www.kidney-international.org ba s i c re sea r ch

Exclusive expression of transmembrane TNFaggravates acute glomerulonephritis despitereduced leukocyte infiltration and inflammation

Martin B. Muller1, John M. Hoppe1, Andrei Bideak1, Moritz Lux1, Maja T. Lindenmeyer1, Susanna Muller2,Nuru Eltrich1, Bernhard Ryffel3,4 and Volker Vielhauer1

1Nephrologisches Zentrum, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Ludwig-Maximilians-UniversitätMünchen, Munich, Germany; 2Pathologisches Institut, Medizinische Fakultät, Ludwig-Maximilians-Universität München, Munich,Germany; 3Laboratory of Experimental and Molecular Immunology and Neurogenetics, UMR 7355 CNRS-University of Orleans, Orléans,France; and 4Institute of Infectious Disease and Molecular Medicine, Medical School, University of Cape Town, Cape Town, South Africa

Tumor necrosis factor-a (TNF) is a cytokine mediatinginflammatory kidney diseases such as immune complexglomerulonephritis. Its two receptors, TNFR1 and TNFR2, playdistinct roles in this process, with TNFR2 strongly required forinduction of disease. In contrast to soluble TNF (sTNF),transmembrane TNF robustly activates TNFR2. Thus, weexamined the functional role of transmembrane TNF byinducing heterologous nephrotoxic serum nephritis in wild-type and transgenic TNFD1-9,K11E knock-in mice expressingtransmembrane TNF but no sTNF (memTNF mice). Comparedto wild-type, nephritis was exacerbated in memTNF mice onday 5, indicated by increased albuminuria, higher serum urealevels, and more pronounced glomerular deposits, togetherwith higher numbers of dying and proliferating glomerularcells. This was associated with greater loss of glomerularendothelial cells, increased podocyte stress, and signs ofaugmented necroptosis in memTNF kidneys. Aggravation ofnephritiswas dependent on transmembraneTNFexpression inparenchymal cells, but not leukocytes. Surprisingly, increasedkidney injury was associated with reduced renal leukocyteinfiltration in memTNF mice, which correlated with decreasedrenal mRNA expression of pro-inflammatory mediators. Thiseffect was also present in isolated memTNF glomerulistimulated with interleukin-1b in vitro. Thus, uncleavedtransmembrane TNF is an important mediator of renal tissuedamage characterized by increased renal cell death and loss ofglomerular endothelial cells in murine glomerulonephritis. Incontrast, sTNF predominantly mediates renal leukocyterecruitment and inflammation. These findings highlight theimportance of transmembrane TNF in inflammatory kidneydisease as a possible therapeutic target.Kidney International (2019) 95, 75–93; https://doi.org/10.1016/j.kint.2018.08.012

KEYWORDS: cell death; cytokines; glomerulonephritis; inflammation

Copyright ª 2018, International Society of Nephrology. Published by

Elsevier Inc. All rights reserved.

Correspondence: Volker Vielhauer, Nephrologisches Zentrum, MedizinischeKlinik und Poliklinik IV, Klinikum der Universität München, Ludwig-Max-imilians-Universität München, Ziemssenstr. 1, 80336 Munich, Germany.E-mail: Volker.Vielhauer@med.uni-muenchen.de

Received 22 November 2017; revised 29 July 2018; accepted 2 August2018; published online 30 October 2018

Kidney International (2019) 95, 75–93

P ro-inflammatory cytokines such as TNF play an impor-tant pathophysiologic role and contribute to inflammatoryinjury in glomerulonephritis (GN).1,2 TNF deficiency

ameliorates murine nephrotoxic serum nephritis (NTN),3,4 awidely studied rodent model for immune complex-mediatedGN.5 Interestingly, TNF expressed in intrinsic renal cells ratherthan infiltrating bone marrow-derived leukocytes promotedNTN.4 Consistently, therapy with TNF-blocking agents attenu-ated glomerular inflammation and crescent formation in ratswith crescentic NTN.6,7 Apart from its pro-inflammatory andanti-infectious roles, TNF also possesses immunoregulatoryfunctions, including suppression of autoimmunity by TNF-induced apoptosis of activated T-cells.8–10 The ability of TNFto limit autoinflammation is illustrated by the appearance oflupus-like symptoms and development of GN in patients withrheumatoid arthritis or inflammatory bowel disease treatedwith anti-TNF agents.11,12 Therefore, it is crucial to better under-stand the molecular basis of inflammatory tissue injury andimmunoregulatory functions mediated by TNF in GN beforeTNF-targeting therapies can be implemented.

We have previously characterized distinct roles of the 2 TNFreceptors (TNFRs), TNFR1 and TNFR2, in murine NTN, withTNFR2 strongly required for disease induction.13 TNFR2 wasprominently expressed in glomerular endothelial cells innephritickidneys with NTN,13 and its expression was readily upregulatedby inflammatory stimuli in mouse glomeruli in vitro.14 Further-more, experiments with TNFR2-chimeric mice indicated thatTNFR2 expressed by intrinsic renal cells is critical for the in-duction of NTN.13 TNF exists in 2 biologically active forms, bothexpressed on intrinsic renal cells upon stimulation.10 Themembrane-bound precursor molecule (memTNF) can bereleased into circulating sTNF via cleavage of the ectodomain bythe metalloproteinase TNF-a-converting enzyme (TACE/ADAM17).15,16 Both memTNF and preferably sTNF activateTNFR1 in the picomolar range, whereas TNFR2 is only robustlyactivated by memTNF.17,18 In line with this data, we have previ-ously demonstrated in isolated mouse glomeruli in vitro thatinduced expression of inflammatory mediators upon stimulationwith sTNF was mediated by TNFR1, but not TNFR2.14

Based on our previous findings identifying expression ofTNFR2 in intrinsic renal cells as a critical step in inducing

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Entrez Gene ID Gene Symbol Alias IgAN (vs. LD) SLE (vs. LD) RPGN (vs. LD) MCD (vs. LD) FSGS (vs. LD) DN (vs. LD)7124 TNF TNFα 1.176 1.186 1.549 1.139 1.133 1.5547132 TNFRSF1A TNFR1 0.895 n.s. 1.308 n.s. n.s. 1.1227133 TNFRSF1B TNFR2 2.205 2.985 3.315 1.269 2.174 2.076

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Figure 2 | Increased mortality and aggravated renal functional impairment in mice with knock-in of 2 uncleavable D1-9,K11E tumornecrosis factor (TNF) alleles (memTNF mice) with heterologous nephrotoxic serum nephritis (NTN). (a) Survival proportions in wild-type(WT) and memTNF mice after induction of NTN (n ¼ 17–18 per group). (b) Serum interleukin-6 (IL-6) levels in WT and memTNF mice at day 5 ofNTN measured by enzyme-linked immunosorbent assay (n ¼ 4 per group). The dashed line indicates mean values in healthy WT mice. (c)Albuminuria was evaluated in spot urine samples at baseline and day 5 after injection of nephrotoxic serum and is expressed as urinaryalbumin-to-creatinine ratio (n ¼ 13 per group). (d) Serum levels of urea were determined at day 5 of NTN. The dashed line indicates meanbaseline values for normal WT mice (n ¼ 7–8 mice per group). Data are expressed as mean � SEM. *P < 0.05, **P < 0.01.

MB Müller et al.: Transmembrane TNF in GN bas i c re sea r ch

complement-dependent kidney injury,13 we hypothesized thatTNFR2 activation via memTNF mediates murine NTN. As aspecific role for memTNF in inflammatory kidney disease hasnot been investigated, we induced heterologous NTN inC57BL/6 wild-type mice and mice with knock-in of 2uncleavable D1-9,K11E TNF alleles (memTNF mice).memTNF mice can only express the transmembrane form of

=Figure 1 | Expression of tumor necrosis factor (TNF) in human and mfrom micro-dissected glomeruli of human renal biopsies with IgA nephroglomerulonephritis (RPGN; n ¼ 23), minimal change disease (MCD; n ¼nephropathy (DN; n ¼ 14). Microarray expression experiments and analymap summarizes significantly regulated gene expression, with glomerulaupregulated in RPGN. Numbers represent fold changes compared withtransplantation biopsies of living donors (LD; n ¼ 42). n.s., non-significagranulomatosis with polyangiitis (GPA), MCD, FSGS, and membranous nglomerular TNF expression. In the glomerulonephritides (IgAN, SLE, GPAadditional expression in podocytes (black arrowheads). A prominent exp(MCD, FSGS, MN; black arrowheads), which lacked endothelial TNF (whitepretransplant biopsies obtained from living kidney donors. As a technicaldiluent alone, but no primary antibody, followed by incubation with secbar ¼ 50 mm. (c) A similar protein expression of TNF in glomerular endokidneys at day 5 of heterologous nephrotoxic serum nephritis (NTN). TNFin of 2 uncleavable D1-9,K11E TNF alleles (memTNF) compared with wilfication x400, bar ¼ 50 mm. To optimize viewing of this image, please s

Kidney International (2019) 95, 75–93

TNF (i.e., memTNF due to a modification on the cleavage sitefor TACE) and lack secreted sTNF.19 Importantly, memTNF isfunctional, normally regulated and expressed in these mice.19

Our results demonstrate that memTNF is an importantmediator of renal tissue damage in NTN by induction ofglomerular and tubulointerstitial cell death, and loss ofglomerular endothelial cells and podocyte integrity. In

urine glomerulonephritis. (a) Gene expression data were obtainedpathy (IgAN; n ¼ 27), lupus nephritis (SLE; n ¼ 32), rapidly progressive14), focal segmental glomerular sclerosis (FSGS; n ¼ 23), and diabeticsis were performed as described in Materials and Methods. The heatr TNF and TNF receptor 2 (TNFR2) expression being most prominentlyexpression levels in healthy control glomeruli obtained from pre-nt. (b) In human glomerulonephropathies including IgAN, SLE,ephropathy (MN), immunohistochemistry demonstrated a prominent) TNF localized to glomerular endothelial cells (black arrows), withression of TNF in podocytes was also present in nephrotic diseasearrows). Only background staining was present in healthy glomeruli ofcontrol for unspecific staining, tissue was incubated with the antibodyondary antibody and detection reagents. Original magnification x400,thelium (arrows) and podocytes (arrowheads) was found in murinestaining was more prominent in nephritic kidneys of mice with knock-d-type (WT), and absent in healthy control kidneys. Original magni-ee the online version of this article at www.kidney-international.org.

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Cel

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Glo

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*

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n.s.

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bas i c re sea r ch MB Müller et al.: Transmembrane TNF in GN

78 Kidney International (2019) 95, 75–93

MB Müller et al.: Transmembrane TNF in GN bas i c re sea r ch

contrast, NFkB-mediated expression of pro-inflammatorymediators and recruitment of infiltrating leukocytes intonephritic kidney is dependent on the presence of sTNF, asthese were significantly reduced in memTNF mice despiteincreased renal injury. These data indicate that memTNF andsTNF play distinct roles in mediating inflammatory kidneydisease and has important implications for designing TNFtargeting therapies in GN.

RESULTSInduced expression of TNF in human glomerulonephritisWe first investigated glomerular expression levels of TNFanalyzing mRNA expression data from human biopsy samplesof the European Renal cDNA Bank - Kröner-Fresenius BiopsyBank.20 Compared with healthy controls, glomerular expres-sion of TNF and predominantly its receptor TNFR2 wassignificantly induced in various glomerular diseases, includingglomerulonephritides like IgA nephropathy, lupus nephritis,and most prominently rapidly progressive GN (Figure 1a).Within glomeruli immunohistochemistry localized TNFprotein expression to endothelial cells and podocytes, with astaining pattern consistent with a cell membrane–associatedlocalization particularly in glomerular endothelial cells.Interestingly, nephrotic disease with predominant podocyteinjury lacked endothelial TNF expression (Figure 1b), indi-cating that TNF expression associates with the primarilyinjured cell type. We found a similar expression in murinekidneys with heterologous NTN (Figure 1c). These findingssuggested that memTNF may contribute to glomerular injuryin GN of humans and mice.

Increased mortality despite reduced systemic inflammation inmemTNF miceFollowing induction of heterologous NTN, memTNF micedisplayed increased mortality in the early phase of the diseasecompared with wild-type controls. At day 1 and 2 after in-jection of nephrotoxic serum, 35.3% of memTNF micerequired killing due to moribund appearance, whereas mor-tality was only 5.6% in wild-type mice (Figure 2a). Toinvestigate whether an increased systemic inflammatoryresponse toward the injected nephrotoxic serum could

=Figure 3 | Increased glomerular injury and cell death in mice with kalleles (memTNF mice) with heterologous nephrotoxic serum nephri(PAS)-stained kidney sections from wild-type (WT) and memTNF mice ilquantitative scoring revealed more extensive glomerular injury in memTnick end-labeling (TUNEL) staining demonstrated increased glomerularassociated with increased numbers of proliferating cell nuclear antigenregenerative cell proliferation. (d) Podocytes were identified by immunoand nephrin. Podocyte numbers similarly decreased in WT and memTNFWT mice. Instead, renal nephrin mRNA expression was significantly reduindicating increased podocyte stress. (e) Immunofluorescence staining foglomerular endothelial cell injury compared with WT glomeruli. (f) Glomcomplement C3d was comparable in both genotypes, consistent with srepresentative glomeruli from each group; original magnification x400,formed as described in Materials and Methods. Data are expressed as mpendently performed experiments. *P < 0.05; n.s., not significant. To optiat www.kidney-international.org.

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explain excessive mortality in memTNF mice, we measuredinterleukin (IL)-6 serum levels in nephritic mice at day 5 ofNTN. In both genotypes levels of serum IL-6 increased inNTN mice, but despite higher mortality IL-6 serum con-centrations were significantly lower in nephritic memTNFmice (Figure 2b). In contrast, 50.0% of memTNF micecompared with 22.2% of wild-type mice developed hematu-ria, and moribund mice manifested ascites, which suggestedthat acute kidney failure was a potential cause of increasedmortality in memTNF mice.

Exclusive expression of memTNF worsens renal functionalparameters in acute NTNTo investigate a potential role of memTNF in mediating renalinjury in NTN, we first analyzed albuminuria and serum urealevels in nephritic wild-type and memTNF mice. At day 5 ofNTN, albuminuria was significantly worse in memTNF mice(Figure 2c). Consistently, renal function deteriorated moresignificantly in memTNF mice, as revealed by higher serumurea levels compared with wild-type at day 5 of NTN(Figure 2d). Thus, exclusive expression of memTNF signifi-cantly aggravated functional parameters of renal injury inacute NTN.

Expression of memTNF aggravates glomerular injury and celldeath in acute NTNIncreased albuminuria suggested more severe glomerularinjury in memTNF mice. Accordingly, glomeruli of memTNFmice showed increased mesangial matrix expansion andfibrinoid necrosis at day 5 of NTN as revealed by semi-quantitative analysis of intraglomerular deposition of periodicacid–Schiff-positive material (Figure 3a). In addition, termi-nal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) staining demonstrated significantlyincreased cell death in memTNF glomeruli (Figure 3b), whichwas associated with increased numbers of proliferating cells ininjured glomeruli (Figure 3c). To analyze further which celltype of the glomerular filtration barrier was affected bymemTNF-mediated injury, podocyte and endothelial cellmarkers were evaluated. Whereas podocyte loss was compa-rable in nephritic wild-type and memTNF mice, reduced

nock-in of 2 uncleavable D1-9,K11E tumor necrosis factor (TNF)tis (NTN). (a) Representative photomicrographs of periodic acid-Schifflustrating glomerular matrix deposition at day 5 of NTN. Semi-NF mice. (b) Terminal deoxynucleotidyl transferase–mediated dUTPcell death in memTNF mice compared with WT mice. (c) This was(PCNA)-positive cells in memTNF glomeruli indicating enhancedfluorescence double-staining for nuclear Wilms tumor antigen (WT)-1mice. The dashed line indicates mean podocyte numbers in healthyced in memTNF mice compared with WT mice at day 5 of NTN,r CD31 was reduced in memTNF glomeruli, demonstrating aggravatederular deposition of injected heterologous sheep antibodies and (g)imilar immune-mediated injury that triggered NTN. Images showbar ¼ 50 mm. Semiquantitative and morphometric analysis was per-ean � SEM of 6 mice per group and are representative for 2 inde-mize viewing of this image, please see the online version of this article

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Figure 4 | Aggravated tubular injury in mice with knock-in of 2 uncleavable D1-9,K11E tumor necrosis factor (TNF) alleles (memTNFmice) with heterologous nephrotoxic serum nephritis (NTN). (a) At day 5 of NTN mRNA expression of the tubular injury markers L-FABP anda-GST were significantly increased in memTNF kidneys compared with wild-type (WT) kidneys, indicating enhanced tubular cell injury. Poly-merase chain reaction results were normalized to 18S rRNA expression used as reference gene. Dashed lines indicate mean baseline expressionin healthy WT kidneys. L-FABP, liver-type fatty acid binding protein; a-GST, a-glutathione S-transferase. (b) Enhanced tubulointerstitial cell deathin memTNF kidneys was visualized by terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) staining. (c) Staining forproliferating cell nuclear antigen (PCNA) revealed a nonsignificant trend toward increased cell proliferation in the tubulointerstitium ofmemTNF kidneys. Original magnification x400, bar ¼ 50 mm. Data are expressed as mean � SEM of 5 to 6 mice per group and are representativefor 2 independently performed experiments. *P < 0.05. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

bas i c re sea r ch MB Müller et al.: Transmembrane TNF in GN

renal mRNA expression of nephrin indicated greater podo-cyte stress in memTNF mice (Figure 3d). Moreover, reducedglomerular staining of CD31 revealed more severe glomerularendothelial cell injury in memTNF mice, whereas CD31expression in nephritic wild-type mice was comparable tothat of healthy controls (Figure 3e). Importantly, glomerulardeposition of injected heterologous sheep antibodies andcomplement C3d was comparable in both genotypes(Figure 3f and g), suggesting that initial immune-mediatedinjury was similar.

While histopathology demonstrated more severe glomer-ular damage in memTNF mice, tubulointerstitial injuryanalyzed in periodic acid–Schiff-stained sections was notsignificantly different in both genotypes at day 5 of NTN,including the extent of tubular dilation, intratubular castformation, tubular cell damage, and tubular vacuolardegeneration (Supplementary Figure S1). However, mRNA

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expression of the 2 tubular injury markers liver-type fatty acidbinding protein (L-FABP) and a-glutathione S-transferase (a-GST) significantly increased in memTNF kidneys as an earlysign of aggravated tubular injury (Figure 4a). Moreover,similar to the glomerular compartment, memTNF kidneysshowed increased cell death and a trend toward enhanced cellproliferation in the tubulointerstitium (Figure 4b). Takentogether, memTNF expression in the absence of sTNF ag-gravates functional and structural renal damage in acuteNTN, and particularly leads to increased cell death andglomerular endothelial cell injury.

Mediators of regulated necrosis are more abundantlyexpressed in nephritic memTNF kidneysTo explore mechanisms of increased cell death in nephriticmemTNF kidneys we analyzed markers of known TNF-induced cell death pathways. Similar to other models of

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Glomerular endothelial cells

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Figure 5 | Role for regulated necrosis but not apoptosis in mediating renal injury of mice with knock-in of 2 uncleavable D1-9,K11Etumor necrosis factor (TNF) alleles (memTNF mice). (a) Protein expression levels of cleaved caspase 3, RIP1, and RIP3 were analyzed innephritic kidneys of wild-type (WT) and memTNF mice at day 5 of nephrotoxic serum nephritis (NTN) by immuoblotting. RepresentativeWestern blot images are shown. b-actin served as a loading control. Data are expressed as mean � SEM, n ¼ 4. (b) Detection of necroptosis in amurine glomerular endothelial cell line after treatment with 300 ng/ml TNF and 10 mM ZVAD-FMK (ZVAD) for 6 hours (MTT assay). Protectionfrom cell death by necrostatin-1s (Nec-1s) treatment (100 mM) confirmed induced necroptosis in treated cells. Data are expressed as mean �SEM of 3 independent experiments. *P < 0.05, **P < 0.01. To optimize viewing of this image, please see the online version of this article atwww.kidney-international.org.

MB Müller et al.: Transmembrane TNF in GN bas i c re sea r ch

acute kidney injury,21,22 in neither wild-type nor memTNFmice could cleaved caspase 3 as an indicator of ongoingapoptosis be detected in nephritic kidneys (Figure 5a). As analternative mechanism of regulated cell death particularlyassociated with inflammatory tissue injury, necroptosis hasbeen identified as an important mediator of renal pathologyin models of acute kidney injury.21–24 We therefore investi-gated protein expression of core components of the

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necroptotic pathway, receptor interacting protein 1 (RIP1)and RIP3, in kidneys at day 5 of NTN. RIP1 protein levelssignificantly increased in nephritic kidneys of memTNF micecompared with wild-type, and a similar trend was noted forRIP3 (Figure 5a). As increased expression of RIP1 and RIP3correlates with the extent of necroptotic cell death in tissue,25

we next determined whether necroptosis could be induced byTNF in glomerular endothelial cells, which were more

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***

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MB Müller et al.: Transmembrane TNF in GN bas i c re sea r ch

severely injured in nephritic memTNF mice. In a murineglomerular endothelial cell line, exposure to high doses ofTNF in combination with the pan-caspase inhibitor Z-VAD-FMK resulted in substantial caspase-independent non-apoptotic cell death. Cell death was significantly attenuated byco-incubation with the necroptosis inhibitor Nec-1s(Figure 5b), indicating that TNF exposure under caspase-inhibiting conditions causes necroptosis in intrinsic glomer-ular cells. Together, these in vivo and in vitro data suggest thatincreased necroptosis contributes to more abundant renal celldeath and kidney injury in memTNF mice with acute NTN.

Renal leukocyte accumulation decreases in memTNF micewith acute NTN despite aggravated renal injuryCell death and tissue injury elicits a necroinflammatoryresponse characterized by leukocyte infiltration andexpression of inflammatory mediators, which leads tofurther tissue damage.26 In acute kidney injury, the severityof tissue damage usually correlates with the extent of renalleukocyte infiltrates. Surprisingly, flow cytometric analysisrevealed a highly significant reduction of renal leukocytenumbers in memTNF kidneys at day 5 of NTN comparedwith wild-type (Figure 6a). We noted significantly reducedinfiltrates of mononuclear phagocyte populations, reducedCD3þ T cells, and a trend toward reduced neutrophilnumbers in nephritic mTNF kidneys (Figure 6a).Compartment-specific evaluation of renal leukocyte infil-tration by immunohistochemistry confirmed reducedglomerular and tubulointerstitial infiltration of neutrophilsand macrophages into nephritic mTNF kidneys, with similartrends seen for CD3þ T cells (Figure 6b). Thus, memTNFexpression causes exacerbated renal injury independent ofrenal leukocyte infiltration through direct effects on renalparenchymal cells.

As rapid infiltration of neutrophils occurs within hoursafter induction of NTN,27 we further analyzed the relation-ship between renal injury and leukocyte infiltration in wild-type and memTNF mice during this early induction phaseof the disease. Two hours after injection of the nephrotoxicserum mice of both genotypes developed substantial butcomparable albuminuria. Instead, serum urea levels and celldeath of tubulointerstitial cells significantly increased inmemTNF mice compared with wild-type mice, indicatingmore severe acute kidney injury in memTNF mice early inNTN (Figure 7a and b). Moreover, renal infiltration of

=Figure 6 | Decreased leukocyte infiltration in kidneys of mice with kalleles (memTNF mice) despite aggravated renal injury. (a) Flow cytomkidneys at day 5 of nephrotoxic serum nephritis (NTN) demonstrated red(WT) mice. Cells were stained for CD45 (pan leukocyte marker), CD11c (Ly6G (neutrophils), CD3 (T-cells), CD3/CD4 (CD4þ T helper cells), and CDvalues for healthy WT mice. Data are expressed as mean � SEM of 11 tmemTNF mice stained for Ly6Gþ neutrophils, Mac2þ glomerular macroOriginal magnification 400x, bar ¼ 50 mm. Glomerular and tubulointerstiMethods. Data are expressed as mean � SEM of 6 to 8 mice per group. *please see the online version of this article at www.kidney-international

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leukocytes and particularly Ly6Gþ neutrophils was signifi-cantly reduced in memTNF kidneys at this time point(Figure 7c). Taken together, these data indicate that memTNFexpression aggravates renal injury despite early and sustainedreductions in leukocyte infiltration in NTN.

Exclusive expression of memTNF in parenchymal cells, butnot leukocytes aggravates renal injury in acute NTNProminent glomerular expression of membrane-associatedTNF in human and murine GN as well as aggravated renalinjury in memTNF mice despite reduced renal leukocyteinfiltration suggested a predominant role of parenchymal cell-expressed memTNF in mediating renal damage. To determinethe extent to which intrinsic cell- versus leukocyte-expressedmemTNF exacerbates disease, we analyzed mice expressingmemTNF only in bone marrow (BM)-derived cells orintrinsic cells that were generated by BM transplantationbetween wild-type (WT) and memTNF-deficient animals.Chimeric mice with wild-type BM transplanted into irradi-ated wild-type recipients and memTNF BM transplanted intomemTNF recipients served as controls. As expected, serumlevels of sTNF were barely detectable in mem-TNF/memTNF chimeras, and greatly reduced inmemTNF/WT and WT/memTNF chimeras comparedwith WT/WT controls (Figure 8a). Consistent with thephenotypes of wild-type and memTNF mice, mem-TNF/memTNF chimeras showed increased albuminuria,more severe hypoalbuminemia, hyperlipidemia, renal func-tional impairment, and glomerular injury compared withWT/WT chimeras (Figure 8), but reduced renal leukocyteinfiltrates (Supplementary Figure S2). A similar aggravationof disease was present in WT/memTNF chimeras, but notin memTNF/WT chimeras (Figure 8). These results clearlyindicate a role for renal cell-expressed but not leukocytememTNF in aggravating heterologous NTN.

Of note, our chimera experiments further support a functionfor sTNF in mediating renal leukocyte infiltration, as onlymemTNF/memTNF chimeras, which completely lack sTNF,showed reduced leukocyte numbers in nephritic kidneys(Supplementary Figure S2). Interestingly, we noted increasedaccumulation of glomerular and interstitial neutrophils in kid-neys of memTNF/WT and WT/memTNF chimeras,respectively. This indicates that in the presence of residual sTNFproduction, memTNF expression in leukocytes or parenchymalcells enhances renal leukocyte infiltration.

nock-in of 2 uncleavable D1-9,K11E tumor necrosis factor (TNF)etric analysis of renal single cell suspensions prepared from nephriticuced leukocyte infiltrates in memTNF mice compared with wild-typeDC like mononuclear phagocytes), F4/80 (mononuclear phagocytes),3/CD8 (CD8þ cytotoxic T cells). Dashed lines indicate mean baselineo 13 mice per group. (b) Representative renal sections of WT andphages, F4/80þ tubulointerstitial phagocytes, and CD3þ T cells.tial leukocyte infiltrates were quantified as described in Materials andP < 0.05, **P < 0.01, ***P < 0.001. To optimize viewing of this image,.org.

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Figure 7 | Impaired renal function and increased cell death despite reduced renal leukocyte infiltrates in mice with knock-in of 2uncleavable D1-9,K11E tumor necrosis factor (TNF) alleles (memTNF mice) with early heterologous nephrotoxic serum nephritis (NTN).Wild-type (WT) and memTNF mice were analyzed 2 hours after injection of nephrotoxic serum. (a) Mice rapidly developed albuminuria, whichwas comparable in both genotypes. Renal functional impairment was only present in memTNF mice, with significantly higher serum urea levelsin memTNF mice compared with WT. (b) Consistently, terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) stainingrevealed aggravated cell death in the tubulointerstitial, but not glomerular compartment of memTNF mice. (c) Numbers of renal CD45þleukocytes and Ly6Gþ neutrophils were significantly lower in memTNF mice. Dashed lines indicate mean baseline values of healthy WT mice.Data are expressed as mean � SEM of 4 mice per group. *P < 0.05, **P < 0.01. To optimize viewing of this image, please see the online versionof this article at www.kidney-international.org.

bas i c re sea r ch MB Müller et al.: Transmembrane TNF in GN

Expression of proinflammatory mediators and NFkBactivation decreases in memTNF kidneys

Our results suggested that increased renal cell death due toregulated necrosis mediates exacerbated renal injury inmemTNF kidneys with acute NTN. However, mechanisms ofimpaired renal leukocyte infiltration despite increased tissueinjury in memTNF mice remained unclear. As expression ofpro-inflammatory cytokines, chemokines, and adhesion

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molecules mediates leukocyte recruitment into diseased kid-neys,28 we compared renal mRNA expression of these in-flammatory mediators in wild-type and memTNF mice. Thisanalysis revealed decreased mRNA expression of the cytokinesIL-1b, IL-6, IL-10, and interferon (IFN)-b, the chemokinesCCL2 and CXCL10, and the adhesion molecule E-selectin innephritic memTNF kidneys, whereas CCL5 expression wassimilar to wild-type (Figure 9a). Interestingly, memTNF

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Figure 8 | Exclusive expression of membrane-bound precursor molecule tumor necrosis factor (memTNF) in parenchymal cells but notleukocytes aggravates heterologous nephrotoxic serum nephritis (NTN). Heterologous NTN was evaluated in chimeric mice with memTNFexpression either in bone marrow (BM) (memTNF/WT chimeras) or parenchymal cells (WT/memTNF chimeras). Wild-type (WT) micetransplanted with WT BM (WT/WT chimeras) and memTNF mice transplanted with memTNF BM (memTNF/memTNF chimeras) served ascontrols. (a) Serum levels of soluble tumor necrosis factor (sTNF) at day 5 of NTN were measured by enzyme-linked immunosorbent assay. (b)Albuminuria at day 5 was significantly worse in WT/memTNF and memTNF/memTNF chimeras, but not memTNF/WT chimeras, comparedwith WT/WT. (c) Consistently, nephrotic hypoalbuminuria, hyperlipidemia, and urea levels significantly increased in WT/memTNF andmemTNF/memTNF chimeras, but not in memTNF/WT chimeras. (d) Representative photomicrographs of periodic acid–Schiff-stained kidneysections at day 5 of NTN. Semiquantitative scoring revealed more extensive glomerular injury in WT/memTNF and memTNF/memTNFchimeras, but not memTNF/WT chimeras, compared with WT/WT. Data are expressed as mean � SEM of 5 mice per group. *P < 0.05, **P <0.01. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

MB Müller et al.: Transmembrane TNF in GN bas i c re sea r ch

kidneys expressed significantly more TNF mRNA, whereasexpression of TNFR1 and TNFR2 was comparable to wild-type (Figure 9b).

As expression of inflammatory mediators is induced by thetranscription factor NFkB, we analyzed NFkB activation inkidneys of wild-type and memTNF mice at day 5 of NTN.Significantly decreased protein levels of phosphorylated in-hibitor of NFkB subunit a (IkB-a), the abundance of whichcorrelates with NFkB pathway activation, confirmed reduced

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NFkB activation in memTNF kidneys (Figure 10a). Asignificantly reduced NFkB2 p52-to-p100 ratio additionallysuggested reduced activity of the alternative NFkB pathway inmemTNF kidneys (Figure 10a). Moreover, mRNA expressionlevels of the NFkB inhibitors IkB-a and TNF-a-inducedprotein 3 (TNFAIP3/A20) as well as the NFkB-associatedanti-apoptotic proteins cIAP1 and XIAP significantlydecreased in memTNF kidneys (Figure 10c). As transcriptionof these negative regulators is induced upon NFkB activation

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Figure 9 | Decreased expression of proinflammatory mediators in nephritic kidneys of mice with knock-in of 2 uncleavable D1-9,K11Etumor necrosis factor (TNF) alleles (memTNF kidneys). (a) Induced mRNA expression of pro-inflammatory cytokines, chemokines, andadhesion molecules in nephritic wild-type (WT) kidneys was significantly attenuated in memTNF kidneys at day 5 of nephrotoxic serum nephritis(NTN). (b) TNFmRNA expression increased in nephritic memTNF kidneys, and expression of TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2) wasnot different in both genotypes. Polymerase chain reaction results were normalized to 18S rRNA as a housekeeping gene. Dashed lines indicatemean baseline expression in healthyWT kidneys, with similar expression in healthymemTNF kidneys (data not shown). Data are expressed asmean� SEM of 6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. CCL, C-C motif chemokine ligand; CXCL, C-X-C motif chemokine ligand; ICAM,intercellular adhesion molecule; IFN, interferon; IL, interleukin; n.s., not significant; VCAM, vascular cell adhesion molecule.

bas i c re sea r ch MB Müller et al.: Transmembrane TNF in GN

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Figure 10 | Reduced NFkB activation in nephritic kidneys of mice with knock-in of 2 uncleavable D1-9,K11E tumor necrosis factor (TNF)alleles (memTNF kidneys). (a) Western blot analysis of IkB phosphorylation and NFkB2 degradation in nephritic wild-type (WT) and memTNFkidneys at day 5 of nephrotoxic serum nephritis (NTN) demonstrating reduced canonical and noncanonical NFkB activation in memTNF mice.b-actin is shown as loading control. Data are expressed as mean � SEM; n ¼ 3. (b) Decreased mRNA expression levels of the NFkB inhibitorsIkB-a and TNFAIP3/A20, and OF NFkB-associated anti-apoptotic proteins cIAP1 and XIAP in memTNF kidneys. Polymerase chain reaction resultswere normalized to 18S rRNA as a housekeeping gene. Dashed lines indicate mean baseline expression in healthy WT kidneys. Data areexpressed as mean � SEM of 6 mice per group. *P < 0.05. cIAP, cellular inhibitor of apoptosis protein; IkB, inhibitor of NFkB; TNFAIP,TNF-a-induced protein; XIAP, X-linked inhibitor of apoptosis protein. To optimize viewing of this image, please see the online version ofthis article at www.kidney-international.org.

MB Müller et al.: Transmembrane TNF in GN bas i c re sea r ch

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Figure 11 | Reduced expression of inflammatory mediators in isolated glomeruli of mice with knock-in of 2 uncleavable D1-9,K11Etumor necrosis factor (TNF) alleles (memTNF glomeruli) in vitro. Glomeruli were isolated from healthy wild-type (WT) and memTNF mice asdescribed in Materials and Methods and stimulated with 10 ng/ml interleukin (IL)-1b for 24 hours. (a) Levels of TNF, IL-6, and C-C motifchemokine ligand (CCL2) in the supernatant were measured by enzyme-linked immunosorbent assay. As expected, TNF secretion was absent inmemTNF glomeruli. Production of IL-6 and CCL2 was significantly reduced in memTNF glomeruli. (b) Analysis of mRNA expression levelsrevealed comparable levels of TNF transcripts in WT and memTNF glomeruli, but reduced IL-6 and CCL2 expression in memTNF glomeruli.Polymerase chain reaction results were normalized to 18S rRNA as a housekeeping gene. Data are expressed as mean � SEM of 4 indepen-dently performed experiments. *P < 0.05, **P < 0.01, ***P < 0.001. n.d., not detected.

bas i c re sea r ch MB Müller et al.: Transmembrane TNF in GN

to form a negative feedback loop, reduced expressionindicates decreased NFkB activity in memTNF kidneys. Thus,reduced leukocyte infiltration into memTNF kidneys corre-lates with reduced NFkB-dependent expression of inflam-matory mediators in nephritic kidneys.

Expression of inflammatory mediators decreases in memTNFglomeruli in vitroReduced NFkB-mediated expression of inflammatory medi-ators in memTNF kidneys could result in reduced renalleukocyte infiltration, but may also be the consequence ofdecreased intrarenal accumulation of inflammatory leuko-cytes. Therefore, we analyzed inflammatory responses inintact glomeruli isolated from healthy wild-type andmemTNF mice in vitro, independently of infiltrating leuko-cytes. Isolated tissue was stimulated with IL-1b, which in vivoupon secretion by leukocytes induces TNF expression inintrinsic renal cells, which are a major source of TNFcontributing to glomerular injury in NTN.4,29 As expected,glomeruli of memTNF mice did not secret TNF upon IL-1bstimulation, which readily induced TNF secretion in wild-type glomeruli. IL-1b stimulation also induced secretion ofIL-6 and CCL2 in both wild-type and memTNF glomeruli,but IL-6 and CCL2 levels were significantly lower in

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stimulated memTNF glomeruli lacking sTNF (Figure 11a).Moreover, whereas IL-1b induced mRNA expression of TNFsimilarly in both genotypes, induced IL-6 and CCL2 mRNAlevels were again significantly lower in memTNF glomeruliupon IL-1b stimulation (Figure 11b). In summary, these re-sults indicate that decreased renal inflammation in nephriticmemTNF mice is caused by reduced expression of inflam-matory mediators by intrinsic renal cells, which subsequentlyresults in reduced renal leukocyte recruitment into damagedrenal tissue. Moreover, these data suggest that secreted sTNF,which is absent in memTNF tissue, contributes to NFkB-dependent expression of inflammatory mediators and sub-sequent leukocyte recruitment into nephritic kidneys.

DISCUSSIONTNF is an important mediator of GN, and TNF-targetingtherapies have been successfully applied in rodentmodels.3,4,6,7,30 However, next to its classic pro-inflammatoryfunctions TNF also possesses immunoregulatory activity,which can limit autoimmune responses and explains occur-rence of autoimmune disease and even GN in patients treatedwith TNF-blocking agents.1 The 2 TNF receptors, TNFR1 andTNFR2, may differentially mediate these TNF functions, withTNFR2 being robustly activated by memTNF only, but not

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MB Müller et al.: Transmembrane TNF in GN bas i c re sea r ch

sTNF.2,17 Thus, more specific inhibition of TNF signalingpathways targeting 1 individual TNF receptor or memTNFversus sTNF may maintain anti-inflammatory efficacy whilereducing adverse immuno-suppressive and autoimmune ef-fects. Here, we hypothesized that memTNF plays a distinctpathophysiologic role in GN, as development of murineNTN, a model of immune complex-mediated GN, dependson the presence of the memTNF receptor TNFR2 expressedby intrinsic renal cells, but not on TNFR1 expression.13 Ourstudies using transgenic TNFD1-9,K11E knock-in mice withexclusive expression of functional memTNF in the absence ofsTNF confirm this concept and further demonstrate thatmemTNF aggravates NTN by enhancing renal cell death,whereas sTNF contributes to intrarenal and systemicexpression of inflammatory mediators and renal leukocyteinfiltration. The fact that renal functional deterioration andglomerular injury increased in memTNF mice despitereduced leukocyte infiltrates and renal inflammation un-derlines the importance of memTNF as a mediator of renaltissue damage and identifies the memTNF/TNFR2 axis as apreferential therapeutic target in GN.

Consistent with our results, memTNF-induced tissueinjury has been reported in other disease models. Forexample, memTNF mediates chronic inflammatoryarthritis31,32 and concanavalin A-induced experimental hep-atitis.33 Moreover, inhibition of TNF processing by melphalanwas shown to elicit memTNF on Kupffer cells, which trig-gered cell death of hepatocytes.34 In experimental colitisblocking memTNF and sTNF with a TNF-inhibiting anti-body, but not treatment with a dominant negative TNFmutant exclusively neutralizing sTNF induced remission in T-cell-mediated colitis, suggesting that memTNF is mediatinginflammatory bowel disease.35 In experimental autoimmuneuveitis signaling via memTNF was sufficient to mediate tissuedamage, although leukocyte infiltration into injured organswas dependent on sTNF,36 which is consistent with ourfindings in the NTN model.

We identified increased renal cell death as an underlyingmechanism for aggravated injury in nephritic memTNFkidneys. memTNF-induced cell death and regulated necrosisare well described,37–40 and involve TNFR2 activation andTNFR2-dependent enhancement of TNFR1-mediated cyto-toxicity.38–42 Moreover, reduced NFkB activation in memTNFkidneys was associated with decreased expression of theintracellular survival factors cIAP1 and XIAP. Depletion ofthese proteins facilitates TNF-induced apoptosis and regu-lated necrosis.43 Similar to other models of acute kidneyinjury,21,22 we did not detect ongoing apoptosis in NTNkidneys. Instead, levels of RIP1 and RIP3 proteins wereincreased in nephritic kidneys of memTNF mice comparedwith wild-type. In vivo, more abundant expression of thesemolecules is associated with induction of regulated necrosis,particularly necroptosis,25 which has an important pathogenicrole in acute kidney injury.44 This suggested enhanced nec-roptotic cell death in memTNF kidneys as a potentialmechanism of aggravated renal injury, which included more

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severe damage to the glomerular endothelium as demon-strated by significantly reduced CD31 expression. Consis-tently, our in vitro data confirmed that TNF-exposedglomerular endothelial cells in the presence of caspase inhi-bition can undergo necroptosis. Moreover, glomerularendothelial cells may be particularly vulnerable to memTNF/TNFR2-mediated cell death, potentially in an auto-stimulatory fashion, as we showed that these cells promi-nently express memTNF in GN and readily induce expressionof TNFR2 upon inflammatory stimulation in vitro,14 andduring NTN in vivo.13 Importantly, endothelial dysfunctionhas been recently recognized to play a critical role in thedevelopment and progression of glomerular disease.45–47

Thus, memTNF/TNFR2-mediated glomerular endothelialcell injury may be an important determinant of glomerularpathology in NTN, leading to podocyte stress, damage of theglomerular filtration barrier, albuminuria and glomerularscaring, which was more prominent in memTNF kidneyswith NTN.

In general, necrotic cell death and tissue damage releasedamage-associated molecular patterns (DAMPs) and triggerleukocyte influx and sterile inflammation leading to the self-perpetuating process of necroinflammation.26 Leukocyteaccumulation and inflammation usually correlate with theextent of tissue injury in affected organs including nephritickidneys with NTN.13,48 Paradoxically, memTNF kidneys withNTN displayed exacerbated tissue injury but significantlyreduced renal leukocyte infiltration and inflammation.Firstly, this suggested that aggravated memTNF-driven tissueinjury is independent of infiltrating leukocytes and increasedlocal inflammation and is mediated through mechanismsinvolving intrinsic renal cells. Our studies in BM-chimericmice confirmed that parenchymal cell- but not leukocyte-expressed memTNF aggravates renal injury. Moreover, ithas been shown that expression of TNF4 as well as TNFR213

in parenchymal renal cells predominantly contributes toNTN. Secondly, these data indicate that sTNF, which is ab-sent in memTNF mice, mediates leukocyte infiltration andinflammatory responses in nephritic kidneys. sTNF bindingto TNFR1 induces classic pro-inflammatory TNF effects viaNFkB activation, including expression of cytokines, chemo-kines and adhesion molecule that facilitate leukocyte infil-tration.1,2 Consistently, we demonstrated impaired activationof NFkB in nephritic memTNF kidneys, which explainsreduced renal expression of inflammatory mediators.Importantly, our in vitro experiments prove that absent sTNFsecretion in memTNF glomeruli reduced expression ofchemokines and cytokines, independently of altered numbersof infiltrating leukocytes in vivo. Lastly, reduced NFkB-mediated expression of cellular survival factors may enhancememTNF-induced cell death as discussed above. Of note,next to impaired TNFR1 signaling due to absent sTNF,binding of memTNF to TNFR2 may directly contribute toinhibition of pro-inflammatory NFkB activity in memTNFkidneys as TNFR2 activation can lead to degradation of theintracellular adapter molecule TRAF2, which is essential in

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Table 1 | Primers used for real-time quantitative polymerasechain reaction

Gene target Primer sequence

Nephrin 50-ACCCTCCAGTTAACTTGTCTTTGG-30 (forward),50-ATGCAGCGGAGCCTTTGA-30 (reverse)

L-FABP 50-AGGAGTGCGACCTGGAGACCAT-30 (forward),50-GCTTCCATTGAGTTCAGTCACGG-30 (reverse)

a-GST 50-CGCAGCACTGAATCCGCACC-30 (forward),50-ACACCGCCCTCGAACTGGGAA-30 (reverse)

CCL2 50-CCTGCTGTTCACAGTTGCC-30 (forward),50-ATTGGGATCATCTTGCTGGT-30 (reverse)

CCL5 50-CCACTTCTTCTCTGGGTTGG-30 (forward),50-GTGCCCACGTCAAGGAGTAT-30 (reverse)

CXCL10 50-GGCTGGTCACCTTTCAGAAG-30 (forward),50-ATGGATGGACAGCAGAGAGC-30 (reverse)

IL-1b 50-TTCCTTGTGCAAGTGTCTGAAG-30 (forward),50-CACTGTCAAAAGGTGGCATTT-30 (reverse)

IL-6 50-TGATGCACTTGCAGAAAACA-30 (forward),50-ACCAGAGGAAATTTTCAATAGGC-30 (reverse)

IL-10 50-ATCGATTTCTCCCCTGTGAA-30 (forward),50-TGTCAAATTCATTCATGGCCT-30 (reverse)

IFN-b 50-CTCAGGGTGTCGATGAGGTC-30 (forward),50-CCCAGTGCTGGAGAAATTGT-30 (reverse)

IFN-g 50-ACAGCAAGGCGAAAAAGGAT-30 (forward),50-TGAGCTCATTGAATGCTTGG-30 (reverse)

TNF 50-CCACCACGCTCTTCTGTCTAC-30 (forward),50-AGGGTCTGGGCCATAGAACT-30 (reverse)

TNFR1 50-GCAACAGCACCGCAGGAGCTGA-30 (forward),50-GTGCGTCCCTTGCAGCCACT-30 (reverse)

TNFR2 50-CAAGACAACCTGGGTGGGCA-30 (forward),50-CTGGGTCGCGCTGGTCTTGC-30 (reverse)

E-selectin 50-CCATGTGCCTTCTTACAACG-30 (forward),50-TTAAGCAGGCAAGAGGAACC-30 (reverse)

ICAM-1 50-GTCACCGTTGTGATCCCTG-30 (forward),50-AACAGTTCACCTGCACGGAC-30 (reverse)

VCAM-1 50-GCTATGAGGATGGAAGACTCTGG-30 (forward),50-ACTTGTGCAGCCACCTGAGATC-30 (reverse)

IkB-a 50-CAGCAGCTCACGGAGGAC-30 (forward),50-GTCTCCCTTCACCTGACCAA-30 (reverse)

TNFAIP3/A20 50-CAGAAGGAGGTCTGTCTGGA-30 (forward),50-TGCATTCCGCTTAGCTTTCT-30 (reverse)

cIAP1 50-TGATGGTGGCTTCAGATG-30 (forward),50-CCCTCCATCCGTATCAAG-30 (reverse)

XIAP 50-CATCCGGGAGCAGCTATCTA-30 (forward),50-TCTTGTAGGCGCCTTAGCTG-30 (reverse)

18S rRNA 50-GCAATTATTCCCCATGAACG-30 (forward),50-AGGGCCTCACTAAACCATCC-30 (reverse)

a-GST, a-glutathione S-transferase; CCL, C-C motif chemokine ligand; CCR, C-Cchemokine receptor; cIAP, cellular inhibitor of apoptosis protein; CXCL, C-X-C motifchemokine ligand; ICAM, intercellular adhesion molecule; IFN, interferon; IkB, in-hibitor of NFkB; IL, interleukin; L-FABP, liver-type fatty acid binding protein; qRT-PCR,quantitative reverse transcriptase-PCR; TNF, tumor necrosis factor-a; TNFAIP, TNF-a-induced protein; TNFR, tumor necrosis factor receptor; VCAM, vascular cell adhesionmolecule; XIAP, X-linked inhibitor of apoptosis protein.

bas i c re sea r ch MB Müller et al.: Transmembrane TNF in GN

inducing canonical NFkB responses upon TNF receptorligation.41

In conclusion, we show that parenchymal expression ofmemTNF increases renal cell death and aggravates renalinjury in NTN, despite reduced renal leukocyte influx andinflammation due to absent sTNF. Our results indicate thatuncleaved transmembrane TNF plays an important patho-physiologic role in GN. Therapeutic targeting of specificallythe memTNF/TNFR2 axis49 may be more beneficial thanunspecific TNF blockade preserving TNF-mediated immu-noregulative effects and antimicrobial functions.

MATERIALS AND METHODSExpression of TNF mRNA and protein in humanglomerulopathiesHuman renal biopsies from patients and controls were collectedwithin the framework of the European Renal cDNA Bank - Kröner-Fresenius Biopsy Bank.20 Diagnostic renal biopsies were obtainedfrom patients after informed consent and with approval of the localethics committees. Glomeruli were microdissected, total RNA iso-lated, linearly amplified, and hybridized to Affymetrix HG-U133Aand HG-U133 Plus 2.0 microarrays (Affymetrix, Santa Clara, CA)as reported previously.50 Pre-transplantation kidney biopsies fromliving donors were used as control tissue. Fragmentation, hybridi-zation, staining, and imaging were performed according to theAffymetrix expression analysis technical manual (Affymetrix). Theraw data were normalized using robust multi-array average andannotated by Human Entrez Gene custom CDF annotation version18 (http://brainarray.mbni.med.umich.edu/Brainarray/default.asp).The log-transformed dataset was corrected for batch effect usingComBat from the GenePattern pipeline (http://www.broadinstitute.org/cancer/software/genepattern/).51 To identify differentiallyexpressed genes the SAM (significance analysis of microarrays)method was applied using TiGR (MeV, Version 4.8.1).52 A q-valuebelow 5% was considered to be statistically significant. Paraffinsections of human kidney biopsies were stained with 1:120 rabbitpolyclonal anti-TNFa antibody (ab183896; Abcam, Cambridge,UK). For detection of TNF protein in murine kidney sections apolyclonal goat anti-mouse TNFa antibody (AF-410-NA, 1:200;R&D Systems, Nordenstadt, Germany) was used.

MiceTransgenic memTNFD1-9,K11E mice with knock-in of 2 uncleavableDelta1-9,K11E TNF alleles on the C57BL/6J background have beendescribed previously19 and were originally provided by BernhardRyffel (University of Orléans, France). All experiments were per-formed in 8- to 10-week-old male memTNF mice and wild-typelittermate controls. Mice were maintained under specific pathogen-free conditions. All experimental procedures were performed ac-cording to the German animal care and ethics legislation and wereapproved by the local government authorities.

BM transplantationWe generated memTNF BM chimeric mice by transplantingmemTNF BM into lethally irradiated wild-type recipients(memTNF/WT chimeras) or vice versa (WT/memTNF chi-meras). WT/WT and memTNF/memTNF chimeras served ascontrols. Irradiation and BM reconstitution was carried out asdescribed.53 To assess the efficiency of BM replacement, CD45.1congenic C57BL/6J wild-type mice were used to distinguish

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CD45.2þ memTNF leukocytes from CD45.1þ wild-type leukocytesby flow cytometry. Fow cytometry of peripheral blood samples,spleens, and kidneys at the end of the experiment revealed that morethan 95% of leukocytes in the chimeras were of donor origin.

Induction of heterologous NTNHeterologous NTN was induced in male mice by i.v. tail vein in-jection of 100 ml of sheep nephrotoxic serum (Probetex, San Anto-nio, TX).5 Spot urine samples were taken at days 0 and 5 of theexperiment. At day 5 mice were killed, peripheral blood was collectedby retro-orbital bleeding and phosphate-buffered saline–perfused

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kidneys were harvested for further analysis. Four mice of each groupwere analyzed 2 hours after injection of nephrotoxic serum.

Functional assessment of renal injuryAlbuminuria in spot urine samples was determined as described.13

Serum urea levels were measured with an Olympus AU-640 auto-analyzer at Synlab.vet (Augsburg, Germany).

Histological evaluation of renal injuryRenal injury was assessed on 2-mm formalin-fixed, paraffin-embedded renal sections stained with periodic acid-Schiff reagent.Fifty glomeruli per mouse were analyzed, applying a semi-quantitative score for glomerular matrix deposition and sclerosis asdescribed.54 Tubulointerstitial injury was quantified by superposing agrid containing 100 sampling points on images of 20 periodic acid-Schiff–stained cortical high power fields (400x) as described.55 Pointsoverlaying tubular spaces, casts, flattened and necrotic tubular cells,and vacuolar degenerated tubuli were counted. Renal cell death wasdetected with an in situ cell death detection (TUNEL) kit (Roche,Mannheim, Germany). For immunostaining applying standardtechniques48 the following primary antibodies were used: mono-clonal mouse anti-PCNA (clone PC-10; Cell Signaling Technology,Danvers, MA), polyclonal rat anti-WT1 (Santa Cruz Biotechnology,Dallas, TX), polyclonal hamster anti-nephrin (Acris Antibodies,Herford, Germany), monoclonal rat anti-CD31 (clone RM0032-1D12; Abcam), polyclonal goat-anti sheep IgG (Vector Labora-tories, Burlingame, CA), and polyclonal goat anti-C3d (Cederlane,Ontario, Canada). Stained cells were quantified in 20 glomeruli and20 high-power fields (�400) per mouse. Glomerular deposition ofsheep IgG and complement C3d was quantified as the fraction ofstained glomerular area using ImageJ (National Institutes of Health,Bethesda, MD). All assessments were performed in a blindedprotocol.

Analysis of renal leukocyte infiltration by flow cytometry andimmunohistochemistryQuantitative assessment of renal leukocyte infiltration by flowcytometry was performed as described.14 For immunohistochem-istry, 2-mm paraffin-embedded renal sections were stained with thefollowing primary antibodies: rat anti-CD3 (clone CD3-12), rat anti-F4/80 (clone Cl:A3-1), and rat anti-Mac 2 (clone M3/38; all fromAbD Serotec, Oxford, UK). Leukocyte infiltration was analyzed in 50glomeruli and 20 cortical high-power fields (�400) per mouse.

Quantitative real-time polymerase chain reactionTotal RNAwas extracted from whole kidneys using the Purelink RNAMini Kit (Thermo Fisher Scientific, Waltham, MA). SYBRGreenmaster mix (Thermo Fisher Scientific) were used to performquantitative real-time polymerase chain reaction on a Light Cycler480 (Roche, Mannheim, Germany). Primers used for amplification(Metabion, Martinsried, Germany) are listed in Table 1. All sampleswere run in duplicates and normalized to 18s rRNA.

ImmunoblottingFor Western blotting the following antibodies were used: rabbitpolyclonal anti-cleaved caspase 3 (Cell Signaling Technology), mouseanti-RIP1 (clone 7H10, Abcam), polyclonal rabbit anti-RIP3(Abcam), rabbit anti-phospho-IkB-a (clone 14D4, Cell SignalingTechnology), and rabbit anti-NF-kB2 p100/p52 (clone 18D10, CellSignaling Technology). b-actin (rabbit polyclonal serum; CellSignaling Technology) levels were measured as loading control.

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Protein bands were visualized using a chemiluminescence kit(Amersham ECL Prime; GE Healthcare, Little Chalfont, UK).Densitometry analysis was performed with ImageJ software.

Cell culture studiesA murine glomerular endothelial cell line14 was grown in Dulbecco’smodified Eagle’s medium with GlutaMax (Thermo Fisher Scientific)supplemented with 10% fetal calf serum (FCS; Biochrom, Berlin,Germany), 100 U/ml penicillin, and 100 mg/ml streptomycin (PAALaboratories, Pasching, Austria). After transfer to 96-well plates,10,000 cells/well were incubated for 6 hours in vehicle control(dimethylsulfoxide) or 300 ng/ml recombinant murine TNF(Thermo Fisher Scientific) and 10 mM Z-VAD-FMK (InvivoGen, SanDiego, CA) to induce cell death in the presence or absence of 100 mMof the necroptosis inhibitor necrostatin-1s (BioVision, Milpitas, CA)as indicated. The CellTiter 96 nonradioactive cell proliferation assay(MTT) kit (Promega, Mannheim, Germany) was used to evaluatecell survival following the manufacturer’s instructions. Results wereexpressed as percentage of the vehicle control.

Isolation and stimulation of glomeruliParamagnetic isolation of glomeruli from healthy wild-type andmemTNF mice was performed as described previously.14 Then 5000intact glomeruli were resuspended in 3 ml Roswell Park MemorialInstitute medium (RPMI) 1640 supplemented with 15% FCS andwere incubated in 6-well plates at 37 �C for 24 hours. After serumstarving for an additional 24 hours, glomeruli were stimulated with10 ng/ml IL-1b (Thermo Fisher Scientific) in RPMI 1640 for 24hours. Supernatant was collected for protein measurements usingcommercially available enzyme-linked immunosorbent assay kits forTNF (BioLegend, San Diego, CA), CCL2 (R&D Systems), and IL-6(R&D Systems). Cells were lysed and used for RNA extraction asdescribed above.

Statistical analysisResults are presented as mean � SEM as indicated. Differences be-tween 2 individual experimental groups were compared using 2-tailed Student’s t-test. Comparisons of multiple groups were per-formed with Kruskal-Wallis test followed by pairwise Mann-WhitneyU tests. Survival curves were compared by log-rank test. P < 0.05was considered to be statistically significant.

DISCLOSUREAll the authors declared no competing interests.

ACKNOWLEDGMENTSThe expert technical assistance of Dan Draganovici and JanaMandelbaum is gratefully acknowledged. This study was supportedby grants from the Faculty of Medicine, Ludwig-Maximilians-Universität München to MBM and VV. Portions of this work wereprepared by MBM as part of his doctoral thesis at the Faculty ofMedicine, Ludwig-Maximilians-Universität München.

SUPPLEMENTARY MATERIALFigure S1. Tubulointerstitial injury in wild-type (WT) and memTNFmice with heterologous nephrotoxic serum nephritis (NTN). Repre-sentative photomicrographs of periodic acid–Schiff (PAS) –stainedkidney sections from wild-type and memTNF mice illustrating tubu-lointerstitial injury at day 5 of NTN. Semiquantitative scoring oftubular dilation, intratubular casts, flattened and necrotic tubularcells, and tubuli with vacuolar degeneration was performed as

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described in Material and Methods. Arrows indicate respectivepathological lesions. Original magnification �400, bar ¼ 50 mm. Datarepresent mean � SEM of 6 mice per group; n.s., not significant.Figure S2. Renal leukocyte infiltration in wild-type (WT) and memTNFbone marrow (BM)-chimeric mice with heterologous nephrotoxicserum nephritis (NTN). Representative renal sections of chimeric micewith memTNF BM (memTNF/WT) or memTNF expressing paren-chymal cells (WT/memTNF) at day 5 of NTN. WT mice transplantedwith WT BM (WT/WT chimeras) and memTNF mice transplantedwith memTNF BM (memTNF/memTNF) served as controls. Tissuewas stained for Ly6Gþ neutrophils, Mac2þ glomerular macrophages,F4/80þ tubulointerstitial phagocytes, and CD3þ T cells. Comparedwith WT/WT chimeras, renal accumulation of neutrophils, glomer-ular macrophages, interstitial phagocytes, and interstitial T cells wassignificantly reduced in memTNF/memTNF chimeras only. Incontrast, glomerular and interstitial neutrophil numbers increased inmemTNF/WT and WT/memTNF chimeras, respectively. Originalmagnification �400, bar ¼ 50 mm. Data represent mean � SEM of 5mice per group. *P < 0.05, **P < 0.01.Supplementary material is linked to the online version of the paper atwww.kidney-international.org.

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