Towards label-free non-invasive autofluorescence multispectral imaging for melanoma diagnosis.

This study focuses on the use of cellular autofluorescence which visualizes the cell metabolism by monitoring endogenous fluorophores including NAD(P)H and flavins. It explores the potential of multispectral imaging of native fluorophores in melanoma diagnostics using excitation wavelengths ranging from 340 nm to 510 nm and emission wavelengths above 391 nm. Cultured immortalized cells are utilized to compare the autofluorescent signatures of two melanoma cell lines to one fibroblast cell line. Feature analysis identifies the most significant and least correlated features for differentiating the cells. The investigation successfully applies this analysis to pre-processed, noise-removed images and original background-corrupted data. Furthermore, the applicability of distinguishing melanomas and healthy fibroblasts based on their autofluorescent characteristics is validated using the same evaluation technique on patient cells. Additionally, the study tentatively maps the detected features to underlying biological processes. This research demonstrates the potential of cellular autofluorescence as a promising tool for melanoma diagnostics.

Emerging clinical applications in oncology for non-invasive multi- and hyperspectral imaging of cell and tissue autofluorescence.

Hyperspectral and multispectral imaging of cell and tissue autofluorescence is an emerging technology in which fluorescence imaging is applied to biological materials across multiple spectral channels. This produces a stack of images where each matched pixel contains information about the sample's spectral properties at that location. This allows precise collection of molecularly specific data from a broad range of native fluorophores. Importantly, complex information, directly reflective of biological status, is collected without staining and tissues can be characterised in situ, without biopsy. For oncology, this can spare the collection of biopsies from sensitive regions and enable accurate tumour mapping. For in vivo tumour analysis, the greatest focus has been on oral cancer, whereas for ex vivo assessment head-and-neck cancers along with colon cancer have been the most studied, followed by oral and eye cancer. This review details the scope and progress of research undertaken towards clinical translation in oncology.

Clinical applications of non-invasive multi and hyperspectral imaging of cell and tissue autofluorescence beyond oncology.

Hyperspectral and multispectral imaging of cell and tissue autofluorescence employs fluorescence imaging, without exogenous fluorophores, across multiple excitation/emission combinations (spectral channels). This produces an image stack where each pixel (matched by location) contains unique information about the sample's spectral properties. Analysis of this data enables access to a rich, molecularly specific data set from a broad range of cell-native fluorophores (autofluorophores) directly reflective of biochemical status, without use of fixation or stains. This non-invasive, non-destructive technology has great potential to spare the collection of biopsies from sensitive regions. As both staining and biopsy may be impossible, or undesirable, depending on the context, this technology great diagnostic potential for clinical decision making. The main research focus has been on the identification of neoplastic tissues. However, advances have been made in diverse applications-including ophthalmology, cardiovascular health, neurology, infection, assisted reproduction technology and organ transplantation.

Pterygium and Ocular Surface Squamous Neoplasia: Optical Biopsy Using a Novel Autofluorescence Multispectral Imaging Technique.

In this study, differentiation of pterygium vs. ocular surface squamous neoplasia based on multispectral autofluorescence imaging technique was investigated. Fifty (N = 50) patients with histopathological diagnosis of pterygium (PTG) and/or ocular surface squamous neoplasia (OSSN) were recruited. Fixed unstained biopsy specimens were imaged by multispectral microscopy. Tissue autofluorescence images were obtained with a custom-built fluorescent microscope with 59 spectral channels, each with specific excitation and emission wavelength ranges, suitable for the most abundant tissue fluorophores such as elastin, flavins, porphyrin, and lipofuscin. Images were analyzed using a new classification framework called fused-classification, designed to minimize interpatient variability, as an established support vector machine learning method. Normal, PTG, and OSSN regions were automatically detected and delineated, with accuracy evaluated against expert assessment by a specialist in OSSN pathology. Signals from spectral channels yielding signals from elastin, flavins, porphyrin, and lipofuscin were significantly different between regions classified as normal, PTG, and OSSN (p < 0.01). Differential diagnosis of PTG/OSSN and normal tissue had accuracy, sensitivity, and specificity of 88 ± 6%, 84 ± 10% and 91 ± 6%, respectively. Our automated diagnostic method generated maps of the reasonably well circumscribed normal/PTG and OSSN interface. PTG and OSSN margins identified by our automated analysis were in close agreement with the margins found in the H&E sections. Such a map can be rapidly generated on a real time basis and potentially used for intraoperative assessment.

Radiodynamic Therapy Using TAT Peptide-Targeted Verteporfin-Encapsulated PLGA Nanoparticles.

Radiodynamic therapy (RDT) is a recent extension of conventional photodynamic therapy, in which visible/near infrared light irradiation is replaced by a well-tolerated dose of high-energy X-rays. This enables greater tissue penetration to allow non-invasive treatment of large, deep-seated tumors. We report here the design and testing of a drug delivery system for RDT that is intended to enhance intra- or peri-nuclear localization of the photosensitizer, leading to DNA damage and resulting clonogenic cell kill. This comprises a photosensitizer (Verteporfin, VP) incorporated into poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) that are surface-functionalized with a cell-penetrating HIV trans-activator of transcription (TAT) peptide. In addition to a series of physical and photophysical characterization studies, cytotoxicity tests in pancreatic (PANC-1) cancer cells in vitro under 4 Gy X-ray exposure from a clinical 6 MV linear accelerator (LINAC) showed that TAT targeting of the nanoparticles markedly enhances the effectiveness of RDT treatment, particularly when assessed by a clonogenic, i.e., DNA damage-mediated, cell kill.

Machine learning reveals mesenchymal breast carcinoma cell adaptation in response to matrix stiffness.

Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition (MET), are believed to play key roles in facilitating the metastatic cascade. Metastatic lesions often exhibit a similar epithelial-like state to that of the primary tumour, in particular, by forming carcinoma cell clusters via E-cadherin-mediated junctional complexes. However, the factors enabling mesenchymal-like micrometastatic cells to resume growth and reacquire an epithelial phenotype in the target organ microenvironment remain elusive. In this study, we developed a workflow using image-based cell profiling and machine learning to examine morphological, contextual and molecular states of individual breast carcinoma cells (MDA-MB-231). MDA-MB-231 heterogeneous response to the host organ microenvironment was modelled by substrates with controllable stiffness varying from 0.2kPa (soft tissues) to 64kPa (bone tissues). We identified 3 distinct morphological cell types (morphs) varying from compact round-shaped to flattened irregular-shaped cells with lamellipodia, predominantly populating 2-kPa and >16kPa substrates, respectively. These observations were accompanied by significant changes in E-cadherin and vimentin expression. Furthermore, we demonstrate that the bone-mimicking substrate (64kPa) induced multicellular cluster formation accompanied by E-cadherin cell surface localisation. MDA-MB-231 cells responded to different substrate stiffness by morphological adaptation, changes in proliferation rate and cytoskeleton markers, and cluster formation on bone-mimicking substrate. Our results suggest that the stiffest microenvironment can induce MET.

Non-invasive assessment of exfoliated kidney cells extracted from urine using multispectral autofluorescence features.

Optimally preserved urinary exfoliated renal proximal tubule cells were assessed by multispectral imaging of cell autofluorescence. We demonstrated different multispectral autofluorescence signals in such cells extracted from the urine of patients with healthy or diseased kidneys. Using up to 10 features, we were able to differentiate cells from individuals with heathy kidneys and impaired renal function (indicated by estimated glomerular filtration rate (eGFR) values) with the receiver operating characteristic area under the curve (AUC) of 0.99. Using the same method, we were also able to discriminate such urine cells from patients with and without renal fibrosis on biopsy, where significant differences in multispectral autofluorescence signals (AUC = 0.90) were demonstrated between healthy and diseased patients (p < 0.05). These findings show that multispectral assessment of the cell autofluorescence in urine exfoliated proximal tubule kidney cells has the potential to be developed as a sensitive, non-invasive diagnostic method for CKD.

Non-invasive real-time imaging of reactive oxygen species (ROS) using auto-fluorescence multispectral imaging technique: A novel tool for redox biology.

Detecting reactive oxygen species (ROS) that play a critical role as redox modulators and signalling molecules in biological systems currently requires invasive methods such as ROS -specific indicators for imaging and quantification. We developed a non-invasive, real-time, label-free imaging technique for assessing the level of ROS in live cells and thawed cryopreserved tissues that is compatible with in-vivo imaging. The technique is based on autofluorescence multispectral imaging (AFMI) carried out in an adapted fluorescence microscope with an expanded number of spectral channels spanning specific excitation (365 nm-495 nm) and emission (420 nm-700 nm) wavelength ranges. We established a strong quantitative correlation between the spectral information obtained from AFMI and the level of ROS obtained from CellROX staining. The results were obtained in several cell types (HeLa, PANC1 and mesenchymal stem cells) and in live kidney tissue. Additioanly,two spectral regimes were considered: with and without UV excitation (wavelengths > 400 nm); the latter being suitable for UV-sensitive systems such as the eye. Data were analyzed by linear regression combined with an optimization method of swarm intelligence. This allowed the calibration of AFMI signals to the level of ROS with excellent correlation (R = 0.84, p = 0.00) in the entire spectral range and very good correlation (R = 0.78, p = 0.00) in the limited, UV-free spectral range. We also developed a strong classifier which allowed us to distinguish moderate and high levels of ROS in these two regimes (AUC = 0.91 in the entire spectral range and AUC = 0.78 for UV-free imaging). These results indicate that ROS in cells and tissues can be imaged non-invasively, which opens the way to future clinical applications in conditions where reactive oxygen species are known to contribute to progressive disease such as in ophthalmology, diabetes, kidney disease, cancer and neurodegenerative diseases.

PGRMC1 effects on metabolism, genomic mutation and CpG methylation imply crucial roles in animal biology and disease.

BACKGROUND: Progesterone receptor membrane component 1 (PGRMC1) is often elevated in cancers, and exists in alternative states of phosphorylation. A motif centered on PGRMC1 Y180 was evolutionarily acquired concurrently with the embryological gastrulation organizer that orchestrates vertebrate tissue differentiation. RESULTS: Here, we show that mutagenic manipulation of PGRMC1 phosphorylation alters cell metabolism, genomic stability, and CpG methylation. Each of several mutants elicited distinct patterns of genomic CpG methylation. Mutation of S57A/Y180/S181A led to increased net hypermethylation, reminiscent of embryonic stem cells. Pathways enrichment analysis suggested modulation of processes related to animal cell differentiation status and tissue identity, as well as cell cycle control and ATM/ATR DNA damage repair regulation. We detected different genomic mutation rates in culture. CONCLUSIONS: A companion manuscript shows that these cell states dramatically affect protein abundances, cell and mitochondrial morphology, and glycolytic metabolism. We propose that PGRMC1 phosphorylation status modulates cellular plasticity mechanisms relevant to early embryological tissue differentiation.

Non-destructive, label free identification of cell cycle phase in cancer cells by multispectral microscopy of autofluorescence.

BACKGROUND: Cell cycle analysis is important for cancer research. However, available methodologies have drawbacks including limited categorisation and reliance on fixation, staining or transformation. Multispectral analysis of endogenous cell autofluorescence has been shown to be sensitive to changes in cell status and could be applied to the discrimination of cell cycle without these steps. METHODS: Cells from the MIA-PaCa-2, PANC-1, and HeLa cell lines were plated on gridded dishes and imaged using a multispectral fluorescence microscope. They were then stained for proliferating cell nuclear antigen (PCNA) and DNA intensity as a reference standard for their cell cycle position (G1, S, G2, M). The multispectral data was split into training and testing datasets and models were generated to discriminate between G1, S, and G2 + M phase cells. A standard decision tree classification approach was taken, and a two-step system was generated for each line. RESULTS: Across cancer cell lines accuracy ranged from 68.3% (MIA-PaCa-2) to 73.3% (HeLa) for distinguishing G1 from S and G2 + M, and 69.0% (MIA-PaCa-2) to 78.0% (PANC1) for distinguishing S from G2 + M. Unmixing the multispectral data showed that the autofluorophores NADH, FAD, and PPIX had significant differences between phases. Similarly, the redox ratio and the ratio of protein bound to free NADH were significantly affected. CONCLUSIONS: These results demonstrate that multispectral microscopy could be used for the non-destructive, label free discrimination of cell cycle phase in cancer cells. They provide novel information on the mechanisms of cell-cycle progression and control, and have practical implications for oncology research.

Impact of maternal e-cigarette vapor exposure on renal health in the offspring.

Maternal smoking during pregnancy is a significant risk factor of renal pathology in the offspring. E-cigarettes are perceived to be a safe option and are increasingly used by pregnant women either continuously during pregnancy or as a replacement for tobacco cigarettes. This study aimed to determine the effects of replacing tobacco cigarettes with e-cigarettes during pregnancy, and continuous e-cigarette use during pregnancy on the offspring's kidneys. Female Balb/c mice were exposed to either air (sham) or tobacco cigarette smoke (SE) for 6 weeks prior to mating, during gestation and lactation. A subset of the "SE group" received e-cigarette vapor (containing nicotine) after mating until pups weaned. Additional female mice were continuously exposed to e-vapor (either with or without nicotine) for 6 weeks prior to mating until pups weaned. Kidneys and urine from the male offspring were assessed at postnatal day 1, day 20 (weaning), and 13 weeks of age (adulthood). E-cigarette replacement was less detrimental to renal development and albuminuria than continuous SE during pregnancy. However, continuous e-vapor exposure during pregnancy increased markers of oxidative stress, inflammation, and fibrosis in the adult offspring, independent of nicotine. E-cigarette use during pregnancy confers future risk to the offspring's kidneys.

A Mitochondrial Specific Antioxidant Reverses Metabolic Dysfunction and Fatty Liver Induced by Maternal Cigarette Smoke in Mice.

Maternal smoking leads to glucose and lipid metabolic disorders and hepatic damage in the offspring, potentially due to mitochondrial oxidative stress. Mitoquinone mesylate (MitoQ) is a mitochondrial targeted antioxidant with high bioavailability. This study aimed to examine the impact of maternal cigarette smoke exposure (SE) on offspring's metabolic profile and hepatic damage, and whether maternal MitoQ supplementation during gestation can affect these changes. Female Balb/c mice (eight weeks) were either exposed to air or SE for six weeks prior to mating and throughout gestation and lactation. A subset of the SE dams were supplied with MitoQ in the drinking water (500 µmol/L) during gestation and lactation. Intraperitoneal glucose tolerance test was performed in the male offspring at 12 weeks and the livers and plasma were collected at 13 weeks. Maternal SE induced glucose intolerance, hepatic steatosis, mitochondrial oxidative stress and related damage in the adult offspring. Maternal MitoQ supplementation reduced hepatic mitochondrial oxidative stress and improved markers of mitophagy and mitochondrial biogenesis. This may restore hepatic mitochondrial health and was associated with an amelioration of glucose intolerance, hepatic steatosis and pathological changes induced by maternal SE. MitoQ supplementation may potentially prevent metabolic dysfunction and hepatic pathology induced by intrauterine SE.

Novel automated non invasive detection of ocular surface squamous neoplasia using multispectral autofluorescence imaging.

PURPOSE: Diagnosing Ocular surface squamous neoplasia (OSSN) using newly designed multispectral imaging technique. METHODS: Eighteen patients with histopathological diagnosis of Ocular Surface Squamous Neoplasia (OSSN) were recruited. Their previously collected biopsy specimens of OSSN were reprocessed without staining to obtain auto fluorescence multispectral microscopy images. This technique involved a custom-built spectral imaging system with 38 spectral channels. Inter and intra-patient frameworks were deployed to automatically detect and delineate OSSN using machine learning methods. Different machine learning methods were evaluated, with K nearest neighbor and Support Vector Machine chosen as preferred classifiers for intra- and inter-patient frameworks, respectively. The performance of the technique was evaluated against a pathological assessment. RESULTS: Quantitative analysis of the spectral images provided a strong multispectral signature of a relative difference between neoplastic and normal tissue both within each patient (at p < 0.0005) and between patients (at p < 0.001). Our fully automated diagnostic method based on machine learning produces maps of the relatively well circumscribed neoplastic-non neoplastic interface. Such maps can be rapidly generated in quasi-real time and used for intraoperative assessment. Generally, OSSN could be detected using multispectral analysis in all patients investigated here. The cancer margins detected by multispectral analysis were in close and reasonable agreement with the margins observed in the H&E sections in intra- and inter-patient classification, respectively. CONCLUSIONS: This study shows the feasibility of using multispectral auto-fluorescence imaging to detect and find the boundary of human OSSN. Fully automated analysis of multispectral images based on machine learning methods provides a promising diagnostic tool for OSSN which can be translated to future clinical applications.

MitoQ supplementation prevent long-term impact of maternal smoking on renal development, oxidative stress and mitochondrial density in male mice offspring.

To investigate the effect of maternal MitoQ treatment on renal disorders caused by maternal cigarette smoke exposure (SE). We have demonstrated that maternal SE during pregnancy increases the risk of developing chronic kidney disease (CKD) in adult offspring. Mitochondrial oxidative damage contributes to the adverse effects of maternal smoking on renal disorders. MitoQ is a mitochondria-targeted antioxidant that has been shown to protect against oxidative damage-related pathologies in many diseases. Female Balb/c mice (8 weeks) were divided into Sham (exposed to air), SE (exposed to cigarette smoke) and SEMQ (exposed to cigarette smoke with MitoQ supplemented from mating) groups. Kidneys from the mothers were collected when the pups weaned and those from the offspring were collected at 13 weeks. Maternal MitoQ supplementation during gestation and lactation significantly reversed the adverse impact of maternal SE on offspring's body weight, kidney mass and renal pathology. MitoQ administration also significantly reversed the impact of SE on the renal cellular mitochondrial density and renal total reactive oxygen species in both the mothers and their offspring in adulthood. Our results suggested that MitoQ supplementation can mitigate the adverse impact of maternal SE on offspring's renal pathology, renal oxidative stress and mitochondrial density in mice offspring.

Photoresponsive endosomal escape enhances gene delivery using liposome-polycation-DNA (LPD) nanovectors.

Lipid-based nanocarriers with stimuli responsiveness have been utilized as controlled release systems for gene/drug delivery applications. In our work, by taking advantage of the high complexation capability of polycations and the light triggered properties, we designed a novel photoresponsive liposome-polycation-DNA (LPD) platform. This LPD carrier incorporates verteporfin (VP) in lipid bilayers and the complex of polyethylenimine (PEI)/plasmid DNA (pDNA) encoding EGFP (polyplex) in the central cavities of the liposomes. The liposomes were formulated with cationic lipids, PEGylated neutral lipids and cholesterol molecules, which improve their stability and cellular uptake in the serum-containing media. We evaluated the nanocomplex stability by monitoring size changes over six days, and the cellular uptake of the nanocomplex by imaging the intracellular route. We also demonstrated that light triggered the cytoplasmic release of pDNA upon irradiation with a 690 nm LED light source. Furthermore, this light triggered mechanism has been studied at the subcellular level. The activated release is driven by the generation of reactive oxygen species (ROS) from VP after light illumination. These ROS oxidize and destabilize the liposomal and endolysosomal membranes, leading to the release of pDNA into the cytosol and subsequent gene transfer activities. Light-triggered endolysosomal escape of pDNA at different time points was confirmed by a quantitative analysis of colocalization between pDNA and endolysosomes. The increased expression of the reporter EGFP in human colorectal cancer cells was also quantified after light illumination at various time points. The efficiency of this photo-induced gene transfection was demonstrated to be more than double compared to non-irradiated controls. Additionally, we observed a reduced cytotoxicity of the LPDs compared with the polyplexes alone. This study has thus shown that light-triggered and biocompatible LPDs enable an improved control of efficient gene delivery, which will be beneficial for future gene therapies.

Programmable LED-based integrating sphere light source for wide-field fluorescence microscopy.

Wide-field fluorescence microscopy commonly uses a mercury lamp, which has limited spectral capabilities. We designed and built a programmable integrating sphere light (PISL) source which consists of nine LEDs, light-collecting optics, a commercially available integrating sphere and a baffle. The PISL source is tuneable in the range 365-490nm with a uniform spatial profile and a sufficient power at the objective to carry out spectral imaging. We retrofitted a standard fluorescence inverted microscope DM IRB (Leica) with a PISL source by mounting it together with a highly sensitive low- noise CMOS camera. The capabilities of the setup have been demonstrated by carrying out multispectral autofluorescence imaging of live BV2 cells.

Quantitative non-invasive cell characterisation and discrimination based on multispectral autofluorescence features.

Automated and unbiased methods of non-invasive cell monitoring able to deal with complex biological heterogeneity are fundamentally important for biology and medicine. Label-free cell imaging provides information about endogenous autofluorescent metabolites, enzymes and cofactors in cells. However extracting high content information from autofluorescence imaging has been hitherto impossible. Here, we quantitatively characterise cell populations in different tissue types, live or fixed, by using novel image processing and a simple multispectral upgrade of a wide-field fluorescence microscope. Our optimal discrimination approach enables statistical hypothesis testing and intuitive visualisations where previously undetectable differences become clearly apparent. Label-free classifications are validated by the analysis of Classification Determinant (CD) antigen expression. The versatility of our method is illustrated by detecting genetic mutations in cancer, non-invasive monitoring of CD90 expression, label-free tracking of stem cell differentiation, identifying stem cell subpopulations with varying functional characteristics, tissue diagnostics in diabetes, and assessing the condition of preimplantation embryos.

Medically relevant assays with a simple smartphone and tablet based fluorescence detection system.

Cell phones and smart phones can be reconfigured as biomedical sensor devices but this requires specialized add-ons. In this paper we present a simple cell phone-based portable bioassay platform, which can be used with fluorescent assays in solution. The system consists of a tablet, a polarizer, a smart phone (camera) and a box that provides dark readout conditions. The assay in a well plate is placed on the tablet screen acting as an excitation source. A polarizer on top of the well plate separates excitation light from assay fluorescence emission enabling assay readout with a smartphone camera. The assay result is obtained by analysing the intensity of image pixels in an appropriate colour channel. With this device we carried out two assays, for collagenase and trypsin using fluorescein as the detected fluorophore. The results of collagenase assay with the lowest measured concentration of 3.75 µg/mL and 0.938 µg in total in the sample were comparable to those obtained by a microplate reader. The lowest measured amount of trypsin was 930 pg, which is comparable to the low detection limit of 400 pg for this assay obtained in a microplate reader. The device is sensitive enough to be used in point-of-care medical diagnostics of clinically relevant conditions, including arthritis, cystic fibrosis and acute pancreatitis.

Oxidative stress, mitochondrial perturbations and fetal programming of renal disease induced by maternal smoking.

An adverse in-utero environment is increasingly recognized to predispose to chronic disease in adulthood. Maternal smoking remains the most common modifiable adverse in-utero exposure leading to low birth weight, which is strongly associated with chronic kidney disease (CKD) in later life. In order to investigate underlying mechanisms for such susceptibility, female Balb/c mice were sham or cigarette smoke-exposed (SE) for 6 weeks before mating, throughout gestation and lactation. Offspring kidneys were examined for oxidative stress, expression of mitochondrial proteins, mitochondrial structure as well as renal functional parameters on postnatal day 1, day 20 (weaning) and week 13 (adult age). From birth throughout adulthood, SE offspring had increased renal levels of mitochondrial-derived reactive oxygen species (ROS), which left a footprint on DNA with increased 8-hydroxydeoxyguanosin (8-OHdG) in kidney tubular cells. Mitochondrial structural abnormalities were seen in SE kidneys at day 1 and week 13 along with a reduction in oxidative phosphorylation (OXPHOS) proteins and activity of mitochondrial antioxidant Manganese superoxide dismutase (MnSOD). Smoke exposure also resulted in increased mitochondrial DNA copy number (day 1-week 13) and lysosome density (day 1 and week 13). The appearance of mitochondrial defects preceded the onset of albuminuria at week 13. Thus, mitochondrial damage caused by maternal smoking may play an important role in development of CKD at adult life.

L-Carnitine reverses maternal cigarette smoke exposure-induced renal oxidative stress and mitochondrial dysfunction in mouse offspring.

Maternal smoking is associated with metabolic disorders, renal underdevelopment, and a predisposition to chronic kidney disease in offspring, yet the underlying mechanisms are unclear. By exposing female Balb/c mice to cigarette smoke for 6 wk premating and during gestation and lactation, we showed that maternal smoke exposure induced glucose intolerance, renal underdevelopment, inflammation, and albuminuria in male offspring. This was associated with increased renal oxidative stress and mitochondrial dysfunction at birth and in adulthood. Importantly, we demonstrated that dietary supplementation of l-carnitine, an amino acid shown to increase antioxidant defenses and mitochondrial function in numerous diseases, in smoke-exposed mothers during pregnancy and lactation significantly reversed the detrimental maternal impacts on kidney pathology in these male offspring. It increased SOD2 and glutathione peroxidase 1, reduced ROS accumulation, and normalized levels of mitochondrial preprotein translocases of the outer membrane, and oxidative phosphorylation complexes I-V in the kidneys of mouse progeny after intrauterine cigarette smoke exposure. These findings support the hypothesis that oxidative stress and mitochondrial dysfunction are closely linked to the adverse effects of maternal smoking on male offspring renal pathology. The results of our study suggest that l-carnitine administration in cigarette smoke-exposed mothers mitigates these deleterious renal consequences.