Visualization of renal rotenone accumulation after oral administration and in situ detection of kidney injury biomarkers via MALDI mass spectrometry imaging

The examination of drug accumulation within complex biological systems offers valuable insights into the molecular aspects of drug metabolism and toxicity. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is an innovative methodology that enables the spatial visualization and quantification of biomolecules as well as drug and its metabolites in complex biological system. Hence, this method provides valuable insights into the metabolic profile and any molecular changes that may occur as a result of drug treatment. The renal system is particularly vulnerable to adverse effects of drug-induced harm and toxicity. In this study, MALDI MSI was utilized to examine the spatial distribution of drug and renal metabolites within kidney tissues subsequent to a single oral dosage of the anticancer compound rotenone. The integration of ion mobility spectrometry with MALDI MSI enhanced the data acquisition and analysis, resulting to improved mass resolution. Subsequently, the MS/MS fragment ions of rotenone reference drug were detected and characterized using MALDI HDMS/MS imaging. Notably, drug accumulation was observed in the cortical region of the representative kidney tissue sections treated with rotenone. The histological examination of treated kidney tissues did not reveal any observable changes. Differential ion intensity of renal endogenous metabolites was observed between untreated and rotenone-treated tissues. In the context of treated kidney tissues, the ion intensity level of sphingomyelin (D18:1/16:0), a sphingolipid indicator of glomerular cell injury and renal damage, was found to be elevated significantly compared to untreated kidney tissues. Conversely, the ion intensities of choline, glycero-3-phosphocholine (GPC), inosine, and a lysophosphatidylcholine LysoPC(18:0) exhibited a significant decrease. The results of this study demonstrate the potential of MALDI MSI as a novel technique for investigating the in situ spatial distribution of drugs and renal endogenous molecules while preserving the anatomical integrity of the kidney tissue. This technique can be used to study drug-induced metabolism and toxicity in a dynamic manner.


Figure S1 .
Figure S1.DriftScope display of the two-dimensional (2D) map of (m/z:drift time) of the main fragment ion of rotenone and its isobaric peak.At 65.27 bin, the fragment ion (m/z 192.0779) aligned with the parent precursor ion (m/z 395.1503).A similar peak was detected at m/z 192.0750 but has a drift time of 27.16 bin.The MALDI MS images were acquired at 200 µm spatial resolution, and processed using the inverted Weather1 gradient and log scale image composition.

Figure S2 .
Figure S2.MALDI MS imaging analysis of possible rotenone detection across treated and untreated kidney tissue sections.The ion images of protonated rotenone [M+H] + in (A) drug-treated kidney tissue sections showed a localized distribution in the renal cortex as compared with the (B) untreated kidney tissues.(C) The summary of MALDI MSI analysis demonstrated that detected rotenone in drug-treated kidney tissues has a mass accuracy of less than 6 ppm and a drift time difference of less than 1 bin.These indicators may suggest a plausible rotenone detection in drug-treated kidney tissues.

Figure S7 .
Figure S7.Principal component analysis (PCA) score plot of endogenous metabolites from control and rotenone-treated kidney tissues.The color represented the classes of endogenous compounds, while the shape indicated the experimental groups.Data for each plotted metabolite is the average of the seven cryosections obtained from rat kidneys.The PCA analysis was performed using EZinfo v3.0 (Waters Corporation, Manchester, U.K.).

Figure S8 .
Figure S8.Principal component analysis (PCA) score plot of endogenous biomarkers with significant metabolic alterations in control and rotenone-treated kidney tissues.The color represented the endogenous compounds in the two experimental groups.Data for each plotted metabolite is the average of the seven cryosections obtained from rat kidneys.The PCA analysis was performed using EZinfo v3.0 (Waters Corporation, Manchester, U.K.).(Abbreviation: GPC = glycero-3-phosphocholine; LysoPC = lysophosphatidylcholine).

TABLE S2 .
Normalized ion intensities of rotenone standard solutions used in MALDI qMSI….4

Drift Time (bin) Adduct Ion Theoreti cal m/z Measured m/z Delta ppm
↑ indicates higher ion intensity level than that of the control group; ↓ indicates lower ion intensity level than that of the control group; organonitrogen compounds ; carboxylic acid and derivatives ; purine nucleosides ; glycerophospholipids and sphingolipid ; (*p <0.05 ; **p <0.01) ; ***p <0.001)

Table S2 . Normalized ion intensities of rotenone standard solutions used in MALDI qMSI. Rotenone Drug Concentration (ng/µL) Normalized Ion Intensities of Rotenone Spotted on Blank Kidney Tissues*
*Data from three independent trials with triplicates per calibrant was summarized as mean ( ̅) ± standard deviation ().