Eur. J. Immunol. 2012. 42: 2441–2451 ImmunomodulationDOI: 10.1002/eji.201242434 2441

Regulatory T cells participate in CD39-mediatedprotection from renal injury

Yuan Min Wang1, Jennifer L. McRae2, Simon C. Robson3, Peter J.Cowan2,4, Geoff Yu Zhang1, Min Hu1, Tania Polhill1, Yiping Wang5,Guoping Zheng5, Ya Wang5, Vincent W.S. Lee5, Robert J. Unwin6,David C.H. Harris5, Karen M. Dwyer2,4 and Stephen I. Alexander1

1 Centre for Kidney Research, Children’s Hospital at Westmead, Sydney, Australia2 Immunology Research Centre, St. Vincent’s Hospital, Melbourne, Australia3 Transplant Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston,

MA, USA4 Department of Medicine, The University of Melbourne, Melbourne, Australia5 Centre for Transplantation and Renal Research, University of Sydney at Westmead

Millennium Institute, Sydney, Australia6 UCL Centre for Nephrology (Royal Free Campus), University College London, London,

United Kingdom

CD39 is an ecto-enzyme that degrades extracellular nucleotides, such as ATP, and ishighly expressed on by the vasculature and circulating cells including Foxp3+ regula-tory T (Treg) cells. To study the role of purinergic regulation in renal disease, we usedthe adriamycin nephropathy (AN) mouse model of chronic renal injury, using humanCD39-transgenic (hCD39Tg) and wild-type (WT) BALB/c mice. Effects of CD39 expressionby Treg cells were assessed in AN by adoptive transfer of CD4+CD25+ and CD4+CD25−

T cells isolated from hCD39Tg and WT mice. hCD39Tg mice were protected from renalinjury in AN with decreased urinary protein and serum creatinine, and significantly lessrenal injury compared with WT mice. While WT CD25+ and hCD39Tg CD25− T cells con-ferred some protection against AN, hCD39Tg CD25+ Treg cells offered greater protection.In vitro studies showed direct pro-apoptotic effects of ATP on renal tubular cells. In con-clusion, hCD39 expressed by circulating leukocytes and intrinsic renal cells limits innateAN injury. Specifically, CD39 expression by Treg cells contributes to its protective rolein renal injury. These findings suggest that extracellular nucleotides mediate AN kidneyinjury and that CD39, expressed by Treg cells and other cells, is protective in this model.

Keywords: Adriamycin nephropathy (AN) � ATP � CD39 � Foxp3 � Treg cells

Introduction

ATP exists in high concentrations intracellularly and is the energycurrency for cellular metabolism. Under pathophysiological con-

Correspondence: Professor Stephen I. Alexandere-mail: StephenA@chw.edu.au

ditions, such as when cells are injured or die, ATP is releasedinto the extracellular space. As extracellular ATP is normallypresent in only very low concentrations, this heightened releaseof nucleotides has a major role in inducing cellular injury andimmune activation.

In such situations, ATP functions as a marker of inflammationor injury [1–3], as it promotes inflammation and thrombosis sig-naling via purinergic type 2 (P2) receptors. P2X7, a member of

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2442 Yuan Min Wang et al. Eur. J. Immunol. 2012. 42: 2441–2451

the P2X receptor subtype family, is widely expressed and func-tions as a “sensor” of extracellular ATP. A number of downstreamevents occur following engagement with ATP, which culminatein cell death. P2X7 is a ligand-gated ion channel receptor thatcan activate the NALP3 inflammasome, leading to macrophageactivation and IL-1 release [4–6]. This receptor is also involvedin pain sensation and is increasingly recognized to have a majorrole in inflammation [7]. As compared with other P2 receptors,P2X7 requires a high level of ATP for activation, which means thata significant amount of cellular injury must occur before levelsof released cellular ATP lead to its stimulation [8]. Furthermore,expression on epithelial cells, including renal cells, in addition tomacrophages, suggests that it may act locally, as well as throughcirculating immune cells, to exacerbate renal injury [9,10].

The extracellular enzyme CD39 tightly regulates extracellularATP levels. CD39 hydrolyzes ATP to ADP and then further dephos-phorylates ADP to AMP. CD73 is another ecto-enzyme which isoften coexpressed and promotes conversion of AMP to adenosine;the anti-inflammatory and anti-thrombotic effects of this cascadehave been confirmed in a number of disease models [11–14]. Inrenal ischemia-reperfusion injury (IRI), deletion of CD39 [15,16],but not CD73 [16,17], exacerbates renal injury, implicating ATPas the mediator of ongoing inflammation. Conversely, when CD39is overexpressed, renal injury is mitigated [15]. CD39 overexpress-ing mice have enhanced capacity to metabolize ATP, as indicatedby increased production of AMP and adenosine [18]. Chronickidney disease is often cumulative and may involve direct toxicinjury and progressive immunological injury [19]. A murine modelthat mimics human renal disease is adriamycin (ADR, doxoru-bicin) nephropathy (AN), where an initial toxic injury in BALB/cmice leads to progressive renal injury. This model has a signifi-cant immune component that closely mimics human chronic pro-teinuric renal diseases such as focal segmental glomerulosclerosis(FSGS). ADR causes direct toxic injury to podocytes and tubu-lar cells, likely leading to ATP release. We have shown previ-ously that regulatory T cells (CD4+CD25+ T cells) are protectiveagainst renal injury in AN. Treg cells induced by transductionwith Foxp3 (the critical transcriptional molecule in the Treg lin-eage) also protect against renal injury in this model [20,21]. Thefinding that Treg cells can also reduce renal injury in immuno-deficient mice in which AN is more severe, suggested that alter-native pathways were being utilized by Treg cells to suppressrenal injury [21]. The recent description of CD39 expression byTreg cells raised the possibility that this might be a mechanismby which ATP is removed, protecting against immune and non-immune injury [13,22,23].

Constitutive expression of CD39 by Treg cells has been shownin humans [23,24] and in mice [23,25]. The expression of CD39on Treg cells may define particular effector subpopulations inhumans [18,26]. The role of CD39 on Treg cells may be multi-functional, including the removal of ATP, which plays a major rolein Treg-mediated immunosuppression [23,26–28] and the gener-ation of adenosine [25]. Recently, CD39 expression has been usedto distinguish memory populations of CD4+CD25+ Foxp3 Tregcells from IL-17 producing pathogenic T cells, supporting its link

to functional regulatory T cells [18,26]. The high expression ofCD39 on Treg cells and its ability to define a functional subset ofTreg cells and its role in removing ATP, a potential mediator ofinjury, make it a major potential mechanism for protection againstrenal injury.

The role of purine nucleotides and their P2 receptors has beenstudied in a variety of animal models, and in human diseaseinvolving chronic injury and fibrosis [9,14,29–31]. In the uni-lateral ureteric obstruction (UUO) model, there was significantlyless injury and macrophage infiltration in P2X7−/− mice [32].In another murine model of acute antibody-mediated nephro-toxic nephritis (NTN) on the C57BL/6 background, significantlyless injury was observed with P2X7 receptor deletion and inthe rat model by pharmacological blockade [33]. However, it isunclear from these models whether this was a function of reducedmacrophage and monocyte priming through the P2X7 receptor, orof reduced direct injury to renal cells. It is therefore important toevaluate whether removal of ATP by CD39 is protective againstrenal tubular injury.

To study the role of ATP in AN, the human CD39 transgenicmouse (hCD39Tg), which is on the required BALB/c backgroundfor this model of AN, has been utilized. These mice have beenshown to overexpress hCD39 generally including on Treg cellsand that this is functional in producing adenosine [11]. Thesestudies were performed in hCD39Tg mice to evaluate the role ofCD39 in Treg-cell function in affording protection in this model ofAN.

Results

hCD39Tg mice were protected against renal injury

AN was induced in both BALB/c hCD39 transgenic mice(hCD39Tg) and BALB/c wild-type mice (WT). All mice wereanalyzed at 4 weeks after ADR when effects of injury are mostnotable. Urinary protein excretion was significantly reduced inhCD39Tg/AN mice compared with WT/AN mice (p < 0.05,Fig. 1A). Serum creatinine was also significantly lower inhCD39Tg/AN mice than in WT/AN mice (p < 0.05, Fig. 1B). Therewere similar levels of proteinuria and serum creatinine betweennormal WT and normal hCD39Tg mice (Fig. 1A and B).

In WT mice with AN, there was a flattening and vacuolizationof tubular epithelial cells and a moderate interstitial monocyteinfiltrate. Glomeruli were reduced in size with severe vacuoliza-tion and mesangial expansion on periodic acid-Schiff (PAS) stain-ing (Figs. 1C and 2A). However, by comparison, there was onlymild damage of glomeruli and tubules in hCD39Tg mice withAN (Fig. 2A). Trichrome staining showed no fibrosis in both nor-mal WT and normal CD39Tg mice, and a significant reduction infibrosis in hCD39Tg mice compared with WT mice (p < 0.001,Figs. 1D and 2B). The immunohistochemical assay for kidney sec-tions showed reduced interstitial monocyte infiltration consistingof macrophages, CD8+ and CD4+ cells in CD39Tg/AN mice com-pared with WT/AN mice (Fig. 2C). Quantitative analysis showed

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Figure 1. hCD39Tg mice were protected againstrenal injury. (A) Proteinuria and (B) serum creati-nine levels were measured in both hCD39Tg withAN (hCD39Tg/AN) and WT mice with AN (WT/AN).(C) Morphological changes in the glomeruli andtubules were evaluated in a semiquantitativemanner. (D) Fibrosis was scored and evaluated ina quantitative manner.(A–D) Data are shown asmean + SD of 5–6 mice per group and are repre-sentative of one out of three independent experi-ments performed. *p < 0.05, **p < 0.01, ***p < 0.001,Student’s t-test.

that there was a significant increase in the number of infiltratingcells in WT AN mice compared with CD39Tg AN mice (Fig. 2D).

Immunohistochemical staining for Foxp3 showed an equivalentnumber of Foxp3+ Treg cells in kidneys and spleens of WT andhCD39Tg mice following AN. (Fig. 3A and B); furthermore, therewas an equivalent proportion of Foxp3+ Treg cells in the CD4+

T cells in normal WT (10 ± 1%), normal hCD39Tg mice(10 ± 1%), hCD39Tg/AN, and WT/AN mice (9 ± 0.6%) (Fig. 3C).There was no significant difference for the absolute numbers ofCD4+Foxp3+ cells within spleens between normal WT (1.5 ±0.2 × 106) and CD39Tg mice (1.5 ± 0.2 × 106).

hCD39 overexpression on Treg cells can limit renalinjury in AN

We have evaluated expression of hCD39 in kidney and spleenin both BALB/c WT and hCD39Tg mice (Fig. 4A), and foundhCD39 expressed throughout the spleen and kidney most markedin the interstitium and glomeruli in hCD39Tg mice. Foxp3+ Tregcells from either WT or hCD39Tg mice showed high expressionof mouse CD39. However, only Treg cells from hCD39Tg miceshowed high levels of human CD39 (Fig. 4B).

CD4+CD25+ T cells and CD4+CD25− T cells were isolatedfrom hCD39Tg/AN. CD4+CD25+ T cells were isolated from WTmice (purities of the transferred cells were 97–98%, Fig. 4C),then the cells were transferred into WT mice. Flow cytometryconfirmed that 95–96% of the isolated CD4+ CD25+ T cellswere Foxp3+ T cells (Fig. 4D). AN was induced 3 days aftercell transfer. Mice were divided into four groups (see Methods).CD4+CD25+ cells from all groups of mice had high levels ofFoxp3 expression, whereas Foxp3 expression was minimal inCD4+CD25− T cells from both groups of mice (Fig. 4E). Although

CD4+CD25+ T cells from WT mice protected against renal injury(p < 0.05), CD4+CD25+ cells isolated from hCD39Tg were themost potent in limiting renal injury (p < 0.05 compared withWT 25+ and hCD39Tg25− groups; p < 0.01 compared with ADRgroup). Proteinuria (Fig. 5A) and serum creatinine (Fig. 5B)were diminished in mice that received CD4+CD25+ T cells fromhCD39Tg mice (p < 0.05 and p < 0.01, respectively); mice thatreceived CD4+CD25− T cells from hCD39Tg mice also showedless severe disease than ADR mice (P < 0.05 compared with ADRonly). Therefore, protection by overexpression of hCD39 can actwith, but also independently of, Treg cells to limit renal injury.The most severe tubular cell atrophy, tubular dilatation, glomeru-losclerosis, and infiltration of mononuclear cells were observedin WT mice treated with ADR (Fig. 5C and E); mice that receivedhCD39 CD4+ CD25+ T cells were least affected (p < 0.01). Asdescribed previously, WT CD4+ CD25+ cells were also protective,but so were hCD39 CD4+ CD25− T cells (p < 0.05). Quantitativeanalysis showed that glomerulosclerosis, tubular damage, andinterstitial monocyte infiltration were significantly lower in thehCD39Tg CD25+ group of mice (Fig. 5C, p < 0.05 and p < 0.01,respectively). Furthermore, mice injected with CD25+ cells fromhCD39Tg mice did not suffer the weight loss seen in the otherADR-treated groups (Fig. 5D, p < 0.05 and p < 0.01 respectively).

CD39Tg primary tubular cells were protected fromATP-induced apoptosis

Apoptosis was assessed using TUNEL staining in a renal tubu-lar cell line (C1.1) and primary tubular cells from both WT andCD39Tg mice. There was a dose-dependent response to ATP withpeak apoptosis occurring at 3 mM ATP [12,34]. A time-courseusing 3 mM ATP found apoptotic cells were first detected at

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Figure 2. hCD39Tg mice were protected against histological renal injury, fibrosis, and cellular infiltration. (A) Histological changes were evaluatedby PAS staining in untreated WT, untreated CD39Tg, WT/AN and CD39Tg/AN mice (six mice in each group, magnification, ×200). (B) Fibrosis (blue)was measured by trichrome staining in four groups (six mice in each group; magnification: ×200). (C) CD4+ and CD8+ T cells, and macrophagesinfiltration were performed by immunohistochemical staining in WT, WT/AN, and CD39Tg/AN mice (six mice in each group; magnification: ×400).(D) The number of infiltrating cells were evaluated by quantitative analysis in WT, WT/AN, and CD39Tg/AN mice per microscopy field (MF). Dataare shown as mean + SD of 5–6 mice per group and are representative of one out of three independent experiments performed. *p < 0.05, Student’st-test.

90 min and reached their peak at 2 h (Fig. 6A). There is less apop-tosis in tubular cells from CD39Tg mice compared with WT miceafter ATP treatment (Fig. 6B and C). Furthermore, quantificationfor primary cell apoptosis demonstrated that extracellular ATP-induced tubular cell apoptosis (TUNEL-positive nuclei) that is timedependent and dose related, and reduced apoptosis in tubularcells from CD39Tg mice compared with WT mice (Fig. 6E and F,**p < 0.01). As a control, tubular cells isolated from both WT andCD39Tg mice showed equivalent apoptosis after exposure to H2O2

(Fig. 6D and F).

Discussion

We have shown that overexpression of hCD39 in BALB/c miceprotected against renal injury in mice with (AN). There was lessproteinuria, weight loss, and elevation of serum creatinine in thehCD39Tg mice. Histological examination of these mice confirmedthat there was less structural injury with less glomerulosclerosis,tubular atrophy, and fibrosis. A substantive role of tissue expres-sion of hCD39 is in keeping with findings of protection againstinjury in vascularized transplant models where overexpression of

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Figure 3. An equivalent proportion of CD4+Foxp3+ cells in WT, hCD39Tg, WT/AN, and CD39Tg/AN mice. (A) Immunohistochemical staining ofFoxp3+ cells in kidneys and spleens for four groups of mice. Magnification, ×400. (B) Foxp3+ T cells per MF were quantitatively analyzed in spleenand kidney. (C) CD4+Foxp3+ cells from splenocytes were analyzed by intracellular flow cytometric staining. (A–C) Data are shown as mean + SD of5–6 mice per group and are representative of one out of three independent experiments performed.

hCD39 in the transplant is protective in cardiac xenografts, renalallotransplants, and during IRI [11,15].

We have previously shown that Treg cells protect against renalinjury in AN and that their depletion exacerbates AN[20]. Fur-ther this effect occurs in SCID mice that lack T and B cells withAN suggesting that Treg cells have an intrinsic protective capacityindependent of other T cells[21]. The high level of CD39 on Tregcells suggested a molecular mechanism operative in immunodefi-cient mice [18].

To address the role of CD39 in Treg-cell function, adoptivetransfer experiments were performed using WT Treg cells,CD39Tg Treg cells, and non-Treg CD4+ cells overexpressing

CD39. These experiments showed that while CD39 expressed bynon-Treg cells on circulating cells can provide some renoprotec-tion, overexpression of CD39 on Treg cells was the more potent inreducing renal injury. These results suggest that CD39 bioactivitymay limit renal injury given the ability of TgCD39 CD4+CD25−

T cells to protect against AN. Decreased levels of ATP are themost likely mechanism of action of CD39 expression, potentiallyreducing signaling through the ATP receptor P2X7 that leadsto NALP3 inflammasome activation and inflammatory cytokinerelease and apoptosis[35,36]. Thus, CD39 appears to play a rolein Treg function that may explain their efficacy in ADR SCID micewhere there are no cognate immune cells [21].

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Figure 4. hCD39 was constitutively expressed in CD39Tg mice and mouse CD39 was highly expressed on Foxp3+ Treg cells from both WT andCD39Tg mice. (A) Immunohistochemical staining of hCD39 in spleen and kidney for both WT and CD39Tg mice. Data are representative of oneout of two independent experiments performed, n = 5; magnification, ×400). (B) Mouse CD39, hCD39, and Foxp3+ Treg cells were analyzed by flowcytometry in both WT and CD39Tg mice (C) CD4+ CD25+ T cells were isolated from both WT mice and CD39Tg mice. (D) Foxp3+ expression wasanalyzed by flow cytometry for the isolated CD4+ CD25+ T cells. (E) Foxp3 expression was evaluated by real-time PCR in BALB/c WT and CD39Tgmice. (B–E) Data are representative of one out of three independent experiments performed, n = 5–6. **p < 0.01, Student’s t-test.

The mechanism of protection of CD39 expression may be a con-sequence of ATP removal and/or adenosine generation. Adenosineis recognized as a potent anti-inflammatory molecule and is pro-tective in a number of small animal models. However, CD73 dele-tion protects against renal IRI suggesting that extracellular ATPper se may be more important than the generation of AMP andadenosine, in keeping with our results [17]. Others have notedthat ATP can lead to Treg apoptosis which is limited by CD39[37,38]. We, however, saw no difference in Treg-cell numbers inWT or hCD39tg mice as shown in Fig. 3. Of interest TGF-β whichis secreted by Treg cells increases adenosine generation [39] andsome Treg cells express chemokine receptors such as CXCR3 thatmay lead to recruitment to the inflamed kidney [40].

As previously reported, we also found P2X7 expression by renaltubular cells by RTPCR (data not shown)[10]. In vitro, increasingdoses of ATP led to apoptosis of cultured renal tubular cells. Ashas been shown previously, the effect of overexpression of CD39

in vivo may be due to a reduction in ATP-induced injury in ANthat is a direct P2X7-mediated effect, or it may be dependent onother cells such as macrophages that also express this receptor.Interestingly, P2X7 is functionally different between C57BL/6 andBALB/c mice, providing another likely explanation for why ADRcauses severe injury in BALB/c mice, in which the receptor isfully functional, whereas C57BL/6 mice that have a less potentreceptor do not develop AN at standard doses [41]. This is inkeeping with our functional data in primary tubular cells (Fig. 6)that demonstrate that tubular cell expression of CD39 protectsagainst ATP-mediated apoptosis.

These data suggest a key role for purinergic signaling inimmune renal disease. They raise the possibility of a role for P2X7in immune activation and injury in the kidney, as has been shownin nephrotoxic nephritis in C57BL/6 mice. Our studies also demon-strate the innate immune protective functions of Treg cells in renalinjury involving CD39. Many forms of progressive proteinuric

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Figure 5. hCD39 overexpression on Treg cells can limit renal injury in AN. (A) Proteinuria, (B) serum creatinine levels, (C) morphological changes,and (D) body weight changes were measured in four groups ADR mice (WT25+: mice received CD4+ CD25+ T cells from WT; CD39 25+: micereceived CD4+ CD25+ T cells from CD39Tg mice; CD39 25−: mice received CD4+ CD25− T cells from CD39Tg mice; ADR only: mice had AN inducedwithout any cell treatment). (E) Histological changes were evaluated by PAS staining in four groups of mice (six mice in each group, magnification,×200. (A–E) Data are representative of one out of three independent experiments performed.

renal disease have features that suggest a combination of non-immune injury, followed by secondary immune progression, as isseen in AN. Our results indicate that nucleotides, in particular ATP,may have a major role in injury and immune activation, and thatthe enzymes that degrade these inflammatory mediators mighthave an important protective and potentially therapeutic role.

Materials and methods

Mice

Human CD39 transgenic mice were generated and were back-crossed to BALB/c for 12 generations as described previously [11].

All experiments were carried out with the approval of the AnimalEthics Committee of Westmead Hospital, Sydney. Male BALB/cmice were obtained from ARC, (Perth, Australia). All mice weremaintained free of pathogens. Eight to 10-week-old mice (weight20–22 g) were used in all groups. All hCD39Tg mice used in thisstudy were homozygous [11].

Induction of ADR nephropathy (AN)

AN was induced in both BALB/c human CD39 transgenic mice(hCD39Tg) and BALB/c wild-type mice (WT). ADR (DoxorubicinHydrochloride, Pharmacia & Upjohn Pty Ltd., Perth, Australia)was injected once via tail vein of each nonanesthetized mouse

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Figure 6. CD39Tg primary tubular cells had less apoptosis induced by ATP. (A) Tubular cell line (C1.1), (B) WT primary tubular cells, and (C) CD39Tgprimary tubular cells were treated with extracellular ATP for the time indicated, DNA fragmentation was determined using TUNEL staining. (D)Cells were treated with either normal medium or 1 mmol/L H2O2 for 24 h before performing the assessment of cell apoptosis by TUNEL. TUNEL-positive nuclei/per MF was measured by quantitative analysis under indicated (E) time and (F) doses. (A–F) Data are representative of one out offour independent experiments performed. Magnification, ×400; **p < 0.01, Student’s t-test.

(9.8 mg/kg). Mice in the normal control group were treated withsaline only and mice were culled 4 weeks after ADR injection(6 mice per group in a single experiment).

Adoptive transfer of CD4+CD25+ and CD4+CD25−

T cells isolated from hCD39Tg and WT mice

CD4+CD25+ and CD4+CD25− T cells were purified from spleno-cytes of hCD39Tg and BALB/c mice using MACS CD4+CD25+

isolation kits (Miltenyi Biotec, Germany). The purity of the cellswas confirmed by flow cytometry, and 2 × 106 of the isolated cellswere injected into the tail vein of each mouse. Mice were divided

into four groups (five mice in each group) and injected with i).CD4+CD25+ cells from WT mice (WT 25+); ii). CD4+CD25+

cells from hCD39Tg mice (Tg 25+); iii). CD4+CD25− cells fromhCD39Tg mice (Tg 25−); iv). ADR only (ADR). Cells were injected3 days before injection of ADR. ADR was injected via the tail veinof each nonanesthetized mouse (9.8 mg/kg).

Renal function and RNA isolation and quantitativereal-time PCR

Renal function was assessed by the measurement of urine pro-tein and serum creatinine, as described previously [20]. RNA was

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isolated from mouse kidney and spleen using TRIZOL Reagent(Invitrogen, Life Technologies, Australia). The total RNA wasreverse transcribed into cDNA and then was subjected to real-timequantitative PCR analyses as described previously [20]. Sampleswere run in triplicate.

Flow cytometric analysis

Antibodies used for flow cytometry included anti-human CD39APC (Clone TU66, BD Biosciences, Australia); Anti-mouse CD39PE (Clone 24DMS1, BD Biosciences, Australia); fluoresceinisothiocyanate (FITC)-conjugated anti-mouse CD25 (Clone 7D4,BD Biosciences, Australia); phycoerythrin (PE)-conjugated anti-mouse Foxp3 (Clone FJK-16S, eBiscience); and APC-conjugatedanti-mouse CD4 with permeabilization as per manufacturer’sinstructions (Clone RM4-5, BD Biosciences, Australia). All sampleswere analyzed on a FACScan analyzer (Becton Dickinson, Moun-tain View, CA, USA). CellQuest software was used for acquisitionand analysis (Becton Dickinson, Australia).

Histology, morphometric evaluation, and quantitationof fibrosis using trichrome staining

Sagittal slices of renal tissue were fixed in formalin and embeddedin paraffin for evaluation of pathology. Five μm slices were stainedwith PAS reagent and assessed by light microscopy. In each biopsy,a semiquantitative score from two blinded trained observers wasused to evaluate the degree of renal injury. The degree of renalinjury was estimated by evaluating the percentage of renal injuryper field and was graded on a scale of 0 to 4 [20]. Quantitationfor interstitial α-SMA and Gomori trichrome staining of fibrosiswere performed by a modified grid-counting method adapted fromVielhauer [42].

Primary tubular cell isolation from WT and CD39 andculture

Kidneys were placed into ice-cold DMEM medium (Invitrogen,Australia) and the cortex was trimmed and minced. The mincedtissues was digested by collagenase solution (collagenase, deoxy-ribonuclease, bovine serum albumin) for 10 min 37◦C two to threetimes and then were washed and concentrated by 45% Percolsolution (GE Health). The suspension of tubular cells were thenwashed twice and cultured with K1 serum medium at 37◦C onCO2 overnight. The cells were grown to confluence in K1 mediumfor 1 week and then were analyzed for apoptosis.

Immunohistochemistry

Immunohistochemical staining was performed to evaluate theexpression of hCD39 and Foxp3 in spleen and kidney, and theinfiltration of CD4+, CD8+ T cells, and macrophages in the kidney.

Primary antibodies used in immunohistochemistry were: mouseanti-human CD39 (Clone A1, BioLegend, Australia Biosearch)and purified anti-Foxp3 (Clone Poly6238, BioLegend, AustraliaBiosearch); CD4+ (Clone GK1.5, BioLegend, Australia Biosearch);CD8+ (Clone 53–6.7, BD Biosciences, Australia), and macrophages(Clone M3/84, BD Biosciences, Australia). The secondary antibod-ies were biotinylated goat anti-mouse IgG (ABCAM) and biotinyl-ated goat anti-rat immunoglobulin (BD Biosciences, Australia).Immunohistochemical staining was performed and interstitialinfiltration was assessed as described previously.

In situ detection of DNA fragmentation in tubular cellapoptosis after ATP or H2O2 treatment (TUNEL assay)

In situ detection of fragmented DNA in cultured tubular cellswas performed using the terminal deoxynucleotidyl transferase(TdT)-mediated deoxyuridine-triphosphatase nick end labeling(TUNEL) method. TUNEL-positive nuclei/per MF were countedby fluorescence microscopy. Adherent cultured tubular cells,C1.1, (ATCC Manassas, VA, USA) primary tubular cells fromboth WT and CD39Tg mice were grown to subconfluence on4-well chamber slides (BD Biosciences, Australia). The cellswere stimulated with ATP (Sigma, 1 mM to 3 mM) or PBS forvarious times up to 3 h. For H2O2 treatment experiment, cellswere treated with either normal medium or 1 mmol/L H2O2,

in normal medium for 24 h [43]. The cells were washed twicewith PBS, and fixed in 4% paraformaldehyde for 60 min at roomtemperature. After rinsing with PBS, the slides were incubatedin permeabilization solution (0.1% Triton X-100 in 0.1% sodiumcitrate) for 2 min on ice. The cells were processed for TUNEL assay(Roche) following the manufacturer’s instructions and analyzedunder reverse fluorescence microscopy (magnification, ×400).

Statistical analysis

Statistical analysis was performed by (ANOVA) for multiple com-parisons. Results are expressed as the group mean ± SD. Twogroup differences were analyzed by Student’s t-test. The p value(two-tailed) of less than 0.05 was considered statistically signifi-cant.

Acknowledgements: This work was supported by researchgrants from the National Health and Medical Research Coun-cil of Australia (NHMRC grant 1030463, 571343, and 632519)and HL094400 NIH USA. We thank the animal house staff in theWestmead Hospital Animal House Facility for care of the ani-mals. Thanks to Fredrick Tam (Imperial College), Anthony JFd’Apice (St. Vincent’s Hospital), and Dr Huiling Wu (Universityof Sydney, Australia) for advice. We thank, Jimmy Jianheng Zhou

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2450 Yuan Min Wang et al. Eur. J. Immunol. 2012. 42: 2441–2451

(Children’s Hospital at Westmead), SoRa Yes Lee, Thian Kui Tan,and Tim Tzu-Ting Hsu (Westmead Hospital, Australia) for techni-cal assistances.

Conflict of Interests: The authors declare no financial or com-mercial conflict of interest.

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Abbreviations: ADP: adenosine diphosphate · ADR: Adriamycin ·AMP: adenosine monophosphate · AN: Adriamycin nephritis · ATP:

adenosine-5′-triphosphate · IRI: ischemia reperfusion injury · MF:

microscopic field · PAS: periodic acid-Schiff

Full correspondence: Assistant Professor Stephen I. Alexander, Centrefor Kidney Research, Children’s Hospital at Westmead, WestmeadNSW 2145, Sydney, Australia.Fax: +98-45-3038e-mail: StephenA@chw.edu.au

Received: 2/2/2012Revised: 4/4/2012Accepted: 22/5/2012Accepted article online: 11/6/2012

C© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu