Perfluoroalkyl sulfonate induces cardiomyocyte apoptosis via endoplasmic reticulum stress activation and autophagy flux inhibition.

Perfluoroalkyl sulfonate (PFOS) is a commonly used chemical compound that often found in materials such as waterproofing agents, food packaging, and fire retardants. Known for its stability and persistence in the environment, PFOS can enter the human body through various pathways, including water and the food chain, raising concerns about its potential harm to human health. Previous studies have suggested a cardiac toxicity of PFOS, but the specific cellular mechanisms remained unclear. Here, by using AC16 cardiomyocyte as a model to investigate the molecular mechanisms potential the cardiac toxicity of PFOS. Our findings revealed that PFOS exposure reduced cell viability and induces apoptosis in human cardiomyocyte. Proteomic analysis and molecular biological techniques showed that the Endoplasmic Reticulum (ER) stress-related pathways were activated, while the cellular autophagy flux was inhibited in PFOS-exposed cells. Subsequently, we employed strategies such as autophagy activation and ER stress inhibition to alleviate the PFOS-induced apoptosis in AC16 cells. These results collectively suggest that PFOS-induced ER stress activation and autophagy flux inhibition contribute to cardiomyocyte apoptosis, providing new insights into the mechanisms of PFOS-induced cardiomyocyte toxicity.

Polystyrene microplastics induce anxiety via HRAS derived PERK-NF-κB pathway.

Exposure to environmentally hazardous substances is recognized as a significant risk factor for neurological associated disorders. Among these substances, polystyrene microplastics (PS-MPs), widely utilized in various consumer products, have been reported to exhibit neurotoxicity. However, the potential association of PS-MPs with abnormal anxiety behaviors, along with the underlying molecular mechanisms and key proteins involved, remains insufficiently explored. Here, we delineated the potential mechanisms of PS-MPs-induced anxiety through proteomics and molecular investigations. We characterized the PS-MPs, observed their accumulation in the brain, leading to anxiety-like behavior in mice, which is correlated with microglia activation and pro-inflammatory response. Consistent with these findings, our studies on BV2 microglia cells showed that PS-MPs activated NF-κB-mediated inflammation resulting in the upregulation of pro-inflammatory cytokines such as TNFα and IL-1ß. Of particular significance, HRAS was identified as a key factor in the PS-MPs induced pro-inflammatory response through whole proteomics analysis, and knockdown of H-ras effectively inhibited PS-MPs induced PERK-NF-κB activation and associated pro-inflammatory response in microglia cells. Collectively, our findings highlight that PS-MPs induce anxiety of mice via the activation of the HRAS-derived PERK-NF-κB pathway in microlglia. Our results contribute valuable insights into the molecular mechanisms of PS-MPs-induced anxiety, and may offer implications for addressing neurotoxicity and prevention the adverse effects of environmentally hazardous substances, including microplastics.

Perfluorooctanoic acid induces Leydig cell injury via inhibition of autophagosomes formation and activation of endoplasmic reticulum stress.

Perfluorooctanoic acid (PFOA) is a man-made chemical broadly distributed in various ecological environment and human bodies, which poses potential health risks. Its toxicity, especially the male reproduction toxicity has drawn increasing attention due to declining birth rates in recent years. However, how PFOA induces male reproductive toxicity remains unclear. Here, we characterize PFOA-induced cell injury and reveal the underlying mechanism in mouse Leydig cells, which are critical to spermatogenesis in the testes. We show that PFOA induces cell injury as evidenced by reduced cell viability, cell morphology changes and apoptosis induction. RNA-sequencing analysis reveals that PFOA-induced cell injury is correlated with compromised autophagy and activated endoplasmic reticulum (ER) stress, two conserved biological processes required for regulating cellular homeostasis. Mechanistic analysis shows that PFOA inhibits autophagosomes formation, and activation of autophagy rescues PFOA-induced apoptosis. Additionally, PFOA activates ER stress, and pharmacological inhibition of ER stress attenuates PFOA-induced cell injury. Taken together, these results demonstrate that PFOA induces cell injury through inhibition of autophagosomes formation and induction of ER stress in Leydig cells. Thus, our study sheds light on the cellular mechanisms of PFOA-induced Leydig cell injury, which may be suggestive to human male reproductive health risk assessment and prevention from PFOA exposure-induced reproductive toxicity.

Triptolide induces apoptosis and cytoprotective autophagy by ROS accumulation via directly targeting peroxiredoxin 2 in gastric cancer cells.

Triptolide, a natural bioactive compound derived from herbal medicine Tripterygium wilfordii, has multiple biological activities including anti-cancer effect, which is being tested in clinical trials for treating cancers. However, the exact mechanism by which Triptolide exerts its cytotoxic effects, particularly its specific protein targets, remains unclear. Here, we show that Triptolide effectively induces cytotoxicity in gastric cancer cells by increasing reactive oxygen species (ROS) levels. Further investigations reveal that ROS accumulation contributes to the induction of Endoplasmic Reticulum (ER) stress, and subsequently autophagy induction in response to Triptolide. Meanwhile, this autophagy is cytoprotective. Interestingly, through activity-based protein profiling (ABPP) approach, we identify peroxiredoxins-2 (PRDX2), a component of the key enzyme systems that act in the defense against oxidative stress and protect cells against hydroperoxides, as direct binding target of Triptolide. By covalently binding to PRDX2 to inhibit its antioxidant activity, Triptolide increases ROS levels. Moreover, overexpression of PRDX2 inhibits and knockdown of the expression of PRDX2 increases Triptolide-induced apoptosis. Collectively, these results indicate PRDX2 as a direct target of Triptolides for inducing apoptosis. Our results not only provide novel insight into the underlying mechanisms of Triptolide-induced cytotoxic effects, but also indicate PRDX2 as a promising potential therapeutic target for developing anti-gastric cancer agents.

Deep learning enables the discovery of a novel cuproptosis-inducing molecule for the inhibition of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common and deadly cancers in the world. The therapeutic outlook for HCC patients has significantly improved with the advent and development of systematic and targeted therapies such as sorafenib and lenvatinib; however, the rise of drug resistance and the high mortality rate necessitate the continuous discovery of effective targeting agents. To discover novel anti-HCC compounds, we first constructed a deep learning-based chemical representation model to screen more than 6 million compounds in the ZINC15 drug-like library. We successfully identified LGOd1 as a novel anticancer agent with a characteristic levoglucosenone (LGO) scaffold. The mechanistic studies revealed that LGOd1 treatment leads to HCC cell death by interfering with cellular copper homeostasis, which is similar to a recently reported copper-dependent cell death named cuproptosis. While the prototypical cuproptosis is brought on by copper ionophore-induced copper overload, mechanistic studies indicated that LGOd1 does not act as a copper ionophore, but most likely by interacting with the copper chaperone protein CCS, thus LGOd1 represents a potentially new class of compounds with unique cuproptosis-inducing property. In summary, our findings highlight the critical role of bioavailable copper in the regulation of cell death and represent a novel route of cuproptosis induction.

Insight into Tetrabromobisphenol A-Associated Liver Transcriptional Landscape via Single Cell RNA Sequencing.

In recent years, there has been growing concern over the rising incidence of liver diseases, with increasing exposure to environmental toxins as a significant contributing factor. However, the mechanisms of liver injury induced by environmental pollutants are largely unclear. Here, using tetrabromobisphenol A (TBBPA), a widely used brominated flame retardant, as an example, environmental toxin-induced liver toxicity in mice is characterized via single-cell sequencing technology. Heterogeneous gene expression profiles after exposure to TBBPA in major cell types of the liver are demonstrated. In hepatocytes, pathway analysis of differentially expressed genes reveals the enhanced interferon response and diminished metabolic processes. The disrupted endothelial functions in TBBPA-treated cells are then shown. Moreover, the activation of M2-polarization in Kupffer cells, as well as activated effector T and B cells are unveiled in TBBPA-treated cells. Finally, ligand-receptor pair analysis shows that TBBPA disrupts cell-cell communication and induces an inflammatory microenvironment. Overall, the results reveal that TBBPA-induced dysfunction of hepatocytes and endothelial cells may then activate and recruit other immune cells such as Kuffer cells, and T/NK cells into the liver, further increasing inflammatory response and liver injury. Thus, the results provide novel insight into undesiring environmental pollutant-induced liver injury.

A single-cell landscape of triptolide-associated testicular toxicity in mice.

Triptolide is a key active component of the widely used traditional Chinese herb medicine Tripterygium wilfordii Hook. F. Although triptolide exerts multiple biological activities and shows promising efficacy in treating inflammatory-related diseases, its well-known safety issues, especially reproductive toxicity has aroused concerns. However, a comprehensive dissection of triptolide-associated testicular toxicity at single cell resolution is still lacking. Here, we observed testicular toxicity after 14 days of triptolide exposure, and then constructed a single-cell transcriptome map of 59,127 cells in mouse testes upon triptolide-treatment. We identified triptolide-associated shared and cell-type specific differentially expressed genes, enriched pathways, and ligand-receptor pairs in different cell types of mouse testes. In addition to the loss of germ cells, our results revealed increased macrophages and the inflammatory response in triptolide-treated mouse testes, suggesting a critical role of inflammation in triptolide-induced testicular injury. We also found increased reactive oxygen species (ROS) signaling and downregulated pathways associated with spermatid development in somatic cells, especially Leydig and Sertoli cells, in triptolide-treated mice, indicating that dysregulation of these signaling pathways may contribute to triptolide-induced testicular toxicity. Overall, our high-resolution single-cell landscape offers comprehensive information regarding triptolide-associated gene expression profiles in major cell types of mouse testes at single cell resolution, providing an invaluable resource for understanding the underlying mechanism of triptolide-associated testicular injury and additional discoveries of therapeutic targets of triptolide-induced male reproductive toxicity.

Novel Insight into Functions of Transcription Factor EB (TFEB) in Alzheimer's Disease and Parkinson's Disease.

A key pathological feature of neurodegenerative diseases (NDs) such as Alzheimer's disease (AD) and Parkinson's disease (PD) is the accumulation of aggregated and misfolded protein aggregates with limited effective therapeutic agents. TFEB (transcription factor EB), a key regulator of lysosomal biogenesis and autophagy, plays a pivotal role in the degradation of protein aggregates and has thus been regarded as a promising therapeutic target for these NDs. Here, we systematically summarize the molecular mechanisms and function of TFEB regulation. We then discuss the roles of TFEB and autophagy-lysosome pathways in major neurodegenerative diseases including AD and PD. Finally, we illustrate small molecule TFEB activators with protective roles in NDs animal models, which show great potential for being further developed into novel anti-neurodegenerative agents. Overall, targeting TFEB for enhancing lysosomal biogenesis and autophagy may represent a promising opportunity for the discovery of disease-modifying therapeutics for neurodegenerative disorders though more in-depth basic and clinical studies are required in the future.

Tributyltin chloride (TBTCL) induces cell injury via dysregulation of endoplasmic reticulum stress and autophagy in Leydig cells.

Tributyltin chloride (TBTCL), a commonly used antiseptic substance, is commonly found in the environment. Human exposure to TBTCL through the consumption of contaminated seafood, fish, or drinking water has aroused concern. It is well-characterized that TBTCL has multiple detrimental effects on the male reproductive system. However, the potential cellular mechanisms are not fully elucidated. Here, we characterized molecular mechanisms of TBTCL-induced cell injury in Leydig cells, a critical supporter for spermatogenesis. We showed that TBTCL induces apoptosis and cell cycle arrest in TM3 mouse Leydig cells. RNA sequencing analyses revealed that endoplasmic reticulum (ER) stress and autophagy were potentially involved in TBTCL-induced cytotoxicity. We further showed that TBTCL causes ER stress and inhibited autophagy flux. Notably, the inhibition of ER stress attenuates not only TBTCL-induces autophagy flux inhibition but also apoptosis and cell cycle arrest. Meanwhile, the activation of autophagy alleviates, and inhibition of autophagy exaggerates TBTCL-induced apoptosis and cell cycle arrest flux. These results suggest that TBTCL-induced ER stress and autophagy flux inhibition contributed to apoptosis and cell cycle arrest in Leydig cells, providing novel understanding into the mechanisms of TBTCL-induced testis toxicity.

A single-cell transcriptomic landscape of mouse testicular aging.

INTRODUCTION: Advanced paternal age of reproduction is an increasing trend, especially in developed countries and areas. This trend results in elevated risks of adverse reproductive outcomes such as reduced fertility rates, increased pregnancy loss, and poor childhood health. Yet, a systematic profiling of aging-associated molecular and cellular alterations in testicular tissue is still missing. OBJECTIVES: We aimed to dissect aging-associated molecular characteristics in testes of mice. METHODS: Single-cell transcriptomic sequencing and analysis were conducted in testes of young (2 months old) and old mice (24 months old). Immunofluorescences and immunochemistry were used to characterize aging-associated phenotypes and verify single cell sequence results. RESULTS: Here, we constructed the first single-cell transcriptomic atlases of testes of young and old mice. In-depth dissection of aging-dependent transcriptional alterations in specific cell types revealed multiple dysregulated biological processes such as increased 'senescence-associated secretory phenotype' and 'inflammation', which were major aging-associated characteristics. Further analysis of aging-related differentially expressed genes uncovered a disrupted balance of undifferentiated and differentiated spermatogonia stem cells in spermatogonia, indicative of a potential role of spermatogonia stem cells in aging-associated subfertility. Importantly, for the first time, our results identified an increased subtype of aging-specific macrophages, which may contribute to a hostile proinflammatory microenvironment during testicular aging. CONCLUSION: Taken together, our findings depict the distinct single-cell transcriptional features of the aged mouse testes and provide enormous resources for a comprehensive understanding of the cell-type-specific molecular mechanisms underlying mouse testicular aging, which may shed light on developing novel potential diagnostic biomarkers and therapeutic targets for age-associated male subfertility.

Modulation of Atg genes expression in aged rat liver, brain, and kidney by caloric restriction analyzed via single-nucleus/cell RNA sequencing.

Dysregulation of macroautophagy/autophagy has been closely implicated in aging. Caloric restriction (CR) is an effective intervention of aging partially via activation of autophagy. Recently, a high-throughput single-cell RNA-seq technique has been employed to detect the comprehensive transcriptomes of individual cells. However, the transcriptional networks of ATG (autophagy related) genes in the aging process and the modulation of ATG genes expression by CR at the single-cell level have not been elucidated. Here, by performing data analysis of single nucleus/cells RNA sequencing in rats undergoing aging and the modulation by CR, we demonstrate that the transcription patterns of Atg genes in different cell types of rat liver, brain, and kidney are highly heterogeneous. Importantly, CR reversed aging-induced changes of multiple Atg genes across different cell types in the brain, liver, and kidney. In summary, our results, for the first time, provide comprehensive information on Atg gene expression in specific cell types of different organs in a mammal during aging and give novel insight into the protective role of autophagy and CR in aging at the single-cell resolution.Abbreviations: ATG genes: autophagy-related genes; Atg5: autophagy related 5; Atg7: autophagy related 7; CR: caloric restriction; DEATG: differentially expressed autophagy-related; NAFLD: nonalcoholic fatty liver disease; ScRNA-seq: single-cell RNA sequencing.

CDH17 nanobodies facilitate rapid imaging of gastric cancer and efficient delivery of immunotoxin.

BACKGROUND: It is highly desirable to develop new therapeutic strategies for gastric cancer given the low survival rate despite improvement in the past decades. Cadherin 17 (CDH17) is a membrane protein highly expressed in cancers of digestive system. Nanobody represents a novel antibody format for cancer targeted imaging and drug delivery. Nanobody targeting CHD17 as an imaging probe and a delivery vehicle of toxin remains to be explored for its theragnostic potential in gastric cancer. METHODS: Naïve nanobody phage library was screened against CDH17 Domain 1-3 and identified nanobodies were extensively characterized with various assays. Nanobodies labeled with imaging probe were tested in vitro and in vivo for gastric cancer detection. A CDH17 Nanobody fused with toxin PE38 was evaluated for gastric cancer inhibition in vitro and in vivo. RESULTS: Two nanobodies (A1 and E8) against human CDH17 with high affinity and high specificity were successfully obtained. These nanobodies could specifically bind to CDH17 protein and CDH17-positive gastric cancer cells. E8 nanobody as a lead was extensively determined for tumor imaging and drug delivery. It could efficiently co-localize with CDH17-positive gastric cancer cells in zebrafish embryos and rapidly visualize the tumor mass in mice within 3 h when conjugated with imaging dyes. E8 nanobody fused with toxin PE38 showed excellent anti-tumor effect and remarkably improved the mice survival in cell-derived (CDX) and patient-derived xenograft (PDX) models. The immunotoxin also enhanced the anti-tumor effect of clinical drug 5-Fluorouracil. CONCLUSIONS: The study presents a novel imaging and drug delivery strategy by targeting CDH17. CDH17 nanobody-based immunotoxin is potentially a promising therapeutic modality for clinical translation against gastric cancer.

Dissection of cellular and molecular mechanisms of aristolochic acid-induced hepatotoxicity via single-cell transcriptomics.

Background: Aristolochic acids (AAs), a class of carcinogenic and mutagenic natural products from Aristolochia and Asarum plants, are well-known to be responsible for inducing nephrotoxicity and urothelial carcinoma. Recently, accumulating evidence suggests that exposure to AAs could also induce hepatotoxicity and even hepatocellular carcinoma, though the mechanisms are poorly defined. Methods: Here, we aimed to dissect the underlying cellular and molecular mechanisms of aristolochic acid I (AAI)-induced hepatotoxicity by using advanced single-cell RNA sequencing (scRNA-seq) and proteomics techniques. We established the first single-cell atlas of mouse livers in response to AAI. Results: In hepatocytes, our results indicated that AAI activated NF-κB and STAT3 signaling pathways, which may contribute to the inflammatory response and apoptosis. In liver sinusoidal endothelial cells (LSECs), AAI activated multiple oxidative stress and inflammatory associated signaling pathways and induced apoptosis. Importantly, AAI induced infiltration of cytotoxic T cells and activation of proinflammatory macrophage and neutrophil cells in the liver to produce inflammatory cytokines to aggravate inflammation. Conclusions: Collectively, our study provides novel knowledge of AAs-induced molecular characteristics of hepatotoxicity at a single-cell level and suggests future treatment options for AAs associated hepatotoxicity.

18beta-glycyrrhetinic acid induces ROS-mediated apoptosis to ameliorate hepatic fibrosis by targeting PRDX1/2 in activated HSCs.

Hepatic stellate cells (HSCs) are essential drivers of fibrogenesis. Inducing activated-HSC apoptosis is a promising strategy for treating hepatic fibrosis. 18beta-glycyrrhetinic acid (18ß-GA) is a natural compound that exists widely in herbal medicines, such as Glycyrrhiza uralensis Fisch, which is used for treating multiple liver diseases, especially in Asia. In the present study, we demonstrated that 18ß-GA decreased hepatic fibrosis by inducing the apoptosis in activated HSCs. 18ß-GA inhibited the expression of α-smooth muscle actin and collagen type I alpha-1. Using a chemoproteomic approach derived from activity-based protein profiling, together with cellular thermal shift assay and surface plasmon resonance, we found that 18ß-GA covalently targeted peroxiredoxin 1 (PRDX1) and peroxiredoxin 2 (PRDX2) proteins via binding to active cysteine residues and thereby inhibited their enzymatic activities. 18ß-GA induced the elevation of reactive oxygen species (ROS), resulting in the apoptosis of activated HSCs. PRDX1 knockdown also led to ROS-mediated apoptosis in activated HSCs. Collectively, our findings revealed the target proteins and molecular mechanisms of 18ß-GA in ameliorating hepatic fibrosis, highlighting the future development of 18ß-GA as a novel therapeutic drug for hepatic fibrosis.

Autophagy activation by dietary piceatannol enhances the efficacy of immunogenic chemotherapy.

Immunogenic cell death (ICD) promotes the immune antitumor response via releasing damage-associated molecular patterns (DAMPs) from dying tumor cells. The induction of autophagy improves the efficacy of multiple immunogenic chemotherapies. Here, we show that piceatannol, a dietary phenolic compound that is widely distributed in multiple fruits and vegetables such as grapes, blueberries, and mushrooms, induces autophagy and enhances oxaliplatin (OXA)-induced anticancer immune response. Specifically, piceatannol enhanced OXA-induced release of DAMPs, several key hallmarks of ICD including ATP release, cell surface exposure of calreticulin, and high-mobility group box 1 (HMGB1) release. Mechanistically, piceatannol promoted autophagy via activating TFEB/TFE3, two key transcription factors of the autophagy-lysosome pathway, and inhibiting autophagy attenuated piceatannol plus OXA-induced ATP release. Furthermore, piceatannol induced endoplasmic reticulum stress, which is critical for its role in enhancing OXA-induced cell surface exposure of calreticulin, another key hallmark of ICD. Consistently, the combination of piceatannol with OXA promoted the anticancer effects in immunocompetent mice. Taken together, our results indicate the importance and great potential of dietary piceatannol in cancer immunotherapy. Therefore, piceatannol may be used as an ICD enhancer that improves the efficacy of chemotherapeutics such as OXA in cancer treatment with minimized toxicity.

LncRNA MIR9-3HG enhances LIMK1 mRNA and protein levels to contribute to the carcinogenesis of lung squamous cell carcinoma via sponging miR-138-5p and recruiting TAF15.

The aberrantly expressed long non-coding RNAs (lncRNAs) are closely correlated with the malignant progression of cancer cells. In our study, we identified lncRNA MIR9-3 host gene (MIR9-3HG) as the research target and explored its roles in lung squamous cell carcinoma (LUSC). RT-qPCR was conducted to reveal that MIR9-3HG was observably overexpressed in LUSC cells. Functional assays encompassing colony formation, 5-ethynyl-2'-deoxyuridine (EdU) staining, transwell and flow cytometry assays and western blot detecting related proteins demonstrated that MIR9-3HG depletion hampered cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) while accelerating cell apoptosis in LUSC. Subcellular fractionation assay were performed to demonstrate that MIR9-3HG was prominently distributed in the cytoplasm of LUSC cells. Luciferase reporter, RNA-binding protein immunoprecipitation (RIP), immunofluorescence (IF), fluorescent in situ hybridization (FISH) and RNA pull down assays were implemented to confirm that MIR9-3HG modulates LIM domain kinase 1 (LIMK1) mRNA and protein levels by sequestering microRNA-138-5p (miR-138-5p) and recruiting TATA-box binding protein associated factor 15 (TAF15) protein. Taken together, our research determined that MIR9-3HG up-regulated LIMK1 mRNA and protein levels to promote LUSC carcinogenesis, which offers a novel insight into mechanisms of LUSC.

Celastrol enhances transcription factor EB (TFEB)-mediated autophagy and mitigates Tau pathology: Implications for Alzheimer's disease therapy.

Alzheimer's disease (AD), characterized by the accumulation of protein aggregates including phosphorylated Tau aggregates, is the most common neurodegenerative disorder with limited therapeutic agents. Autophagy plays a critical role in the degradation of phosphorylated Tau aggregates, and transcription factor EB (TFEB) is a master regulator of autophagy and lysosomal biogenesis. Thus, small-molecule autophagy enhancers targeting TFEB hold promise for AD therapy. Here, we found that celastrol, an active ingredient isolated from the root extracts of Tripterygium wilfordii (Lei Gong Teng in Chinese) enhanced TFEB-mediated autophagy and lysosomal biogenesis in vitro and in mouse brains. Importantly, celastrol reduced phosphorylated Tau aggregates and attenuated memory dysfunction and cognitive deficits in P301S Tau and 3xTg mice, two commonly used AD animal models. Mechanistical studies suggest that TFEB-mediated autophagy-lysosomal pathway is responsible for phosphorylated Tau degradation in response to celastrol. Overall, our findings indicate that Celastrol is a novel TFEB activator that promotes the degradation of phosphorylated Tau aggregates and improves memory in AD animal models. Therefore, Celastrol shows potential as a novel agent for the treatment and/or prevention of AD and other tauopathies.

Integrated single-cell transcriptomics and proteomics reveal cellular-specific responses and microenvironment remodeling in aristolochic acid nephropathy.

Aristolochic acid nephropathy (AAN) is characterized by acute proximal tubule necrosis and immune cell infiltration, contributing to the global burden of chronic kidney disease and urothelial cancer. Although the proximal tubule has been defined as the primary target of aristolochic acids I (AAI), the mechanistic underpinning of gross renal deterioration caused by AAI has not been explicitly explained, prohibiting effective therapeutic intervention. To this point, we employed integrated single-cell RNA-Seq, bulk RNA-Seq, and mass spectrometry-based proteomics to analyze the mouse kidney after acute AAI exposure. Our results reveal a dramatic reduction of proximal tubule epithelial cells, associated with apoptotic and inflammatory pathways, indicating permanent damage beyond repair. We found the enriched development pathways in other nephron segments, suggesting activation of reparative programs triggered by AAI. The divergent response may be attributed to the segment-specific distribution of organic anion channels along the nephron, including OAT1 and OAT3. Moreover, we observed dramatic activation and recruitment of cytotoxic T and macrophage M1 cells, highlighting inflammation as a principal contributor to permanent renal injury. Ligand-receptor pairing revealed that critical intercellular crosstalk underpins damage-induced activation of immune cells. These results provide potentially novel insight into the AAI-induced kidney injury and point out possible pathways for future therapeutic intervention.