Protection of Human Podocytes from Shiga Toxin 2-InducedPhosphorylation of Mitogen-Activated Protein Kinases and Apoptosisby Human Serum Amyloid P Component

Anne K. Dettmar,a Elisabeth Binder,b Friederike R. Greiner,a Max C. Liebau,c,d,e Christine E. Kurschat,d,e Therese C. Jungraithmayr,b

Moin A. Saleem,f Claus-Peter Schmitt,g Elisabeth Feifel,h Dorothea Orth-Höller,i Markus J. Kemper,a Mark Pepys,j Reinhard Würzner,i

Jun Oha

Department of Pediatrics, University Medical Center Hamburg-Eppendorf, Hamburg, Germanya; Department of Pediatrics, Innsbruck Medical University, Innsbruck,Austriab; Department of Pediatrics, University of Cologne, Cologne, Germanyc; Nephrological Research Laboratory, University of Cologne, Cologne, Germanyd; ExcellenceCluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germanye; Children’s Renal Unit and Academic Renal Unit, Universityof Bristol, Bristol, United Kingdomf; Department of Pediatrics, University of Heidelberg, Heidelberg, Germanyg; Department of Physiology and Medical Physics, InnsbruckMedical University, Innsbruck, Austriah; Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austriai; Centre for Amyloidosis and AcutePhase Proteins, University College London, London, United Kingdomj

Hemolytic uremic syndrome (HUS) is mainly induced by Shiga toxin 2 (Stx2)-producing Escherichia coli. Proteinuria can occurin the early phase of the disease, and its persistence determines the renal prognosis. Stx2 may injure podocytes and induce pro-teinuria. Human serum amyloid P component (SAP), a member of the pentraxin family, has been shown to protect against Stx2-induced lethality in mice in vivo, presumably by specific binding to the toxin. We therefore tested the hypothesis that SAP canprotect against Stx2-induced injury of human podocytes. To elucidate the mechanisms underlying podocyte injury in HUS-asso-ciated proteinuria, we assessed Stx2-induced activation of mitogen-activated protein kinases (MAPKs) and apoptosis in immor-talized human podocytes and evaluated the impact of SAP on Stx2-induced damage. Human podocytes express Stx2-bindingglobotriaosylceramide 3. Stx2 applied to cultured podocytes was internalized and then activated p38� MAPK and c-Jun N-termi-nal kinase (JNK), important signaling steps in cell differentiation and apoptosis. Stx2 also activated caspase 3, resulting in anincreased level of apoptosis. Coincubation of podocytes with SAP and Stx2 mitigated the effects of Stx2 and induced upregula-tion of antiapoptotic Bcl2. These data suggest that podocytes are a target of Stx2 and that SAP protects podocytes against Stx2-induced injury. SAP may therefore be a useful therapeutic option.

Shiga toxin (Stx)-associated hemolytic uremic syndrome(HUS) is a life-threatening disease characterized by hemolytic

anemia, thrombocytopenia, and renal failure (1). It is the mostfrequent cause of acute renal failure in childhood and is usuallycaused by Stx2-producing enterohemorrhagic Escherichia coli(EHEC), but to a lesser extent by Stx1-producing EHEC (2). Stx1and Stx2 induce apoptosis and activate stress response pathways inendothelial cells (3, 4), causing the clinical picture of thromboticmicroangiopathy (TMA) (1). A direct toxic effect of Stx2 on non-endothelial cells has recently been suggested. Stx2 binds to suscep-tible cells via the unique toxin-binding glycosphingolipid receptorgalactose-�1,4-glucose-ceramide (Gb3), which is generated byGb3 synthase (5, 6). After internalization, Stx2 activates variousintracellular stress pathways, such as the endoplasmic stress re-sponse or ribotoxic stress response (3). This may lead to apoptosisvia activation of various cell death signals (7), such as downregu-lation of antiapoptotic Bcl2 (8), activation of mitogen-activatedprotein kinases (MAPKs) p38� and c-Jun N-terminal kinase(JNK) (7, 9), and caspase 3-dependent apoptosis (10, 11).

In endothelial cells, this leads to TMA, which can result in acuterenal failure and neurological injury (1, 12). It has been suggestedthat neurological involvement may be due to deficiency or im-paired function of an Stx2-neutralizing factor (13) in affected pa-tients, in which case plasma supply and exchange might be bene-ficial. Various candidates for such a neutralizing factor have beeninvestigated, and serum amyloid P component (SAP) appears tobe the most promising (14, 15). SAP is one member of the pen-traxin family; the other member is C-reactive protein (CRP) (16).

Although the gene-coding, amino acid sequences, homopentam-eric molecular assembly, and calcium-dependent ligand-bindingproperties of SAP are phylogenetically conserved, the baselineplasma concentration, acute-phase behavior, and binding affinityof SAP proteins vary between species. No deficiencies or structuralvariants of human SAP protein have yet been described, and itsphysiological functions are therefore not completely understood.Nevertheless, there is experimental evidence that SAP can contrib-ute to host defense against certain bacterial infections (17). SAPneutralizes Stx2, but not Stx1, in vitro, and human SAP protectsmice from Stx2-induced disease (14). The Stx2-neutralizing effectis not present in the sera of nonhuman species tested so far (15),consistent with the much higher binding avidity of human SAPthan mouse SAP to other known ligands (18).

Microalbuminuria is seen in rats soon after intraperitoneal in-jection of Stx2 (19), and it is also an early sign of renal involvementin patients with EHEC-HUS. Following recovery from the acutephase of the disease, proteinuria may persist; such persistence cor-relates with a poor long-term renal prognosis (20–22). Injury of

Received 12 February 2014 Accepted 14 February 2014

Published ahead of print 24 February 2014

Editor: S. R. Blanke

Address correspondence to Jun Oh, j.oh@uke.de.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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podocytes has emerged as the key mechanism underlying this glo-merular dysfunction (23) and may have a critical impact on thecourse of the disease and renal outcome. Severe podocyte injuryresults in impaired actin cytoskeleton dynamics, foot process ef-facement, and proteinuria (24, 25). There is increasing evidencethat both Stx1 and -2 injure podocytes. Injection of Stx1 results infoot process effacement in animal models (26). In one child withEHEC-induced HUS, Stx1 was detected in mesangial and endo-thelial cells, as well as in podocytes (27). Stx1 binds to Gb3 inpodocytes, resulting in inhibition of protein synthesis and celldeath (28). Stx2 results in activation of mitogen-activated proteinkinases and actin remodeling in podocytes (29, 30).

We therefore hypothesized that human SAP neutralizes someof the harmful effects of Stx2 on human podocytes. The rationalewas to gain detailed information on the glomerular damagecaused by binding of Stx2 to podocytes early in HUS.

MATERIALS AND METHODSCell culture and media. Conditionally immortalized human podocyteswere produced by transfection with the temperature-sensitive simian vi-rus 40 (SV40) T gene (31). Cells were seeded on collagen type I-coatedplates (BD Bioscience, Franklin Lakes, NJ, USA) at 32°C. RPMI 1640culture medium (PAA, Pasching, Austria) was supplemented with 100U/ml penicillin, 0.1 mg/ml streptomycin, glutamine, and insulin, trans-ferrin, and sodium selenite mixture (ITS) (Sigma, St. Louis, MO, USA)and with 10% fetal bovine serum (Calbiochem via Merck, Darmstadt,Germany). To induce growth arrest and differentiation, podocytes weremaintained under nonpermissive conditions at 37°C for 2 to 3 weeks atabout 70% confluence.

Stx2. Stx2 was provided courtesy of H. Karch, Institute of Hygiene,University of Münster, Münster, Germany. Purification of Stx2 was per-formed as described previously (32). To investigate the effects of Stx2 onhuman podocytes, cell cultures were exposed to medium containing Stx2at a final concentration of 1.5 or 15 ng/ml for up to 48 h. Optimal workingconcentrations of Stx2 were identified with a dose-mortality curve (datanot shown). All studies were performed before cell layers exceeded 80%confluence. RPMI 1640 was used as the incubation medium and wassupplemented with 10% fetal calf serum (FCS), which does not containhuman SAP, with or without the addition of purified SAP, or with 10%human serum instead of FCS. The medium was provided to the cellsbefore Stx2 was added.

Coincubation with SAP. Isolated �99.9% pure, intact, fully func-tional human SAP from healthy blood donors was purified and charac-terized as described previously (33) and added to FCS-free incubationmedium at a final concentration of 3 mg/liter. Alternatively, the mediumwas supplemented with 10% (vol/vol) serum from one healthy donor witha known SAP concentration of 47 mg/liter (final concentration of SAP, 4.7mg/liter during incubation) or from a pool of normal human sera (NHS)from healthy laboratory staff.

SAP-free incubation. SAP-free incubations were performed with 10%FCS or SAP-depleted NHS from the individual mentioned above. Deple-tion of SAP in NHS was performed using sepharose-phosphoethano-lamine (34).

Lipid analysis for detection of Gb3. Neutral and acidic lipids wereseparated by anion-exchange chromatography using DEAE-Sephadex ac-cording to published methods (35). About 107 differentiated podocyteswere homogenized in 1 ml of water using the Homogenizator Precellys 24(Peqlab, Erlangen, Germany) at 6,500 rpm for 30 s. After extraction oflipids and separation of cell debris by solvent filtration, the sample wasevaporated in a stream of nitrogen. Interfering glycerolipids were de-graded by alkaline hydrolysis with 2.5 ml of 100 mM sodium hydroxide inmethanol for 2 h at 37°C. This was followed by neutralization with 15 �l ofacetic acid, and the lipid extract was desalted with reversed-phase C18

(RP-18) chromatography. High-performance thin-layer chromatography

(TLC) Silica Gel 60 plates (Merck) were washed and dried before applica-tion of the lipid fraction. Chloroform-methanol-water (70:30:4 [vol/vol/vol]) was used as the solvent system. Standard lipids (Neutral Glycosph-ingolipid Mix; Biotrend, Cologne, Germany) were applied to the TLCplates as markers. The plates were sprayed with a phosphoric acid-coppersulfate reagent {15.6 g of CuSO4(H2O)5 and 9.4 ml of H3PO4 (85% [wt/vol]) in 100 ml of water} and charred at 180°C for 10 min to detect lipidbands (36). Quantification was achieved with densitometry using theTLC-Scanner 3 (Camag, Berlin, Germany) at a wavelength of 595 nm.

Immunofluorescence for detection of Gb3 and Hoechst staining forassessment of apoptosis. Podocytes were cultured on glass slides, fixedwith 4% paraformaldehyde, and permeabilized with 0.05% Triton X-100.Immunofluorescence staining was performed with an anti-Gb3 primaryantibody (BD Pharmingen, Heidelberg, Germany) and DyLight 649-con-jugated AffiniPure donkey anti-mouse IgM (Jackson ImmunoResearch,West Grove, PA, USA) as the secondary antibody. Slides were thenmounted with a commercially available antifade kit (Invitrogen, Darm-stadt, Germany) and examined with immunofluorescence microscopy(Axiovert 200 with Apotome System; Zeiss, Jena, Germany). No stainingwas observed with an isotype-matched naive primary antibody (data notshown). Apoptosis was identified with Hoechst 33258 staining (Calbi-ochem via Merck) according to the manufacturer’s instructions. The per-centage of apoptotic cells was determined by counting condensed nucleiin 10 different visual fields. Images were taken, and the cells were countedby blinded observers.

Stx2 internalization and morphological analysis with confocal mi-croscopy. Podocytes were cultured in 8-well chambered cover glasses(Nalge-Nunc International, Rochester, NY, USA). Oyster 488-labeledStx2 (200 ng/ml) was added to the cells. Stx2 labeling was performed withan Oyster 488 antibody labeling kit (Luminartis, Münster, Germany).Staining with Oyster 488-labeled isotype-matched primary antibodyshowed only minor background staining (data not shown). Confocal laserscanning microscopy was performed with a TCS SP5 microscope (Leica,Mannheim, Germany) equipped with an HCX PL APO lambda blue 63.0/1.20 water UV objective. Images were processed with LAS AF software(version 2.4.1; Leica, Mannheim, Germany).

Western blot analysis. Cells were harvested using tissue protein ex-traction buffer (Thermo Scientific 78510) supplemented with completeMini Inhibitor Cocktail (Roche, Basel, Switzerland), 1 mM calyculin A(Cell Signaling Technologies, Danvers, MA, USA), 1 mM NaF, and 1 mMNaVO3. After centrifugation, the supernatant was collected and supple-mented with 4� NuPAGE LDS Sample Buffer (Invitrogen) and 0.1 Mdithiothreitol (DTT). After boiling, the samples were loaded on NuPAGENovex 4% to 12% Bis-Tris Gel (1.0 mm; Invitrogen). IRDye (Li-Cor,Lincoln, NE, USA) was used as a marker. Proteins were transferred ontoImmobilon-FL polyvinylidene difluoride (PVDF) membranes (Millipore,Billerica, MA, USA) and electrophoresed with an XCell SureLock Mini-Cell electrophoresis system (Invitrogen). The membranes were incubatedwith blocking buffer (Li-Cor) and primary antibody overnight at 4°C.Primary antibodies against phospho-p38� (p-p38�), p-JNK, Bcl2, extra-cellular signal-regulated kinase 1/2 (ERK1/2), and cleaved caspase 3 werepurchased from Cell Signaling Technologies; GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was from Santa Cruz Biotechnology (SantaCruz, CA, USA); and p38� was from R&D Systems (Wiesbaden, Ger-many). The following secondary antibodies were used: IRDye 800CWagainst mouse IgG and goat or IRDye 680RD against rat and rabbit (Li-Cor). Fluorescence was measured with an Odyssey Clx Blot Scanner andImage Studio software (Li-Cor). All experiments were performed at leastthree times in duplicate. Blots were quantified with ImageJ 1.47v. Graphswere made and statistical analysis was performed with Prism 5.0b.

Conventional PCR. Cells were harvested using complete medium andtrypsin-EDTA and then centrifuged at 300 � g for 10 min at 15°C. Thepellet was washed with phosphate-buffered saline (PBS) and centrifuged,and RNA was then isolated using an innuPrep RNA Mini Kit (AnalytikJena, Biometra, Jena, Germany). The RNA concentration was measured

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with a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). To-tal RNA (100 ng/�l) was used for cDNA synthesis. PCR was accomplishedusing the Mesa Green qPCR MasterMix Plus for SYBR Assay (Eurogentec,Cologne, Germany) with an Applied Biosystems StepOnePlus system andStepOnePlus 2.0 software for evaluation. The primer sequences for Gb3synthase were 5=-CACGACGCACGAGGCCATGA-3= and 5=-CCCGGGCCCTCAATCTTGCC-3= (Life Technologies via Invitrogen). The PCRproduct was loaded onto a 1.5% agarose gel for size determination. ABiomol 100-bp DNA Ladder (Biomol GmbH, Hamburg, Germany) wasused as a marker.

Caspase 3 assay. An EnzChek Caspase 3 Assay Kit 2 (Invitrogen) wasused according to the manufacturer’s instructions. Caspase 3 activity wasmeasured by the absorbance at 535 nm using a microplate reader (GeniosPlus; Tecan, Maennedorf, Switzerland). The protein content was mea-sured by the bicinchoninic acid method (Thermo Scientific). The amountof converted enzyme was related to the total amount of protein, and aratio of sample and untreated control was calculated to allow better com-parability of each experiment. Controls were normalized to a ratio of 1.

Statistical analysis and images. Statistical analyses were done forcaspase 3 activity, the densitometry of Western blots, and counting ofnuclei in Hoechst staining, using a paired Wilcoxon test (with Prism 5) ora paired t test (with SPSS). P values of �0.05 were regarded as significant.Controls were normalized to a ratio of 1. All experiments were performedat least three times in duplicate. Images were arranged in MicrosoftPowerPoint, and minimal processing of brightness, contrast, and colorbalance was carried out.

RESULTSGb3 expression by human podocytes. We started to investigatethe effects of Stx2 on podocytes by determining whether podo-cytes express Stx2-binding Gb3 and the corresponding synthase.We therefore performed PCR for Gb3 synthase mRNA, Gb3 im-munofluorescence staining, and lipid analyses. Our mRNA stud-ies found that human podocytes show transcription of the Gb3synthase gene (Fig. 1A). Lipid analyses and immunofluorescencestaining confirmed that podocytes can synthesize Gb3, which was

mostly localized along the cell membrane and in the cytosol (Fig.1B and C).

Internalization of Stx2 by human podocytes. To test whetherStx2 could be internalized by human podocytes, we performedStx2 stimulation experiments. Confocal microscopy revealedbinding and immediate uptake of fluorescence-labeled Stx2 bydifferentiated human podocytes. After 40 min of exposure, themajority of the Stx2 was already located within the cell, where itwas widely distributed throughout the cytoplasm (Fig. 2).

Blockage by SAP of Stx2-induced phosphorylation of MAPKp38� and JNK. SAP has emerged as a potential blocker of Stx2effects on various cell types. We therefore tested whether SAP hadbeneficial effects on Stx2-stimulated human podocytes by inves-tigating the extent to which MAPK phosphorylation was evidentin cell lysates. Stx2 (15 ng/ml) induced phosphorylation of MAPKp38� at 60 min. Activation was highest under the influence of 15ng/ml; phosphorylation was maximal at 180 min and was stillpresent at 480 min. Human SAP, which was added at the sametime in physiological concentrations (3 mg/liter), significantly in-hibited Stx2-dependent phosphorylation of p38� at 180 and 480min (Fig. 3A). Similarly, Stx2 induced phosphorylation of JNK at60, 180, and 480 min. SAP significantly inhibited this phosphory-lation at 60 and 180 min (Fig. 3B). In contrast, MAPK p38�,MAPK, and ERK1/2 were not activated at any stage of incubation(data not shown).

Inhibition of Stx2-induced caspase 3 activation by SAP. Tomeasure apoptosis, we determined caspase 3 activity with an en-zyme-linked immunosorbent assay (ELISA). After 24 h and 48 h,Stx2 (1.5 ng/ml) induced significantly greater caspase 3 activity inpodocytes than in control cells (10.5 � 11.1 arbitrary units [AU]versus 1.3 � 0.9 AU; P � 0.05) (Fig. 4A). When the medium wassupplemented with SAP, the Stx2-induced increase in caspase 3activity was significantly diminished (3.7 � 3.5 AU versus 10.5 �

FIG 1 Expression of Gb3 synthase and Gb3 in human podocytes. (A) Expression of Gb3 synthase in untreated human podocytes (huPo) by conventional PCR(lane 3). (B) Lipid analysis for the presence of Gb3 in untreated human podocytes. (C) Localization of Gb3 in differentiated untreated podocytes along themembrane (arrows) and within the cytoplasm by immunofluorescence (pink) and in cell nuclei (blue). All experiments were repeated at least four times inindependent experiments (scale bars � 15 �m).

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11.1 AU; P � 0.05) (Fig. 4A) but was still higher than in the controlgroup (1.3 � 0.9; P � 0.05) (Fig. 4A). Consistent with these re-sults, the quantity of cleaved caspase 3 was elevated and reached apeak after 24 h of incubation with Stx2. When SAP was added tomedium containing Stx2, cleaved caspase 3 was not detected after24 or 48 h (Fig. 4B).

Prevention of Stx2-induced condensation of nuclei by SAP.Following Stx2 incubation (1.5 ng/ml) for 24 h, Hoechst stainingdemonstrated significantly enhanced formation of fragmentednuclei and condensed chromatin (39.5% � 16.4% versus14.0% � 5.8%; P � 0.05). Addition of SAP inhibited the Stx2-induced apoptosis, so that only 16.7% � 7.4% of podocyte nucleishowed condensed chromatin (P � 0.05 versus Stx2; P 0.05versus control) (Fig. 5).

Upregulation of Bcl2 expression by SAP. Endoplasmic stressenhances the expression of CHOP, which in turn diminishes anti-apoptotic Bcl2, so we investigated this effector molecule, which iscritical for cell survival. We observed significantly enhanced Bcl2expression after coincubation of Stx2 with SAP at 60 and 180 min(Fig. 6, top row on right) compared to incubation without SAP(Fig. 6, top row on left). There was no significant increase in Bcl2at any time point after incubation with Stx alone.

DISCUSSION

Our study demonstrates the protective effect of human SAP onStx2-injured podocytes. Our data are consistent with the results ofprevious studies that have analyzed the effects of Stx2 on human(29) and murine (29, 30) podocytes. No previously reported stud-ies on the effects of Stx2 on human podocytes have provided anynovel therapeutic information. We show here for the first time

that direct interactions between Stx2 and human SAP have novelfeatures and important functional effects in vitro. These mayprove to be clinically significant in future.

Although proteinuria is not a major symptom in patients withEHEC-HUS, increased glomerular protein loss influences the re-nal outcome of patients and is often associated with a progressivedecline in renal function (20). Even though podocytes may not bethe main target of Stx2-induced renal damage, the data of Morigiet al. indicate that Stx2 can induce vasoactive endothelin 1 (ET-1)expression in cultured murine podocytes, which emphasizes thepossible important cross talk between endothelial cells and podo-cytes (30). Vascular endothelial growth factor (VEGF) is a proteinsecreted by podocytes that is necessary for the survival of endo-thelial cells, podocytes, and mesangial cells. Stx2 mediated a de-cline in VEGF release by human podocytes. As decreased podocyteVEGF has been demonstrated to cause glomerular thromboticmicroangiopathy in mice and in humans, the mechanism of Stx2-mediated reduction of VEGF may contribute to HUS clinically(37). Substantial deterioration of the glomerular barrier can occurin HUS and is of particular prognostic importance due to the lowregenerative capacity of podocytes.

Stx2 is internalized and routed to the endoplasmic reticulum(ER) (3) and induces multiple signaling pathways. Some of thesecontribute to apoptosis, such as activation of MAPKs and caspase3 or downregulation of antiapoptotic Bcl2 (7). We confirmed thatimmortalized human podocytes express Gb3 (which is requiredfor Stx2 binding and uptake) and observed rapid binding to thecell membrane and internalization of Stx2. Once internalized,Stx2 induced phosphorylation of p38� and JNK and apoptosis viathe caspase 3 pathway. Such mechanisms have been previously

FIG 2 Internalization of labeled Stx2 (white) in podocytes after 40 min of incubation. Distribution is predominantly within the cytoplasm. No Stx2 staining wasfound in the cell nucleus. The cell outline (dotted lines) and nucleus (dashed line) are marked (scale bar � 15 �m). The experiment was repeated at least threetimes independently.

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described in endothelial cells (3) and in podocytes (29). In humanpodocytes, the nonselective caspase inhibitor Q-VD-OPH rescuedover 75% of the cytotoxic Stx2 effects (29).

The MAPK family regulates a wide variety of cellular processes(inflammation, cell cycle regulation, apoptosis, differentiation, se-nescence, and tumorigenesis). Both p38� and JNK contribute toapoptosis (38, 39). Inhibiting the activation of p38� can decreaseStx2-induced apoptosis in intestinal epithelial cells. Apoptosis ismainly initiated by caspases, which are proteases and effector mol-

ecules in programmed cell death. Caspase 3 is a key enzyme in thiscascade (40), which is activated in the cytoplasm by an increase incalcium concentration. Calcium is released from the ER after pro-longed endoplasmic stress (3). Persistent ER stress also inducesdownregulation of Bcl2. The Bcl2 family comprises importantregulators of apoptosis. It prevents apoptosis in various ways, suchas stabilization of mitochondria and inhibition of caspase activa-tion (41).

In recent years, investigators have tried to identify the Stx2-

FIG 3 Western blot and densitometry of phosphorylation of MAPK p38� and JNK. P-p38� (A) and p-JNK (B) after treatment with 15 ng/ml Stx2 and incombination with SAP (3 mg/liter) after 60, 180, or 480 min. Below are shown loading controls with GAPDH. *, P � 0.05. Means and standard errors of the meansof five independent experiments, performed in duplicate, are shown.

FIG 4 Caspase 3 activity and cleaved caspase 3. (A) Caspase 3 activity (in AU) after Stx2 incubation (1.5 ng/ml) for 24 or 48 h compared to untreated cells andafter coincubation with SAP (3 mg/liter) as evaluated by ELISA. For statistical analysis, the values at 24 and 48 h were compared. (B) Amounts of cleaved caspase3 after Stx2 incubation with or without addition of SAP after 24 and 48 h. Below is shown loading control with GAPDH. *, P � 0.05. Means and standard errorsof the means of four independent experiments, performed in duplicate, are shown.

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neutralizing factor in normal human serum (13). Armstrong andcolleagues identified SAP as a potential neutralizing factor (14).SAP is synthesized in the liver and circulates in the blood at levelsof 8 to 55 mg/liter (42). It is a universal constituent of amyloiddeposits (due to its specific calcium-dependent binding to amy-loid fibrils) and contributes to the pathogenesis and persistence ofthese deposits (43). SAP is also a constituent of the extracellularmatrix and is present in the glomerular basement membrane (44)and the microfibrillar mantle of elastic fibers throughout the body(45). However, the function of human SAP is not fully under-stood. Despite reports that it can opsonize in vitro (46), binding ofSAP to bacteria is potently antiopsonic (17). Furthermore, theubiquity of SAP in amyloid deposits is definitely not opsonic, sincethese deposits are almost universally ignored by phagocytic cells(43). On the other hand, SAP clearly contributes to host resistanceagainst bacterial infections (17, 44, 45), although the mechanismsare still unknown. However, human SAP is known to bind to Stx2,neutralizing its toxic effects (14, 15). We showed that SAP mark-

edly reduced MAPK activation and apoptosis of Stx2-injuredpodocytes, implying that SAP may protect podocytes from severeStx2-induced damage. These observations in cultured podocytessuggest a mechanism by which SAP may confer clinical protectionon patients with an EHEC infection.

Our findings raise the question as to whether susceptibility toEHEC-HUS is related to plasma SAP levels and whether new ther-apeutic approaches may emerge with the availability of recombi-nant SAP, which has already been administered to patients withpulmonary fibrosis (47). The efficacy of plasma therapy in EHEC-HUS is currently controversial (22). There is evidence that SAPmay not be able to prevent thrombotic microangiopathy becauseStx2 binds to human neutrophils by a mechanism independent ofGb3 (48), but our data suggest that SAP may be able to attenuatethe toxic effects of Stx2 on podocytes. Prevention of proteinuriacould have a positive influence on the long-term renal outcome.

In conclusion, Stx2 induces activation of MAPK and apoptosisin human podocytes. Such effects may trigger progressive deteri-

FIG 5 Apoptosis, measured via the amount of condensed or apoptotic nuclei after Stx2 incubation. (A) Untreated control. (B) Nuclei of podocytes aftertreatment with 1.5 ng/ml Stx2. (Left) Typical enclosures/cysts (arrowheads) and indentations (arrows). (Right) An apoptotic body. (C) Content of condensednuclei after Stx2 treatment compared to control or coincubation of Stx2 and SAP (3 mg/liter). *, P � 0.05. Means and standard deviations of three independentexperiments are shown.

FIG 6 Induction of Bcl2 after Stx2 treatment (15 ng/ml) with or without coincubation with SAP (3 mg/liter) after 60, 180, and 480 min. Below is shown loadingcontrol with GAPDH. *, P � 0.05. Means and standard errors of the means of five independent experiments, performed in duplicate, are shown.

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oration of renal function in patients with EHEC-HUS. The pro-tective effect of SAP on Stx2-stressed podocytes may be a potentialtherapeutic option for EHEC-HUS patients by administration ofSAP itself or Stx-binding derivatives.

ACKNOWLEDGMENTS

This study was supported by the Georg und Jürgen Rickertsen Stiftung,Hamburg, Germany.

We thank G. Gstraunthaler (Innsbruck, Austria) for laboratory spaceand helpful scientific discussions. We thank H. Karch and M. Bielasze-wska, Münster, Germany, for providing Stx2. We owe special thanks to A.Rosales and S. Ehrlenbach (Innsbruck, Austria) and S. Brodesser (Co-logne, Germany) for supplementary data.

M.P. reports grants from the United Kingdom Medical ResearchCouncil and work independent of the National Institute of Health Re-search. M.P. also has a patent on work done with Pentraxin TherapeuticsLtd. C.E.K. and M.C.L. report grant funding from the Deutsche For-schungsgemeinschaft and Shire HGT during the conduct of this study.The other authors have nothing to disclose.

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