1

The Effects of Shiga Toxin Type 2 on a Bioengineered 3D Model of Human Renal Tissue 1

Teresa M. DesRochersa*

, Erica Palma Kimmerlinga, Dakshina M. Jandhyala

bc, Wassim El-2

Jounid, Jing Zhou

d, Cheleste Thorpe

c, John M. Leong

b, and David L. Kaplan

a# 3

Department of Biomedical Engineering, Tufts University, Medford, MA, USAa; Department of 4

Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, USAb; 5

Division of Geographic Medicine and Infectious Disease, Tufts Medical Center, Boston, MA, 6

USAc, Harvard Medical School, Boston, MA

d 7

Running Head: Exposure of 3D Renal Tissue to Shiga Toxin Type 2 8

#Address correspondence to David L. Kaplan, david.kaplan@tufts.edu 9

*Present Address: Teresa M. DesRochers, KIYATEC, Inc., Greenville, SC, USA 10

11

IAI Accepts, published online ahead of print on 13 October 2014Infect. Immun. doi:10.1128/IAI.02143-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

2

Abstract 12

Shiga toxins (Stx) are a family of cytotoxic proteins that can cause Hemolytic Uremic Syndrome 13

(HUS), a thrombotic microangiopathy, following infections by Shiga toxin producing 14

Escherichia coli (STEC). Renal failure is a key feature of HUS and a major cause of childhood 15

renal failure worldwide. There are currently no specific therapies for STEC-associated HUS and 16

the mechanism of Stx-induced renal injury is not well understood primarily due to a lack of fully 17

representative animal models and an inability to monitor disease progression on a molecular or 18

cellular level in humans at early stages. 3D tissue models have been shown to be more in vivo-19

like in their phenotype and physiology than 2D cultures for numerous disease models including 20

cancer and polycystic kidney disease. It is unknown if exposure of a 3D renal tissue model to Stx 21

will yield a more in vivo-like response than 2D cell culture. In this study, we have characterized 22

Stx2-mediated cytotoxicity in a bioengineered 3D human renal tissue model previously shown to 23

be a predictor of drug-induced nephrotoxicity and compared its response to Stx2 exposure in 2D 24

cell culture. Our results demonstrate that although many mechanistic aspects of cytotoxicity were 25

similar between 3D and 2D, treatment of the 3D tissues with Stx resulted in an elevated secretion 26

of the kidney injury marker 1 (Kim-1) and the cytokine IL-8 compared to the 2D cell cultures. 27

This study represents the first application of 3D tissues for the study of Stx-mediated kidney 28

injury. 29

30

3

Introduction 31

Shiga toxin (Stx) is a family of AB5 protein cytotoxins expressed by Shigella dysenteriae 32

and Shiga toxin producing Escherichia coli (STEC) (1). Infections by Stx producing organisms 33

can result in the onset of a disease termed the Hemolytic Uremic Syndrome (HUS) that is 34

characterized by renal failure, hemolytic anemia, and thrombocytopenia. Shiga toxins are 35

considered the key STEC virulence factor required for the onset of HUS with Stx2 being the 36

toxin most often associated with severe disease (2-4). In the United States, HUS is the most 37

common cause of acute renal failure in children under 5 years of age (5-7). The majority of HUS 38

cases are caused by STEC, and in the U.S., the O157:H7 serotype is most frequently associated 39

with this severe disease (7). Although the underreporting of cases precludes precise calculation 40

of the incidence of STEC infection, the CDC has estimated that 73,000 cases of O157:H7 41

infection occur annually in the U.S. (http://www.cdc.gov/pulsenet/pathogens/ecoli.html) (8). 42

Approximately two-thirds of children who develop HUS will require dialysis (9), and 43

many will go on to permanent renal insufficiency or renal failure. Excluding supportive therapies 44

such as hemodialysis and volume expansion with intravenous fluids, there are no specific 45

therapies for STEC-associated HUS. While Stxs are the primary STEC pathogenicity factors 46

responsible for the onset of HUS by STEC, the mechanism(s) by which Stxs promote this deadly 47

thrombotic microangiopathy in patients is still not well-understood. 48

Shiga toxins are AB5 toxins composed of 5 B-subunits which bind to the host-cell surface 49

receptor globotriaosylceramide (Gb3), and a single A-subunit with a specific N-glycosidase 50

activity that affects protein synthesis (10-12). Following binding to Gb3, Stx undergoes 51

endocytosis and retrograde trafficking to the endoplasmic reticulum (ER) where the A-subunit 52

4

separates from the B-subunits and is then transported across the ER membrane to the cytosol 53

(13). Once in the cytosol, the A-subunit catalyzes the cleavage of an adenine from the 28S rRNA 54

thus inhibiting protein synthesis and activating pro-inflammatory signaling (11,12). Certain types 55

of protein synthesis inhibitors, including Stx, which interact with and/or damage the 28S rRNA, 56

have been shown to activate the mitogen activated protein kinases (MAPKs), jun-N-terminal 57

kinases (JNKs) and p38 (14,15). This activation of MAPKs by Stx and other protein synthesis 58

inhibitors has been termed the ribotoxic stress response or RSR. Stx-mediated activation of the 59

RSR has been shown to contribute to induction of cytokines such as IL-8 at both the 60

transcriptional and post-transcriptional level in intestinal epithelial cells (16,17). 61

While renal failure is a key feature of HUS, and renal samples from patients with HUS show 62

extensive damage (2), the temporal pattern of Stx-mediated renal damage has not been 63

determined and it remains unclear which renal cells are the primary targets during disease. This 64

is largely due to an inability to monitor renal damage on a molecular and cellular level during the 65

course of disease in human patients and to access patients during the early stages of disease 66

progression. To overcome these limitations, investigators have used either animal models (18,19) 67

or 2D monolayer cell cultures to study Stx-mediated disease (20-22). 2D cell culture systems, 68

while simple and low in cost, lack the architecture required to play a major role in cell-cell and 69

cell-ECM interactions, and are thus incapable of recapitulating the complexity of the in vivo 70

environment (23,24) as well as inadequate at predicting cellular toxicity (25,26). Studies using 71

2D human renal proximal tubule cells have demonstrated that Stx increases apoptosis in a time- 72

and dose-dependent manner along with the secretion of IL-1β, IL-6, and IL-8, inhibition of 73

protein synthesis, and water absorption (20,21,27,28). Animal models are incapable of fully 74

recapitulating human disease for numerous reasons, including differences in physiology and 75

5

experimental conditions (29). Importantly, differences in Stx receptor expression patterns 76

between mice and humans contribute to the failure of murine models of HUS to completely 77

mimic human sequelae (30). While nonhuman primates have been shown to recapitulate the 78

clinical manifestation of HUS and respond to therapeutics (31), these models are expensive and 79

require specialized staff and facilities. 80

The advantages of using 3D human tissues over 2D cell culture have already been 81

demonstrated in studies of cancer progression, signal transduction, and drug toxicity (23,32-35). 82

They have revealed advantages of 3D tissue models over 2D cell cultures due to differences in 83

cell-cell signaling, ECM interactions, ECM composition, gene expression, and epigenetic gene 84

regulation (23,36). Bioengineered 3D kidney tissues produced in our lab using human cells have 85

been shown to better reflect human renal physiology than 2D cell culture (37). These tissues are 86

composed of human telomerase reverse transcriptase (hTERT) immortalized human renal 87

proximal tubule cells embedded within a hydrogel. Following 2 weeks of culture, they form an 88

interconnecting network of tubules (Fig. 1A). These bioengineered kidney tissues are capable of 89

revealing toxic effects of both acute and chronic exposure to insult as they are viable for up to 8 90

weeks, unlike 2D cell cultures, which are viable and relevant for only days before increased cell 91

density results in cell death. 92

The goal of this study was to adapt our 3D bioengineered human renal tissue model for the study 93

of Stx-induced renal toxicity and to compare the responses with responses by 2D cultures. We 94

examined cytotoxicity, activation of the RSR, renal injury biomarker expression, and 95

inflammatory cytokine secretion. While the overall cytotoxicity profile, protein synthesis 96

inhibition, and RSR activation were similar between 2D and 3D cultures, there were differences 97

in the IC50 at different time points, the secretion of renal injury biomarkers, and cytokine 98

6

secretion between the two systems. Importantly, treatment of the 3D tissues with Stx resulted in 99

an elevated secretion of the kidney injury marker 1 (Kim-1) and the cytokine IL-8 compared to 100

the 2D cell cultures. IL-8 secretion levels have been shown to significantly increase in the urine 101

of baboons following Stx2 treatment (31) and from intestinal epithelial cells exposed to Stx2 102

(38). Kim-1 has been shown to be induced upon infection of mice with Stx-producing 103

Citrobacter rodentium, a murine model for EHEC disease (39); the current study is the first to 104

show a Kim-1 renal injury biomarker response to Stx2 in vitro. This study is the first utilization 105

of a 3D renal tissue model for the study of Stx-mediated nephrotoxicity and reveals significant 106

benefits of a 3D tissue model compared to 2D cell culture. 107

Materials and Methods 108

Cell culture. The NKi-2 cells used in all studies were derived by the serial passaging in low 109

serum media of hTERT immortalized human renal cortical cells. Cells were grown in high 110

glucose DMEM/F12 (Invitrogen) containing 2% FBS (Invitrogen), 20ng/mL hEGF (Invitrogen), 111

72 ng/mL T3 (Sigma), 1% ITS (Invitrogen), 100 ng/mL hydrocortisone (Sigma), and 1% 112

penicillin/streptomycin (Invitrogen). For 2D studies, cells were seeded at a density of 25,000 113

cells per well of a 12-well tissue culture plate 24 hours before exposure to Stx2. Every effort was 114

taken to ensure matching passages were used for 2D and 3D cultures. 115

Shiga Toxin. Stx2 was purchased from Dr. Anne Kane of the Phoenix Laboratory at Tufts 116

Medical Center. The toxin is purified by P1 glycoprotein affinity chromatography based on the 117

method of Donahue-Rolfe et al., 1989(40). Lipopolysaccharide (LPS) was removed using Pierce 118

High-capacity Endotoxin Removal Resin (Thermo) on a column with a 4 hour incubation. The 119

removal of lipopolysaccharides (LPS) was confirmed with the ToxinSensorTM

Chromogenic 120

LAL Endotoxin Assay Kit (Genscript). As a control, Stx2 was inactivated by boiling for 8 121

7

hours. For dosing, Stx2 was reconstituted in sterile PBS at 1mg/mL and then diluted in cell 122

culture media for concentrations ranging from 1fg/mL to 1μg/mL. For 2D cultures, 2 mL of Stx2 123

containing media was applied to each well and 350μL was removed at each time point. For 3D 124

cultures, 1.5 mL of Stx2 culture media was applied below the tissue and 0.5 mL on top of the 125

tissue. 350μL of media was removed from the bottom at each time point. The cellular 126

supernatants were stored at -80°C and used for LDH, Kim-1, NGAL, and cytokine assays. 127

3D tissue constructs. 3D tissue constructs were formed as previously describes (37). Briefly, 128

100,000 NKi-2 cells were combined with a 50:50 mix of Matrigel and rat tail collagen I at a final 129

concentration of 1 mg/mL in a 12-well transwell insert. The tissues were allowed to polymerize 130

at 37°C for 1 hour after which NKi-2 cell culture media was added to both the bottom well of the 131

transwell and inside the insert. Tissues were grown at 37°C, 5% CO2 with media changes every 2 132

days. 133

Cytotoxicity. LDH secretion was assayed in 100μL of cell culture supernatant from both 2D 134

NKi-2 cells and 3D tissues using LDH cytotoxicity assay from Clontech and following the 135

manufacturer’s instructions. 2% Triton was used as the positive control for cell death. Percent 136

cytotoxicity was calculated as (Stx2 treated – untreated)/(2% Triton treated – untreated)x100. 137

Samples from 3 independent experiments were analyzed and graphed using GraphPad Prism 5 138

software. The data are expressed as averages ± standard errors of the mean (SEM). 139

IC50 calculations. IC50, the dosage at which 50% toxicity was achieved, was calculated using 140

LDH data following 48 and 72 hours of treatment for 2D cells and 3D tissues. Values were 141

calculated with GraphPad Prism 5 software using nonlinear regression analysis. 142

Protein synthesis inhibition. Protein synthesis inhibition was determined by measuring the 143

decrease in 3H-leucine incorporation. For 2D culture, cells were plated in 96-well plates at a 144

8

density of 0.6-1 x 105 cells/well. The following day media was removed, and fresh media 145

without toxin or with Stx2 at concentrations ranging from 1g/ml-1pg/ml was added and the 146

cells were incubated overnight. The next day (24-26 hours post-intoxication) the media was 147

removed, and the cells were gently washed two times with pre-warmed PBS. To the cells, media 148

made 1:1 with RPMI w/25 mM HEPES w/o leucine (Chemicon International R-078-B) and 149

DMEM low glucose w/o Leu, Lys, Arg, (Sigma cat. # SLBH1080) and containing 5 ul/ml of 3H-150

Leucine (Leucine [3, 4, 5-3H(N)], Perkin Elmer cat # NGT460001MC, 1 mCi, 106.2 Ci/mMol) 151

was added and the cells incubated at 37C in 5% CO2 for 1 hour. Following this incubation, 152

media was removed and the cells were washed two times with 200 l of PBS. 50 l of 0.2 M 153

KOH was added to each well of cells and incubated at room temperature for 15 minutes followed 154

by the addition of 150 l of 10 % trichloroacetic acid (TCA). The plates were then incubated at 155

room temperature for 30 minutes followed by transfer of the well contents to a 96-well vacuum 156

manifold-coupled filter plate (Millipore cat # MAHVN4550). Wells were washed two times with 157

200 l of 5% TCA transferring the each wash to the appropriate filter well on the vacuum 158

manifold. Filters were washed one time with 5% TCA 200 l/filter followed by a wash with 200 159

l of 1 % acetic acid. Filters were allowed to dry and then were punched into vials for 160

scintillation counting. 161

The 3D tissues in 12-well plates were treated similar to 2D-cultures except for the 162

following: 1) Following removal of toxin containing media from the wells, tissues were washed 163

2 times in prewarmed PBS and then incubated in leucine-free media for 90 min to dilute out 164

leucine containing media from the matrix prior to adding the 3H-leucine containing media. 2)

3H-165

leucine containing media was incubated for 7:30 hours to allow for sufficient detectable 166

incorporation of radioactivity. Tissues were then washed 2 times with PBS. 3) 300 l of Dispase 167

9

(BD Scientific cat # 354235) containing 100 g/ml collagenase type I was added to the upper 168

chamber of the tissue containing transwell to breakdown the tissue containing matrix. This was 169

incubated for 1:30 hours at 37C, 5 % CO2. 4) The protein in the upper chamber was then 170

precipitated by adding 100 % TCA to a final concentration of 25 %, incubating this at 4oC for 30 171

minutes and spinning in a microfuge for 5 minutes at maximum speed. One ml of ice cold 172

acetone was added to the pellet and the tubes were centrifuged at maximum speed for 5 minutes. 173

The resulting pellet was allowed to dry and suspended in scintillation fluid prior to being 174

transferred to a scintillation vial for counting. 175

Immunohistochemistry. Following 72 hours of Stx2 exposure, tissues were fixed with 10% 176

buffered formalin for 48 hours at 4°C and embedded in paraffin. H&E staining was performed on 177

8 µm tissue sections and images were captured with Leica Application Suite v4, using a Leica 178

DMIL microscope equipped with a Leica DFC340FX camera for fluorescent images and a Leica 179

DFC295FX camera for brightfield images. 180

Kidney injury biomarkers. Kim-1 and NGAL secretion was assayed in 50uL of the cell culture 181

supernatant from both the NKi-2 cells and 3D tissues using ELISA assays from R&D systems 182

and following the manufacturer’s instructions. All measurements were performed with Softmax 183

Pro 5.4 software on a SpectraMax M2 plate reader (Molecular Devices). Experiments were 184

analyzed and graphed using GraphPad Prism 5 software and are expressed as mean concentration 185

± SEM. Statistical significance was assessed at p<0.05 by two-way ANOVA with Bonferroni 186

posttest. 187

Cytokine secretion. Cytokine secretion was measured in 25uL of the cell culture supernatant 188

using the Human Cytokine/Chemokine Milliplex® MAP kit from Millipore customized to 189

measure IL-1β, IL-6, IL-8, TNFα, and MCP-1 and following the manufacturer’s instructions. 190

10

Experiments were analyzed and graphed using GraphPad Prism 5 software and are expressed as 191

mean concentration ± SEM. Statistical significance was assessed at p<0.05 by two-way ANOVA 192

with Bonferroni posttest. 193

MAPK assays. Phospho-JNKs and Phospho-p38 levels were determined using 194

immunoprecipitation based assays by Cell Signaling Technology (cat. #s 8794 and 9820 for 195

Phospho JNKs and Phospho-p38 respectively) according to the manufacturer’s instructions. To 196

200 g of protein-containing cell lysates were added phospho-specific JNKs or p38 antibody-197

conjugated beads and incubated overnight. The beads were then washed twice in lysis buffer and 198

twice in kinase reaction buffer followed by resuspension of the beads in kinase reaction buffer 199

containing 200 M ATP and 1 g kinase substrate (c-Jun fusion protein for phosphor-JNKs and 200

ATF-2 fusion protein for phsopho-p38). 201

MTT Assay. Cells were plated in 96-well tissue culture plates at a density of 0.6 x 105 cells/well. 202

The next day Stx2 was added at concentrations varying from 1 pg/ml to 1 g/ml. After 24 and 48 203

hours of incubation at 37oC, 5 % CO2, media was removed and 100 l of 0.5 mg/ml 3-(4,5-204

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in PBS was added to each well. 205

Following incubation at 37oC, 5 % CO2 for 1 hour, the MTT solution was removed and 50 l of 206

isopropanol containing 4 mM HCL and 0.1 % NP40 Nonidet was added followed by gentle 207

shaking at room temperature for 15 min. Optical density measurements were made at 550 nm 208

and 620 nm. 209

210

Results 211

Shiga Toxin Induced Nephrotoxicity 212

11

In order to validate our bioengineered 3D human renal model for the study of Stx-213

induced nephrotoxicity, we began by comparing cytotoxicity in 3D to cytotoxicity in the same 214

cells grown in 2D (Fig. 1). 3D tissues containing immortalized renal proximal tubule cells (NKi-215

2) were formed and allowed to grow for 2 weeks prior to treatment with Stx2 while 2D cultures 216

of the NKi-2 cells were seeded 24 hours prior to Stx2 treatment. Stx2 was purified free of LPS, 217

which has been used as a co-stimulus in a murine model of HUS (41,42), and may potentiate a 218

Stx response by increasing the inflammatory response or increasing Gb3 receptor on the cell 219

surface (43-46). Both the cells and the tissues were treated with a range of Stx2 concentrations 220

(1fg -1μg /mL) for up to 72 hours with supernatant sampling occurring at 6, 24, 48, and 72 hours 221

post-treatment (Fig. 1B). 222

To first test whether Stx2 had the predicted inhibitory effect on protein synthesis, we 223

added increasing concentrations of the toxin to either 2D or 3D cultures. Protein synthesis of 224

both cultures, measured by 3H-leucine incorporation (see Materials and Methods), was inhibited 225

in a similar dose-dependent manner (Fig. 1C). The predicted Stx2-induced cytotoxicity was 226

demonstrated for 3D cultures by H&E staining, which revealed the loss of nuclei at 72 h post-227

exposure (Fig. 1D), and for 2D cultures by an MTT cell viability assay (Fig. S1). Importantly, 228

cell viability at 24 hours after Stx2 exposure was greater than 50% for all Stx2 concentrations 229

tested, indicating that the inhibition of protein synthesis observed at 24 h (Fig. 1C) was not 230

simply a reflection of cell death. Heat-treatment of purified Stx2 abrogated the cytotoxic effect 231

(data not shown), suggesting that cytotoxicity was not a reflection of possible contamination by 232

LPS. To discern possible dramatic differences in the kinetics or dose-dependence of cytoxicity 233

between 2D and 3D cultures, we measured the IC50 for lactate dehydrogenase (LDH) release at 234

various times after Stx2 exposure. We found that at 48 h post-exposure, 3D cultures were 235

12

somewhat (seven-fold) less sensitive than 2D cultures; at 72 h, 3D cultures were somewhat 236

(three-fold) more sensitive (Fig. 1E). We conclude that although the cytotoxic responses of the 237

two cultures may differ somewhat in kinetics and relative sensitivity, Stx2 inhibits protein 238

synthesis and triggers cytotoxicity similarly in both 2D and 3D cultures.. 239

Finally, since Stx-mediated damage to actively translating ribosomes results in activation 240

of the RSR, we investigated whether a RSR was induced in 2D and 3D renal cultures. Whole cell 241

lysates were made from 2D and 3D cultures untreated, treated with heat-inactivated-Stx2 (HI), 242

Stx2 without LPS removed, and Stx2 following LPS removal for 6 hours. Phospho-JNKs were 243

immunoprecipitated from whole cell lysates and immunoprecipitates were incubated with c-jun 244

substrate and phosphorylated c-jun was determined by western blot. Activation of JNKs (Fig. 245

2A) was increased in Stx2 treated 2D and 3D samples, but not in HI samples. Similarly, 246

immunoprecipitation of phosphorylated p38 (Fig. 2B) demonstrates that p38 activation was also 247

increased by Stx2 in both 2D and 3D cultures. Taken together, these data indicate that there are 248

similarities in the general cytotoxic response to Stx2 between 2D and 3D cultures in terms of 249

dose response, protein synthesis inhibition, and activation of the RSR. 250

Kidney Injury Marker Secretion in Response to Stx2 Treatment 251

A number of biomarkers have been identified to monitor kidney damage. These include 252

both Kim-1 and neutrophil gelatinase-associated lipocalin (NGAL). Kim-1 secretion in the urine 253

of patients or by kidney epithelial cells in response to Stx2 has not been examined and only two 254

studies have examined the secretion of NGAL in patient urine and serum in response to Stx 255

(47,48). NGAL has been examined for its ability to predict renal injury in HUS (48) and the need 256

for renal replacement therapy following Stx exposure (47). We have previously shown its utility 257

as a marker of drug-induced nephrotoxicity in 3D tissues and shown that it was predictive of 258

13

renal proximal tubule cytotoxicity for highly toxic agents at high doses but incapable of 259

distinguishing more subtle differences in toxicity in vitro due to high expression from untreated 260

controls (37). None of the Stx2 doses resulted in NGAL secretion in excess of untreated controls 261

in either 3D or 2D cultures (Fig. 3A). Interestingly, the highest doses of Stx2, 1μg/mL and 262

1ng/mL, which resulted in almost 100% cytotoxicity in 3D by LDH secretion (Fig. 1C), resulted 263

in a significant decrease in NGAL secretion (p<0.001) 72 hours post-treatment. This may be due 264

to the almost complete inhibition in protein synthesis seen at these doses by 24 hours post-265

treatment. These data raise the possibility that low levels of NGAL, rather than high levels, may 266

be predictive of Stx-induced cytotoxicity in vitro. 267

Kim-1 has been shown to be a very sensitive biomarker of kidney injury and we have 268

previously shown its utility in measuring cytotoxicity in 3D with highly nephrotoxic compounds 269

(37). However, we have also shown that secreted Kim-1 increases over time in untreated control 270

cultures (37) making it only useful at extremely toxic concentrations and not sensitive or precise 271

enough to reveal subtle changes in cytotoxicity in vitro. Consistent with these previous findings, 272

Kim-1 increased in expression in both the 2D and 3D untreated controls over time (data not 273

shown). When assayed at 72 h after exposure to 1 pg/ml Stx2 or greater, Kim-1 production by 274

2D cultures was inhibited, perhaps due to the protein synthesis inhibition induced by the toxin, 275

whereas 3D cultures were largely unaffected by Stx2 exposure (Fig. 3B). When assayed at an 276

earlier time point, i.e. 24 h after exposure, 1μg/mL Stx2 induced Kim-1 production increased 277

almost two-fold above the untreated controls (Fig. 3C). In contrast, at 24 h post-exposure no 278

induction of Kim1 was observed for 2D cultures at any Stx2 concentration (Fig. 3B). The Stx2-279

induced Kim-1 secretion response by 3D but not 2D cultures suggest that 3D tissues may provide 280

a better experimental model for examining renal damage using this marker. 281

14

Stx2-Induced Cytokine Secretion 282

Since cytokines have been shown to play a role in Stx2-induced renal damage (for review 283

(49)), are secreted by a number of different cell types in response to Stx2 (17,20,50-52), and are 284

differentially secreted based upon the toxin sensitivity of the cell (20,53), we examined whether 285

3D culturing would elicit a different response compared to 2D in the secretion of a set of 286

cytokines previously shown to be secreted by cells in response to Stx, IL-1β, IL-6, IL-8, MCP-1, 287

and TNFα (Fig. 4) (20,21,31,54-56). All cytokines were measured at 24 and 48 hours after 288

treatment with increasing concentrations of Stx2. IL-1β was not detectable in either 2D or 3D 289

cultures (data not shown), and untreated controls had baseline secretion of IL-6, IL-8, TNFα, and 290

MCP-1 that increased between 24 and 48 hours for both 2D and 3D cultures (Fig. 4A-D; see 291

open and filled bars, “untreated”). 292

Upon intoxication of either model with increasing concentrations of Stx2, MCP-1 293

secretion was not induced. However, MCP-1 production exhibited a dramatic difference between 294

the two models. MCP-1 was almost undetectable in 2D cultures, and while the secreted levels 295

did increase over time, the levels never exceeded the levels of the untreated controls (Fig. 4A). 296

In striking contrast, MCP-1 was produced at levels 20- to 40-fold higher in 3D cultures, a result 297

that may reflect the observation that increased MCP-1 is detected in the urine of children with 298

HUS (57). 299

For both 3D and 2D cultures, the secretion of IL-6 (Fig. 4B), TNFα (Fig. 4C) and IL-8 300

(Fig. 4D) were each induced 48 hours after exposure to 1pg/mL Stx2. Cytokines were not 301

induced by 1ng/mL or 1g/ml Stx2. The fact that a significant change in expression was only 302

seen at 1pg/mL may be due to the fact that at the higher concentrations protein synthesis 303

15

inhibition prevented cytokine production immediately. In contrast, 1pg/mL is most similar to the 304

IC50 values determined in Figure 1B and may not intoxicate cells rapidly enough to prevent the 305

production and secretion of inflammatory factors. That Stx2 induces cytokine production despite 306

promoting protein synthesis inhibition is consistent with the ability of Stx to induce gene 307

expression and protein production via signaling pathways not directly activated by protein 308

synthesis inhibition (16,58,59). 309

The 3D tissue IL-8 responses to Stx2 deserves particular note. First, unlike IL-6 and 310

TNFα, but like MCP-1, basal production of IL-8 in the absence of Stx2 was 4- to 8- fold higher 311

in 3D tissues compared to 2D cells (Fig. 4D). Second, this already high level of secretion of IL-8 312

was further induced ~1.8-fold by 1pg/mL Stx2, resulting in IL-8 levels higher than that induced 313

in 2D cultures by Stx2. This increase in 3D versus 2D could indicate a property of the 3D culture 314

system that up-regulates both basal and induced IL-8 levels or it could reflect a difference in cell 315

numbers able to secrete IL-8 at this time point. Importantly, while the exact role of IL-8 in HUS 316

is unknown, increases in concentration have been noted in the urine of children with HUS, the 317

urine of Stx2 treated baboons, and in human intestinal epithelial cells following Stx2 exposure 318

(31,38,57). 319

320

Discussion 321

In this study, we have shown the utility of a 3D bioengineered human renal tissue model for the 322

examination of the mechanisms of Stx-induced renal cytotoxicity. While many responses were 323

similar between the 2D and 3D cultures, including overall cytotoxicity, IC50, protein synthesis 324

inhibition, RSR, and IL-6 and TNFα secretion, there were differences indicating that a 3D 325

16

system may provide a more physiologically relevant model than a 2D system for examination of 326

cellular responses to Stx2. Two distinct and notable differences were observed between the 2D 327

and 3D systems. First, higher basal levels of Kim-1, NGAL, MCP-1, and IL-8 were observed 328

from the 3D tissues compared to the 2D cells indicating that these inflammatory or renal injury 329

markers may be better expressed in the 3D tissues. Second, higher induction levels of Kim-1 and 330

IL-8 were observed from the 3D tissues compared to the 2D cells indicating that the 3D tissues 331

maybe be a better system for monitoring Stx2-mediated renal injury in vitro through renal 332

biomarker secretion (Kim-1) and characteristic cytokine secretion (IL-8). Taken together, these 333

data suggest differential cellular mechanisms functioning between 2D and 3D cultures in 334

response to Stx2. 335

Initially, the general features of cytotoxicity of Stx2 appeared to be very similar between 336

the 2D and 3D cultures. For example, several studies have demonstrated that intoxication with 337

Stx activates pro-inflammatory and pro-apoptotic signaling pathways (60,61), in part through the 338

activation of the mitogen-activated protein kinase (MAPK) pathway (62-64). This activation of 339

the RSR contributes to both pro-inflammatory and pro-apoptotic signaling in cultured cells 340

(62,63,65,66). We showed that human renal 2D and 3D tissue both underwent an RSR following 341

Stx2 treatment suggesting that this signaling cascade may participate in Stx-associated 342

inflammation and cell death in the human kidney. Whether the kinetics of the RSR in 2D and 3D 343

models differs will be a subject of future studies as will defining the relative contribution of the 344

RSR to cytokine production and cell death. 345

A second similarity was the general trend of inflammatory cytokine expression in the cell 346

culture supernatant. Inflammatory signaling is associated with HUS, as patients show increased 347

urinary levels of pro-inflammatory cytokines, and renal biopsies have revealed infiltration of 348

17

neutrophils and monocytes into the kidney during disease (57,67-69). In 2D cell culture models, 349

Stx treatment of human proximal tubular cells has been shown to induce the secretion of IL-1β, 350

IL-6, and IL-8 (27), and evidence suggests that IL-1, IL-8, TNFα, and IL-6 may be elevated in 351

the serum of D+HUS patients (55,56). Also, IL-1 and TNF have been shown to upregulate the 352

Stx cell surface receptor globotriaosylceramide (Gb3) and increase cellular sensitivity to Stx 353

cytotoxicity in endothelial cells (70,71). In nonhuman primates treated with Stx, kidney tissue 354

had up-regulated levels of IL-8, MCP-1, and TNFα mRNA along with increased urinary levels of 355

IL-6, IL-8, and MCP-1 (31). Our studies revealed an increase in IL-6, IL-8, and TNFα over time 356

following Stx2 treatment in both 2D and 3D models. The three detectable cytokines all showed 357

an increase above untreated controls at 48 hours with 1pg/mL Stx2 while the higher 358

concentrations did not secrete any cytokines above the level of the untreated control. This was 359

most likely due to the relatively high rate of cytotoxicity and protein synthesis inhibition seen 360

with the higher doses; in contrast, the 1pg/mL dose was close to the calculated IC50 and therefore 361

did not result in complete cytotoxicity of the cultures. Studies designed to examine cytokine 362

secretion at earlier time points following Stx2 treatment may reveal increased secretion at higher 363

doses. However, studies of cytokine secretion from HK-2 cells at early time points (<4 hours) 364

did not reveal an increase in IL-1β, TNFα, or IL-8 protein levels although increases in mRNA 365

transcripts were observed (20). 366

Examination of the IC50 at both 48 and 72 hours revealed a difference between the 2D 367

and 3D culture systems. In particular, there was a difference in the response rate to Stx2 between 368

the 2D and 3D cultures. The 2D cultures were seven-fold more sensitive to Stx2 than the 3D 369

cultures at 48 hours post-treatments, but by 72 hours the two cultures exhibited roughly similar 370

Stx2 sensitivity. This difference does not simply reflect delayed diffusion of Stx2 into the 3D 371

18

culture, because at 24 hours post-treatment Stx2 induced a similar degree of protein synthesis 372

inhibition in both the 2D and 3D cultures. Thus it may be that the difference in cellular 373

organization and structure has an impact upon Stx2-mediated cytotoxicity. Interestingly, HK-2 374

cells, a HPV immortalized human renal proximal tubule cell line, were shown to have an IC50 of 375

20 pg/mL at 72 hours post-treatment with Stx2, compared to 2.2 pg/mL and 0.7 pg/mL at the 376

same time point for 2D and 3D NKi-2 cells respectively in this study. HK-2 cells have also been 377

shown to lack sensitivity to drug-induced toxicity (26) indicating they may not be the best cell 378

line for studying renal cytotoxicity. 379

A second significant difference between the 2D and 3D cultures was the inability to 380

detect MCP-1 in the 2D cultures, and while it was detectable in the 3D cultures and increased 381

with time, following Stx2 treatment it was secreted at levels significantly lower than the 382

untreated controls. Interestingly, while MCP-1 mRNA has been shown to be elevated in the 383

kidneys of nonhuman primates exposed to Stx2 and the protein has been shown to be elevated in 384

the urine, its mRNA was barely detectable in cultured renal endothelial cells (31). This may 385

indicate that the secreted MCP-1 is originating from a different cell type or the specifically high 386

basal levels of MCP-1 might also reflect a physiologic response to the accumulation of metabolic 387

waste products. In vivo, tubules exist adjacent to both a vasculature and a lumen through which 388

metabolites can be either taken up into the blood stream, or discarded through the urine. Since 389

our current 3D-tubule system is not perfused, it is reasonable to hypothesize that the increase in 390

MCP-1 over the course of 48 hours reflects a normal physiological response to the accumulation 391

of metabolic waste products. In this scenario, the tubular epithelium is signaling for resident 392

tissue phagocytes to aid in the removal of these waste products in an effort to maintain a 393

homeostatic environment. Furthermore, the high basal MCP-1 level does not appear to be part of 394

19

an inflammatory response since neither TNFα nor IL-6 appear to be upregulated in our model in 395

the absence of Stx. This hypothesis is supported by the observation that the kidney maintains 396

distinct populations of resident mononuclear phagocytes that produce anti-inflammatory and 397

homeostatic factors (72), and therefore, the high basal levels of MCP-1 might represent a 398

homeostatic response not present in 2D cell culture. 399

A third significant difference between the 2D and 3D cultures was the secretion of IL-8. 400

While the secretion of IL-8 increased from 24 to 48 hours for both the 3D tissues and the 2D 401

cells, the amounts were approximately four-fold higher from the 3D tissues compared to the 2D 402

cells. Importantly, this induction occurred in response to 1 pg/mL Stx2, which may be present in 403

sera of STEC-infected patients at very low levels, i.e., often lower than the 6 pg/ml limit of 404

detection (73). IL-8 has been shown in the urine of nonhuman primates 48 hours after exposure 405

to Stx2 at approximately 600 pg/mL (31). Our cells in 2D culture did not reach that amount at 406

any time or dose while the 3D tissues more than doubled that amount at 48 hours with a 1 pg/mL 407

dose. Thus, the 3D tissue system may be more representative of the in vivo response than the 408

cells in 2D. 409

Biomarkers of renal injury are extremely important for the prediction of drug-induced 410

nephrotoxicity and the early assay of acute renal injury. A number of potential markers have 411

been identified and are in different stages of validation and use. Kim-1 and NGAL are two such 412

markers. While they have been shown to be predictive of drug-induced nephrotoxicity in vivo 413

and have been examined as biomarkers for HUS (47,48,74), we have previously shown that they 414

may only be minimally useful to assess toxicity in cultured renal cells in vitro primarily due to 415

the high levels secreted by untreated controls (37). What was notable in our studies was an 416

increase in Kim-1 secretion at 1μg/mL Stx2 compared to the untreated controls at 24 hours that 417

20

then later decreased. This spike was only evident in the 3D cultures. The 2D cultures never 418

reached a secretion level above the untreated controls. This may indicate an initial early spike in 419

secretion in relation to Stx2-mediated cytotoxicity that then decreases due to the inhibition of 420

protein synthesis which we show occurred by 24 hours post-treatment. Further studies at early 421

time points need to be conducted. This spike would never have been noticed if we had only been 422

examining the cells in 2D culture. Our 3D studies indicate that Kim-1 has the potential to act as 423

an early biomarker of renal injury for infected patients and in vivo studies need to be performed 424

to confirm this. If it is confirmed in vivo, then our studies also indicate a larger relevance of the 425

3D system over 2D cell culture in mimicking Stx2-mediated renal injury. 426

In conclusion, we have presented the first use of a 3D bioengineered human kidney tissue 427

model to study renal damage upon Stx2 exposure. We have shown that the 3D tissue responds to 428

Stx2 with a dose- and time-dependent cytotoxicity that is related to the inhibition of protein 429

synthesis. We have also shown changes in renal injury biomarkers in a dose-dependent manner 430

along with changes in the secretion of cytokines and activation of the RSR. Key differences 431

between the 3D tissue over 2D cell culture included differences in the IC50 and the ability to 432

measure significantly high levels of kidney injury biomarkers and cytokines, particularly IL-8, 433

MCP-1 and Kim-1. Further use of this model may save on the use of inaccurate animal models 434

while making in vitro studies more relevant to in vivo conditions. 435

436

437

Acknowledgments 438

21

This work was supported by the NIH (P41 EB002520, R01 AI46454, R37 DK51050, and R01 439

DK099532) along with the Tufts Collaborates! Seed Grant Program. We would like to thank Dr. 440

Anne Kane of the Phoenix Laboratory for her assistance with the Stx2 and Alexander Histed for 441

his insight on MCP-1. We would also like to thank Milva Ricci, Carmen Preda, and Darin 442

Goodwin for their assistance. 443

Figure Legends 444

Figure 1: Shiga toxin mediated cytotoxicity. (A) A representative image of the interconnecting 445

tubules present in the 3D bioengineered human kidney tissue model. (B) Timeline of tissue 446

formation, treatment, and sampling. (C) 3H-leucine incorporation assay was used to determine 447

the sensitivity of 2D and 3D cultures to Stx2 at 24 hours post treatment. (D) H&E of 448

representative 3D tissues following 72 hours of no treatment or treatment with noted 449

concentrations of Stx2. Arrows indicate the hematoxylin stained cells within the matrix. (E) IC50 450

values at specific time points for 2D and 3D tissues. 451

Figure 2: Ribotoxic Stress Response. Activation of JNKs (A) or p38 (B) was determined by 452

western blot, comparing in vitro phosphorylation of c-jun (JNKs) or ATF-2 (p38) following 453

precipitation of phosphoyrlated JNKs or p38 from whole cell lysates. Bar graphs represent blot 454

band intensity of P-c-jun or P-ATF-2 as normalized against loading controls. 455

Figure 3: Kidney Injury Markers. (A) NGAL production in response to Stx2 at 72 hours post-456

toxin treatment. Kim-1 production in response to Stx2 is shown at (B) 24 hours and (C) 72 hours 457

post-toxin treatment. 458

22

Figure 4: Cytokine expression. (A) MCP-1, (B) IL-6, (C) TNFα, and (D) IL-8 secretion into 459

the cell culture supernatant following 24 and 48 hour Stx2 treatment of 3D and 2D cultures. 460

*p<0.05, **p<0.01, ***p<0.001. 461

462

23

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