Physiological Reports ISSN 2051-817X ORIGINAL RESEARCH Candidate genes for limiting cholestatic intestinal injury identified by gene expression profiling Samuel M. Alaish1, Jennifer Timmons1, Alexis Smith1, Marguerite S. Buzza2, Ebony Murphy1, Aiping Zhao3, Yezhou Sun4, Douglas J. Turner5, Terez Shea-Donahue2,3, Toni M. Antalis1,2, Alan Cross3 & Susan G. Dorsey6 1 Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland 2 Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 3 Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 4 Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, Maryland 5 Baltimore Veterans Administration Medical Center, Baltimore, Maryland 6 University of Maryland School of Nursing, Baltimore, Maryland Keywords Abstract Cholestasis, growth hormone, intestine, lipocalin, microarray. The lack of bile flow from the liver into the intestine can have devastating complications including hepatic failure, sepsis, and even death. This pathologic Correspondence condition known as cholestasis can result from etiologies as diverse as total Samuel M. Alaish, Department of Surgery, parenteral nutrition (TPN), hepatitis, and pancreatic cancer. The intestinal University of Maryland School of Medicine, injury associated with cholestasis has been shown to result in decreased intesti- Room N4E35, 22 South Greene Street, nal resistance, increased bacterial translocation, and increased endotoxemia. Baltimore, MD 21201. Tel: (410) 328-5730 Anecdotal clinical evidence suggests a genetic predisposition to exaggerated Fax: (410) 328-0652 injury. Recent animal research on two different strains of inbred mice demon- E-mail: [email protected] strating different rates of bacterial translocation with different mortality rates supports this premise. In this study, a microarray analysis of intestinal tissue Funding Information following common bile duct ligation (CBDL) performed under general anes- Research reported in this publication was thesia on these same two strains of inbred mice was done with the goal of supported by the National Institutes of identifying the potential molecular mechanistic pathways responsible. Over 500 General Medical Sciences of the National Institutes of Health under award number genes were increased more than 2.0-fold following CBDL. The most promising K08GM081701. The content is solely the candidate genes included major urinary proteins (MUPs), serine protease-1- responsibility of the authors and does not inhibitor (Serpina1a), and lipocalin-2 (LCN-2). Quantitative polymerase chain necessarily represent the official views of the reaction (qPCR) validated the microarray results for these candidate genes. In National Institutes of Health. an in vitro experiment using differentiated intestinal epithelial cells, inhibition of MUP-1 by siRNA resulted in increased intestinal epithelial cell permeability. Diverse novel mechanisms involving the growth hormone pathway, the acute Received: 17 June 2013; Revised: 22 July phase response, and the innate immune response are thus potential avenues for 2013; Accepted: 1 August 2013 limiting cholestatic intestinal injury. Changes in gene expression were at times doi: 10.1002/phy2.73 found to be not only due to the CBDL but also due to the murine strain. Should further studies in cholestatic patients demonstrate interindividual vari- Physiol Rep, 1 (4), 2013, e00073, doi: ability similar to what we have shown in mice, then a “personalized medicine” 10.1002/phy2.73 approach to cholestatic patients may become possible. Introduction cal etiologies, such as an obstructing pancreatic cancer or biliary stricture. In the pediatric population, cholestasis Cholestasis, defined as little or no bile flow from the liver resulting from prolonged parenteral nutrition is by far the into the intestine, is a complex pathologic condition that most common etiology. Cholestatic injury has not only a can develop from either functional etiologies, such as hepa- hepatic component but also an intestinal one. Failure of the tic parenchymal disease secondary to hepatitis, or mechani- intestinal barrier with decreased intestinal resistance, ª 2013 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of 2013 | Vol. 1 | Iss. 4 | e00073 the American Physiological Society and The Physiological Society. Page 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Candidate Genes for Cholestatic Intestinal Injury S. M. Alaish et al. increased bacterial translocation, and increased episodes of cycles. All mice weighed 18–25 g at the time of operation. sepsis has been well described (Campillo et al. 1999; Pasc- Matriptase (St14) hypomorphic C57BL/6J mice (List et al. ual et al. 2003; Frances et al. 2004); however, the exact 2007) were bred in the Antalis laboratory. Animal studies mechanisms remain poorly understood. were conducted according to protocols reviewed and Common bile duct ligation (CBDL) is a standard approved by the University of Maryland School of Medi- model of cholestasis in the literature (Georgiev et al. cine Institutional Animal Care and Use Committee and 2008). CBDL in mice leads to both hepatic and intestinal adhered to guidelines promulgated by the National Insti- injuries which are precisely interrelated. We have previ- tutes of Health. In accordance with these guidelines, we ously found differences in the systemic inflammatory used the minimum number of animals to meet the rigor responses and outcome following CBDL between two necessary for this series of experiments. inbred mouse strains, C57BL/6J (B6) and A/J, suggesting a genetic contribution (Alaish et al. 2005). In particular, Experimental design B6 mice were significantly more likely to develop ascites following 1 week of CBDL (Alaish et al. 2005). In concor- CBDL operative procedure dance with this observation, the frequency of mortality after CBDL was significantly higher in B6 mice compared Mice were anesthetized by inhaled isoflurane anesthesia. to A/J mice on days following CBDL (Alaish et al. 2005). The abdomen was clipped and then prepared in sterile Interestingly, although both strains demonstrated mark- fashion with 70% ethyl-ethanol followed by betadine. A edly elevated plasma liver function tests following CBDL, transverse upper abdominal incision was performed. The no difference was noted in liver histology between the CBD was dissected away from the portal vein and was two ligated strains. More recently, our laboratory has ligated near its junction with the duodenum using aneu- shown decreased intestinal resistance and increased bacte- rysm clips engineered with a precisely standardized open- rial translocation following CBDL in these same two ing/closing mechanism. The abdominal wall was then strains of inbred mice. Furthermore, we found genetic closed in a two-layer fashion using absorbable sutures. variation in the intestinal resistance and bacterial translo- Sham-operated mice were treated identically but without cation rates, which correlated with mortality following dissection or ligation of the CBD. Postoperatively, animals CBDL in different strains of inbred mice (Alaish et al. were resuscitated with warmed subcutaneous injections of 2013). Further analysis implicated an IFN-c-mediated saline (1 mL) to replace losses. Mice were returned to apoptotic-independent mechanism of tight junction dis- clean cages where food and water were provided ad libi- ruption, which has been well described in vitro (Madara tum. Buprenorphine, 0.05–0.1 mg/kg was given subcuta- and Stafford 1989; Marano et al. 1998; Youakim and Ah- neously at the time of surgery and then every 8–12 h to dieh 1999; Bruewer et al. 2003; Clayburgh et al. 2004), as treat postoperative pain for 48–72 h. a mechanism possibly responsible for the genetic varia- tion. Nevertheless, the 2.5-fold changes in IFN-c gene RNA extraction expression following CBDL, albeit significant, did not seem monumental enough to fully explain the striking Seven days following the surgery, the mice underwent genetic influence on mortality following CBDL in the deep general anesthesia and euthanasia by thoracotomy mice. In order to uncover other potential mechanisms and cardiac exsanguination. Postoperative day 7 was cho- including novel pathways, we embarked on a whole-gen- sen because this time point corresponded to our earlier ome microarray analysis of jejunal tissue in these two dif- finding of decreased intestinal transepithelial electrical ferent strains of inbred mice following either a sham resistance (TEER) after CBDL (Alaish et al. 2013). In operation or CBDL. The differentially expressed genes addition, this time point exhibited differences in TEER reported here constitute a resource of candidate genes for based on the genetic background of the mouse (Alaish roles in cholestatic intestinal injury. et al. 2013). These TEER findings were found in both the jejunum and ileum and correlated with differences in bac- Material and Methods terial translocation and mortality. Further studies on jeju- nal tissue demonstrated differences in tight junction protein expression between CBDL and sham animals and Animals between the strains (Alaish et al. 2013). Therefore, in this Male A/J and C57BL/6J (B6) mice (8 weeks old) were study, we chose jejunum once again; the intestinal