INFECTION AND IMMUNITY, Mar. 2009, p. 959–969 Vol. 77, No. 30019-9567/09/$08.00�0 doi:10.1128/IAI.00679-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Shiga Toxin 2 Targets the Murine Renal Collecting Duct Epithelium�

Mitchell A. Psotka,1 Fumiko Obata,1 Glynis L. Kolling,1 Lisa K. Gross,1 Moin A. Saleem,2,3

Simon C. Satchell,2 Peter W. Mathieson,2 and Tom G. Obrig1*Department of Internal Medicine, Division of Nephrology, University of Virginia, Charlottesville, Virginia1; Academic Renal Unit,

University of Bristol, Southmead Hospital, Bristol, United Kingdom2; and Children’s Renal Unit, University of Bristol,Southmead Hospital, Bristol, United Kingdom3

Received 30 May 2008/Returned for modification 26 August 2008/Accepted 29 December 2008

Hemolytic-uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli infection is a leadingcause of pediatric acute renal failure. Bacterial toxins produced in the gut enter the circulation and cause asystemic toxemia and targeted cell damage. It had been previously shown that injection of Shiga toxin 2 (Stx2)and lipopolysaccharide (LPS) caused signs and symptoms of HUS in mice, but the mechanism leading to renalfailure remained uncharacterized. The current study elucidated that murine cells of the glomerular filtrationbarrier were unresponsive to Stx2 because they lacked the receptor glycosphingolipid globotriaosylceramide(Gb3) in vitro and in vivo. In contrast to the analogous human cells, Stx2 did not alter inflammatory kinaseactivity, cytokine release, or cell viability of the murine glomerular cells. However, murine renal cortical andmedullary tubular cells expressed Gb3 and responded to Stx2 by undergoing apoptosis. Stx2-induced loss offunctioning collecting ducts in vivo caused production of increased dilute urine, resulted in dehydration, andcontributed to renal failure. Stx2-mediated renal dysfunction was ameliorated by administration of thenonselective caspase inhibitor Q-VD-OPH in vivo. Stx2 therefore targets the murine collecting duct, and thisStx2-induced injury can be blocked by inhibitors of apoptosis in vivo.

Shiga toxin-producing Escherichia coli (STEC) is the princi-pal etiologic agent of diarrhea-associated hemolytic-uremicsyndrome (HUS) (42, 60, 66). Renal disease is thought to bedue to the combined action of Shiga toxins (Shiga toxin 1 [Stx1]and Stx2), the primary virulence factors of STEC, and bacteriallipopolysaccharide (LPS) on the renal glomeruli and tubules(6, 42, 60, 66). Of these, Stx2 is most frequently associated withthe development of HUS (45). Shiga toxin enters susceptiblecell types after binding to the cell surface receptor glycosphin-golipid globotriaosylceramide (Gb3) and specifically depuri-nates the 28S rRNA, thereby inhibiting protein synthesis (42,60, 66). The damage initiates a ribotoxic stress response con-sisting of mitogen-activated protein (MAP) kinase activation,and this response can be associated with cytokine release andcell death (21, 22, 25–27, 61, 69, 73). This cell death is oftencaspase-dependent apoptosis (18, 61). Gb3 is expressed by hu-man glomerular endothelial cells, podocytes, and multiple tu-bular epithelial cell types, and damage markers for these cellscan be detected in urine samples from HUS patients (10–12,15, 49, 73). Shiga toxin binds to these cells in renal sectionsfrom HUS patients, and along with the typical fibrin-rich glo-merular microangiopathy, biopsy sections demonstrate apop-tosis of both glomerular and tubular cell types (9, 29, 31).

Concomitant development of the most prominent featuresof HUS: anemia, thrombocytopenia, and renal failure, requiresboth Shiga toxin and LPS in the murine model (30, 33). Nev-ertheless, our previous work demonstrated that renal failure ismediated exclusively by Stx2 (33). While it is established that

Gb3 is the unique Shiga toxin receptor (46), the current liter-ature regarding the mechanism by which Shiga toxin causesrenal dysfunction in mice is inconsistent. Even though Gb3 hasbeen localized to some murine renal tubules and tubular dam-age has been observed (19, 23, 46, 53, 65, 68, 72, 74), thespecific types of tubules affected have been incompletely char-acterized. Although multiple groups have been unable to lo-cate the Shiga toxin receptor Gb3 in glomeruli in murine renalsections (19, 53), one group has reported that murine glomer-ular podocytes possess Gb3 and respond to Stx2 in vitro (40),and another group has reported that renal tubular capillariesexpress the Gb3 receptor (46). Furthermore, murine glomeru-lar abnormalities, including platelet and fibrin deposition, oc-cur in some murine HUS models (28, 30, 33, 46, 59, 63). Wedemonstrate here that murine glomerular endothelial cells andpodocytes are unresponsive to Stx2 because they do not pro-duce the glycosphingolipid receptor Gb3 in vitro or in vivo.Further, murine renal tubules, including collecting ducts, ex-press Gb3 and undergo Stx2-induced apoptosis, resulting indysfunctional urine production and dehydration.

MATERIALS AND METHODS

Shiga toxin and LPS. Shiga toxin 2 was purified by immunoaffinity chroma-tography from cell lysates (generously provided by Alison O’Brien) of E. coliDH5� containing the Stx2-producing pJES120 plasmid (39). Lysates were pro-cessed using 11E10 antibody (48) immobilized with an AminoLink Plus kit, andendotoxin was removed using De-toxi-Gel (Pierce Biotechnology, Rockford, IL).The purity of Stx2 was assessed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis and determined to be endotoxin free by a Limulus amebocyteassay, and activity was measured in a Vero cell cytotoxicity assay. E. coli O55:B5LPS purified by gel filtration chromatography and gamma irradiation was pur-chased from Sigma-Aldrich (St. Louis, MO).

Cell lines. Human umbilical vein endothelial cells (HUVEC) were purchasedfrom VEC Technologies (Rensselaer, NY) and grown in MCDB 131 medium(Mediatech, Herndon, VA), supplemented as described previously (62). Primaryhuman renal proximal tubule epithelial cells (RPTEC) were purchased from

* Corresponding author. Present address: Department of Microbi-ology and Immunology, University of Maryland, Baltimore, 660 Red-wood St., Baltimore, MD 21201. Phone: (410) 706-6917. Fax: (410)706-2129. E-mail: tobrig@som.umaryland.edu.

� Published ahead of print on 5 January 2009.

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Clonetics (San Diego, CA) and grown in REGM (Lonza, Walkersville, MD)(73). Conditionally immortalized human glomerular endothelial cells were grownin EBM-2 MV medium with provided supplements (Lonza) (55). Primary murineaortic endothelial cells graciously provided by Lynn Hedrick (University of Vir-ginia, Charlottesville) were grown in Dulbecco modified Eagle medium(DMEM) (Gibco, Grand Island, NY) and supplemented as described previously(7). Simian virus 40 (SV40) large-T-antigen-immortalized murine proximal tu-bular epithelial cells (TKPTS) kindly provided by Mark Okusa (University ofVirginia, Charlottesville) were grown in DMEM:F12 (Gibco) supplemented asdescribed previously (73). Conditionally immortalized human podocytes weregrown in RPMI 1640 (Mediatech) with appropriate supplements (54). Condi-tionally immortalized murine podocytes generously provided by John Sedor(Case Western Reserve University, Cleveland, OH) and conditionally immortal-ized murine glomerular endothelial cells provided by Michael Madaio (TempleUniversity, Philadelphia, PA) were cultured in a 3:1 mixture of DMEM:F12(Gibco), supplemented as described previously (2, 4, 41).

Cells were grown at 37°C with 5% CO2 and 90% humidity in Falcon 75-cm2

flasks (BD Biosciences, Bedford, MA) except for the conditionally immortalizedcell lines maintained at the permissive temperature (33°C). Only the condition-ally immortalized murine cell lines were given 10 U of mouse gamma interferon(Sigma) per ml at 33°C. Conditionally immortalized cell lines maintained at thepermissive 33°C were considered undifferentiated. Undifferentiated cells weremoved to the nonpermissive temperature (37°C) (and gamma interferon wasremoved) 2 weeks prior to experimental use, after which point they were con-sidered differentiated (2, 41, 54, 55). Cells were seeded at 5 � 105 per well in6-well plates or 2 � 104 per well in 96-well plates. All experiments were per-formed on 6- or 96-well plates (Corning, Corning, NY) coated with rat tailcollagen I (BD Biosciences) in serum-free RPMI 1640 (Mediatech). All exper-iments were performed in serum-free RPMI supplemented with L-glutamine.Except for cytotoxicity assays, cells were challenged with either no toxin, Stx2, 1�g/ml LPS, or Stx2 and LPS, with 1 pM and 1 nM Stx2 employed for humanglomerular and murine glomerular cells, respectively.

TLC Shiga toxin overlay. Each cell type grown to confluence in one 75-cm2

flask was trypsinized, and neutral glycolipids were isolated (51). For some stud-ies, cells were incubated with 1 �g/ml LPS (E. coli O55:B5; Sigma-Aldrich) 24 hprior to trypsinization. Gb3 content was analyzed by thin-layer chromatography(TLC) with Stx1B overlay (43). Total neutral lipids on a duplicate plate werevisualized using CuSO4 along with neutral glycosphingolipid standards (MatreyaLLC, Pleasant Gap, PA) (1). Images are representative of triplicate experiments.

Immunoblotting. Cells were incubated with toxins for 0.5 to 12 h. Afterincubation, cells were rinsed twice with phosphate-buffered saline and lysed inmodified radioimmunoprecipitation assay buffer containing 50 mM Tris, 150 mMNaCl, 1% Igepal CA-630, 0.5% deoxycholate, 100 �M leupeptin, 1 mM phenyl-methylsulfonyl fluoride, 0.15 U/ml aprotinin, and 1 mM vanadate as previouslydescribed (56). Lysate protein was quantified with a BCA protein assay (Pierce).Total cell lysate was loaded at 5 �g per well, resolved by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene diflu-oride membrane, and probed with the following antibodies (according to thedilutions suggested by the manufacturer): anti-p38 MAP kinase and anti-phos-pho-p38 antibodies (BD Biosciences), anti-�-actin (Abcam, Cambridge, MA),and anti-SV40 T antigen (Calbiochem, San Diego, CA). Anti-mouse horseradishperoxidase-tagged antibody (Amersham, Piscataway, NJ) was used as the sec-ondary antibody. Bound horseradish peroxidase was detected by chemilumines-cence (Perkin Elmer, Waltham, MA). Images are representative of triplicateexperiments.

Cytotoxicity assay. Cells were treated with Stx2 at a concentration between 1fM and 10 nM for 24 h. CCK-8 cell viability assays were performed to determinethe 50% cytotoxic dose (CD50) (Dojindo Molecular Technologies, Gaithersburg,MD). Stx2 coincubation with 1 �g/ml LPS was tested in all cell types butenhanced only HUVEC cytotoxicity (62). Caspases were inhibited with 100 �MQ-VD-OPH (MP Biochemicals, Solon, OH) suspended in dimethyl sulfoxide(DMSO) for 1 h before and after the addition of Stx2 (8). The final concentrationof DMSO was 0.5%. Data are from quadruplicate experiments.

Extracellular signaling molecule quantification. Cells were incubated withtoxins for 12 h. Extracellular monocyte chemoattractant protein 1, interleukin 6(IL-6), vascular endothelial growth factor (VEGF), human IL-8, and murineCXCL1/KC release was quantified using species-specific DuoSet enzyme-linkedimmunosorbent assay kits (R&D Systems, Minneapolis, MN). Because mice donot produce IL-8, CXCL1/KC was used as the murine functional homolog (35).Extracellular proteins measured in picograms per milliliter were normalized tovalues from control cells (grown in media only) and expressed as changes (aspercentages) from those control values. Data are from triplicate experiments.

Caspase activity assay. Cells were challenged with toxins for 12 h. Lysates werecollected as described above and tested for caspase activity with the caspase 3/7assay kit (Upstate, Lake Placid, NY) using the fluorogenic caspase substrateAc-DEVD-AMC (where Ac is N-acetyl, DEVD is Asp-Glu-Val-Asp, and AMCis 7-amino-4-methylcoumarin). Data are from triplicate experiments.

Murine model. Male C57BL/6 mice weighing 22 to 24 g were purchased fromCharles River (Wilmington, MA). Food and water were provided ad libitum.Mice were injected intraperitoneally with 300 �g of LPS (O55:B5; Sigma-Al-drich) per kg of body weight, 225 ng of Stx2/kg, or both as described previously(33). Mice were weighed every 12 h after injection, and weight loss was expressedas percent change from the weight of the mouse at time zero. At 0, 24, 48, 60, and72 h after injection, mice were euthanized. The kidneys and blood and urine werecollected from each mouse. To prevent apoptosis in vivo, mice were intraperi-toneally injected with two 18-mg/kg doses of Q-VD-OPH in 100 �l of 50%DMSO at 24 and 48 h after injection of Stx2 plus LPS (38). Q-VD-OPH formsan irreversible thioether bond with the active site cysteine of the caspase, dis-placing the 2,6-diflurophenol group to inhibit caspase activity. No toxicity wasobserved in mice receiving only Q-VD-OPH. In separate experiments, healthymice were dehydrated for 20 h by withholding access to water. Urine samples forvolume measurements were collected from mice housed in individual metaboliccages. To determine Gb3 localization in mouse kidney, C3H/HeN and CD-1 mice(Charles River), C3H/HeJ and BALB/c mice (Jackson, Bar Harbor, ME), andC57BL/6 mice were used. All animal procedures were done in accordance withUniversity of Virginia Animal Care and Use Committee policies (Charlottes-ville, VA).

FIG. 1. Human and murine glomerular cell expression of SV40large T antigen and Gb3. (A) Whole-cell lysates from human glomer-ular cells and murine glomerular cells grown at the permissive tem-perature (33°C) compared to human and murine glomerular cellsgrown at the nonpermissive temperature (37°C). Differentiated cellsgrown for 2 weeks at the nonpermissive temperature degraded theSV40 temperature-sensitive large T antigen (T-Ag). HUVEC humanprimary endothelial cells (ENDO) were used as a control. PODO,podocytes. (B) (Top) Thin-layer chromatography of total neutral lipidswith Stx1B overlay to specifically detect Gb3. (Bottom) Total neutrallipids visualized by cupric sulfate to demonstrate similar loads in thelanes. RPTEC are human proximal tubule cells, whereas TKPTS aremurine proximal tubule cells. The glycosphingolipid standards in-cluded the following: ceramide monohexoside (glucosylceramide)(CMH), ceramide dihexoside (lactosylceramide) (CDH), globotri-aoslyceramide (Gb3), and globotetraosylceramide (Gb4). Glom, glo-merular; Endo, endothelial.

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Blood analysis and urinalysis. Blood was collected with heparinized capillarytubes (Fisher, Pittsburgh, PA) by retro-orbital bleed and centrifuged at 840 � gfor 15 min at 4°C to collect the plasma layer. The level of blood urea nitrogen(BUN) was determined spectrophotometrically with VetScan (Idexx Corpora-tion, Westbrook, ME). Urine was collected by direct bladder puncture, andosmolality was determined with the Vapro vapor pressure osmometer (Wescor,Logan, UT). Each data point represents the average for eight mice.

Immunohistochemistry. C57BL/6, C3H/HeN, and CD-1 male mice were fromCharles River (Wilmington, MA). C3H/HeJ and BALB/c mice were from Jack-son (Bar Harbor, ME). Control C57BL/6 mice, C57BL/6 mice challenged withStx2 plus LPS, and untreated CD-1, BALB/c, C3H/HeN, and C3H/HeJ micewere used. Kidneys were fixed in 4% paraformaldehyde, processed in acetone forGb3 or ethanol for terminal deoxynucleotidyltransferase biotin-dUTP nick endlabeling (TUNEL) staining, and embedded in paraffin as described previously(34). Ethanol has been previously demonstrated to both remove endogenous Gb3

and cause false-positive Gb3 staining (34). Sections were incubated with anti-Gb3/CD77 immunoglobulin M (IgM) (Beckman Coulter, Fullerton, CA) at adilution of 1:40, isotype-matched rat IgM (Millipore, Billerica, MA), anti-acti-vated caspase 3 (Cell Signaling, Danvers, MA) at a dilution of 1:200 or isotype-matched rabbit IgG (Chemicon), and anti-rat IgM biotin conjugate (AmericanQualex, San Clemente, CA) at a dilution of 1:500. TUNEL was performed withthe ApopTag peroxidase in situ apoptosis detection kit (Chemicon, Temecula,CA), with postweaning female rat mammary gland as a positive control. Immu-noreactivity was detected using Vectastain Elite ABC kit (Vector Laboratories,Burlingame, CA). Hematoxylin was the counterstain. Renal apoptosis was quan-tified by counting the number of apoptotic cell nuclei per 16 fields (at a magni-fication of �200) spread among the cortex and outer and inner medulla. Renalapoptosis was expressed as the average number of apoptotic nuclei per kidney.Each data point represents the average for eight mice.

Immunofluorescence. Kidney tissue was isolated following perfusion fixationin 4% paraformaldehyde. Sections were blocked with anti-rat IgM or secondaryantibody-matched normal sera, incubated with anti-Gb3/CD77 at a dilution of1:100, anti-CD31 (BD Pharmingen) at 1:50, anti-aquaporin-1 (AQP-1; Chemi-con) at 1:1,000, anti-aquaporin-2 (AQP-2; Chemicon) at 1:800, or isotype controlantibody (Chemicon) at the equivalent concentration in normal goat serum,washed, and incubated with fluorescent secondary antibodies (Invitrogen, Carls-bad, CA). Specimens were examined with an LSM 510 microscope (Zeiss,Thornwood, NY) and analyzed using LSM Image Browser software (Zeiss).

Statistics. All data are expressed as means � standard deviations. Statisticswere performed using two-sample Student’s t test assuming unequal variances. AP of �0.05 was considered significant.

RESULTS

Murine glomerular cells do not express Gb3. Human HUS isthought to result from glomerular damage by Shiga toxin (10,66). We began investigating the mechanism of renal failure inthe mouse model by determining the sensitivity of murineglomerular filtration barrier cells to Stx2. Conditionally immor-talized human and murine glomerular endothelial cells andpodocytes have been described in detail (2, 41, 54, 55). Thesecells grow indefinitely at 33°C. When differentiated at 37°C,these cells express cell type-specific markers and slow theirproliferation. Whole-cell lysates were derived from cells grownat permissive and nonpermissive temperatures and analyzed by

FIG. 2. Western immunoblots of activated p38 (phospho-38) and total p38 from differentiated human and murine glomerular cells over a 12-htime course. Cells were incubated in media alone as a control (Cont) or challenged with Stx2 (Stx), 1 �g/ml LPS, or Stx2 plus LPS (Stx LPS). Forhuman cells, 1 pM Stx2 was employed, while 1 nM Stx2 was used for the murine cells.

TABLE 1. Summary of Stx2 CD50 and immortalization status data for cell types used in this studya

Cell typeHuman cells Murine cells

Cell type CD50 at 24 h Cell type CD50 at 24 h

Glomerular endothelium Conditionally immortalized 0.1 pM Conditionally immortalized Insensitiveb

Glomerular podocytes Conditionally immortalized 0.5 pM Conditionally immortalized InsensitiveProximal tubulesc Primary 10 pM Immortalized InsensitiveLarge vesseld endothelium Primary 1 nM Primary Insensitive

a The cells were treated with 1 fM to 10 nM Stx2 for 24 h.b Insensitive cells did not respond to the maximum dose of 10 nM Stx2 for 48 h.c The human proximal tubule cells were RPTEC, and the murine proximal tubule cells were TKPTS.d The human large vessel endothelial cells were HUVEC, and the murine large vessel endothelial cells were primary aortic cells.

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immunoblotting (Fig. 1A). These cells were compared toHUVEC that do not express the temperature-sensitive SV40large T antigen. When incubated at the nonpermissive temper-ature (37°C), all conditionally immortalized cell types appro-priately degraded the transgene and slowed or stopped prolif-eration. Differentiated human and murine podocytes alsodeveloped typical arborizations (data not shown) (41, 54).

To test the murine glomerular cells for the presence of theStx2 receptor Gb3, cellular lipids were isolated and separatedby TLC. Total neutral lipids were visualized on the TLC plateby staining with cupric sulfate (Fig. 1B, bottom panel), and Gb3

was specifically identified by overlay with Stx1B (Fig. 1B, toppanel). Stx1B bound to no other glycolipid bands on theseplates. Although the lipid profiles differed for the various celltypes, similar loads were verified by the slowly migrating gly-colipid band at the bottom of the TLC plate. Human glomer-ular endothelial cells and podocytes expressed very high levelsof Gb3, and human RPTEC and HUVEC produced moderatelevels, consistent with previous reports (15, 49). In contrast,murine glomerular endothelial cells, podocytes, and primarycells failed to express detectable Gb3. When these cells wereincubated with LPS for 24 h to test for cytokine-mediated Gb3

upregulation, only HUVEC produced more Gb3 (data notshown) (62).

Murine glomerular cells are insensitive to Stx2. Stx2 hasbeen demonstrated to have multiple effects on susceptible celltypes; these effects include initiation of inflammatory intracel-lular signaling, cytokine release, and cellular apoptosis (21, 22,25–27, 61, 69). Table 1 summarizes the immortalization statusand CD50 data for the human and mouse cells used in thisstudy after the cells were treated with Stx2 for 24 h. Humanglomerular cells were extremely sensitive to the cytotoxic ef-fects of Stx2, while murine glomerular cells were insensitive toStx2, even at a dose of 10 nM. Primary cells from both specieswere used as controls for the conditional immortalization. Pri-mary HUVEC and RPTEC were sensitive, as previously re-ported, while primary murine aortic endothelial cells were not(62, 73). The sensitivities of cells to the cytotoxic effect of Stx2generally correlated with their level of Gb3 expression (Table1 and Fig. 1B).

Stx2 increases inflammatory mediator release and MAP ki-nase activation in some cell types, even in the absence ofcytotoxicity (25, 61, 69). Even though the murine cells were notsensitive to the cytotoxic effects of Stx2, they might respond byactivating intracellular kinases or by releasing extracellular sig-naling molecules. To test this, human and murine glomerularcells were treated with Stx2 or LPS over a 12-h time course,and lysates were immunoblotted for total and activated p38MAP kinase (Fig. 2) and JNK (Jun N-terminal protein kinase)(data not shown). p38 was activated in the human cells by bothLPS and 1 pM Stx2, though the kinetics of activation differed.LPS caused a rapid increase in p38 activation from 1 to 2 h,while Stx2 began to exert its effect by 2 to 6 h and continuedthroughout the experiment. In contrast, the murine glomerularcells responded only to LPS, even at a Stx2 concentration of 1nM (Fig. 2). The murine glomerular cells were more sensitivethan the human glomerular cells to the effects of LPS, showinga more rapid response between 0.5 and 2 h, but no significantincrease in p38 phosphorylation was observed in response toStx2. The qualitative results of JNK activation due to Stx2 and

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LPS were similar to those of p38 activation in all cell types(data not shown).

Table 2 presents the relative changes in extracellular signal-ing molecule release by human and murine glomerular cellsafter 12 h with Stx2 and LPS. LPS increased cytokine releaseinto the supernatant by these cell types. In contrast, Stx2 af-fected only the Gb3-expressing human cells and did so bydecreasing the signaling molecules released. Cytokines upregu-lated by LPS were reduced by Stx2, as was basal VEGF secre-tion by human podocytes. Stx2 mediated 15% � 3% and25% � 5% cytotoxicity of human podocytes and endothelialcells, respectively, at the 24-h time point. Human glomerularcells incubated with the same dose of LPS but a 10-fold-lowerdose of Stx2 (100 fM) did not exhibit significantly decreasedcell viability or LPS-induced cytokine release by 12 h (data notshown). This suggested that the Stx2 inhibition of inflamma-tory mediator release was secondary to Stx2-mediated celldeath.

Stx2 mediates caspase-dependent apoptosis. Shiga toxin me-diates caspase-dependent apoptotic cell death in certain celltypes (18, 61). Caspases are cytoplasmic cysteine proteases

essential to the destructive phase of apoptosis (67). Thus, ac-tivity of the major effector caspase 3 was measured in lysatesfrom cells treated for 12 h with Stx2, LPS, or both. Caspase 3activity was increased in human glomerular endothelial cells(Fig. 3A) and podocytes (Fig. 3B) in response to Stx2, but notin murine glomerular cells (data not shown). LPS did not havea significant impact on caspase 3 activity in any cell type (Fig.3A and B). In human cells, the nonselective caspase inhibitorQ-VD-OPH rescued 80% of the cytotoxic effect of Stx2 onendothelial cells (Fig. 3C) and 100% of the effect on podocytes(Fig. 3D). Although higher and lower concentrations of thecaspase inhibitor were tested, 100 �M provided the maximumnontoxic effect (data not shown).

Murine renal tubules produce Gb3. It has previously beenshown that Stx2 causes renal failure in mice (3, 33, 47). Havingdemonstrated that Stx2 does not directly affect the murinerenal glomerular filtration barrier cells in vitro, we sought todetermine the Stx2 target cells in the mouse kidney. Healthymouse renal tissue subjected to immunohistochemistry withanti-Gb3 antibody demonstrated Gb3 only on cortical and med-ullary tubular cells and not in glomeruli or blood vessels (Fig.

FIG. 3. Human glomerular cell caspase 3 activity and cell death inhibition by the nonselective caspase inhibitor Q-VD-OPH. Humanglomerular endothelial cells (A) and human glomerular podocytes (B) were treated with Stx2, LPS, or Stx2 plus LPS for 12 h, and lysates wereisolated. The viability of cells after incubation of human glomerular endothelial cells (C) and human glomerular podocytes (D) with Q-VD-OPH(QVDOPH) was compared to that of cells grown in media alone (control) or in media containing 0.5% DMSO (vehicle). Values that weresignificantly different are indicated as follows: *, P � 0.05 compared to the value for the control (not challenged with Stx2); **, P � 0.05 comparedto the value for mice challenged with Stx2 alone (without Q-VD-OPH).

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4). Similar qualitative staining was observed in all mousestrains tested, including C57BL/6, CD-1, BALB/c, C3H/HeN,and C3H/HeJ mice (data not shown), as noted previously (19,34). Morphologically, Gb3 appeared to localize to specific celltypes in the three different areas of the murine kidney (Fig. 4B,C, and D).

To identify the most abundant Gb3-positive cell types, im-munofluorescence colocalization for Gb3 and aquaporin-1(AQP-1) and AQP-2 were performed (16). Aquaporins arecytoplasmic and membrane proteins that mediate water reab-sorption from the renal tubular lumen (16). Proximal tubulesin the cortex and the thin descending loops of Henle in themedulla specifically express AQP-1, while collecting ducts inthe cortex, medulla, and papilla express AQP-2 (16). Gb3 wasexpressed on some AQP-1-producing cortical proximal tubules(Fig. 5A), although expression of the two markers appeared tobe mostly localized to different sites within the same cell. It wasnoted that not every AQP1-positive proximal tubule expressedGb3 (Fig. 5A), and no medullary AQP-1-positive loops ofHenle were Gb3 positive (Fig. 5B). In contrast, some AQP-1-negative cortical tubules robustly expressed Gb3 (Fig. 5A).High-power cortical and medullary images of Gb3 and AQP-2showed colocalized staining: Gb3 was found on the outer mem-brane, consistent with antibody binding the outer carbohydratemoiety, and AQP-2 was distributed in both the membrane andcytoplasm (Fig. 6A). Low-power images of the murine renalmedulla stained for Gb3 and AQP-2 (Fig. 6B) showed thatalmost all Gb3-expressing medullary tubules were collectingducts. Gb3 staining did not colocalize with the endothelialmarker CD31 in the mouse kidney (data not shown and ref-erence 34).

Stx2 causes murine tubular apoptosis. To determinewhether the cells found to produce Gb3 in vivo undergoapoptosis, TUNEL staining was performed on renal sectionsfrom mice 72 h after the mice were injected with Stx2 plus LPS.Positive TUNEL stain was found only in tubular cell nuclei(Fig. 7A, inset), and only rare TUNEL-positive cells wereobserved in renal sections from healthy mice. Consistent withthe absence of Gb3, there were no apoptotic cells visualized in

FIG. 4. Gb3 localization in untreated mouse renal tissue by immu-nohistochemistry. (A) Isotype control shows no renal staining. Sectionsfrom cortex (B), papilla (C), and medulla (D) demonstrate anti-Gb3staining of some population of tubules. No staining was observed inglomeruli. Asterisks in panel C are examples of Gb3-positive tubules inthis region. All images shown are representative of images from eightmice and are at a magnification of �200.

FIG. 5. Immunofluorescence staining of healthy murine renal tissue for Gb3 and AQP-1. Cortical (A) and medullary (B) tubules stained withanti-Gb3 and anti-AQP-1, and the merged image is shown, with overlap colored in yellow. Some cortical tubules express only AQP-1 (single whitearrow), some express only Gb3 throughout (two white arrows), while others express AQP-1 and Gb3 in a punctuate pattern (large whitearrowhead). Bar, 20 �m.

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the glomeruli or renal vasculature of mice challenged with Stx2plus LPS. The qualitative immunohistochemical results for ac-tivated caspase 3 were similar to the qualitative TUNEL results(data not shown). Analysis of kidneys from mice between 0 and72 h after Stx2-plus-LPS injection demonstrated increasedTUNEL-positive cells at 60 and 72 h postinjection (Fig. 7A).LPS alone did not increase renal apoptosis above the baselinelevel (data not shown).

Renal tubular apoptosis correlates with renal dysfunction.As murine cortical tubular damage (23, 46, 53, 65, 68, 72, 74)and proximal tubular physiologic dysfunction manifest by glu-cosuria had been previously described (47), we determined theeffect of collecting duct dysfunction. BUN values after 24 h inmice injected with Stx2 plus LPS increased with a time coursesimilar to that of tubular apoptosis (Fig. 7B). Previous studiesdemonstrated that LPS mediated the initial 24-h weight lossand Stx2 mediated the later weight loss in these mice (33).Because the collecting ducts are responsible for water reab-sorption, we tested whether these mice had a defect in urineconcentration that might cause dehydration and weight loss(16). Mice challenged with Stx2 plus LPS developed brief poly-uria (increased urine volume) during the first 12 h that led toa compensatory increase in urine osmolality when measured at24 h postinjection (Fig. 7C). The initial increase in urineosmolality was reproduced by injection with LPS alone (data notshown), as published previously (17). Mice given Stx2 plus LPSor Stx2 alone developed polyuria and osmotically dilute urinebetween 48 and 72 h postinjection (Fig. 7C). Stx2-plus-LPS-challenged mice produced 2.7 � 0.8 ml of urine between 48and 72 h compared to 1.1 � 0.5 ml of urine from nonchal-lenged controls (P � 0.05; n � 8). Polyuria from 48 to 72 hcorrelated with the weight loss and observed signs of dehydra-tion in mice given Stx2 plus LPS (Fig. 7D). This decreasedurine osmolality contrasts with 20-h dehydration of normalmice by water restriction. Water-restricted mice lost 10% �

0.5% of their body weight and produced a minimal volume ofurine, and all of the urine had a high osmolality of 3,485 � 762mmol/kg (n � 4).

Renal tubular apoptosis contributes to renal dysfunction.To test whether tubular apoptosis caused collecting duct dys-function, dehydration, and renal failure, mice injected withStx2 plus LPS were given two divided doses of the nonselectiveapoptosis inhibitor Q-VD-OPH at 24 and 48 h. At 72 h afterinjection with Stx2 plus LPS, these mice demonstrated signif-icantly decreased numbers of renal tubular apoptotic cells,increased urine osmolality, and decreased BUN levels (Fig. 8Ato C). Although these mice developed the initial polyuria andweight loss mediated by LPS, they exhibited a significant re-covery in body weight during the time when Stx2 normallycaused a loss in body weight (Fig. 8D).

DISCUSSION

Previous studies examining the location of murine renal Gb3

have provided conflicting results (19, 34, 46, 53, 68). Our datasupport the conclusion that the primary Gb3-producing struc-ture and Stx2 target in the murine kidney is the tubular system.We did not detect Gb3 expression by murine endothelial cells,and it is noteworthy that the previous study that reportedmurine renal endothelial production of Gb3 did not performdirect colocalization (46). Even though not all collecting ductcells appeared TUNEL positive at any single time point afterStx2 challenge, it is likely that more cells died than were visu-alized because apoptotic cells stain TUNEL positive for only 3hours (20). Collecting duct dysfunction is in agreement withfindings for other murine models of Shiga toxin-mediated in-jury and with microarray analysis in this model, which revealedStx2-mediated downregulation of collecting duct-specific tran-scripts (9, 31, 33, 53, 58, 64). LPS has been previously shownnot to cause tubular damage when administered at similar

FIG. 6. Immunofluorescence staining of healthy murine renal tissue for Gb3 and AQP-2. (A) High-power images of a medullary tubule stainedwith anti-Gb3 and anti-AQP-2, and the merged image is shown, with overlap colored in yellow. The inset shows magnified view of the boxed region.(B) Low-power images of renal medulla stained with anti-Gb3 and anti-AQP-2 and the merged image. Bars, 10 �m (A) and 100 �m (B).

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doses over this time course (23, 33). Although functional col-lecting duct damage in response to Shiga toxin was postulatedin prior reports (47, 53), it was probably not observed becauselittle morphological change occurs. In support of our findings,production of dilute urine has recently been reported for miceinoculated with STEC (13). The increased murine BUN levelin response to Stx2 challenge may be secondary to dehydrationcaused by collecting duct dysfunction. Significant dehydrationcan decrease renal perfusion and raise the BUN value.

The present study confirmed that murine glomerular podo-cytes lack Gb3 and are insensitive to Stx2. Even though iden-tical conditionally immortalized mouse podocytes were previ-ously reported to produce Gb3 and respond to Stx2, we failedto detect Gb3 by a more specific method or to demonstrate aresponse to Stx2, even at 500 times the reported dose (40, 41).These cells are known to express TLR4 (Toll-like receptor 4)and release cytokines in response to LPS (5), and our cellsresponded to LPS by activating p38 in a time course similar tothat detailed for Stx2 (40). Additionally, we have demonstratedthat these cells lack Gb3 in vivo. Therefore, the effects previ-ously ascribed to Stx2 in murine podocytes may be due to asmall contaminating dose of LPS. Furthermore, the murineglomerular endothelial cells displayed similar responses to LPS

and insensitivity to Stx2, suggesting that murine models report-ing glomerular damage are likely due to LPS or indirect effectsof Stx2; only those models that use live STEC or inject micewith Shiga toxin plus LPS observe glomerular defects (13, 28,33, 59, 63). Although the Stx2-induced HUS mouse modellacks glomerular damage, we believe this difference fromhuman disease does not preclude the utility of this system.Challenging mice with Stx2 plus LPS results in anemia,leukocytosis, thrombocytopenia, and cytokine-dependent fi-brin deposition, and their relationships to HUS patient find-ings remain to be investigated (32, 33).

The human glomerular cells studied here were exquisitelysensitive to the cytotoxic effects of Stx2. Whereas it was previ-ously reported that human glomerular epithelial cells weresensitive to Shiga toxin in vitro only at a much higher dose (27),the cells used prior were likely to be glomerular parietal epi-thelial cells rather than podocytes. This supposition is sup-ported by their isolation using a sieving procedure shown tocreate cultures of nonpodocyte glomerular epithelial cells,their adoption of cobblestone as opposed to arborized mor-phology, and their lack of expression of the podocyte markerWT-1 (41, 75, 76).

Human tubular damage does occur in HUS patients, though

FIG. 7. Quantification of renal apoptosis, renal failure, urine osmolality, and weight loss in mice injected with Stx2 plus LPS. (A) The totalnumber of TUNEL-stained nuclei were counted per 16 fields (at a magnification of �200) per tissue section and averaged. Renal apoptosisincreased over the time course and became significant starting 60 h after injection. An example of TUNEL stain at 72 h after Stx2-plus-LPSinjection is shown in the inset. The black arrowheads indicate TUNEL-positive nuclei. (B) Increased BUN levels occurred over a time coursesimilar to that of tubular apoptosis. (C) Urine osmolality was decreased late in the time course, although these mice demonstrated an initialincrease in urine solute concentration. (D) Mice lost substantial weight over the time course. Values that were significantly different (P � 0.05)from those of the control are indicated by an asterisk.

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the glomerular dysfunction may be predominant (10, 31). Weshowed that Stx2 is more toxic to human glomerular cells thanto tubular cells. This supports studies that have failed to findcases of renal disease in the absence of microvascular andhemolytic symptoms following bloody diarrhea caused bySTEC (37, 52). In contrast to the polyuria and dilute urine ofthe mice challenged with Stx2 plus LPS, most HUS patients areoligoanuric (66). However, two case reports detail Shiga toxin-mediated HUS associated with polyuria and persistent produc-tion of isosmotic urine (29, 57). Thus, direct tubular insult byStx2 may participate in HUS-associated renal failure, and wehypothesize that collecting duct damage may facilitate dehy-dration that contributes to worse outcomes in some patients(24, 44). Although not without technical difficulty (66), testingprodromal HUS patients for urine-concentrating defects mayidentify those with severe disease and at greater risk for dehy-dration with a worse outcome.

The findings reported here have specific implications forunderstanding and treating human HUS. In contrast to theother human endothelial and epithelial cells described pre-viously (22, 25–27, 70), the response of the human glomer-ular filtration barrier to Stx2 appeared distinctly noninflam-matory. Despite causing a ribotoxic stress response in the

human glomerular cells, Shiga toxin did not increase releaseof the inflammatory mediators tested. This may explain whyHUS patients often report a fever during the diarrheal pro-drome, presumably due to increased circulating inflamma-tory mediators, but are afebrile upon HUS presentation (36,50, 66, 69, 70). However, Stx2 mediated a decline in humanpodocyte VEGF release. As decreased podocyte VEGF hasbeen demonstrated to cause renal glomerular thromboticmicroangiopathy in mice and in human patients, this mech-anism of Shiga toxin-mediated reduction in VEGF may con-tribute to HUS clinically (14). Finally, we have also de-scribed how blocking apoptosis can rescue direct Stx2 renalinsult in vivo and how Stx2-induced human glomerularendothelial and podocyte apoptosis can be inhibited by the sameantiapoptotic agent in vitro. Thus, a clinically approvedcaspase inhibitor may be able to block Shiga toxin-mediatedapoptosis in patients (9, 31, 71).

ACKNOWLEDGMENTS

This research was supported by U.S. Public Health Service grantsAI024431 and AI075778 (T.G.O.) and Wellcome Trust Fellowship075731 (S.C.S.).

We thank Sanford Feldman for expert animal advice.

FIG. 8. Quantification of renal apoptosis, renal failure, urine osmolality, and weight loss in mice injected with Stx2 plus LPS alone or with thenonselective caspase inhibitor Q-VD-OPH (WVDOPH) or with the DMSO vehicle alone. Mice challenged with Stx2 plus LPS and treated withthe caspase inhibitor demonstrated decreased numbers of apoptotic nuclei on TUNEL-stained renal tissue sections (A), increased urine osmolality(B), decreased BUN levels (C), and less weight loss (D) than mice challenged with Stx2 plus LPS alone. Values that were significantly differentare indicated as follows: *, P � 0.05 compared to the values for the control; **, P � 0.05 compared to the values for mice challenged with Stx2plus LPS alone.

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