Gadolinium accumulates intracellularly in response to GBCA exposure

To analyze the agent- and time-dependent accumulation of gadolinium, a surrogate measurand for GBCA cellular uptake, MRPTEpiCs were treated with a physiological concentration of 2 mM (using estimated diagnostic dose) (Friebe et al. 2018) for 1, 4, 24, and 48 h of either linear or macrocyclic GBCA (Fig. 1a). Overall bulk analysis of gadolinium concentrations by ICP-MS between untreated and GBCA treated groups displayed remarkable differences in gadolinium levels, as gadolinium is a non-physiological element. As early as 1 h of exposure to either class of GBCA, gadolinium levels were significantly increased, suggesting early GBCA uptake. Analysis of each time point, up to 48 h exposure, confirms gadolinium accumulation, indicating the intracellular uptake of GBCAs (Fig. 1b). This cellular association of gadolinium suggests a rate of uptake that increases in parallel with the incubation periods, irrespective of GBCA class. This data speaks in favor of intracellular uptake of the extracellularly designed agents.

Fig. 1

Intracellular accumulation of linear and macrocyclic gadolinium-based contrast agents. a. Chemical structures, chemical names, and generic and brand names of the linear and macrocyclic gadolinium-based contrast agents used in this study. b. Gadolinium concentrations in MRPTEpiCs at 1, 4, 24, and 48 h GBCA exposure, as assayed by ICP-MS. Gadolinium levels were obtained from 1 × 106 cells, with each value representing the mean of triplicate experiments ± SEM (n = 3 per group). The p-values are denoted numerically, from Untreated, by One-way ANOVA, Tukey honesty significant difference post-hoc testing

GBCAs are injurious to renal proximal tubules in a time-dependent manner

For the remainder of the study, we focused on time points up to 24 h GBCA exposure. An XTT colorimetric assay was used to monitor the metabolic activity of GBCA-treated cells and indicate cell viability. The conversion of the yellow tetrazolium salt to an orange-colored formazan product was used to quantify metabolically active cells (Fig. 2a). The 1 and 4 h time points showed no significant changes in the activity of the GBCA-treated cells. However, 24-h exposure to linear GBCAs resulted in a reduced metabolic capacity. To further determine whether these agents were injurious to MRPTEpiCs, kidney injury molecule-1 (KIM-1), a biomarker of drug-induced nephrotoxicity, was used to monitor proximal tubule injury. Expression of KIM-1 in normal renal tubular cells is low, but upon insult, overexpression of KIM-1 correlates with the extent of kidney damage, making it a valuable biomarker for toxicity (Song et al. 2019). After 24-h linear GBCA treatment, protein levels of KIM-1 increased compared to untreated controls; treatment with macrocyclic GBCA showed no increase in protein expression of the biomarker (Fig. 2b). These data indicate that linear GBCAs are injurious to renal proximal tubule epithelial cells in a time-dependent manner.

Fig. 2

Time- and agent-dependent effects of GBCA exposures on MRPTEpiC function. a. Impact of 1 h, 4 h, and 24 h GBCA exposures on cell viability using the XTT assay. Cell viability is expressed as the percentage of metabolically active cells after the indicated GBCA exposures. Results are represented as mean ± SEM (n = 3 per group). The p-values are denoted numerically, ns = not significant from Untreated, by One-way ANOVA, Tukey honesty significant difference post-hoc testing. b. Quantitative western blot analysis of relative kidney injury molecule-1 (KIM-1) following 24 h GBCA exposures. Data are represented as mean ± SEM (n = 4). c. Representative confocal images of LysoTracker Red-DND-99 (LTR) probing at 1 h, 4 h, and 24 h GBCA exposure. Cell loaded with 65 nM LTR and nuclei counterstained with 4',6-diamidino-2-phenylindole (DAPI). Leica TCS SP8. Bars = 10 µm. d. Quantification of the corrected total cellular fluorescence of LTR compared to untreated at the respective time points. Results are represented as mean ± SEM from ≥ 65 cells (n = 3). The p-values are denoted numerically, ns = not significant from Untreated, by One-way ANOVA, Tukey honesty significant difference post-hoc testing

Linear contrast agent treatment, in vivo, results in distinct morphological changes of tubular injury characterized by vacuolization, accumulation of unilamellar vesicles, profound mitochondrial injury, and the biosynthesis of gadolinium nanoparticles (DeAguero et al. 2023). Additionally, in vivo, treatment with macrocyclic contrast agents resulted in similar morphological changes, including alterations to mitochondrial ultrastructure (Supplementary Fig. S1a, upper) and enlarged endosomes with lipid and electron-dense cargo (Supplementary Fig. S1a, middle). These morphological changes are further characterized by the intracellular accumulation of unilamellar vesicles (Supplementary Fig. S1a, lower). In addition, treatment of either class of GBCAs resulted in the development of concentric structures referred to as lamellar bodies (Supplementary Fig. S1b), a hallmark of phospholipidosis. This in vivo data suggests that critical organelles, particularly the mitochondria and endolysosomes, are either the initiator or target of GBCAs. Lysosomotropic dye, LysoTracker Red DND-99 (LTR), accumulates in intact lysosomes and other acidic cellular compartments by acidotropsim and was used to monitor the integrity of the endolysosomal system in this exposure model. LTR cellular fluorescence was evaluated following 1, 4, and 24 h exposures to either agent (Fig. 2c). A 1 h exposure to the linear GBCA resulted in a nominal decrease in LTR staining, with a slight LTR fluorescence increase in the macrocyclic group (Fig. 2c). After 4 h exposures to both agents, there was a marked decrease in LTR staining compared to untreated controls, whereas after 24 h exposure to either agent resulted in a highly pronounced LTR fluorescence as seen in Fig. 2d. The decrease in LTR fluorescence at 4 h suggests alterations to lysosomal integrity. To address the drastic increase in LTR fluorescence after 24 h exposure, populations of LTR-positive puncta were quantified. Increases in LTR staining could indicate changes in endolysosomal volume, as lysosomal biogenesis is often a compensatory mechanism for cell survival in response to lysosomal insult (Papadopoulos et al. 2020). LTR-positive puncta population density increased at least twofold in both exposure groups at 24 h compared to untreated controls (Supplementary Fig. S2). In addition, to address whether this increase in LTR-positive puncta populations is a transient response to the 24-h GBCA exposure, an additional series of experiments was conducted. Following the established 24 h exposure model, cells were afforded a 24 h washout period. Linear exposure with washout (Linear-WO) treated cells sustained the increase in LTR puncta compared to the untreated washout control, albeit reduced to linear alone (Supplementary Fig. S2b). Macrocyclic exposure with washout (Macrocyclic-WO) maintained a similar trend but to a lesser extent than the linear groups, as shown in Supplementary Fig. 2. Thus far, these data suggest not only that linear GBCAs are injurious to renal proximal tubule epithelia but also that GBCAs alter lysosomal functionality.

Exposure to GBCAs induce endolysosomal enlargement

Functional lysosomes are essential to maintaining cellular homeostasis. Dysfunctional lysosomes are involved in various diseases, including lysosomal storage diseases, neurodegeneration, and cancer (Cao et al. 2021). Changes in the expression or activity of lysosomal enzymes, alterations in the size, number, and position of lysosomes, and destabilization of the lysosomal membrane are critical features of lysosomal dysfunction (Wang et al. 2018a, b). Innumerable external and internal stimuli, including lysosomotropic agents, can lead to this dysfunction. Because of the apparent changes to the endolysosomal system, as observed by changes in LTR staining patterns seen in Fig. 2, it was pertinent to understand the effect of GBCAs on lysosome and lysosomal-related organelle structure. Lysosomal-associated membrane protein 1 (LAMP-1), a well-characterized lysosomal membrane protein, was used as a marker of lysosomes in this exposure model (Fig. 3a). To monitor changes in LAMP-1 positive lysosomes at 4 h and 24 h post-exposure, we quantitively analyzed the size of these organelles. A 4 h exposure period increased the diameter of LAMP-1 lysosomes in both GBCA groups compared to the untreated control (Fig. 3b, left). Lysosomes in the 24 h exposure groups are further characterized by a continual enlargement of LAMP-1 organelles, as seen in Fig. 3b, albeit with linear exposed lysosomes being characteristically larger. At this point, the data confirms GBCA exposure leads to enlargement of the lysosome, but whether this enlargement is detrimental to the lysosomes remains unclear.

Fig. 3

GBCA-induced lysosomal enlargement and injury. a. Lysosomal-associated membrane protein 1 (LAMP-1) (green) expression and distribution in untreated and GBCA exposed cells. Nuclei counterstained with DAPI. Higher magnification of the region of interest is denoted by the dashed box and displayed adjacent to the respective image. Leica TCS SP8. Bars = 10 µm b. Quantification of LAMP-1 granule diameter in untreated and treated cells following 4 h and 24 h exposures. Results are represented as mean ± SEM from ≥ 50 cells (n = 3). The p-values are denoted numerically and determined by Two-way ANOVA, Tukey honesty significant difference post-hoc testing. c. Representative images of galectin-3 translocation assay in GBCA-exposed MRPTEpiCs at the indicated time points. Higher magnification of the merged region of interest is denoted by the dashed box and displayed adjacent to the respective image: Galectin-3 (Upper), LAMP-1 (Middle), Merge (Lower). Leica TCS SP8. Bars = 10 µm. d. Quantification of galectin-3 positive cells in untreated and treated MRPTEpiCs at 4 h and 24 h GBCA exposure. Results are represented as mean ± SEM from ≥ 75 cells (n = 3). The p-values are denoted numerically and determined by Two-way ANOVA, Tukey honesty significant difference post-hoc testing. e. Transmission electron microscopy images of MRPTEpiCs following 4 and 24 h exposure highlighting vacuolization (magenta arrows). Nuclear fragmentation (black arrow). n ≥ 75 cells per group. Hitachi HT7000 TEM, AMT 16-megapixel digital camera. Bars = 2.5 µm

Intracellular galectins are well-studied as markers of endolysosomal damage. They form distinct puncta as part of the multi-tiered response to endomembrane damage (Aits et al. 2015a, b). Galectin-3 (Gal-3) functions during lysosomal damage and was used as a damage marker in this study, as changes from a diffuse cytoplasmic staining pattern to distinct puncta are observable by confocal microscopy (Fig. 3c). LAMP-1 was again used as a lysosomal marker to determine the proximity of Gal-3 staining. The percentage of cells expressing endogenous, cytoplasmic Gal-3 puncta was quantified. Cells positive for Gal-3 puncta staining were observable at 4 h and 24 h exposure, regardless of the agent (Fig. 3d). Interestingly, more Gal-3 positive cells were detected at the 4 h exposure time point compared to 24 h exposure. This suggests that not only does 4 h GBCA exposure induce lysosomal enlargement, but it also prompts lysosomal injury. Furthermore, the decrease in Gal-3 positive cells at 24 h may indicate lysosomal repair and removal. We used TEM to observe these morphological changes to further visualize the abnormal enlargement of LAMP-1 lysosomes at 4 and 24 h exposure. As early as 4 h exposure, in conjunction with the confocal microscopy data, cell exposed to either class of GBCA displayed the aggregation of numerous enlarged vesicles (Fig. 3e, upper). Cells exposed to either agent for 24 h exhibited swelling of organelles and profound formation of vacuoles (Fig. 3e, lower). Linear exposed cells displayed a more drastic response and signs of nuclear fragmentation, indicating GBCA-induced cell death. In addition, at the 24-h time point, cells were co-stained for markers of the endolysosomal system: late endosome marker RAB-7 and lysosomal marker LAMP-1. Consistent with the TEM studies, confocal microscopy further highlights that GBCA exposure in kidney cells affects the endolysosomal system and the development of enlarged endolysosomal structures (EELs) (Supplementary Fig. S3). These results, taken together, suggest that 4 h exposure to either linear or macrocyclic is the initiating time point at which lysosomal injury and enlargement of LAMP-1-positive lysosomes are present.

GBCAs induce lysosomal dysfunction and partial lysosomal membrane permeabilization

To investigate how GBCAs might alter lysosomal function and whether GBCA-induced lysosomal enlargement is a pre-requisite for lysosomal membrane destabilization, we monitored the expression and localization of cysteine protease cathepsin B (CTSB) and aspartic protease cathepsin D (CTSD) in response to GBCA exposure. CTSB and CTSD are the most abundant lysosomal proteases and play critical cellular functions, including late-stage roles in autophagy (Yadati et al. 2020). The cellular outcome of lysosomal stress often depends upon the extent of LMP influenced by the size of the pores formed in the lysosomal membrane. The degree of LMP will dictate the selective translocation of lysosomal content into the cytoplasm and the compensatory mechanisms initiated by the cell (Wang et al. 2018a, b). To examine how GBCA exposure and potential LMP affect the processing of CTSB, intracellular levels of the protein were assessed by western blot at 4 h (Fig. 4a) and 24 h (Fig. 4d) exposure. ProCTSB autoproteolytically activates itself into mCTSB within endo/lysosomes (Turk et al. 2001). Exposure to both linear and macrocyclic agents for 4 h significantly impaired the maturation of CTSB (mCTSB) (Fig. 4c). GBCA exposure at either time point produced no significant changes to proCTSB levels. However, mCTSB protein levels were restored to a certain degree when compared to untreated levels following 24 h exposure to both agents (Fig. 4f). Our data show that a 4 h exposure to either type of agent results in early lysosomal dysfunction.

Fig. 4

Effect of GBCAs on cathepsin B processing and lysosomal membrane permeabilization. a. Representative western blot showing levels of CTSB in MRPTEpiCs following 4 h exposure. b-c. Quantitative analysis of relative proCTSB and mCTSB levels in untreated and exposed groups, respectively. d. Western blot displaying the expression levels of CTSB after 24 h linear or macrocyclic GBCA exposure. e–f. Quantification of relative protein levels of proCTSB and mCTSB following exposure. b-c-e–f. Data represented as mean ± SEM (n = 4 per group). The p-values are denoted numerically, ns = not significant from Untreated, by One-way ANOVA, Tukey honesty significant difference post-hoc testing. g. Representative confocal images of CTSB and LAMP-1 staining patterns in GBCA-exposed MRPTEpiCs from at least three independent experiments. Inset dashed boxes indicate regions of interest highlighted by magnified adjacent images: CTSB (Upper), LAMP-1 (Middle), and Merge (Lower). Nuclei were counterstained with DAPI. Leica TCS SP8. Bars = 10 µm

To further determine whether LMP occurs in GBCA-exposed cells, cathepsin localization was monitored by confocal microscopy as lysosomal proteases will translocate from the lysosomal lumen to the cytosol in response to LMP. Immunostaining data from the untreated controls showed a consistent colocalization of CTSB with LAMP-1 at both the 4 h and 24-h time points (Fig. 4g, left), indicating that the LAMP-1 positive lysosomes in this cell type are positive for lysosomal proteases and that these organelles have a degradative capacity. A bright, diffuse staining pattern was observed at both the 4 h and 24 h time points in linear GBCA exposed cells (Fig. 4g, middle), indicating a degree of LMP. Macrocyclic exposed cells displayed a brighter punctate staining pattern with increased cytoplasmic diffusion at 24 h compared to 4 h (Fig. 4g, right), suggesting a time-dependent release of CTSB.

CTSD is trafficked as a pro-form via the endocytic pathway to the lysosome, where it undergoes proteolysis-mediated maturation to mCTSD by CTSB and cathepsin L. Intracellular protein levels of CTSD were also assessed by western blotting at 4 h (Fig. 5a) and 24 h (Fig. 5d). Quantitative analysis revealed that levels of proCTSD decreased in both linear and macrocyclic exposed cells at 4 h compared to untreated protein levels (Fig. 5b). At the same time, mCTSD levels in linear 4 h-exposed cells showed a similar response (Fig. 5c). Macrocyclic exposure displayed a similar response but to a lesser degree. At the 24-h time point, proCTSD and mCTSD returned to near untreated levels, as seen in Fig. 5e-f. At 4 h exposure, levels of pro-forms of CTSD were significantly altered in both linear and macrocyclic GBCA-exposed groups. In contrast, only linear exposure for 4 h significantly reduced mCTSD protein levels, indicating altered cathepsin processing. This response indicates potential linear-induced degradation of CTSD or less transport of proCTSD to the lysosome, resulting in reduced formation of mCTSD or reduced proteolysis of proCTSD into mCTSD by CTSB. Given the dynamic role of cathepsin B in the turnover of proteins, particularly the maturation of cathepsin D, it was important to assess the activity of CTSB at 4 and 24 h exposure. A 4 h GBCA exposure resulted in a non-significant increase in CTSB activity (Supplementary Fig. S4a). Further exposure to either agent proceeded to significantly increase CTSB activity at 24 h exposure (Supplementary Fig. S4b). These data suggest the effect of GBCA exposure on CTSD protein expression levels is multifactorial. Reduced levels of proCTSD at 4 h and near-normal CTSB activity suggest reduced endocytic trafficking of the pro-peptide to the lysosome. Additionally, GBCA-induced low levels of mCTSB at 4 h further indicate reduced processing of proCTSD into mCTSD, highlighting reduced lysosomal stability at 4 h exposure, leading to dysfunctional lysosomes. Altogether these data indicate that the intracellular accumulation of GBCAs leads to the dysregulation of cathepsins.

Fig. 5

Impact of GBCA exposure on cathepsin D processing and release from lysosomal lumen. a. Western blot showing levels of CTSD in 4 h exposed MRPTEpiCs. b-c. Quantification of relative proCTSD and mCTSD levels in untreated and exposed groups. d. Representative western blot displaying expression levels of CTSD following 24 h GBCA exposures. e–f. Quantitative analysis of relative protein levels of proCTSD and mCTSD after exposure. b-c-e–f. Results are represented as mean ± SEM (n = 4 per group). The p-values are denoted numerically, ns = not significant, by One-way ANOVA, Tukey honesty significant difference post-hoc testing. g. Confocal images of GBCA-exposed MRPTEpiCs highlighting CTSD staining patterns relative to LAMP-1 staining from at least three independent experiments. Inset dashed boxes indicate regions of interest emphasized by magnified adjacent images: CTSD (Upper), LAMP-1 (Middle), and Merge (Lower). Nuclei were counterstained with DAPI. Leica TCS SP8. Bars = 10 µm

Like CTSB in untreated controls, immunofluorescence of CTSD shows confinement to LAMP-1-positive lysosomes at both time points (Fig. 5g, left). Exposure to linear GBCA resulted in an intensively stained, immensely diffuse cytosolic pattern at 4 h, with a similar yet reduced response at 24 h (Fig. 5g, middle). A 4-h macrocyclic GBCA exposure resulted in a brightly diffuse staining pattern compared to untreated controls; following 24-h exposure, CTSD staining displayed reduced diffusion but limited co-staining with LAMP-1 (Fig. 5g, right). Smaller cleaved CTSD (~ 27 kDa) is released first from the lysosome, followed by the larger CTSB (~ 37 kDa) (Wang et al. 2018a, b), indicating size exclusivity of released lysosomal content. The differences in immunofluorescent staining patterns of CTSB and CTSD in this exposure model suggest that GBCAs induce partial lysosomal membrane permeabilization. These findings indicate that GBCA-induced pLMP is a dynamic process.

Linear GBCA-induced pLMP impacts mitochondrial function and cell viability

Cell homeostasis depends upon properly functioning organelles working in concert to adapt to changes in the cellular environment (Petkovic et al. 2021). Given that in vivo data demonstrates that not only are endolysosomes impacted by GBCA exposures but mitochondria as well (Supplementary Fig. S1) (DeAguero et al. 2023), it was essential to understand whether the organelle injury occurred in parallel or was interconnected. We also monitored mitochondrial dynamics and network morphology in response to GBCA exposures to explore this potential relationship. Renal tubular cells are rich in mitochondria, with a filamentous, interconnected network that facilitates essential transport functions (Zhan et al. 2013). To evaluate the impact of GBCA exposure on the mitochondrial network of MRPTEpiCs, we utilized live-cell confocal imaging of MitoTracker Red-FM-stained cells to quantitate changes in mitochondrial structure. A 4 h exposure to either the linear or macrocyclic contrast agent resulted in no changes to the characteristic filamentous mitochondrial morphology as mitochondria’s mean area, mean perimeter, and aspect ratio are maintained across untreated and treatment groups (Supplementary Fig. S5a-b). Mitochondrial membrane potential was also measured using fluorescent tetramethylrhodamine ethyl ester (TMRE) dye as it is a direct indicator of mitochondrial health owing to the capacity of positively charged dye to accumulate in the mitochondrial matrix dependent upon mitochondrial membrane potential (ΔψM). When MRPTEpiCs are exposed to either GBCA for 4 h, the TMRE relative fluorescence is consistent across all groups, indicating intact mitochondrial membrane potential (Supplementary Fig. S5c). The data suggests that 4-h GBCA exposure does not directly impact mitochondrial health.

Given that we see a lysosomal-mediated response as early as 4 h GBCA exposure and that the mitochondria are intact and functional at that time point, we speculated that pLMP occurs upstream of cell injury and that the release of lysosomal proteases plays a role in the loss of cell viability at 24 h (see Fig. 2). To examine the impact of GBCAs and lysosomal proteases on mitochondrial health and cell viability, we studied the effect of GBCAs in tandem with a broad specificity lysosomal protease inhibitor cocktail (PI) and the selective cathepsin B inhibitor CA-074Me for the 24 h exposure period. Parameters of the mitochondrial network were quantified in live-cell images. There is a visible structural shortening of the mitochondria in response to either linear or macrocyclic GBCA exposure at 24 h, while linear displayed increased shortening (Fig. 6a). Co-incubation of the GBCAs with PI or CA-074Me blocked this effect. Quantifying mitochondrial morphological parameters highlights that 24 h exposure to linear GBCA significantly reduces the mean area, mean perimeter, and aspect ratio of mitochondria, indicating increased mitochondrial fragmentation (Fig. 6b). PI and CA-07Me co-treatment with linear GBCA blocked this fragmentation. In contrast, PI co-treatment preserved mitochondrial mean area to a greater degree. Macrocyclic GBCA exposure resulted in a significant decrease in mitochondrial mean area. In contrast, the mean perimeter and aspect ratio were non-significantly changed, demonstrating a shortening of the mitochondrial network rather than its fragmentation (Fig. 6b). Either PI or CA-07Me co-treatment blocked this macrocyclic-induced shortening effect to that of untreated controls.

Fig. 6

Effect of lysosomal protease inhibition on the mitochondria structure and function in GBCA-treated MRPTEpiCs. a. Representative confocal images of labeled mitochondria following 24 h GBCA exposure plus co-treatment with the indicated lysosomal protease inhibitors. Live cells loaded with 250 nM MitoTracker Red-FM. Leica TCS SP8. Bars = 10 µm. Insets are processed, binary images produced in FIJI/Image J. b. Mitochondrial network analysis of MitoTracker Red live probed cells using the FIJI/ImageJ Mitochondria Analyzer plugin. Distribution of the mean mitochondrial area and mean mitochondrial perimeter in treated cells. Mitochondrial aspect ratio (length/width) was calculated using the plugin. Results are represented as mean ± SEM from ≥ 75 cells (n = 3). The p-values are denoted numerically, ns = not significant, denoted by line in the graph, or from Untreated, by One-way ANOVA, Tukey honesty significant difference post-hoc testing. c. Quantitative analysis of mitochondrial membrane potential (ΔΨm) using tetramethylrhodamine ethyl ester (TMRE). Results of relative fluorescence are represented as mean ± SEM (n = 3). ns = not significant from Untreated, by One-way ANOVA, Tukey honesty significant difference post-hoc testing. d. Effect of lysosomal protease inhibitor co-treatment with GBCAs on the metabolic capacity of exposed cells as a measure of cell viability. Data are represented as mean ± SEM (n = 3 per group). The p-values are denoted numerically, ns = not significant from Untreated, by One-way ANOVA, Tukey honesty significant difference post-hoc testing. Protease inhibitor cocktail (PI) (P1860, Sigma-Aldrich). CA-074 methyl ester (CA-074Me) (S7420, SelleckChem)

Next, we investigated if these structural changes to the mitochondrial network impacted mitochondrial activity. Only 24 h linear GBCA exposure resulted in decreased TMRE relative fluorescence, indicating a collapse of the mitochondrial membrane potential (Fig. 6c). This reduction in mitochondrial membrane potential was rescued in the presence of either lysosomal protease inhibitor, indicating that linear GBCA-induced pLMP impacted mitochondrial function. Macrocyclic GBCA exposure, with or without lysosomal protease inhibitors, displayed no impact on mitochondrial membrane potential. As we found that 24-h linear GBCA exposure resulted in reduced metabolic capacity of the treated cells, we monitored cell viability in the GBCA and lysosomal protease inhibitor groups. Both PI and CA-074Me co-incubation with linear GBCA salvaged the metabolic capacity of the exposed cells, while CA-07Me returned viability near that of untreated controls (Fig. 6d). Macrocyclic GBCA and co-incubation with PI had no impact on viability. However, co-incubation of the macrocyclic agent with CA-07Me drastically increased cell viability. Altogether, the lysosomal damage caused by GBCA exposures, primarily linear GBCA, induced functional damage to the mitochondrial network and impacted overall MRPTEpiC health, which appears to be cathepsin dependent.