Cystic Fibrosis

Cystic Fibrosis (CF)-related hepatobiliary disease is seen in ~20-30% of the CF patients, and is the second leading cause of death in this population. The only cell type in the liver that expresses the cystic fibrosis transmembrane conductance regulator (CFTR) protein is the intrahepatic biliary epithelial (IBE) cells that line the bile ducts. Wild-type CFTR functions as a cAMP-dependent protein kinase A-regulated apical membrane Cl- channel. The most common mutation of CFTR in North America is the DF508 deletion, which results in the absence of CFTR from the apical membrane. Presently, it is not clear how the lack of CFTR in the apical plasma membrane of IBE cells causes hepatobiliary disease. Progress in CF-related hepatobiliary disease has significantly lagged behind the advances made in CF-related airway disease mainly due to the lack of appropriate model systems for study. This laboratory has overcome this major problem by producing the only CF-IBE and matched normal IBE cell lines derived from humans. The primary function of IBE cells is to respond to secretin stimulation by secreting copious amounts of HCO3- into the lumen of the bile ducts, probably through the apical Cl-/HCO3- anion exchanger (AE). We have demonstrated that wild type CFTR expression is important for the regulation of the Cl-/HCO3- anion exchanger function in CF-IBE cells. Cl-/HCO3- exchanger activity is significantly reduced in CF-IBE cells but can be normalized by CFTR-complemention of CF-IBE cells. These findings, together with data from other laboratories in studies of airway epithelial cells, support the idea that wild type CFTR plays a role in the regulation of the function of other transporter(s) and channel(s) by some yet-to-be defined mechanism. Altered secretion of HCO3- into the bile in CF could lead to significant changes in bile composition and flow which in turn can initiate injury to the liver. Therefore, our observation of misregulated Cl-/HCO3- exchanger functions represents the first testable hypothesis with which to begin to approach the long-term goals of this research, that is, the delineation of the cellular basis of the pathobiology of CF-related hepatobiliary disease. It is our hypothesis that CF-associated hepatobiliary disease results from the combined effects of both deficient Cl- secretion as well as altered regulation of heterologous ion transporter(s) caused by the absence of CFTR in the apical membrane. We further hypothesize that CFTR interacts, either directly or indirectly via another protein(s), with the Cl-/HCO3- exchanger to regulate its activity in the plasma membrane. The goals of our studies are to identify the protein-partners of CFTR that are necessary for the regulation of anion exchanger activity and to and ultimately to investigate the mechanisms by which these interactions take place. Delineating the regulatory function of CFTR for the activity of the Cl-/HCO3- exchanger, at both a physiological and biochemical level, will produce insight into the pathobiology of CF-related hepatobiliary disease.

Autosomal Dominant Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is an inheritable disorder characterized by progressive bilateral enlargement of the kidneys due to accumulation of fluid in large numbers of cysts scattered throughout the renal parenchyma. Hepatic cysts are derived from the biliary epithelium and are the most common extrarenal manifestation of ADPKD, affecting 60% of these patients. It has been demonstrated that mutations in PKD1 or PKD2 genes produce aberrant polycystin proteins that generate the ADPKD phenotype. However, the specific cellular defects underlying the pathology of ADPKD have not yet been elucidated. Determination of how mutations in polycystin 1 and polycystin 2 produces the associated ADPKD phenotypes is central to understanding the disease and creating meaningful approaches for its study and ultimately, treatment.

A new focus of the laboratory is to identify the underlying mechanisms responsible for hepatic cystogenesis in ADPKD by examining how inactivation of polycystin protein generates the ADPKD phenotype, using normal and PKD biliary cell lines and physiologic markers defined in this laboratory. These studies are utilizing a novel technology, chromophore-assisted laser inactivation (CALI), to damage extracellular and/or intracellular domains of polycystin 1 and/or 2 and assess, in real time, the effects of these alterations (collaboration with Dr. D. Jay). Inactivation is accomplished using chromophore-conjugated antibodies to a protein of interest. This approach creates knockout phenotype in living cells in real time, thus preventing compensatory mechanisms that may override or mask the effects of protein inactivation. Furthermore, this technique will also be used to inactivate the reported protein-partners of polycystin, to help dissect out the role that these protein complexes play in the described ADPKD phenotypes, as well as in the normal physiology of biliary cells. The cell lines that are being used in these studies were developed by Dr. Jefferson and his collaborators,and provide a unique in vitro model that can be used to examine the mechanism(s) responsible for the ADPKD phenotype. Using these cell lines, the Jefferson laboratory has identified several ADPKD-associated phenotypic markers. Presently they are assessing changes in Cl/HCO3 anion exchanger activity and intracellular Ca2+ regulation in normal cells to determine if inactivation of extracellular and/or intracellular domains of polycystin 1 and polycystin by CALI elicits the ADPKD phenotype. Changes in these physiological functions may involved changes in signaling or protein/protein interactions, but occur over a short time frame, making them uniquely suitable as endpoint markers for CALI. These studies will lead to a greater understanding of the function and physiology of the polycystin proteins.