This article presents the experimental datasets obtained from the histological/histochemical studies of endocrine disrupting effects of graphene oxide (GO) on thyroid follicles and gas gland (GG) cells of Japanese medaka larvae at the onset of maturity. The experiment was conducted on one day-post hatch (dph) starved fries (orange-red variety) immersed in different concentrations of GO (2.5-20.0 mg/L) and no GO (controls) in embryo-rearing medium (ERM) for 96 h under laboratory conditions (25 ± 1 °C; light cycle 16 h light: 8 h dark). After treatment, larvae were maintained in balanced salt solution (BSS) with food and allowed depuration for 6 more weeks in a GO-free environment. On 47 dph, the larvae were anesthetized in MS 222 and their total lengths (mm) and weights (mg) were measured, and they were then cut into three small pieces (head, trunk, and tail). Head and trunk regions were fixed in 4% PFA in 20 mM PBS for 48 h at room temperature and the post-anal tail was preserved in TRI reagent and kept at −20 °C until analysis. Tissues in 4% PFA were used for cutting 5µm thick paraffin sections in a manual rotary microtome. Sections of head regions were evaluated for thyroid follicles after hematoxylin-eosin (HE) or Periodic acid-Schiff (PAS) staining. Trunk sections were used for swim bladder (SB) inflation studies and for phenotypic sex (ovary and testis) of the larvae after HE staining. Genetic sex assessment was made from tail DNA by genotyping Y chromosome-specific male sex-determining gene dmy . Digital images were captured by using either an Olympus B-max 40 microscope attached to a camera with Q-capture Pro 7 software or an Olympus CKX53 microscope with DP22 camera and CellSens software. Images of thyroid follicles and GG cells were analyzed using imagej software. HE stained histological sections of thyroid follicles near the heart and branchial regions were captured and the area (µm 2 ) of individual follicles (minimum 3) available in the entire section were measured. The heights of thyrocytes (µm) were determined directly. Manual counting of GG cells was made from the digital images captured in several regions of the SB avoiding blood cells and other cells which have indistinct nucleus and pale cytoplasm; results were expressed as the number of GG cells/mm 2 . Data were analyzed by GraphPad prism version 7.04. For normally distributed data, one-way ANOVA followed by post-hoc Tukey's test or unpaired parametric “t” test including Welch's correction was used. Otherwise, Kruskal-Wallis test followed by nonparametric Mann-Whitney's test as a post hoc test was used. Data were expressed as means ±SEM and the level of significance was set at p < 0.05.
Background: Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism and polycystic ovaries. Renal injury, increased central adiposity and higher circulating levels of the adipokine leptin are common characteristics of PCOS. Leptin has direct renal fibrotic, hypertrophic, and albuminuric effects and can cause renal mitochondrial dysfunction and oxidative stress leading to chronic kidney disease development. Despite increased circulating leptin level in both lean and obese PCOS women, its role in PCOS-mediated renal damage remains unknown. In a well-characterized mouse model of PCOS, we aimed to test the hypothesis that hyperandrogenemia increases central adiposity leading to hyperleptinemia, which via leptin receptors induces intrarenal mitochondrial oxidative stress and dysfunction causing renal injury. Methods: Three-week-old peripubertal female mice were implanted with Silastic tubes filled with the non-aromatizable androgen dihydrotestosterone (DHT, 8 mg) or vehicle for 12 weeks. Eight weeks post-Silastic tubes implantation, the animals were treated with the leptin receptor antagonist (LepR-Ant) pegylated leptin (2 mg/kg, ip, 3x/week) for 4 weeks. Body weight, fat mass (EchoMRI), kidney weight (gravimetry), plasma leptin (ELISA), the glomerular filtration rate (GFR, transcutaneous fluorescence), as well as the renal injury markers urinary albumin to creatinine ratio (UACR, clinical chemistry analyzer), NGAL, and KIM1 (ELISA) were measured. Freshly isolated kidney mitochondria were used to measure mitochondrial reactive oxygen species (mtROS) by Amplex Red assay. Moreover, mitochondrial complex I and complex II-driven respiration and complex IV activity were assessed using Oroboros Fluorespirometer. Results: DHT significantly (p<0.05) increased body weight (31.6 ± 2.3 vs. 23.6 ± 0.5 g), fat mass (3.5 ± 0.6 vs. 1.9 ± 0.4 g), kidney weight (365.6 ± 13.1 vs. 257.1 ± 3.7 mg), leptin (3.7-fold), UACR (1013.3 ± 56.4 vs. 658.9 ± 103.6 μg albumin/g creatinine), NGAL (6-fold), and KIM1 (3.7-fold). Moreover, PCOS mice had significantly (p<0.05) lower GFR (1067.2 ± 59.9 vs. 1335.6 ± 60.3 uL/min/100g body weight), higher mtROS driven by complexes I (3.8 ± 0.8 vs. 2.0 ± 0.3 % electron leak) and III (8.0 ± 1.8 vs. 3.7 ± 0.9 % electron leak), as well as lower complexes I, II, and IV respiration (43-71%). The LepR-Ant had no effect on kidney weight or GFR; however, it abolished DHT-induced increases in body weight and fat mass. Excitingly, the LepR-Ant inhibited mtROS generation and completely restored complex I and II respiration, which was associated with a significant reduction in the urinary renal injury markers UACR, NGAL, and KIM-1 levels (40-60%, p<0.05). Conclusion and significance: Our findings suggest that leptin via its receptor activation plays a significant role in the renal outcomes in PCOS by inducing mitochondrial dysfunction. Leptin receptor blockade could be a novel therapeutic approach to ameliorate renal injury in PCOS. Supported by NIH grants NIGMS P20GM121334 to KSE, LLYC, and DGR, NIGMS P20GM104357, NHLBI P01HL51971, and American Heart Association Predoctoral Fellowship 903804 (A.M.H.). This is the full abstract presented at the American Physiology Summit 2023 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.
Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women. PCOS women often have increased central adiposity, insulin resistance, and renal injury. Hyperandrogenism is prevalent in PCOS women and is positively associated with metabolic dysfunction. Androgens bind to the androgen receptor (AR) to impart downstream transcriptional activity. Targeting the AR may alleviate PCOS complications, however the use of AR blockers is limited by their severe adverse effects in females thus safer alternative approaches are highly needed. ARV-110, a novel AR Proteolysis Targeting Chimera (PROTAC) degrader demonstrates high efficacy to prevent AR signaling in prostate cancer, however its efficacy in PCOS is unknown. Using a well-established hyperandrogenemic mouse model of PCOS, we aim to test the hypothesis that decreasing AR protein levels with ARV-110 will ameliorate androgen-mediated derangements in PCOS. Methods: Three-week old female mice (C57BL/6N) were implanted with Silastic tubes filled with the androgen dihydrotestosterone (DHT, 8mg, S.C., 90 days) or vehicle (n=6/grp). Four weeks post-DHT administration, mice were treated with ARV-110 (1 mg/kg/day, S.C.) for an additional 8 weeks. Body composition (EchoMRI) and the oral glucose tolerance test (OGTT) were assessed. Retroperitoneal fat (RPF) and kidney weights were measured by gravimetry. AR and extracellular matrix (ECM) remodeling marker MMP9 protein levels were assessed by Western blot, while the pro-fibrotic marker TGF-β was assessed by ELISA. Results: DHT mice showed significant (p<0.05) increases in body weight (27.3±0.6 vs 23.9±0.26 g), fat mass (4.2±0.6 vs 2.1±0.1 g), lean mass (21.6±0.2 vs 19.8±0.2 g), as well as RPF (244.4±37.4 vs 50.0±1.8 mg) and kidney weights (491.3±7.69 vs 303.1±5.5 mg) compared to controls. DHT increased fasting glucose (183.2±25.2 vs 141.5±9.9 mg/dL, p<0.05) and impaired OGTT (AUC:34557±6255 vs 23459±1896 mg.min/dL, p<0.05). ARV-110 treatment ameliorated all aforementioned DHT-mediated changes. On the molecular level, DHT mice had higher AR expression in RPF (1.5-fold) and kidney (2.5-fold), and higher MMP9 in both tissues (2- and 1.5-fold, respectively) indicating ECM remodeling. DHT had no significant effect on TGF-β in RPF but it upregulated TGF-β level in the kidney (2-fold, p<0.05). ARV-110 successfully decreased both RPF and renal AR protein levels by 70% in DHT mice compared to vehicle-treated DHT. Excitingly, while ARV-110 did not modulate RPF nor renal TGF-β levels, it completely abolished DHT-mediated MMP9 increase in both tissues. Conclusion and significance: Our findings suggest that DHT-mediated WAT expansion, kidney hypertrophy, and associated ECM remodeling are ameliorated by ARV-110 in DHT mice. These results highlight the pivotal role of AR in androgen-mediated and tissue-specific derangements common in PCOS. AR PROTACs could be a novel therapeutic approach to treat metabolic and renal dysfunction in PCOS. Supported by National Institute of General Medical Sciences of the National Institutes of Health grant P20GM121334 (L.L.Y.C. and D.G.R.), and American Heart Association Predoctoral Fellowship 903804 (A.M.H.) This is the full abstract presented at the American Physiology Summit 2023 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.
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