This study was carried out to screen PANoptosis-related biomarker genes and the involved underlying mechanism in clear cell renal cell carcinoma (ccRCC). Differentially expressed (DE) PANoptosis-related genes were identified. Then prognostic biomarker genes were screened, and used to construct a prognostic model. The predictive ability of the model was explored by receiver operating characteristic (ROC) curves. Then the correlation between risk score with clinical features and immune cell infiltration were evaluated. The expression level and prognosis of gelsolin (GSN) were examined in datasets. Furthermore, in vivo and in vitro experiments were employed to investigate underlying mechanisms. ScRNA-seq analysis uncovered that PANoptosis scores varied significantly across 9 cell types in the tumor microenvironment. A total of 26 DE-PANoptosis-related genes were identified by intersecting DEGs and PANoptosis-related genes. Then 3 prognosis biomarker genes were screened and used to build the prognostic model, namely TLR3, GSN and TNFRSF1A. The risk score was associated with tumor grade, stage (I, II, III, IV), pathologic T-Stage, etc, and also significantly correlated with immune cells and expression of immune checkpoint proteins. In addition, GSN was upregulated in ccRCC, and high expression of GSN indicated a favorable OS. In vivo and in vitro experiments revealed that GSN liquid-liquid phase separation (LLPS) directly induces PANoptosis in ccRCC by inhibiting YAP-dependent glycolysis. In conclusion, using TLR3, GSN and TNFRSF1A, the established PANoptosis-related prognostic model was established and showed good predictive performance for predicting prognosis of ccRCC patients. GSN LLPS directly induces PANoptosis in ccRCC by inhibiting YAP-dependent glycolysis.
Diabetes and obesity are associated with poorer outcomes after ischemic stroke; however, it remains unclear whether this results from increased neuronal susceptibility to injury or from vascular dysfunction induced by metabolic syndrome. To minimize the contribution of vascular factors, we used a model of photoinduced thrombosis (PT) in cortical vessels, which generates lesions of reproducible size and is less dependent on collateral blood flow. PT was induced in wild-type (WT) mice, as well as in <i>ob</i>/<i>ob</i> (leptin-deficient) and <i>db</i>/<i>db</i> (leptin receptor-deficient) mice. Magnetic resonance imaging (MRI) revealed that PT produced comparable infarct volumes in all mouse groups. Several genes associated with inflammation and activation of microglia and macroglia in the peri-infarct area <i>(Cst7</i>, <i>Ccl3</i>, <i>Tlr2</i>, <i>Gfap</i>) exhibited similar expression patterns across all three mouse strains, while transcriptional response to cerebral ischemia of <i>Tnfa</i>, <i>Cxcl9</i>, <i>Il6</i>, <i>Cox2</i>, <i>Mmp3</i>, and <i>Bdnf</i> genes depended on the genotype. Overall, despite individual differences in the expression profiles of certain genes, disruption of leptin signaling (whether due to leptin deficiency or leptin receptor deficiency) caused no genotype-specific exacerbation of stroke-induced injury. Assessment of post-stroke neurological deficits revealed substantial differences in absolute scores between WT and <i>ob</i>/<i>ob</i> or <i>db</i>/<i>db</i> mice, attributable to baseline disparities in body weight and motor activity. In <i>db</i>/<i>db</i> mice, normalization of post-stroke neurological status scores to pre-injury values revealed a more pronounced relative functional decline compared to <i>ob</i>/<i>ob</i> mice, suggesting impairments in early compensatory mechanisms and an important role of leptin signaling in neuroplasticity rather than in the extent of acute neuronal damage. Thus, under conditions that minimize vascular complications, neither leptin deficiency nor leptin receptor deficiency exacerbated acute ischemic brain damage or neuroinflammation.
We explored the possibility of antioxidant and antifibrotic effects of panthenol (PL) associated with modulation of coenzyme A (CoA) biosynthesis in the liver in a rat model of chronic obstructive cholestasis induced by bile duct ligation (BDL). We found that PL increased alcohol dehydrogenase (ADH) activity in the liver of BDL rats. PL and its analog pantethine increased pantothenate kinase (PANK) activity, restored hepatic CoA levels reduced by BDL, lowered protein-bound CoA, and normalized impaired mitochondrial functions associated with induced oxidative stress after BDL. These effects were accompanied by decreased collagen deposition and improved morphological features of hepatocytes. In contrast, PANK inhibitor, hopantenic acid (HPA), reduced hepatic CoA levels, aggravated hepatocellular damage, and promoted fibrosis. In the human hepatic stellate cell line LX-2, PL exhibited no cytotoxicity over a wide concentration range, increased intracellular CoA levels, decreased reactive oxygen species (ROS) production, and attenuated collagen accumulation associated with oxidative stress in vitro. Importantly, inhibition of ADH by 4-methylpyrazole completely abolished the protective effects of panthenol, indicating that its activity depends on metabolic pathways involving CoA. Notably, PL did not directly reduce H<sub>2</sub>O<sub>2</sub> or superoxide anion radical production in cell-free systems but significantly suppressed lipid peroxidation in liposomes and red blood cells in vitro. Ultimately, these findings indicate that the antioxidant and antifibrotic effects of PL are associated with modulation of CoA metabolism and enhanced resistance of biological membranes to oxidative damage.
Caloric restriction (CR) is known to activate a broad spectrum of cytoprotective signaling pathways and enhance tissue tolerance to various stressors, including those associated with the cytotoxic effects of pharmaceutical agents. Nephrotoxic drugs, such as aminoglycoside antibiotics, remain a major clinical concern due to their frequent use and potential to cause acute kidney injury (AKI), for which effective preventive strategies are still limited. In this study, we investigated whether CR applied for 5 weeks (4-week pretreatment + 1-week concurrent with AKI induction) can alleviate AKI triggered by the antibiotic gentamicin, with a focus on evaluating changes in antioxidant-related parameters and autophagy-associated signaling during CR-mediated nephroprotection. CR's nephroprotective effects were evaluated using diagnostic assays, Western blotting, and histological analysis. Additionally, oxidative stress markers and mitochondrial integrity were assessed to analyze the impact of CR on antioxidant-related pathways. CR significantly improved renal function and structure, with reduced kidney injury markers (KIM-1, NGAL) and alleviated histological damage. Critically, CR mitigated oxidative stress, evidenced by decreased thiobarbituric acid reactive substances (TBARS) and protein carbonylation, as well as increased levels of the reduced form of glutathione and activity of glutathione peroxidase (GPx). A lowered Bcl-X<sub>L</sub>/X<sub>S</sub> ratio was consistent with reduced apoptotic signaling, while reduced leukocyte infiltration reflected attenuated renal inflammation. Additionally, a reduction in mitochondrial DNA (mtDNA) lesions suggested that CR was associated with modulation of mitochondrial and metabolism-related pathways, with concurrent improvements in mitochondrial stability. Our findings demonstrate that CR attenuated gentamicin-induced AKI and was associated with changes in antioxidant-related parameters, reduced mtDNA damage, a decrease in inflammatory cell infiltration, and modulation of autophagy-related signaling.
<h4>Background/objectives</h4>Inflammation and oxidative stress are key factors contributing to the initiation and progression of liver fibrosis in chronic obstructive cholestasis. Pantothenic acid (PA) and some of its derivatives have been reported to exhibit moderate anti-inflammatory, antioxidant, and regenerative effects. This study aimed to evaluate the redox-modulating effects of PA derivatives-panthenol (PL), pantethine (PT), and hopantenic acid (HPA) in a rat model of chronic obstructive cholestasis induced by common bile duct ligation (BDL).<h4>Methods</h4>Macroscopic, histological, and ultrastructural alterations in the liver were assessed, along with molecular markers of oxidative stress, inflammation, and parameters of the glutathione (GSH) system.<h4>Results</h4>BDL-induced liver injury was associated with enhanced lipid peroxidation, mitochondrial structural alterations, depletion of GSH, increased levels of protein S-glutathionylation (PSSG), and elevated thiobarbituric acid-reactive substances in mitochondria. Treatment with PL and, to a lesser extent, PT was associated with attenuation of hepatocellular ultrastructural damage, reduced bile duct hyperplasia, decreased inflammatory and necrotic changes, and moderate improvement in fibrosis-related parameters. In contrast, HPA (a PA antagonist) did not demonstrate hepatoprotective effects and it was associated with more pronounced liver injury.<h4>Conclusions</h4>Chronic BDL is accompanied by suppression of glutathione redox capacity and enhanced oxidative stress. PL and PT, but not HPA, were associated with reduced levels of protein S-glutathionylation and partial restoration of redox balance. The protective effects of PL and PT may contribute to their antifibrotic activity, potentially through direct antioxidant capacity or redox-modulating mechanisms associated with the GSH system.
Drug-induced nephrotoxicity is a leading cause of acute kidney injury (AKI) and subsequent chronic kidney disease. Nephrotoxicity often develops as a consequence of treatment with commonly prescribed aminoglycoside antibiotics, and remains a significant clinical challenge. One approach to treating AKI and its associated complications is caloric restriction or its pharmacological mimetics. This study aimed to evaluate the effects of caloric restriction mimetic hydroxycitrate (HC) in gentamicin-induced nephrotoxicity, with particular focus on the influence of treatment duration and the underlying molecular mechanisms. In vitro renal tubular epithelial cells models were used to assess HC's effects on viability, proliferation, and autophagy activation. For in vivo validation, rats with gentamicin-induced AKI received HC treatment via two distinct regimens (3-week and 7-week administration). Experiments on renal tubule cells showed that HC significantly increased cell viability and proliferation and led to the activation of autophagy. In the rat model, only the 7-week administration of HC demonstrated significantly attenuated renal dysfunction in gentamicin-induced AKI. Moreover, it reduced macrophage infiltration, increased renal cell tolerance to apoptosis, activated autophagy, and reduced oxidative stress. Thus, our results indicate that 7-week HC administration could be used as a prophylactic strategy against antibiotic nephrotoxicity, exerting its effects by promoting autophagy, resisting apoptosis, and attenuating oxidative damage.
Hemorrhagic shock (HS) is a life-threatening condition that leads to multiple organ failure due to centralization of blood flow and impaired blood clotting. In this study, we investigated the acute and delayed effects of HS on the brain, kidneys, and liver of rats to identify molecular targets for therapy of the consequences of shock. Blood acid-base balance and electrocardiography (ECG) parameters were studied in rats in the acute phase of HS. Gene expression of antioxidant enzymes (Gpx1, Sod1, Cat, Nfe2l2) and inflammatory markers (Ptprc, Cxcl1, Cd86, Itgal, Il1b, Il6, Tnf, Tlr2, Cox2, Cst7, Ccl3, Il10) in brain, kidney, and liver tissues was analyzed, as well as the amount of protein markers for kidney damage (NGAL, KIM-1) in urine 24 h after HS. In addition, markers for the activation of astrocytes (Gfap) and microglia (Aif1) as well as neuronal markers (Eno2, Tubb3) in brain tissue were analyzed. Biochemical markers for liver and kidney damage and total antioxidant activity were determined in blood serum. Acute HS caused decompensated lactic acidosis, arterial hypotension and characteristic changes in the ECG. Although no pronounced inflammatory response was detected in brain, kidney and liver tissue in the late phase after acute blood loss, the brain and liver tissue were more susceptible to the adverse effects of acute blood loss than the kidneys according to a number of indicators. This points to the need to develop targeted strategies to protect organs in the postresuscitation period by targeting specific molecular targets in specific tissues.
In this study, in a culture of renal epithelial cells, we identified those expressing nestin, a cytoskeletal protein associated with stem/progenitor/activated/proliferating cell states. A mouse expressing GFP under the nestin promoter was used, followed by cell isolation and culture. It is hypothesized that this can be used to assess the stem/progenitor/activated/proliferating cell level in a mixed kidney cell culture. Both nestin-positive and nestin-negative cells were demonstrated to be present in the culture. After visualization, cells were attached to a glass slide with a grid, fixed, and prepared for electron microscopy analysis, with each cell visually identified by light microscopy being analyzed. Electron microscopy revealed tight interactions between nestin-positive and nestin-negative cells. Significant differences in the ultrastructure of nestin-positive and nestin-negative cells were observed. Nestin-positive cells were distinguished by a high ribosome content, indicating high protein-synthesizing activity. In the nestin-GFP-high (sorted) population examined by electron microscopy, vesicle-containing protrusions were frequently observed. These cells could contain multiple nuclei of varying sizes and had a high content of lysosomes. No significant differences in mitochondrial ultrastructure were observed in nestin-positive and -negative cells, although functional characteristics evaluated by the membrane potential probe differed.
Ischemic diseases represent a broad and complex group of clinical pathologies caused by acute or chronic tissue perfusion disorders [...].