Exposure to Radiocontrast Agents Induces Pancreatic Inflammation by Activation of Nuclear Factor-κB, Calcium Signaling, and Calcineurin

doi:10.1053/j.gastro.2015.05.004

Gastroenterology

Volume 149, Issue 3, September 2015, Pages 753-764.e11

https://doi.org/10.1053/j.gastro.2015.05.004 Get rights and content

Background & Aims

Radiocontrast agents are required for radiographic procedures, but these agents can injure tissues by unknown mechanisms. We investigated whether exposure of pancreatic tissues to radiocontrast agents during endoscopic retrograde cholangiopancreatography (ERCP) causes pancreatic inflammation, and studied the effects of these agents on human cell lines and in mice.

Methods

We exposed mouse and human acinar cells to the radiocontrast agent iohexol (Omnipaque; GE Healthcare, Princeton, NJ) and measured intracellular release of Ca²⁺, calcineurin activation (using a luciferase reporter), activation of nuclear factor-κB (NF-κB, using a luciferase reporter), and cell necrosis (via propidium iodide uptake). We infused the radiocontrast agent into the pancreatic ducts of wild-type mice (C57BL/6) to create a mouse model of post-ERCP pancreatitis; some mice were given intraperitoneal injections of the calcineurin inhibitor FK506 before and after infusion of the radiocontrast agent. CnAβ^-/- mice also were used. This experiment also was performed in mice given infusions of adeno-associated virus 6–NF-κB–luciferase, to assess activation of this transcription factor in vivo.

Results

Incubation of mouse and human acinar cells, but not HEK293 or COS7 cells, with iohexol led to a peak and then plateau in Ca²⁺ signaling, along with activation of the transcription factors NF-κB and nuclear factor of activated T cells. Suppressing Ca²⁺ signaling or calcineurin with BAPTA, cyclosporine A, or FK506 prevented activation of NF-κB and acinar cell injury. Calcineurin Aβ–deficient mice were protected against induction of pancreatic inflammation by iohexol. The calcineurin inhibitor FK506 prevented contrast-induced activation of NF-κB in pancreata of mice, this was observed by live imaging of mice given infusions of adeno-associated virus 6–NF-κB–luciferase.

Conclusions

Radiocontrast agents cause pancreatic inflammation in mice, via activation of NF-κB, Ca²⁺ signaling, and calcineurin. Calcineurin inhibitors might be developed to prevent post-ERCP pancreatitis in patients.

Section snippets

Reagents and Animals

RC refers primarily to iohexol (Omnipaque 300; GE Healthcare, Princeton, NJ), which is categorized as a low-osmolality (672 mOsm/kg water), nonionic, iodinated (300 mg/iodine/mL) contrast medium. A second RC, iopamidol (Isovue 300; Bracco Diagnostics, Monroe Township, NJ), which is in the same category as iohexol, was used to verify key findings from the study, and its use is specified in the text. Nuclear factor of activated T cells (NFAT) luciferase (Qiagen, Valencia, CA), NF-κB luciferase

Intraductal Infusion of RC Induces Pancreatic Injury in Mice

We developed a PEP model in mice by performing a transduodenal cannulation of the CBD as detailed in the Materials and Methods section (Figure 1A). Infusions were channeled into the pancreatic duct by clamping the proximal CBD and thus preventing retrograde flow into the liver.

The histologic severity of pancreatitis, graded by the presence of edema, inflammatory infiltrate, and necrosis, was assessed from the head of the pancreas 24 hours after the surgical procedure (Figure 1B and

Discussion

The current study shows, using the context of pancreatic injury, that RC exposure causes NF-κB activation and results in epithelial and organ damage by inducing aberrant Ca²⁺ signals and activating calcineurin.

We developed a novel model of PEP in mice. Previous models in larger animals such as dogs used an endoscopic cannulation of the biliopancreatic ducts.41, 42 More recent models in smaller animals have instilled RC by performing a laparotomy and achieving transduodenal access.22, 43 The

Acknowledgments

The authors acknowledge Drs Mark Lowe and Peter Lucas for technical assistance.

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There is an unmet clinical need for effective, targeted interventions to prevent post-ERCP pancreatitis (PEP). We previously demonstrated that the serine-threonine phosphatase, calcineurin (Cn) is a critical mediator of PEP and that the FDA-approved calcineurin inhibitors, tacrolimus (Tac) or cyclosporine A, prevented PEP. Our recent observations in preclinical PEP models demonstrating that Cn deletion in both pancreatic and hematopoietic compartments is required for maximal pancreas protection, highlighted the need to target both systemic and pancreas-specific Cn signaling. We hypothesized that rectal administration of Tac would effectively mitigate PEP by ensuring systemic and pancreatic bioavailability of Tac. We have tested the efficacy of rectal Tac in a preclinical PEP model and in cerulein-induced experimental pancreatitis.
C57BL/6 mice underwent ductal cannulation with saline infusion to simulate pressure-induced PEP or were given seven, hourly, cerulein injections to induce pancreatitis. To test the efficacy of rectal Tac in pancreatitis prevention, a rectal Tac suppository (1 mg/kg) was administered 10 min prior to cannulation or first cerulein injection. Histological and biochemical indicators of pancreatitis were evaluated post-treatment. Pharmacokinetic parameters of Tac in the blood after rectal delivery compared to intravenous and intragastric administration was evaluated.
Rectal Tac was effective in reducing pancreatic injury and inflammation in both PEP and cerulein models. Pharmacokinetic studies revealed that the rectal administration of Tac helped achieve optimal blood levels of Tac over an extended time compared to intravenous or intragastric delivery.
Our results underscore the effectiveness and clinical utility of rectal Tac for PEP prophylaxis.
Preclinical safety evaluation of calcineurin inhibitors delivered through an intraductal route to prevent post-ERCP pancreatitis demonstrates endocrine and systemic safety
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There is an urgent need for safe and targeted interventions to mitigate post-ERCP pancreatitis (PEP). Calcineurin inhibitors (CnIs) offer therapeutic promise as calcineurin signaling within acinar cells is a key initiating event in PEP. In previous proof-of-concept studies using experimental models, we showed that concurrent intra-pancreatic ductal administration of the CnIs, tacrolimus (Tac) or cyclosporine A (CsA) with the ERCP radiocontrast agent (RC) prevented PEP. To translate this finding clinically, we investigated potential toxic effects of intraductal delivery of a single-dose RC-CnI formulation on endocrine pancreas function and systemic toxicities in a preclinical PEP model.
C57BL/6J mice underwent ductal cannulation and received a single, intra-pancreatic ductal infusion of RC or RC with Tac or CsA (treatment groups) or underwent ductal cannulation without infusion (‘sham’ group). To assess endocrine function, intraperitoneal glucose tolerance test (IPGTT) was performed at two days before infusion and on day 2 and 14 post-surgery. To evaluate off-target tissue toxicities, renal and hepatic function-related parameters including blood urea nitrogen, plasma creatinine, potassium, aspartate aminotransferase, alanine aminotransferase, and total bilirubin were measured at the same time-points as IPGTT. Histological and biochemical indicators of pancreas injury and inflammation were also evaluated.
No abnormalities in glucose metabolism, hepatic or renal function were observed on day 2 or 14 in mice administered with intraductal RC or RC with Tac or CsA.
Intraductal delivery of RC-CnI formulation was safe and well-tolerated with no significant acute or subacute endocrine or systemic toxicities, underscoring its clinical utility to prevent PEP.
Unraveling corticotropin-releasing factor family-orchestrated signaling and function in both sexes
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Stress responses to physical, psychological, environmental, or cellular stressors, has two arms: initiation and recovery. Corticotropin-releasing factor (CRF) is primarily responsible for regulating and/or initiating stress responses via, whereas urocortins (UCNs) are involved in the recovery response to stress via feedback inhibition. Stress is a loaded, polysemous word and is experienced in a myriad of ways. Some stressors are good for an individual, in fact essential, whereas other stressors are associated with bad outcomes. Perceived stress, like beauty, lies in the eye of the beholder, and hence the same stressor can result in individual-specific outcomes. In mammals, there are two main biological sexes with reproduction as primary function. Reproduction and nutrition can also be viewed as stressors; based on a body of work from my laboratory, we propose that the functions of all other organs have co-evolved to optimize and facilitate an individual's nutritional and reproductive functions. Hence, sex differences in physiologically relevant outcomes are innate and occur at all levels- molecular, endocrine, immune, and (patho)physiological. CRF and three UCNs are peptide hormones that mediate their physiological effects by binding to two known G protein-coupled receptors (GPCRs), CRF₁ and CRF₂. Expression and function of CRF family of hormones and their receptors is likely to be sexually dimorphic in all organs. In this chapter, based on the large body of work from others and my laboratory, an overview of the CRF family with special emphasis on sex-specific actions of peripherally expressed CRF₂ receptor in health and disease is provided.
Intraductal pressure in experimental models of acute and chronic pancreatitis in mice
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Citation Excerpt :
A p values less than 0.05 were considered as statistically significant. To assess the magnitude of the intraductal pressure in a mouse model of post-ERCP pancreatitis or PEP, we induced PEP, as previously reported, by retrograde infusion of normal saline or radiocontrast at the constant rate of 10 or 20 μL/min [16,17]. The cannula was attached to a rate-regulated infusion pump, along with a pressure transducer that allow us to record the changes of pancreatic intraductal pressure while inducing PEP (Fig. 1A).
Pancreatic intraductal pressure is related to the development of pancreatitis, including post-ERCP (endoscopic retrograde cholangiopancreatography) pancreatitis. In this study, we investigate pancreatic intraductal pressure in various mouse models of acute and chronic pancreatitis.
Post-ERCP pancreatitis was induced by retrograde infusion of normal saline or radiocontrast at the constant rate of 10 or 20 μL/min. Obstructive pancreatitis was induced by ligation of the pancreatic duct followed by a single injection of caerulein and the changes of intraductal pressure were recorded in day 3 for obstructive acute pancreatitis and day 14 for obstructive chronic pancreatitis. Non-obstructive pancreatitis was induced by repetitive intraperitoneal injections of caerulein. The changes of intraductal pressure were recorded right after the last caerulein injection for non-obstructive acute pancreatitis and after the completion of 4-week caerulein injections for non-obstructive chronic pancreatitis.
Elevated pancreatic intraductal pressure was observed in both normal saline and radiocontrast infusion groups and was furtherly indicated that was positively correlated with the viscosity of solution but not genders. In the models of obstructive pancreatitis, a rise in intraductal pressure was observed in both acute and chronic pancreatitis; whereas in the models of non-obstructive pancreatitis, a rise in intraductal pressure was only observed in chronic, but not acute pancreatitis.
During ERCP, the elevations in pancreatic intraductal pressure are induced by increasing rate or viscous solution of infusion. During different forms of experimental acute and chronic pancreatitis, obstructive or non-obstructive etiologies of pancreatitis also induces the elevations in pancreatic intraductal pressure.
A review of the rationale for the testing of the calcineurin inhibitor tacrolimus for post-ERCP pancreatitis prevention
2022, Pancreatology
Endoscopic retrograde cholangiopancreatography (ERCP) is commonly performed for the management of pancreaticobiliary disorders. The most troublesome ERCP-associated adverse event is post-ERCP pancreatitis (PEP), which occurs in up to 15% of all patients undergoing ERCP. A substantial body of preclinical data support a mechanistic rationale for calcineurin inhibitors in preventing PEP. The findings are coupled with recent clinical data suggesting lower rates of PEP in patients who concurrently use the calcineurin inhibitor tacrolimus (e.g., solid organ transplant recipients). In this review, we will firstly summarize data in support of testing the use of tacrolimus for PEP prophylaxis, either in combination with rectal indomethacin or by itself. Secondly, we propose that administering tacrolimus through the rectal route could be favorable for PEP prophylaxis over other routes of administration.
Radiocontrast agent and intraductal pressure promote the progression of post-ERCP pancreatitis by regulating inflammatory response, cellular apoptosis, and tight junction integrity
2022, Pancreatology
Citation Excerpt :
However, contrast agent concentration is not a critical factor in terms of regulating inflammation. An increasing body of evidence suggests that the activation of NF-κB p65 and STAT3 pathways contributes to acute pancreatitis progression [17,20,26,27]. Thus, we tested whether contrast agents and increased hydrostatic pressure can regulate p65 and STAT3 activation/phosphorylation using immunohistochemistry and Western blot analysis (Fig. 3A–C).
Post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP) is the most common complication following ERCP and the mechanism is not fully understood. This study evaluated the changes in the inflammatory response, cellular apoptosis, and tight junction integrity in a rat model of pancreatitis to explore the underlying mechanism.
PEP was induced in rats by retrograde biliopancreatic ductal infusion of contrast agents or saline. Pancreatic tissues were harvested and evaluated by histopathologic, immunohistochemical, immunofluorescence, and Western blot analyses. In addition, amylase and proinflammatory cytokines in plasma were quantified by ELISA assay.
PEP rats developed more severe acute pancreatitis than the sham group after injection of the contrast agent or isotonic saline. PEP rats exhibited increased tissue damage, plasma amylase, proinflammatory cytokines, necrosis, inflammatory infiltrates, apoptosis, and tight junction disruption. At the molecular level, contrast agent and isotonic saline-injected PEP rats demonstrated elevated NF-κB p65 and STAT3 pathways activation, altered expression and activation of apoptosis-related proteins, and suppressed expression of tight junction molecules. However, the contrast agent concentration had no effect on these changes.
In models of acute pancreatitis induced using contrast agent and hydrostatic pressure, the contrast agent and high hydrostatic pressure easily induced the inflammatory response, apoptosis, and tight junction disruption. It is noteworthy that no significant difference in damaged pancreatic acinar cells was observed with different concentrations of the contrast agent.

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: Conflicts of interest The authors disclose no conflicts.

: Funding This work was supported by National Institutes of Health grants DK083327, DK093491, and DK03002 (S.Z.H.).

: Author names in bold designate shared co-first authors.

View full text

Original ResearchFull Report: Basic and Translational—PancreasExposure to Radiocontrast Agents Induces Pancreatic Inflammation by Activation of Nuclear Factor-κB, Calcium Signaling, and Calcineurin

Background & Aims

Methods

Results

Conclusions

Section snippets

Reagents and Animals

Intraductal Infusion of RC Induces Pancreatic Injury in Mice

Discussion

Acknowledgments

Gastrointest Endosc

Gastrointest Endosc

Clin Gastroenterol Hepatol

Gastrointest Endosc

Gastroenterology

J Biol Chem

J Biol Chem

J Virol Methods

Lab Invest

J Biol Chem

Gastrointest Endosc

Trends Pharmacol Sci

Gastroenterology

Gastroenterology

Am J Pathol

Immunity

Am J Pathol

J Biol Chem

J Biol Chem

Curr Biol

Trends Cell Biol

Biochim Biophys Acta

Trends in the utilization of endoscopic retrograde cholangiopancreatography (ERCP) in the United States

Am J Gastroenterol

Post-ERCP pancreatitis and its prevention

Nat Clin Pract Gastroenterol Hepatol

Incidence rates of post-ERCP complications: a systematic survey of prospective studies

Am J Gastroenterol

A randomized trial of rectal indomethacin to prevent post-ERCP pancreatitis

N Engl J Med

Calcineurin/NFAT coupling participates in pathological, but not physiological, cardiac hypertrophy

Circ Res

FK-506- and CsA-sensitive activation of the interleukin-2 promoter by calcineurin

Nature

Defective T cell development and function in calcineurin Abeta-deficient mice

Proc Natl Acad Sci USA

Experimental acute biliary pancreatitis induced by retrograde infusion of bile acids into the mouse pancreatic duct

Nat Protoc

Cholecystokinin activates pancreatic calcineurin-NFAT signaling in vitro and in vivo

Mol Biol Cell

Glucocorticoids increase amylase mRNA levels, secretory organelles, and secretion in pancreatic acinar AR42J cells

J Cell Biol

A simplified purification method for AAV variant by polyethylene glycol aqueous two-phase partitioning

Bioengineered

Original Research
Full Report: Basic and Translational—Pancreas
Exposure to Radiocontrast Agents Induces Pancreatic Inflammation by Activation of Nuclear Factor-κB, Calcium Signaling, and Calcineurin