Sayantap Datta

Introduction: Nicotine has been shown to induce Nlrp3 (nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3) inflammasome activation, thereby contributing to podocyte injury. However, the molecular mechanisms underlying nicotine-induced Nlrp3 inflammasome activation and subsequent podocyte dysfunction remain largely unknown. The present study investigated whether membrane raft (MR)-associated redox signalling plays a critical role in nicotine-induced Nlrp3 inflammasome activation and podocyte injury. Methods and Results: Nicotine treatment induced membrane raft clustering in podocytes in a dose-dependent manner. Upon nicotine stimulation, the NADPH oxidase subunits gp91 ^phox and p47 ^phox were recruited and aggregated within membrane raft clusters, leading to the formation of a membrane raft redox signalling platform. The formation of this signalling platform was significantly inhibited by pretreatment with the membrane raft disruptor methyl-β-cyclodextrin (MCD), the NADPH oxidase inhibitor diphenyleneiodonium (DPI), or the acid sphingomyelinase (Asm) inhibitor amitriptyline. Nicotine also markedly enhanced the colocalization of Nlrp3 with ASC and caspase-1, indicating inflammasome assembly, and significantly increased caspase-1 activity, IL-1β production, desmin expression, podocyte permeability, and apoptosis compared with control cells. These effects were significantly attenuated by pretreatment with MCD, DPI, the caspase-1 inhibitor WEHD, or amitriptyline. Furthermore, immunofluorescence analysis demonstrated that nicotine significantly reduced podocin expression, whereas pretreatment with MCD, DPI, WEHD, or amitriptyline largely preserved podocin expression. Conclusion: Our findings demonstrate that membrane raft-associated redox signalling serves as a critical upstream mechanism driving nicotine-induced NLRP3 inflammasome activation and podocyte injury.

Objective: Gut microbe metabolite, Trimethylamine-N-Oxide (TMAO), has been identified as an independent risk factor for adverse cardiovascular events. However, the molecular mechanisms of TMAO-induced cardiovascular diseases (CVDs) is not clearly understood. In recent years extracellular vesicles has gained attention because of their role in physiological and pathological processes in CVDs and their potential as a biomarker in prognosis of CVDs. It is unclear whether TMAO trigger the formation and release of exosomes from endothelial cells (ECs). Based on our preliminary results, we hypothesized that TMAO stimulation results in enhanced exosomal release from ECs and these EC-derived exosomes have the potential to induce endothelial dysfunction. Methods: Exosomes were isolated from the EC culture and plasma with or without the treatment of TMAO, using exosome isolation kit. Exosomes markers like CD63, CD9 were analysed by western blot. Isolated exosomes were characterized by Nano Sight NS300 analyser. Exosome’s structure and morphology were determined by transmission electron microscope (TEM) and Cryo-TEM. Immunophenotyping of exosomes was measured by nano-flow cytometry, where the exosomes were labelled with antibodies targeting CD63, CD9 proteins. ECs were treated with these isolated exosomes for 24 hr and endothelial barrier integrity was determined by FITC-dextran permeability and trans-endothelial electrical resistance (TEER) from confluent endothelial monolayers. Cytokine production in ECs was measured by ELISA. In addition, tight junctional disruption was examined by immunofluorescent staining. Results: TMAO treatment enhanced both CD9 and CD63 protein levels in ECs as compared to control (n=5/group; p<0.05). Nanoparticle tracking analysis determined the particle size and revealed enhanced release of exosomes upon TMAO treatment in ECs. Additionally, TMAO-induced exosome secretion was confirmed by TEM analysis. EC-derived exosomes induced damage to the endothelial barrier integrity which was demonstrated by ECs permeability assay and TEER experiments. TMAO induced exosomal release also resulted in enhanced production of interleukin-1β in ECs (n=4/group; p<0.05). Immunofluorescence revealed that TMAO induced exosomal release markedly reduced tight junctional proteins such as ZO-1, ZO-2, and VE-cadherin (n=4/group; p<0.05). Conclusion: In conclusion, TMAO induced formation and release of exosomes from ECs both in vitro and in vivo. These activated EC-derived exosomes contribute to endothelial dysfunction and vascular injury. NIH R01HL148711 to Dr. Saisudha Koka This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Background: Previous studies strongly identify nicotine as a predisposing factor for chronic kidney disease (CKD) onset and progression to end-stage renal disease (ESRD), clear mechanistic understanding of nicotine induced renal dysfunction remains less understood. The Thioredoxin (TRX) system, comprising NADPH, TRX reductase and TRX, is central to cellular redox balance and protection against cellular oxidative stress. Upregulation in Thioredoxin interacting protein (TXNIP) expression sequesters reduced TRX and affects the TRX cellular disulfide oxidoreductase system. Our recent study postulated that NADPH oxidase dependent O2•− production (reactive oxygen species (ROS)) mediates nicotine-induced NLRP3 inflammasome activation and leads to podocyte injury. However, it remains unknown how NADPH oxidase-derived ROS is sensed by NLRP3 inflammasomes in podocytes upon nicotine stimulation. Hence, the current study assessed whether TXNIP mediates nicotine-induced NLRP3 inflammasome activation and consequent podocyte injury. Methods: TXNIP/NLRP3 molecular interaction was assessed via co-immunoprecipitation; Inflammasome formation and immunofluorescence expressions were analyzed via confocal microscopy; IL-1β production was measured using ELISA; Caspase-1 enzymatic activity was measured by colorimetric assay; Apoptotic induction was assessed by fluorescence-based TUNEL assay. Results: Co-immunoprecipitation based molecular interaction studies showed that TXNIP strongly interacts with NLRP3 upon nicotine stimulation as compared to control podocytes. However, pre-treatment with TXNIP-specific inhibitor SRI-37330 attenuates TXNIP/NLRP3 interaction. Confocal microscopic analysis suggests that nicotine significantly augments the colocalization of NLRP3 with apoptosis associated speck-like protein containing a caspase recruitment domain (ASC), NLRP3 with caspase-1 and NLRP3 with TXNIP in podocytes compared to control cells. However, pre-treatment with TXNIP inhibiting SRI-37330 abolished the nicotine-induced NLRP3/ASC, NLRP3/Caspase-1 and NLRP3/TXNIP co-localization. Nicotine treatment significantly increases active caspase-1 expression (6-fold), caspase-1 enzymatic activity and IL-1β production compared to control cells. However, prior treatment with TXNIP inhibiting SRI-37330 attenuated the nicotine-induced active caspase-1 expression, caspase-1 activity and IL-1β production. Further immunofluorescence studies confirm that nicotine significantly decreases podocin and nephrin (2.5-fold) expression (markers of podocyte damage) compared to control cells. However, pretreatment with TXNIP inhibiting SRI-37330 attenuated the nicotine-induced podocin and nephrin downregulation. Moreover, confocal and biochemical analysis showed that nicotine treatment significantly increased desmin expression, podocyte permeability and apoptosis relative to control cells. However, pre-treatment with TXNIP inhibiting SRI-37330 significantly attenuates nicotine-induced desmin expression, cell permeability and apoptotic induction. Conclusion: Cumulatively, our studies suggest that NLRP3/TXNIP interaction is an important signaling mediator of nicotine-induced NLRP3 inflammasome activation and subsequent podocyte injury. This project was supported by NIH grant, R01HL148711. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Background: Recent studies demonstrate the role of NLRP3 inflammasome activation in nicotine-induced podocyte injury, making it possible to target the intracellular machinery of inflammation for treatment of end stage renal disease. However, since Nlrp3 inflammasomes are activated in the cytosol and their products may not be secreted out of podocytes via a classical and Golgi apparatus-mediated delivery pathway, it remains unclear how NLRP3 inflammasome activation-derived products such as HMGB1, IL-1 β or IL-18 are released out to trigger the inflammatory response and ultimately cause podocyte injury or glomerular injury. Hence, we tested the hypothesis whether acid sphingomyelinase (Asm) contributes to robust release of inflammatory exosomes in podocytes and contributes to the glomerular injury or sclerosis. Methods: Exosome morphology and structural characterization was analyzed using transmission electron microscopy (cryo-TEM). Cytek Aurora flow cytometry was utilized to analyze and quantify the exosome release. Nanoparticle Tracking Analysis (NTA) was done using NanoSight NTA3.2 to determine exosome particle size distribution. Uninephrectomized Asm knockout (Asm ^-/- ) and wild type (Asm ^+/+ ) mice were fed a normal chow for 5 weeks with or without nicotine treatment to produce glomerular injury. Results: By confocal microscopy, exosome release in glomeruli was confirmed by colocalization of exosome markers (annexin A2 vs. alkaline phosphate, ALP) in glomeruli of nicotine treated Asm+/+ mice, but not in Asm-/- mice. Nanoparticle Tracking Analysis (NTA) showed a largely increased urinary exosomes or microvesicles (90-120 nm) in Asm+/+ mice upon nicotine treatment, but not in Asm-/- mice. Biochemical analyses showed that Nlrp3 inflammasome product, HMGB1 or IL-1β was significantly increased in glomeruli of nicotine treated Asm+/+ mice, but not in Asm-/- mice. In functional studies, we found that urinary protein excretion was significantly higher in nicotine treated Asm+/+ mice compared to vehicle treated mice. The Asm -/- mice had significantly decreased nicotine-induced increase in urinary protein excretion. In addition, nicotine treated Asm+/+ mice produced significant morphological changes in the glomeruli, such as mesangial expansion, capillary collapse and increased cellularity, which was significantly attenuated in the Asm-/- mice. Furthermore, the nicotine-induced glomerular damage index (GDI) was significantly attenuated in Asm- /- mice. In invitro studies, using nanoparticle tracking analysis (NTA) we found that nicotine stimulation increased exosomes secretion (60-120 nm) from podocytes in dose dependent manner. Cytek Aurora flow cytometry analysis showed that nicotine stimulation increased the CD9/CD63 expression (exosome markers) in podocytes compared to control cells. However, prior treatment Asm inhibitor, amitriptyline blocked the nicotine-induced exosome secretion (CD9/CD63 expression) in podocytes. In conclusion, our observations demonstrate that nicotine enhanced Nlrp3 inflammasomes activation and at the same time increased exosome release from podocytes, which work together to trigger podocyte glomerular inflammation and injury. This project was supported by NIH grant, R01HL148711. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Cardiometabolic risk and associated dysfunctions contribute largely to the recent rise in mortality globally. Advancements in multi-omics in recent years promise a better understanding of potential biomarkers that enable an early diagnosis of cardiometabolic dysfunction. However, the molecular mechanisms driving the onset and progression of cardiometabolic disorders remain poorly understood. Adipokines are adipocyte-specific cytokines that are central to deleterious cardiometabolic alterations. They exhibit both pro-inflammatory and anti-inflammatory effects, complicating their association with cardiometabolic disturbances. Thus, understanding the cardiometabolic association of adipokines from a molecular and signaling perspective assumes great importance. This review presents a comprehensive outline of the most prominent adipokines exhibiting pro-inflammatory and/or anti-inflammatory functions in cardiometabolic dysfunction. The review also presents an insight into the pathophysiological implications of such adipokines in different cardiometabolic dysfunction conditions, the status of adipokine druggability, and future studies that can be undertaken to address the existing scientific gap. A clear understanding of the functional and mechanistic role of adipokines can potentially improve our understanding of cardiovascular disease pathophysiology and enhance our current therapeutic regimen in the years to come.

Targeting oncogenes and their interactive partners is an effective approach to developing novel targeted therapies for cancer and other chronic diseases. We and others have long suggested the MDM2 oncogene being an excellent target for cancer therapy, based on its p53-dependent and -independent oncogenic activities in a variety of cancers. The MYC family proteins are transcription factors that also regulate diverse biological functions. Dysregulation of MYC, such as amplification of MYCN, is associated with tumorigenesis, especially for neuroblastoma. Although the general survival rate of neuroblastoma patients has significantly improved over the past few decades, high-risk neuroblastoma still presents a poor prognosis. Therefore, innovative and more potent therapeutic strategies are needed to eradicate these aggressive neoplasms. This review focuses on the oncogenic properties of MYCN and its molecular regulation and summarizes the major therapeutic strategies being developed based on preclinical findings. We also highlight the potential benefits of targeting both the MYCN and MDM2 oncogenes, providing preclinical evidence of the efficacy and safety of this approach. In conclusion, the development of effective small molecules that inhibit both MYCN and MDM2 represents a promising new strategy for the treatment of neuroblastoma and other cancers.

Introduction: The oncogene MDM2 has garnered attention not only for its role in cancer as a negative regulator of the tumor suppressor p53 but also for its p53-independent oncogenic activities. MDM2 also involves metabolic reprogramming, such as serine metabolism, respiration, mitochondrial functions, the folate cycle, and redox balance. Traditional MDM2 inhibitors blocking the protein-protein binding between MDM2 and p53 have shown limited clinical success in various stages of clinical trials, most likely due to low efficacy, drug toxicity, and drug resistance, highlighting the need for a novel, p53-independent strategy to inhibit MDM2. The present study investigated the antitumor effects of MA242, a novel MDM2 and NFAT1 inhibitor, in breast cancer models.Methods: The anticancer activity and underlying mechanisms of MA242 were evaluated in vitro using breast cancer cell lines with different p53 backgrounds and in vivo using orthotopic and patient-derived xenograft models.Results: We demonstrated that MA242 significantly inhibited cell viability and induced apoptosis in breast cancer cells, regardless of p53 status. Metabolic analysis revealed that MA242 notably disrupted nicotinamide metabolism, modified nucleotide metabolism, and elevated cellular oxidative stress by disturbing the redox balance. Furthermore, in animal models, MA242 reduced MDM2 expression and effectively inhibited tumor growth dependent on MDM2 expression without causing host toxicity.Discussion: These findings highlight the potential of MA242 as a modulator of cancer metabolism and support its further development as a therapeutic option for aggressive breast cancers.

Background: Recent studies have shown that Nlrp3 inflammasome activation is importantly involved in podocyte dysfunction induced by nicotine. The present study was designed to test whether acid sphingomyelinase (Asm) and ceramide signaling play a role in mediating nicotine-induced Nlrp3 inflammasome activation and subsequent podocyte damage. Methods and Results: Nicotine treatment significantly increased the Asm expression and ceramide production compared to control cells. However, prior treatment with amitriptyline, an Asm inhibitor significantly attenuated the nicotine-induced Asm expression and ceramide production. Confocal microscopic and biochemical analyses showed that nicotine treatment increased the colocalization of NLRP3 with Asc, Nlrp3 vs. caspase-1, IL-1β production, caspase-1 activity, and desmin expression in podocytes compared to control cells. Pretreatment with amitriptyline abolished the nicotine-induced colocalization of NLRP3 with Asc, Nlrp3 with caspase-1, IL-1β production, caspase-1 activity and desmin expression. Immunofluorescence analyses showed that nicotine treatment significantly decreased the podocin expression compared to control cells. However, prior treatment with amitriptyline attenuated the nicotine-induced podocin reduction. In addition, nicotine treatment significantly increased the cell permeability, O₂ production, and apoptosis compared to control cells. However, prior treatment with amitriptyline significantly attenuated the nicotine-induced cell permeability, O₂ production and apoptosis in podocytes. Conclusions: Asm is one of the important mediators of nicotine-induced inflammasome activation and podocyte injury. Asm may be a therapeutic target for the treatment or prevention of glomerulosclerosis associated with smoking.