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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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GFP and NTR-GFP expression under the 3.5-kb podocin promoterA. Constructs used to generate the podocin driven GFP (left panel) and NTR-GFP (right panel) transgenic lines are shown respectively. B. GFP expression in glomeruli from transgenic fish Tg(podocin:GFP)(left panel, arrow) and Tg(podocin:NTR-GFP)(right panel, arrow). C. GFP expression (panels in B) overlaps with nephrin expression in glomeruli from Tg(podocin:GFP) (left panel, arrow) and Tg(podocin:NTR-GFP) (right panel, arrow) embryos. D. NTR is expressed in glomeruli from Tg(podocin:NTR-GFP)(right panel, arrows), but absent in glomeruli from Tg(podocin:GFP) embryos (left panel).
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Figure 1: GFP and NTR-GFP expression under the 3.5-kb podocin promoterA. Constructs used to generate the podocin driven GFP (left panel) and NTR-GFP (right panel) transgenic lines are shown respectively. B. GFP expression in glomeruli from transgenic fish Tg(podocin:GFP)(left panel, arrow) and Tg(podocin:NTR-GFP)(right panel, arrow). C. GFP expression (panels in B) overlaps with nephrin expression in glomeruli from Tg(podocin:GFP) (left panel, arrow) and Tg(podocin:NTR-GFP) (right panel, arrow) embryos. D. NTR is expressed in glomeruli from Tg(podocin:NTR-GFP)(right panel, arrows), but absent in glomeruli from Tg(podocin:GFP) embryos (left panel).

Mentions: We isolated a 3.5-kb DNA fragment located upstream of the podocin gene that has previously been found to contain the mouse podocin promoter (30, 31). We subsequently ligated GFP and GFP-NTR under the control of this promoter in the Tol2 transposon vector and injected zebrafish embryos with these constructs (32)(Fig. 1A). By out-crossing with wild type fish, we identified 4 independent founders for both transgenic fish lines, Tg(podocin:GFP) and Tg(podocin:NTR-GFP) respectively. Embryos from each of the founders displayed identical expression patterns in which GFP was expressed exclusively in the region of the pronephric glomerulus from 60 hours post-fertilization (hpf) by fluorescence microscopy (Fig. 1B). The founders with the strongest GFP expression were used to collect embryos for study and line maintenance. Consistent with these lines expressing GFP in podocytes, we found that the GFP signal localized to the site of nephrin expression (Fig. 1C), and they both colocalized with the site of NTR expression in Tg(podocin:NTR-GFP) laval fish (Fig. 1D).


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)

GFP and NTR-GFP expression under the 3.5-kb podocin promoterA. Constructs used to generate the podocin driven GFP (left panel) and NTR-GFP (right panel) transgenic lines are shown respectively. B. GFP expression in glomeruli from transgenic fish Tg(podocin:GFP)(left panel, arrow) and Tg(podocin:NTR-GFP)(right panel, arrow). C. GFP expression (panels in B) overlaps with nephrin expression in glomeruli from Tg(podocin:GFP) (left panel, arrow) and Tg(podocin:NTR-GFP) (right panel, arrow) embryos. D. NTR is expressed in glomeruli from Tg(podocin:NTR-GFP)(right panel, arrows), but absent in glomeruli from Tg(podocin:GFP) embryos (left panel).
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Related In: Results  -  Collection

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Figure 1: GFP and NTR-GFP expression under the 3.5-kb podocin promoterA. Constructs used to generate the podocin driven GFP (left panel) and NTR-GFP (right panel) transgenic lines are shown respectively. B. GFP expression in glomeruli from transgenic fish Tg(podocin:GFP)(left panel, arrow) and Tg(podocin:NTR-GFP)(right panel, arrow). C. GFP expression (panels in B) overlaps with nephrin expression in glomeruli from Tg(podocin:GFP) (left panel, arrow) and Tg(podocin:NTR-GFP) (right panel, arrow) embryos. D. NTR is expressed in glomeruli from Tg(podocin:NTR-GFP)(right panel, arrows), but absent in glomeruli from Tg(podocin:GFP) embryos (left panel).
Mentions: We isolated a 3.5-kb DNA fragment located upstream of the podocin gene that has previously been found to contain the mouse podocin promoter (30, 31). We subsequently ligated GFP and GFP-NTR under the control of this promoter in the Tol2 transposon vector and injected zebrafish embryos with these constructs (32)(Fig. 1A). By out-crossing with wild type fish, we identified 4 independent founders for both transgenic fish lines, Tg(podocin:GFP) and Tg(podocin:NTR-GFP) respectively. Embryos from each of the founders displayed identical expression patterns in which GFP was expressed exclusively in the region of the pronephric glomerulus from 60 hours post-fertilization (hpf) by fluorescence microscopy (Fig. 1B). The founders with the strongest GFP expression were used to collect embryos for study and line maintenance. Consistent with these lines expressing GFP in podocytes, we found that the GFP signal localized to the site of nephrin expression (Fig. 1C), and they both colocalized with the site of NTR expression in Tg(podocin:NTR-GFP) laval fish (Fig. 1D).

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