A zebrafish model of conditional targeted podocyte ablation and regeneration
2013; Elsevier BV; Volume: 83; Issue: 6 Linguagem: Inglês
10.1038/ki.2013.6
ISSN1523-1755
AutoresJianmin Huang, Mary McKee, Hong D. Huang, Alice Xiang, Alan J. Davidson, Hua Lu,
Tópico(s)Genetic and Kidney Cyst Diseases
ResumoPodocytes 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. 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. The kidney is a vital organ that performs a number of essential functions, including blood filtration and clearance of endogenous waste products. Podocytes are specialized epithelial cells that contribute critically to the kidney’s ‘filtration apparatus.’ Podocyte dysfunction and/or damage has been associated with both acute and chronic glomerular diseases, including focal segmental glomerulosclerosis, diabetic nephropathy, and HIV nephropathy.1.Asanuma K. Mundel P. The role of podocytes in glomerular pathobiology.Clin Exp Nephrol. 2003; 7: 255-259Crossref PubMed Scopus (223) Google Scholar, 2.Fogo A.B. Mechanisms of progression of chronic kidney disease.Pediatr Nephrol. 2007; 22: 2011-2022Crossref PubMed Scopus (180) Google Scholar, 3.Moreno J.A. Sanchez-Nino M.D. Sanz A.B. et al.A slit in podocyte death.Curr Med Chem. 2008; 15: 1645-1654Crossref PubMed Scopus (18) Google Scholar, 4.Wiggins R.C. 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Podocyte injury in focal segmental glomerulosclerosis: lessons from animal models (a play in five acts).Kidney Int. 2008; 73: 399-406Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 8.Matsusaka T. Xin J. Niwa S. et al.Genetic engineering of glomerular sclerosis in the mouse via control of onset and severity of podocyte-specific injury.J Am Soc Nephrol. 2005; 16: 1013-1023Crossref PubMed Scopus (213) Google Scholar, 9.Macary G. Rossert J. Bruneval P. et al.Transgenic mice expressing nitroreductase gene under the control of the podocin promoter: a new murine model of inductible glomerular injury.Virchows Arch. 2010; 456: 325-337Crossref PubMed Scopus (15) Google Scholar Understanding how podocytes respond to injury and whether they are capable of regeneration will provide valuable information for the development of new therapies that seek to replace damaged or lost podocytes.10.Ortmann J. Amann K. Brandes R.P. et al.Role of podocytes for reversal of glomerulosclerosis and proteinuria in the aging kidney after endothelin inhibition.Hypertension. 2004; 44: 974-981Crossref PubMed Scopus (131) Google Scholar The zebrafish is a widely used vertebrate model organism for the study of developmental mechanisms and disease pathologies for many organs. It combines many advantages including genetic tractability of both forward and reverse genetics, accessibility to observation and manipulation during organogenesis, and a great capability for regeneration after injury.11.Fishman M.C. Stainier D.Y. Cardiovascular development. Prospects for a genetic approach.Circ Res. 1994; 74: 757-763Crossref PubMed Scopus (56) Google Scholar, 12.Ingham P.W. Zebrafish genetics and its implications for understanding vertebrate development.Hum Mol Genet. 1997; 6: 1755-1760Crossref PubMed Scopus (40) Google Scholar, 13.Houdebine L.M. Chourrout D. 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Zebrafish kidney development.Methods Cell Biol. 2010; 100: 233-260Crossref PubMed Scopus (88) Google Scholar, 20.Drummond I. Making a zebrafish kidney: a tale of two tubes.Trends Cell Biol. 2003; 13: 357-365Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 21.Drummond I.A. Majumdar A. Hentschel H. et al.Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function.Development. 1998; 125: 4655-4667Crossref PubMed Google Scholar, 22.Wingert R.A. Davidson A.J. The zebrafish pronephros: a model to study nephron segmentation.Kidney Int. 2008; 73: 1120-1127Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar Despite the structural simplicity of the zebrafish pronephros, consisting of a single glomerulus in connection with two pronephric tubules, it possesses a glomerular filtration apparatus with a similar complexity to that of the mammalian kidney.21.Drummond I.A. Majumdar A. Hentschel H. et al.Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function.Development. 1998; 125: 4655-4667Crossref PubMed Google Scholar,23.Drummond I.A. Kidney development and disease in the zebrafish.J Am Soc Nephrol. 2005; 16: 299-304Crossref PubMed Scopus (151) Google Scholar More recently, it has been utilized as an alternative in vivo model for studying kidney injury and regeneration.16.Diep CQ, Ma D, Deo RC et al. Identification of adult nephron progenitors capable of kidney regeneration in zebrafish. Nature 470: 95–100.Google Scholar, 24.Hentschel D.M. Park K.M. Cilenti L. et al.Acute renal failure in zebrafish: a novel system to study a complex disease.Am J Physiol Renal Physiol. 2005; 288: F923-F929Crossref PubMed Scopus (143) Google Scholar, 25.Hentschel D.M. Mengel M. Boehme L. et al.Rapid screening of glomerular slit diaphragm integrity in larval zebrafish.Am J Physiol Renal Physiol. 2007; 293: F1746-F1750Crossref PubMed Scopus (83) Google Scholar, 26.Zhou W. Boucher R.C. Bollig F. et al.Characterization of mesonephric development and regeneration using transgenic zebrafish.Am J Physiol Renal Physiol. 2010; 299: F1040-F1047Crossref PubMed Scopus (98) Google Scholar The zebrafish kidney has a remarkable ability to regenerate after injury, and kidney stem/progenitor cells have been identified in adults.16.Diep CQ, Ma D, Deo RC et al. Identification of adult nephron progenitors capable of kidney regeneration in zebrafish. Nature 470: 95–100.Google Scholar,26.Zhou W. Boucher R.C. Bollig F. et al.Characterization of mesonephric development and regeneration using transgenic zebrafish.Am J Physiol Renal Physiol. 2010; 299: F1040-F1047Crossref PubMed Scopus (98) Google Scholar Similar to a recent report,27.Zhou W. Hildebrandt F. Inducible podocyte injury and proteinuria in transgenic zebrafish.J Am Soc Nephrol. 2012; 23: 1039-1047Crossref PubMed Scopus (97) Google Scholar we have independently established two transgenic zebrafish lines where green fluorescence protein (GFP) and a fusion protein of GFP and the bacterial nitroreductase (NTR) are expressed in podocytes under the control of the podocin promoter. In the Tg(podocin:GFP) line, the podocytes are fluorescently tagged allowing them to be visualized, isolated, and tracked in vivo, whereas the Tg(podocin:NTR–GFP) line utilizes bacterial NTR to convert the nontoxic pro-drug metronidazole (Mtz) into a cytotoxic, DNA crosslinking agent that induces cell death.28.Curado S. Stainier D.Y. Anderson R.M. Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies.Nat Protoc. 2008; 3: 948-954Crossref PubMed Scopus (251) Google Scholar,29.Pisharath H. Rhee J.M. Swanson M.A. et al.Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. coli nitroreductase.Mech Dev. 2007; 124: 218-229Crossref PubMed Scopus (279) Google Scholar Here we report that specific podocyte ablation and glomerular dysfunction occurs in Tg(podocin:NTR–GFP) embryos after treatment with Mtz. Interestingly, following Mtz washout, there is a recovery of glomerular filtration barrier function that is associated with podocyte proliferation in the glomerulus and a restoration of normal podocyte foot process architecture. These findings suggest that zebrafish podocytes are capable of regeneration following depletion and establish the Tg(podocin:NTR–GFP) line as a useful model to identify new therapeutic targets involved in the response of podocytes to injury. 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.Shigehara T. Zaragoza C. Kitiyakara C. et al.Inducible podocyte-specific gene expression in transgenic mice.J Am Soc Nephrol. 2003; 14: 1998-2003PubMed Google Scholar,31.He B. Ebarasi L. Hultenby K. et al.Podocin-green fluorescence protein allows visualization and functional analysis of podocytes.J Am Soc Nephrol. 2011; 22: 1019-1023Crossref PubMed Scopus (26) Google Scholar We subsequently ligated GFP and GFP–NTR under the control of this promoter in the Tol2 transposon vector and injected zebrafish embryos with these constructs32.Kawakami K. Transgenesis and gene trap methods in zebrafish by using the Tol2 transposable element.Methods Cell Biol. 2004; 77: 201-222Crossref PubMed Scopus (181) Google Scholar (Figure 1a). By outcrossing with wild-type fish, we identified four 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 60h after fertilization by fluorescence microscopy (Figure 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 (Figure 1c) and they both colocalized with the site of NTR expression in Tg(podocin:NTR–GFP) larval fish (Figure 1d). 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 h after fertilization were incubated with Mtz for 12–48h at concentrations ranging from 1 to 20mmol/l. Exposure to Mtz for 12h resulted in pericardial edema in Tg(podocin:NTR–GFP) larval fish, consistent with renal failure (Figure 2a). The extent of pericardia edema was more pronounced with increasing Mtz concentration or prolonged exposure even when low (2mmol/l) 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 manner (Figure 2b). A robust effect was found when Tg(podocin:NTR–GFP) embryos were exposed to Mtz at 4 or 10mmol/l for 12h, with ∼95% (n=41/43) of the animals showing a marked reduction or loss of GFP fluorescence in the glomerulus (Figure 2b and cB). No effects on GFP signal or the appearance of pericardial edema was observed in Mtz-treated Tg(podocin:GFP) embryos for 12 or 48h (Figure 2a and cA, left panel). When Mtz concentrations of >20mmol/l were used, we observed nonspecific 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 (Figure 2cD and Figure 4aJ and K) and podocin in the glomerulus (Figure 4aF and G). Despite significant edema and reduced expression of GFP/nephrin/podocin induced by Mtz in Tg(podocin:NTR–GFP) animals, we did not detect 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 revealed the presence of podocyte foot process effacement (Figure 3aB). A more severe disruption in foot process architecture and areas of podocyte denudation was detected in animals following exposure to Mtz for 72h (Figure 3aD). Consistent with this, quantitation of the podocytopathy by classifying the areas of injury into mild, moderate, severe, and denuded (Figure 3b), according to established methods,25.Hentschel D.M. Mengel M. Boehme L. et al.Rapid screening of glomerular slit diaphragm integrity in larval zebrafish.Am J Physiol Renal Physiol. 2007; 293: F1746-F1750Crossref PubMed Scopus (83) Google Scholar confirmed that 72h of Mtz treatment caused greater injury than 12h of Mtz treatment (Figure 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 (Figure 3aD), indicating that the NTR-mediated cell damage is confined to podocytes.Figure 3Ultrastructural examination of podocytes in metronidazole (Mtz)-treated Tg(podocin:GFP) and Tg(podocin:NTR–GFP) fish larvae. (a) 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 12h 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 12h after 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 72h (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). At 4 days after Mtz washout, foot process–like structures appear attached to the glomerular basement membrane in the glomerulus (arrows in E). At 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.Hentschel D.M. Mengel M. Boehme L. et al.Rapid screening of glomerular slit diaphragm integrity in larval zebrafish.Am J Physiol Renal Physiol. 2007; 293: F1746-F1750Crossref PubMed Scopus (83) Google Scholar (c) Quantitation of the damage of podocytes after Mtz treatment and podocyte recovery at day 4 (D7) and day 7 (D7) after Mtz washout. BS, Bowman’s space; Cap, capillary space; Cont, control; endo, endothelial cell; GFP, green fluorescence protein; NTR, nitroreductase; podo, podocyte.View Large Image Figure ViewerDownload (PPT) NTR is known to induce cell death by converting Mtz into a DNA crosslinking agent.33.Felmer R.N. Clark J.A. The gene suicide system Ntr/CB1954 causes ablation of differentiated 3T3L1 adipocytes by apoptosis.Biol Res. 2004; 37: 449-460Crossref PubMed Scopus (17) Google Scholar In line with this, chromatin condensation, a hallmark of the onset of apoptosis, was clearly detected by electron microscopy in some of the podocytes in Mtz-treated Tg(Podocin:NTR–GFP) larval fish (Figure 3aC). We further investigated Mtz-induced podocyte apoptosis using the terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) assay (Figure 2cE and F). Although no apoptotic cells were detected in the glomeruli of Mtz-treated wild-type (data not shown) and Tg(Podocin:GFP) (Figure 2cE) controls (n=30, respectively), we observed strong apoptotic signals in the glomeruli of Mtz-treated Tg(Podocin:NTR–GFP) animals (n=30) (Figure 2cF). These apoptotic cells costained with the anti-panCrb antibody, which recognizes the Crumbs protein Crb2b on podocytes,34.Ebarasi L. He L. Hultenby K. et al.A reverse genetic screen in the zebrafish identifies crb2b as a regulator of the glomerular filtration barrier.Dev Biol. 2009; 334: 1-9Crossref PubMed Scopus (56) Google Scholar confirming that the dying cells were podocytes (Figure 2cF). Taken together, these results indicate that apoptosis of podocytes is responsible for the loss of GFP fluorescence and nephrin/podocin expression in the glomeruli of Mtz-treated Tg(Podocin:NTR–GFP) animals. However, we cannot rule out that NTR-induced injury downregulates podocin and nephrin expression independently of podocyte cell death or that podocyte cell death causes secondary damage to surrounding cells such as the endothelium, pronephric tubules, and mesangial cells. Interplays between podocyte, endothelial cells, and mesangial cell have been suggested to be critical for the survival and/or development of the complex architecture and function of the glomerulus.35.Ma J. Rossini M. Yang H.C. et al.Effects of podocyte injury on glomerular development.Pediatr Res. 2007; 62: 417-421Crossref PubMed Scopus (12) Google Scholar, 36.Fogo A.B. Kon V. The glomerulus—a view from the inside—the endothelial cell.Int J Biochem Cell Biol. 2010; 42: 1388-1397Crossref PubMed Scopus (48) Google Scholar, 37.St John P.L. Abrahamson D.R. Glomerular endothelial cells and podocytes jointly synthesize laminin-1 and -11 chains.Kidney Int. 2001; 60: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 38.Lee H.S. Song C.Y. Differential role of mesangial cells and podocytes in TGF-beta-induced mesangial matrix synthesis in chronic glomerular disease.Histol Histopathol. 2009; 24: 901-908PubMed Google Scholar Albuminuria is considered to be a hallmark of glomerulopathy and has been widely used as an important clinical marker for diagnosis, monitoring disease progression, and remission in patients with chronic kidney diseases.39.Cravedi P. Ruggenenti P. Remuzzi G. Proteinuria should be used as a surrogate in CKD.Nat Rev Nephrol. 2012; 8: 301-306Crossref PubMed Scopus (107) Google Scholar,40.Fink H.A. Ishani A. Taylor B.C. et al.Screening for, monitoring, and treatment of chronic kidney disease stages 1 to 3: a systematic review for the U.S. Preventive Services Task Force and for an American College of Physicians Clinical Practice Guideline.Ann Intern Med. 2012; 156: 570-581Crossref PubMed Scopus (144) Google Scholar To functionally assess the effects of podocyte depletion on the glomerular filtration barrier of Mtz-treated Tg(Podocin:NTR–GFP) animals, we injected rhodamine-conjugated albumin into the circulation of larval fish. If the glomerular filtration barrier is compromised, the albumin tracer will pass into the pronephros and be taken up by proximal tubule cells. At 5–6h after injection, rhodamine–albumin-containing vesicles were detected inside the cells of the pronephric tubules in Mtz-treated Tg(Podocin:NTR–GFP) larval fish (Figure 4b, middle), but not in Tg(Podocin:NTR–GFP) larval fish without Mtz treatment (Figure 4b, left panel) or in Mtz-treated Tg(Podocin:GFP) fish (data not shown). Although still controversial in mammals, recent studies have shown that the zebrafish kidney contains renal stem/progenitor cells and regenerates after acute kidney injury.16.Diep CQ, Ma D, Deo RC et al. Identification of adult nephron progenitors capable of kidney regeneration in zebrafish. Nature 470: 95–100.Google Scholar,26.Zhou W. Boucher R.C. Bollig F. et al.Characterization of mesonephric development and regeneration using transgenic zebrafish.Am J Physiol Renal Physiol. 2010; 299: F1040-F1047Crossref PubMed Scopus (98) Google Scholar To investigate the regenerative capacity of podocytes after NTR/Mtz-mediated ablation, we conducted Mtz washout experiments. After treatment with 4mmol/l Mtz for 12h, Mtz was washed away and the fish were observed periodically under light and fluorescence microscope to assess their pericardial edema and glomerular GFP fluorescence. At 4 days after Mtz washout, weak but visible GFP signal reappeared in the glomeruli of Tg(podocin:NTR–GFP) animals. By 7 days after Mtz washout, the glomerular GFP fluorescence had become more intense, and both nephrin and podocin transcripts could be redetected (Figure 4aD). In addition, the pericardial edema had resolved (data not shown). To determine whether the reappearance of GFP fluorescence and expression of podocin and nephrin also results in a corresponding recovery in the structure of the previously ablated glomerulus, we examined podocyte ultrastructure by electron microscopy. A reestablishment of slit diaphragms was found in Mtz-treated Tg(podocin:NTR–GFP) animals starting at 4 days after Mtz washout, although the majority of podocytes displayed a ‘moderate’ degree of effacement at this stage (Figure 3aE). At 7 days after Mtz washout, well-formed foot processes linked by slit diaphragms were found, with the majority of podocytes showing only a ‘mild’ degree of effacement (Figure 3aF and C). A recovery in the glomerular barrier function was examined by the rhodamine-conjugated bovine albumin filtration. In Tg(podocin:NTR–GFP) animals at 7 days after Mtz washout, no rhodamine–albumin-positive vesicles were detected in the proximal tubules (Figure 4b, right panel), consistent with a functional recovery of the NTR/Mtz-damaged glomerulus. To understand whether podocyte proliferation contributes to the recovery of the NTR/Mtz-damaged glomerulus, cell proliferation was examined by 5-bromo-2-deoxyuridine (BrdU) labeling. BrdU was injected into circulation and 2–4h later, larval fish were fixed and BrdU incorporation detected by immunofluorescence staining using anti-BrdU antibody. A low level of BrdU incorporation was seen in glomeruli from Tg(podocin:NTR–GFP) larval fish without Mtz treatment (Figure 4c, left panel). Despite the presence of BrdU labeling in neighboring cells of the glomerulus and pronephric tubules, almost no BrdU incorporation was detected in the glomeruli in Tg(Podocin:NTR–GFP) larval fish treated with Mtz for 12h. The GFP fluorescence signal was also significantly reduced in the glomerulus of these animals (Figure 4c, middle panel). However, considerably increased BrdU labeling was observed in Tg(podocin:NTR–GFP) animals at 7 days after Mtz washout. The majority of these BrdU-labeled cells also expressed GFP, consistent with being podocytes and implicating podocyte proliferation in the glomerular recovery from NTR-induced injury. In summary, we have shown here the utility of the Mtz/NTR system to cause glomerular damage by using the podocin promoter to specifically restrict the apoptotic-inducing effects of NTR to podocytes. This system is temporally inducible, highly efficient, and the duration of the ablation can be controlled by washout experiments. Using this genetic tool, we demonstrated that podocyte depletion leads to effacement and a loss of slit diaphragms and glomerular barrier function. Remarkably, about a week following Mtz washout, the glomerulus is able to recover functionality, restoring foot process architecture and filtration integrity. This regeneration is associated with podocyte proliferation, suggesting that zebrafish podocytes can reenter the cell cycle and replenish cells lost to injury. Alternatively, new podocytes may be derived from a resident stem/progenitor cell population, such as the putative CD133+CD24+CD106+ stem cells identified in the Bowman’s capsule of human glomeruli.41.Angelotti M.L. Ronconi E. Ballerini L. et al.Characterization of renal progenitors committed toward tubular lineage and their regenerative potential in renal tubular injury.Stem Cells. 2012; 30: 1714-1725Crossref PubMed Scopus (234) Google Scholar By combining this tool with forward genetic or chemical screens, it should now be possible to identify new genes and novel compounds that accelerate podocyte recovery from injury. Wild-type and transgenic zebrafish (Danio rerio) embryos, larvae, and adult fish were raised and maintained under standard laboratory conditions.42.Westerfield M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio), 3rd edn. 1995Google Scholar Rhodamine B isothiocyanate–conjugated bovine albumin, fluorescein isothiocyanate (FITC)–dextran 10kDa, BrdU, and Mtz were purchased from Sigma-Aldrich (St Louis, MO). The TUNEL-In Situ Cell Death Detection Kit, TMR red, was from Roche (Indianapolis, IN) (catalog no. 12156792910). Mouse anti-BrdU antibody was purchased from Invitrogen (Grand Island, NY). The rabbit anti-panCrb antibody was a kind gift from Dr J Malicki (Sheffield, UK).34.Ebarasi L. He L. Hultenby K. et al.A reverse genetic screen in the zebrafish identifies crb2b as a regulator of the glomerular filtration barrier.Dev Biol. 2009; 334: 1-9Crossref PubMed Scopus (56) Google Scholar,43.Pellikka M. Tanentzapf G. Pinto M. et al.Crumbs, the Drosophila homologue of human CRB1/RP12, is essential for photoreceptor morphogenesis.Nature. 2002; 416: 143-149Crossref PubMed Scopus (349) Google Scholar Fluorescence-conjugated secondary antibodies were obtained from the Jackson Laboratory (Bar Harbar, ME). A 3540-bp DNA fragment from zebrafish genomic DNA corresponding to the 5′ end of the zebrafish podocin gene was cloned by PCR amplification using the primers (forward) 5′-TACGCTTGAGCAACTAAATGAATGGC-3′ and (reverse) 5′-GTGAAGTGTCCTCTGGTGTTTGG-3′. The PCR fragment was subsequently cloned into a pGEM-T Easy vector (pGEM-PodP). To generate the constructs for Tg(podocin:NTR–GFP) and Tg(podocin:GFP) transgenic fish, we use the pTol2-Slc2a15b-NTR/GFP and pTol2-Slc2a15b-GFP constructs (Dr Davidson, Auckland, New Zealand) as templates to replace the Slc2a15b promoter with podocin promoter fragment. After obtaining pTol2-podocin:GFP and pTol2-podocin:NTR–GFP constructs, they were coinjected with Tol2 transposase RNA32.Kawakami K. Transgenesis and gene trap methods in zebrafish by using the Tol2 transposable element.Methods Cell Biol. 2004; 77: 201-222Crossref PubMed Scopus (181) Google Scholar into two-cell-stage zebrafish embryos for genomic integration and generating stable transgenic lines. Adult carriers of Tg(podocin:GFP) and Tg(podocin:NTR–GFP) were identified by screening their progeny for GFP fluorescence. Adult fish expressing transgene were outcrossed to wild-type fish to obtain the germline transgenics. Zebrafish embryos were collected from timed pair mating of F1 Tg(podocin:GFP), Tg(podocin:GFP–NTR), and wild-type fish ∼60–70h after fertilization (with visible GFP fluorescence in the glomerulus) and dechorionated. Mtz was freshly prepared in 0.1% ethonol and added to fish water. The larval fish were treated with various concentrations of Mtz for 12, 24, 48, and 72h in the dark. For recovery experiment, the Mtz-containing medium was replaced with three to four changes of fresh embryo medium, and embryos/larvae were returned to 28°C, and monitored every 6–12h. Minimal 15 embryos/larvae were in each group under each treatment condition. Each experiment was repeated for at least three times. The control experiment was set up as following: wild-type+ethonol (control 1); Tg(Podocin:NTR–GFP)+ethonol (control 2); wild-type+Mtz (control 3); Tg(podocin:GFP) +Mtz (control 4); and Tg(podocin:NTR–GFP) +Mtz (experiment). The morphology of the fish and the intensity of the fluorescence signal in the glomerulus were monitored by stereomicroscope and fluorescence microscope, respectively. Whole-mount in situ hybridization was performed as previously reported,44.Thisse B. Thisse C. High Resolution Whole-Mount In Situ Hybridization. University of Oregon Press, Eugene1998Google Scholar with the modification of longer proteinase K treatment of 30min for 4 days after fertilization embryos and 1h of treatment for fish at or beyond 7 days after fertilization. Zebrafish larvae were first fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS) at 4°C overnight, transferred to 2.0% glutaraldehyde in 0.1M sodium cacodylate buffer, pH 7.4 (Electron Microscopy Sciences, Hatfield, PA) overnight at 4°C, and then rinsed in cacodylate buffer and postfixed in 1.0% osmium tetroxide in cacodylate buffer for 1h at room temperature, followed by dehydration through a graded series of ethanol to 100%. They were then infiltrated with Epon resin (Ted Pella, Redding, CA) in a 1:1 solution of Epon:100% ethanol overnight on a rotator. The following day they were embedded in fresh Epon at 60°C overnight. Thin sections were cut on a Leica EM UC7 ultramicrotome (Buffalo Grove, IL), collected onto formvar-coated grids, and stained with uranyl acetate and lead citrate. All grids were examined in a JEOL JEM 1011 transmission electron microscope (Peabody, MA) at 80kV. Images were collected using an AMT digital imaging system (Advanced Microscopy Techniques, Danvers, MA). The TUNEL assay was performed using the In Situ Cell Death Detection Kit, TMR red (Roche; catalog no. 12156792910), following the manufacturer’s instruction with modification. After staining, fish embryos were embedded in 2% agarose block, dehydrated, and embedded in JB-4 resin (Polysciences, Warrington, PA). After polymerization, the resin block was cut into 5μmol/l-thick section, mounted, and viewed under fluorescence microscopy. Zebrafish larvae aged 3.5 to 4 days after fertilization were anesthetized with Tricaine before injections. Rhodamine B isothiocyanate–conjugated bovine albumin (Sigma, St Louis, MO) was diluted in PBS to make a final concentration of 1mg/ml. FITC–dextran 10kDa was diluted to 10mg/ml in PBS. Approximately 23nl of rhodamine–albumin or FITC–dextran solution was injected into retro-orbital vasculature in each animal. At 6h after injection, animals were harvested and fixed in 4% paraformaldehyde/PBS, and processed for embedding in JB-4 resin as mentioned previously. Finally, the block was sectioned and viewed directly under fluorescence microscopy. In addition to rhodamine-conjugated albumin, FITC–dextran of 10kDa (10mg/ml, 23nl total) was also injected into each animal as an internal control. In some of the recovery experiments, 10kDa FITC–dextran was avoided because of the interference of FITC–dextran signal with the reappearance of GFP signal in recovered podocytes. The 10kDa FITC–dextran is freely filtered by the glomerulus into the tubule system and is uptaken by tubular cells. However, the rhodamine-labeled albumin will not be filtered under normal circumstance by the glomerulus and therefore will not appear in the tubular system/tubular cells, unless there is a leakiness/destruction of the glomerular filtration barrier. This assay has frequently been used as a readout of the permeability/barrier function of the glomerulus in animals.45.Pugliese G. Ricci C. Iacobini C. et al.Glomerular barrier dysfunction in glomerulosclerosis- resistant Milan rats with experimental diabetes: the role of renal haemodynamics.J Pathol. 2007; 213: 210-218Crossref PubMed Scopus (14) Google Scholar BrdU (Sigma) was diluted in PBS to make a final concentration of 100μmol/l. Approximately 23nl of the BrdU solution was injected into each animal. At 2h after BrdU injection, animals were harvested and fixed in 4% paraformaldehyde/PBS and processed for JB-4 embedding. After section, tissue slices were stained with anti-BrdU antibody following the manufacturer’s instructions (Invitrogen). We thank Dr Dennis Brown for assistance with electron microscopy analysis; Dr Yawei Kong for assistance with generating the constructs and transgenic zebrafish; Drs J Malicki, Lem (Tufts University, Boston), Tepass, and Silva-Gagliardi (University of Toronto, Canada) for providing anti-crumbs antibody (panCrb); and Dr Iain Drummond for assistance with image analysis of zebrafish and providing constructs for probe generation. We thank Renee Ethier and David Machon in the zebrafish facility at Massachusetts General Hospital for their dedicated support and Dr Patricia K Donahoe for providing support for image acquisition and analysis. Dr AJD is supported by the Rutherford Foundation, Marsden Fund, and the Auckland Medical Research Foundation. HJL is supported by NIH KO8 grant DK075940, NIH RO3 DK084295, and a Gottschalk research grant from the American Society of Nephrology (ASN). The zebrafish facility is supported by the Center for Regenerative Medicine (MGH). The Program in Membrane Biology receives additional support from the Boston Area Diabetes and Endocrinology Research Center (NIH DK-57521) and from the Center for the Study of Inflammatory Bowel Disease (NIH DK-43351).
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