Vehicle-treated Group Research Articles

In Vitro and In Vivo Antiurolithic Effect of Betulinic Acid Obtained from Citharexylum mirianthum.

The study aimed to investigate the potential antiurolithic effects of extracts, fractions, and betulinic acid (BA) from Citharexylum mirianthum. In vitro analysis involved precipitating calcium oxalate (CaOx) crystals in urine. For in vivo studies, rats were divided into four groups: naive; vehicle; potassium citrate (KC); and BA. Urolithiasis was induced using ethylene glycol and ammonium chloride. After seven days, urine, blood, and kidney tissues were evaluated. The results showed that methanolic extract, hexane, dichloromethane, and ethyl acetate fractions, as well as BA, reduced CaOx crystal formation. In vivo, the vehicle-treated group exhibited reduced urinary volume and Na+ excretion, while the BA-treated group showed restored urinary volume and Na+ excretion similar to the naive group. BA also significantly reduced urinary monohydrate and dihydrate crystal formation, comparable to the KC group. Other urinary parameters remained unchanged, but plasma analysis revealed decreased Na+, K+, and Ca2+ in the KC group. Renal tissue analysis indicated reduced lipid hydroperoxides and increased reduced glutathione in all urolithiasis groups, with unchanged nitrite levels. BA treatment also improved renal corpuscle morphology. Overall, our findings demonstrate that treatment with BA effectively prevented kidney damage induced by EG+AC ingestion, thereby improving renal function in the urolithiasis model.

5-Methoxy-2-aminoindane Reverses Diet-Induced Obesity and Improves Metabolic Parameters in Mice: A Potential New Class of Antiobesity Therapeutics.

The escalating prevalence of obesity and its related disorders represents a daunting global health challenge. Unfortunately, current pharmacological interventions for obesity remain limited and are often associated with debilitating side effects. Against this backdrop, the psychoactive aminoindane derivative 5-methoxy-2-aminoindane (MEAI) has gained considerable attention for its ability to induce a pleasurable, alcohol-like sensation while curbing alcohol consumption. Given the potential impact of MEAI on food addiction and energy homeostasis, we examined its metabolic efficacy on appetite regulation, obesity, and related comorbidities under acute and chronic settings, utilizing a mouse model of diet-induced obesity (DIO). Our results demonstrated that MEAI treatment significantly reduced DIO-induced overweight and adiposity by preserving lean mass and decreasing fat mass. Additionally, MEAI treatment exhibited positive effects on glycemic control by attenuating DIO-induced hyperglycemia, glucose intolerance, and hyperinsulinemia. Furthermore, MEAI reduced DIO-induced hepatic steatosis by decreasing hepatic lipid accumulation and lowering liver triglyceride and cholesterol levels, primarily by inhibiting de novo lipid synthesis. Metabolic phenotyping revealed that MEAI increased energy expenditure and fat utilization while maintaining food consumption similar to that of the vehicle-treated group. Lastly, MEAI normalized voluntary locomotion actions without any overstimulatory effects. These findings provide compelling evidence for the antiobesity effects of MEAI treatment and call for further preclinical testing. In conclusion, our study highlights the potential of MEAI as a novel therapeutic approach for treating obesity and its associated metabolic disorders, offering hope for the development of new treatment options for this global health challenge.

Liraglutide Impacts Iron Homeostasis in a Murine Model of Hereditary Hemochromatosis.

Classic hereditary hemochromatosis (HH) is an autosomal recessive iron-overload disorder resulting from loss-of-function mutations of the HFE gene. Patients with HH exhibit excessive hepatic iron accumulation that predisposes these patients to liver disease, including the risk for developing liver cancer. Chronic iron overload also poses a risk for the development of metabolic disorders such as obesity, type 2 diabetes, and insulin resistance. We hypothesized that liraglutide, GLP1 receptor agonist, alters iron metabolism while also reducing body weight and glucose tolerance in a mouse model of HH (global HFE knockout, HFE KO) and diet-induced obesity and glucose intolerance. The total body HFE KO and wild-type control mice were fed high-fat diet for 8 weeks. Mice were subdivided into liraglutide and vehicle-treated groups and received daily subcutaneous administration of the respective treatment once daily for 18 weeks. Liraglutide improved glucose tolerance and hepatic lipid markers and reduced body weight in a mouse model of HH, the HFE KO mouse, similar to wild-type controls. Importantly, our data show that liraglutide alters iron metabolism in HFE KO mice, leading to decreased circulating and stored iron levels in HFE KO mice. These observations highlight the potential that GLP1 receptor agonist could be used to reduce iron overload in addition to reducing body weight and improving glucose regulation in HH patients.

The dual amylin and calcitonin receptor agonist KBP-336 elicits a unique combination of weight loss, antinociception and bone protection – a novel disease-modifying osteoarthritis drug

BackgroundDespite the extensive research to provide a disease-modifying osteoarthritis drug (DMOAD), there is still no approved DMOAD. Dual amylin and calcitonin receptor agonists (DACRA) can provide metabolic benefits along with antinociceptive and potential structural preserving effects. In these studies, we tested a DACRA named KBP-336 on a metabolic model of OA in meniscectomised (MNX) rats.MethodsWe evaluated KBP-336’s effect on pain-like symptoms in Sprague Dawley (SD) rats on high-fat diet (HFD) that underwent meniscectomy using the von Frey test to measure the 50% paw withdrawal threshold (PWT) and analyzed using one-way ANOVA. Short in vivo studies and in vitro cell receptor expression systems were used to illustrate receptor pharmacology.ResultsAfter 30 weeks on HFD, including an 8-week treatment, female MNX animals receiving KBP-336 4.5 nmol/Kg/72 h had lower body weight and smaller adipose tissues than their vehicle-treated counterparts. After 20 weeks on HFD, including an 8-week treatment, male rats receiving KBP-336 had lower body weight than the vehicle group. In both the female and male rats, the MNX groups on KBP-336 treatment had a higher PWT than the vehicle-treated MNX group. Aiming to identify the receptor influencing pain alleviation, KBP-336 was compared to the long-acting human calcitonin (hCTA). Single-dose studies on 12-week-old male rats showed that hCTA lowers CTX-I without affecting food intake, confirming its calcitonin receptor selectivity. On the metabolic OA model with 18 weeks of HFD, including 6-week treatment, hCTA at 100 nmol/Kg/24 h and KBP-336 at 0.5, 1.5, and 4.5 nmol/Kg/72 h produced significantly higher PWT in MNX animals compared to MNX animals on vehicle treatment. hCTA and KBP-336 at 0.5 nmol/Kg did not affect body weight and fat tissues.ConclusionOverall, KBP-336 improved the pain observed in the metabolic OA model. Calcitonin receptor activation proved to be essential in this antinociceptive effect.

Rhei Undulati Rhizoma attenuates memory decline and reduces amyloid-β induced neuritic dystrophy in 5xFAD mouse

BackgroundAlzheimer's disease (AD) is a common type of dementia characterized by amyloid-β (Aβ) accumulation, lysosomal dysfunction, and tau hyperphosphorylation, leading to neurite dystrophy and memory loss. This study aimed to investigate whether Rhei Undulati Rhizoma (RUR), which has been reported to have anti-neuroinflammatory effect, attenuates Aβ-induced memory impairment, neuritic dystrophy, and tau hyperphosphorylation, and to reveal its mode of action.MethodsFive-month-old 5xFAD mice received RUR (50 mg/kg) orally for 2 months. The Y-maze test was used to assess working memory. After behavioral testing, brain tissue was analyzed using thioflavin S staining, western blotting, and immunofluorescence staining to investigate the mode of action of RUR. To confirm whether RUR directly reduces Aβ aggregation, a thioflavin T assay and dot blot were performed after incubating Aβ with RUR.ResultsRUR administration attenuated the Aβ-induced memory impairment in 5xFAD mice. Furthermore, decreased accumulation of Aβ was observed in the hippocampus of the RUR-treated 5xFAD group compare to the vehicle-treated 5xFAD group. Moreover, RUR reduced the dystrophic neurites (DNs) that accumulate impaired endolysosomal organelles around Aβ. In particular, RUR treatment downregulated the expression of β-site amyloid precursor protein cleaving enzyme 1 and the hyperphosphorylation of tau within DNs. Additionally, RUR directly suppressed the aggregation of Aβ, and eliminated Aβ oligomers in vitro.ConclusionsThis study showed that RUR could attenuate Aβ-induced pathology and directly regulate the aggregation of Aβ. These results suggest that RUR could be an efficient material for AD treatment through Aβ regulation.Graphical

Characterization of an Uncommon Finding in the Basal Nuclei in Beagle Dogs: A Novel Potential Background Lesion in the Canine Brain.

Degenerative lesions specific to the basal nuclei have not been described as a background finding in Beagle dogs. This report comprises a documentation of seven cases. In the context of a nonclinical safety studies, the authors suggest documenting the lesion descriptively as degeneration neuropil, basal nuclei, bilateral as it is characterized by (1) vacuolation, neuropil; (2) gliosis (astro- and/or microgliosis); and (3) demyelination. This novel lesion is considered a potential new background change for several reasons: (1) It occurred in animals from test item-treated and also vehicle-treated groups; (2) no dose dependency was observed; (3) in one of six affected test item-treated dogs, the given compound was shown not to penetrate the blood-brain barrier; and (4) statistical comparison between the proportions of affected dogs in the treatment and control groups did not yield a statistically significant difference. The etiology remains unknown and is subject to further investigations.

Mechanisms contributing to inhibition of retinal ganglion cell death by cell permeable peptain-1 under glaucomatous stress

This study assesses the neuroprotective potential of CPP-P1, a conjugate of an anti-apoptotic peptain-1 (P1) and a cell-penetrating peptide (CPP) in in vitro, in vivo, and ex vivo glaucoma models. Primary retinal ganglion cells (RGCs) were subjected to either neurotrophic factor (NF) deprivation for 48 h or endothelin-3 (ET-3) treatment for 24 h and received either CPP-P1 or vehicle. RGC survival was analyzed using a Live/Dead assay. Axotomized human retinal explants were treated with CPP-P1 or vehicle for seven days, stained with RGC marker RBPMS, and RGC survival was analyzed. Brown Norway (BN) rats with elevated intraocular pressure (IOP) received weekly intravitreal injections of CPP-P1 or vehicle for six weeks. RGC function was evaluated using a pattern electroretinogram (PERG). RGC and axonal damage were also assessed. RGCs from ocular hypertensive rats treated with CPP-P1 or vehicle for seven days were isolated for transcriptomic analysis. RGCs subjected to 48 h of NF deprivation were used for qPCR target confirmation. NF deprivation led to a significant loss of RGCs, which was markedly reduced by CPP-P1 treatment. CPP-P1 also decreased ET-3-mediated RGC death. In ex vivo human retinal explants, CPP-P1 decreased RGC loss. IOP elevation resulted in significant RGC loss in mid-peripheral and peripheral retinas compared to that in naive rats, which was significantly reduced by CPP-P1 treatment. PERG amplitude decline in IOP-elevated rats was mitigated by CPP-P1 treatment. Following IOP elevation in BN rats, the transcriptomic analysis showed over 6,000 differentially expressed genes in the CPP-P1 group compared to the vehicle-treated group. Upregulated pathways included CREB signaling and synaptogenesis. A significant increase in Creb1 mRNA and elevated phosphorylated Creb were observed in CPP-P1-treated RGCs. Our study showed that CPP-P1 is neuroprotective through CREB signaling enhancement in several settings that mimic glaucomatous conditions. The findings from this study are significant as they address the pressing need for the development of efficacious therapeutic strategies to maintain RGC viability and functionality associated with glaucoma.

DIPG-38. VAL-083 IN COMBINATION WITH AZD1775 FOR THE TREATMENT OF DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse midline gliomas (DMGs) are malignant pediatric brain tumors characterized by H3K27M mutation and global genome hypomethylation. This results in enhanced transcription of MGMT, a DNA-repair enzyme, rendering alkylating chemotherapeutics ineffective. Therefore, we investigated the efficacy of a functionally different DNA damaging agent, Val-083, that induces double strand breaks and triggers S/G2 phase cell cycle arrest for homologous DNA repair. We further evaluated its combined efficacy with Wee1 (G2/M checkpoint) inhibitor, AZD1775, to override cell cycle arrest and propel cells with DNA damage into premature mitosis and apoptosis. METHODS DMG cells were treated with increasing concentration of Val-083 and/or AZD1775 and their viability was evaluated using CellTiter-Glo luminescent assay. Next, the drugs’ mechanisms of action were investigated by immunoblotting for biomarkers involved in DNA damage response and cell cycle arrest. Flow cytometry was used to investigate cell cycle profile and apoptosis. Additionally, in vivo tumor growth inhibition and survival benefit were evaluated using zebrafish and mouse xenograft DMG models. RESULTS The IC50 values of Val-083 range between 0.69 and 4.38 µM while the IC50 values of AZD1775 range between 0.22 to 0.98 µM. Combination of both drugs showed high areas of synergy on multiple DMG lines, DNA double strand breaks and significant increase in cell population at the S (p<0.01) and G2/M (p<0.05) phases. Importantly, on zebrafish xenograft tumor model, combination treatment significantly (p<0.001) inhibited tumor growth in comparison to control and single treatment groups. Furthermore, the combined treatment demonstrated significant (p<0.01) survival benefit (median survival (MS) = 62 days) on tumor-bearing nude mice models, in comparison to vehicle (MS= 44 days), Val-083 (MS= 54.5 days) and AZD1775 (MS= 47 days) treatment groups. CONCLUSION Val-083 in combination with AZD1775 demonstrate promising efficacy in DMG preclinical models, providing a strong rationale for positioning these arms for clinical testing.

Evaluating the Antidepressant-like Properties of Melatonin and Vitamin D3 Combination in Mice Subjected to Restraint Stress: Investigating the Involvement of Oxidative Stress

Background: Increased levels of oxidative stress are connected with depression. Due to the melatonin's antioxidant effects and Vitamin D3 (Vit D3)'s regulatory effect on the biosynthesis of neurotransmitters and neurotrophic factors, the present study investigated the possible protective effect of melatonin and Vit D3 combination on restraint stress-induced depression-like behaviors in mice. Methods: After being subjected to restraint stress, mice were divided into six groups using a randomization process. These groups included non-stressed and stressed vehicle-treated groups, as well as groups treated with Vit D3 (25 μg/kg/day), melatonin (10 mg/kg) or fluoxetine. A group also received a combination of both melatonin and Vit D3. The Forced Swimming Test (FST), and Open Field Test (OFT) were conducted to evaluate behavioural changes. The Malondialdehyde (MDA) level and Catalase (CAT), Superoxide Dismutase (SOD) activity, and ADP/ATP ratio were evaluated in the hippocampus of mice. Results: Restraint stress lengthened the immobility period in FST, while melatonin, Vit D3, and their combination all significantly reversed this impact. Co-administration of melatonin and Vit D3 was more effective than melatonin or Vit D3 administration alone at reducing immobility time. The exposure of mice to restraint stress has been linked to an elevation in the ADP/ATP ratio and oxidative stress in their hippocampus; however, these effects are reversed by the administration of melatonin and Vit D3 (10 mg/kg) alone or in combination. Melatonin and Vit D3 combination increased the hippocampus CAT activity compared with melatonin and Vit D3 alone. Conclusion: The current study's findings suggested that Vit D3 may enhance melatonin's potential as an antidepressant in FST.

The Combination of Auranofin and Celecoxib Suppresses Local Synovial Sarcoma Progression In Vivo.

Synovial sarcoma (SS) is a rare malignant tumor with a poor survival rate. We previously reported that a combination of auranofin (AUR), a thioredoxin reductase inhibitor, and celecoxib (CE), an anti-inflammatory drug, significantly impedes the local progression of osteosarcoma (OS). However, the role of redox regulation in SS remains to be elucidated. This study aimed to investigate the efficacy of combined treatment of AUR and CE on the local progression of SS in vivo. Nu/nu mice were implanted with the human SS cell line, Aska-SS, and treated with vehicle control, AUR, or a combination of AUR and CE (AUR-CE). Primary tumor size and weight were evaluated for the study duration and upon resection, respectively. Hematoxylin and eosin (H&E) and Ki-67 staining were performed to assess the local progression of SS. A statistically significant reduction in tumor size and weight was observed in the AUR- and AUR-CE-treated groups upon excision compared to that in the vehicle-treated group. The AUR-CE-treated group showed synergistic inhibition of local tumor growth. H&E staining of local SS tumors revealed decreased cell density and nuclear deformation in the AUR- and AUR-CE-treated groups compared to those in the vehicle-treated group. Immunohistochemical staining revealed a statistically significant decrease in Ki-67-positive cells in the AUR-CE-treated group compared to the vehicle-treated group. The combination of AUR and CE showed significant potential for delaying the local progression of SS. These findings support the repurposing of AUR and CE as early treatment options for SS.

Role of NOD1 in atrial myopathy associated with heart failure

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funding: Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (ISCIII [PI17/01344 and PI20/01482]), Ministry of Education (FPU19/01973), SEC, Proyecto Traslacional 2019, FEDER, FSE, CIBER-CV, a network funded by ISCIII Introduction Heart failure (HF) is a complex syndrome that constitutes one of the leading causes of cardiovascular death worldwide. Although multifactorial mechanisms appear to be responsible of atrial and ventricular HF-myopathy, the innate immune system and the derived inflammatory response have gained much attention in the last years. Previous studies from our group have demonstrated that the nucleotide-binding oligomerization domain 1 (NOD1), a receptor of the innate immune system, plays a role in HF left ventricular (LV) dysfunction. However, its role in HF-atrial myopathy remains completely unknown. The main purpose of our study is to determine the role of NOD1 in human and experimental HF-atrial myopathy. Methods and Results The study was performed in two cohorts of individuals who underwent heart valve repair surgery, 51 non-failing individuals (NF) with LV ejection fraction (LVEF)>50% and NT-ProBNP levels<200pg/mL and 41 subjects with medium to severe reduced EF (HF)<50% and NT-ProBNP levels>200pg/mL. An informed consent was signed by all patients. Atrial myocardial tissue was excised from the atrial appendage and prepared for biochemical and histological studies. Echocardiography analysis showed that left and right atrial areas (LA and RA) were significantly increased in the HF group compared to NF, while LA and RA reservoir functions were significantly decreased in HF. Immunostaining demonstrated NOD1 location in atrial cardiomyocytes, being the protein levels of NOD1 and its specific adaptor RIP2 significantly increased in atrial tissue obtained from HF vs. NF. Although the prevalence of atrial fibrillation (AF) was significantly higher in the HF group compared to NF, the upregulation of NOD1 was not conditioned by the presence of AF. Similarly, pigs that underwent transverse aortic constriction (TAC) or proximal left circumflex coronary artery occlusion involving the LA branch (LAI), showed an upregulation of NOD1 pathway in atrial tissue, compared with the sham operated animals. Interestingly, an atrial upregulation of NOD1 axis was also observed in wild-type TAC mice, whereas Nod1-/- knockout mice showed a prevention in both structural and functional atrial remodelling induced by TAC, mainly by a prevention of intracellular calcium mishandling in isolated atrial cardiomyocytes. Finally, wild-type mice injected i.p. for 3 days with 3.3 mg/kg with a selective activator of NOD1, showed increased structural atrial remodelling and intracellular calcium mishandling compared to the vehicle-treated group. Conclusions Our results demonstrate that NOD1 axis is upregulated in human, swine and murine atrial failing myocardium. Genetic deletion of NOD1 prevented HF-atrial structural and functional remodelling, mainly by avoiding intracellular calcium mishandling, whereas its pharmacological activation induced atrial dilation and intracellular Ca2+ mishandling. All these data uncover NOD1 as a new player in atrial HF-remodelling.

#977 Etelcalcetide improve left ventricular hypertrophy by suppressing the renin-angiotensin-aldosterone system and cardiac fibroblast growth factor 23

Abstract Background and Aims Calcimimetics are allosteric activators of the calcium-sensing receptor (CaSR) and are employed as therapeutic agents for managing secondary hyperparathyroidism in chronic kidney disease (CKD) patients undergoing dialysis. Although they have shown potential in reducing cardiovascular diseases and suppressing left ventricular hypertrophy (LVH), the mechanisms underlying these effects are not fully elucidated. In this study, we focused on exploring the mechanisms behind the suppression of LVH by calcimimetics using CKD, LVH, and CKD+LVH model rats. Specifically, we investigated the impact on CKD-mineral bone disorder (CKD-MBD) parameters, the renin-angiotensin-aldosterone system (RAAS), and calcineurin/nuclear factor of activated T cells (CaN/NFAT) pathway. Method In this study, we used etelcalcetide (ET) as a calcimimetic agent. At 8 weeks of age, LVH was induced in rats through transverse aortic coarctation, and CKD was induced by 5/6 nephrectomy at 9 and 10 weeks of age. The rats were divided into the four main groups: the sham group, the CKD group, the LVH group, and the CKD+LVH group. Each of the latter three groups were further subdivided into a vehicle-treated group and an ET-treated group for a duration of eight weeks. At 19 weeks of age, echocardiography was conducted on all rats prior to sacrifice. Subsequently, the rats were sacrificed, and blood and urine analyses, mRNA expression analysis, and renal histological analysis were performed. We evaluated LVH, CKD-MBD, RAAS, and CaN/NFAT pathways across these seven groups. Results In both the CKD and CKD+LVH groups, serum levels of parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) were significantly elevated, and these elevations were mitigated by ET. However, in the LVH group, serum PTH and FGF23 levels were not elevated, and ET did not induce any changes. No significant alterations in serum levels of phosphorus, calcium, 25(OH)vitamin, and 1,25(OH)2vitaminD levels due to ET were observed in any of the groups. Interestingly, ET suppressed the increase in myocardial weight/body weight ratios and the area of cardiomyocytes not only in the CKD and CKD+LVH group but also in the LVH group, despite no changes in serum PTH and FGF23 levels. Regarding the RAAS, ET attenuated the increase in serum aldosterone levels, expressions of cardiac angiotensin-converting enzyme (ACE), cardiac angiotensinogen, and cardiac angiotensin II in the LVH group and the CKD+LVH group. The expression of cardiac FGF23, which increased in the LVH group and the CKD+LVH group, was decreased by ET. Moreover, there was a significant correlation between cardiac RAAS parameters and cardiac FGF23 expressions. No alteration was observed in the CaN/NFAT pathway in response to ET. Conclusion Our findings indicated that ET effectively suppressed LVH in the CKD group and the CKD+LVH group, and it was also effective in the LVH group, independent of changes in serum CKD-MBD parameters. This suggests that ET may exert its LVH-suppressing effects through the RAAS pathway and cardiac FGF23.

#1603 A potent protein inhibitor of HB-EGF protects kidneys from glomerular lesions in a pig model of crescentic glomerulonephritis

Abstract Background and Aims Crescentic glomerulonephritis (CGN) is a severe disease that rapidly destroys the glomerular architecture. CGN is due to immuno-inflammatory aggressions triggered by anti-neutrophil cytoplasmic autoantibody-associated vasculitides, anti-glomerular basement membrane (GBM) autoantibodies, or immune complexes deposition as occurs in systemic lupus erythematosus, IgA nephropathy or infections. Treatments are based on aggressive immunosuppression. Although required, immunosuppression doesn't directly target the deleterious glomerular response to inflammation and leads to significant morbidity. Moreover, most patients still develop chronic or end-stage kidney failure, indicating that complementary strategies are needed. We showed previously that local over-expression of the growth factor HB-EGF (Heparin-binding EGF-like growth factor) and subsequent activation of EGFR lead to dedifferentiation and proliferation of parietal epithelial cells and podocytes, leading to crescentic lesions that irreversibly obliterate the glomeruli. We aimed to evaluate the therapeutic potential of a protein inhibitor of HB-EGF in a pig model of CGN. Method We engineered a protein inhibitor of HB-EGF, DTR8, from the receptor-binding domain of diphtheria toxin, a highly selective natural ligand of HB-EGF. Twelve mutations improved solubility, led to pM affinity, and reduced immunogenicity and antigenicity. Pigs received decomplemented nephrotoxic serum (NTS) from sheep immunized against pig GBM to mimic anti-GBM antibodies disease responsible for one form of CGN. Ten pigs were treated with 0.3 mg/kg of DTR8 twice a day by intramuscular route for 20 days, and 12 pigs received vehicle only starting 6 hours after NTS. Animals were sacrificed on day 21, and blood, urine and kidneys were analyzed. Five animals that had received pre-immune sheep serum served as controls. Results Pigs developed a nephritic syndrome with high proteinuria, hypoalbuminemia, kidney failure with increased BUN and blood creatinine levels, anasarca and crescentic lesions typical of CGN along with de novo HB-EGF glomerular overexpression. The vehicle-treated group had only 24.8% of histologically normal glomeruli, while the DTR8-treated group had 57.3% of healthy glomeruli. DTR8 treatment significantly reduced the percentage of crescentic lesions (P = .0365) and preserved podocyte differentiation as assessed by the expression of nephrin that was markedly reduced in vehicle-treated animals but maintained close to normal in DTR8-treated animals (P = .0028). DTR8 administration reduced EGFR phosphorylation in glomerular cells (P = .0161), suggesting local inhibition of the HB-EGF/EGFR cascade. Lymphocyte infiltration was reduced (P = .2543). Notably, the treatment group also displayed a global reduction in interstitial fibrosis (P = .0313). Conclusion The therapeutic protein candidate DTR8 protects kidney glomeruli in a CGN model in pigs.

#694 Effects of nonsteroidal mineralocorticoid receptor antagonists on kidney of cirrhotic rats

Abstract Background and Aims Aldosterone binds to renal mineralocorticoid receptors (MRs) to regulate fluid and electrolytes homeostasis. In addition, aldosterone can be deleterious as a driver of inflammation and tissue fibrosis in several organs. Thus, blockade of aldosterone represents an important option to mitigate the target-organ damage. Mineralocorticoid receptor antagonists (MRAs) have been widely used as diuretic drugs for the management of hypertension, cirrhosis, and heart failure. However, the use of MRAs is limited by adverse events such as hyperkalemia. More recently, novel nonsteroidal selective MRAs with improved safety profiles have been developed. This nonsteroidal MRA (finerenone) has higher selectivity and stronger affinity for the MR, leading to lower risk of hyperkalemia. Recent evidences showed that finerenone reduces albuminuria, risk of CKD progression, and cardiovascular events in patients with chronic kidney disease and type 2 diabetes with a low incidence of hyperkalemia related treatment discontinuation. The pathophysiology of cirrhosis is very similar to that of cardiac failure. Decompensated cirrhosis is frequently treated with the MRAs. In addition to fluid management, MRAs showed hepatoprotective actions via reducing oxidative stress and inflammatory responses in CCl4-induced and common bile duct ligation (CBDL)-induced liver injury models. The potential beneficial effects of nonsteroidal MRA on cirrhosis had never been studied. This study aims to explore the renal protective role of nonsteroidal selective MRA and associated mechanisms in common bile duct ligation-related liver injury in rat model. Method CBDL was used to create liver injury in the Male Sprague-Dawley Drats. Finerenone (10 mg/kg/day) or vehicle was administered since the 1st day after CBDL. The effects of nonsteroidal selective MRA on kidneys were surveyed in two paralleled series on the 7th day (inflammation) and the 28th day (cirrhosis), respectively. The following parameters were evaluated: (a) hemodynamic parameters; (b) serum potassium, liver and renal function; (c) in-situ kidney perfusion; (d) western analyses of renal cortex and medulla. Results Compared with the vehicle-treated group, there was significantly lower renal vascular responsiveness to ET-1 in CBDL rats receiving 28 days of MRA (p<0.05) (Fig. 1B), but not in those receiving 7 days of MRA (Fig. 1A). MRA treatment led to no significant change in mean arterial pressure, portal pressure, serum potassium, liver, and renal function in CBDL rats (Table 1). The expressions of vasoactive mediators, such as iNOS, eNOS, ETA, and ETB in both renal cortex and medulla showed no significant change in CBDL rats following 28 days of MRA treatment (Fig. 2). Conclusion MRA treatment led to no significant effects on hemodynamics, liver, and renal function, but lower renal vascular responsiveness to ET-1 in CBDL rats receiving 28 days of MRA. The exact mechanisms remained to be further studied.

Multiscale effects of the calcimimetic drug, etelcalcetide on bone health of rats with secondary hyperparathyroidism induced by chronic kidney disease

Chronic kidney disease-induced secondary hyperparathyroidism (CKD-SHPT) heightens fracture risk through impaired mineral homeostasis and elevated levels of uremic toxins (UTs), which in turn enhance bone remodeling. Etelcalcetide (Etel), a calcium-sensing receptor (CaSR) agonist, suppresses parathyroid hormone (PTH) in hyperparathyroidism to reduce excessive bone resorption, leading to increased bone mass. However, Etel's effect on bone quality, chemical composition, and strength is not well understood. To address these gaps, we established a CKD-SHPT rat model and administered Etel at a human-equivalent dose concurrently with disease induction. The effects on bone and mineral homeostasis were compared with a CKD-SHPT (vehicle-treated group) and a control group (rats without SHPT). Compared with vehicle-treated CKD-SHPT rats, Etel treatment improved renal function, reduced circulating UT levels, improved mineral homeostasis parameters, decreased PTH levels, and prevented mineralization defects. The upregulation of mineralization-promoting genes by Etel in CKD-SHPT rats might explain its ability to prevent mineralization defects. Etel preserved both trabecular and cortical bones with attendant suppression of osteoclast function, besides increasing mineralization. Etel maintained the number of viable osteocytes to the control level, which could also contribute to its beneficial effects on bone. CKD-SHPT rats displayed increased carbonate substitution of matrix and mineral, decreased crystallinity, mineral-to-matrix ratio, and collagen maturity, and these changes were mitigated by Etel. Further, Etel treatment prevented CKD-SHPT-induced deterioration in bone strength and mechanical behavior. Based on these findings, we conclude that in CKD-SHPT rats, Etel has multiscale beneficial effects on bone that involve remodeling suppression, mineralization gene upregulation, and preservation of osteocytes.

Evaluation of haematinic activity of <i>Tapinanthus globiferus</i> (A. Rich.) van Tiegh leaf extract and fractions in phenylhydrazine-induced anaemic rats

Anaemia is a leading cause of morbidity and mortality in both males and females of all ages. Anaemia was induced by intraperitoneal (i.p.) administration of phenylhydrazine (60 mg/kg) for a period of 3 days. Anaemic rats were allotted to five (5) groups; group I served as the negative control, group II served as the positive control received Astyfer syrup (0.15 mg/kg p.o) , groups III-V served as the test groups that received Tapinanthus globiferus methanol extract, ethyl acetate and n-hexane fractions (100, 200 and 400 mg/kg, p.o.), respectively. Packed cell volume (PCV), haemoglobin concentration (HB) and red blood cell count (RBC) were analysed as indices of anaemia at 7-day intervals for 21 days. The mean cell volume (MCV) and mean cell haemoglobin concentration (MCHC) were calculated accordingly. Leaf extract and fractions of Tapinanthus globiferus significantly (P<0.001) increased the PCV, the HB concentration and the RBC, which had been originally decreased by phenylhydrazine (P<0.001) administration within one week of treatment. The extract and fractions were compared with rats administered with the positive and vehicle-treated group. The results suggested that Tapinanthus globiferus leaves have haematinicactivities.

Impact of the Oral Administration of Polystyrene Microplastics on Hepatic Lipid, Glucose, and Amino Acid Metabolism in C57BL/6Korl and C57BL/6-Lepem1hwl/Korl Mice.

The impact of microplastics (MPs) on the metabolic functions of the liver is currently unclear and not completely understood. To investigate the effects of the administration of MPs on the hepatic metabolism of normal and obese mice, alterations in the lipid, glucose (Glu), and amino acid regulation pathways were analyzed in the liver and adipose tissues of C57BL/6Korl (wild type, WT) or C57BL/6-Lepem1hwl/Korl mice (leptin knockout, Lep KO) orally administered polystyrene (PS) MPs for 9 weeks. Significant alterations in the lipid accumulation, adipogenesis, lipogenesis, and lipolysis pathways were detected in the liver tissue of MP-treated WT and Lep KO mice compared to the vehicle-treated group. These alterations in their liver tissues were accompanied by an upregulation of the serum lipid profile, as well as alterations in the adipogenesis, lipogenesis, and lipolysis pathways in the adipose tissues of MP-treated WT and Lep KO mice. Specifically, the level of leptin was increased in the adipose tissues of MP-treated WT mice without any change in their food intake. Also, MP-induced disruptions in the glycogenolysis, Glu transporter type 4 (GLUT4)-5' AMP-activated protein kinase (AMPK) signaling pathway, levels of lipid intermediates, and the insulin resistance of the liver tissues of WT and Lep KO mice were observed. Furthermore, the levels of seven endogenous metabolites were remarkably changed in the serum of WT and Lep KO mice after MP administrations. Finally, the impact of the MP administration observed in both types of mice was further verified in differentiated 3T3-L1 adipocytes and HepG2 cells. Thus, these results suggest that the oral administration of MPs for 9 weeks may be associated with the disruption of lipid, Glu, and amino acid metabolism in the liver tissue of obese WT and Lep KO mice.

Chloroquine Affects Frequency-Dependent Endocytosis in Diaphragm Muscle of Old Mice

Chloroquine is an antimalarial agent commonly used as an autophagy inhibitor by preventing autolysosomal fusion. Autophagy is an important cellular process through which defective cytoplasmic structures are recycled and degraded that has been implicated in aging neuromuscular effects. We recently showed that acute chloroquine treatment blunts the maximal forces generated by the diaphragm muscle, likely by impairing neuromuscular transmission generally consistent with aging effects in the diaphragm muscle. In addition, old mice show increased accumulation of autophagy proteins in phrenic motor neurons, consistent with impaired autophagy flux. In the present study, we hypothesized that chloroquine treatment results in accumulation of bulk endosomes pre-synaptically. Each hemidiaphragm with attached phrenic nerve was excised from 24-month-old male and female C57BL/6 mice to evaluate frequency dependent (0 vs 80 Hz stimulation) uptake of the styryl dye FM4-64, given that 80-Hz stimulation frequency induces bulk endocytosis after synaptic vesicle retrieval. Diaphragm-phrenic nerve preparations were incubated in 5 μM FM4-64 for 1 h prior to 6-min nerve stimulation (0.5 ms supramaximal pulses in 80 Hz, 200 ms trains delivered at 40% duty cycle) followed by a 5-min post-stimulation incubation. FM4-64 uptake at diaphragm neuromuscular junctions (NMJs) was measured using confocal microscopy. Preparations were randomly assigned to co-incubation with chloroquine (50 μM) or vehicle (Rees-Simpson solution). There was minimal FM4-64 uptake in the absence of phrenic nerve stimulation, consistent with the activity-dependent uptake of the styryl dye. With 80 Hz stimulation, high levels of FM4-64 uptake were evident in both the chloroquine and vehicle-treated groups without significant differences across groups. FM4-64 fluorescence was evident throughout the presynpatic terminal with few areas of dense accumulation, consistent with retention of endosomes. There was no evidence of greater endosome accumulation with chloroquine treatment. Of note, a slight increase in FM4-64 uptake was evident in the chloroquine treated groups compared to vehicle in the non-stimulated NMJs. These results are consistent with FM4-64 uptake reflecting bulk endocytosis that mainly occurs during high frequency stimulation. The impairment in autophagy flux following chloroquine treatment is expected to result in endosome accumulation but it is possible that the stimulation protocol was insuffcient to elucidate this possibility. Collectively, the findings of this study are consistent with impaired autophagy at diaphragm presynaptic terminals which may underlie neuromuscular deficits observed in old age. Supported by NIH grant R01 AG057052 and the Mayo Clinic. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Role of Adiponectin in the Androgen-Mediated Cardiometabolic Complications in a Rat Model of Polycystic Ovary Syndrome

Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women and is associated with a high prevalence of obesity, adipose insulin resistance (adipose IR), and insulin resistance (IR). Adipose IR is an early metabolic defect in the development of whole-body IR. Specific evaluation of adipose IR and its consequences on the whole-body metabolic state in women with PCOS has not been thoroughly investigated. Adiponectin is a peptide secreted by adipocytes that increases insulin sensitivity. PEG-BHD1028 is a novel potent peptide adiponectin receptor agonist. Despite that PEG-BHD1028 modulates insulin sensitivity in different models of diabetes, the potential of PEG-BHD1028 to attenuate androgen-mediated adipose IR and obesity remains unknown. Hypothesis: We tested the hypothesis that PEG-BHD1028 attenuates adipose IR and obesity in a rat model of PCOS. Methods: To induce PCOS, four-week-old female Sprague Dawley rats were implanted with dihydrotestosterone (DHT) silastic tubes (7.5 mg) or placebo (PBO) for 90 days (n=4-8/group). During the final three weeks of DHT treatment, rats received PEG-BHD1028 (25 μg/kg/day) subcutaneously. Body weight (BW) by gravimetry, fat and lean mass were measured by Echo-MRI. Fasting plasma insulin was measured by ELISA. Fasting plasma glucose and non-esterified fatty acids (NEFA) were measured with a clinical chemistry analyzer. HOMA-IR, an index of systemic IR, and adipose IR (fasting insulin x fasting NEFA) were calculated. Protein levels of insulin signaling markers including IRS1, p-IRS1 (Ser1101), PI3 kinase p110, Akt, p-Akt (SER473), GLUT4, mTOR, p-mTOR (Ser2481) were quantified in the retroperitoneal fat by Western-blot. Results: DHT significantly (p<0.05) increased BW (352.67± 6.93 vs 250.88 ± 5.26 g), fat mass (20.82 ± 1.48 vs 9.44 ± 1.81 g), and lean mass (316.92 ± 4.15 vs 224.45 ± 4.79 g), compared to PBO group. DHT significantly (p<0.05) increased HOMA-IR and adipose IR by 75% and 80%, respectively, compared to PBO group. DHT downregulated retroperitoneal fat p-IRS-1, IRS-1, p-mTOR, mTOR, GLUT4, PI3 kinase and Akt protein expression by 50% to 70% while upregulating p-Akt by 1.8- fold compared to the PBO group. PEG-BHD1028 significantly decreased BW, fat mass and lean mass while attenuating IR by 34.7% reduction in HOMA-IR and significantly (p<0.05) decreased adipose IR by 51.6% in PCOS rats. Interestingly, PEG-BHD1028 significantly (p<0.05) upregulated p-IRS-1, IRS-1, p-mTOR, mTOR and p-AKT levels by 2.6-, 3-, 2.2-, 4-, 2.6- fold, respectively with no impact on the other insulin signaling markers compared to vehicle-treated group. Conclusion: PCOS rats exhibit obesity and lower adiponectin levels that are associated with both adipose and whole-body insulin resistance. Activating adiponectin receptors with PEG-BHD1028 attenuated PCOS-associated obesity and adipose IR. Significance: Targeting adiponectin signaling could be a novel and effective therapeutic approach to mitigate metabolic dysfunction in females with excess androgens. Supported by NIH-NIGMS grant P20GM121334 (L.L.Y.C. and D.G.R.), NIH-NIGMS grant P20GM121334, P20GM104357, NIH-NHLBI grant P01HL51971, NIH-NIMHD-P50MD017338 and University of Mississippi Medical Center Medical Student Research Program (R.C.) and the Robert M. Hearin Foundation (R.C.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exploring the potential effect of electroacupuncture on cardiovascular function and lipid profiles in spontaneously hypertensive rats

BackgroundInvestigating the effects of electroacupuncture (EA) treatment on cardiovascular function and aortic lipid profiles in spontaneously hypertensive rats (SHR) constitutes the foundational focus of this study. The overarching goal is to comprehensively elucidate the alterations brought about by EA treatment and to assess its potential as an alternative therapy for hypertension. MethodsConsecutive EA treatments were administered to SHR, and the effects on systolic blood pressure, cardiac function, and hypertension-related neuronal signals were assessed. Aortic lipid profiles in vehicle-treated SHR and EA-treated SHR groups were analyzed using mass spectrometry-based lipid profiling. Additionally, the expression of Cers2 and GNPAT, enzymes involved in the synthesis of specific aortic lipids, was examined. ResultsThe study demonstrated that consecutive EA treatments restored systolic blood pressure, improved cardiovascular function, and normalized hypertension-related neuronal signals in SHR. Analysis of the aortic lipid profiles revealed distinct differences between the vehicle-treated SHR group and the EA-treated SHR group. Specifically, EA treatment significantly altered the levels of aortic sphingomyelin and phospholipids, including very long-chain fatty acyl-ceramides and ether phosphatidylcholines. These changes in aortic lipid profiles correlated significantly with systolic blood pressure and cardiac function indicators. Furthermore, EA treatment significantly altered the expression of Cers2 and GNPAT. ConclusionsThe findings suggest that EA may influence cardiovascular functions and aortic lipid profiles in SHR.