The use of electronic nicotine delivery systems (ENDS) such as e-cigarettes and one-time use vaping devices has become popular among adolescents in recent years [1]. ENDS devices heat e-liquids so that they may be vaporized and inhaled by the user. These e-liquids often contain solvents such as propylene glycol (PG), vegetable glycerin (VG), flavors, and nicotine. Although many of the ingredients found within e-liquids are classified by the Food and Drug Administration (FDA) as “generally recognized as safe” (GRAS), this classification only refers to the oral route of ingestion. E-liquids are heated and vaporized for inhalation however, and therefore this route of exposure is not well characterized in terms of safety for the user.

In a large-scale survey, it was recently reported that e-cigarettes are among the most used tobacco product in middle and high school students [2]. Flavorings found in e-liquids have also been shown to augment vaping behavior by affecting how often vaping occurs and amount and length of puffs taken by the user [3,4]. The common flavoring aldehyde vanillin (VAN, Fig. 1) is a popular e-liquid flavoring ingredient that can be found in many e-liquid products, especially flavors labeled as “desserts”, “sweets”, or “coffee” types [5,6]. E-liquids that use VAN as an ingredient can become unstable prior to heating, leading to acetal formation that can then be inhaled by the user and lead to subsequent activation of the transient receptor potential (TRP) ion channels, which act as receptors for irritant aldehydes within the airway [7]. Exposure to VAN has also been shown to reduce nitric oxide (NO) production and increase interleukin-6 (IL-6) production in human aortic endothelial cells (HAECs) [8], as well as decrease parasympathetic activity within the heart in mice after long-term (10 week) exposure [9]. A surge in reports of e-cigarette or vaping use-associated lung injury (EVALI) cases in recent years led to the Centers for Disease Control (CDC) to recommend discontinuing use of vaping products that contain vitamin E acetate, which was heavily linked to the onset of EVALI in affected patients [10]. Well-documented medical cases of EVALI patients have reported diffuse interstitial fibrosis in the lung, worsening oxygen needs, presence of ground-glass opacities (GGOs), and leukocytosis [11]. These reports indicate that e-liquid components which are inhaled should be characterized and the toxicity of exposure to them should be assessed.

Studies on effects of e-liquids and their flavoring components have been increasing in recent years, however effects within the kidney are still not well characterized. Many studies focus on effects within the respiratory tract and lung because that is the first point of exposure after inhalation. However, once e-liquid components have passed through the lung and into the bloodstream, it is necessary to understand their subsequent organ targets and effects. The kidney expends large amounts of energy to filter the blood, and also receives approximately 25 % of cardiac output. A survey study in adolescents and young adults suffering from chronic kidney disease (CKD) who frequently used e-cigarettes reported increased proteinuria, although it was determined that occasional e-cigarette use was not a strong risk factor for CKD progression [12]. High-fat diet (HFD) together with vaping has been shown to cause deleterious effects in the kidney, leading to altered mitochondrial phosphorylation (OXPHOS) complex expression as well as altered inflammatory, oxidative stress, and pro-fibrotic markers [13]. In addition, rats treated by intraperitoneal (ip) injection to nicotine and non-nicotine e-liquids e exhibited reduced renal oxidative stress defense enzymes while also having increased renal proteins and thiols, as well as darkened and condensed nuclei with reduced cytoplasm within the collecting ducts. Although studies on e-liquid effects in the kidney are few, these results indicate that the kidney is a potential site of toxicity after exposure.

This study focused on identifying potential targets of renal toxicity after exposure to the e-liquid flavoring vanillin. Human proximal tubule (HK-2) epithelial cells were exposed to VAN and changes in cellular stress markers as well as energy processes were determined. Markers for apoptosis, lipid peroxidation-derived oxidative stress, endoplasmic reticulum (ER), and autophagy/mitophagy were evaluated by western blot. Glycolytic and mitochondrial energy processes were quantified using a Seahorse XFp Analyzer after exposure to VAN. Additionally, MTT assay was performed on cells to assess mitochondrial health. Finally, trypan blue exclusion was performed on samples after VAN exposure to determine cell viability and membrane integrity.