mtz for 12 hours resulted in pericardial edema in Tg(podocin:NTR-GFP) larval fish, consistent with renal failure (Fig. 2A). The extent of pericardia edema was more pronounced with increasing Mtz concentration or prolonged exposure even when low (2 mM) concentration of Mtz was used (data not shown). Concomitant with the presence of pericardial edema, the intensity of the GFP signal in the glomerulus of Mtz treated Tg(podocin:NTR-GFP) larval fish was significantly reduced in a dose dependent fashion (Fig 2B). A robust effect was found when Tg(podocin:NTR-GFP) embryos were exposed to Mtz at 4 or 10 mM for 12 hours with ~95% (n=41/43) of the animals showing a dramatic reduction or loss of GFP fluorescence in the glomerulus (Fig. 2B and 2C, b). No effects on GFP signal or the appearance of pericardial edema was observed in Mtz treated Tg(podocin:GFP) embryos for 12 or 48 hr (Fig. 2A left panel, and 2C, a). When Mtz concentrations >20 mM were used we observed non-specific toxicity, characterized by necrosis of the larva without significant pericardial edema in all groups (Tg(podocin:NTR-GFP), Tg(podocin:GFP) and wild type fish; data not shown). Whole mount in situ hybridization showed that the loss of GFP fluorescence induced by Mtz was concomitant with loss of the expression of nephrin (Fig. 2C, d and Fig. 4A, j and k) and podocin in the glomerulus (Fig. 4A, f and g). Despite significant edema and reduced expression of GFP/nephrin/podocin induced by Mtz in Tg(podocin:NTR-GFP) animals, we did not detected any abnormalities or change of gene expression in Mtz treated Tg(podocin:GFP) and wild type larval fish, or in Tg(podocin:NTR-GFP) larval fish without Mtz treatment. Ultrastructural examination of the glomerulus from Mtz treated Tg(podocin:NTR-GFP) larval fish by electron microscopy (EM) revealed the presence of podocyte foot process effacement (Fig. 3A, b). A more severe disruption in foot process architecture and areas of podocyte denudation was detected in animals following exposure to Mtz for 72 hours (Fig. 3A, d). Consistent with this, quantitation of the podocytopathy by classifying the areas of injury into mild, moderate, severe, and denuded (Fig. 3B), according to established methods (25), confirmed that 72 hrs of Mtz treatment caused greater injury than 12 hrs of Mtz treatment (Fig. 3C). Interestingly, despite severe damage of podocytes in some of the Mtz treated fish, the morphology of the glomerular basement membrane and the endothelium remained well preserved (Fig. 3A, d), indicating that the NTR mediated cell damage is confined to podocytes.


A zebrafish model of conditional targeted podocyte ablation and regeneration.

Huang J, McKee M, Huang HD, Xiang A, Davidson AJ, Lu HA - Kidney Int. (2013)

Ultrastructural examination of podocytes in Mtz treated Tg(podocin:GFP) and Tg(podocin:NTR-GFP) fish larvaeA. Electron microscopy examination of Mtz treated Tg(podocin:GFP) larval fish shows normal podocyte morphology, intact foot processes and normal appearing glomerular basement membrane (a). Examination of Tg(podocin:NTR-GFP) fish treated for 12 hours with Mtz reveals the presence of foot process enfacement (b and c, indicated by arrows). Chromatin condensation and early nuclear fragmentation in podocytes is clearly seen at 12 hours post exposure to Mtz indicating podocyte apoptosis (c). A complete loss of foot process and significant podocyte destruction are observed in fish treated with Mtz for 72 hours (d), however, the morphology of neighboring endothelial cells appear grossly normal with intact intercellular junctional structures and glomerular basement membrane (b and d, arrowheads indicate intercellular junctional structures). 4 days after Mtz washout, foot process-like structures appear attached to the glomerular basement membrane in the glomerulus (arrows in e). 7 days after washout, near complete recovery of foot processes and slit diaphragms are found in the glomerulus (f). B. The change in podocyte ultrastructure in response to Mtz treatment and subsequent recovery after Mtz washout is categorized into mild, moderate, severe and denuded injuries based on established methods (25). C. Quantitation of damage of podocytes after Mtz treatment and podocyte recovery 4 day and 7 days after Mtz washout. Podo, podocyte. Endo, endothelial cell. BS, Bowman’s space. Cap, capillary space.
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Figure 3: Ultrastructural examination of podocytes in Mtz treated Tg(podocin:GFP) and Tg(podocin:NTR-GFP) fish larvaeA. Electron microscopy examination of Mtz treated Tg(podocin:GFP) larval fish shows normal podocyte morphology, intact foot processes and normal appearing glomerular basement membrane (a). Examination of Tg(podocin:NTR-GFP) fish treated for 12 hours with Mtz reveals the presence of foot process enfacement (b and c, indicated by arrows). Chromatin condensation and early nuclear fragmentation in podocytes is clearly seen at 12 hours post exposure to Mtz indicating podocyte apoptosis (c). A complete loss of foot process and significant podocyte destruction are observed in fish treated with Mtz for 72 hours (d), however, the morphology of neighboring endothelial cells appear grossly normal with intact intercellular junctional structures and glomerular basement membrane (b and d, arrowheads indicate intercellular junctional structures). 4 days after Mtz washout, foot process-like structures appear attached to the glomerular basement membrane in the glomerulus (arrows in e). 7 days after washout, near complete recovery of foot processes and slit diaphragms are found in the glomerulus (f). B. The change in podocyte ultrastructure in response to Mtz treatment and subsequent recovery after Mtz washout is categorized into mild, moderate, severe and denuded injuries based on established methods (25). C. Quantitation of damage of podocytes after Mtz treatment and podocyte recovery 4 day and 7 days after Mtz washout. Podo, podocyte. Endo, endothelial cell. BS, Bowman’s space. Cap, capillary space.
Mentions: We next determined the conditions under which Mtz will induce conditional ablation of podocytes. Wild type, Tg(podocin:GFP) and Tg(podocin:NTR-GFP) larval fish at 70 hpf were incubated with Mtz for 12–48 hours at concentrations ranging from 1–20 mM. Exposure to Mtz for 12 hours resulted in pericardial edema in Tg(podocin:NTR-GFP) larval fish, consistent with renal failure (Fig. 2A). The extent of pericardia edema was more pronounced with increasing Mtz concentration or prolonged exposure even when low (2 mM) concentration of Mtz was used (data not shown). Concomitant with the presence of pericardial edema, the intensity of the GFP signal in the glomerulus of Mtz treated Tg(podocin:NTR-GFP) larval fish was significantly reduced in a dose dependent fashion (Fig 2B). A robust effect was found when Tg(podocin:NTR-GFP) embryos were exposed to Mtz at 4 or 10 mM for 12 hours with ~95% (n=41/43) of the animals showing a dramatic reduction or loss of GFP fluorescence in the glomerulus (Fig. 2B and 2C, b). No effects on GFP signal or the appearance of pericardial edema was observed in Mtz treated Tg(podocin:GFP) embryos for 12 or 48 hr (Fig. 2A left panel, and 2C, a). When Mtz concentrations >20 mM were used we observed non-specific toxicity, characterized by necrosis of the larva without significant pericardial edema in all groups (Tg(podocin:NTR-GFP), Tg(podocin:GFP) and wild type fish; data not shown). Whole mount in situ hybridization showed that the loss of GFP fluorescence induced by Mtz was concomitant with loss of the expression of nephrin (Fig. 2C, d and Fig. 4A, j and k) and podocin in the glomerulus (Fig. 4A, f and g). Despite significant edema and reduced expression of GFP/nephrin/podocin induced by Mtz in Tg(podocin:NTR-GFP) animals, we did not detected any abnormalities or change of gene expression in Mtz treated Tg(podocin:GFP) and wild type larval fish, or in Tg(podocin:NTR-GFP) larval fish without Mtz treatment. Ultrastructural examination of the glomerulus from Mtz treated Tg(podocin:NTR-GFP) larval fish by electron microscopy (EM) revealed the presence of podocyte foot process effacement (Fig. 3A, b). A more severe disruption in foot process architecture and areas of podocyte denudation was detected in animals following exposure to Mtz for 72 hours (Fig. 3A, d). Consistent with this, quantitation of the podocytopathy by classifying the areas of injury into mild, moderate, severe, and denuded (Fig. 3B), according to established methods (25), confirmed that 72 hrs of Mtz treatment caused greater injury than 12 hrs of Mtz treatment (Fig. 3C). Interestingly, despite severe damage of podocytes in some of the Mtz treated fish, the morphology of the glomerular basement membrane and the endothelium remained well preserved (Fig. 3A, d), indicating that the NTR mediated cell damage is confined to podocytes.

Bottom Line: Treatment of these transgenic zebrafish with metronidazole results in podocyte apoptosis, a loss of nephrin and podocin expression, foot process effacement, and a leaky glomerular filtration barrier.Following metronidazole washout, proliferating cells were detected in the glomeruli of recovering transgenic fish with a restoration of nitroreductase-GFP fluorescence, nephrin and podocin expression, a reestablishment of normal foot process architecture, and glomerular barrier function.Thus, our studies show that zebrafish podocytes are capable of regenerating following depletion, and establish the Tg(podocin:NTR-GFP) fish as a new model to study podocyte injury and repair.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems Biology, Program in Membrane Biology, Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.

ABSTRACT
Podocytes are specialized cells that contribute critically to the normal structure and function of the glomerular filtration barrier. Their depletion plays an important role in the pathogenesis of glomerulosclerosis. Here, we report generation of a genetic model of conditional podocyte ablation and regeneration in zebrafish using a bacterial nitroreductase strategy to convert a prodrug, metronidazole, into a cytotoxic metabolite. A transgenic zebrafish line was generated that expresses green fluorescence protein (GFP) and the nitroreductase fusion protein under the control of the podocin promoter Tg(podocin:nitroreductase-GFP). Treatment of these transgenic zebrafish with metronidazole results in podocyte apoptosis, a loss of nephrin and podocin expression, foot process effacement, and a leaky glomerular filtration barrier. Following metronidazole washout, proliferating cells were detected in the glomeruli of recovering transgenic fish with a restoration of nitroreductase-GFP fluorescence, nephrin and podocin expression, a reestablishment of normal foot process architecture, and glomerular barrier function. Thus, our studies show that zebrafish podocytes are capable of regenerating following depletion, and establish the Tg(podocin:NTR-GFP) fish as a new model to study podocyte injury and repair.

Show MeSH
Related in: MedlinePlus