Kip Hermann

The pathogenic mechanisms underlying acute pancreatitis are not clear. Two key pathologic acinar cell responses of this disease are vacuole accumulation and trypsinogen activation. We show here that both result from defective autophagy, by comparing the autophagic responses in rodent models of acute pancreatitis to physiologic autophagy triggered by fasting. Pancreatitis-induced vacuoles in acinar cells were greater in number and much larger than those induced with fasting. Degradation of long-lived proteins, a measure of autophagic efficiency, was markedly inhibited in in vitro pancreatitis, while it was stimulated by acinar cell starvation. Further, processing of the lysosomal proteases cathepsin L (CatL) and CatB into their fully active, mature forms was reduced in pancreatitis, as were their activities in the lysosome-enriched subcellular fraction. These findings indicate that autophagy is retarded in pancreatitis due to deficient lysosomal degradation caused by impaired cathepsin processing. Trypsinogen activation occurred in pancreatitis but not with fasting and was prevented by inhibiting autophagy. A marker of trypsinogen activation partially localized to autophagic vacuoles, and pharmacologic inhibition of CatL increased the amount of active trypsin in acinar cells. The results suggest that retarded autophagy is associated with an imbalance between CatL, which degrades trypsinogen and trypsin, and CatB, which converts trypsinogen into trypsin, resulting in intra-acinar accumulation of active trypsin in pancreatitis. Thus, deficient lysosomal degradation may be a dominant mechanism for increased intra-acinar trypsin in pancreatitis.

The pathogenic mechanisms underlying acute pancreatitis are not clear. Two key pathologic acinar cell responses of this disease are vacuole accumulation and trypsinogen activation. We show here that both result from defective autophagy, by comparing the autophagic responses in rodent models of acute pancreatitis to physiologic autophagy triggered by fasting. Pancreatitis-induced vacuoles in acinar cells were greater in number and much larger than those induced with fasting. Degradation of long-lived proteins, a measure of autophagic efficiency, was markedly inhibited in in vitro pancreatitis, while it was stimulated by acinar cell starvation. Further, processing of the lysosomal proteases cathepsin L (CatL) and CatB into their fully active, mature forms was reduced in pancreatitis, as were their activities in the lysosome-enriched subcellular fraction. These findings indicate that autophagy is retarded in pancreatitis due to deficient lysosomal degradation caused by impaired cathepsin processing. Trypsinogen activation occurred in pancreatitis but not with fasting and was prevented by inhibiting autophagy. A marker of trypsinogen activation partially localized to autophagic vacuoles, and pharmacologic inhibition of CatL increased the amount of active trypsin in acinar cells. The results suggest that retarded autophagy is associated with an imbalance between CatL, which degrades trypsinogen and trypsin, and CatB, which converts trypsinogen into trypsin, resulting in intra-acinar accumulation of active trypsin in pancreatitis. Thus, deficient lysosomal degradation may be a dominant mechanism for increased intra-acinar trypsin in pancreatitis.

Metastatic renal cell carcinoma (mRCC) is nearly incurable and accounts for most of the mortality associated with RCC. Von Hippel Lindau (VHL) is a tumour suppressor that is lost in the majority of clear cell RCC (ccRCC) cases. Its role in regulating hypoxia-inducible factors-1α (HIF-1α) and -2α (HIF-2α) is well-studied. Recent work has demonstrated that VHL knock down induces an epithelial-mesenchymal transition (EMT) phenotype. In this study we showed that a CRISPR/Cas9-mediated knock out of VHL in the RENCA model leads to morphologic and molecular changes indicative of EMT, which in turn drives increased metastasis to the lungs. RENCA cells deficient in HIF-1α failed to undergo EMT changes upon VHL knockout. RNA-seq revealed several HIF-1α-regulated genes that are upregulated in our VHL knockout cells and whose overexpression signifies an aggressive form of ccRCC in the cancer genome atlas (TCGA) database. Independent validation in a new clinical dataset confirms the upregulation of these genes in ccRCC samples compared to adjacent normal tissue. Our findings indicate that loss of VHL could be driving tumour cell dissemination through stabilization of HIF-1α in RCC. A better understanding of the mechanisms involved in this phenomenon can guide the search for more effective treatments to combat mRCC.

) gene knockout RENCA cell line in BALB/c mice, generating an improved model of immunocompetent, metastatic renal cell carcinoma (RCC).

Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism. Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism.