CLINICAL RESEARCH www.jasn.org

B7–1 Blockade Does Not Improve Post–TransplantNephrotic Syndrome Caused by Recurrent FSGS

Marianne Delville,*† Emilie Baye,‡ Antoine Durrbach,§ Vincent Audard,| Tomek Kofman,|

Laura Braun,¶ Jérôme Olagne,¶** Clément Nguyen,‡ Georges Deschênes,†† Bruno Moulin,¶

Michel Delahousse,‡‡ Gwenaëlle Kesler-Roussey,§§ Séverine Beaudreuil,§ Frank Martinez,||

Marion Rabant,¶¶ Philippe Grimbert,| Morgan Gallazzini,*‡ Fabiola Terzi,*‡

Christophe Legendre,*|| and Guillaume Canaud*‡||

*Université Paris Descartes, Sorbonne Paris Cité, †Service de Biothérapie, ‡Institut National de la Santé et de laRecherche Médicale U1151, Institut Necker Enfants Malades, ||Service de Néphrologie Transplantation Adultes, and¶¶Service d’Anatomie et Cytologie Pathologiques, Hôpital Necker-Enfants Malades, Paris, France; §Service deNéphrologie et Transplantation, Hôpital Bicêtre, Le Kremlin-Bicêtre, Institut National de la Santé et de la RechercheMédicale 1197, Université Paris-Saclay, Le Kremlin Bicêtre, France; |Groupe Hospitalier Henri Mondor AlbertChenevier, Service de Néphrologie et Transplantation, Université Paris Est Créteil, Creteil, France; ¶Service deNéphrologie et Transplantation, Hôpitaux Universitaires de Strasbourg, Nouvel Hôpital Civil, Strasbourg, France;**Laboratoire d’Anatomo-Pathologie, Hôpitaux Universitaires de Strasbourg, Hôpital de Hautepierre, Strasbourg,France; ††Service de Néphrologie pédiatrique, Hôpital Robert Debré, Paris, France; ‡‡Service de NéphrologieTransplantation, Hôpital Foch, Suresnes, France; and §§Service de Néphrologie pédiatrique, Centre HospitalierUniversitaire de Nantes, Nantes, France

ABSTRACTFSGS is a common glomerular disorder that has a high propensity for recurrence after kidney transplant.The pathophysiology of FSGS is unknown, but podocytes seem to be the target of one or severalcirculating factors that lead to cytoskeleton reorganization and proteinuria. Research on podocytes hasidentified B7–1 as an important factor in podocyte biology and a new therapeutic target in renal disease.Indeed, in four patients with recurrent FSGS after transplant, treatment with the B7–1 blocker abataceptwas associated with proteinuria remission. Here, we prospectively treated nine patients with recurrentFSGS after transplant using either abatacept or belatacept, a B7–1 blocker with higher affinity, and did notinduce proteinuria remission. Furthermore, we did not detect B7–1 expression by immunofluorescence inpodocytes of biopsy specimens from these or other kidney grafts or podocytes of native kidney biopsyspecimens. In conclusion, B7–1 blockade did not induce FSGS remission after transplant in our study.

J Am Soc Nephrol 27: ccc–ccc, 2015. doi: 10.1681/ASN.2015091002

FSGS is a frequent cause of end stage renal failure inadults and one of the leading causes of transplan-tation (Tx) in children. After Tx, its recurrence isfrequent (#50%), and it is associated with poorallograft outcomes.1–3 Its pathophysiology is stillcontroverted and supposed to be the consequenceof one or several circulating factors that target po-docytes.4 There is no consensus on how to treat therecurrence, and current treatment is mainly on thebasis of plasmapheresis exchange and the adminis-tration of high-dose steroids, calcineurin inhibi-tors, and/or rituximab.5 Such treatments are ofteneffective but unfortunately, also frequently associ-ated with adverse events and hospitalization.

Recently, B7–1, a costimulation molecule that isinvolved in lymphocyte activation, was found to beexpressed in stressed podocytes in mice and humans

Received September 9, 2015. Accepted November 18, 2015.

A.D. and V.A. contributed equally to this work.

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

Correspondence: Dr. Guillaume Canaud, Renal Division, NeckerHospital, Paris Descartes University, Institut National de la Santéet de la Recherche Médicale U1151, 149 rue de Sèvres, 75015Paris, France. Email: guillaume.canaud@inserm.fr

Copyright © 2015 by the American Society of Nephrology

J Am Soc Nephrol 27: ccc–ccc, 2015 ISSN : 1046-6673/2708-ccc 1

in several proteinuric states, including diabetic nephropathy.6,7

B7–1 was described to regulate the actin cytoskeleton of podo-cytes, participating in podocyte foot process effacement andpro-teinuria occurrence.6 Basically, such findings encouraged Yuet al.8 to treat patients with recurrent FSGS with abatacept, acostimulation blocker. This treatment led four patients with a

resistant form of recurrent FSGS into remissionwith only one ortwo abatacept infusions.8 Therefore, B7–1 was positioned as amajor player in podocyte biology during pathologic conditionsand the main molecule to target for treating proteinuria.

However, although this first report was exciting,8 few casereports using abatacept or belatacept, another B7–1 blocker

Figure 1. Abatacept does not improve albuminuria in patients with FSGS recurrence after Tx. Charts showing the post–Tx serumcreatinine levels (black) and albuminuria-to-creatinine ratios (red) of (A) patient 1, (B) patient 2, (C) patient 3, (D) patient 4, and (E)patient 5. The orange triangles represent abatacept infusion. The purple triangles represent rituximab infusion. The light blue rect-angles represent plasmapheresis. The dark blue rectangles represent IA, whereas the yellow rectangles represent iv cyclosporin, andthe green rectangles represent oral cyclosporin. The red rectangle represents hemodialysis.

2 Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc, 2015

CLINICAL RESEARCH www.jasn.org

with a twofold higher affinity for B7–1, have failed to induceFSGS remission after Tx.9,10

We decided to prospectively administer either abatacept orbelatacept to nine consecutive patients with a resistant form ofrecurrentFSGSafterTx.Wedidnot induce any remission in thepatients even using several drug infusions. Additionally, we didnot detect B7–1 expression in the patients’ biopsies. Lastly, weexplored several kidney biopsies from patients with variousforms of podocyte injuries and concluded that B7–1 is notexpressed in podocytes.

RESULTS

Neither Abatacept nor Belatacept Induces FSGSRemission after Kidney TxAll nine patients had a history of biopsy–proven primary FSGSon native kidneys. The primary FSGS was either initially ste-roid sensitive and became steroid dependent (n=8) or steroidresistant (n=1). In all patients, other immunosuppressivedrugs (cyclosporin, cyclophosphamide, and mycofenolatemofetil) had a transient or no effect on proteinuria, andpatients developed progressive end stage renal failure. Theearly recurrence after Tx in all patients confirmed the primaryform of FSGS. Of note, two patients had systematic genetictesting for NPHS2 andWilms Tumor 1. We did not detect anymutation or polymorphism in these two patients.

Patient 1A 22-year-old womanwhose first kidney Tx failed because ofFSGS recurrence received a second kidney transplant fromher mother (Figure 1A, Table 1). After the Tx, FSGS recurredat day 7 (albuminuria-to-creatinine ratio of 8 g/g). She had 2weeks of intravenous (iv) cyclosporin followed by oral cyclo-sporin, plasmapheresis, corticosteroid pulses, and rituximabinfusion. Her proteinuria decreased to 3–4 g/g. Because of in-complete remission, we decided to add two abatacept infusions(10mg/kg) at 1month after Tx. The two abatacept infusions hadno effect on the proteinuria, which remained in the nephroticrange. Plasmapheresis was then switched to immunoadsorption(IA) for 1 month without success, and the patient reached endstage renal failure 12 months after the Tx.

Patient 2A 49-year-old manwhose first kidney Tx failed because of FSGSrecurrence received a second kidney Tx from a deceased donor(Figure 1B, Table 1). FSGS recurrence occurred 2 months afterthe Tx (24-hour albuminuria was 4.7 g/d). He received treat-ment that included iv cyclosporin followed by oral cyclosporin,plasmapheresis, and corticosteroid pulses. The proteinuria re-mained nephrotic and was between 1.5 and 13 g/d. He wasswitched to IA, but the proteinuria remained abundant. Wethen decided to add two infusions of abatacept (10 mg/kg) at11 months after Tx. This treatment did not improve the rate ofalbuminuria after 12 months of follow-up. Ta

ble

1.Cha

racteristics

ofnine

patientswithFS

GS

Variables

Patient

1Patient

2Patient

3Patient

4Patient

5Patient

6Patient

7Patient

8Patient

9

Agean

dsex

22-yr-old

wom

an49

-yr-old

man

49-yr-old

man

12-yr-oldgirl

5-yr-old

girl

56-yr-old

man

36-yr-old

man

54-yr-oldman

30-yr-old

man

Gen

etics

Not

perform

edNot

perform

edNot

perform

edNPH

S2:n

eg;

WT1

:neg

Not

perform

edNPH

S2:n

egNot

perform

edNot

perform

edNot

perform

ed

Kidne

ydon

or

LRD

DD

DD

DD

DD

DD

DD

DD

DD

Txrank

22

12

11

11

1Re

curren

ceon

apreviou

sgraft

Yes

Yes

N/A

Yes

N/A

N/A

N/A

N/A

N/A

Trea

tmen

tfor

FSGSrecu

rren

cebefore

abatacep

t/belatac

ept

CsA

,steroids,

PE,ritu

ximab

CsA

,steroids,

PE,IA

PE,C

sARituximab

,IA

Rituximab

,IA,ivIg

CsA

,steroids,PE

PE,ritu

ximab

PE,ritu

ximab

N/A

Abatacep

t/belatacep

ttherap

yAbatac

ept:

twoinfusion

sAbatac

ept:

twoinfusion

sAbatac

ept:

twoinfusions

Abatac

ept:

five

infusion

sAbatacep

t:threeinfusion

sBelatac

ept:

five

infusion

sBelatacep

t:.10

infusion

sBelatacep

:.10

infusions

N/A

FSGSremission

after

abatacep

t/belatacep

t(partia

lorc

omplete)

No

No

No

No

No

No

No

No

Occurrenc

eof

FSGSwith

belatacep

ttrea

tmen

tNPH

S-2,

pod

ocin;n

eg,n

omutationor

polym

orphism

iden

tified

;WT1

,Wilm

sTu

mor

1;LR

D,livingrelateddon

or;D

D,d

ecea

seddon

or;N

/A,n

ota

pplicab

le;C

sA,c

yclosp

orin;P

E,plasm

aphe

resisex

chan

ge.

J Am Soc Nephrol 27: ccc–ccc, 2015 B7–1 in Podocytes 3

www.jasn.org CLINICAL RESEARCH

Patient 3A 49-year-old man received his first kidney Tx from a deceaseddonor (Figure 1C, Table 1). FSGS recurrence occurred on day 7after the Tx (albuminuria-to-creatinine ratio of 10 g/g). Wepromptly initiated plasmapheresis in association with anabatacept infusion. The patient received two infusions of aba-tacept (10 mg/kg). The rate of albuminuria remainedunchanged (albuminuria-to-creatinine ratio of 8.8 g/g) after8 months of follow-up.

Patient 4A 12-year-old girl whose first kidney Tx failed because ofFSGS recurrence received a second kidney transplant from adeceased donor (Figure 1D, Table 1). FSGS recurred on day7 after the Tx. We started IA and rituximab. Using thistreatment, she achieved complete remission. However, afterIA interruption, her albuminuria dramatically increased to5 g/g. The proteinuria decreased to 0.2 g/g under weekly IA.To allow IA weaning, we decided to add five abataceptinfusions (10 mg/kg). Unfortunately, the albuminuriarose up to 2 g/g with this treatment. She is currently IAdependent.

Patient 5A 5-year-old girl received her first kidney Tx from a deceaseddonor (Figure 1E, Table 1). FSGS recurrence occurred on day 1after the Tx (albuminuria-to-creatinine ratio of 6 g/g). Westarted IA, iv Ig, and rituximab. Because of the absence ofremission, we decided to add three abatacept infusions. The

abatacept infusions did not improve therate of albuminuria, which remained be-tween 1 and 5 g/g.

Patient 6A 56-year-old man received his first kidneyTx from a deceased donor (Figure 2A,Table 1). FSGS recurrence was observedon day 14 after the Tx (5–10 g/g). Hereceived iv cyclosporin followed by oralcyclosporin, plasmapheresis, and cortico-steroid pulses without any albuminuriaimprovement.We decided to switch the cy-closporin for belatacept on day 71 after Tx.He received five infusions. Unfortunately,this treatment had no effect on the rate ofalbuminuria with a follow-up of 10months.

Patient 7A 36-year-old man received his first kidneyTx from a deceased donor (Figure 2B, Table1). FSGS recurrence occurred on day 30after the Tx (albuminuria-to-creatinine ra-tio of 7 g/g).We started plasmapheresis andone rituximab infusion. The rate of albu-

minuria decreased to 4 g/g. To further reduce the rate of al-buminuria, we decided to introduce belatacept. After 15months of follow-up, the belatacept had no effect on the levelof albuminuria.

Patient 8A 54-year-oldman received his first kidney Tx from a deceaseddonor (Figure 2C, Table 1). FSGS recurrence was observed onday 120 after the Tx. We then added plasmapheresis and rit-uximab. The albuminuria remained nephrotic, and therefore,we decided to switch to belatacept. Unfortunately, the albu-minuria dramatically rose up to 10 g/g, and we failed to induceremission.

Patient 9A 30-year-oldman received his first kidney Tx from a deceaseddonor (Figure 2D, Table 1). FSGS recurrencewas diagnosed onthe first day after the Tx (albuminuria-to-creatinine ratio of21 g/g). He received iv cyclosporin followed by oral cyclosporin,plasmapheresis, and corticosteroid pulses. With this treat-ment, the patient reached complete remission. Eighteenmonths after the Tx, the patient was still in completeremission. A transplant biopsy was performed and showedcalcineurin inhibitor toxicity. We then decided to switchfrom using cyclosporin to using belatacept. Immediately afterthe switch, the patient developed nephrotic-range proteinuria(albuminuria-to-creatinine ratio of 5 g/g). Belatacept wasstopped, and he received oral cyclosporin, plasmapheresis,and rituximab infusion, which led to partial remission.

Figure 2. Belatacept does not improve albuminuria in patients with FSGS recurrenceafter Tx. Charts showing the post–Tx serum creatinine levels (black) and albuminuria-to-creatinine ratios (red) of (A) patient 6, (B) patient 7, (C) patient 8, and (D) patient 9.The brown triangles represent belatacept infusion. The purple triangles representrituximab infusion. The light blue rectangles represent plasmapheresis. The yellowrectangles represent iv cyclosporin, and the green rectangles represent oral cyclo-sporin.

4 Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc, 2015

CLINICAL RESEARCH www.jasn.org

Figure 3. B7–1 is not expressed in podocytes in patients with FSGS recurrence after Tx. (A) Coimmunostaining of nephrin (green) andB7–1 (red) on transplant biopsies performed at the time of FSGS recurrence. (B) Coimmunostaining between nephrin (green) and B7–1(red) on transplant biopsies performed at the time of FSGS recurrence showing positive cells for B7–1 in the interstitium. (C) Westernblot of B7–1 in HeLa cells transfected with plasmids containing either an empty vector or B7–1 cDNA. (D) B7–1 immunostaining in HeLacells transfected with plasmids containing either an empty vector or B7–1 cDNA. (E) Coimmunostaining of CD20 (green) and B7–1 on

J Am Soc Nephrol 27: ccc–ccc, 2015 B7–1 in Podocytes 5

www.jasn.org CLINICAL RESEARCH

B7–1 Is Not Expressed in the Podocytes of Patientswith FSGS RecurrenceThe absence of treatment efficiency led us to explore B7–1protein expression by immunostaining in the biopsies ofnine patients with FSGS recurrence. We performed coimmu-nostaining of nephrin, a podocyte marker, and anti-B7–1antibody (Figure 3A). We did not detect B7–1 expression inpodocytes of nine biopsies performed at the time of recur-rence. Importantly, in several biopsies, we could detect someinterstitial inflammatory infiltrating cells that were expressingB7–1 (Figure 3B). Intrigued by the results, we decided to per-form similar experiments using two other commercially avail-able anti-B7–1 antibodies. These experiments gave similarresults to the previous result, confirming that B7–1 was notdetected in podocytes (not shown). To ensure the validity ofthe anti-B7–1 antibodies, we checked if they were able to de-tect B7–1 expression in HeLa cells transfected with plasmidsencoding either an empty vector or B7–1 cDNA. Western blotexperiments as well as immunofluorescence studies confirmedthat anti-B7–1 antibodies were able to detect B7–1 expression(Figure 3, C and D). To convince us that they were also workingin human tissues and specific for B7–1, we explored B7–1 ex-pression in tonsils and spleens. Coimmunostaining showed thatactivated B cells were expressing CD20 and B7–1 in the tonsils(Figure 3E, Supplemental Figure 1A). Additionally, we observedthat B7–1 staining in human tonsils and spleens by three differ-ent antibodies was similar (Figure 3F, Supplemental Figure 1B).Importantly, kidney biopsies, spleens, and tonsils were fixedwith the same fixative and processed in the same way.

We then considered that one explanation of the drug’s in-effectiveness could be related to the fact that we administeredabatacept and belatacept to patients who were not B7–1 pos-itive. To test this hypothesis, we did a colocalization studyexamining B7–1 and nephrin in the biopsies of 22 patientswith recurrent FSGS (Supplemental Figure 1C). Analyses ofthe biopsies revealed that B7–1 was not detectable in podo-cytes in any of the biopsies, regardless of the time of recurrenceafter Tx. This suggests that B7–1 is not expressed in podocytesduring FSGS recurrence.

B7–1 Is Not Expressed in Other Forms of HumanProteinuric DiseasesFinally, we were interested in determining whether B7–1 wasdetectable in other proteinuric diseases of native kidney. Weperformed colocalization studies between nephrin and B7–1in several pathologic conditions of native kidneys. We testedprimary and secondary forms of FSGS (n=7), diabetic ne-phropathy (n=7), membranous nephropathy (n=7), ANCA

vasculitis (n=7), and lupus nephritis (classes 3 and 4; n=7).As shown Figure 4, B7–1 was not detectable in podocytes inany of these patients and was only found in circulating inflam-matory cells.

We, therefore, conclude that B7–1 is not expressed duringpodocyte injury in human kidney biopsies.

DISCUSSION

In this study, we observed that B7–1 blockade was ineffective atinducing proteinuria remission in adults and children with re-current FSGS after Tx. Furthermore, our coimmunostaining ex-periments revealed that B7–1 was not expressed in podocytes inthe post-transplant biopsies of these patients. We confirmed thatB7–1 was undetectable in podocytes in an independent valida-tion cohort that included 22 patients with recurrent FSGS afterTx and also, several other proteinuric diseases of native kidneys.

In nine patients who were studied, B7–1 blockade was in-effective. The treatment was used systematically in patientswho were resistant to usual therapy and always combinedwith other drugs. Indeed, such as in the study by Yu et al.,8

patients received a large number of therapies before B7–1blockade. We mostly used abatacept, administering severalinfusions of the drug because of its primary ineffectiveness.In some patients, we tested belatacept, because belatacept has ahigher affinity for B7–1.11 The structure of belatacept is dif-ferent from that of abatacept, with a fourfold increase in B7–2binding affinity and a twofold increase in B7–1 binding.11 Thisincrease in binding affinities to the B7 ligand family resultedin a 10-fold increased inhibition of T cell activation comparedwith abatacept in vitro.11 Unfortunately, belatacept was alsounable to achieve proteinuria remission in patients. Further-more, in one patient, we observed the occurrence of nephroticsyndrome after we introduced belatacept. We acknowledgethat belatacept was not reported to induce proteinuria remis-sion in the work by Yu et al.,8 and a possible mechanism ofactions cannot be excluded. However, if these results are incontrast with the recent report by Yu et al.,8 they are consistentwith a few case reports showing the ineffectiveness of B7–1 block-ade at inducing post–transplant proteinuria remission.9,10 In thepost-transplant course, Yu et al.8 used one or two infusions ofabatacept, a sufficient dosage to induce prolonged proteinuriaremission. Following this approach, we did not induce any re-mission. The absence of efficiency led us to increase the numbersof abatacept/belatacept infusions without success. Unfortunately,at that point, we did not have any scientific explanation for thediscrepancy between our study and the report by Yu et al.8

human tonsils at (left panel) low or (right panel) higher magnification. (A–E) For all of these experiments, the anti-B7–1 antibody fromSanta Cruz Biotechnology (SC-9091) was used. (F) B7–1 immunostaining in human tonsil using three different commercially availableantibodies (AF140 from R&D Systems Europe, SC-9091 from Santa Cruz Biotechnology, and MA5–15512 from Fisher Scientific). DAPI,49,6-diamidino-2-phenylindole. Scale bar, 10 mm.

6 Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc, 2015

CLINICAL RESEARCH www.jasn.org

The detection of B7–1 antigen in the kidney using immuno-histochemistry or immunofluorescent studies was subject to con-troversy with a supposed instability of B7–1 epitopes.12,13 Here,we provide evidence to rule out such arguments. Indeed, wetested three different commercially available anti-B7–1 antibodiesand observed that B7–1 immunostaining of human kidneybiopsies is feasible and reliable. We first confirmed in vitro thatthese antibodies were effective in detecting B7–1. Then, we con-firmed that these antibodies were able to work in human tissuesby detecting activated B cells in the tonsils that were expressingB7–1. The B7–1 staining was similar in the tonsils and the spleensindependent of the antibody used. Very importantly, all samples(kidney, tonsils, and spleens) were fixed with the same fixativereagents and processed in the sameway, and the coimmunostain-ing was performed in all samples in the same time to avoid anyvariability. Finally,wewere able to detect B7–1-expressing inflam-matory cells in the kidney, a good internal control. Indeed, ourdata do not support the epitope instability hypothesis. Convincedby our immunostaining,we thenverified the B7–1 expression in alarge number of kidney biopsies either after Tx or on native kid-ney andobserved that B7–1was never expressed in podocytes.Weused paraffin-embedded sections instead of the frozen sectionsused in the report by Yu et al.,8 which could partially explain thediscrepancies, but our results are consistent with a recent publi-cation showing that B7–1 is not detectable in human podocytesduring diabetic nephropathy.14 This does not support the use ofB7–1 immunostaining as a biomarker to treat patients withabatacept.

In conclusion, our data do not support the use of B7–1inhibition in patients with proteinuric diseases. B7–1 is

possibly expressed at low levels in podo-cytes, and other factors play more impor-tant roles in this complicated disease. Morestudies aremandatory before use of the B7–1 blocker in patients who are proteinuric.

CONCISE METHODS

PatientsNine transplant recipients who received abatacept

or belatacept treatment are described in Results.

Briefly, the patients were treated according to

hospital guidelines. The study included two chil-

dren (Nantes: n=1 and Paris: n=1) and seven

adults (Paris: n=6 and Strasbourg: n=1). The

study started January 1, 2014 and ended June

30, 2015.

Morphologic AnalysesHuman kidney transplant biopsies were fixed in

an alcohol-formalin-acetic acid solution and

then, embedded in paraffin. All of the biopsies

were performed at the time of proteinuria re-

currence. Native kidney biopsies werefixed in an

alcohol-formalin-acetic acid solution and then, embedded in paraffin.

For each pathologic condition, we tested seven different biopsies from

seven different patients. As positive controls, we used tonsil and spleen

samples that were fixed in an alcohol-formalin-acetic acid solution

and then, embedded in paraffin. Therefore, importantly, all samples,

transplant biopsies, native kidney biopsies, spleens, and tonsils were

fixed using the same fixative and processed in the same way.

Urine and Plasma AnalysesFor human samples, urinary creatinine and albuminwere determined

using a Hitachi 917 Analyzer (Roche Diagnostics, Indianapolis, IN),

whereas plasma creatinine was evaluated using a Synchron Cx4

Autoanalyzer (Beckman Coulter, Inc., Brea, CA).

ImmunofluorescenceFour-micrometer paraffin-embedded sections were incubated with

three different anti-B7–1 (Santa Cruz Biotechnology, Santa Cruz, CA;

Fisher Scientific, Waltham, MA; R&D Systems Europe, Lille, France),

anti-Nephrin (Progen, Heidelberg, Germany), and CD20 (Dako) an-

tibodies (Santa Cruz Biotechnology) after antigen retrieval. In detail,

the sections were deparaffinized and rehydrated; then, the slides were

brought to a boil in Tris-EDTA buffer (10 mMTris base, 1 mM EDTA

solution, and 0.05% Tween 20, pH 9.0) maintained at a sub-boiling

temperature (95°C) for 40 minutes. This step was followed by a cool-

ing time on the bench for 30minutes. Then, each sectionwas blocked

with Tris-buffered saline and Tween 20/5% normal goat serum for

30minutes. All anti-B7–1 antibodies were used at the dilution of 1:50

and incubated on the sections for 12 hours at 4°C. The primary

antibodies were revealed with the appropriate Alexa 488– or Alexa

555–conjugated secondary antibodies (Molecular Probes, San Diego,

Figure 4. B7–1 is not expressed in podocytes in patients with proteinuric diseases ofnative kidneys. Representative coimmunostaining of nephrin (green) and B7–1 (red;using anti-B7–1 antibody SC-9091 from Santa Cruz Biotechnology) in native kidneybiopsies from patients with primary FSGS, secondary FSGS, diabetic nephropathy,membranous nephropathy, ANCA vasculitis, or lupus nephritis classes 3 or 4. Humantonsils served as positive controls. The arrow shows circulating B7–1-positive cells.DAPI, 49,6-diamidino-2-phenylindole. Scale bar, 10 mm.

J Am Soc Nephrol 27: ccc–ccc, 2015 B7–1 in Podocytes 7

www.jasn.org CLINICAL RESEARCH

CA) used at the dilution of 1:200 for 2 hours at room temperature.

Immunofluorescence staining was visualized using a Zeiss LSM 700

Confocal Microscope. Importantly, all sections (kidney, tonsil, and

spleen) were stained at the same time. For each biopsy, we evaluated

all glomeruli.

Cell ExperimentsB7–1 TransfectionHeLa cells were transfected with pcDNA3.1/N-DYK B7–1

(NM_005191, open reading frame sequence, 861 bp; GenScript HK

Limited, Hong Kong) plasmids using Lipofectamine 2000 according

to the supplier’s instructions (Invitrogen, Carlsbad, CA).

Cell Immunofluorescence StudiesHeLa cells were fixed in 4% paraformaldehyde for 20 minutes,

permeabilized in PBS containing 0.2% Triton X-100, incubated

with anti-B7–1 antibody (Santa Cruz Biotechnology), and revealed

with Alexa 555–conjugated secondary antibody (Molecular

Probes). Immunofluorescence staining was visualized using a Zeiss

LSM 700 Confocal Microscope.

Western BlotWestern blots were performed as previously described.15,16 Briefly,

protein extracts from HeLa cells were resolved by SDS-PAGE before

being transferred onto the appropriate membranes and incubated

with anti-B7–1 (Santa Cruz Biotechnology) and anti–b-actin

(Sigma-Aldrich, St. Louis, MO) antibodies followed by the appropri-

ate peroxidase–conjugated secondary antibodies. Chemilumines-

cence was acquired using a Fusion FX7 Camera (Vilbert Lourmat),

and densitometry was performed using ImageJ software.

ACKNOWLEDGMENTS

This work was supported by the Fondation Maladies Rénales and

Transplantations, Recherche and Innovation Thérapeutique and the

Day Solvay Foundation.

This project has received funding from the European Research

Council under the European Union’s Horizon 2020 research and

innovation programme Grant 679254.

DISCLOSURESNone.

REFERENCES

1. Habib R, Lévy M, Gubler MC: Clinicopathologic correlations in thenephrotic syndrome. Paediatrician 8: 325–348, 1979

2. Cameron JS: The enigma of focal segmental glomerulosclerosis. Kid-ney Int Suppl 57: S119–S131, 1996

3. CanaudG, Dion D, Zuber J, Gubler MC, Sberro R, Thervet E, Snanoudj R,Charbit M, Salomon R, Martinez F, Legendre C, Noel LH, Niaudet P: Re-currence of nephrotic syndrome after transplantation in a mixed pop-ulation of children and adults: Course of glomerular lesions and value ofthe Columbia classification of histological variants of focal and segmentalglomerulosclerosis (FSGS).Nephrol Dial Transplant 25: 1321–1328, 2010

4. Cravedi P, Kopp JB, Remuzzi G: Recent progress in the pathophysiologyand treatment of FSGS recurrence. Am J Transplant 13: 266–274, 2013

5. Canaud G, Zuber J, Sberro R, Royale V, Anglicheau D, Snanoudj R,Gaha K, Thervet E, Lefrère F, Cavazzana-Calvo M, Noël LH, Méjean A,Legendre C, Martinez F: Intensive and prolonged treatment of focaland segmental glomerulosclerosis recurrence in adult kidney transplantrecipients: A pilot study. Am J Transplant 9: 1081–1086, 2009

6. Reiser J, von Gersdorff G, Loos M, Oh J, Asanuma K, Giardino L,Rastaldi MP, Calvaresi N, Watanabe H, Schwarz K, Faul C, Kretzler M,DavidsonA, SugimotoH, Kalluri R, SharpeAH, Kreidberg JA,Mundel P:Induction of B7-1 in podocytes is associatedwith nephrotic syndrome. JClin Invest 113: 1390–1397, 2004

7. Fiorina P, Vergani A, Bassi R, Niewczas MA, Altintas MM, Pezzolesi MG,D’Addio F, Chin M, Tezza S, Ben Nasr M, Mattinzoli D, Ikehata M,Corradi D, Schumacher V, Buvall L, YuCC,Chang JM, La Rosa S, Finzi G,Solini A, Vincenti F, Rastaldi MP, Reiser J, Krolewski AS, Mundel PH,Sayegh MH: Role of podocyte B7-1 in diabetic nephropathy. J Am SocNephrol 25: 1415–1429, 2014

8. Yu CC, Fornoni A, Weins A, Hakroush S, Maiguel D, Sageshima J, ChenL, CiancioG, FaridiMH, BehrD, Campbell KN, Chang JM,ChenHC,OhJ, Faul C, Arnaout MA, Fiorina P, Gupta V, Greka A, Burke GW 3rd,Mundel P: Abatacept in B7-1-positive proteinuric kidney disease.N Engl J Med 369: 2416–2423, 2013

9. Alachkar N, Carter-Monroe N, Reiser J: Abatacept in B7-1-positiveproteinuric kidney disease. N Engl J Med 370: 1263–1264, 2014

10. Grellier J, Del Bello A, Milongo D, Guilbeau-Frugier C, Rostaing L,Kamar N: Belatacept in recurrent focal segmental glomerulosclerosisafter kidney transplantation. Transpl Int 28: 1109–1110, 2015

11. Larsen CP, Pearson TC, Adams AB, Tso P, Shirasugi N, Strobert E,AndersonD, Cowan S, Price K,Naemura J, Emswiler J, Greene J, Turk LA,Bajorath J, Townsend R, Hagerty D, Linsley PS, Peach RJ: Rational de-velopment of LEA29Y (belatacept), a high-affinity variant ofCTLA4-Igwithpotent immunosuppressiveproperties.AmJTransplant5: 443–453, 2005

12. Benigni A, Gagliardini E, Remuzzi G: Abatacept in B7-1-positive pro-teinuric kidney disease. N Engl J Med 370: 1261–1263, 2014

13. Mundel P, Greka A: Developing therapeutic ‘arrows’ with the precisionof William Tell: The time has come for targeted therapies in kidneydisease. Curr Opin Nephrol Hypertens 24: 388–392, 2015

14. Gagliardini E, Novelli R, Corna D, Zoja C, Ruggiero B, Benigni A,Remuzzi G: B7-1 is not induced in podocytes of human and experi-mental diabetic nephropathy [published online ahead of print August28, 2015]. J Am Soc Nephrol doi:ASN.2015030266

15. Canaud G, Bienaimé F, Tabarin F, Bataillon G, Seilhean D, Noël LH,Dragon-Durey MA, Snanoudj R, Friedlander G, Halbwachs-Mecarelli L,Legendre C, Terzi F: Inhibition of the mTORC pathway in the anti-phospholipid syndrome. N Engl J Med 371: 303–312, 2014

16. Canaud G, Bienaimé F, Viau A, Treins C, BaronW, Nguyen C, Burtin M,Berissi S, Giannakakis K, Muda AO, Zschiedrich S, Huber TB,Friedlander G, Legendre C, Pontoglio M, Pende M, Terzi F: AKT2 isessential to maintain podocyte viability and function during chronickidney disease. Nat Med 19: 1288–1296, 2013

This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2015091002/-/DCSupplemental.

8 Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc, 2015

CLINICAL RESEARCH www.jasn.org