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Shiga toxin 2-induced endoplasmic reticulum stress is minimized by activated protein C but does not correlate with lethal kidney injury - PubMed

  • ️Thu Jan 01 2015

Shiga toxin 2-induced endoplasmic reticulum stress is minimized by activated protein C but does not correlate with lethal kidney injury

Caitlin S L Parello et al. Toxins (Basel). 2015.

Abstract

Enterohemorrhagic Escherichia coli produce ribotoxic Shiga toxins (Stx), which are responsible for kidney injury and development of hemolytic uremic syndrome. The endoplasmic reticulum (ER) stress response is hypothesized to induce apoptosis contributing to organ injury; however, this process has been described only in vitro. ER stress marker transcripts of spliced XBP1 (1.78-fold), HSP40 (4.45-fold) and CHOP (7.69-fold) were up-regulated early in kidneys of Stx2 challenged mice compared to saline controls. Anti-apoptotic Bcl2 decreased (-2.41-fold vs. saline) and pro-apoptotic DR5 increased (6.38-fold vs. saline) at later time points. Cytoprotective activated protein C (APC) reduced early CHOP expression (-3.3-fold vs. untreated), increased later Bcl2 expression (5.8-fold vs. untreated), and had early effects on survival but did not alter DR5 expression. Changes in kidney ER stress and apoptotic marker transcripts were observed in Stx2-producing C. rodentium challenged mice compared to mice infected with a non-toxigenic control strain. CHOP (4.14-fold) and DR5 (2.81-fold) were increased and Bcl2 (-1.65-fold) was decreased. APC reduced CHOP expression and increased Bcl2 expression, but did not alter mortality. These data indicate that Stx2 induces renal ER stress and apoptosis in murine models of Stx2-induced kidney injury, but decreasing these processes alone was not sufficient to alter survival outcome.

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Figures

Figure 1
Figure 1

Stx2 challenge increases kidney injury marker expression. Mice were challenged with 0.05 ng Stx2/g body weight (n = 4–12) by i.p. injection and were euthanized on either Day 2, Day 3 or upon reaching euthanasia criteria typically by Day 4–6. Control mice (n = 5) were challenged with sterile saline and kidneys were harvested by Day 5–10. Mean values ± S.D. are shown. Measurement of kidney injury marker gene transcripts for neutrophil gelatinase-associated lipocalin (NGAL) (A) and kidney injury marker 1 (KIM1) (B) were quantified after RNA isolation and qPCR. Significance was determined by Kruskal-Wallis test with Dunn’s multiple comparison test. * p < 0.05, ** p < 0.001, *** p < 0.001.

Figure 2
Figure 2

Murine kidney injury following Stx2 challenge is accompanied by ER stress. Mice were challenged with 0.05 ng Stx2/g body weight (n = 3–6) by i.p. injection and were euthanized on either Day 2, Day 3, or upon reaching euthanasia criteria typically by Day 4–6. Control mice (n = 3–5) were challenged with sterile saline. Kidneys were processed for downstream analysis of ER stress marker transcripts as described in Section 4, Materials and Methods. Mean ± S.D. are shown. (A) Measurement of XBP1 mRNA splicing with time; (B) Measurement of kidney HSP40 mRNA expression with time; (C) Measurement of kidney CHOP expression with time. Significance was determined by Kruskal-Wallis test with Dunn’s multiple comparisons test. Significance is compared to saline. * p < 0.05, ** p < 0.01, n.s. not significant.

Figure 3
Figure 3

Changes in anti- and pro-apoptotic markers in kidneys. Mice were challenged with 0.05 ng Stx2/g body weight (n = 4–6) or saline (n = 5) by i.p. injection and were euthanized on either Day 3 or upon reaching euthanasia criteria by Day 4–6. Kidneys were processed for downstream analysis of (A) anti-apoptotic marker Bcl2 and (B) pro-apoptotic marker DR5 transcripts as described in Materials and Methods. Mean ± S.D. are shown. Significance was determined by Kruskal-Wallis test with Dunn’s multiple comparison test. ** p < 0.01.

Figure 4
Figure 4

APC treatment reduces early ER stress markers and delays anti-apoptotic patterns. Stx2 challenged mice (solid, ●) were treated with 20 μg activated protein C daily on Days 0–3 (horizontal stripes, ■) or with 125 μg Z-VAD-FMK (vertical stripes, ▲) and were euthanized on either Day 3 or upon reaching euthanasia criteria. Kidneys were harvested and processed for mRNA markers. (A) ER stress marker CHOP (n = 3–6) and (B) HSP40 (n = 5); (C) Kidney mRNA expression of anti-apoptotic marker Bcl2 (n = 5) and (D) pro-apoptotic marker DR5 (n = 4–5); (E) Kaplan-Meier curve of survival of challenged animals (Stx2, solid, n = 44; APC + Stx2, dashed, n = 10; Z-VAD-FMK + Stx2, dotted, n = 4); (F) Plasma BUN was measured in challenged animals as a marker of kidney injury; (G) PAS stained kidney of challenged animals. Renal tubular epithelial injury predominated and no pathology differences were observed between cortex or medulla tissue. Representative images are shown; (H) Histology score of Stx2 challenged animals; n = 2 per group with six images per animal. All images were blinded prior to analysis. Significance was determined by either Kruskal Wallis test with Dunn’s multiple comparison test (A–D) or repeated measures ANOVA with Bonverroni post-test (F), and is compared to Stx2 only unless otherwise indicated. * p < 0.05, ** p < 0.01, n.s. not significant.

Figure 5
Figure 5

ER stress accompanies Stx2 producing C. rodentium induced lethal kidney injury, and is abolished through APC co-treatment. Mice were challenged with ~1 × 109 CFU of Stx2 producing Citrobacter rodentium (n = 9–19; solid, ■) either alone or with 20 μg APC treatment (n = 3; dashed, ▲). Control mice were challenged with non-toxigenic C. rodentium (n = 10; dotted, ●). Means ± S.D. are shown. (A) Kidney mRNA expression of ER stress marker CHOP; (B) anti-apoptotic Bcl2 and (C) pro-apoptotic marker DR5; (D) Kaplan-Meier analysis of survival; (E) Plasma BUN was measured with time in challenged animals as a marker of kidney injury; (F–G) Kidney mRNA expression of injury markers NGAL (F) and KIM1 (G). Significance was determined by Kruskal-Wallis test with Dunn’s multiple comparison test (A–C,F,G) or repeated measures ANOVA with Bonferroni post-test (E) and is compared to C.r-control unless otherwise specified. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. not significant.

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