Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

An update on the use of animal models in diabetic nephropathy research

In the current review, we discuss limitations and recent advances in animal models of diabetic nephropathy (DN). As in human disease, genetic factors may determine disease severity with the murine FVB and DBA/2J strains being more susceptible to DN than C57BL/6J mice. On the black and tan, brachyuric (BTBR) background, leptin deficient (ob/ob) mice develop many of the pathological features of human DN. Hypertension synergises with hyperglycemia to promote nephropathy in rodents. Moderately hypertensive endothelial nitric oxide synthase (eNOS(−/−)) deficient diabetic mice develop hyaline arteriosclerosis and nodular glomerulosclerosis and induction of renin-dependent hypertension in diabetic Cyp1a1mRen2 rats mimics moderately severe human DN. In addition, diabetic eNOS(−/−) mice and Cyp1a1mRen2 rats recapitulate many of the molecular pathways activated in the human diabetic kidney. However, no model exhibits all the features of human DN; therefore, researchers should consider biochemical, pathological, and transcriptomic data in selecting the most appropriate model to study their molecules and pathways of interest

Kynurenine pathway enzymes in guinea pigs

In some animals, the administration of repeated doses of tryptophan can cause death. It has been reported that guinea pig does not survive repeated doses of tryptophan, due to the absence of the hormonal induction mechanism of liver tryptophan 2,3-dioxygenase (TDO). Therefore, it was of interest to investigate if guinea pig is an animal model suitable for studying tryptophan metabolism. The activities of the enzymes of the kynurenine pathway were determined. Liver TDO was present only as a holoenzyme; kynurenine 3-monooxygenase showed similar, but very high, activity in both liver and kidney. Liver and kidney kynureninase values were also similar, whereas kynurenine-oxoglutarate transaminase activity was higher in kidney than in liver. 3-Hydroxyanthranilate 3,4-dioxygenase gave similar, but very high, values in both liver and kidney, whereas aminocarboxymuconate-semialdehyde decarboxylase activity was double in kidney with respect to liver, but much lower than that of 3-hydroxyanthranilate 3,4-dioxygenase. Total and free tryptophan concentrations in serum were also determined. The free fraction was about 10% of total tryptophan