Panax quinquefolium (American ginseng) is a perennial understory herb that has been widely used as a medicinal plant in China and other countries. Autotoxicity has been reported to be one of the major problems hindering the consecutive cultivation of American ginseng. However, the potential autotoxic compounds produced by the root of American ginseng are less well known. Here, we report the isolation and characterization of five groups of autotoxic compounds from aqueous extracts of the fibrous roots of American ginseng. Ether extracts of the water-soluble compounds were further analyzed and separated into seven fractions. Among them, the most autotoxic fraction (Fraction V) was subjected to GC/MS analysis, and 44 compounds were identified. Based on literature information, 14 individual compounds were selected and their autotoxic effects on seedling growth were further tested. The results revealed that, of these 14 compounds, 9 phenolic compounds significantly reduced the growth of seedlings in a concentration-dependent manner, while 5 aliphatic compounds showed modest inhibition at all three concentrations tested. Furthermore, we verified the existence of the autotoxic compounds in the plow layer soil of commercially cultivated American ginseng fields, and the concentration of these compounds as determined by HPLC analysis was in line with the concentration determined to be bioactive. Taken together, our study established a functional link between the compounds produced by American ginseng and their autotoxic effects.

The aquaporin 2 (AQP2) plays a critical role in water reabsorption to maintain water homeostasis. AQP2 mutation leads to nephrogenic diabetes insipidus (NDI), characterized by polyuria, polydipsia, and hypernatremia. We previously reported that a novel AQP2 mutation (G215S) caused NDI in a boy. In this study, we aimed to elucidate the cell biological consequences of this mutation on AQP2 function and clarify the molecular pathogenic mechanism for NDI in this patient. First, we analyzed AQP2 expression in Madin-Darby canine kidney (MDCK) cells by AQP2-G215S or AQP2-WT plasmid transfection and found significantly decreased AQP2-G215S expression in cytoplasmic membrane compared with AQP2-WT, independent of forskolin treatment. Further, we found co-localization of endoplasmic reticulum (ER) marker (Calnexin) with AQP2-G215S rather than AQP2-WT in MDCK cells by immunocytochemistry. The functional analysis showed that MDCK cells transfected with AQP2-G215S displayed reduced water permeability compared with AQP2-WT. Visualization of AQP2 structure implied that AQP2-G215S mutation might interrupt the folding of the sixth transmembrane α-helix and/or the packing of α-helices, resulting in the misfolding of monomer and further impaired formation of tetramer. Taken together, these findings suggested that AQP2-G215S was misfolded and retained in the ER and could not be translocated to the apical membrane to function as a water channel, which revealed the molecular pathogenic mechanism of AQP2-G215S mutation and explained for the phenotype of NDI in this patient.

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