Evaluation of CFTR Expression and Localisation in Human Pancreas

Introduction: Cystic fibrosis-related diabetes (CFRD) is the most common co-morbidity in people with CF, occurring in 40-50% of adults. Whilst it is established that β-cell dysfunction in cystic fibrosis (CF) leads to diabetes, the mechanism by which the CF transmembrane conductance regulator (CFTR) channel influences insulin secretion remains debated. Currently, three major hypotheses have been proposed: 1. Intrinsic CFTR-dependent pathways of insulin secretion 2. Pancreas-extrinsic CFTR defects 3. Remodeling of islets following loss of exocrine tissue due to inflammation. Since the contribution of each to the pathogenesis of CFRD remains largely unknown, we sought to determine CFTR localisation within human pancreas using novel and highly sensitive approaches. Methods: Expression of chromogranin A (ChrA), CFTR and keratin 19 (K19) was assessed by immunofluorescence (IF) staining of tissue obtained from deceased donors without diabetes (n=10, age: 23-71 years). Two CFTR antibodies (ab576 and ab590) obtained from the CF Foundation were used to confirm specificity, with ductal and endocrine cells determined by K19 and ChrA respectively. CFTR RNA expression was determined by RNAscope® in situ hybridisation (ISH) and combined with either ChrA or insulin immunohistochemistry (IHC). Results: IF staining of pancreatic tissues indicated co-localisation of CFTR in K19 + ductal cells, but not in ChrA + endocrine cells. These observations were confirmed by combined RNAscope® ISH and IHC (ChrA and insulin), which demonstrated the absence of CFTR RNA in human islets. Conclusion: Employment of these highly sensitive techniques has demonstrated absence of CFTR within normal β-cells or any other islet endocrine cell types. This is in line with recent observations in isolated human islets. We conclude that CFTR abnormalities do not directly impact beta-cell function and that CFRD is mediated by factors extrinsic to the pancreatic endocrine compartment. Disclosure R.R. Maheshwari: None. C.J. Jones: None. J.A.M. Shaw: Advisory Panel; Self; Novo Nordisk A/S. M.G. White: None.