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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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Dynamic changes in gene expression, glomerular function, and cell proliferation during Mtz induced podocyte injury and recovery after Mtz washoutA. GFP fluorescence is reduced and then absent after Mtz treatment for 12 and 48 hours, respectively (b–c), but reappears at 7 days after Mtz wash out (d). Arrows indicate the glomerulus. Whole mount in situ hybridization for podocin (f–h) and nephrin (j–l) transcripts reveals the concomitant loss and re-appearance of expression corresponding with the change in GFP fluorescence in the glomerulus of Mtz-treated Tg(podocin:NTR-GFP) animals. No change in GFP expression (a), or podocin (e) and nephrin (i) expression is seen in the glomerulus of Tg(podocin:NTR-GFP) animals without Mtz treatment. B. Assessment of glomerular filtration function using the rhodamine-conjugated albumin filtration assay. 5–6 hours after retro-orbital injection of rhodamine-albumin, rhodamine-albumin containing vesicles are detected inside pronephric tubule cells of Mtz treated Tg(Podocin:NTR-GFP) fish (middle panel, inset shows a higher magnified view of the tubule with arrowheads indicating red rhodamine-albumin containing vesicles inside the proximal tubular cells). Rhodamine-positive vesicles are not seen in untreated Tg(Podocin:NTR-GFP) control fish, although the uptake of freely filtered 10 kDa FITC-dextran is detected in these animals (left panel, arrowhead in inset indicates green FITC-dextran containing vesicles). No rhodamine-positive vesicles are seen in the pronephric tubule cells in recovered Tg(Podocin:NTR-GFP) animals at 7 days post Mtz washout (right panel). C. Detection of proliferating cells by BrdU incorporation. A small number of BrdU-positive cells are seen in the glomerulus of untreated Tg(podocin:NTR-GFP) laval fish (left panel, arrow indicates the red-colored BrdU signal in the nucleus. Green fluorescence marks NTR-GFP expressing podocytes). In Tg(Podocin:NTR-GFP) fish larvae treated with Mtz for 12 hours, despite the presence of BrdU labeling in neighboring cells of the glomerulus and pronephric tubules (arrows), almost no BrdU incorporation is detected in the glomerulus and the GFP fluorescence of podocytes is significant reduced (middle panel). Greatly increased BrdU staining is detected in the glomerulus of Tg(podocin:NTR-GFP) fish at 7 days post Mtz washout (right panel) (arrows). Overall GFP fluorescence is also increased in the glomerulus of these recovered animals. Some of the BrdU labeled cells are apparently also expressing GFP. N, notochord. Pt, pronephric tubule. * indicates glomerulus*.
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Figure 4: Dynamic changes in gene expression, glomerular function, and cell proliferation during Mtz induced podocyte injury and recovery after Mtz washoutA. GFP fluorescence is reduced and then absent after Mtz treatment for 12 and 48 hours, respectively (b–c), but reappears at 7 days after Mtz wash out (d). Arrows indicate the glomerulus. Whole mount in situ hybridization for podocin (f–h) and nephrin (j–l) transcripts reveals the concomitant loss and re-appearance of expression corresponding with the change in GFP fluorescence in the glomerulus of Mtz-treated Tg(podocin:NTR-GFP) animals. No change in GFP expression (a), or podocin (e) and nephrin (i) expression is seen in the glomerulus of Tg(podocin:NTR-GFP) animals without Mtz treatment. B. Assessment of glomerular filtration function using the rhodamine-conjugated albumin filtration assay. 5–6 hours after retro-orbital injection of rhodamine-albumin, rhodamine-albumin containing vesicles are detected inside pronephric tubule cells of Mtz treated Tg(Podocin:NTR-GFP) fish (middle panel, inset shows a higher magnified view of the tubule with arrowheads indicating red rhodamine-albumin containing vesicles inside the proximal tubular cells). Rhodamine-positive vesicles are not seen in untreated Tg(Podocin:NTR-GFP) control fish, although the uptake of freely filtered 10 kDa FITC-dextran is detected in these animals (left panel, arrowhead in inset indicates green FITC-dextran containing vesicles). No rhodamine-positive vesicles are seen in the pronephric tubule cells in recovered Tg(Podocin:NTR-GFP) animals at 7 days post Mtz washout (right panel). C. Detection of proliferating cells by BrdU incorporation. A small number of BrdU-positive cells are seen in the glomerulus of untreated Tg(podocin:NTR-GFP) laval fish (left panel, arrow indicates the red-colored BrdU signal in the nucleus. Green fluorescence marks NTR-GFP expressing podocytes). In Tg(Podocin:NTR-GFP) fish larvae treated with Mtz for 12 hours, despite the presence of BrdU labeling in neighboring cells of the glomerulus and pronephric tubules (arrows), almost no BrdU incorporation is detected in the glomerulus and the GFP fluorescence of podocytes is significant reduced (middle panel). Greatly increased BrdU staining is detected in the glomerulus of Tg(podocin:NTR-GFP) fish at 7 days post Mtz washout (right panel) (arrows). Overall GFP fluorescence is also increased in the glomerulus of these recovered animals. Some of the BrdU labeled cells are apparently also expressing GFP. N, notochord. Pt, pronephric tubule. * indicates glomerulus*.

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)

Dynamic changes in gene expression, glomerular function, and cell proliferation during Mtz induced podocyte injury and recovery after Mtz washoutA. GFP fluorescence is reduced and then absent after Mtz treatment for 12 and 48 hours, respectively (b–c), but reappears at 7 days after Mtz wash out (d). Arrows indicate the glomerulus. Whole mount in situ hybridization for podocin (f–h) and nephrin (j–l) transcripts reveals the concomitant loss and re-appearance of expression corresponding with the change in GFP fluorescence in the glomerulus of Mtz-treated Tg(podocin:NTR-GFP) animals. No change in GFP expression (a), or podocin (e) and nephrin (i) expression is seen in the glomerulus of Tg(podocin:NTR-GFP) animals without Mtz treatment. B. Assessment of glomerular filtration function using the rhodamine-conjugated albumin filtration assay. 5–6 hours after retro-orbital injection of rhodamine-albumin, rhodamine-albumin containing vesicles are detected inside pronephric tubule cells of Mtz treated Tg(Podocin:NTR-GFP) fish (middle panel, inset shows a higher magnified view of the tubule with arrowheads indicating red rhodamine-albumin containing vesicles inside the proximal tubular cells). Rhodamine-positive vesicles are not seen in untreated Tg(Podocin:NTR-GFP) control fish, although the uptake of freely filtered 10 kDa FITC-dextran is detected in these animals (left panel, arrowhead in inset indicates green FITC-dextran containing vesicles). No rhodamine-positive vesicles are seen in the pronephric tubule cells in recovered Tg(Podocin:NTR-GFP) animals at 7 days post Mtz washout (right panel). C. Detection of proliferating cells by BrdU incorporation. A small number of BrdU-positive cells are seen in the glomerulus of untreated Tg(podocin:NTR-GFP) laval fish (left panel, arrow indicates the red-colored BrdU signal in the nucleus. Green fluorescence marks NTR-GFP expressing podocytes). In Tg(Podocin:NTR-GFP) fish larvae treated with Mtz for 12 hours, despite the presence of BrdU labeling in neighboring cells of the glomerulus and pronephric tubules (arrows), almost no BrdU incorporation is detected in the glomerulus and the GFP fluorescence of podocytes is significant reduced (middle panel). Greatly increased BrdU staining is detected in the glomerulus of Tg(podocin:NTR-GFP) fish at 7 days post Mtz washout (right panel) (arrows). Overall GFP fluorescence is also increased in the glomerulus of these recovered animals. Some of the BrdU labeled cells are apparently also expressing GFP. N, notochord. Pt, pronephric tubule. * indicates glomerulus*.
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Figure 4: Dynamic changes in gene expression, glomerular function, and cell proliferation during Mtz induced podocyte injury and recovery after Mtz washoutA. GFP fluorescence is reduced and then absent after Mtz treatment for 12 and 48 hours, respectively (b–c), but reappears at 7 days after Mtz wash out (d). Arrows indicate the glomerulus. Whole mount in situ hybridization for podocin (f–h) and nephrin (j–l) transcripts reveals the concomitant loss and re-appearance of expression corresponding with the change in GFP fluorescence in the glomerulus of Mtz-treated Tg(podocin:NTR-GFP) animals. No change in GFP expression (a), or podocin (e) and nephrin (i) expression is seen in the glomerulus of Tg(podocin:NTR-GFP) animals without Mtz treatment. B. Assessment of glomerular filtration function using the rhodamine-conjugated albumin filtration assay. 5–6 hours after retro-orbital injection of rhodamine-albumin, rhodamine-albumin containing vesicles are detected inside pronephric tubule cells of Mtz treated Tg(Podocin:NTR-GFP) fish (middle panel, inset shows a higher magnified view of the tubule with arrowheads indicating red rhodamine-albumin containing vesicles inside the proximal tubular cells). Rhodamine-positive vesicles are not seen in untreated Tg(Podocin:NTR-GFP) control fish, although the uptake of freely filtered 10 kDa FITC-dextran is detected in these animals (left panel, arrowhead in inset indicates green FITC-dextran containing vesicles). No rhodamine-positive vesicles are seen in the pronephric tubule cells in recovered Tg(Podocin:NTR-GFP) animals at 7 days post Mtz washout (right panel). C. Detection of proliferating cells by BrdU incorporation. A small number of BrdU-positive cells are seen in the glomerulus of untreated Tg(podocin:NTR-GFP) laval fish (left panel, arrow indicates the red-colored BrdU signal in the nucleus. Green fluorescence marks NTR-GFP expressing podocytes). In Tg(Podocin:NTR-GFP) fish larvae treated with Mtz for 12 hours, despite the presence of BrdU labeling in neighboring cells of the glomerulus and pronephric tubules (arrows), almost no BrdU incorporation is detected in the glomerulus and the GFP fluorescence of podocytes is significant reduced (middle panel). Greatly increased BrdU staining is detected in the glomerulus of Tg(podocin:NTR-GFP) fish at 7 days post Mtz washout (right panel) (arrows). Overall GFP fluorescence is also increased in the glomerulus of these recovered animals. Some of the BrdU labeled cells are apparently also expressing GFP. N, notochord. Pt, pronephric tubule. * indicates glomerulus*.
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.

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Related in: MedlinePlus