The Roles of MRP9 in cholangiocyte metabolism - PROJECT SUMMARY/ABSTRACT Cholangiopathies are liver disorders that primarily injure the cholangiocytes, leading to cholestasis, biliary fibro- sis/cirrhosis, and cholangiocarcinoma. These conditions cause significant morbidity and mortality and are a major indication for liver transplantation. Developing effective treatments is challenging due to the heterogene- ous nature of cholangiopathies, many of which are orphan diseases. About 30% of patients with suspected in- herited cholestasis, including many with cholangiopathies, lack pathogenic variants in known disease-causing genes. This lack of molecular diagnosis hinders investigation into disease pathogenesis and development of personalized treatment. Our long-term goal is to understand the etiology and molecular genetics of chronic cholestatic liver diseases and improve patient diagnosis and treatment. We recently identified a homozygous deleterious variant in ABCC12, which encodes the ATP-binding cassette protein MRP9, in a patient with chronic intrahepatic cholestasis. Studies of the patient and two animal models indicate that the loss of MRP9 renders cholangiocytes more susceptible to cell death. The overall objective of this application is to delineate how MRP9 deficiency causes cholangiocyte injury. Our preliminary studies revealed mitochondrial damage in a human cholangiocyte cell line, as well as in the cholangiocytes of larval zebrafish and neonatal mice lacking MRP9. The mitochondrial phenotype occurred prior to changes in bile duct morphology or cholangiocyte cell number and was not observed in hepatocytes in the same animals. In abcc12-/- zebrafish, bile ducts accumu- lated the heme intermediate protoporphyrin IX (PPIX) ectopically. Restoring wild-type MRP9 in mutant cholan- giocytes, but not hepatocytes, partially suppressed PPIX accumulation and bile duct loss. Both neonatal Abcc12-/- mouse cholangiocytes and human H69 cholangiocyte cells carrying the patient variant showed in- creased PPIX accumulation and cell death when treated with porphyrin precursor aminolevulinic acid. Our overarching hypothesis is that MRP9 maintains cholangiocyte integrity by mediating mitochondrial metabolism. We propose two complementary specific aims: 1) Elucidate the molecular and cellular mechanisms underlying cholangiocyte death caused by MRP9 deficiency, and 2) Define the function of MRP9 in cholangiocyte metabo- lism. Aim 1 will test if MRP9 deficiency causes PPIX accumulation and mitochondrial damage within cholangio- cytes, resulting in their death. Aim 2 will determine if MRP9 transports porphyrins. We will identify PPIX-inter- acting proteins in human and mouse cholangiocytes and study how MRP9 cooperates with other porphyrin transporters to maintain cholangiocyte porphyrin homeostasis and health. The proposed study is highly innova- tive, because it integrates both in vitro and in vivo models to study the function of a completely new gene in cholangiocyte metabolism and reveals a novel etiology for cholestatic cholangiopathis. It also investigates an unexplored concept: porphyrin homeostasis in cholangiocytes. The proposed research is significant, because it will reveal new susceptible factors and therapeutic targets for cholestatic cholangiopathies.
