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A Novel Role for GATA3 in Mesangial Cells inGlomerular Development and Injury

Irina V. Grigorieva,1 Andre Oszwald,1 Elena F. Grigorieva,1 Helga Schachner,1

Barbara Neudert,1 Tammo Ostendorf,2 Juergen Floege,2 Maja T. Lindenmeyer,3

Clemens D. Cohen,3 Ulf Panzer,4 Christof Aigner ,5 Alice Schmidt,5 Frank Grosveld ,6

Rajesh V. Thakker,7 Andrew Jackson Rees,1 and Renate Kain 1

1Department of Pathology and 5Division of Nephrology and Dialysis, Department of Medicine III, Medical UniversityVienna, Vienna, Austria; 2Division of Nephrology and Clinical Immunology, Rheinisch-Westfälische TechnischeHochschule Aachen University, Aachen, Germany; 3Nephrological Center, Medical Clinic and Policlinic IV, Universityof Munich, Munich, Germany; 4III. Medical Clinic, University Medical Center Hamburg-Eppendorf, Hamburg,Germany; 6Department of Cell Biology, Dr. Molewaterplein 50, Rotterdam, The Netherlands; and 7Oxford Centre forDiabetes, Endocrinology and Metabolism, Churchill Hospital, University of Oxford, Oxford, UK

ABSTRACTBackground GATA3 is a dual-zinc finger transcription factor that regulates gene expression in manydeveloping tissues. In the kidney, GATA3 is essential for ureteric bud branching, and mice without it failto develop kidneys. In humans, autosomal dominant GATA3mutations can cause renal aplasia as part ofthe hypoparathyroidism, renal dysplasia, deafness (HDR) syndrome that includes mesangioproliferativeGN. This suggests that GATA3 may have a previously unrecognized role in glomerular developmentor injury.

Methods To determine GATA3’s role in glomerular development or injury, we assessed GATA3 expres-sion in developing and mature kidneys from Gata3 heterozygous (+/2) knockout mice, as well as injuredhuman and rodent kidneys.

Results We show that GATA3 is expressed by FOXD1 lineage stromal progenitor cells, and a subset ofthese cells mature intomesangial cells (MCs) that continue to expressGATA3 in adult kidneys. Inmice, weuncover that GATA3 is essential for normal glomerular development, and mice with haploinsufficiencyof Gata3 have too few MC precursors and glomerular abnormalities. Expression of GATA3 is maintainedin MCs of adult kidneys and is markedly increased in rodent models of mesangioproliferative GN and inIgA nephropathy, suggesting that GATA3 plays a critical role in the maintenance of glomerularhomeostasis.

Conclusions These results provide new insights on the roleGATA3plays inMCdevelopment and responseto injury. It also shows that GATA3 may be a novel and robust nuclear marker for identifying MCs in tissuesections.

JASN 30: ccc–ccc, 2019. doi: https://doi.org/10.1681/ASN.2018111143

GATAbinding protein 3 (GATA3) is amember of anevolutionary conserved family of six dual zinc-fingertranscription factors (GATA1–6) with essential rolesin cell lineage commitment and differentiation.GATA3 binds and remodels inaccessible chroma-tin,1 which is essential for cellular reprogrammingduring embryonic development and in response toinjury. GATA3 is crucial for the normal development

Received November 21, 2018. Accepted May 1, 2019.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Irina V. Grigorieva or Dr. Renate Kain,Department of Pathology, Medical University of Vienna, Wäh-ringer Gürtel 18-20, 1090 Vienna, Austria. E-mail: grigorievai@cardiff.ac.uk or renate.kain@meduniwien.ac.at

Copyright © 2019 by the American Society of Nephrology

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of many fetal tissues, including breast, parathyroid glands,kidney, and inner ear.227 In adults, GATA3 is required formaintenance of cell differentiation; for example, in mammaryluminal epithelial cells and in parathyroid chief cells.2,3 GATA3is also the master regulator of leukocyte differentiation intoTh2 lymphocytes and group 2 innate lymphoid cells (ILC2),both of which modulate inflammation and promote tissuerepair.8–10

In the kidney, GATA3 is essential for formation of thenephric duct and ureteric buds that give rise to branchingof the ureteric tree and subsequently the collecting ducts.4,5

Deletion of Gata3 in mice leads to failed kidney develop-ment,6 whereas specific inactivation in the nephric duct cau-ses ectopic ureteric budding and a spectrum of urogenitalmalformations.4 Mutations of GATA3 in humans cause syn-dromic autosomal dominant hypoparathyroidism, sensori-neural deafness, and renal dysplasia (HDR).11213 HDR oftenresults in progressive kidney disease caused by congenital re-nal aplasia, hypoplasia, or dysplasia (41%); vesicoureteral re-flux (16%); and cysts or pelvicalyceal deformities (11%).14

More recently, HDR has been associated with nephroticsyndrome and isolated cases of GN,15,16 whereas Gata3 hypo-morphic mutant mice rescued with a yeast artificial chromo-some Gata3 transgene, whose expression was limited to renaltubules and not glomeruli, have been shown to suffer glomer-ular mesangial cell (MC) defects.17 In genome-wide tran-scriptome studies, GATA3 has been identified as a highlyenriched transcript in MCs,18,19 but the functional signifi-cance of GATA3 expression in MCs in health and diseasehas not been explored. Moreover, GN in HDR could reflectthe effect of GATA3 deficiency on immunity because overex-pression in T cells protects against lupus nephritis in BXSB/MpJ-Yaamice,20 and on ILC2s, which are reported to be cen-tral to renal repair.8 Alternatively it could be evidence of anovel role for GATA3 in glomerular development or homeo-stasis; this study was designed to determine whether this isthe case.

We show that GATA3 is expressed by FOXD1+ stromal cells,and that a subset, which mature into MCs, continues to ex-press GATA3 in adult kidneys. Inmice, we uncover an essentialrole for GATA3 in glomerular development and documentmarked changes in mesangial GATA3 expression in GN(both rodent models and IgA nephropathy [IgAN]). The re-sults provide novel insights into MC development and re-sponses to injury, identifying GATA3 as a new and robustmarker for MCs in tissue sections.

METHODS

AnimalsStudies with congenic Gata3+/taulacZ (Gata3+/2) mice21 on anFVB/N background were approved by the University of Ox-ford Ethical Review Committee and were licensed under theAnimal (Scientific Procedures) Act 1986, issued by the UK

Government Home Office Department. Kidneys were dis-sected and fixed overnight in 4% paraformaldehyde at 4°C.Embryos were collected with the day of the vaginal plugdesignated as embryonic day (E) 0.5 and staged by morpho-logic criteria, which included somite number and eye and limbmorphology.

Mesangial Proliferative Anti-Thy1.1 Nephritis ModelAnti-Thy1.1nephritiswas induced inmaleWistar rats (CharlesRiver Laboratories, Wilmington, MA) by injection of specific(OX7) antibody as described.22

Nephrotoxic Nephritis Mouse ModelNephrotoxic serumnephritiswas induced in 8- to 10-week-oldmale C57BL/6 wild-type mice by intraperitoneal injection of2.5 mg of nephrotoxic sheep serum per gram of mouse bodyweight, as described.23 Controls were injected intraperitone-ally with an equal amount of nonspecific sheep IgG.

Human Kidney Biopsy SamplesThe use of patient material was approved by the Ethics Com-mitteeofMedicalUniversityofVienna (ethical approval no.EK1673/2013) before commencement of the study, and patientsprovided informed written consent in agreement with theDeclaration of Helsinki. Human kidney biopsy samples eitherfrom time-zero renal allograft biopsy samples (n=12) orthose that were diagnosed with steroid-responsive minimalchange disease (n=5), membranous nephropathy (MGN)(n=7), lupus nephritis class IV (LN) (n=13), or IgAN(n=25) (Supplemental Tables 1–3) were processed for prepa-ration of 2-mm sections for immunohistologic evaluation.

MC CulturePrimaryMC cultures were established from glomeruli isolatedfrom kidney cortex tissue (sampled from patients undergoingnephrectomy because of renal cell carcinoma) by the differen-tial sieving method described before.24,25 MCs outgrowthswere grown in RPMI 1640 medium containing 10% FCS,1% penicillin/streptomycin (Gibco), and ITS (Sigma-Aldrich). Cells were harvested on days 7, 21, 29, and 43 afterseeding glomeruli for preparation of RNA and protein lysates.

Significance Statement

Mesangial cells play a crucial role in maintaining glomerular ho-meostasis and injuries to these cells often result in progression toCKD like IgA and diabetic nephropathies. However, the transcrip-tion factors involved inmesangial cell development and function arelargely unknown. The authors describe the role transcription factorGATA3 plays in mesangial cells in embryonic kidneys and healthyand injured adult glomeruli. Mice with haploinsufficiency of GATA3have too few MC precursor cells and glomerular abnormalities.GATA3 expression increases in mesangial cells in mesangial pro-liferative GN in humans and rodent models suggesting GATA3 isimportant for glomerular homeostasis and response to injury.GATA3 also may be a useful a nuclear marker of human mesangialcells.

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Conditionally immortalized human glomerular MCs werekindly provided by Professor Moin Saleem (University ofBristol) and grown as described.25 Cells were growth-arrestedin serum-free medium for 24 hours before addition ofPDGF-BB (5–60 ng/ml), TGF-b (2 ng/ml), TNF-a (10 ng/ml),IL4 (25 ng/ml), and INF-g (20 ng/ml). After 24–48 hours cellswere harvested for preparation of RNA or protein lysates.

Quantitative RT-PCRTotalRNAwas extracted fromfrozenkidney tissue andprimarymesangial or conditionally immortalized MCs with RNeasymini kit (Qiagen), quantified by NanoDrop spectrophotome-ter and used to prepare cDNA with SuperScript III reversetranscription (Invitrogen). Quantitative PCR was performedusing KAPA SYBR FAST master mix (Kapa Biosystems) andspecific primers (Supplemental Table 4). Samples were ana-lyzed on a qTOWER3 thermal cycler (Analytik Jena), and therelative level of GATA3 expression was normalized to the levelof GADPH expression using the 22DDCTmethod.

Microarray AnalysisHuman kidney biopsy specimens for Affymetrix microarrayexpression data (HG-U133A Array) were obtained within thescope of the European Renal cDNA Bank Kröner-FreseniusBiopsy Bank.26,27 Diagnostic renal biopsy specimens were re-ceived from patients after an informed written consent, ap-proved by the local ethics committees. After renal biopsy, thetissue was transferred to RNase inhibitor and microdissectedinto glomerular and tubulointerstitial compartments. TotalRNA was isolated from microdissected glomeruli, reversetranscribed, and linearly amplified as described.28 Microarrayexpression data presented in this study was obtained fromindividual patients with IgAN (n=27). Pretransplantationkidney biopsy specimens from living donors (n=6) wereused as controls. Fragmentation, hybridization, staining,and imaging were performed with the Affymetrix HG-U133A system (Affymetrix, Santa Clara, CA), according tothe manufacturer’s instructions. CEL file normalizationwas performed with the Robust Multichip Average methodusing RMAExpress (version 1.0.5) and the human Entrez-Gene custom CDF annotation from Brain Array, version 18(http://brainarray.mbni.med.umich.edu/Brainarray/default.asp).To identify differentially expressed genes, the significanceanalysis of microarrays) method was applied using TiGR(MeV, version 4.8.1).29 A q-value ,5% was considered to bestatistically significant.

Western Blot AnalysisTotal proteinwas extracted in lysis buffer (50mMTris, 150mMNaCl, 0.1% SDS, 0.5% sodiumdeoxycholate, 1%Triton3100,1 mM PMSF, 1% protease inhibitor cocktail) and separated by10% SDS-PAGE, followed by transfer onto polyvinylidene di-fluoride membranes (Immobilon-FL). Membranes wereblocked in Odyssey buffer (LI-COR) for 1 hour at room tem-perature, followed by incubation with primary antibodies

against GATA3 and a-tubulin (Supplemental Table 5) dilutedinOdyssey buffer overnight at 4°C. After washing, membraneswere incubated with secondary antibodies targeting rabbitor mouse IgG (IRDye 680RD and IRDye 800CW conjugates;LI-COR). Detection was performed using LI-COR Odysseyinfrared scanner and quantification performed using LI-CORImage Studio Lite.

Histology, Immunohistochemistry, andImmunofluorescenceFixed mouse kidneys or embryos were processed for embed-ding in paraffin and cut into 2–6 mm sections for hematoxylinand eosin staining, periodic acid–Schiff staining, or for im-munostaining. Deparaffinized kidney sections were rehydra-ted in graded alcohols (100%, 96%, 70%, and 50%), andantigen retrieval was performed in citrate buffer in the auto-clave at 120°C for 20 minutes. For IHC signal detection, en-dogenous peroxidase activity was quenched by incubationin 3% (vol/vol) H2O2 for 10 minutes. Where biotinylated sec-ondary antibodies were used, sections were incubated withavidin block for 10 minutes. Sections were blocked in 10%goat serum for 30minutes at room temperature and incubatedovernight at 4°Cwith primary antibodies (Supplemental Table5). For IHC detection, the immobilized antibodies weredetected by UltraVision-LP HRP Polymer detection systemspecific for anti-mouse and anti-rabbit IgG (ThermoFisherScientific), or with biotinylated secondary antibodies andAB reagents (Vector Laboratories) according to manufac-turer’s instructions. DAB (Vector Laboratories) and hematox-ylin were used as the chromogen and the nuclear counterstain,respectively. In negative controls, primary antibodies wereomitted. For IF detection the following secondary antibodieswere used: Alexa Fluor 488 goat anti-mouse IgG1, Alexa Fluor488 goat anti-mouse IgG (H+L), Alexa Fluor 546 goat anti-mouse IgG2A, Alexa Fluor 546 goat anti-rabbit IgG, AlexaFluor 546 goat anti-rat IgG, Alexa Fluor 647 goat anti-rabbitIgG, Alexa Fluor 594 donkey anti-goat IgG (ThermoFisherScientific). Dapi was used to visualize the nuclei.

Quantitative Image AnalysisImmunostained tissue slides were visualized and digitized us-ing Zeiss AxioCam512 color camera attached to a Zeiss ImagerM2 microscope; by a confocal laser scanning microscope(LSM700; Carl Zeiss); or by digital slide scanner (3DHISTECHPannoramic 250 Flash equipped with Adimec Q-12A-180Fccamera). Images were analyzed with the ZEN2012 software(Carl Zeiss) and quantification was performed with ImageJ(National Institutes of Health) and CellProfiler software.30

Cell Profiler pipelines are available upon request. Briefly, glo-merular cross section (GCS) areas were outlined and cells wereidentified by nuclear DAPI staining. To determine the percent-age of positively stained cells, IF intensity thresholds for eachstain (nuclear or cytoplasmic) were determined either auto-matically using an inbuilt classifier (Cell Profiler Analyst)or by plotting mean intensity values per cell on a scatter plot

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Figure 1. Expression of GATA3 in healthy adult kidneys. (A) IHC for GATA3 (brown) in time-zero renal allograft biopsy samples fromhealthy adults demonstrated strong nuclear expression of GATA3 in a subset of tubular epithelial cells. CaSR (red ) was used as anephron segment marker53 in double staining, which showed that GATA3 was absent from proximal tubules (PT) and thick ascendinglimbs (TAL), but was expressed in distal/connecting nephron segments (DT/CN) and in principal cells of the CDs where it was absentfrom intercalated cells (arrow). IF staining for GATA3 (green) and CaSR (red) confirmed the cell specific expression of GATA3 inprincipal cells of the CD, which do not express CaSR. Scale bars, 20 mm. (B) Glomerular GATA3 expression was detected by IHC

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(example shown in Supplemental Material). The number ofpositively stained cells were expressed as percentage of totalDapi+ cells per GCS area in more than ten glomeruli per an-imal/genotype. Measurements of immunohistochemicallystained areas were expressed as a percentage of total GCS areas(mm2) in more than ten glomeruli per animal/genotype.

Statistical AnalysesAll values are expressed as means6SD. Statistical significance(defined as P,0.05) was evaluated using t tests, Mann–WhitneyU tests, or ANOVA for multiple group comparisons. Statisticalanalysis was performed using GraphPad Prism 6.0 (GraphPadSoftware Inc., San Diego, CA).

RESULTS

Characterization of GATA3 Expression in Healthy AdultKidneysGATA3 has an established role in kidney development, butwhether GATA3 is expressed in mature kidneys, or involvedin homeostasis as in other tissues, is unknown.2,3,9 Using renalallograft biopsy specimens from healthy adults to analyzeGATA3 expression showed strong expression in collectingduct (CD) epithelium (Figure 1A) (originating fromGATA3-expressing ureteric bud progenitors) and in distaland connecting tubules. In CDs, GATA3 was found in princi-pal but not intercalated cells, as demonstrated by the ab-sence of coexpression with calcium sensing receptor (CaSR)(Figure 1A). By contrast, GATA3 was uniformly undetectable inproximal tubular epithelium arising from progenitors in themesenchymal cap, as do podocytes and glomerular parietal ep-ithelial cells. Thus, renal epithelial expression of GATA3 ismain-tained in cells that arise from ureteric bud progenitors,suggesting a function in homeostasis.

There was strong nuclear GATA3 expression inMCs iden-tified by colocalization with PDGF receptor b (PDGFR-b)and a-smoothmuscle actin (a-SMA). GATA3 did not coloc-alize with Wilms tumor protein (WT-1) or CD31, demon-strating it was not expressed by podocytes, parietal epithelialcells, or endothelial cells (Figure 1B). In healthy humankidneys, 28.42%62.18% (mean6SD) of the cells per GCSexpressed GATA3, consistent with the expected proportionof MCs in the glomerular tuft (Figure 1C). GATA3 expres-sion and its colocalization with cell-specific markerswas conserved between human, mouse, and rat kidneys(Supplemental Figure 1).

GATA3 was detected in renin-expressing cells of thejuxtaglomerular apparatus, revealing a novel role of GATA3in renal endocrine cells. GATA3 was also expressed in renalsmooth muscle cells (VSMCs) colocalized with a-SMA,and in a subset of peritubular fibroblasts (Figure 1D). MCs,renin-secreting cells, VSMCs, and some renal fibroblasts haverecently been reported to share a common renal stromal pro-genitor,31234 and so it is striking that they all express GATA3 inthe adult kidney. This led us to analyze embryonic mouseand human kidneys to investigate GATA3 expression in renalstromal cells during development.

GATA3 Is Expressed in Embryonic Renal StromalMesenchyme and Is a Marker of Mesangial ProgenitorCellsGATA3 spatio-temporal expression in renal stromal mesen-chyme and its cell derivatives was examined inmouse embryosat various time points. At E12.5, strong GATA3 expressionwaslimited to the ureteric buds and tips; it was very weakly ex-pressed in interstitial stromal cells (Figure 2A), but absent fromcap mesenchyme that provides progenitor cells for the neph-ron. By E13.5, GATA3 was strongly expressed in a subset ofspindle-shaped mesenchymal cells occupying spaces betweenureteric bud branches and induced nephrons interior to thenephrogenic zone (Figure 2, A and B). These stromal cells arecharacterized by expression of forkhead-box d1 (FOXD1)transcription factor from E10.5,34,35 and a subset of them ex-pressed GATA3 from E13.5 (Figure 2B). GATA3-expressingcells were observed at the entrance of the vascular cleft ofthe comma-shaped body, within the cleft of the s-shaped(SS) body, and in the cap-stage glomeruli (Figure 2C).GATA3 colocalized with PDGFR-b and a-SMA in the coreof the capillary tuft, but not with WT-1 or CD31 (Figure 2D).This identifies GATA3 as an early marker for MC precursors.Interestingly, FOXD1 expressionwas no longer detected in theseMC precursors, suggesting downregulated expression duringdifferentiation from stromal progenitors (Figure 2B). Therewas no colocalization between GATA3 and CD31 either in glo-meruli or arterioles (Figure 2D), demonstrating its expressionin the renal vasculature was confined to VSMCs.

A similar pattern of GATA3 expression was observed in23-week human fetal kidneys. GATA3-expressing cells weredetected in the vascular cleft of the SS body andwithin capillaryloop-stage glomeruli where GATA3 colocalized with a-SMAand PDGFR-b, demonstrating its expression by MC precur-sors in the core of the renal corpuscle (Figure 2, E and F,Supplemental Figure 2). GATA3 was also expressed in

(brown) and by IF (green) where it was confined to MCs costaining with MCmarkers PDGFR-b and a-SMA (red). GATA3 did no colocalizewith WT-1 or CD31, markers of epithelial (podocyte and parietal) and endothelial cells, respectively. Scale bars, 20 mm. (C) Quantificationof the number of GATA3-expressing cells in GCS areas (n$10) from healthy human kidneys (n=6). (D) GATA3 colocalizedwith the aspartyl-protease renin (red) in juxtaglomerular cells (arrows) and with a-SMA (red) in smooth muscle cells of the afferent arterioles (A). Peritubularexpression of GATA3 was detected by IHC in a subset of interstitial fibroblasts (arrows). Arterioles indicated by dashed lines. Scale bars,20 mm. Daltonized versions of Immunofluorescence pictures are provided in Supplemental Appendix 1. G, glomerulus.

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Figure 2. Expression of GATA3 in developing mouse and human kidneys. (A) ICH for GATA3 (brown) in wild-type mouse embryosat E12.5 and E13.5. GATA3 expression is strong in ureteric bud (ub) cell nuclei, but absent from cap mesenchyme (cm). Arrows in-dicate increased expression of GATA3 in stromal cells (st) in E13.5 kidneys. Scale bars, 50 mm. (B) IHC for FOXD1 (left panel) and

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developing renal VSMCs where it colocalized with a-SMA,PDGFR-b, and renin, which is expressed throughout the de-veloping renal vasculature (Supplemental Figure 2).31 GATA3expression preceded renin expression in renal VSMCs in bothhuman and mouse embryos (data not shown).

Unexpectedly, expressionofGATA3was alsoobserved in thedistal SS body (Figure 2, C and E, Supplemental Figure 3),revealing that GATA3 is also a marker of distal-tubule precur-sors; its persistent expression in the distal and connectingnephron segments in the mature kidney (Figure 1) suggeststhat GATA3 is required for differentiation and maturation ofthese distal-tubule epithelial cell precursors.

GATA3 Expression Is Downregulated in MCs in vitroGATA3was uniformly highly expressed in humanMCs duringdevelopment and in mature adult glomeruli, as well as in ro-dents. GATA3 expression (quantitative RT-PCR and Westernblot) was similarly high in glomeruli freshly isolated fromhuman kidneys, but was rapidly downregulated in primarycultures of MCs derived from them: reduced in the early(7-day) outgrowths and negligible after 21 days (SupplementalFigure 4A). These unexpected results were supported by theabsence of GATA3 in a conditionally immortalized humanMC line,25 both in their proliferative, “permissive” tempera-ture (33°C), and after growth arrest and differentiation at 37°C(Supplemental Figure 4B). GATA3 expression, assessedby protein or mRNA, could not be rescued by addition ofcytokines, including PDGF-BB (activates MC proliferation36

), TNF-a, TGF-b, IL4 (potent stimulus of T cell GATA3), orINF-g (Supplemental Figure 4B). The loss of GATA3 fromprimary MC cultures precludes further analysis of GATA3functions in vitro, and raises more general issues aboutusing cultured MC to study MC-specific cellular pathways.

MC Defects in Gata3-Deficient MiceHomozygous Gata3 knockout (Gata32/2) mouse embryoshad gross developmental abnormalities with absent metaneph-ric kidneys at E12.5 and died shortly thereafter (Figure 3A).In contrast, kidneys of Gata3+/2 mice at E12.5 and E13.5were of similar size and morphology to those of age-matchedwild-type littermates with similar numbers of ureteric buds,comma-shaped/SS bodies and developing glomeruli, and sub-sequently developed into mature kidneys (Figure 3, A–C).

However, analysis of GCS areas in both developing and maturekidneys revealed a significant reduction inGata3+/2mice com-pared with wild-type littermates (Figure 3, B–D). Periodicacid–Schiff staining showed that Gata3+/2 mice had weakerglomerular basement membrane staining and a higher fre-quency of glomerular capillary loop dilation (Figure 3C).Although plasma parameters for kidney function, includingcreatinine, urea, albumin, and total protein concentrations,were not significantly different between 6-week-old Gata3+/2

mice and age- and sex-matched wild-type littermates, a subsetof male Gata3+/2 mice exhibited elevated plasma creatinineconcentrations (Figure 3E, Supplemental Table 6). Immuno-histochemical analysis of glomerular GATA3 expression andquantification of GATA3+ cells revealed that Gata3+/2 micehad fewer MCs (Figure 3, F and G), which was confirmedby a reduction in PDGFR-b staining (Figure 3, H and I).

We next analyzed the effect of GATA3 haploinsufficiency onglomerular maturation. By E15.5, developing glomeruli areorganized into discrete cellular compartments, and analysisof GATA3 and PDGFR-b expression showed that Gata3+/2

mice had significantly fewer MC precursors than wild-typelittermates and smaller total GCS areas (Figure 4, A–C).Despite normal CD31+ endothelial and WT-1+ podocyte cellnumbers, the mesangial cores were significantly smallerin the Gata3+/2 mice and capillaries were poorly organized(Figure 4, A, D, and E). Quantification of the PDGFR-b IFsignal intensity indicated that expression of PDGFR-b wasattenuated in MCs of Gata3+/2 compared with wild-typemice, as was the GATA3 IF signal, confirming mesangialGATA3 haploinsufficiency, albeit by approximately 30%reduction of the wild-type levels. Although PDGFR-b expres-sion preceded GATA3 expression in stromal mesenchyme(Supplemental Figure 5), these observations suggest thatGATA3 regulates PDGFR-b expression in MCs in maturingglomeruli.

Next we determined the mechanisms to explain reducedMC number in Gata3+/2 mice by investigating recruitmentof MC precursors into the developing glomeruli and MCproliferation rates in maturing glomeruli. Quantification ofGATA3+ PDGFR-b+ cells in the vascular cleft of SS bodiesrevealed that fewer MC precursors migrated into the cleft inGata3+/2 mice compared with age-matched wild-type litter-mates (Figure 4, H and I, Supplemental Figure 6). Moreover,

GATA3 (right panel) in E13.5 mouse kidney serial sections. Expression of FOXD1 and GATA3 overlaps in a subset of stromal cells in thecapsular stoma (cs). FOXD1expression is absent fromMCswhereGATA3 is strongly expressed (arrow). Highermagnification images of theboxed regions are shown. (C)At E13.5,GATA3-expressing stromal cells (arrows) canbe seenmigrating into the vascular cleft of the comma-shaped (cs) and SS bodies and reside within the core of the glomerulus (g). Smooth muscle cells of the developing arterioles (a) alsoexpress GATA3. Scale bars, 50 mm. (D) Co-IF with anti-GATA3 (green) and anti–WT-1 (podocyte marker), anti-CD31 (endothelial marker),anti–PDGFR-b, and anti–a-SMA (mesangial markers) antibodies in mouse E13.5 kidneys showing localization of GATA3 in MCs of thedeveloping glomeruli (g, outlined) and in smooth muscle cells of arterioles (a). Scale bars, 20 mm. (E) IHC for GATA3 (brown) in 23-weekhuman fetal kidney demonstrating GATA3 expression in renal stromal cells (arrows) located within the vascular cleft of the SS bodies andthe core of the glomeruli (g). Scale bars, 50 mm. (F) Co-IF in 23-week human fetal kidney with anti-GATA3 (green) and anti–PDGFR-b (red,left panel) and anti–a-SMA (red, right panel) antibodies demonstrating GATA3 expression in MCs of glomeruli. Scale bars, 20 mm. ms,medullary stroma; nz, nephrogenic zone.

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Figure 3. Characterization of embryonic and mature kidneys from Gata3 deficient mice. (A) Hematoxylin and eosin (H&S) stainedsections of mouse embryonic kidneys at E12.5 and E13.5 show that Gata3+/2 mice have kidneys of similar size and morphology tothose of age-matched wild-type littermates (Gata3+/+), whereas Gata32/2 mice have gross developmental abnormalities and ab-sent metanephric kidneys at E12.5. Corresponding higher magnification image of boxed region (left panel). Scale bars, 100 mm.

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in wild-type embryos, GATA3+ MC precursors closelyfollowed CD31+ endothelial progenitors, whereas this associ-ation was disrupted in Gata3+/2 mice and GATA3+ MC pro-genitors were found to lag behind migrating endothelial cellsand group together around the entrance to the cleft, presum-ably as a result of attenuated PDGFR-b expression (Figure 4H,Supplemental Figure 6). Analysis of proliferation rates withinthe cap-stage maturing glomeruli using the Ki67 prolifera-tionmarker demonstrated that although total glomerular pro-liferation rates were unaffected in Gata3+/2 mice, reflectingnormal endothelial cell proliferation, the proportion ofGATA3+ cells that costained for Ki67 was significantly reducedrevealing perturbed mesangial progenitor cell proliferationrates in Gata3+/2 mice (Figure 4, H, J, and K).

Thus, Gata3+/2 mice have impaired MC progenitor mi-gration and proliferation during development resultingin a paucity of MCs in adult kidneys. These findingsidentify a nonredundant role for GATA3 in normal glomer-ular development; its persistent expression in matureMCs suggests a potential contribution to homeostasisand the response to injury. We tested this by analyzingGATA3 expression in rodent GN models and in human re-nal biopsy samples.

GATA3 Expression Is Increased in MCs in Thy.1.1Nephritis in Rats and Nephrotoxic Nephritis in MiceGlomerular GATA3 expression was quantified in rats withThy1.1 nephritis. On day 8 postdisease induction (maximalMC activation and proliferation), there was a striking increasein the number of GATA3-expressing nuclei in glomeruli com-pared with control rats, and also of the proportion of totalGATA3-positiveGCS area, although the extent varied betweenglomeruli (Figure 5, A and B). The nuclear staining waslocalized to cells that expressed PDGFR-b and a-SMA, con-firming they were MCs (Figure 5C). There were significantlymore GATA3+ MCs in the nephritic rats than in controls(mean6SD: 60.6%63.3% and 34.5%65.2%, respectively;P,0.001) (Figure 5D). Moreover, the nuclei of the GATA3+

MCs in nephritic rats were significantly enlarged, indicatingcellular hypertrophy (Figure 5E). MC proliferation assessedby GATA3 and Ki67 double-staining was rare in healthy

rats: less than a quarter of glomeruli contained a single dou-ble-positive cell, and only 1.55%63.06% of GATA3+ MCsexpressed Ki67 (Figure 5, C and F). ProliferatingMCs accounted for 0.55%61.04% of total cells per GCS(Figure 5G) and a quarter of total proliferating cells perGCS, most of which were endothelial cells (SupplementalFigure 7). GATA3+Ki67+ MCs were much more common inThy1.1 nephritis, accounting for 10.94%64.24% of total cellsper GCS area, 20.89%68.33% of total GATA3+ MCs, and54.78%615.92% of the total glomerular proliferating cells(Figure 5, C, F, and G). Again, endothelial cells accountedfor most of the remainder (Supplemental Figure 7). Interest-ingly, 18.01%610.17% of MCs of Thy1.1 rats had GATA3 IFintensity above the highest control GATA3 IF signal (Figure 5H,Supplemental Figure 8), indicating more GATA3 protein. Sig-nificantly more GATA3HIGH MCs were proliferating (Ki67+)than the GATA3NORMAL cells (49.90%617.58% comparedwith 24.14%66.99%, respectively; P,0.05) and the meanGATA3 IF intensity was greater in proliferating (GATA3+Ki67+)compared with nonproliferating Thy1.1 MCs (Figure 5H). Im-munoblotting of glomerular protein lysates revealed thatGATA3 abundance was almost two-fold higher in glomerulifrom Thy1.1 compared with control rats (Figure 5I). ThusGATA3 expression is upregulated in proliferating MCs ininjured glomeruli.

To confirm that changes in GATA3 expression were notunique to glomeruli with extensivemesangiolysis, we repeatedthe analyses in a nephrotoxic nephritis mouse model. On days3 and 5 after anti-GBM antibody injection, glomeruli fromnephritic mice had significantly more GATA3-expressing cells.Staining intensity was significantly greater, and the GATA3-expressing nuclei were enlarged (Figure 6). Later the numberof GATA3-expressing MCs decreased, although some persistedeven in severely scarred glomeruli.

GATA3 as a MC Marker in Human MesangialProliferative DiseaseWe next examined GATA3 expression in kidney biopsy spec-imens from patients with four different glomerular diseaseswith and without mesangial proliferation: systemic LN,37

IgAN, MGN, and steroid-responsive minimal change

(B) Morphologic characterization of embryonic kidneys from E13.5 Gata3+/2 (n=4) and wild-type (n=3) embryos was performed byquantifying the number of ureteric bud tips, comma-shaped (CS) and S-shaped (ss) bodies, cap-stage glomeruli in .15 4-mm sectionspassing through the midline plane per kidney; GCS areas were measured in at least ten cap-stage glomeruli per embryo (*P,0.05; un-paired t test). (C) Kidney sections from adult mice stained with periodic acid–Schiff (PAS) show that Gata3+/2 mice developed maturekidneys with similar numbers of glomeruli. Higher magnification images reveal capillary loop dilation and thickened glomerular basementmembrane in Gata3+/2 mice. (D) Glomeruli were counted in .50 fields (5003500 mm) of digitized images of whole kidney sections(n=4 per kidney) from age-matched Gata3+/2 (n=3) and wild-type (n=3) adult mice. (E) Quantification of GCS areas (n.20 per kidney) inGata3+/2 (n=3) and wild-type (n=3) adult mice (***P,0.001). (F) Plasma creatinine measurements in 6-week old male (M) and female (F)Gata3+/+ (n=12) andGata3+/2 (n=16) mice. (G) IHC for GATA3 (brown) in adult kidneys from wild-type andGata3+/2mice show reducednumber of GATA3+ MCs in Gata3+/2 mice. (H) Quantification of the number of GATA3+ cells within glomeruli (n.20 per animal) ofwild-type (n=3) and Gata3+/2 (n=3) mice (***P,0.001). (I) Confirmation of reduced MC number using an independent mesangial markerPDGFR-b by IHC (brown) in adult kidneys from wild-type and Gata3+/2 mice. (J) Quantification of the area occupied by PDGFR-b stainwithin glomeruli (n.10 per animal) of wild-type (n=3) and Gata3+/2 (n=3) mice (*P,0.05). Scale bars, 20 mm.

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Figure 4. Gata3+/2 mice have MC defects. (A) IHC for GATA3 (brown) in developing glomeruli of E15.5 wild-type and Gata3+/2

embryos reveals a reduction in the number of MCs in Gata3+/2 mice (top panel). IHC for the endothelial marker CD31 (brown) showsabnormalities in capillary loop organization and smaller mesangium cores (outlined) in glomeruli of Gata3+/2 mice (middle panel). Co-IF with anti-GATA3 (green) and anti–PDGFR-b (red) antibodies (lower panel) in cap-stage glomeruli. Scale bars, 20 mm. (B) The numberof GATA3-expressing cells within glomeruli (n$12 per genotype) of Gata3+/2 (n=4) embryos are significantly reduced compared with

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disease. There was no difference in the number of glomerularGATA3-expressing nuclei in biopsy specimens from time-zerorenal allograft controls, MGN, or minimal change disease(Figure 7A). By contrast there were significantly moreGATA3-expressing nuclei in all lupus nephritis biopsy speci-mens and a subset of IgAN cases (Figure 7B). There were twoIgAN groups segregated by the degree of glomerular GATA3expression: those with increased mesangial GATA3+ cell num-ber and those with normal expression (Figure 7C). To verifythe bimodal distribution, we analyzed renal biopsy specimensfrom a second group of nine patients with IgAN who had asecond biopsy and inwhomwe could correlate the results withclinical data. Glomerular GATA3 expression increased in fivepatients and was normal in four, whether assessed by areaoccupied by GATA3+ nuclei-to-total GCS ratio, correctedGATA3+ cell number, or the mean nuclear GATA3 IF intensity(Figure 7, A–C). IgAN GATA3HIGH expressers also had signif-icantly more MC proliferation (Ki67+) (GATA3 HIGH: 0.44%60.62%; GATA3NORMAL: 0.12%60.16%; P,0.05) (Figure 7,G and H). These GATA3HIGH patients with IgAN had sus-tained heavy proteinuria either at the time of the biopsy ordeveloped it within 6 months and three had increased serumcreatinine concentrations (progressive group, SupplementalTable 3). Of note, in this progressive group we also observedextraglomerular GATA3+ cell expansion often localized to thejuxtaglomerular region, and in several cases, expansion ofGATA3+ cells in the periglomerular fibrotic regions, associatedboth with ostensibly normal as well as highly sclerotic glomer-uli where GATA3 expression persisted in the few remainingMCs (Supplemental Figure 9A). The GATA3NORMAL patients,however, had a normal serum creatinine both when biopsiedand 6 months later (indolent group). However, all four hadclinical relapses between 10 and 21 years later with increases inserum creatinine that provoked another renal biopsy (indo-lent: relapse). All four had increased GATA3+ MC number,MC proliferation, and GATA3 expression, demonstrating acorrelation with progressive disease (Figure 7, D–H). Group-ing the patients according to their Oxford Mesangial and

Endocapillary hypercellularity, Segmental glomerulosclerosis,Tubular atrophy score of mesangial hypercellularity (M, zero,or one) showed a significant difference in GATA3 expressiondemonstrating the utility of GATA3 as a MC marker whenevaluating MC expansion and proliferation (SupplementalFigure 9).

Recent transcriptomic analysis of differentially expressedgenes in IgAN glomeruli identified GATA3 as an upregulatedgene.38 We interrogated the cDNA expression data from mi-crodissected glomeruli (European Renal cDNA Bank) andrevealed a 2.1-fold increase (q,0.001) in GATA3 expression inIgAN (n=27) compared with living donors (n=6), providingindependent confirmation that GATA3 expression is upregu-lated in IgAN.

DISCUSSION

MCs are descendants of FOXD1-expressing stromal progen-itor cells in the developing kidney,31 but the transcriptionfactors responsible for this specification are largely unknown.Here, we identify GATA3 as one such transcription factor indeveloping kidneys by demonstrating its expression inFOXD1-stromal cells, that its first expression is coincidentwith stromal cell differentiation, and that GATA3 deficiencyin Gata3+/2 mice reduces the frequency of mesangial precur-sors and their proliferation in embryonic mouse kidneys, re-sulting in small glomeruli containing fewer MCs in adultmice. Furthermore, expression of GATA3 in MCs of maturekidneys increases in mesangial proliferative GN in humansand rodent models, suggesting a homeostatic importance af-ter injury, and thus a potential therapeutic target. In addition,GATA3 has practical utility as a simple, robust way to quantifyMCs in GN.

Kidney organogenesis involves three major progenitorpopulations: the well characterized RET1+/WNT11+ uretericbud, SIX2+/CITED1+ cap mesenchymal cells, and less welldescribed FOXD1+ stromal mesenchymal cells.34,35,39

wild-type (n=3) (*P,0.05). (C) Quantification of the number of PDGFR-b–expressing cells within glomeruli (n$15 per genotype) of wild-type (n=3) andGata3+/2 (n=4) embryos confirms reduced number of MCs inGata3+/2 mice (*P,0.05). (D) Areas occupied by CD31 stainwithin glomeruli (n$20 per genotype) of wild-type (n=3) and Gata3+/2 (n=4) embryos are similar, showing that endothelial cell numbersare not affected inGata3+/2mice. (E) Quantification of the number of podocytes within glomeruli (n$30 per genotype) of wild-type (n=3)andGata3+/2 (n=4) embryos shows that podocyte numbers inGata3+/2mice are similar towild-type. (F) Themean cytoplasmic intensity ofthe PDGFR-b IF signal is significantly reduced in glomeruli (n$15 per genotype) of Gata3+/2 (n=4) compared with wild-type (n=3) em-bryos. *P,0.05. (G) The mean nuclear intensity of the GATA3 IF signal is significantly reduced in MCs of Gata3+/2 mice (*P,0.05). (H) Inwild-type kidneys cells expressing high levels of GATA3 (green) and PDGFR-b (red)migrate from the stroma into the vascular cleft of the SSbody (arrows, top panel); these cells are closely associated with CD31+ endothelial progenitors (red arrows, middle panel). In Gata3+/2

littermates fewer GATA3+PDGFR+ cells can be seen within the vascular cleft; instead they group around the entrance to the cleft and aredissociated from CD31+ cells. Co-IF for GATA3 and Ki67 (bottom panel) in cap-stage glomeruli shows that fewer GATA3+ MCs areproliferating (i.e., GATA3+Ki67+) in Gata3+/2 kidneys, but the number of Ki67+ endothelial or podocyte cells are unaffected. (I) Quan-tification of the number of GATA3+ cells in the vascular cleft (VC; n$15 per genotype) of the SS bodies in Gata3+/+ (n=3) and Gata3+/2

(n=4) kidneys (***P,0.001). (J) The percentage of Ki67+ cells per total cells within GCS areas (n$15 per genotype) are similar betweenwild-type (n=3) and Gata3+/2 (n=4) kidneys. (K) A smaller proportion of GATA3+ cells in cap-stage glomeruli are proliferating (i.e.,GATA3+Ki67+, yellow arrow) in Gata3+/2 kidneys (*P,0.05).

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Figure 5. Increased GATA3 expression in the Thy1.1 mesangial proliferative nephritis model. (A) IHC for GATA3 (brown) in kidneysections of rats with Thy1.1 mesangial proliferative nephritis (day 8 post antibody administration) and control rats. Scale bar,50 mm. (B) Quantification of the area occupied by GATA3 stain, corrected for GCS area (expressed as a % of GCS) (each dotrepresents a single glomerulus; mean6SEM shown). (C) Colocalization of GATA3 (green) with PDGFR-b (red, top panel) and a-SMA(red, middle panel) in glomerular MCs and with Ki67 (red, bottom panel) in proliferating MCs (arrows). (D) Quantitative analysis ofdigitized images of sections stained by IF revealed an increase in the number of GATA3-expressing nuclei per total cell number(Dapi stained) per GCS area in Thy1.1 rat kidneys compared with control kidneys (***P,0.001; n=3 rats per group, 10–15 glomeruliper rat). (E) Measurement of the nuclear area (Dapi stained) showed that GATA3-expressing cells in Thy1.1 rat glomeruli had

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FOXD1 expression occurs from E10.5 in stromal cells, pre-cursors of VSMCs, MCs, renin-producing cells, pericytes, andinterstitial fibroblasts,35,40 including myofibroblasts in CKD.41

Deletion of Foxd1 causes severe kidney malformations, includ-ing reduced ureteric bud branching and decreased nephronnumbers; aberrant vessel-patterning; and fewer mesangialand renin cells.42,43 Identification of GATA3 in two of the

major progenitor populations (ureteric bud and stroma)and continued GATA3 expression by all descendant cellpopulations in mature kidneys (Figure 8), highlights its im-portance to renal development and initiation and mainte-nance of cell phenotype, explaining the wide spectrum ofkidney abnormalities associated with GATA3 deficiency inpatients with HDR. Renal expression of GATA3 is controlled

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Figure 6. Glomerular expression of GATA3 in mice induced with nephrotoxic nephritis. (A) IHC for GATA3 (brown) in kidney sections ofmice that were administered nephrotoxic antibodies to induce nephritis by days 3 and 10; and in corresponding control mice. Scalebar, 50 mm. (B) Co-IF with anti-GATA3 (green) and anti–PDGFR-b (red) antibodies demonstrating an increase in GATA3 IF signal in MCscoexpressing PDGFR-b at day 3 post disease induction (boxed region). Scale bar, 50 mm. (C) Quantification of the number of GATA3-expressing nuclei within a GCS area (n$20) reveals an increase in GATA3+ MC number on the third and fifth day post disease in-duction, and a reduction in the number of GATA3+ cells by day 10. *P,0.05.

enlarged nuclei compared with GATA3-expressing cells in control rats or GATA3-negative cells, indicating MC hypertrophy in Th1.1nephritis (**P,0.01; n=3 rats per group, 10–15 glomeruli per rat). (F) Quantification of the proportion of MCs (GATA3+) undergoingproliferation (Ki67+GATA3+) per GSC area (***P,0.001; n=3 rats per group, 10–15 glomeruli per rat). (G) Proliferation rates assessed byKi67 and GATA3 coimmunostaining. (***P,0.001; n=3 rats per group, 10–15 glomeruli per rat). (H) The mean nuclear intensity ofthe GATA3 IF signal significantly increased in nuclei of MCs (GATA3+) in Thy1.1 rats compared with controls (***P,0.001), indicating thatGATA3 protein expression is upregulated in MCs in mesangial proliferative nephritis. Proliferating MCs (GATA3+, Ki67+) had a higherGATA3 IF signal intensity compared with nonproliferating MCs (GATA3+, Ki672) (***P,0.001; n=3 rats per group, 10–15 glomeruli perrat). (I) Western blot analysis of glomerular lysates from Thy1.1 nephritic and control rats reveals profound upregulation of GATA3 protein(50 kDa). GATA3 protein levels normalized to total actin are shown (*P,0.05).

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Figure 7. Glomerular expression of GATA3 in patient renal biopsy samples. (A) IHC for GATA3 (brown) in sections of kidney biopsysamples from unaffected individuals (control) and patients with minimal change disease (MCD), membranous GN (MGN), and systemiclupus nephritis (LN). Scale bars, 50 mm. (B) IHC for GATA3 in sections of kidney biopsy samples from patients with IgA nephritis (IgAN)(brown, right panel), and dual ICH for GATA3 and CD10, a podocyte marker (red, left panel). Scale bars, 50 mm. (C) Numbers ofGATA3+ cells were quantified in glomeruli of normal (n=4), MCD (n=5), MGN (n=7), LN (n=13), and IgAN (n=7) kidney biopsy samples,corrected for GCS area (in mm2; average GCS area in control is 0.02760.007 mm2, mean6SD) and expressed as a ratio (GATA3+ cell

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by enhancer elements that are uniquely active in the twoprogenitor populations, and although a recent studyshowed that GATA3 expression can be directed to uretericbud-derived renal tubules,17 the location of the stromal/mesangial-specific GATA3 regulatory elements are yetto be identified. Absence of CD malformation in Gata3+/2

mice supports previous reports17,44 that threshold levelsfor GATA3 activ ity are cell-specific, and that MCsrequire a high level of GATA3 activity such that a 30% re-duction in protein expression, as we see in Gata3+/2 MCs(Figure 4G), is enough to result in their aberrant develop-ment, whereas ureteric bud–derived tubules are unaffectedby this level of GATA3 deficiency. Indeed, recent single-cellRNA-sequencing analysis on isolated MCs identifiedGATA3 as the third most enriched transcript expressed byevery single MC.19

The paucity of MCs in Gata3+/2 mice is reminiscentof parathyroid defects reported in these mice with smallerparathyroid-primordia containing fewer CaSR-expressingcells, and failure to initiate proliferation to enlarge the para-thyroid mass in response to a low calcium/vitamin D diet.3

Our current findings demonstrate that MC progenitors fromGata3+/2 mice have reduced proliferations rates and thatGATA3 expression is upregulated in proliferating MCs in bi-opsy specimens of patients with experimental mesangial pro-liferative GN and IgAN, suggesting that transcriptionalGATA3 activity is required for proliferation in response tosignals promoting MC activation in injured glomeruli. Inbreast and neuroblastoma tumor cells GATA3 promotes cellproliferation by facilitating G1/S transition through tran-scriptional regulation of the CCND1 gene.45,46 PDGF-B,TGF-b, and IL-6 signaling pathways have pivotal roles inMC proliferation.36,47,48 Whether GATA3 is involved inthese pathways remains to be elucidated. Our findingsthat fewer MC precursors enter the vascular cleft of theSS body are reminiscent of the MC defects reported inmice with stromal-mesenchyme specific deletion ofRBP-J, a downstream effector of Notch signaling.32,49

RPB-J has been shown to directly regulate GATA3 expressionin T-helper 2 cells,50,51 which indicates that the Notch-RBP-J-GATA3 regulatory axis may be involved in specificationand differentiation of MCs from FOXD1+ stromalmesenchyme.

Using GATA3 as a nuclear mesangial marker allowed us toprecisely evaluateMCexpansion andmesangial proliferationrates in mesangial proliferative GN, and demonstrate inter-glomerular variation. Inconsistencies in reportingmesangialcellularity are common when applying the Mesangial andEndocapillary hypercellularity, Segmental glomerulosclero-sis, Tubular atrophy score in IgAN biopsy specimens.52

Using GATA3 as a nuclear marker specific for MCs may beof great utility in quantification of mesangial hypercellu-larity and evaluation of disease progression. Indeed, expan-sion of GATA3-expressing nuclei was more prominent inbiopsy samples from patients with aggressive IgAN andhigher serum creatinine. Mesangial proliferation in thesebiopsy specimens was elevated compared with biopsy sam-ples from patients with indolent IgAN and lower serumcreatinine. Moreover, quantifying GATA3 expressionshowed, in agreement with the Thy1.1 model, thatGATA3 expression increased in MCs from biopsy speci-mens with higher mesangial proliferation. This suggeststhat aberrant GATA3 expression is associated with activa-tion of MCs and abnormal proliferation and extracellu-lar matrix deposition. Transcriptomic-profiling of MCsisolated from stromal-specific Gata3-deficient mice withinduced experimental mesangial injury will identify thegenes and pathways regulated by this integral MC tran-scription factor.

In summary, we identified a novel, nonredundant role forGATA3 in MCs from their first appearance in embryonickidney to mature glomeruli in health and disease. Further-more, GATA3 expression increases inMCs in mesangial pro-liferative PGN and haploinsufficiency of GATA3 results inMC defects, suggesting its importance for glomerularhomeostasis and response to injury. Moreover, GATA3 is a

number divided by GCS area). Each dot represents a mean of at least eight GCS areas per patient and mean6SEM per disease group isshown. Two populations were evident in the IgAN group (outlined). (D–F) Kidney biopsy samples from a second cohort of patients withIgAN with aggressive (n=5) or indolent (n=4; paired biopsy samples taken at first diagnosis and at relapse) disease were analyzed forGATA3 expression and time-zero renal allograft biopsy samples were used as controls. Areas occupied by GATA3 IHC stain, expressed asa percentage GCS area, were markedly increased in the aggressive IgAN group (P,0.01), but not in indolent IgAN group (D). Similarly,analysis of sections stained by IF revealed an increase in the number of GATA3-expressing nuclei per total cell number (Dapi stained) perGCS area in the aggressive IgAN group, but not in the indolent group, which only showed an increase in the repeat biopsy after relapse(E). The mean nuclear intensity of the GATA3 IF signal significantly increased in nuclei of MCs (GATA3+) in the aggressive IgAN group (F).Each dot represents a mean of at least eight GCS areas per patient and mean6SEM per group is shown. For each measurement, dif-ferences between means were considered statistically significant at *P,0.05; **P,0.01; ***P,0.001. (G) Assessment of the total (Ki67+)and mesangial (Ki67+, GATA3+) proliferation rates (expressed as a percentage of total cells per GCS area) revealed that patients withaggressive IgAN (n=5) had increased MC proliferation rates compared with controls (0.44%60.62% versus 0.12%60.16%; *P,0.05).A least eight GCS areas per patient were analyzed and mean6SEM per group is shown. (H) Representative sections of kidney biopsysamples from patients with IgAN stained by IHC for GATA3 (brown, top panel), and by IF for GATA3 and Ki67 showing localization ofGATA3 in MCs and proliferating MCs (arrows), respectively. Scale bars, 20 mm. ns, not significant.

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new, clinically applicable nuclearmarker ofMCs, superior tocurrent markers.

ACKNOWLEDGMENTS

We thank Dr. Enikoe Kallay for kindly providing the CaSR antibody,

Anton Jäger for assistance with the graphical abstract, and Dr. Robert

Steadman for his valuable comments on the manuscript. We also

thank all participating centers of the European Renal cDNA Bank,

Kröner–Fresenius Biopsy Bank (ERCB-KFB), and their patients for

their cooperation. Dr. Grigorieva, Prof. Rees, and Prof. Kain designed

the study. Prof. Thakker, Prof. Ostendorf, Prof. Floege, and

Dr. Panzer made substantial contributions to acquisition of data

and revised the article critically for important intellectual content.

Dr. Grigorieva, Dr. Grigorieva,Ms. Schachner, andMs. Neudert carried

out experiments. Dr. Grigorieva, Dr. Oszwald, and Dr. Lindenmeyer

analyzed the data. Dr. Grigorievamade the figures. Dr. Grigorieva, Prof.

Rees, and Prof. Kain drafted and revised the paper, all authors approved

the final version of the manuscript.

The opinions expressed in this publication are those of the authors

and donot purport to reflect the official position or views of the EC, its

agencies, or officers.

For a list of active members at the time of the study, see

Shved et al.54

DISCLOSURES

Dr. Aigner reports personal fees from Sanofi, non-financial support fromChiesi, outside the submittedwork. Prof. Thakker reports grants fromMedical

Stromal progenitorsFOXD1+

Cap mesenchyme:SIX2+/CITED1+

GATA3 expressing cells

Ureteric bud:GATA3+/RET1+/WNT11+ Cortical stroma:

GATA3+/FOXD1+

Endothelial:FLIK1+/TIE2+

embrionic

GATA3

MsPGN

epithelial (DT/CN/CD) Mesangial VSMC Renin-producingcell

Fibroblast

GATA3(Gata3+/-)

Mesangial cell deficiencyand smaller glomeruli

adult

Figure 8. Schematic diagram showing location of GATA3 expression (green) in the embryonic and adult kidney. GATA3 is expressed by twomajor renal progenitor populations, ureteric bud and stromal cells, and continues to be expressed by all cell types that originate from them,i.e., epithelial cells of the CDs/DN/CN and stromal cell descendants (mesangial, VSMCs, renin-producing cells, and fibroblasts). Deficiency inGATA3 results in a paucity of MCs and smaller glomeruli, and increased GATA3 expression is associated with mesangial proliferative GN.

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Research Council, grants fromWellcome Trust, grants fromNational Institutefor Health Research, grants from EU Horizon 2020 Marie Curie Actions InitialTraining Networks, grants from Kidney Research UK, grants from National In-stitute for Health Research, Translational Research Collaboration, grants fromMarshall Smith Syndrome Foundation, during the conduct of the study; grantsfromNPS/ Shire Pharmaceuticals, grants fromNovartis PharmaAG, grants fromGlaxoSmithKline, personal fees from Ipsen, personal fees fromAstraZeneca, out-side the submitted work. All of the remaining authors have nothing to disclose.

FUNDING

Dr. Grigorieva was funded by a Seventh Framework Programme of theEuropean Union Marie Curie IEF Fellowship (grant number 302739[2011]). This project has received funding from the European Union Horizon2020 research and innovation programme under grant agreement 668036(RELENT). The European Renal cDNA Bank Kröner-Fresenius Biopsy Bankis supported by the Else Kröner-Fresenius Foundation.

SUPPLEMENTAL MATERIAL

This article contains the following supplemental material online at

http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2018111143/-/

DCSupplemental.

Supplemental Figure 1. GATA3 expression in the kidney is con-

served between human, rat, and mouse.

SupplementalFigure2.ExpressionofGATA3inthehumanfetalkidney.

Supplemental Figure 3. GATA3 is a marker of distal tubule pre-

cursor cells.

Supplemental Figure 4. GATA3 expression is downregulated in

primary MC cultures.

Supplemental Figure 5. Expression of PDGFR-b precedes GATA3

expression in stromal mesenchyme.

Supplemental Figure 6. Impaired recruitment of GATA3+ MC

precursors into the developing glomeruli in Gata3+/2 mice.

Supplemental Figure 7. Proliferating endothelial cells in the Thy1.1

mesangial proliferative nephritis model.

Supplemental Figure 8. Quantification of GATA3 IF intensity.

Supplemental Figure 9. Extraglomerular and crescentic GATA3

expression in renal biopsy samples.

Supplemental Table 1. Patient clinical parameters.

Supplemental Table 2. Clinical parameters of patients in the Lupus

nephritis cohort.

Supplemental Table 3. Clinical parameters of patients in the IgAN

cohort.

Supplemental Table 4. Primer sequences.

Supplemental Table 5. Primary antibodies.

Supplemental Table 6.Gata3+/2mouse kidney function parameters.

Supplemental Appendix 1. Daltonized versions of Figures 1, 2, and

4–7, and Supplemental Figures 2, 7, and 9.

REFERENCES

1. TakakuM, GrimmSA, Shimbo T, Perera L, Menafra R, Stunnenberg HG,et al.: GATA3-dependent cellular reprogramming requires activation-domain dependent recruitment of a chromatin remodeler. GenomeBiol 17: 36, 2016

2. Kouros-Mehr H, Slorach EM, Sternlicht MD, Werb Z: GATA-3 maintainsthe differentiation of the luminal cell fate in the mammary gland.Cell 127: 1041–1055, 2006

3. Grigorieva IV, Mirczuk S, Gaynor KU, Nesbit MA, Grigorieva EF, Wei Q,et al.: Gata3-deficient mice develop parathyroid abnormalities due todysregulation of the parathyroid-specific transcription factor Gcm2.J Clin Invest 120: 2144–2155, 2010

4. Grote D, Boualia SK, Souabni A, Merkel C, Chi X, Costantini F, et al.:Gata3 acts downstream of beta-catenin signaling to prevent ectopicmetanephric kidney induction. PLoS Genet 4: e1000316, 2008

5. GroteD, Souabni A, BusslingerM, BouchardM: Pax 2/8-regulatedGata3 expression is necessary for morphogenesis and guidance of thenephric duct in the developing kidney.Development 133: 53–61, 2006

6. Lim KC, Lakshmanan G, Crawford SE, Gu Y, Grosveld F, Engel JD:Gata3 loss leads to embryonic lethality due to noradrenaline deficiencyof the sympathetic nervous system. Nat Genet 25: 209–212, 2000

7. van der Wees J, van Looij MA, de Ruiter MM, Elias H, van der Burg H,Liem SS, et al.: Hearing loss following Gata3 haploinsufficiency iscaused by cochlear disorder. Neurobiol Dis 16: 169–178, 2004

8. Riedel JH, Becker M, Kopp K, Düster M, Brix SR, Meyer-Schwesinger C,et al.: IL-33-mediated expansion of type 2 innate lymphoid cellsprotects from progressive glomerulosclerosis. J Am Soc Nephrol 28:2068–2080, 2017

9. Hosoya T, Maillard I, Engel JD: From the cradle to the grave: Activi-ties of GATA-3 throughout T-cell development and differentiation.Immunol Rev 238: 110–125, 2010

10. Mjösberg J, Bernink J, Golebski K, Karrich JJ, Peters CP, Blom B, et al.:The transcription factor GATA3 is essential for the function of humantype 2 innate lymphoid cells. Immunity 37: 649–659, 2012

11. Van Esch H, Groenen P, Nesbit MA, Schuffenhauer S, Lichtner P,Vanderlinden G, et al.: GATA3 haplo-insufficiency causes human HDRsyndrome. Nature 406: 419–422, 2000

12. Ali A, Christie PT, Grigorieva IV, Harding B, Van Esch H, Ahmed SF,et al.: Functional characterization of GATA3 mutations causing thehypoparathyroidism-deafness-renal (HDR) dysplasia syndrome: Insightinto mechanisms of DNA binding by the GATA3 transcription factor.Hum Mol Genet 16: 265–275, 2007

13. Gaynor KU, Grigorieva IV, Nesbit MA, Cranston T, Gomes T, Gortner L,et al.: A missense GATA3 mutation, Thr272Ile, causes the hypopara-thyroidism, deafness, and renal dysplasia syndrome. J Clin EndocrinolMetab 94: 3897–3904, 2009

14. Belge H, Dahan K, Cambier JF, Benoit V, Morelle J, Bloch J, et al.:Clinical and mutational spectrum of hypoparathyroidism, deafness andrenal dysplasia syndrome. Nephrol Dial Transplant 32: 830–837, 2017

15. Chenouard A, Isidor B, Allain-Launay E, Moreau A, Le Bideau M,Roussey G: Renal phenotypic variability in HDR syndrome: Glomerularnephropathy as a novel finding. Eur J Pediatr 172: 107–110, 2013

16. Kamezaki M, Kusaba T, Adachi T, Yamashita N, Nakata M, Ota N, et al.:Unusual proliferative glomerulonephritis in a patient diagnosed to havehypoparathyroidism, sensorineural deafness, and renal dysplasia (HDR)syndrome with a novel mutation in the GATA3 gene. Intern Med 56:1393–1397, 2017

17. Moriguchi T, Yu L, Otsuki A, Ainoya K, Lim KC, Yamamoto M, et al.:Gata3 hypomorphic mutant mice rescued with a yeast artificial chro-mosome transgene suffer a glomerular mesangial cell defect.Mol CellBiol 36: 2272–2281, 2016

18. Brunskill EW, Potter SS: Changes in the gene expression programsof renal mesangial cells during diabetic nephropathy. BMC Nephrol13: 70, 2012

19. Lu Y, Ye Y, Yang Q, Shi S: Single-cell RNA-sequence analysis of mouseglomerular mesangial cells uncovers mesangial cell essential genes.Kidney Int 92: 504–513, 2017

20. Yoh K, Shibuya K, Morito N, Nakano T, Ishizaki K, Shimohata H, et al.:Transgenic overexpression of GATA-3 in T lymphocytes improves au-toimmune glomerulonephritis in mice with a BXSB/MpJ-Yaa geneticbackground. J Am Soc Nephrol 14: 2494–2502, 2003

JASN 30: ccc–ccc, 2019 GATA3 in Mesangial Cells 17

www.jasn.org BASIC RESEARCH

21. van Doorninck JH, van Der Wees J, Karis A, Goedknegt E, Engel JD,Coesmans M, et al.: GATA-3 is involved in the development of sero-tonergic neurons in the caudal raphe nuclei. J Neurosci 19: RC12, 1999

22. Floege J, Ostendorf T, Janssen U, Burg M, Radeke HH, Vargeese C,et al.: Novel approach to specific growth factor inhibition in vivo:Antagonism of platelet-derived growth factor in glomerulonephritisby aptamers. Am J Pathol 154: 169–179, 1999

23. Panzer U, Steinmetz OM, Paust HJ, Meyer-Schwesinger C, Peters A,Turner JE, et al.: Chemokine receptor CXCR3 mediates T cell recruit-ment and tissue injury in nephrotoxic nephritis in mice. J Am SocNephrol 18: 2071–2084, 2007

24. Kreisberg JI, Karnovsky MJ: Glomerular cells in culture. Kidney Int 23:439–447, 1983

25. Sarrab RM, Lennon R, Ni L, Wherlock MD, Welsh GI, Saleem MA:Establishment of conditionally immortalized human glomerular me-sangial cells in culture, with unique migratory properties. Am J PhysiolRenal Physiol 301: F1131–F1138, 2011

26. Cohen CD, Frach K, Schlöndorff D, Kretzler M: Quantitative gene ex-pression analysis in renal biopsies: A novel protocol for a high-throughputmulticenter application. Kidney Int 61: 133–140, 2002

27. Martini S, Nair V, Keller BJ, Eichinger F, Hawkins JJ, Randolph A, et al.;European Renal cDNA Bank; C-PROBE Cohort; CKDGen Consortium:Integrative biology identifies shared transcriptional networks in CKD.J Am Soc Nephrol 25: 2559–2572, 2014

28. Cohen CD, Klingenhoff A, Boucherot A, Nitsche A, Henger A, BrunnerB, et al.: Comparative promoter analysis allows de novo identification ofspecialized cell junction-associated proteins. Proc Natl Acad Sci U S A103: 5682–5687, 2006

29. Tusher VG, Tibshirani R, Chu G: Significance analysis of microarraysapplied to the ionizing radiation response. Proc Natl Acad Sci U S A 98:5116–5121, 2001

30. Jones TR, Kang IH, Wheeler DB, Lindquist RA, Papallo A, Sabatini DM,et al.: CellProfiler Analyst: Data exploration and analysis software forcomplex image-based screens. BMC Bioinformatics 9: 482, 2008

31. Sequeira Lopez ML, Gomez RA: Development of the renal arterioles.J Am Soc Nephrol 22: 2156–2165, 2011

32. Lin EE, Sequeira-Lopez ML, Gomez RA: RBP-J in FOXD1+ renal stromalprogenitors is crucial for the proper development and assembly of thekidney vasculature and glomerular mesangial cells. Am J Physiol RenalPhysiol 306: F249–F258, 2014

33. Sequeira-Lopez ML, Lin EE, Li M, Hu Y, Sigmund CD, Gomez RA: Theearliest metanephric arteriolar progenitors and their role in kidneyvascular development. Am J Physiol Regul Integr Comp Physiol 308:R138–R149, 2015

34. Kobayashi A, Mugford JW, Krautzberger AM, Naiman N, Liao J,McMahon AP: Identification of a multipotent self-renewing stromalprogenitor population duringmammalian kidney organogenesis. StemCell Reports 3: 650–662, 2014

35. Hatini V, Huh SO, Herzlinger D, Soares VC, Lai E: Essential role ofstromal mesenchyme in kidney morphogenesis revealed by targeteddisruption of Winged Helix transcription factor BF-2. Genes Dev10: 1467–1478, 1996

36. Floege J, van Roeyen C, Boor P, Ostendorf T: The role of PDGF-Din mesangioproliferative glomerulonephritis. Contrib Nephrol 157:153–158, 2007

37. Weening JJ, D’Agati VD, Schwartz MM, Seshan SV, Alpers CE, AppelGB, et al.: The classification of glomerulonephritis in systemic lupuserythematosus revisited. J Am Soc Nephrol 15: 241–250, 2004

38. Liu P, Lassén E, Nair V, Berthier CC, Suguro M, Sihlbom C, et al.:Transcriptomic and proteomic profiling provides insight intomesangialcell function in IgA nephropathy. J Am Soc Nephrol 28: 2961–2972,2017

39. Little MH, McMahon AP: Mammalian kidney development: Princi-ples, progress, and projections. Cold Spring Harb Perspect Biol 4,2012

40. Li W, Hartwig S, Rosenblum ND: Developmental origins and func-tions of stromal cells in the normal and diseased mammalian kidney.Dev Dyn 243: 853–863, 2014

41. Humphreys BD, Lin SL, Kobayashi A, Hudson TE, Nowlin BT,Bonventre JV, et al.: Fate tracing reveals the pericyte and not epi-thelial origin of myofibroblasts in kidney fibrosis. Am J Pathol 176:85–97, 2010

42. Levinson RS, Batourina E, Choi C, Vorontchikhina M, Kitajewski J,Mendelsohn CL: Foxd1-dependent signals control cellularity in therenal capsule, a structure required for normal renal development.Development 132: 529–539, 2005

43. Hum S, Rymer C, Schaefer C, Bushnell D, Sims-Lucas S: Ablation ofthe renal stroma defines its critical role in nephron progenitor andvasculature patterning. PLoS One 9: e88400, 2014

44. Hasegawa SL, Moriguchi T, Rao A, Kuroha T, Engel JD, Lim KC: Dos-age-dependent rescue of definitive nephrogenesis by a distant Gata3enhancer. Dev Biol 301: 568–577, 2007

45. Shan L, Li X, Liu L, Ding X, Wang Q, Zheng Y, et al.: GATA3 cooperateswith PARP1 to regulate CCND1 transcription through modulating his-tone H1 incorporation. Oncogene 33: 3205–3216, 2014

46. Molenaar JJ, Ebus ME, Koster J, Santo E, Geerts D, Versteeg R, et al.:Cyclin D1 is a direct transcriptional target of GATA3 in neuroblastomatumor cells. Oncogene 29: 2739–2745, 2010

47. Schlöndorff D, Banas B: The mesangial cell revisited: No cell is an is-land. J Am Soc Nephrol 20: 1179–1187, 2009

48. Eitner F, Westerhuis R, Burg M, Weinhold B, Gröne HJ, Ostendorf T,et al.: Role of interleukin-6 inmediatingmesangial cell proliferation andmatrix production in vivo. Kidney Int 51: 69–78, 1997

49. Boyle SC, Liu Z, Kopan R: Notch signaling is required for the formationof mesangial cells from a stromal mesenchyme precursor during kidneydevelopment. Development 141: 346–354, 2014

50. Amsen D, Antov A, Jankovic D, Sher A, Radtke F, Souabni A, et al.:Direct regulation of Gata3 expression determines the T helper differ-entiation potential of Notch. Immunity 27: 89–99, 2007

51. Fang TC, Yashiro-Ohtani Y, Del Bianco C, Knoblock DM, Blacklow SC,Pear WS: Notch directly regulates Gata3 expression during T helper 2cell differentiation. Immunity 27: 100–110, 2007

52. Trimarchi H, Barratt J, Cattran DC, Cook HT, Coppo R, Haas M, et al.;IgAN Classification Working Group of the International IgA Ne-phropathy Network and the Renal Pathology Society; ConferenceParticipants: Oxford classification of IgA nephropathy 2016: An up-date from the IgA nephropathy classification working group. KidneyInt 91: 1014–1021, 2017

53. Graca JA, Schepelmann M, Brennan SC, Reens J, Chang W, Yan P,et al.: Comparative expression of the extracellular calcium-sensing re-ceptor in themouse, rat, and human kidney.Am J Physiol Renal Physiol310: F518–F533, 2016

54. Shved N, Warsow G, Eichinger F, Hoogewijs D, Brandt S, Wild P,et al.: Transcriptome-based network analysis reveals renal cell type-specific dysregulation of hypoxia-associated transcripts. Sci Rep. 7:8576, 2017

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