TNFR2-mediated apoptosis and necrosis in cisplatin-induced acute renal failure - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:47 |只看该作者 |倒序浏览 |打印

作者：Ganesan Ramesh and W. Brian Reeves作者单位：Division of Nephrology, The Pennsylvania State College of Medicine,Hershey 1703 and Lebanon Veterans Affairs Medical Center, Lebanon,Pennsylvania 17042
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      【摘要】2 c6 j' B* I! v# d; a
   Cisplatin produces acute renal failure in humans and mice. Previous studieshave shown that cisplatin upregulates the expression of TNF- in mousekidney and that inhibition of either the release or action of TNF- protects the kidney from cisplatin-induced nephrotoxicity. In this study, weexamined the effect of cisplatin on the expression of TNF receptors TNFR1 andTNFR2 in the kidney and the role of each receptor in mediating cisplatinnephrotoxicity. Injection of cisplatin into C57BL/6 mice led to anupregulation of TNFR1 and TNFR2 mRNA levels in the kidney. The upregulation ofTNFR2 but not TNFR1 was blunted in TNF- -deficient mice, indicatingligand-dependent upregulation of TNFR2. To study the roles of each receptor,we administered cisplatin to TNFR1- or TNFR2-deficient mice. TNFR2-deficientmice developed less severe renal dysfunction and showed reduced necrosis andapoptosis and leukocyte infiltration into the kidney compared with either TNFR1-deficient or wild-type mice. Moreover, renal TNF- expression, ICAM-1 expression, and serum TNF- levels were lower in TNFR2-deficient mice compared with wild-type or TNFR1-deficient mice treated with cisplatin.These results indicate that TNFR2 participates in cisplatin-induced renalinjury in mice and may play an important role in TNF- -mediatedinflammation in the kidney in response to cisplatin.
      【关键词】 acute tubular necrosis gene expression tumor necrosis factor tumor necrosis factor receptor cytokines
   CISPLATIN IS A HIGHLY EFFECTIVE antitumor agent used to treat awide variety of malignancies( 28 ). The key limitation of this chemotherapeutic agent is nephrotoxicity. Approximately 25-35% ofpatients experience a significant decline in renal function after a singledose of cisplatin ( 45 ). Recent evidence indicates that inflammatory mechanisms play an important role in thepathogenesis of cisplatin-induced renal injury. Specifically, severallaboratories have demonstrated that TNF- pathways are activated incisplatin injury ( 15, 23, 44 ). Indeed, TNF- is acentral figure in a large network of chemokines and cytokines expressed in thekidney after cisplatin injection ( 44 ). Moreover, inhibition ofeither TNF- production or its activity ameliorated cisplatin-inducedrenal dysfunction and reduced cisplatin-induced structural damage( 44 ). However, the pathwaythrough which TNF- mediates its toxicity in cisplatin injury is notknown.
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TNF- is a potent proinflammatory cytokine that plays important rolesin chronic inflammation and autoimmune diseases, such as rheumatoid arthritis,autoimmune diabetes, and multiple sclerosis( 2, 10 ). The biological activitiesof TNF- are mediated by two functionally distinct receptors, TNFR1(p55) and TNFR2 (p75). Many of the cytotoxic and proinflammatory actions ofTNF- are mediated by TNFR1( 7, 32 ). TNFR1-deficient mice areresistant to endotoxic shock and show abrogated induction of adhesionmolecules by TNF- ( 38, 46 ). In contrast,TNFR2-deficient mice exhibit only subtle defects( 18 ), and the role of TNFR2 indisease is unclear ( 9, 11 ). TNFR2 is thought tocooperate with TNFR1 by passing ligands to TNFR1( 53 ) or formingheterocomplexes with TNFR1( 40 ). However, the engagementof TNFR2 by TNF- also leads to TNFR1-independent cellular events,including apoptosis of activated T cells( 39, 57 ), thymocyte proliferation ( 52 ), and inhibition of earlyhematopoiesis ( 55 ). In this study, we have used TNFR1-, TNFR2-, and TNF- -deficient mice to examinethe TNF- receptor subtype that mediates cisplatin-induced renal injuryand the regulation of their expression in response to cisplatin. Our resultsindicate that TNFR2 expression in the kidney is regulated in aligand-dependent manner and participates in both necrosis and apoptosis ofrenal epithelial cells in cisplatin nephrotoxicity.2 y& v, I# C5 q0 w# G$ @- v: L

MATERIALS AND METHODS" g, v2 G* ?/ P& k

Animals and drug administration. Experiments were performed on 10-to 12-wk-old male C57BL/6, TNFR1 (C57BL/6-Tnfrs1a tm1mak )-, TNFR2(C57BL/6-Tnfrs1b tm1Mwm )-, or TNF- (B6;129-Tnf tm1Gkl )-deficient mice weighing 30 g. Mice were obtained from JacksonLaboratory (Bar Harbor, ME) and maintained on a standard diet, and water wasfreely available. PCR genotyping was performed on selected animals from eachstrain to confirm the correct genotype. Cisplatin (Sigma-Aldrich, St. Louis,MO) was dissolved in saline at a concentration of 1 mg/ml. Mice were given asingle intraperitoneal injection of either vehicle (saline) or cisplatin (20mg/kg body wt). This dose of cisplatin produces severe renal failure in mice( 44 ). Animals were killed 72 hafter cisplatin injection, and blood and kidney tissues were collected.

Renal function. Renal function was assessed by measurement of ureanitrogen in the serum using a commercially available kit.) M* d9 g' w/ T# S) j
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Quantitation of mRNA by real-time RT-PCR. Total RNA was isolated from kidneys using the TRIzol reagent. Real-time RT-PCR was performed usingthe Applied Biosystems 7700 Sequence Detection System. Total RNA (5 µg) wasreverse transcribed in a reaction volume of 20 µl using Superscript IIreverse transcriptase and random primers. The product was diluted to a volumeof 500 µl, and either 2 (actin)- or 10-µl (all others) aliquots wereused as templates for amplification using the SYBR green PCR amplificationreagent (PE Biosystems, Foster City, CA) and gene-specific primers. The primersets used were actin (forward: CATGGATGACGATATCGCT; reverse:CATGAGGTAGTCTGTCAGGT); TNF- (forward: GCATGATCCGCGACGTGGAA; reverse:AGATCCATGCCGTTG GCCAG); TNFR1 (forward: CCGGGCCACCTGGTCCG; reverse:CAAGTAGGTTCCTTTGTG); TNFR2 (forward: GTCGCGCTGGTCTTCGAACTG; reverse:GGTATACATGCTTGCCTCACAGTC); and ICAM-1 (forward: AGATCACATTCACGGTGCTG; reverse:CTTCAGAGGCAGGAAACAGG).) ]# w( |0 h  |8 b& A

TNF- and soluble TNFR2 quantitation by ELISA. Levels of TNF- and soluble TNFR2 in serum were determined using anELISA assay (Quantikine Mouse TNF- and Quantikine Mouse sTNFRII kits,R&D Systems, Minneapolis, MN) according to the manufacturer'sinstructions.
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Western blot analysis. Kidneys were homogenized in PBS, and theprotein concentration was quantitated (BCA protein assay reagent, Pierce,Rockford, IL). Samples of protein (100 µg) were separated by 10% SDS-PAGEand then transferred onto a polyvinylidene difluoride membrane. Western blotanalysis was performed with an anti-TNFR2 antibody (1: 1,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA). Proteins were detected using enhancedchemiluminescence detection reagents (Amersham Pharmacia Biotech, Piscataway,NJ).
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Histology and immunohistochemistry. Kidney tissue was fixed inbuffered formalin for 12 h and then embedded in paraffin wax. Sections (5µm) were stained with periodic acid-Schiff (PAS) or naphthol AS-Dchloroacetate esterase (Sigma kit 91A). The esterase stain identifiesinfiltrating neutrophils and monocytes. Thirty x 40 fields ofesterase-stained sections of kidney cortex were examined for quantitation ofleukocytes. Tubular injury was assessed in PAS-stained sections using a semiquantitative scale ( 23, 26, 43 ) in which the percentage ofcortical tubules showing epithelial necrosis was assigned a score: 0 = normal;1 = 75%. Apoptosiswas scored by counting the number of apoptotic cells, as defined by chromatincondensation or nuclear fragmentation (apoptotic bodies), on PAS-stainedsections of cortex. The individual scoring the slides was blinded to thetreatment and strain of the animal.0 i! T4 `5 G6 a' a/ G8 w
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Statistical methods. All assays were performed in duplicate. Thedata are reported as means ± SE. Statistical significance was assessedby an unpaired, two-tailed Student t -test for single comparison orANOVA for multiple comparisons.8 i8 y0 c7 S/ G4 O& f) g3 A
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RESULTS: r1 R  F: ]& L" E( w, [
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Cisplatin upregulates TNFR2 in a ligand-dependent manner. TNFR1 iswidely expressed relative to the more restricted distribution of TNFR2( 17 ). The expression of TNFR1and, in particular, TNFR2, which has NF- B and other transcriptionfactor binding sites in its promoter( 47 ), can be altered indisease states. Therefore, we studied the expression of TNFR1 and TNFR2 mRNA in response to cisplatin injection. As shown in Fig. 1, cisplatin increased theexpression of TNFR1 about threefold and increased TNFR2 expression six- toninefold over saline-treated controls in either C57BL/6 or B6;129J mice. Toexamine whether the upregulation of either TNFR1 or TNFR2 is TNF- dependent, receptor mRNA was quantified in cisplatin-treatedTNF- -deficient mice. The expression of TNFR1 was unaltered between thewild-type mice treated with cisplatin and TNF- -deficient mice( Fig. 1 ). However, theupregulation of TNFR2 mRNA was blunted in TNF- -deficient mice. We alsoexamined the effect of cisplatin on kidney TNFR2 protein levels( Fig. 2 ). As shown in Fig. 2 ( top ), kidneyTNFR2 protein levels were increased by cisplatin, and this increase wasblunted in TNF- -deficient mice. These results indicate ligand-dependent regulation of TNFR2 in cisplatin nephrotoxicity. As shown in Fig. 2 ( bottom ), TNFR2protein was measured in C57BL/6 and TNFR1-deficient and TNFR2-deficient mice.Little or no TNFR2 was detectable in kidneys of saline-treated mice.Consistent with the above results, cisplatin produced a marked increase inTNFR2 content in C57BL/6 mice. Moreover, a similar increase occurred inTNFR1-deficient mice. As expected, no TNFR2 protein was present in theTNFR2-deficient kidneys. These results confirm that TNFR2 expression isincreased in cisplatin-treated kidneys and indicate that the upregulation isnot mediated via TNFR1.
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Fig. 1. Regulation of renal tumor necrosis factor (TNF) receptor (TNFR1 and TNFR2)expression by cisplatin. C57BL/6, B6;129J, or TNF- -deficient (TNF-KO)mice were injected with cisplatin (20 mg/kg body wt). Kidneys were harvested72 h after injection, and levels of TNFR1 (hatched bars) and TNFR2 (solidbars) mRNA were determined by RT-PCR as described in MATERIALS AND METHODS. Cisplatin increased the levels of both TNFR1 and TNFR2 mRNA.The increase in TNFR2 mRNA was blunted in TNF- -deficient mice.* P n =3-4/group).; \1 R! b/ F. K

Fig. 2. Regulation of TNFR2 expression by cisplatin. Mice from each indicatedstrain were injected with saline or cisplatin and killed after 72 h. Thecontent of TNFR2 in kidneys was determined by Western blot analysis. Top : cisplatin treatment increased the TNFR2 protein content inB6;129 mice. This increase was blunted in TNF-KO mice. Bottom :cisplatin increased TNFR2 protein content in both C57BL/6 and TNFR1-deficient(TNFR1-KO) mice.

Serum levels of soluble TNFR2 were measured in wild-type mice and in micedeficient in either TNF-, TNFR1, or TNFR2( Fig. 3 ). Treatment of micewith cisplatin increased soluble TNFR2 levels in wild-type mice (eitherB6;129J or C57BL/6 strains) and TNFR1-deficient mice. TNF- -deficientmice had lower levels of soluble TNFR2 in the presence or absence ofcisplatin. As expected, TNFR2-deficient mice had undetectable levels ofsoluble TNFR2. These results are also consistent with TNF- -dependent regulation of TNFR2 expression.

Fig. 3. Effect of cisplatin injection on soluble TNFR2 in serum. Mice from eachindicated strain were injected with either saline or cisplatin (20 mg/kg).Serum was obtained 72 h after injection for measurement of soluble TNFR2protein. ND, not detectable. Cisplatin increased soluble TNFR2 levels in bothB6;129J and C57BL/6 mice (  P n =3-8). Soluble TNFR2 levels were lower in TNF-KO mice than in B6;129Jwild-type mice (* P n = 4).# y# E8 q* V( C' M0 r

Cisplatin upregulates TNF- expression inTNFR1-deficient but not in TNFR2-deficient mice. Cisplatin increaseskidney TNF- expression and serum TNF- levels( 15, 23, 44 ). To determine whether thisupregulation is mediated through TNF- receptors, we measuredTNF- mRNA in kidney and TNF- protein in the serum of TNFR1- orTNFR2-deficient mice after cisplatin injection. As shown in Fig. 4 A,TNFR1-deficient mice showed a similar increase in kidney TNF- mRNA asseen in wild-type mice treated with cisplatin. However, the increase wasblunted significantly in TNFR2-deficient mice. Similarly, serum TNF- protein levels ( Fig.4 B ) were also significantly lower in TNFR2-deficient micethan in wild-type or TNFR1-deficient mice, suggesting TNFR2-dependentregulation of TNF- production in the kidney in response tocisplatin.* d/ K5 R2 ~, N# I

Fig. 4. Effect of cisplatin on renal TNF- mRNA and serum TNF- proteinlevels. Mice from the indicated strains were injected with either saline (openbars) or cisplatin (filled bars). Kidney and blood were obtained 72 h afterinjection for measurement of TNF- mRNA ( A ) and TNF- protein ( B ). Cisplatin caused marked elevations in renal TNF- mRNA and serum TNF- levels in C57BL/6 and TNFR1-KO mice. The levelswere significantly lower in TNFR2-deficient (TNFR2-KO) mice. TNF- mRNAlevels are expressed relative to the levels in saline-injected mice.* P n = 3 for mRNAand n = 8 for protein measurements).2 f) Q5 T. r7 p: e# g) i; s& M

TNFR2-deficient mice are resistant to cisplatin nephrotoxicity. Toaddress the role of TNFR1 and TNFR2 in the pathogenesis of cisplatin-inducedacute renal failure, we examined cisplatin nephrotoxicity in mice withtargeted deletions of either TNFR1 or TNFR2. As shown in Fig. 5, wild-type andTNFR1-deficient mice developed severe renal failure after injection withcisplatin [72-h urea = 135 ± 7 and 161 ± 14 mg/dl for wild-type ( n = 17) and TNFR1-deficient mice ( n = 15), respectively, P = not significant (NS)]. In contrast, TNFR2-deficient mice had better preservation of function (72-h urea = 88 ± 8 mg/dl, n =16, P = 0.015 vs. wild-type). The histological findings also revealedless severe damage in the TNFR2-deficient mice. As shown in Fig. 6, cisplatin treatment ofwild-type and TNFR1-deficient mice resulted in severe tubular injury asreflected by cast formation, loss of brush-border membranes, sloughing oftubular epithelial cells, and dilation of tubules. This injury was present throughout the cortex and outer medulla. These changes were minimal in kidneysfrom TNFR2-deficient mice treated with cisplatin.
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Fig. 5. Role of TNFR1 and TNFR2 in cisplatin nephrotoxicity. Mice from each strainwere injected with saline or cisplatin (CP). Blood urea nitrogen was measuredat the indicated times after injection as a measure of renal function.Cisplatin caused marked elevations of urea in the C57BL/6 and TNFR1-KO mice.TNFR2-KO mice had significantly lower urea levels. * P n = 5-7/group).0 d' W, S6 K' N) b  B

Fig. 6. Kidney histology after cisplatin injection. C57BL/6 ( A and D ), TNFR1-KO ( B and E ), or TNFR2-KO ( C and F ) mice were killed 72 h after cisplatin injection. Kidney cortexfrom C57BL/6 and TNFR1-KO mice showed extensive histological damage, such astubular dilation, cast formation (*) and necrosis, and sloughing of renalepithelial cells (arrowheads). Kidneys from TNFR2-KO mice had better preservedmorphology. Magnification: x 10 ( A - C ); x 40( D - F ).9 N$ A- o. {5 s; C) O% l9 w6 @

TNFR2 induces apoptosis and necrosis in cisplatin nephrotoxicity. Cisplatin produces both necrosis and apoptosis of renal epithelial cells invitro ( 31, 37 ). The contribution ofnecrosis and apoptosis to cisplatin injury in vivo is less clear. Moreover, the contribution of TNFR2 signaling to apoptosis in vivo is not wellestablished. Therefore, we quantitated the extent of cisplatin-inducednecrosis and apoptosis in vivo and determined the role of TNF- andTNFR1 and TNFR2 in both processes. The results in Fig. 7 show that cisplatintreatment resulted in both necrosis and apoptosis in vivo. Deletion ofTNF- resulted in a decrease in both necrosis and apoptosis. Deletion ofTNFR2 also reduced apoptosis and necrosis, although to a lesser extent thandid TNF- deletion. Deletion of TNFR1 resulted in a slight reduction inhistological necrosis but no decrease in apoptosis.

Fig. 7. Role of TNFR2 in cisplatin-induced necrosis and apoptosis. Mice from eachindicated strain were injected with either saline or cisplatin and killed 72 hlater. Kidneys were harvested and processed for light microscopy. Tubularnecrosis ( A ) and apoptosis ( B ) were measured using asemiquantitative scoring method. Cisplatin injection produced a large increasein necrosis and apoptosis in B6;129J, C57BL/6, and TNFR1-KO mice. TNF-KO andTNFR2-KO mice sustained less tubular necrosis and less apoptosis than thecorresponding wild-type mice. TNFR1-KO mice had less tubular necrosis than butapoptosis equivalent to that in C57BL/6 mice. * P P P P

TNFR2 induces leukocyte infiltration and ICAM-1 expression. Cisplatin nephrotoxicity is associated with the infiltration of leukocytesinto the kidney and up-regulation of ICAM-1 ( 15, 23, 44 ). Inhibition of TNF- ( 44 ) or ICAM-1( 23 ) reduces leukocyteinfiltration and also lessens cisplatin nephrotoxicity. Accordingly, weexamined the dependence of leukocyte infiltration and ICAM-1 expression on thepresence of TNFR1 and TNFR2. Leukocyte infiltration was measured using thenaphthol AS-D chloroacetate esterase stain. As shown in Fig. 8, in either C57BL/6 orTNFR1-deficient mice, cisplatin injection produced a large increase inleukocytes within the kidney cortex. In contrast, TNFR2 knockout mice had little or no increase in leukocytes.+ t1 {" u2 p1 P  @

Fig. 8. Role of TNFR1 and TNFR2 in renal neutrophil infiltration. Neutrophils andmonocytes were counted in sections of kidney cortex stained with naphol AS-Dchloroacetate esterase. Cisplatin-treated C57BL/6 and TNFR1-KO mice had alarge number of infiltrating leukocytes. Kidneys from TNFR2-KO mice had fewerinfiltrating leukocytes. * P n = 5).
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A similar pattern was observed for ICAM-1 expression( Fig. 9 ). Namely, cisplatinincreased ICAM-1 expression in wild-type and TNFR1-deficient mice six- toninefold over saline-treated control mice. In contrast, ICAM-1 expression wassignificantly blunted in TNFR2-deficient mice compared with wild-type mice ( n = 4, P+ R- g. T% n6 q9 R, u

Fig. 9. Role of TNFR2 in renal ICAM-1 expression. ICAM-1 mRNA was measured inkidneys from cisplatin-treated C57BL/6, TNFR1-KO, and TNFR2-KO mice usingquantitative RT-PCR. ICAM-1 expression was markedly increased in C57BL/6 mice.Significantly less ICAM-1 expression was seen in TNFR2-KO mice. * P n = 3).3 k- {& ?; K2 M! S2 j+ g: _

DISCUSSION

TNF- is a highly pleiotrophic cytokine that plays a role in immuneinflammatory response. Intracellular signaling through TNF receptors may leadto apoptosis, cell activation, and/or cell proliferation. Whereas TNFR1signaling is clearly involved in a number of pathological states( 41 ), the role of TNFR2 inorgan pathology is not widely established. Recently, a role for TNF- intoxic and ischemic acute renal failure has been recognized( 14, 16, 26, 44, 51 ). The mechanisms whereby TNF- mediates acute renal failure are not clear. We used a clinically relevant model of acute renal failure, cisplatin nephrotoxicity, toinvestigate the TNF- signaling pathways during acute renal injury.Several results are noteworthy.
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First, we found that the expressions of both TNFR1 and TNFR2 areupregulated after cisplatin injection. This upregulation may serve tosensitize the kidney to the effects of TNF-. In this regard, serumlevels of TNF- in cisplatin nephrotoxicity, although increased( 44 ), are lower than thosethat occur in some other disorders mediated by TNF-, such as sepsis. Adissociation between serum TNF- levels and TNFR2 expression has beennoted in other settings ( 1 ),possibly reflecting a need for high levels of TNFR2 expression to mediatebiological actions. In addition, elegant studies by Douni and Kollias( 17 ) and Akassoglou et al.( 3 ) demonstrated that highlevels of TNFR2 expression can mediate inflammation in a ligand-independentfashion. We did not directly test for ligand-independent actions of TNFR2 incisplatin nephrotoxicity. However, the observation that deletion of eitherTNF- ( 44 ) or TNFR2( Fig. 5 ) results in similardegrees of protection suggests that ligand-independent actions of TNFR2 do notplay a major role. Upregulation of kidney TNF receptors has also been reportedin kidney transplant rejection( 4 ). Al-Lamki et al.( 4 ) found relatively little expression of TNFR1 and TNFR2 in normal human kidney allografts. During acutetransplant rejection, however, the expression of TNFR1 and TNFR2 was increasedin areas of lymphocytic infiltration. TNFR2 expression and/or serum levels ofsoluble TNFR2 are also elevated in a number of inflammatory conditions,including inflammatory bowel diseases( 35 ), sepsis( 48 ), acute respiratory distress syndrome ( 33 ), andcerebral malaria ( 33 ).0 z. S  d  n  n" E

We determined that the upregulation of TNFR2 was dependent, in part, onTNF-. TNF-, via receptor-interacting protein (RIP), activatesI B kinase and subsequent NF- B transcriptional activity ( 22 ). Because the promoter ofTNFR2 contains NF- B binding sites ( 47 ), this pathway is aplausible mechanism to account for the observed TNF- -dependentupregulation of TNFR2. Similarly, the expression of TNF- was dependenton TNFR2. TNFR2 activation can result in NF- B activation( 34 ). The presence ofNF- B binding sites within the TNF- promoter( 56 ), then, provides amechanism whereby TNF- can stimulate its own production via TNFR2. The mechanism for the TNF- -independent upregulation of both TNFR1 and TNFR2is unknown. We have found that cisplatin activates multiple signaling pathwaysin the kidney, including ERK, JNK, and p38 (preliminary data). It is possiblethat one or more of these pathways may influence TNF receptor expression.Finally, the actions of TNF- in a heterogenous organ like the kidney are likely determined by both the quantity and spatial distribution of TNFreceptors. Accordingly, it will be informative to determine the specific sitesof TNFR1 and TNFR2 expression in cisplatin nephrotoxicity.
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Second, cisplatin-induced tissue injury is mediated, at least in part, viaTNFR2. In many pathological states, including some kidney diseases (seebelow), the actions of TNF- are mediated through TNFR1. In othersettings, TNFR2 may contribute to tissue injury by enhancing TNFR1-mediatedtoxicity ( 40, 53, 55 ). In contrast, we foundthat TNFR2, independently of TNFR1, was responsible for cisplatinnephrotoxicity. There was a tendency, although not statistically significant,for renal function to be worse in TNFR1-deficient mice than in wild-type mice, raising the possibility that TNFR1 activation may oppose the cytotoxicity ofTNFR2 in this model. Further studies with TNFR1/TNFR2-deficient mice will beneeded to examine the interactions between these receptors.
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Our finding that TNFR2 mediates acute renal injury expands a small butgrowing list of disorders in which TNFR2 has been shown to play an importantrole. For example, TNFR2 is upregulated in intestinal inflammation andTNFR2-deficient mice develop less severe intestinal inflammation( 35 ). TNFR2 also participates in intestinal graft vs. host disease( 10 ) and accelerates the earlyphase of collagen-induced arthritis( 50 ). The recent report byAkassoglou et al. ( 3 )demonstrated that overexpression of TNFR2, in the absence of TNFR1 and evenTNF-, induces vascular inflammation and ischemic necrosis in thecentral nervous system (CNS). Few studies have assigned the actions ofTNF- to a specific receptor in the kidney. In a model of obstructiveuropathy, Guo et al. ( 21 )found that both TNFR1 and TNFR2 contributed to interstitial fibrosis,NF- B activation, and TNF- expression. Cunningham et al.( 13 ) determined thatendotoxin-induced acute renal injury was dependent on intrarenal TNFR1,consistent with the known role of TNFR1 in mediating endotoxic shock( 38 ). There are a number ofpossible explanations for the differences between our results and the resultsof Cunningham et al. ( 13 ). Injection of endotoxin causes rapid and massive release of TNF-, theprime mediator of endotoxic shock. In comparison, TNF- secretion incisplatin nephrotoxicity is much slower and more modest( 44 ). TNFR2 may be moreimportant in cell death induced by low levels of TNF- ( 18 ). TheTNF- -dependent induction of TNFR2 expression may also affect therelative importance of TNFR1 and TNFR2 in this model. Moreover, solubleTNF- is a more efficient agonist of TNFR1( 20 ), whereas membrane-bound TNF- preferentially activates TNFR2( 19 ). Accordingly, with endotoxin injection, the high levels of secreted TNF- might be expectedto act primarily via TNFR1, which has a broader constitutive level ofexpression than TNFR2, whereas, in cisplatin nephrotoxicity, upregulation ofTNFR2 along with local production of TNF- may favor TNFR2 pathways.0 I# e, Y& y1 K% [& K7 f

Third, cisplatin produces both necrotic and apoptotic cell death in vivo,and TNF- /TNFR2 signaling contributes to both processes ( Fig. 7 ). Apoptotic signalinghas been clearly demonstrated to occur through TNFR1( 6 ). However, the role ofTNFR2, which lacks an intracellular death domain, in apoptosis andinflammation is less clear. TNFR2 can potentiate the proapoptotic effects ofTNFR1 activation ( 55, 57 ). TNFR2-mediatedubiquitination and degradation of TNF receptor-associated factor 2 may account for this phenomenon ( 30 ).TNFR2 may also mediate apoptosis and inflammation independently of TNFR1( 3, 39, 57 ). In T cells, the abilityof TNFR2 to initiate apoptosis in vitro was dependent on high levels of RIPexpression ( 39 ). However, invivo demonstrations of TNFR2-dependent apoptosis are lacking. Overexpressionof TNFR2 in the CNS, for example, produced vasculitis and necrosis but notapoptosis, whereas TNFR1 expression resulted in apoptosis of oligodendrocytes( 3 ). In cisplatin-induced acuterenal failure, we found that TNFR2 mediates, either directly or indirectly,both apoptotic and necrotic death of renal epithelial cells. The relativeroles of apoptosis and necrosis in the pathogenesis of renal dysfunction aftercisplatin injection are not known. In ischemic injury, recent evidence pointsto an important role for apoptosis rather than necrosis( 24, 25 ). However, because bothnecrosis and apoptosis were reduced in TNFR2-deficient mice, our results donot allow any conclusions regarding their relative importance in cisplatintoxicity. Further studies are required to determine whether the cells expressing TNFR2 are the cells that subsequently undergo necrotic or apoptoticdeath. Similarly, we do not understand the factors that determine whetherrenal epithelial cells die by necrosis or apoptosis. Intracellular ATP( 36 ) and dGTP concentrations ( 8 ) and the concentration ofcisplatin to which cells are exposed ( 31 ) have all been proposed ascellular determinants of the mode of cell death., n- R% I4 D$ A

Fourth, TNFR2 plays a role in ICAM-1 expression and leukocyte infiltrationin cisplatin acute renal injury. Inflammatory mechanisms are believed to playan important role in the pathogenesis of acute renal failure( 42, 49 ). The inflammatory response is characterized predominantly by an early neutrophil and a late monocyteinflux and is preceded by the expression of adhesion molecules, such asICAM-1, as well as the induction of various chemokines responsible formonocyte and neutrophil migration ( 29, 44 ). Renal ICAM-1 expressionis increased after either ischemic or toxic( 15, 23, 26, 44 ) renal insults. Blockade ofICAM-1 reduces acute renal injury after ischemia( 26 ) or cisplatin exposure( 23 ). The signals that resultin ICAM-1 expression in these settings are unknown. In many systems, induction of ICAM-1 expression occurs via TNFR1( 5, 27, 54 ). In the kidney, however,Burne et al. ( 12 ) demonstratedthat ischemia-induced ICAM-1 expression is independent of TNFR1. Thedependence on TNFR2 was not examined in that study. Our results indicate that TNFR2 contributes to ICAM-1 upregulation in cisplatin and perhaps other formsof acute renal injury. TNFR2-dependent ICAM-1 expression was also noted intransgenic mice overexpressing TNFR2 in the CNS( 3 ).
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In summary, we demonstrated, using TNF- - and TNF receptor-deficient mice, that cisplatin-induced renal inflammation, cell death, and organdysfunction are mediated, in part, through TNFR2. Moreover, the induction ofTNF- and TNFR2 expression is interdependent. We also demonstrated thatboth apoptosis and necrosis of renal epithelial cells were dependent on TNFR2.We conclude that TNFR2 may play a greater role in ischemic and toxic organinjury than had been previously appreciated. Antagonism of TNF- production or action may have a therapeutic benefit in these settings.
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DISCLOSURES; Q& X' ]* O! c! `: A
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This work was supported by the Veterans Affairs Medical Research Serviceand grants from the American Heart Association and the Four Diamonds Fund.! h* M. m, O' ~) `! ^$ s
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