research article atlas of cellular dynamics during zebrafish adult kidney...

Research ArticleAtlas of Cellular Dynamics during Zebrafish AdultKidney Regeneration

Kristen K McCampbell Kristin N Springer and Rebecca A Wingert

Department of Biological Sciences and Center for Zebrafish Research University of Notre Dame Notre Dame IN 46556 USA

Correspondence should be addressed to Rebecca A Wingert rwingertndedu

Received 7 November 2014 Accepted 7 January 2015

Academic Editor Takashi Yokoo

Copyright copy 2015 Kristen K McCampbell et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The zebrafish is a useful animal model to study the signaling pathways that orchestrate kidney regeneration as its renal nephronsare simple yet they maintain the biological complexity inherent to that of higher vertebrate organisms including mammals Recentstudies have suggested that administration of the aminoglycoside antibiotic gentamicin in zebrafish mimics human acute kidneyinjury (AKI) through the induction of nephron damage but the timing and details of critical phenotypic events associated with theregeneration process particularly in existing nephrons have not been characterized Here we mapped the temporal progressionof cellular and molecular changes that occur during renal epithelial regeneration of the proximal tubule in the adult zebrafishusing a platform of histological and expression analysis techniques This work establishes the timing of renal cell death aftergentamicin injury identifies proliferative compartments within the kidney and documents gene expression changes associatedwith the regenerative response of proliferating cells These data provide an important descriptive atlas that documents the seriesof events that ensue after damage in the zebrafish kidney thus availing a valuable resource for the scientific community that canfacilitate the implementation of zebrafish research to delineate the mechanisms that control renal regeneration

1 Introduction

Thevertebrate kidney is comprised of functional units knownas nephrons which are epithelial tubules that cleanse thebloodstream of metabolic waste through vascular filtrationand subsequent urine production [1] Vertebrates form upto three kidney structures that are comprised of nephronsduring development termed the pronephros mesonephrosandmetanephros [1 2] A conserved trait of nephrons amongthese kidney forms across diverse terrestrial and aquaticvertebrate species is that they display a fundamentally similarregional organization along their length containing a renalcorpuscle that serves to filter blood proximal and distaltubule segments that are specialized to perform discrete tasksin solute reabsorption and secretion and a collecting ductthat transports urine out of the organ and modifies salt andwater levels [3 4]

Acute kidney injury (AKI) is a devastating and often le-thal condition in which nephron epithelial cells are destroyed

by damage from ischemia or toxin exposure typically affect-ing proximal tubule segments [5] While there is compellingevidence from work in various fish and mammalian modelsthat vertebrate nephron epithelial tubule cells can be robustlyregenerated after some forms of AKI damage [6 7] there isstill a poor understanding of the mechanisms that mediatethis regeneration response and there are ongoing contro-versies regarding the cell(s) of origin that enable kidneyregeneration in different species [2 8 9]

The zebrafish Danio rerio has emerged as a geneti-cally tractable vertebrate model to study renal biology andassociated medical conditions such as AKI both in theembryonic and in adult settings [10] The zebrafish embryokidney which is functional pronephros consists of a pairof segmented nephrons that share a blood filter and eachcontains two proximal and two distal tubule segments [1112] This structure forms by 1 day post fertilization (dpf)and blood filtration commences at approximately 2 dpf [1314] thus proffering a rapid and anatomically simple system

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for research on nephron patterning [15ndash18] identificationof essential genes [11ndash13 19] tubulogenesis [20 21] andphysiology and diseasemodeling [22ndash26] In comparison theadult zebrafish kidney or mesonephros is a single relativelyflat organ attached to the dorsal body wall that consists ofcharacteristic bilaterally symmetric regions referred to as thehead (or anterior) trunk (or medial) and tail (or posterior)(Figure 1(a)) [27] The mesonephros begins to form betweenabout 12 and 14 dpf with the progressive addition of nephronsto the existing pronephric pair [28] Over the lifespan ofthe zebrafish the mesonephros continues to accumulatenephronsmdasha phenomenon linked to their continual adultgrowth (measured in tip to tail length of the animal) and theassociated increasing excretory demands [28] In the typicalzebrafish adult at around 6 months of age this mesonephrickidney is estimated to possess approximately 450 nephronswith themost densely populated sites of nephrons in the headand trunk [28] The adult nephrons have similar segmentsas found in the pronephros but are grouped in branchedarrangements (Figure 1(a)) [29] which like other fishes donot show a regular orientation of nephrons as seen in thecortex and medulla of the mammalian metanephric kidney[6] Zebrafishmesonephric nephrons commonly have shareddistal tubule segments (Figure 1(a)) and drain into a pair ofmajor collecting ducts that span the length of the organ [1029] The stroma consisting of the cells interspersed betweenmesonephric nephrons is the site of adult hematopoiesis andalso contains an intriguing populace of mesenchymal renalprogenitors [10 28 29] These renal progenitors provide acontinual source of new nephrons that are produced in a pro-cess termed nephron neogenesis or neonephrogenesis whichoccurs during the aforementioned processes of mesonephrosdevelopment and when the mesonephros grows in responseto naturally increasing biomass of the aging fish [10 28 29]

Overall the complexity of the zebrafish mesonephrickidney provides a useful adult setting for renal biology studiesand shows promise for identifying genetic components of therenal regeneration response that can complement researchin traditional mammalian AKI models such as the mouseand rat [10] To date kidney regeneration paradigms in adultzebrafish have included nephrotoxin administration specifi-cally of the aminoglycoside antibiotic gentamicin to inducewidespread nephron tubule damage [10 28 29] as well asthe creation of several transgenic strains that were engineeredto induce ablation of particular epithelial cell types in thenephron blood filter [30 31] Among the former previousstudies have demonstrated that two major events transpirefollowing gentamicin-induced AKI in the adult zebrafishkidney organ (1) neonephrogenesis or the production ofnew nephrons due to the activation of the aforementionedstromal renal progenitors which is detectable by histologybased on the appearance of basophilic nephron units [1028 29] and (2) partial functional restoration in existingnephrons around 4 days post injury (dpi) suggestive that thedamaged nephron tubule epithelium recovers rather rapidlyfrom chemical insult [29] Despite these observations theprecise temporal sequence of molecular events that transpirewithin zebrafish nephrons during AKI including cell deathand proliferation has received relatively little scrutiny Some

alterations in gene expression have been annotated in priorwork such as the transient abrogation of transcripts encodingslc20a1a [29] a sodium-dependent phosphate transporterthat is normally localized to the proximal tubule epitheliumbut further observations have been quite limited

A significant impediment toward the pursuit of charac-terizing kidney regeneration aspects in zebrafish has beenthe paucity of histological and other molecular labelingmethodologies tailored for use in this model organismMore recently we have adapted a number of techniques toilluminate renal structures that can now be utilized [32] Forexample we demonstrated that the various proximal tubulesegments could be distinguished by unique combinationsof lectin staining dextran uptake and alkaline phosphatase(AP) reactivity (Figure 1(a)) [32] Further distal tubules aredistinguished by labeling with a different lectin (Figure 1(a))[32]

Here to obtain a more detailed understanding of theregeneration events associated with zebrafish renal injuryand identify cellular attributes enabling the demarcationof tubule segments we performed extensive histologicaland immunofluorescence studies to annotate the sequenceof tissue changes that result following gentamicin nephro-toxicity For this work we used andor modified severaltraditional histology protocols for use with zebrafish renaltissues and also implemented a number of our recentlydeveloped nephron labeling methodologies [32] With thesetools we now demonstrate for the first time that followinggentamicin-inducedAKI nephron proximal tubule epithelialregeneration proceeds over approximately one week Wehave now documented the detailed succession of cell deathand proliferation in proximal tubules during this intervalThrough additional structural and functional assays we showthat proximal tubules throughout the post-AKI zebrafishkidney regain absorptive capacity between 14 and 21 dpiFurther we show that neonephrogenesis occurs in a partiallyoverlapping time frame as nephron epithelial regenerationbeginning around 5 dpi and progressing over the subsequenttwo weeks and show that regenerating populations in bothexisting and new nephrons express the renal transcriptionfactor Pax2 Taken together these descriptive and functionalstudies provide an essential foundation for future workaimed at elucidating the mechanisms that regulate kidneyregeneration following AKI in the adult zebrafish

2 Results

The aminoglycoside antibiotic gentamicin is an establishednephrotoxin that has been used to model AKI by inducingrenal tubule damage in the adult zebrafish [10 28 29] as wellas other fish species such as goldfish and medaka [6 33]In the latter studies the histology of nephron phenotypesafter gentamicin administration was documented includingassessment of cell proliferation by suchmeasures as detectionof proliferating cell nuclear antigen (PCNA) labeling andthe incorporation of 5-bromo 21015840deoxyuridine (BrdU) [633] Previous studies in zebrafish have documented theappearance of gentamicin-damaged nephrons at 1 dpi andthe incorporation of BrdU in immature nephrons [29] but

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Figure 1 Anatomy of the zebrafish kidney and the identification of histological stains to distinguish renal structures (a) The adult zebrafishkidney is comprised of arborized arrangements of nephrons that share common distal late tubule segments and drain into major collectingducts (schematic adapted from [32]) ((b)ndash(d)) Histological staining in the zebrafish kidney revealed similarities to that of mammalian organstructure (b) HampE and (c) PAS staining of wild-type zebrafish and mouse kidney tissue emphasized the brush border and elongated cellscharacteristic of the proximal tubule (PT yellow label) compared to the pale pink hue of the distal tubule (DT yellow label) Blood filters orglomeruli (G yellow label) were as indicated (d) Silver staining highlighted the brush border in a deep brown hue and additionally stainedhyaline droplets located in the PT while DT structures lacked the brown labeling Zebrafish tissue sections 60x mouse tissue sections 40x((e)ndash(i)) Fluorescent labeling of zebrafish tubules and collecting duct with perimeters of each respective structure outlined in white dots andnuclei ((e)ndash(h)) labeled with DAPI Scale bars 25 120583m (e) LTL (green) stained the PT while (f) DBA (red) stained the DT and these tubulepopulations are mutually exclusive (h) (g) LTL also stained the collecting ducts which were distinguished by a myosin VI antibody (i) Intransgenic Tgenpepegfp zebrafish PT structures labeled with ELF-97 to detect alkaline phosphatase were mutually exclusive to DBA-staineddistal tubules and tubules were identified by labeling with anti-eGFP (representative panel reprinted with permission from [32])


the sequence of cellular alterations in gentamicin-damagednephrons over timehas not been determined To examine anddocument such cellular changes we first adapted histologicalmethods that would enable characterization of renal struc-tures (Figure 1) and then utilized these and other approachesto explore the spatial and temporal sequence of proximaltubule cell death and proliferation (Figures 2ndash8)

21 Histological Stains Distinguish Renal Structures Theadultzebrafish kidney is comprised of pinwheel-like arrangementsof nephrons in which several nephrons connect to individualbranched distal tubules that drain into the collecting ducts(Figure 1(a)) [10 29 32] Hematoxylin and eosin (HampE)staining is a basic method that distinguishes the proximaltubule from the distal tubule based partly on the presence ofa brush border the proximal tubule possesses a brush borderwhereas distal tubules do not [34] The brush border foundon the luminal side of the proximal tubule epithelial cellsis lined with densely packed microvilli forming a surfacethat greatly increases the surface area of the cells facilitatingtheir reabsorption functions When paraffin sections of thekidney from healthy adult zebrafish were stained with HampEthe brush border was prominent as a thick pink stainedband at the apical side of dark-pink stained proximal tubulecells which showed characteristic elongated shapes andformed a dilated lumen (Figure 1(b)) In comparison thecells of the distal tubule had a narrow lumen and stainedwith a much lighter pink hue allowing clear distinction ofsegment identity (Figure 1(b)) HampE staining in the healthyadult murine kidney revealed a comparable staining result(Figure 1(b))

Periodic acid-Schiff (PAS) is a staining method used todetect polysaccharides in tissues and the reagents in this stainhave an affinity for the brush border of themammalian proxi-mal tubule [35] Adult zebrafish kidney paraffin sections werestained with PAS which revealed that their nephrons pos-sessed numerous characteristics conserved with the murinekidney (Figure 1(c)) Namely the zebrafish proximal tubuleswere discernible as their brush borders stained a very deepshade of magenta and the cell cytoplasm was dark pink(Figure 1(c)) The basement membranes of the glomerularcapillary loops and tubular epithelium were also stained witha similar deep shade of magenta while epithelial cells of thedistal tubule were stained pale pink in color (Figure 1(c))

Methenamine silver stains are able to detect proteins andhave been documented as a marker for basement membranesin mammals [36 37] Applying this stain to tissue paraffinsections of the zebrafish kidney the basement membranesof tubules and glomeruli were visualized by a dark brownhue and the brush borders of the proximal tubules were alsostained dark brown (Figure 1(d)) In addition the silver stainrevealed the presence of hyaline droplets in the proximaltubules which has also been documented in mammals [38]

22 Tubule and Duct Compartments Can Be DistinguishedFurther with Labeling Methods Using Lectins Myosin VIExpression and Alkaline Phosphatase Reactivity with ELF-97 Lectins are sugar-binding proteins of nonimmune originthat are expressed throughout nature [39] Specifically in the

kidney organ Lotus tetragonolobus lectin (LTL) marks theproximal tubules and the targets of Dolichos biflorus agglu-tinin (DBA) include the distal tubules and collecting ducts[39] Previous studies requiring the ability to distinguishproximal versus distal tubules and quantify such structures inmice have utilized LTL and DBA staining with great success[39ndash42] Similarly for the identification of tubular segmentsin a medaka fish model of polycystic kidney disease LTL wasused as a proximal tubule marker and DBA was used as adistal tubule marker [43] Tissue cryosections of zebrafishadult kidneys were subjected to staining with LTL and DBA(Figures 1(e)ndash1(i)) as well as whole mount staining (data notshown) The binding specificity of the lectins was conservedin zebrafish with LTL and DBA labeling distinct tubules(Figures 1(e) and 1(f) resp) and these labels were mutuallyexclusive both in cryosection (Figure 1(h)) and in wholemount preparations (data not shown) [32] Interestingly themajor collecting ducts in the zebrafish kidney which are apair of large drainage ducts that extend the entire length ofthe organ [28] were distinguished via colabeling of LTL andan antibody to detect myosin VI (Figure 1(g)) These wereuniquely identified as each zebrafish kidney contained onlytwo such structures one on each symmetric side of the organ(data not shown)

As proximal tubules possess a brush border a fluores-cence-basedmethod known as ELF- (enzyme labeled fluores-cence-) 97 was used to determine if this activity couldbe localized in adult zebrafish kidneys The brush bordersof epithelial cells in the intestine are known to expresshigh levels of endogenous alkaline phosphatase activity [44]One previous study reported ELF-97 reactivity in the adultzebrafish kidney suggesting that ELF-97 staining may bea viable way to label proximal tubule cells [45] Tissuecryosections of adult zebrafish kidneys stained with the ELF-97 phosphatase were counterstained with the distal tubulemarker DBA The ELF-97 signals were localized to theproximal tubules which have very prominent brush bordersof microvilli that project into the tubule lumens (Figure 1(i)data not shown) [32] In contrast tubules that were identifiedbyDBAcompletely lacked any ELF-97 precipitate (Figure 1(i)data not shown) and these observations were confirmed inwhole mount kidney preparations as well (data not shown)[32]These observations are in agreementwith the knowledgethat vertebrate nephron distal segments do not possess abrush border and would therefore not be stained with ELF-97 [4 46]

23 Histological Characterization of Gentamicin Injury TimeCourse Next we utilized these various histological tools toanalyze the phenotype of zebrafish nephrons following AKIAn intraperitoneal injection of gentamicin was administeredto induce AKI in adult zebrafish based on a previouslyestablished dosage [28 29 47] The injected fish weresacrificed at several time points and their kidneys fixedsectioned and then stained with HampE (Figure 2(a)) At 1 dpisufficient nephron damage was induced that resulted in adenuded basement membrane and an immense amount ofintraluminal cellular debrisThe proximal tubular epitheliumhad become vacuolated and massive disorganization was


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Figure 2 Histological staining reveals the process of regeneration after gentamicin-induced injury in adult zebrafish (a) HampE and (b) PASstaining demonstrated extensive nephron damage and tubule destruction followed by regenerative events that were completed by 21 dpiTheformation of basophilic cellular aggregates (darkest purple cellular staining) was indicative of new nephron formation Both time courseswere completed over a three-week period 60x DT distal tubule G glomerulus PT proximal tubule Yellow labels asterisk (lowast) indicatesluminal debris double asterisk (lowastlowast) indicates restoration of cells to tubules arrowhead indicates neonephrogenic cluster arrow indicatesneonephron with visible lumen


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Figure 3 Dynamics of slc20a1a expression over the course of zebrafish adult kidney injury and regeneration (a) Whole mount in situhybridization of adult kidneys over a two-week injury time course revealed the initial absence of slc20a1a transcripts followed by an increasein transcript levels and domains to near-normal levels by 14 dpi Throughout the time course new nephrons first appeared as small cellularaggregates that form coils and eventually elongated into PCT segments that are indistinguishable from preexisting regenerated PCT tubules3x magnification (b) Representative images of the progressive stages of nephron formation Scale bar 25 120583m (c) Quantification shows thatslc20a1a positive structures during regeneration Labels lowastlowast indicates 119875 lt 001 lowast lowast lowast indicates 119875 lt 0001 black indicates aggregate greenindicates coil purple indicates segment


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Figure 4 Detection of proximal tubule cell death after gentamicin injury (a) Kidney tissue was assayed for TUNEL-positive cells in uninjuredkidneys and at 1 3 5 7 and 10 dpi The peak of TUNEL reactivity in cells was identified within LTL-positive proximal tubules at 1 3 and5 days after kidney injury By 7 and 10 days the level of TUNEL-positive cells decreased returning to basal levels previously established inuntreated kidneys Proximal tubules were labeled by LTL (b) Quantification of TUNEL labeling in nephron proximal segments Scale bar25 120583m Labels lowast indicates 119875 lt 005 lowastlowast indicates 119875 lt 001

evident Cellular disruption was still apparent at 3 dpi butwithin the tubular spaces cells with amesenchymal structurewere congregated At 5 dpi cells within the tubules weremoreorganized appearing as a visually intact single layer of cellswith a hint of a luminal opening In addition a small numberof basophilic dark purple cellular clusters had emergedwhich is a trait of neonephrogenesis in fish [6] By 7 dpinumerous basophilic cellular aggregates had formed somecontaining a lumen and a majority of cellular debris wascleared Tubular lumens continued to form in the aggregatesat 10 dpi and lumens that were evident at 7 dpi had widenedIn addition tubule cells displayed shades of pink similarto that of proximal and distal tubules By 14 dpi many ofthe aggregates possessed obvious brush borders indicativeof a proximal segment and the kidney tissue overall wasanalogous to that of an uninjured adult fish Kidney tissuestaining at 21 dpi revealed a similarwild-type appearancewithnonexistent cellular aggregates

A second histological analysis was completed to stainzebrafish kidneys with PAS (Figure 2(b)) PAS was utilizedbecause the stain emphasizes the brush borders of the proxi-mal tubule more effectively than HampE and thus we hypoth-esized that this staining method could provide additionalinsight into the establishment of epithelial character in regen-erated proximal tubules At 1 dpi the appearance of PAS-positive intraluminal cellular debris was readily observedin distal tubules as observed in HampE stained samplesInterestingly hyaline casts have previously been documented

as being PAS-positive in the injured kidney of other ver-tebrate species [48] Thus this suggests that the luminalcellular debris was the result of cell death and subsequentsloughing within the renal tubules in areas located upstreamof the distal segments presumably localized to the proximalregions Kidney tissue at 3 5 and 7 dpi corresponded closelyto that of previously described tissue in the HampE timecourse Interestingly dark magenta linings were noted inmany tubules that had only subtle lumens suggesting theywere putative newly regenerated proximal tubules At 10 dpinumerous aggregates that have formed possessed a PAS-positive brush border and by 14 dpi the kidney tissue wasindistinguishable from wild-type tissue Again 21 dpi tissuestaining revealed an absence of cellular aggregates suggestingthat the regeneration process of neonephrogenesis had beencompleted Interestingly the location of basophilic cellu-lar aggregates that appeared throughout both histologicaltime courses was closely juxtaposed to a nephron tubule(Figure 2) Particularly at later time points (eg 7ndash10 dpi)each aggregate was associated with a plumbing event into apreexisting proximal tubule based on PAS reactivity thoughthese events were detectable as early as the 5 dpi time pointas well (Figure 2 data not shown) Further the aggregatesthemselves displayed the characteristic of PAS reactivity withdark magenta apical staining (Figure 2 data not shown)This staining character of the basophilic aggregates suggeststhe largely intriguing notion that new nephrons possess aproximal nature


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Figure 5 Cell proliferation in the regenerating proximal tubule epithelium and in neonephrogenic clusters Kidney tissue was assayed forPCNA-positive nuclei (red) and proximal tubules were identified by LTL labeling (green) Nuclei were stained with DAPI (blue) (a) Mostepithelial proliferation occurred at 3 5 and 7 days after kidney injury Scale bar 25120583m (b) Quantification of PCNA expression in nephronproximal tubular segments Labels lowastlowast indicates 119875 lt 001 lowast lowast lowast indicates 119875 lt 0001 (c) Epithelial proliferation marked by PCNA (red)occurred at high levels in cellular aggregates at 3 and 5 dpi At 7 dpi aggregates that have formed lumens continued to express intense levelsof PCNA At 10 dpi PCNA was still abundant in tubules that have become LTL-positive (green)

24 slc20a1a Expression Time Course in the Injured KidneyThe solute transporter slc20a1a which marks the proximalconvoluted tubule (PCT) segment in the adult nephron(Figure 1(a)) as well as the embryo has been used as a markerof proximal tubule segments after nephrotoxin injury in thezebrafish [29] To correlate spatiotemporal alterations of thisgene with our histology time courses gentamicin-injectedzebrafish kidneys were examined using in situ hybridizationwith slc20a1a (Figure 3) At 1 dpi the gentamicin-inducednephron damage was so catastrophic that slc20a1a transcriptexpressionwas completely abrogated (Figure 3(a)) consistentwith prior observations [29] The appearance of a smallnumber of slc20a1a+ cellular aggregates at 3 dpi (058 plusmn 028)was followed by substantial increases in this number at 5 dpi(6666 plusmn 39) and 7 dpi (745 plusmn 94) (Figures 3(a) and 3(b))

Small coiled structures first appeared at 5 dpi (23 plusmn 055)and were numerous throughout the kidney by 7 dpi (306 plusmn65) (Figures 3(a) and 3(b)) A high number of aggregatesalso persisted between 7 and 10 dpi however the quantitydeclined asmore coils appeared (Figure 3(a) data not shown)Further the emergence of segment structures which closelyphenocopied uninjured PCT segments appeared at 7 dpiwith an incidence of 79 plusmn 26 structures per kidney (Figures3(a) and 3(b)) At 14 dpi slc20a1a expression was analogousto that of wild-type adult kidneys with the majority ofstructures stained resembling segments (Figure 3(a)) Tofurther analyze these observations we performed ANOVAstatistical analyses to compare the number of aggregatescoils and segments which revealed that there was a signifi-cant increase in aggregates between 3 and 7 dpi accompanied


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Figure 6 Regenerating proximal tubules show BrdU incorporation after gentamicin injury (a) Kidney tissue was assayed for BrdU-positivenuclei (red) and proximal tubules were identified by LTL labeling (green) Nuclei were stained with DAPI (blue) (b) Quantification showedthat BrdU incorporation occurs progressively at 3 4 and 5 dpi

by the emergence of coils and segments at 5 and 7 dpiwhich was also significant (Figure 3(c)) This suggests thatthroughout the time period following nephrotoxicant injurynew nephrons first appear as small cellular aggregates thateventually coil and elongate into healthy normal functioningnephrons in keeping with the qualitative observations ofbasophilic aggregates and coils in histological data as wellas previous observations of nephron formation [28 29]The populace of coils and segments likely also includes thepopulation of existing nephrons that regenerate the damagedproximal tubule epithelium most likely at time points from5 dpi onward However additional studies are needed todistinguish new nephrons that express slc20a1a from existingnephrons that show tubular regeneration

25 Cellular Death and Proliferation during Kidney Regen-eration The TUNEL method is a useful and specific labelfor nuclear DNA fragmentation [49] which is a signatureof cellular apoptosis Previous AKI studies with gentamicinin zebrafish have not examined the timing and location ofnephron tubule cell death though this agent is well known todestroy renal proximal tubule cells To confirm this directlyand to assess whether cell death occurred in other tubulesegments we implemented a combination of TUNEL andLTL labeling to localize when and where cell death occurredin proximal tubules compared to other segments followinggentamicin exposure While uninjured kidneys showed verylow levels of TUNEL-positive cells (095 plusmn 043) TUNELreactivity escalated dramatically in the nuclei of tubular cellsspecifically within LTL-positive proximal tubules at 1 and3 dpi (Figures 4(a) and 4(b)) At 1 dpi 2878 plusmn 112 ofcells in LTL-positive proximal tubules were TUNEL-positivewhich climbed to an incidence of 4411 plusmn 603 of proximal

tubule cells at 3 dpi (Figures 4(a) and 4(b)) By 5 dpi only782 plusmn 105 of proximal tubule cells were TUNEL-positive(Figures 4(a) and 4(b)) By 7 and 10 days after injection thelevel of TUNEL-positive cells in proximal tubules decreasedeven further returning to approximately basal levels that hadbeen established in kidneys that were untreated (Figures 4(a)and 4(b)) The rapid elevation in TUNEL reactivity from1 to 3 dpi followed by rapid decline over 3 to 10 dpi wasstatistically significant (Figure 4(b)) Renal tubules that wereLTL-negative were not found to be TUNEL-positive (data notshown) Similar trends were observed in independent studiesin which TUNEL staining was performed in conjunctionwith ELF-97 to label the proximal tubule (data not shown)Overall these data show that cell death was occurring largelyin the proximal tubule and that it transpires in a wave thatpeaks at approximately 3 dpi

Next we evaluated the dynamics of cell proliferation fol-lowing gentamicin exposure using PCNA labeling (Figure 5)PCNA is found in varying concentrations within the cellduring the cell cycle and is in maximum quantities duringthe S phase [50] As with the cell death analysis the use ofLTL labeling in conjunction with the use of an antibody todetect PCNA allowed for examination of cell proliferation inproximal nephron tubules compared to the rest of the renaltubules and ducts At basal levels in untreated kidneys 097plusmn 021 of cells were positive in the LTL-positive proximaltubules (Figures 5(a) and 5(b)) At 1 dpi no cells were foundto express PCNA (Figures 5(a) and 5(b)) However by 3 dpi286 plusmn 169 of LTL-positive tubular cells were positivefor PCNA (Figures 5(a) and 5(b)) The percentage of PCNApositive cells in proximal tubules peaked at 5 days after injuryreaching 609 plusmn 216 of all proximal tubule cells Thisincidence declined to 241 plusmn 051 at 7 dpi and then further


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Figure 7 Changes in ELF-97 staining and proximal tubule functionality after gentamicin injury Whole mount labeling of (a) alkalinephosphatase reactivity and (b) uptake of fluoro-ruby dextran in the zebrafish kidney during recovery from AKI (a) Untreated kidneyscontained discrete PCT-PST structures (white box) that were destroyed at 1 dpi (white arrowheads) Restoration of discrete PCT-PSTstructures was completed by 14 dpi (b) Nephron reabsorption was visualized in injured kidneys in which the PCT was demarcated by uptakeof fluoro-ruby dextran while at 1 dpi such structures were absent (white arrowheads) Partial uptake was sporadically observed at 5ndash7 dpibut was not consistent throughout the kidney organ until 21 dpi when PCT segments were distinctly visualized (white box)


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Figure 8The Pax2 transcription factor marks cells in the regenerating proximal tubule and neonephrogenic clusters (a) Expression of Pax2in the regenerating kidney tubule Pax2-positive nuclei (red) were identified in kidney tubule cryosections at baseline and over a two-weekspan after injury Elevated Pax2 expression was detected at 5 7 and 10 dpi Nuclei were stained with DAPI (blue) Scale bar 25120583m (b) IntensePax2 immunoreactivity demarcated neonephrogenic units High levels of Pax2 expression (red) were present in coil-like structures at 5 dpiAt 10 dpi Pax2 continued to stain profound numbers of cells in the coil-like neonephron structures Nuclei are stained with DAPI (blue)Scale bar 25 120583m


to 174plusmn 039by 10 dpi PCNAstainingwas also completedin combination with the proximal tubule marker ELF-97which showed similar dynamics in the regenerating nephronepithelium with the proliferation at 3 5 and 7 dpi (data notshown)

Interestingly high levels of PCNA were observed inneonephrogenic kidney structures during this time courseas well (Figure 5(c)) Beginning at 3 dpi when aggregatesare forming intense PCNA expression was found in theentire structure colocalizing with DAPI-stained nuclei (Fig-ure 5(c)) By 5 and 7 dpi when aggregates have formedlumens and transform into coil-like neonephrons high levelsof PCNA were still present (Figure 5(c)) At 10 dpi PCNAcontinued to be abundant in these neonephrogenic structuresthat now had a brush border that stained positive for LTL(Figure 5(c)) Based on these observations it appears as if theintensely stained PCNA-positive neonephron structures havebecome proximal tubules However genetic fate mappingis needed to definitively track the progression of thesestructures and label them as having a proximal fate

As another measure to evaluate cell proliferation we per-formed BrdU pulse-chase experiments in zebrafish followinggentamicin-induced AKI (Figure 6) For this examinationan intraperitoneal injection of BrdU was administered touninjured zebrafish and gentamicin-injected zebrafish 24hours prior to each timepoint for analysis over a 5-day courseand kidneys were then analyzed by immunofluorescenceto detect BrdU-positive cells in proximal tubules labeledwith LTL (Figure 6) In uninjured kidneys we found that125 plusmn 032 of proximal tubule cells had incorporatedthe BrdU label (Figures 6(a) and 6(b)) After gentamicininjection at 1 2 and 3 dpi there was a low incidenceof BrdU incorporation (044 plusmn 019 042 plusmn 018083 plusmn 037 resp) (Figures 6(a) and 6(b)) At 4 and5 dpi however the incidence of BrdU-positive cells in LTL-stained proximal tubules increased to 509 plusmn 116 and742 plusmn 173 respectively (Figures 6(a) and 6(b)) WhileANOVA statistical analysis failed to show that the increasein BrdU incorporation was significant the overall trend ofelevated BrdU incorporation over time in the regeneratingproximal tubules is consistent with the observations of PCNAincorporation over time (Figure 5) Taken together thesedata show that proliferation in regenerating proximal tubulesoccurs within the first week following injury

26 Restoration of Additional Proximal Tubule StructuralCharacteristics and Absorptive Functions following Gentami-cin-Induced AKI To further visualize the regeneration ofproximal tubule structures following gentamicin-inducedinjury and to assess the restoration of proximal tubule phys-iological function we utilized alkaline phosphatase stainingand dextran uptake assays respectively [32] In whole mountkidney preparations these assays enable a three-dimensionalassessment of nephrons throughout the organ (Figure 7)[32] Labeling of alkaline phosphatase activity with ELF-97specifically enables the visualization of the entire proximaltubule both the PCT and PST segments which are connectedand have distinguishing diameters with the PCT beingdistinctively wide compared to the narrow diameter of the

attached PST (Figure 7(a)) [32] At 1 and 3 dpi alkaline phos-phatase reactivity was diminished and dispersed throughoutthe kidney with few PCT structures evident (Figure 7(a))At 5 dpi and 7 dpi tubules were more distinctly labeled withalkaline phosphatase but wide PCT-like tubules were rarelyobserved (Figure 7(a)) By 14 dpi there was a clear restorationof PCT-PST structures labeled with alkaline phosphatasereactivity (Figure 7(a))

In parallel we examined renal uptake of fluorescentlylabeled dextran moieties an assay that determines whetherthe PCT epithelial cells are capable of endocytosis [32] Unin-jured kidney tubules evinced PCT-specific uptake of fluoro-ruby dextran or fluorescein dextran while this propertywas abrogated following gentamicin injury at 1 and 3 dpi(Figure 7(b) data not shown) Between 5 dpi and 19 dpi PCTuptake of fluoro-ruby or fluorescein dextran was sporadicwith labeling detected in only a few nephron tubules andnot until the 21 dpi time point was PCT uptake consistentacross nephrons of the entire organ (Figure 7(b) data notshown) This suggests that regeneration of PCT functionalityrequires up to three weeks even though at two weeksthe nephron tubules appear structurally intact by alkalinephosphatase labeling (Figure 7(a)) and other histologicalmethods (Figure 2)

27 Pax2 Expression Marks Regenerating Tubular EpithelialCells and Neonephrogenic Structures The process of nephro-genesis is controlled by specific genes that can either enhanceor inhibit cell survival and direct subsequent proliferationand differentiation events [51 52] One such gene is thePax2 transcription factor During development when renalcells undergo a mesenchymal to epithelial transition intocondensed cellular aggregates and differentiate into nephrontubules they express an increased level of Pax2 later innephrogenesis the transcription factor is downregulated [53ndash55] In order to restore organ or tissue function in adultanimals after undergoing physical damage or injury it hasbeen suggested that the regeneration processmay recapitulatespecific developmental processes [56 57] In keeping withthis notion previous research has demonstrated that Pax2is reexpressed in nephron tubular cells following AKI in theadult mouse [58] as well as in zebrafish embryonic nephronssubsequent to gentamicin-induced AKI [59]

In order to test the hypothesis that developmental genesare reexpressed during regenerative events and explorewhether zebrafish adult nephrons similarly show tubularPax2 expression following AKI an antibody to Pax2 was usedfor immunolabeling during the adult zebrafish injury andrepair time course (Figure 8) Analysis of Pax2 expressionin the injured kidney revealed that this developmental tran-scription factor was expressed in the epithelial tubules at alow level in the uninjured nephron tubule and was morestrongly expressed in tubular cells at multiple time pointsover a two-week time span after injury (Figure 8(a)) Notablya higher expression level of Pax2 was present between 5and 7 dpi (Figure 8(a)) A low level was still detectable inthe repaired tubules by 14 dpi comparable to the untreatedkidney (Figure 8)


Further similar to PCNA expression patterns in neone-phrogenic structures Pax2 was expressed at high levelsin the cells of coiled bodies and other neonephrogenesisstructures at 5 and 10 dpi respectively (Figure 8(b)) Thetubules containing Pax2-positive cells were distinguishedfrom the neonephrogenic structures based on lumen diam-eter Tubules that were undergoing repair possessed a largerlumen representative of a tubule that was previously estab-lished and functioning in the kidney In contrast lumens ofthe neonephrons were very small and appeared to expandin time beyond that which is documented in this timecourse Taken together these data demonstrate that expres-sion of Pax2 accompanies regeneration of the proximal tubuleepithelium as well as neonephrogenesis in the zebrafishkidney

3 Discussion

To date three modes of kidney regeneration have been char-acterized following exposure to nephrotoxins or mechanicalinjuries (1) tubular epithelium regeneration in which exist-ing nephrons are repopulated after cells have been destroyed(2) compensatory renal hypertrophy in which remainingkidney structures enlarge typically observed following uni-lateral nephrectomy and (3) nephron neogenesis from renalmesenchymal progenitorstem cells [60] Vertebrate speciesvary with regard to whether they can perform several orall three of these feats [6 60] For example humans andother mammals are incapable of developing new nephronsfollowing either gestation or the neonatal period a featureknown to be associated with renal stem cell exhaustionduring metanephros ontogeny [52 61ndash67] In contrast fishand amphibians have versatile regenerative traits throughoutjuvenile stages as well as adulthood that include the for-mation of entirely new nephrons [10] While various fishspecies including goldfish medaka skate trout tilapia andtoadfish can undergo kidney regeneration [33 68ndash71] thezebrafish provides an advantage to discover the pathwaysand signaling events involved in kidney regeneration dueto their genetic tractability Before undertaking traditionalgenetic or chemical screens using zebrafish to identify the castof components in renal regeneration however it is vital tohave a thorough understanding of the progression of tissuechanges that transpires following toxicant exposure

Here we have further characterized the spatiotemporalsequence of cellular and gene expression changes associatedwith regeneration of the zebrafish nephron tubular epithe-lium and also annotated a number of features associatedwith neonephrogenesis In sum our work has revealedthat the injured nephron tubule epithelium is regeneratedwithin one week of damage involving partially overlappingwaves of cell death and proliferation that is accompanied byPax2 expression (Figure 9) Functionality of the regeneratednephrons is subsequently restored between 2 and 3 weeksfollowing damage In agreement with prior studies wefound that neonephrogenesis commences by approximately5 dpi with nephron clusters forming new nephrons overthe subsequent week and show for the first time that thenew nephrons possess the proximal tubule feature of PAS

reactivity (Figure 9) While this suggests that new nephronshave proximal character genetic fate mapping studies areneeded to ascertain what functional segment(s) the newnephrons come to possess Given the highly branched natureof the nephron arrangements in the zebrafish mesonephrosit is an attractive hypothesis that new nephrons commonlyplumb into preexisting proximal segments thus adding tothe filtration and bulk reabsorption functions of the kidneywhile utilizing the existing distal and collecting duct systemsfor fine-tuning of salt balance in the urinary stream

31 Toolkit for Cellular and Molecular Renal Studies in Zebra-fish These studies provide a new and important descriptiveatlas of the cellular changes that transpire during zebrafishadult kidney regeneration Moreover in the pursuit of thesestudies we have refined a number of histological methodsfor their application in the adult zebrafish kidney Togetherthis set of information andmethodologies provide a resourcefor further studies in this promising regeneration modelThree histological stains that have proven to be valuableinclude HampE stain PAS stain and silver stainTheHampE staindistinguishes the proximal tubules from the distal tubulesbased essentially on the presence of a brush border (a markedfeature of proximal tubules) The reagents in the PAS stainhave a high affinity for the brush borders of proximal tubulesand allow a more effective characterization of the varyingstructures within the kidney tissueThe silver stain also stainsthe brush borders a discernable dark brown color allowingdistinction from distal tubules Notably the silver stain alsolabels hyaline droplets formed from protein reabsorptionwhich are located specifically in the proximal tubule The useof lectin stains to distinguish tubule compartments based onsugar-binding proteins also plays a vital role in this noveltoolkit LTL is a robust proximal tubule marker labeling thebrush borders of the tubule DBA is a marker for the distaltubules Finally the ELF-97 staining method which detectshigh levels of endogenous alkaline phosphatase activity inbrush borders is a consistent technique to differentiate theproximal tubules from the distal tubules A major limitationofworkingwith these andothermarkers noted above involvesincompatibilities with kidney tissue fixation requirementsWe have found the most success in zebrafish renal histologyprocedures by fixing the organ in two different ways fixa-tion in paraformaldehyde (with or without antigen retrievalprior to immunolabeling) or ethanolic formaldehyde Thesemethodologies should prove to be useful to further studyrenal regeneration in the adult kidney as well as establishedand emerging models of embryonic nephron injury andregeneration [22 59 72ndash75] However not all markers workwith both fixatives in combination with immunofluorescentantibodies and a current limitation in the zebrafish systemis the paucity of commercially available antibodies Futurework will mostly likely benefit by examinations of geneexpression by additional in situhybridization studies inwholemount or sections [32]These methods enable the spatial andtemporal localization of gene transcripts which is feasiblefor any gene(s) of interest because the zebrafish genomehas been sequenced and because appropriate reagents arecommercially available for gene expression studies


Healthy nephron tubule

Day 0 Day 3 Day 5

Injury

Process 2neonephrogenesis

Cell death

Cell proliferation Pax2+


Days 7ndash14

epithelialProcess 1

regeneration

Figure 9 Summary of major cellular events in zebrafish kidney regeneration After injury to proximal tubules of nephrons within the adultzebrafish kidney successive and overlapping waves of cell death and cell proliferation occur Cell proliferation is accompanied by Pax2expression Neonephrogenesis entails abundant Pax2 expression and cell proliferation in neonephron structures during the regenerationprocess

32 Stem Cells and Their Roles in Zebrafish Adult KidneyRegeneration Studies in various mammalian species havedemonstrated that intratubular proliferation occurs in thehealthy nephron tubule [76ndash78] Similarly in the presentwork we have documented a low level of proliferation inthe uninjured zebrafish proximal tubule based on PCNAreactivity and BrdU incorporation in LTL+ nephron cellsFate mapping studies in the mouse have clearly demon-strated that intratubular nephron populations replenish theinjured nephron [79 80] However the nature of theseregenerating cells in the damagedmammalian kidney tubulesremains a topic of intense debate At present two hypothesesexist that describe the cellular attributes of this intratubu-lar cell source One scenario involves the dedifferentia-tion of epithelial cells that will migrate and proliferateto repair injured tubules The second scenario posits thatstemprogenitor cells located within the tubule undergodivision and amplification in response to tissue damageThere is experimental evidence among mammalian modelsthat supports both modelsmdashfate mapping studies in themouse support dedifferentiation [8] while there is alsoopposing evidence from human kidney research that uniquesubpopulations of renal cells with features suggestive of stemcell character are located among the nephron epithelial cellsand that these cells fuel tubular regeneration [9] Whetherthere are differences across mammalian species or whetheradditional studies will eventually reconcile these conflict-ing data remains a fascinating area of current nephrologyresearch

As such an important aspect of future renal regen-eration research with regard to the zebrafish model willbe to clarify the origin of reparative epithelia in existingnephrons through transgenic genetic fate mapping Suchlineage analysis will be vital for evaluating the origins ofthe cells in repaired tubules Further since renal cells can

be isolated by flow cytometry and then operationally testedthrough transplantation procedures [81] in vivo experimentsto test the replicative and differentiation potential of putativeintratubular stemprogenitor cells in zebrafish if they areidentified are likely to become feasible

33 Complexities and Benefits in Comparing Zebrafish andMammals Ultimately the degree to which the zebrafishkidney is ldquouniquerdquo from higher vertebrates including mam-mals is an important biological question to understand Forexample the zebrafish adult kidney stroma is the site ofhematopoiesismdashthus the microenvironment in the zebrafishkidney is arguably quite distinct from mammalian counter-parts in which blood production ensues elsewhere Researchthat uncovers these or other differences and how they impactrenal regeneration capacities may provide vital insights intomethods that could be used to stimulate similar reparativeresponses to treat kidney injury and disease or assist indesigning reprogramming strategies [82ndash84] The data pre-sented here provide a valuable foundation for researchersthat aim to embark on such genetic and cellular studiesto ascertain the identities of the molecules and signalingpathways that activate and regulate renal regeneration inzebrafish

4 Experimental Procedures

41 Zebrafish Strain and Maintenance Adult zebrafish ofthe Tubingen wild-type strain were raised and maintainedat 285∘C on a 14-hour light 10-hour dark cycle at anaverage luminance of 200 lux [85] in the Center for ZebrafishResearch at the Notre Dame Freimann Life Science CenterAll protocols were approved by the IACUC of the Universityof Notre Dame animal protocols 13-021 and 16-025


42 Gentamicin Injections and Kidney Dissections For gen-tamicin injections zebrafish were anesthetized with a dilutedworking solution of 002 Tricaine made using a 02Tricaine pH 70 stock for approximately 2-3 minutes andtransferred to an injection mold Fish received an intraperi-toneal injection of 25mgmL gentamicin and were returnedto a clean system tank At various time points adults wereeuthanized with an overdose of Tricaine and fixed witheither 4 paraformaldehyde1X PBS01 dimethyl sulfoxide(DMSO) or 9 1 ethanolic formaldehyde (100 ethanol 37formaldehyde) The kidneys of adult fish were dissected aspreviously described [27 32] Briefly fish were euthanizedwith 02 Tricaine pH 70 for approximately 5 minutesDissection needles were used to pin open the body wallsby attaching them to a dissection tray that contained 4paraformaldehyde1X PBS01 DMSO The samples werefixed overnight at 4∘C The following day the fixative wasremoved from the tray and fine forceps were used to detachthe kidney from the dorsal wall

43 Whole Mount In Situ Hybridization Whole mountin situ hybridization (WISH) on adult kidneys was per-formed as previously described [27 32] Briefly kidneyswere fixed in 4 paraformaldehyde1X PBS01 DMSOand pigmentation in the organ was removed by hydrogenperoxide treatment Kidneys underwent permeabilizationand hybridization steps in a humidified chamber at 70∘Covernight Samples were then incubated in blocking buffer atroom temperature (10 bovine serum albumin and 5 fetalcalf serum) and following extensive washes digoxigenin-labeled probes were detected with alkaline phosphatase con-jugated to an antidigoxigenin antibody NBTBCIP (Sigma-Aldrich) served as the enzymatic substrate for the pur-ple color reaction Color reactions were stopped with 4paraformaldehyde1X PBS

44 Tissue Cryosections As described previously [32] adultfish were fixed in either 4 paraformaldehyde1X PBS01DMSO or 9 1 ethanolic formaldehyde overnight at 4∘Cand the kidneys were dissected out the next day Sampleswere washed with 5 sucrose1X PBS cryoprotected in 30sucrose1X PBS overnight at 4∘C and then subsequentlywashed in 1 1 tissue freezing medium (TFM TriangleBiomedical Sciences) 30 sucrose1X PBS overnight at 4∘CThe following day samples were embedded in 100 TFMSerial sections of approximately 12ndash14 120583m thickness weretransversely cut through the entire kidney Frozen cryosec-tions were mounted onto glass microscope slides (TruBond380 Microscope Slides Tru Scientific) and allowed to air-dryfor 1 hour at 50∘C Slides were stored at minus80∘C until use

45 Histology Analysis For all histological stains adult fishwere euthanized at various time points by an overdose ofTricaine and fixed overnight in 4 paraformaldehyde1XPBS01DMSO Kidneys were dissected out washed in 70ethanol at 4∘C and then were paraffin-embedded and seriallysectioned on a microtome After slides were deparaffinizedand rehydrated sections were stained with hematoxylin andeosin periodic acid-Schiff or methenamine silver (Notre

Dame Integrated Imaging FacilityndashHistology Core) Mousekidney sections (a generous gift from the Notre DameHistology Core) were treated with the same histologicalprotocol

46 BrdU Incorporation Cell proliferation was assayedthrough BrdU incorporation Adult zebrafish were anes-thetized in 002 Tricaine pH 70 and intraperitoneallyinjectedwith 5mMBrdU (Molecular Probes) 24 hours beforesacrifice Cells that incorporated BrdU were visualized byimmunofluorescence analysis

47 Immunofluorescence Slides were thawed for 30 minutesat 50∘C and then rehydrated in 1X PBS005 Tween-20Cryosections were incubated at room temperature in block-ing solution 1X PBS005Tween-2010 fetal calf serum1DMSO for 2 hours and then placed at 4∘C for overnight pri-mary antibody incubation Primary antibodies were dilutedin block and included mouse anti-Green Fluorescent Pro-tein monoclonal antibody (1 500 Sigma-Aldrich) rabbitanti-Pax2 polyclonal antibody (1 50 Covance) rabbit anti-myosin VI antibody (1 50 Sigma-Aldrich) mouse anti-BrdU (1 50Molecular Probes) andmouse anti-proliferatingcell nuclear antigen (PCNA) polyclonal antibody (1 1000Sigma-Aldrich) Following primary antibody incubationcryosections were washed in 1X PBS005 Tween-20 andincubated in secondary antibody solution for 2 hours at roomtemperature Secondary antibodies were diluted 1 500 in 1XPBS005 Tween-20 and included Alexa Fluor 488 and 568goat anti-mouse IgG and 594 goat anti-rabbit IgG (MolecularProbes) Nuclei were labeled with DAPI (Molecular Probes)for 5 minutes Cryosections were washed with 1X PBSand mounted with Vectashield Mounting Medium (VectorLaboratories) Antigen retrieval was performed by incubatingslides between 95∘C and 100∘C for 40 minutes in preheated10mM sodium citrate buffer for Pax2 and PCNA labelingor by incubating cryosections with 2M HCl at 37∘C for 30minutes for BrdU labeling Sections were then washed andimmunolabeled as described above

48 Identification of Sectioned Tubule Segments Tubularsegments of the kidney were identified by utilizing the fol-lowing markers fluorescein Lotus tetragonolobus lectin (LTLVector Laboratories) diluted 1 100 in 1X PBS for 2 hoursto label the proximal tubule enzyme labeled fluorescence-(ELF-) 97 (Molecular Probes) diluted 1 20 in detectionbuffer (included in kit) for 1 hour to label the proximaltubule rhodamine Dolichos biflorus agglutinin (DBA VectorLaboratories) diluted 1 100 in 1X PBS for 2 hours to labelthe distal tubule [32] If colabeling with an antibody LTLandor DBA stains were applied directly after the secondaryantibody incubation DAPI immunolabeling followed LTLandor DBA as described above For ELF-97 colabeling [32]substrate solution was applied directly after the secondaryantibody solutionThe reaction was stopped with wash buffer1XPBS25mMEDTA5mM levamisol pH80 incubating thecryosectionswith fresh buffer 3 times for 15minutes each andthen visualized in ELF-97 mounting medium


49 Whole Mount Kidney Morphology Assays

491 Dextran Labeling Whole mount labeling of proximalconvoluted tubule segments in the kidney was followed aspreviously described [32] In short adult zebrafish wereanesthetized and injected intraperitoneally with 50mgmLfluoro-ruby dextran (Invitrogen) and returned to a cleansystem tank The next day the fish were sacrificed and thekidney was dissected out for fluorescent tubule visualization

492 ELF-97 Labeling Whole mount labeling of pan-proximal segments was performed as previously described[32] Briefly kidneys were subjected to fixation dissectionpermeabilization and pigmentation removal The kidneyswere blocked and then incubated inELF-97 substrate solutionfor 30 minutes Once the reaction was stopped multiplewashes were performed and the fluorescent proximal seg-ments were visualized

493 LTL and DBA Labeling Whole mount labeling of pan-distal segments in the kidney was conducted as previouslydescribed [32] In brief kidneys were fixed dissected andpermeabilized and pigment was removed After blockingkidneys were incubated in the respective staining solutionOnce the staining solution was removed with several washesthe fluorescent signal(s) could be visualized

410 TUNEL Assay Apoptotic cells were identified with theTUNEL assay using theApoAlert DNAFragmentationAssayKit (Clontech Laboratories) and the TACS 2 TdT ReplenisherKit (Trevigen) Adult zebrafish were fixed in 9 1 ethanolicformaldehyde and their kidneys were dissected embeddedand cryosectioned as previously described Cryosectionswere thawed at 50∘C for 1 hour permeabilized with 1X PBSfor 20 minutes 01 sodium citrate buffer01 Triton X-100 for 2 minutes and again with 1X PBS for 5 minutes atroom temperature Equilibration buffer was applied directlyto the cryosections for 10 minutes followed by the additionof biotinylatedDNTPs and TdT enzyme (both at a concentra-tion of 1 50 in equilibration buffer) for 2 hours at 37∘C Thelabeling reaction was terminated by incubating cryosectionsin 2X SSC stop buffer for 15 minutes Positive nuclei werevisualized by applying Alexa Fluor 568 Streptavidin dilutedin 1X PBS (1 200 Molecular Probes) for 1 hour

411 Statistical Analysis Statistical significance among exper-imental groups was analyzed using the one-way ANOVAfollowed by Tukeyrsquos HSD multiple comparisons test using Rversion 303 Data shown are mean plusmn SEM Significance wasaccepted at 119875 lt 005 or greater

Key Findings

(i) A suite of histological stains was characterized toprovide tools to identify distinguishing features ofzebrafish adult kidney anatomy including nephronproximal tubule traits

(ii) Cell death and proliferation in the injured proximaltubule are dynamic and transpire in successive wavesof activity the first week following injury while func-tional restoration occurs over the subsequent weeks

(iii) Spatiotemporal immunofluorescence studies revealedthat Pax2 is expressed both in the epithelial popula-tion in regenerating nephrons and in neonephrogenicclusters that are associated with the production of denovo nephrons after AKI

Abbreviations

AKI Acute kidney injuryAP Alkaline phosphataseBrdU 5-Bromo 21015840deoxyuridineCD Collecting ductdpi Days after injuryDAPI 410158406-Diamidino-2-phenylindoleDBA Dolichos biflorus agglutininDE Distal earlyDL Distal lateDT Distal tubuleELF-97 Enzyme labeled fluorescence-97G GlomerulusHampE Hematoxylin and eosinLTL Lotus tetragonolobus lectinN NeckPAS Periodic acid-SchiffPax2 Paired box gene 2PCNA Proliferating cell nuclear antigenPCT Proximal convoluted tubulePST Proximal straight tubulePT Proximal tubuleT TubuleTUNEL Terminal deoxynucleotidyl transferase

dUTP nick end labelingWISH Whole mount in situ hybridization

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Dr Clay Connor for kindly providinghis zebrafish histology and immunofluorescence expertiseduring the refinement of methodologies that were employedin the study They also thank Shanna Newton Elvin Moralesand Charles Renner for performing exploratory studiesrelated to this project They thank the entire staff of theDepartment of Biological Sciences for ongoing support andthe Center for Zebrafish Research at Notre Dame for theirdedication and care of our zebrafish aquarium They espe-cially thank the Notre Dame Integrated Imaging Facility andHistology Core for reagents and other research supportsFinally they thank the members of our research lab for theircomments discussions and insights about this work The


research is supported by National Institutes of Health (NIH)Grant nos DP2OD008470 K01DK083512 and R01DK100237and March of Dimes Basil OrsquoConnor Starter Scholar GrantAward no 5-FY12-75 Support was also provided from startupfunding to RAW from the University of Notre Dame Collegeof Science and Department of Biological Sciences as wellas a Gift to the University of Notre Dame from Elizabethand Michael Gallagher to support stem cell research KristinSpringer was supported by summer fellowship funding fromtheGlynn FamilyHonors Program and theCollege of Scienceat the University of Notre Dame

References

[1] L Saxen Organogenesis of the Kidney Cambridge UniversityPress Cambridge UK 1987

[2] K K Mccampbell and R A Wingert ldquoRenal stem cells fact orscience fictionrdquo Biochemical Journal vol 444 no 2 pp 153ndash168 2012

[3] P Romagnani ldquoToward the identification of a lsquorenopoieticsystemrsquordquo Stem Cells vol 27 no 9 pp 2247ndash2253 2009

[4] R A Wingert and A J Davidson ldquoThe zebrafish pronephrosa model to study nephron segmentationrdquo Kidney Internationalvol 73 no 10 pp 1120ndash1127 2008

[5] J V Bonventre and L Yang ldquoCellular pathophysiology ofischemic acute kidney injuryrdquo Journal of Clinical Investigationvol 121 no 11 pp 4210ndash4221 2011

[6] R Reimschuessel ldquoA fish model of renal regeneration anddevelopmentrdquo ILAR Journal vol 42 no 4 pp 285ndash291 2001

[7] A Benigni M Morigi and G Remuzzi ldquoKidney regenerationrdquoThe Lancet vol 375 no 9722 pp 1310ndash1317 2010

[8] B D Humphreys ldquoKidney injury stem cells and regenerationrdquoCurrent Opinion in Nephrology and Hypertension vol 23 pp25ndash31 2013

[9] P Romagnani L Lasagni and G Remuzzi ldquoRenal progenitorsan evolutionary conserved strategy for kidney regenerationrdquoNature Reviews Nephrology vol 9 no 3 pp 137ndash146 2013

[10] K K McCampbell and R A Wingert ldquoNew tides usingzebrafish to study renal regenerationrdquo Translational Researchvol 163 no 2 pp 109ndash122 2014

[11] R AWingert R Selleck J Yu et al ldquoThe cdx genes and retinoicacid control the positioning and segmentation of the zebrafishpronephrosrdquo PLoS Genetics vol 3 no 10 pp 1922ndash1938 2007

[12] R A Wingert and A J Davidson ldquoZebrafish nephrogenesisinvolves dynamic spatiotemporal expression changes in renalprogenitors and essential signals from retinoic acid and irx3brdquoDevelopmental Dynamics vol 240 no 8 pp 2011ndash2027 2011

[13] I A Drummond A Majumdar H Hentschel et al ldquoEarlydevelopment of the zebrafish pronephros and analysis of muta-tions affecting pronephric functionrdquo Development vol 125 no23 pp 4655ndash4667 1998

[14] G F Gerlach and R A Wingert ldquoKidney organogenesisin the zebrafish insights into vertebrate nephrogenesis andregenerationrdquo Wiley Interdisciplinary ReviewsmdashDevelopmentalBiology vol 2 no 5 pp 559ndash585 2013

[15] R W Naylor A Przepiorski Q Ren J Yu and A J DavidsonldquoHNF1120573 is essential for nephron segmentation during nephro-genesisrdquo Journal of the American Society of Nephrology vol 24no 1 pp 77ndash87 2013

[16] Y Li C N Cheng V A Verdun and R A Wingert ldquoZebrafishnephrogenesis is regulated by interactions between retinoicacid mecom and notch signalingrdquo Developmental Biology vol386 no 1 pp 111ndash122 2014

[17] CN Cheng Y Li AMarra V Verdun andR AWingert ldquoFlatmount preparation for observation and analysis of zebrafishembryo specimens stained by whole mount in situ hybridiza-tionrdquo Journal of Visualized Experiments vol 89 Article IDe51604 2014

[18] C N Cheng and R A Wingert ldquoNephron proximal tubulepatterning and corpuscles of Stannius formation are regulatedby the sim1a transcription factor and retinoic acid in zebrafishrdquoDevelopmental Biology vol 399 no 1 pp 100ndash116 2015

[19] P T Kroeger Jr S J Poureetezadi R McKee et al ldquoProductionof haploid zebrafish embryos by in vitro fertilizationrdquo Journal ofVisualized Experiments no 89 Article ID e51708 2014

[20] G F Gerlach and R A Wingert ldquoZebrafish pronephros tubu-logenesis and epithelial identity maintenance are reliant on thepolarity proteins Prkc iota and zetardquoDevelopmental Biology vol396 no 2 pp 183ndash200 2014

[21] R McKee G F Gerlach J Jou C N Cheng and R AWingertldquoTemporal and spatial expression of tight junction genes duringzebrafish pronephros developmentrdquo Gene Expression Patternsvol 16 no 2 pp 104ndash113 2014

[22] D M Hentschel K M Park L Cilenti A S Zervos I Drum-mond and J V Bonventre ldquoAcute renal failure in zebrafish anovel system to study a complex diseaserdquoTheAmerican Journalof PhysiologymdashRenal Physiology vol 288 pp F923ndashF929 2005

[23] C C Cosentino B L Roman I A Drummond and N AHukriede ldquoIntravenous microinjections of zebrafish larvae tostudy acute kidney injuryrdquo Journal of Visualized Experimentsno 42 Article ID e2079 2010

[24] S A Rider C S Tucker J del-Pozo et al ldquoTechniques for thein vivo assessment of cardio-renal function in zebrafish (Daniorerio) larvaerdquo Journal of Physiology vol 590 no 8 pp 1803ndash1809 2012

[25] L Ebarasi A Oddsson K Hultenby C Betsholtz and K Tryg-gvason ldquoZebrafish a model system for the study of vertebraterenal development function and pathophysiologyrdquo CurrentOpinion in Nephrology and Hypertension vol 20 no 4 pp 416ndash424 2011

[26] L M Swanhart C C Cosentino C Q Diep A J DavidsonM de Caestecker and N A Hukriede ldquoZebrafish kidneydevelopment basic science to translational researchrdquo BirthDefects Research CmdashEmbryo Today Reviews vol 93 no 2 pp141ndash156 2011

[27] G F Gerlach L N Schrader and R A Wingert ldquoDissection ofthe adult zebrafish kidneyrdquo Journal of Visualized Experimentsno 54 Article ID e2839 2011

[28] W Zhou R C Boucher F Bollig C Englert and F Hilde-brandt ldquoCharacterization of mesonephric development andregeneration using transgenic zebrafishrdquoThe American Journalof PhysiologymdashRenal Physiology vol 299 no 5 pp F1040ndashF1047 2010

[29] C Q Diep D Ma R C Deo et al ldquoIdentification ofadult nephron progenitors capable of kidney regeneration inzebrafishrdquo Nature vol 470 no 7332 pp 95ndash100 2011

[30] W Zhou and F Hildebrandt ldquoInducible podocyte injury andproteinuria in transgenic zebrafishrdquo Journal of the AmericanSociety of Nephrology vol 23 no 6 pp 1039ndash1047 2012

[31] J HuangMMcKeeHDHuang A Xiang A J Davidson andH A J Lu ldquoA zebrafishmodel of conditional targeted podocyte


ablation and regenerationrdquo Kidney International vol 83 no 6pp 1193ndash1200 2013

[32] K K McCampbell K N Springer and R AWingert ldquoAnalysisof nephron composition and function in the adult zebrafishkidneyrdquo Journal of Visualized Experiments no 90 Article IDe51644 2014

[33] N Watanabe M Kato N Suzuki et al ldquoKidney regenerationthrough nephron neogenesis in medakardquo Development Growthand Differentiation vol 51 no 2 pp 135ndash143 2009

[34] P Vinay A Gougoux and G Lemieux ldquoIsolation of a puresuspension of rat proximal tubulesrdquo The American Journal ofPhysiology vol 241 no 4 pp F403ndashF411 1981

[35] J B Longley and E R Fisher ldquoA histochemical basis for changesin renal tubular function in young micerdquo Quarterly Journal ofMicroscopical Science vol 97 part 2 pp 187ndash195 1956

[36] CM Tsai andC E Frasch ldquoA sensitive silver stain for detectinglipopolysaccharides in polyacrylamide gelsrdquo Analytical Bio-chemistry vol 119 no 1 pp 115ndash119 1982

[37] K Amann and C S Haas ldquoWhat you should know about thework-up of a renal biopsyrdquoNephrology Dialysis Transplantationvol 21 no 5 pp 1157ndash1161 2006

[38] D B Jones ldquoNephrotic glomerulonephritisrdquo The AmericanJournal of Pathology vol 33 no 2 pp 313ndash329 1957

[39] L Watson J Vassallo G G Cantor et al ldquoLectin histochem-istry an alternative to immunohistochemistry for identifyingspecific structures in rat renal papillary necrosisrdquo HistoLogicvol 41 pp 28ndash31 2008

[40] F Cachat B Lange-Sperandio A Y Chang et al ldquoUreteralobstruction in neonatal mice elicits segment-specific tubularcell responses leading to nephron lossrdquo Kidney Internationalvol 63 no 2 pp 564ndash575 2003

[41] H-T Cheng M Kim M T Valerius et al ldquoNotch2 butnot Notch1 is required for proximal fate acquisition in themammalian nephronrdquoDevelopment vol 134 no 4 pp 801ndash8112007

[42] R G B Bonegio L H Beck R K Kahlon W Lu and DJ Salant ldquoThe fate of Notch-deficient nephrogenic progenitorcells duringmetanephric kidney developmentrdquoKidney Interna-tional vol 79 no 10 pp 1099ndash1112 2011

[43] E Mochizuki K Fukuta T Tada et al ldquoFish mesonephricmodel of polycystic kidney disease in medaka (Oryzias latipes)pc mutantrdquo Kidney International vol 68 no 1 pp 23ndash34 2005

[44] H Harris ldquoThe human alkaline phosphatases what we knowand what we donrsquot knowrdquo Clinica Chimica Acta vol 186 no 2pp 133ndash150 1990

[45] W G Cox and V L Singer ldquoA high-resolution fluorescence-based method for localization of endogenous alkaline phos-phatase activityrdquo Journal of Histochemistry and Cytochemistryvol 47 no 11 pp 1443ndash1455 1999

[46] R F Reilly R E Bulger and W Kriz ldquoStructural-functionalrelationships in the kidneyrdquo in Diseases of the Kidney andUrinary Tract LippincottWilliams ampWilkins Philadelphia PaUSA 8th edition 2007

[47] I A Drummond and A J Davidson ldquoZebrafish kidney devel-opmentrdquoMethods in Cell Biology vol 100 pp 233ndash260 2010

[48] C Ronco R Bellomo and J Kellum Critical Care NephrologyElsevier Saunders Philadelphia Pa USA 2nd edition 2009

[49] Y Gavrieli Y Sherman and S A Ben-Sasson ldquoIdentificationof programmed cell death in situ via specific labeling of nuclearDNA fragmentationrdquo Journal of Cell Biology vol 119 no 3 pp493ndash501 1992

[50] K M Connolly and M S Bogdanffy ldquoEvaluation of prolifer-ating cell nuclear antigen (PCNA) as an endogenous markerof cell proliferation in rat liver a dual-stain comparison with5-bromo-21015840-deoxyuridinerdquo Journal of Histochemistry and Cyto-chemistry vol 41 no 1 pp 1ndash6 1993

[51] A S Woolf ldquoMultiple causes of human kidney malformationsrdquoArchives of Disease in Childhood vol 77 no 6 pp 471ndash473 1997

[52] M H Little and A P McMahon ldquoMammalian kidney develop-ment principles progress and projectionsrdquoCold Spring HarborPerspectives in Biology vol 4 no 5 Article ID a008300 2012

[53] G R Dressler U Deutsch K Chowdhury H O Nornes and PGruss ldquoPax2 a newmurine paired-box-containing gene and itsexpression in the developing excretory systemrdquo Developmentvol 109 no 4 pp 787ndash795 1990

[54] P J D Winyard R A Risdon V R Sams G R Dresslerand A S Woolf ldquoThe PAX2 transcription factor is expressedin cystic and hyperproliferative dysplastic epithelia in humankidney malformationsrdquo Journal of Clinical Investigation vol 98no 2 pp 451ndash459 1996

[55] R Attar F Quinn P J D Winyard et al ldquoShort-term urinaryflow impairment deregulates PAX2 and PCNA expression andcell survival in fetal sheep kidneysrdquo The American Journal ofPathology vol 152 no 5 pp 1225ndash1235 1998

[56] R Bacallao and L G Fine ldquoMolecular events in the orga-nization of renal tubular epithelium from nephrogenesis toregenerationrdquoTheAmerican Journal of PhysiologymdashRenal Fluidand Electrolyte Physiology vol 257 no 6 pp F913ndashF924 1989

[57] A Wallin G Zhang T W Jones S Jaken and J LStevens ldquoMechanism of the nephrogenic repair responsestudies on proliferation and vimentin expression after 35S-12-dichlorovinyl-L-cysteine nephrotoxicity in vivo and in culturedproximal tubule epithelial cellsrdquo Laboratory Investigation vol66 no 4 pp 474ndash484 1992

[58] M Imgrund E Grone H-J Grone et al ldquoRe-expression of thedevelopmental gene Pax-2 during experimental acute tubularnecrosis in micerdquo Kidney International vol 56 no 4 pp 1423ndash1431 1999

[59] C C Cosentino N I Skrypnyk L L Brilli et al ldquoHistonedeacetylase inhibitor enhances recovery after AKIrdquo Journal ofthe American Society of Nephrology vol 24 no 6 pp 943ndash9532013

[60] Y Li and R AWingert ldquoRegenerative medicine for the kidneystem cell prospects amp challengesrdquo Clinical and TranslationalMedicine vol 2 no 1 article 11 2013

[61] D Herzlinger C Koseki T Mikawa and Q Al-AwqatildquoMetanephric mesenchyme contains multipotent stem cellswhose fate is restricted after inductionrdquo Development vol 114no 3 pp 565ndash572 1992

[62] J Qiao D Cohen and D Herzlinger ldquoThe metanephricblastema differentiates into collecting system and nephronepithelia in vitrordquo Development vol 121 no 10 pp 3207ndash32141995

[63] M Self O V Lagutin B Bowling et al ldquoSix2 is required forsuppression of nephrogenesis and progenitor renewal in thedeveloping kidneyrdquo EMBO Journal vol 25 no 21 pp 5214ndash5228 2006

[64] S Boyle A Misfeldt K J Chandler et al ldquoFate mapping usingCited1-CreERT2 mice demonstrates that the cap mesenchymecontains self-renewing progenitor cells and gives rise exclu-sively to nephronic epitheliardquo Developmental Biology vol 313no 1 pp 234ndash245 2008


[65] A Kobayashi M T Valerius J W Mugford et al ldquoSix2 definesand regulates a multipotent self-renewing nephron progenitorpopulation throughout mammalian kidney developmentrdquo CellStem Cell vol 3 no 2 pp 169ndash181 2008

[66] F Costantini and R Kopan ldquoPatterning a complex organbranchingmorphogenesis and nephron segmentation in kidneydevelopmentrdquo Developmental Cell vol 18 no 5 pp 698ndash7122010

[67] C Hendry B Rumballe K Moritz and M H Little ldquoDefiningand redefining the nephron progenitor populationrdquo PediatricNephrology vol 26 no 9 pp 1395ndash1406 2011

[68] R Reimschuessel and D Williams ldquoDevelopment of newnephrons in adult kidneys following gentamicin-inducednephrotoxicityrdquo Renal Failure vol 17 no 2 pp 101ndash106 1995

[69] C J Salice J S Rokous A S Kane and R Reimschuessel ldquoNewnephron development in goldfish (Carassius auratus) kidneysfollowing repeated gentamicin-induced nephrotoxicosisrdquoCom-parative Medicine vol 51 no 1 pp 56ndash59 2001

[70] J Augusto B Smith S Smith J Robertson and R Reimschues-sel ldquoGentamicin-induced nephrotoxicity and nephroneogene-sis in Oreochromis nilotica a tilapian fishrdquo Disease of AquaticOrganisms vol 26 no 1 pp 49ndash58 1996

[71] M Elger H Hentschel J Litteral et al ldquoNephrogenesis isinduced by partial nephrectomy in the elasmobranch Leucorajaerinaceardquo Journal of the American Society of Nephrology vol 14no 6 pp 1506ndash1518 2003

[72] C S Johnson N F Holzemer and R A Wingert ldquoLaserablation of the zebrafish pronephros to study renal epithelialregenerationrdquo Journal of Visualized Experiments no 54 ArticleID e2845 2011

[73] E D de Groh L M Swanhart C C Cosentino et alldquoInhibition of histone deacetylase expands the renal progenitorcell populationrdquo Journal of the American Society of Nephrologyvol 21 no 5 pp 794ndash802 2010

[74] S J Poureetezadi and R A Wingert ldquoCongenital and acutekidney disease translational research insights from zebrafishchemical geneticsrdquo General Medicine vol 1 no 3 article 1122013

[75] A Palmyre J Lee G Ryklin et al ldquoCollective epithelialmigration drives kidney repair after acute injuryrdquo PLoS ONEvol 9 no 7 Article ID e101304 2014

[76] T Nadasdy Z Laszik K E Blick L D Johnson and F G SilvaldquoProliferative activity of intrinsic cell populations in the normalhuman kidneyrdquo Journal of the American Society of Nephrologyvol 4 no 12 pp 2032ndash2039 1994

[77] A Vogetseder A Karadeniz B Kaissling andM L Hir ldquoTubu-lar cell proliferation in the healthy rat kidneyrdquo Histochemistryand Cell Biology vol 124 no 2 pp 97ndash104 2005

[78] A Vogetseder T Palan D Bacic B Kaissling and M Le HirldquoProximal tubular epithelial cells are generated by division ofdifferentiated cells in the healthy kidneyrdquoTheAmerican Journalof PhysiologymdashCell Physiology vol 292 no 2 pp C807ndashC8132007

[79] F Lin A Moran and P Igarashi ldquoIntrarenal cells not bonemarrow-derived cells are the major source for regeneration inpostischemic kidneyrdquo Journal of Clinical Investigation vol 115no 7 pp 1756ndash1764 2005

[80] B D Humphreys M T Valerius A Kobayashi et al ldquoIntrinsicepithelial cells repair the kidney after injuryrdquoCell Stem Cell vol2 no 3 pp 284ndash291 2008

[81] C Q Diep and A J Davidson ldquoTransplantation of cellsdirectly into the kidney of adult zebrafishrdquo Journal of VisualizedExperiments no 51 article 2725 2011

[82] C Sagrinati E Ronconi E Lazzeri L Lasagni and P Romag-nani ldquoStem-cell approaches for kidney repair choosing theright cellsrdquo Trends in Molecular Medicine vol 14 no 7 pp 277ndash285 2008

[83] C Hopkins J Li F Rae and M H Little ldquoStem cell options forkidney diseaserdquo Journal of Pathology vol 217 no 2 pp 265ndash2812009

[84] E E Morales and R A Wingert ldquoRenal stem cell reprogram-ming prospects in regenerative medicinerdquo World Journal ofStem Cells vol 6 no 4 pp 458ndash466 2014

[85] M Westerfield ldquoRecipesrdquo in The Zebrafish Book chapter 10University ofOregonPress EugeneOreUSA 4th edition 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


for research on nephron patterning [15ndash18] identificationof essential genes [11ndash13 19] tubulogenesis [20 21] andphysiology and diseasemodeling [22ndash26] In comparison theadult zebrafish kidney or mesonephros is a single relativelyflat organ attached to the dorsal body wall that consists ofcharacteristic bilaterally symmetric regions referred to as thehead (or anterior) trunk (or medial) and tail (or posterior)(Figure 1(a)) [27] The mesonephros begins to form betweenabout 12 and 14 dpf with the progressive addition of nephronsto the existing pronephric pair [28] Over the lifespan ofthe zebrafish the mesonephros continues to accumulatenephronsmdasha phenomenon linked to their continual adultgrowth (measured in tip to tail length of the animal) and theassociated increasing excretory demands [28] In the typicalzebrafish adult at around 6 months of age this mesonephrickidney is estimated to possess approximately 450 nephronswith themost densely populated sites of nephrons in the headand trunk [28] The adult nephrons have similar segmentsas found in the pronephros but are grouped in branchedarrangements (Figure 1(a)) [29] which like other fishes donot show a regular orientation of nephrons as seen in thecortex and medulla of the mammalian metanephric kidney[6] Zebrafishmesonephric nephrons commonly have shareddistal tubule segments (Figure 1(a)) and drain into a pair ofmajor collecting ducts that span the length of the organ [1029] The stroma consisting of the cells interspersed betweenmesonephric nephrons is the site of adult hematopoiesis andalso contains an intriguing populace of mesenchymal renalprogenitors [10 28 29] These renal progenitors provide acontinual source of new nephrons that are produced in a pro-cess termed nephron neogenesis or neonephrogenesis whichoccurs during the aforementioned processes of mesonephrosdevelopment and when the mesonephros grows in responseto naturally increasing biomass of the aging fish [10 28 29]

Overall the complexity of the zebrafish mesonephrickidney provides a useful adult setting for renal biology studiesand shows promise for identifying genetic components of therenal regeneration response that can complement researchin traditional mammalian AKI models such as the mouseand rat [10] To date kidney regeneration paradigms in adultzebrafish have included nephrotoxin administration specifi-cally of the aminoglycoside antibiotic gentamicin to inducewidespread nephron tubule damage [10 28 29] as well asthe creation of several transgenic strains that were engineeredto induce ablation of particular epithelial cell types in thenephron blood filter [30 31] Among the former previousstudies have demonstrated that two major events transpirefollowing gentamicin-induced AKI in the adult zebrafishkidney organ (1) neonephrogenesis or the production ofnew nephrons due to the activation of the aforementionedstromal renal progenitors which is detectable by histologybased on the appearance of basophilic nephron units [1028 29] and (2) partial functional restoration in existingnephrons around 4 days post injury (dpi) suggestive that thedamaged nephron tubule epithelium recovers rather rapidlyfrom chemical insult [29] Despite these observations theprecise temporal sequence of molecular events that transpirewithin zebrafish nephrons during AKI including cell deathand proliferation has received relatively little scrutiny Some

alterations in gene expression have been annotated in priorwork such as the transient abrogation of transcripts encodingslc20a1a [29] a sodium-dependent phosphate transporterthat is normally localized to the proximal tubule epitheliumbut further observations have been quite limited

A significant impediment toward the pursuit of charac-terizing kidney regeneration aspects in zebrafish has beenthe paucity of histological and other molecular labelingmethodologies tailored for use in this model organismMore recently we have adapted a number of techniques toilluminate renal structures that can now be utilized [32] Forexample we demonstrated that the various proximal tubulesegments could be distinguished by unique combinationsof lectin staining dextran uptake and alkaline phosphatase(AP) reactivity (Figure 1(a)) [32] Further distal tubules aredistinguished by labeling with a different lectin (Figure 1(a))[32]

Here to obtain a more detailed understanding of theregeneration events associated with zebrafish renal injuryand identify cellular attributes enabling the demarcationof tubule segments we performed extensive histologicaland immunofluorescence studies to annotate the sequenceof tissue changes that result following gentamicin nephro-toxicity For this work we used andor modified severaltraditional histology protocols for use with zebrafish renaltissues and also implemented a number of our recentlydeveloped nephron labeling methodologies [32] With thesetools we now demonstrate for the first time that followinggentamicin-inducedAKI nephron proximal tubule epithelialregeneration proceeds over approximately one week Wehave now documented the detailed succession of cell deathand proliferation in proximal tubules during this intervalThrough additional structural and functional assays we showthat proximal tubules throughout the post-AKI zebrafishkidney regain absorptive capacity between 14 and 21 dpiFurther we show that neonephrogenesis occurs in a partiallyoverlapping time frame as nephron epithelial regenerationbeginning around 5 dpi and progressing over the subsequenttwo weeks and show that regenerating populations in bothexisting and new nephrons express the renal transcriptionfactor Pax2 Taken together these descriptive and functionalstudies provide an essential foundation for future workaimed at elucidating the mechanisms that regulate kidneyregeneration following AKI in the adult zebrafish

2 Results

The aminoglycoside antibiotic gentamicin is an establishednephrotoxin that has been used to model AKI by inducingrenal tubule damage in the adult zebrafish [10 28 29] as wellas other fish species such as goldfish and medaka [6 33]In the latter studies the histology of nephron phenotypesafter gentamicin administration was documented includingassessment of cell proliferation by suchmeasures as detectionof proliferating cell nuclear antigen (PCNA) labeling andthe incorporation of 5-bromo 21015840deoxyuridine (BrdU) [633] Previous studies in zebrafish have documented theappearance of gentamicin-damaged nephrons at 1 dpi andthe incorporation of BrdU in immature nephrons [29] but


DAPI

DAPI

DAPILTL

LTL

LTL

DT

PT GDT

PT

DT

PT

G

DT

PT

PT DT

PT

DT

DAPI


DBA

DBAMyosin VI

DBA


Renal corpuscle

Proximal tubule

Distal tubule LTLAP

AP

PCT PST DE DL CD


Kidney


Head TrunkTail

Collecting duct


MouseZebrafish

Silv

er st

ain

PAS

Hamp

E

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)












Untreated

PT

DT

DTPT

HampE

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

1 dpi 3 dpi 5 dpi


(a)

PAS

Untreated

DT

G

G

DT

PT

PT

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

1 dpi 3 dpi 5 dpi


(b)




(a)


(b)

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast


90

80

70

60

50

40

30

20

10

0

lowastlowastlowast


1dpi 3dpi 5dpi 7dpi

slc20a1a+

struc

ture

s

(c)



Untreated

TUNEL

DAPILTL

1 dpi 3 dpi

5 dpi 7 dpi 10 dpi

(a)

lowast

60

50

40

30

20

10

0

lowastlowast


lowastlowast


NEL

+ce

lls(

)(b)






Untreated

PCNADAPILTL

1 dpi 3 dpi

5 dpi 7 dpi 10 dpi

(a)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



lowastlowastlowast

70

60

50

40

30

20

10

0

lowastlowast

PCN

A+

cells

()


(b)

PCNA

DAPILTL


(c)





Untreated

BrdU

DAPILTL

1 dpi

3 dpi 5 dpi

(a)

BrdU

+ce

lls(

)

0

1

2

3

4

5

6

7

8

9

10


(b)







Untreated


1 dpi 3 dpi

5 dpi 7 dpi 14 dpi

(a)

Untreated

Fluoro-ruby dextran

1 dpi 3 dpi

5 dpi 7 dpi 21 dpi

(b)



Untreated

Pax2DAPI

1 dpi 3 dpi

5 dpi 7 dpi

14 dpi

10 dpi

(a)

Pax2DAPI

5 dpi 10 dpi

(b)













3 Discussion







Day 0 Day 3 Day 5

Injury


Cell death



Days 7ndash14

epithelialProcess 1

regeneration
























Key Findings




Abbreviations





Acknowledgments




References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


DAPI

DAPI

DAPILTL

LTL

LTL

DT

PT GDT

PT

DT

PT

G

DT

PT

PT DT

PT

DT

DAPI


DBA

DBAMyosin VI

DBA


Renal corpuscle

Proximal tubule

Distal tubule LTLAP

AP

PCT PST DE DL CD


Kidney


Head TrunkTail

Collecting duct


MouseZebrafish

Silv

er st

ain

PAS

Hamp

E

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)












Untreated

PT

DT

DTPT

HampE

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

1 dpi 3 dpi 5 dpi


(a)

PAS

Untreated

DT

G

G

DT

PT

PT

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

1 dpi 3 dpi 5 dpi


(b)




(a)


(b)

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast


90

80

70

60

50

40

30

20

10

0

lowastlowastlowast


1dpi 3dpi 5dpi 7dpi

slc20a1a+

struc

ture

s

(c)



Untreated

TUNEL

DAPILTL

1 dpi 3 dpi

5 dpi 7 dpi 10 dpi

(a)

lowast

60

50

40

30

20

10

0

lowastlowast


lowastlowast


NEL

+ce

lls(

)(b)






Untreated

PCNADAPILTL

1 dpi 3 dpi

5 dpi 7 dpi 10 dpi

(a)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



lowastlowastlowast

70

60

50

40

30

20

10

0

lowastlowast

PCN

A+

cells

()


(b)

PCNA

DAPILTL


(c)





Untreated

BrdU

DAPILTL

1 dpi

3 dpi 5 dpi

(a)

BrdU

+ce

lls(

)

0

1

2

3

4

5

6

7

8

9

10


(b)







Untreated


1 dpi 3 dpi

5 dpi 7 dpi 14 dpi

(a)

Untreated

Fluoro-ruby dextran

1 dpi 3 dpi

5 dpi 7 dpi 21 dpi

(b)



Untreated

Pax2DAPI

1 dpi 3 dpi

5 dpi 7 dpi

14 dpi

10 dpi

(a)

Pax2DAPI

5 dpi 10 dpi

(b)













3 Discussion







Day 0 Day 3 Day 5

Injury


Cell death



Days 7ndash14

epithelialProcess 1

regeneration
























Key Findings




Abbreviations





Acknowledgments




References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Untreated

PT

DT

DTPT

HampE

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

1 dpi 3 dpi 5 dpi


(a)

PAS

Untreated

DT

G

G

DT

PT

PT

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

1 dpi 3 dpi 5 dpi


(b)




(a)


(b)

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast


90

80

70

60

50

40

30

20

10

0

lowastlowastlowast


1dpi 3dpi 5dpi 7dpi

slc20a1a+

struc

ture

s

(c)



Untreated

TUNEL

DAPILTL

1 dpi 3 dpi

5 dpi 7 dpi 10 dpi

(a)

lowast

60

50

40

30

20

10

0

lowastlowast


lowastlowast


NEL

+ce

lls(

)(b)






Untreated

PCNADAPILTL

1 dpi 3 dpi

5 dpi 7 dpi 10 dpi

(a)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



lowastlowastlowast

70

60

50

40

30

20

10

0

lowastlowast

PCN

A+

cells

()


(b)

PCNA

DAPILTL


(c)





Untreated

BrdU

DAPILTL

1 dpi

3 dpi 5 dpi

(a)

BrdU

+ce

lls(

)

0

1

2

3

4

5

6

7

8

9

10


(b)







Untreated


1 dpi 3 dpi

5 dpi 7 dpi 14 dpi

(a)

Untreated

Fluoro-ruby dextran

1 dpi 3 dpi

5 dpi 7 dpi 21 dpi

(b)



Untreated

Pax2DAPI

1 dpi 3 dpi

5 dpi 7 dpi

14 dpi

10 dpi

(a)

Pax2DAPI

5 dpi 10 dpi

(b)













3 Discussion







Day 0 Day 3 Day 5

Injury


Cell death



Days 7ndash14

epithelialProcess 1

regeneration
























Key Findings




Abbreviations





Acknowledgments




References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



(a)


(b)

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast


90

80

70

60

50

40

30

20

10

0

lowastlowastlowast


1dpi 3dpi 5dpi 7dpi

slc20a1a+

struc

ture

s

(c)



Untreated

TUNEL

DAPILTL

1 dpi 3 dpi

5 dpi 7 dpi 10 dpi

(a)

lowast

60

50

40

30

20

10

0

lowastlowast


lowastlowast


NEL

+ce

lls(

)(b)






Untreated

PCNADAPILTL

1 dpi 3 dpi

5 dpi 7 dpi 10 dpi

(a)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



lowastlowastlowast

70

60

50

40

30

20

10

0

lowastlowast

PCN

A+

cells

()


(b)

PCNA

DAPILTL


(c)





Untreated

BrdU

DAPILTL

1 dpi

3 dpi 5 dpi

(a)

BrdU

+ce

lls(

)

0

1

2

3

4

5

6

7

8

9

10


(b)







Untreated


1 dpi 3 dpi

5 dpi 7 dpi 14 dpi

(a)

Untreated

Fluoro-ruby dextran

1 dpi 3 dpi

5 dpi 7 dpi 21 dpi

(b)



Untreated

Pax2DAPI

1 dpi 3 dpi

5 dpi 7 dpi

14 dpi

10 dpi

(a)

Pax2DAPI

5 dpi 10 dpi

(b)













3 Discussion







Day 0 Day 3 Day 5

Injury


Cell death



Days 7ndash14

epithelialProcess 1

regeneration
























Key Findings




Abbreviations





Acknowledgments




References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Untreated

TUNEL

DAPILTL

1 dpi 3 dpi

5 dpi 7 dpi 10 dpi

(a)

lowast

60

50

40

30

20

10

0

lowastlowast


lowastlowast


NEL

+ce

lls(

)(b)






Untreated

PCNADAPILTL

1 dpi 3 dpi

5 dpi 7 dpi 10 dpi

(a)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



lowastlowastlowast

70

60

50

40

30

20

10

0

lowastlowast

PCN

A+

cells

()


(b)

PCNA

DAPILTL


(c)





Untreated

BrdU

DAPILTL

1 dpi

3 dpi 5 dpi

(a)

BrdU

+ce

lls(

)

0

1

2

3

4

5

6

7

8

9

10


(b)







Untreated


1 dpi 3 dpi

5 dpi 7 dpi 14 dpi

(a)

Untreated

Fluoro-ruby dextran

1 dpi 3 dpi

5 dpi 7 dpi 21 dpi

(b)



Untreated

Pax2DAPI

1 dpi 3 dpi

5 dpi 7 dpi

14 dpi

10 dpi

(a)

Pax2DAPI

5 dpi 10 dpi

(b)













3 Discussion







Day 0 Day 3 Day 5

Injury


Cell death



Days 7ndash14

epithelialProcess 1

regeneration
























Key Findings




Abbreviations





Acknowledgments




References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Untreated

PCNADAPILTL

1 dpi 3 dpi

5 dpi 7 dpi 10 dpi

(a)


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



lowastlowastlowast

70

60

50

40

30

20

10

0

lowastlowast

PCN

A+

cells

()


(b)

PCNA

DAPILTL


(c)





Untreated

BrdU

DAPILTL

1 dpi

3 dpi 5 dpi

(a)

BrdU

+ce

lls(

)

0

1

2

3

4

5

6

7

8

9

10


(b)







Untreated


1 dpi 3 dpi

5 dpi 7 dpi 14 dpi

(a)

Untreated

Fluoro-ruby dextran

1 dpi 3 dpi

5 dpi 7 dpi 21 dpi

(b)



Untreated

Pax2DAPI

1 dpi 3 dpi

5 dpi 7 dpi

14 dpi

10 dpi

(a)

Pax2DAPI

5 dpi 10 dpi

(b)













3 Discussion







Day 0 Day 3 Day 5

Injury


Cell death



Days 7ndash14

epithelialProcess 1

regeneration
























Key Findings




Abbreviations





Acknowledgments




References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Untreated

BrdU

DAPILTL

1 dpi

3 dpi 5 dpi

(a)

BrdU

+ce

lls(

)

0

1

2

3

4

5

6

7

8

9

10


(b)







Untreated


1 dpi 3 dpi

5 dpi 7 dpi 14 dpi

(a)

Untreated

Fluoro-ruby dextran

1 dpi 3 dpi

5 dpi 7 dpi 21 dpi

(b)



Untreated

Pax2DAPI

1 dpi 3 dpi

5 dpi 7 dpi

14 dpi

10 dpi

(a)

Pax2DAPI

5 dpi 10 dpi

(b)













3 Discussion







Day 0 Day 3 Day 5

Injury


Cell death



Days 7ndash14

epithelialProcess 1

regeneration
























Key Findings




Abbreviations





Acknowledgments




References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Untreated


1 dpi 3 dpi

5 dpi 7 dpi 14 dpi

(a)

Untreated

Fluoro-ruby dextran

1 dpi 3 dpi

5 dpi 7 dpi 21 dpi

(b)



Untreated

Pax2DAPI

1 dpi 3 dpi

5 dpi 7 dpi

14 dpi

10 dpi

(a)

Pax2DAPI

5 dpi 10 dpi

(b)













3 Discussion







Day 0 Day 3 Day 5

Injury


Cell death



Days 7ndash14

epithelialProcess 1

regeneration
























Key Findings




Abbreviations





Acknowledgments




References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Untreated

Pax2DAPI

1 dpi 3 dpi

5 dpi 7 dpi

14 dpi

10 dpi

(a)

Pax2DAPI

5 dpi 10 dpi

(b)













3 Discussion







Day 0 Day 3 Day 5

Injury


Cell death



Days 7ndash14

epithelialProcess 1

regeneration
























Key Findings




Abbreviations





Acknowledgments




References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












3 Discussion







Day 0 Day 3 Day 5

Injury


Cell death



Days 7ndash14

epithelialProcess 1

regeneration
























Key Findings




Abbreviations





Acknowledgments




References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Day 0 Day 3 Day 5

Injury


Cell death



Days 7ndash14

epithelialProcess 1

regeneration
























Key Findings




Abbreviations





Acknowledgments




References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















Key Findings




Abbreviations





Acknowledgments




References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



References
































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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