Receptor Inhibitor Research Articles

Mechanism of protective actions of sparsentan in the kidney: lessons from studies in models of chronic kidney disease.

Simultaneous inhibition of angiotensin II AT1 and endothelin ETA receptors has emerged as a promising approach for treatment of chronic progressive kidney disease. This therapeutic approach has been advanced by the introduction of sparsentan, the first dual AT1 and ETA receptor antagonist. Sparsentan is a single molecule with high affinity for both receptors. It is US Food and Drug Administration approved for immunoglobulin A nephropathy (IgAN) and is currently being developed as a treatment for rare kidney diseases, such as focal segmental glomerulosclerosis. Clinical studies have demonstrated the efficacy and safety of sparsentan in these conditions. In parallel with clinical development, studies have been conducted to elucidate the mechanisms of action of sparsentan and its position in the context of published evidence characterizing the nephroprotective effects of dual ETA and AT1 receptor inhibition. This review summarizes this evidence, documenting beneficial anti-inflammatory, antifibrotic, and hemodynamic actions of sparsentan in the kidney and protective actions in glomerular endothelial cells, mesangial cells, the tubulointerstitium, and podocytes, thus providing the rationale for the use of sparsentan as therapy for focal segmental glomerulosclerosis and IgAN and suggesting potential benefits in other renal diseases, such as Alport syndrome.

Inhibition of β2-adrenergic Receptor regulates necroptosis in prostate cancer cell

As the malignant tumor with the highest incidence in male, prostate cancer poses a significant threat to the reproductive health of elderly men. Our previous studies have shown that promoting necroptosis of cancer cells can effectively inhibit cancer cell proliferation. This study includes lentivirus-mediated knockdown of β2AR which resulted in stable transfectants that exhibited an increased ability to form clones compared to that of the negative control group. In the protein and mRNA levels, necroptosis associated RIP and mixed lineage kinase domain-like (MLKL) were significantly higher in the treatment group than they were in the control group. Furthermore, cells treated with propranolol exhibited necrotic morphology as observed by transmission electron microscopy. The combination of β2AR suppression and necroptosis inhibitors resulted in a more potent suppression of cell proliferation compared to that observed in the control and negative control groups. Additionally, it elevated in the necrosis rate as determined by flow cytometry. Immunofluorescence staining revealed enhanced RIP and MLKL expression in the sh-β2AR group compared to levels in the negative control group. Co-immunoprecipitation experiments detected an interaction between β2AR and RIP. MLKL and RIPK3 levels were significantly higher in xenograft tumor sections from the sh-β2AR group compared to levels in the sh-NC group. To conclude, our research indicates the proliferation of PC-3 and DU-145 cprostate cancer cells can be suppressed by inhibiting β2AR, and this occurs through the RIP/MLKL-mediated pathway of necroptosis.

Synergistic Effects of Neratinib in Combination With Palbociclib or Miransertib in Brain Cancer Cells.

Aberrant expression and activation of epidermal growth factor receptor (EGFR) resulted in approval of several forms of EGFR inhibitors in the treatment of patients with a wide range of epithelial cancers. However, no EGFR inhibitor has yet been approved for the treatment of patients with brain cancer, indicating that targeting EGFR alone may not be sufficient in some patients. In this study, we investigated the role of all members of the EGFR family, other growth factor receptors, cell-cycle proteins, and downstream cell signaling pathways (e.g., mitogen-activated protein kinase (MAPK), serine/threonine protein kinase (AKT), signal transducer and activator of transcription (STAT3), Src, Abelson murine leukemia viral oncogene homolog (Abl)) on the growth of a panel of human brain cancer cell lines (HBCCLs). We examined the growth response of HBCCLs to treatment with 17 targeted agents compared to two cytotoxic drugs. Of the targeted agents, the irreversible pan-human epidermal growth factor receptor (HER) inhibitors neratinib and afatinib were more effective than erlotinib and lapatinib at inhibiting the growth of all HBCCLs, and the cyclin-dependent kinase (CDK)1/2/5/9 inhibitor dinaciclib was the most potent targeted agent. We found that treatment with Src/Abl/c-kit inhibitor dasatinib, signal transducer and activator of transcription (STAT3) inhibitor stattic, Abl/platelet-derived growth factor receptor (PDGFR)α/vascular endothelial growth factor (VEGFR)2/fibroblast growth factor receptor (FGFR)1 inhibitor ponatinib, and the tropomyosin receptor kinase (TRK)/ROS proto-oncogene 1 receptor tyrosine kinase (ROS)/anaplastic lymphoma kinase (ALK) inhibitor entrectinib, also inhibited the growth of all HBCCLs. Interestingly, these agents were more effective in inhibiting growth of HBCCLs when proliferating at a slower rate. In addition to inhibiting the proliferation of HBCCLs, treatment with neratinib, dinaciclib, dasatinib, stattic and trametinib inhibited the migration of brain tumor cell line A172. Notably, we found that treatment with neratinib in combination with palbociclib (CDK4/6 inhibitor), or miransertib (AKT1/2/3 inhibitor) resulted in synergistic growth inhibition of all HBCCLs. Our results support that repurposing drugs like neratinib in combination with the palbociclib or miransertib may be of therapeutic potential in brain cancer and warrants further investigations.

Systemic hypertension with VEGFR inhibitor (axitinib) therapy in cancer patients: A meta-analysis and systematic review of randomized controlled trials

Vascular endothelial growth factor receptor inhibitors (VEGFRi), namely axitinib, are commonly used chemotherapeutic agents in patients with cancer; however, this medication has a significant cardiovascular side effect profile, such as high-grade hypertension. We performed this updated meta-analysis of RCTs to compile cardiovascular adverse events, such as all-grade and high-grade (>3) hypertension, the risk for thrombosis (DVT and PE), and peripheral edema. A systematic search was performed on PubMed, Cochrane, and Embase from inception until October 2023 for studies using axitinib to treat various cancers. Trials with patients randomly allocated for VEGFRi drug therapy with axitinib and reported all-grade hypertension as an outcome were included. Statistical analysis was performed using Cochrane Review Manager to calculate pooled proportions of odds ratios (OR) with a 95 % confidence interval (CI) using the random-effects model, Mantel−Haenszel method. A total of 8 RCTs and 2502 patients were included in the review. Compared with the placebo group, the VEGFRi (Axitinib) therapy group was associated with a higher risk of all-grade and high-grade hypertension, hand–foot syndrome, and fatigue. Furthermore, there was no increased risk of thromboembolism (DVT/PE) or hypothyroidism. However, a lower risk of peripheral edema was noted between the two groups. Screening for patients with preexisting hypertension, identifying risk factors for cardiovascular diseases before the initiation of VEGFRi therapy, and careful monitoring of high-risk patients during VEGFRi therapy, as well as prompt treatment with antihypertensive drugs, will help mitigate the adverse effects. Further evaluation using prospective designs is required to study the clinical significance and develop mitigation strategies.

FDA Approval Summary: Fruquintinib for the Treatment of Refractory Metastatic Colorectal Cancer.

On November 8, 2023, the FDA approved fruquintinib, an inhibitor of vascular endothelial growth factor receptors (VEGFR)-1, -2, and -3, for the treatment of patients with metastatic colorectal cancer (mCRC) who have been previously treated with fluoropyrimidine‑, oxaliplatin‑, and irinotecan‑based chemotherapy, an anti‑VEGF therapy, and, if RAS wild‑type and medically appropriate, an anti EGFR therapy. Approval was based on Study FRESCO-2, a globally-conducted, double-blind, placebo-controlled randomized trial. The primary endpoint was overall survival (OS). The key secondary endpoint was progression-free survival (PFS). A total of 691 patients were randomized (461 and 230 into the fruquintinib and placebo arms, respectively). Fruquintinib provided a statistically significant improvement in OS with a hazard ratio (HR) of 0.66 (95% CI: 0.55, 0.80; p<0.001). The median OS was 7.4 months (95% CI: 6.7, 8.2) in the fruquintinib arm and 4.8 months (95% CI: 4.0, 5.8) for the placebo arm. Adverse events observed were generally consistent with the known safety profile associated with inhibition of the VEGFR. The results of FRESCO-2 were supported by the FRESCO study, a double-blind, single country, placebo-controlled, randomized trial in patients with refractory mCRC who have been previously treated with fluoropyrimidine‑, oxaliplatin‑, and irinotecan‑based chemotherapy. In FRESCO, the OS HR was 0.65 (95% CI: 0.51, 0.83; p<0.001). FDA concluded that the totality of the evidence from FRESCO-2 and FRESCO supported an indication for patients with mCRC with prior treatment with fluoropyrimidine, oxaliplatin-, and irinotecan-based chemotherapy, an anti-VEGF biological therapy, and if RAS wild‑type and medically appropriate, an anti-EGFR therapy.

Effects of oral administration of nonselective Trk inhibitor on bladder overactivity in rodent models of prostatic inflammation.

Our research focused on the assessment of the impact of systemic inhibition of Trk receptors, which bind to nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), on bladder hypersensitivity in two distinct rodent models of prostatic inflammation (PI). Male Sprague-Dawley rats were divided into three groups (n = 6 each): the control group (no PI, vehicle administration), the untreated group (PI, vehicle administration), and the treated group (PI, nonselective Trk inhibitor, GNF 5837, administration). PI in rats was induced by a intraprostatic injection of 5% formalin. Posttreatment, we carried out conscious cystometry and a range of histological and molecular analyses. Moreover, the study additionally evaluated the effects of a nonselective Trk inhibitor on bladder overactivity in a mouse model of PI, which was induced by prostate epithelium-specific conditional deletion of E-cadherin. The rat model of PI showed upregulations of NGF and BDNF in both bladder and prostate tissues in association with bladder overactivity and inflammation in the ventral lobes of the prostate. GNF 5837 treatment effectively mitigated these PI-induced changes, along with reductions in TrkA, TrkB, TrkC, and TRPV1 mRNA expressions in L6-S1 dorsal root ganglia. Also, in the mouse PI model, GNF 5837 treatment similarly improved bladder overactivity. The findings of our study suggest that Trk receptor inhibition, which reduced bladder hypersensitivity and inflammatory responses in the prostate, along with a decrease in overexpression of Trk and TRPV1 receptors in sensory pathways, could be an effective treatment strategy for male lower urinary tract symptoms associated with PI and bladder overactivity.

The effects and mechanisms of Gefitinib on airway inflammation and airway remodeling in C57BL/6 mice with asthma

Objective: To investigate the effects of the epidermal growth factor receptor(EGFR) inhibitor Gefitinib on airway inflammation and airway remodelling in asthmatic C57BL/6 mice, and to analyze its possible mechanisms. Methods: Male C57BL/6 mice, aged 6-8 weeks, were randomly assigned into five groups: Group A (control group), Group B (asthma group), Group C (asthma+20 mg/kg gefitinib group), Group D (asthma+40 mg/kg gefitinib group), and Group E (40 mg/kg gefitinib group), with seven mice per group. Mice were sensitized by intraperitoneal injection of a mixture of 0.2 ml solution containing OVA and Al(OH)3 [20 μg OVA+2 mg Al(OH)3 dissolved in 0.2 ml of physiological saline] at Day 0 and 14. Starting from Day 25 to 31, Group B, C, and D were challenged with nebulization of 1% OVA solution (8 ml) to induce asthma, once a day for approximately 40 minutes, with continuous aerosolization for 7 days. Group C and D were given 0.2 ml of Gefitinib dissolved in 0.5% carboxymethylcellulose sodium (CMCNa) by gavage half an hour before challenging, and Group E was simultaneously given with 0.2 ml of Gefitinib dissolved in 0.5% CMCNa only. Group A and B were given an equivalent volume of 0.5% CMCNa by gavage. After 24 h of final challenge, the bronchoalveolar lavage fluid (BALF) was prepared for the determination of total cell count and eosinophil count. The levels of total immune globulin E (IgE) in serum and interleukin (IL)-4, IL-5 and IL-13 in BALF and lung tissue homogenates were measured by ELISA. The mRNA expression levels of IL-4, IL-5, IL-13 in lung were measured. Immunohistochemistry and Western blot experiments were used to detect the expression levels of EGFR in lung tissues. Results: In Group B, the level of total IgE in serum, total cell count, eosinophil count, the levels of IL-4, IL-5, IL-13 in BALF and the phosphorylation of EGFR and its downstream activation in lung were higher than those in Group A (all P<0.05). The levels of total IgE in serum [(261.32±44.38) ng/ml, (194.09±52.39) ng/ml vs (1 023.70±105.51) ng/ml], total cell count [(23.70±4.08)×105/ml, (14.92±4.06)×105/ml vs (35.36±6.30)×105/ml], eosinophil count [(108.00±13.69)×104/ml, (67.00±17.28)×104/ml vs (147.86±20.06)×104/ml], IL-4 [(36.42±4.48) pg/ml, (30.45±8.12) pg/ml vs (58.72±7.17) pg/ml], IL-5 [(16.20±4.62) pg/ml, (13.38±5.14) pg/ml vs (23.46±5.38) pg/ml], IL-13 [(18.45±7.28) pg/ml, (14.33±7.70) pg/ml vs (104.12±24.66) pg/ml] in BALF of Group C and D were lower than those in Group B (all P<0.05). The levels of IL-4, IL-5, and IL-13 as well as their mRNA levels in the lung tissue of Group C and D were lower than those in Group B (all P<0.05). In Group C and D, the positive expression rate of phosphorylated epidermal growth factor receptor (p-EGFR) in lung tissue [(40.53±6.80)%, (23.60±4.42)% vs (70.78±5.36)%], p-EGFR/EGFR (61.68±7.48, 51.13±5.19 vs 105.90±11.66), phosphorylated extracellular regulated protein kinase (p-Erk)/extracellular regulated protein kinase (Erk) (75.28±7.11, 47.54±4.83 vs 98.76±4.71), and phosphorylated protein kinase B (p-Akt)/protein kinase B (Akt) (96.24±5.40, 68.52±2.73 vs 103.30±4.52) was lower than those of Group B (all P<0.05). There was no statistically significant difference in the relevant indicators between Group A and E (all P>0.05). Conclusion: Gefitinib may alleviate airway inflammation and airway remodeling in asthmatic mice by inhibiting EGFR phosphorylation and affecting the activation of downstream Erk and Akt.

Review of the Ticagrelor Trials Evidence Base.

Ticagrelor is a platelet P2Y12 receptor inhibitor approved for use in patients with acute coronary syndromes, coronary artery disease, and low-moderate risk acute ischemic stroke or high-risk transient ischemic attack. Clinical trials have evaluated the efficacy and safety of ticagrelor on ischemic and bleeding outcomes for different indications and with varying treatment approaches. As a result, there is a large body of clinical evidence demonstrating different degrees of net clinical benefit compared with other platelet inhibitor drugs based on indication, patient characteristics, clinical presentation, treatment duration, and other factors. We provide a review of the major trials of ticagrelor in the context of other randomized trials of clopidogrel and prasugrel to organize the volume of available information, elevate corroborating and conflicting data, and identify potential gaps as areas for further exploration of optimal antiplatelet treatment.

Practical implications of androgen receptor inhibitors for prostate cancer treatment

Antiandrogens have been used for the treatment of prostate cancer as a single agent or in combination with hormone deprivation therapy. New generation antiandrogens act like androgen receptor inhibitors (ARIs). Their binding complex blocks the pathways of cellular proliferation and differentiation of the prostate. Enzalutamide, apalutamide and darolutamide are the new ARIs that demonstrated acceptable tolerability and toxicity, both active in hormone-sensitive and castration-resistant prostate cancer (CRPC). There is no evidence of superiority of one drug over the other, therefore the therapeutic choice depends on the safety profile in relation to the individual patient, their comorbidities and clinical condition. ARIs have also shown promising results in association with new drugs that are active on patients with metastatic CRPC carrying the mutated breast cancer gene (BRCA). Before undergoing new antiandrogenic therapies, patients should be evaluated for cardiological and metabolic risk and possible drug interactions.

Bone-metastatic lung adenocarcinoma cells bearing CD74-ROS1 fusion interact with macrophages to promote their dissemination.

Approximately 40% of patients with lung adenocarcinoma (LUAD) often develop bone metastases during the course of their disease. However, scarcely any in vivo model of LUAD bone metastasis has been established, leading to a poor understanding of the mechanisms underlying LUAD bone metastasis. Here, we established a multiorgan metastasis model via the left ventricular injection of luciferase-labeled LUAD cells into nude mice and then screened out lung metastasis (LuM) and bone metastasis (BoM) cell subpopulations. BoM cells exhibited greater stemness and epithelial-mesenchymal transition (EMT) plasticity than LuM cells and initially colonized the bone and subsequently disseminated to distant organs after being reinjected into mice. Moreover, a CD74-ROS1 fusion mutation (C6; R34) was detected in BoM cells but not in LuM cells. Mechanistically, BoM cells bearing the CD74-ROS1 fusion highly secrete the C-C motif chemokine ligand 5 (CCL5) protein by activating STAT3 signaling, recruiting macrophages in tumor microenvironment and strongly inducing M2 polarization of macrophages. BoM cell-activated macrophages produce a high level of TGF-β1, thereby facilitating EMT and invasion of LUAD cells via TGF-β/SMAD2/3 signaling. Targeting the CD74-ROS1/CCL5 axis with Crizotinib (a ROS1 inhibitor) and Maraviroc (a CCL5 receptor inhibitor) in vivo strongly impeded bone metastasis and secondary metastasis of BoM cells. Our findings reveal the critical role of the CD74-ROS1/STAT3/CCL5 axis in the interaction between LUAD bone metastasis cells and macrophages for controlling LUAD cell dissemination, highlighting the significance of the bone microenvironment in LUAD bone metastasis and multiorgan secondary metastasis, and suggesting that targeting CD74-ROS1 and CCL5 is a promising therapeutic strategy for LUAD bone metastasis.

Exercise-induced changes in left ventricular strain are affected by interleukin-6 activity: An exploratory analysis of a randomised-controlled trial in humans with abdominal obesity.

Whilst the exercise-induced myokine interleukin-6 (IL-6) plays a beneficial role in cardiac structural adaptations, its influence on exercise-induced functional cardiac outcomes remains unknown. We hypothesised that IL-6 activity is required for exercise-induced improvements in left ventricular global longitudinal strain (LV GLS). In an exploratory study 52 individuals with abdominal obesity were randomised to 12weeks' high-intensity exercise or no exercise in combination with IL-6 receptor inhibition (IL-6i) or placebo. LV strain and volume measurements were assessed by cardiac magnetic resonance. Exercise improved LV GLS by -5.4% [95% CI: -9.1% to -1.6%] (P=0.007). Comparing the change from baseline in LV GLS in the exercise + placebo group (-4.8% [95% CI: -7.4% to -2.2%]; P<0.0004) to the exercise + IL-6i group (-1.1% [95% CI: -3.8% to 1.6%]; P=0.42), the exercise + placebo group changed -3.7% [95% CI: -7.4% to -0.02%] (P=0.049) more than the exercise + IL6i group. However, the interaction effect between exercise and IL-6i was insignificant (4.5% [95% CI: -0.8% to 9.9%]; P=0.09). Similarly, the exercise + placebo group improved LV global circumferential strain by -3.1% [95% CI: -6.0% to -0.1%] (P=0.04) more compared to the exercise + IL-6i group, yet we found an insignificant interaction between exercise and IL-6i (4.2% [95% CI: -1.8% to 10.3%]; P=0.16). There was no effect of IL-6i on exercise-induced changes to volume rates. This study underscores the importance of IL-6 in improving LV GLS in individuals with abdominal obesity suggesting a role for IL-6 in cardiac functional exercise adaptations.

The inhibitory impact of Schisandrin on inflammation and oxidative stress alleviates LPS-induced acute kidney injury.

Inflammation and oxidative stress (OS) are the major pathogenic characteristics of acute kidney injury (AKI). Studies have shown that Schisandrin (Sch) could regulate inflammatory disease. However, the function and mechanism of Sch in AKI progression are still unknown. Here, we investigated Sch's potential effects and mechanism on mice's renal damage and macrophages induced by lipopolysaccharide (LPS). Sch decreased LPS-induced inflammatory factor production while increasing the activity of related antioxidant enzymes in macrophages and mouse kidney tissues. Hematoxylin and eosin staining revealed that Sch may have the ability to profoundly inhibit inflammatory cell invasion and tissue damage caused by LPS in renal tissue. Furthermore, Western blot and immunohistochemical studies showed that Sch exerted its effects mainly through up-regulation of nuclear factor erythroid 2-related factor 2/heme oxygenase-1 and inhibition of Toll-like receptor 4‒mitogen-activated protein kinases/nuclear factor-kappa B pathways. Collectively, this study illustrates that Sch suppresses LPS-stimulated AKI by descending inflammation and OS, illuminating prospective AKI treatment options.

Paeoniflorin alleviates TGF-β2-mediated extracellular matrix remodeling and oxidative stress in human trabecular meshwork cells.

The multifunctional profibrotic cytokine transforming growth factor-beta2 (TGF-β2) is implicated in the pathophysiology of primary open angle glaucoma. Paeoniflorin (PAE) is a monoterpene glycoside with multiple pharmacological efficacies, such as antioxidant, anti-fibrotic, and anti-inflammatory properties. Studies have demonstrated that paeoniflorin protects human corneal epithelial cells, retinal pigment epithelial cells, and retinal microglia from damage. Here, the biological role of PAE in TGF-β2-dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment. Primary or transformed (GTM3) human TM (HTM) cells conditioned in serum-free media were incubated with TGF-β2 (5ng/mL). PAE (300μM) was added to serum-starved confluent cultures of HTM cells for 2h, followed by incubation with TGF-β2 for 22h. SB-431542, a TGF-β receptor inhibitor (10μM), was used as a positive control. The levels of intracellular ROS were evaluated by CellROX green dye. Western blotting was used to measure the levels of TGF-β2/Smad2/3 signaling-related molecules. Collagen 1α1, collagen 4α1, and connective tissue growth factor (CTGF) expression was evaluated by RT-qPCR. Immunofluorescence assay was conducted to measure collagen I/IV expression in HTM cells. Phalloidin staining assay was conducted for evaluating F-actin stress fiber formation in the cells. PAE attenuated TGF-β2-induced oxidative stress and suppressed TGF-β2-induced Smad2/3 signaling in primary or transformed HTM cells. Additionally, PAE repressed TGF-β2-induced upregulation of collagen 1α1, collagen 4α1, and CTGF expression and reduced TGF-β2-mediated collagen I/IV expression and of F-actin stress fiber formation in primary or transformed HTM cells. PAE alleviates TGF-β2-induced ECM deposition and oxidative stress in HTM cells through inactivation of Smad2/3 signaling.

Exploring side effects of corticosteroids in myasthenia gravis: Unveiling alternatives for safer treatment

Myasthenia Gravis (MG) is a chronic organ-specific autoimmune disease that weakens voluntary muscles by affecting neuromuscular junctions. The formation of antibodies against acetylcholine receptors is the most prevalent cause. The incidence and prevalence rates of (MG) are increasing exponentially (1). In addition to symptomatic medication, corticosteroids continue to be the first-line treatment for MG due to their affordability, ease of access, and efficacy (2). Although corticosteroids are frequently prescribed, they are not without drawbacks. Long-term corticosteroid use can result in various side effects, including osteoporosis, weight gain, acne, elevated blood pressure, mood fluctuations, and a cushingoid appearance. It has also been linked to rare complications such as gastric and esophageal irritation, compressive fractures, and aseptic femoral head fracture (3). Given the hazards associated with long-term corticosteroid use in treating MG, researchers and healthcare professionals should investigate alternative MG treatments. By doing so, new and safer treatment options can be identified, which could contribute to improved treatment and substantially enhance the quality of life for MG-affected populations. Numerous nonsteroidal medications, including mycophenolate mofetil, methotrexate, and azathioprine, have demonstrated success in clinical trials for kidney transplantation, rheumatoid arthritis, and myasthenia gravis, respectively. To confirm the efficacy of these pharmaceuticals in the treatment of MG, urgent research and trials based on in vitro and animal models are required. Based on well-designed clinical trials, these also require human testing (4). The tapering of prednisone is an alternative option. Prednisone tapering is still a problem in the therapeutic management of patients with generalized MG because, although it is a useful treatment, it has a number of adverse effects that make it necessary to find the lowest possible effective dose as quickly as feasible. Rituximab and azathioprine enable quick tapering. It is necessary to evaluate and contrast the corticosteroid-sparing effect of novel treatments with that of azathioprine and rituximab. Given their impact on MG status, it's possible that these novel therapies will make it possible to drastically cut back on corticosteroids or possibly stop taking them entirely(5) The abnormal immune responses in MG can be managed and resolved with the new drugs that specifically target them. These agents include chimeric antigen receptor T [CART-T] cell therapy, autologous stem cell transplantation, B cell depleting agents (anti-CD 19 and 20, and B cell activating factor [BAFF] inhibitors), proteosome inhibitors, terminal complement C5 inhibitors, Fc receptor inhibitors, T cells, and cytokine-based therapies (subcutaneous immunoglobulin [SCIG]). ---Continue

#457 Sparsentan has direct effects on the glomerular capillary wall to attenuate increased permeability after exposure to nephrotic syndrome plasma

Abstract Background and Aims The glomerular endothelial glycocalyx (eGlx), a luminal layer of proteoglycans, glycoproteins and glycolipids, forms the first part of the glomerular filtration barrier (GFB). Nephrotic syndrome (NS) describes a group of pathologies of the renal glomerulus that result in proteinuria and is associated with glomerular endothelial dysfunction. Current treatments are broad and non-specific. Sparsentan is a single-molecule dual endothelin type-A and angiotensin II type 1 receptor antagonist that has received accelerated approval in the United States for the reduction of proteinuria in adults with IgA nephropathy at high risk of disease progression. Our validated glomerular permeability assay directly measures the albumin permeability (Ps’alb)of capillary loops within individually trapped glomeruli [1]. This ex vivo assay is independent of haemodynamic factors and tubular albumin handling – factors known to affect urine protein concentrations. This work examines whether sparsentan could reduce glomerular albumin permeability in NS, by preserving the eGlx to maintain the GFB. Method Human NS plasma samples were collected under ethical consent from 3 patients that had undergone plasma exchange from periods when they were in relapse (RL), or subsequent remission (RM). Adult male Sprague Dawley rats (175–200 g) were perfused with 4% Ringer BSA solution. Glomeruli were isolated on ice by graded sieving from the cortex of each kidney. Glomeruli were incubated, in the presence of AF488-conjugated BSA (AF488-BSA, 30 μg/ml) and R18 (36.5 μg/ml), with 10% plasma from NS patients for 1 hour, and simultaneously treated with sparsentan (0.1 μM, 1 μM and 10 μM) or vehicle. The glomerular Ps’alb assay was used to measure changes in albumin permeability. We applied a fluorescence profile peak-to-peak confocal imaging technique to treated glomeruli to assess glomerular eGlx thickness [2]. Results Human NS patients in RL had a significantly greater proteinuria compared to RM (RL, 10, 000 ± 1, 354 mg/g; RM, 95.3 ± 59.7 mg/g, P = 0.017). Incubation of rat glomeruli with RL plasma induced a significant increase in Ps’alb (RM+vehicle, 6.0 × 10−7 ± 0.12 × 10−7 cm/s; RL+vehicle, 10.9 × 10−7 ± 0.84 × 10−7 cm/s, P &amp;lt; .001) with a significant reduction in glomerular eGlx thickness (RM+vehicle, 210 ± 21.2 nm; RL+vehicle, 109 ± 7.9 nm, P = .027), compared to rat glomeruli incubated with paired RM plasma. Sparsentan-treated RL incubated glomeruli were protected from both the increase in Ps’alb (RL+spars 10 µM, 6.3 × 10−7 ± 0.19 × 10−7 cm/s, P &amp;lt; .001) and the loss in glomerular eGlx (RL+spars 10 µM, 222 ± 8.5 nm, P = .013), to a level comparable to RM incubated glomeruli. The effect of sparsentan on Ps’alb was dose dependent (RL+spars 1 µM, 8.0 × 10−7 ± 0.46 × 10−7 cm/s; RL+spars 0.1 µM, 9.6 × 10−7 ± 0.22 × 10−7 cm/s) with Ps’alb changes correlating inversely with glomerular eGlx thickness (r² = 0.75, P &amp;lt; .0001). In RM glomeruli, sparsentan alone had no effect on Ps’alb compared with vehicle (RM+spars 10 µM, 6.2 × 10−7 ± 0.25 × 10−7 cm/s, P = .590), suggesting no effect on otherwise healthy capillaries. Conclusion We have shown that dual inhibition of endothelin and angiotensin receptors, with sparsentan, preserves the glomerular eGlx resulting in normalised glomerular permeability in NS. These findings suggest that the direct action of sparsentan on the GFB could help maintain barrier integrity in NS, in particular by glycocalyx protection.

#1610 FGF23-mediated hypertension via augmented calcium entry in vascular smooth muscle cells

Abstract Background and Objectives Ongoing studies are shedding light on the potential connection between FGF23 and hypertension. In experimental settings, the administration of recombinant FGF23 in mice has been demonstrated to induce hypertension by elevating serum sodium, along with an increase in NCC receptors in the kidney. Clinical evidences further supports pro-hypertensive effects of high circulating levels of FGF23. Our group has previously demonstrated a direct influence of FGF23 on increased arterial stiffness by inducing a phenotypic transition in vascular smooth muscle cells (VSMCs) from a contractile to a synthetic phenotype. This study aimed comprehensively to evaluate, both in vivo and in vitro, whether FGF23 directly leads to hypertension through the stimulation of intracellular calcium entry in vascular cells. Methods To ascertain whether FGF23 contributes to hypertension through a direct impact on blood vessels, recombinant FGF23 (15 µg/day) was administered for 28 days via Alzet pumps. Blood pressure was meticulously measured in these animals, and we investigated alterations in vascular remodeling and calcium channels, as well as proteins associated with vascular contraction such as Orai1, STIM1, PKD1, SERCA2a, and AGTR1. In vitro experiments included the evaluation of the effect of elevated levels of recombinant FGF23 (2 ng/ml) over 9 days on intracellular calcium entry stimulated by Ang2 (100 nM) or Thapsigargin (2.5 µM). We used the FURA method to conduct these experiments. To elucidate the in vitro pro-hypertensive mechanisms of FGF23, studies were conducted with the administration of inhibitors targeting FGF Receptors 1-3 (AZD4547, 150 nM), FGF Receptor-4 (BLU9931, 10 nM), and Erk1/2 phosphorylation (PD98059, 10 μM). Additionally, changes in Orai1, STIM1, PKD1, SERCA2a, and AGTR1 were also evaluated in these VSMCs. Results After 28 days of recombinant FGF23 treatment, no significant differences were observed in serum sodium levels, yet there was a marked increase in urinary phosphorus excretion. Systolic and diastolic blood pressure values showed a progressive significant elevation in animals exposed to high levels of FGF23. The expression of AGTR1 and PKD1 levels significantly increased after FGF23 administration for 28 days (Figure). However, Orai1 and STIM1 in the thoracic aorta exhibited no significant differences following FGF23 treatment. In VSMCs, the exposure to recombinant FGF23 for 9 days resulted in increased calcium entry upon Ang2 stimulation (Figure). This effect was markedly diminished after inhibiting FGFR1-3 receptors and Erk1/2 phosphorylation (Figure). Notably, the inhibition of FGF23 receptor 4 (FGFR4) had no impact on the calcium entry induced by FGF23 (Figure). While Orai1, STIM1, IP3R, and TRPV5 in VSMCs remained unaffected by recombinant FGF23 administration, PKD1, SERCA2, and AGTR1 levels were significantly elevated compared to control cells (Figure). The addition of Thapsigargin in a Ca2+-free culture medium to VSMCs incubated with recombinant FGF23 for 9 days led to increased Ca2+ efflux from endoplasmic reticulum. Conclusion Elevated levels of FGF23 directly enhanced calcium entry in VSMCs. This effect is mediated by FGFR1-3 receptors, Erk1/2 phosphorylation, and the upregulation of related protein to cell contraction such as SERCA2a, PKD1 or AGTR1. These modifications could be responsible of FGF23-associated hypertension in the experimental model.

Novel therapeutic perspectives for wet age-related macular degeneration: RGD-modified liposomes loaded with 2-deoxy-D-glucose as a promising nanomedicine

Choroidal neovascularization (CNV), characterized as a prominent feature of wet age-related macular degeneration (AMD), is a primary contributor to visual impairment and severe vision loss globally, while the prevailing treatments are often unsatisfactory. The development of conventional treatment strategies has largely been based on the understanding that the angiogenic switch of endothelial cells is dictated by angiogenic growth factors alone. Even though treatments targeting vascular endothelial growth factor (VEGF), like Ranibizumab, are widely administered, more than half of the patients still exhibit inadequate or null responses, emphasizing the imperative need for solutions to this problem. Here, aiming to explore therapeutic strategies from a novel perspective of endothelial cell metabolism, a biocompatible nanomedicine delivery system is constructed by loading RGD peptide-modified liposomes with 2-deoxy-D-glucose (RGD@LP-2-DG). RGD@LP-2-DG displayed good targeting performance towards endothelial cells and excellent in vitro and in vivo inhibitory effects on neovascularization were demonstrated. Moreover, our mechanistic studies revealed that 2-DG interfered with N-glycosylation, leading to the inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) and its downstream signaling. Notably, the remarkable inhibitory effect on neovascularization and biocompatibility of RGD@LP-2-DG render it a highly promising and clinically translatable therapeutic candidate for the treatment of wet AMD and other angiogenic diseases, particularly in patients who are unresponsive to currently available treatments.

Identifying novel mechanisms of per- and polyfluoroalkyl substance-induced hepatotoxicity using FRG humanized mice.

Per- and polyfluoroalkyl substances (PFAS) such as perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) and perfluoro-2-methyl-3-oxahexanoic acid (GenX), the new replacement PFAS, are major environmental contaminants. In rodents, these PFAS induce several adverse effects on the liver, including increased proliferation, hepatomegaly, steatosis, hypercholesterolemia, nonalcoholic fatty liver disease and liver cancers. Activation of peroxisome proliferator receptor alpha by PFAS is considered the primary mechanism of action in rodent hepatocyte-induced proliferation. However, the human relevance of this mechanism is uncertain. We investigated human-relevant mechanisms of PFAS-induced adverse hepatic effects using FRG liver-chimeric humanized mice with livers repopulated with functional human hepatocytes. Male FRG humanized mice were treated with 0.067mg/L of PFOA, 0.145mg/L of PFOS, or 1mg/L of GenX in drinking water for 28days. PFOS caused a significant decrease in total serum cholesterol and LDL/VLDL, whereas GenX caused a significant elevation in LDL/VLDL with no change in total cholesterol and HDL. All three PFAS induced significant hepatocyte proliferation. RNA-sequencing with alignment to the human genome showed a total of 240, 162, and 619 differentially expressed genes after PFOA, PFOS, and GenX exposure, respectively. Upstream regulator analysis revealed that all three PFAS induced activation of p53 and inhibition of androgen receptor and NR1D1, a transcriptional repressor important in circadian rhythm. Further biochemical studies confirmed NR1D1 inhibition and in silico modeling indicated potential interaction of all three PFAS with the DNA-binding domain of NR1D1. In conclusion, our studies using FRG humanized mice have revealed new human-relevant molecular mechanisms of PFAS including their previously unknown effect on circadian rhythm.

#2549 Real world data of dapagliflozin effect on proteinuria in primary glomerulopathies in Croatia

Abstract Background and Aims SGLT2 inhibitors, namely dapagliflozin, proved to slow progression of kidney disease in randomized clinical trials. Our aim was to assess real world efficacy of dapagliflozin on proteinuria level in primary glomerulopathies (GN). Method adult patients with biopsy proven glomerulonephritis from all university hospitals in Croatia who started dapagliflozin as a standard of care were enrolled. Data were analyzed if patients had at least six months of follow up. None of the patients were on immunosuppression. Data are shown as N (%) or median with interquartile range (IQR) and accompanied p levels. Results Overall 118 patients, predominantly male 84 (71.8%) of average age of 52 years (min–max 20–76), were enrolled. Most of the patients had IgA nephropathy (IgAN) (N = 55;46.6%), followed by membranous nephropathy (N = 27;22.9%), FSGS (N = 19;16.1%), membranoproliferative GN (N = 8;6.8%), Alport syndrome (N = 8;6.8%). 95.8% of patients were on ACE inhibitors or angiotensin receptor blockers and 10.7% were on mineralocorticoid receptor inhibitors and RAAS blockage was not significantly upscaled during follow up. During the median follow up of 12.2 (7.5-18.6) months, proteinuria was significantly reduced from 1139 (534–2265) to 701 (305-1543) mg/dU; p &amp;lt; 0.001. When divided by glomerulonephritis type, statistically significant reduction of proteinuria was observed in patients with IgAN and Alport syndrome, while trend of reduction of proteinuria was observed in all types of GN. There was a slight reduction in eGFR levels (54 (43-78) vs. 52 (39–73) ml/min/1,73 m²; p &amp;lt; 0.001) and rise in creatinine values (122 (89–149) vs. 128 (91–159) μmol/l; p &amp;lt; 0.001). There was a significant reduction in systolic (130 (120-140) vs.125 (115-134) mmHg; p &amp;lt; 0.001) and diastolic blood pressure (80 (75-90) vs. 80 (70-85) mmHg; p &amp;lt; 0.001). Reduction of uric acid level was noticed (377 (313–460) vs.343 (294–422); p &amp;lt; 0.001), while there was no effect on LDL levels (2.6 (2.12–3.53) vs.2.3 (1.9–3.30); p = 0.073). Dapagliflozin was well tolerated, one patient discontinued the therapy due to intolerance, one due to urinary infection and two due to relapse of GN which needed immunosuppressive therapy. One patient was excluded from analysis due to non-adherence. Conclusion SGLT2 inhibitor dapagliflozin has significant effect on reduction of proteinuria in patients with primary glomerulonephritis. Our data support a growing body of evidence that SGLT2 inhibitors should be used as a standard of care in patients with glomerular kidney diseases, especially IgAN. Future research should address earlier use of SGLT2 inhibitors during the treatment of glomerulonephritis alongside immunosuppressive therapy as well as look into the effect of SGLT2 inhibitors on mesangial cells.

#2700 Serum C3/C4 levels in renal antineutrophil cytoplasmic antibody-associated vasculitis: predictors of lung disease and worse prognosis

Abstract Background and Aims Complement activation, specially the alternative pathway, has been implicated in the development of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). This was further corroborated with the efficacy of Avacopan, a selective C5a receptor inhibitor used as a corticosteroids reduction strategy in the treatment of AAV [1]. Low complement factor 3 (C3) levels without real hypocomplementemia are predictors of prognosis in AAV [2]. Low C4 levels have not yet been associated with worst outcomes. We aimed to evaluate the association between circulating C3 and C4 levels at diagnosis of AAV with clinical and biochemical features and with relevant outcomes: relapse, dialysis and death. Method This is a retrospective study including 63 patients with AAV with renal involvement followed between 2003-2023, with C3 and C4 levels measurements at diagnosis. Data was recorded from clinical registries. Patients were grouped according to the median C3 and/or C4 levels, as previously performed [2]. Groups were compared using the chi-square test for categorical variables and non-parametric tests for continuous variables. We created a logistic regression model for the outcomes, adjusting for covariates. Results Mean age at diagnosis was 61.1 ± 13.4 years old and most patients had ANCA MPO specificity (69.8%, n = 44). Only 3 patients had low C3 levels (&amp;lt; 83 mg/dL) and C4 hypocomplementemia (&amp;lt;12 mg/dL) was present in other 3 patients. Median C3 levels were 127.5 mg/dL (107.3-139.0) and median C4 levels were 29.5 mg/dL (22.0-35.0). Groups were defined according to these values: low C3 levels (C3 &amp;lt;127.5 mg/dL, n = 31), low C4 levels (C4 &amp;lt;29.5 mg/dL, n = 31) and low C3 and/or C4 levels, when either of these conditions were met (n = 40). There was a positive correlation between C3 and C4 levels (r = 0.57, p &amp;lt; 0.001). Patients with low C3 levels had more frequent lung involvement than patients with higher C3 levels (71% vs 31.3%, p = 002), as well as alveolar hemorrhage (38.7% vs 12.9%, p = 0.020). Regarding the outcomes, there was a tendency for more patients requiring acute dialysis (first month) and progressing to dialysis during follow-up in the low C3 subgroup. Relapse and death rates were similar between the two groups. Low C4 levels were associated with older age (75 ± 11.8 vs 67.5 ± 13.7 years, p = 0.043), higher relapse rates (50% vs 25.8%, p = 0.05) and faster progression to dialysis (1.0 (1.0-5.0) vs 9.5 (1.8-54) months, p = 0.034). When combining low C3 and/or C4 levels, lung involvement was again more frequent in patients with lower complement levels (62.5% vs 30.4%, p = 0.014). Time to dialysis was lower and relapse rates were higher in the subgroup with low complement levels, but not statistically significant. No association with death was found. Interestingly, acute dialysis requirement was more prevalent in the low C3/C4 levels subgroup (32.5% vs 8.7%, p = 0.033). After adjustment for baseline renal function, low C3/C4 levels showed an increase of 7 times the odds of a patient requiring acute dialysis (OR 6.98; 95% CI 1,196-40,839, p = 0.031, Table 1). Low C3/C4 levels independently predicted the odds of lung disease in AAV (OR = 5,069; 95% CI 1,475-17,612 p = 0.010, Table 2). Conclusion Complement activation in AAV has been previously associated to renal outcomes, but our results also highlight a possible role of complement in the pathophysiology of lung disease. Low C4 levels were associated with higher relapse rates and faster progression to dialysis, revealing that C4 activation impacts outcomes in AAV and that both classic and alternative complement pathways are involved. Combining low C3 and/or C4 levels identified patients at higher risk of acute dialysis requirement, independently of renal function at admission. Further insights in the specific effects of C3/C4 are needed to stipulate the value of monitoring and targeting complement factors in AAV.