Deficient Rats Research Articles

Role of Sulfotransferase 1C2 in Vitamin D Signaling in Ischemic AKI Associated with Vitamin D Deficiency in Rats

Role of Sulfotransferase 1C2 in Vitamin D Signaling in Ischemic AKI Associated with Vitamin D Deficiency in Rats

Regulation of 11β-HSD1 reductase and the HPA axis by long-snake moxibustion in kidney-yang deficiency rats

BackgroundLong-snake moxibustion can improve hypothalamic-pituitary-adrenal (HPA) axis function in patients with kidney-yang deficiency (KYDS). 11β-HSD1 controls the HPA axis by boosting CORT production via reductase activity. However, the interaction and mechanism of long snake moxibustion and 11β-HSD1 remain unknown. This study examined the impact of lengthy snake moxibustion on the hypothalamus-pituitary-adrenal axis in KYDS rats. The potential significance of 11β-HSD1 in this process was explored. MethodsRats were randomly divided into two groups: the blank group and the experimental group. The KYDS model was established with an intramuscular injection of hydrocortisone. Rats were randomly assigned to four groups: model, sham intervention, long snake moxibustion, and long snake moxibustion plus 11β-HSD1 inhibitor. Physical indicators included body weight, toe temperature, rectal temperature, and spontaneous movement. The serum levels of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and CORT were measured. Immunohistochemical examination reveals 11β-HSD1 protein expression in the liver. Western blotting (WB) detected the levels of 11β-HSD1, H6PDH and NADPH/NADP + protein in the liver. ResultsThe experimental rats' body weight, toe temperature, rectal temperature, time and frequency of spontaneous activity all dropped, as did their serum ACTH, CORT, and CRH levels. The protein expressions of 11β-HSD1, H6PDH, and NADPH/NADP+ in the liver decreased significantly. Long-snake moxibustion improved HPA axis function in rats, boosting expression of 11β-HSD1, H6PDH, and NADPH/NADP+. Adding an 11β-HSD1 inhibitor to Long-snake moxibustion decreased its effect on the HPA axis. ConclusionLong-snake moxibustion improves KYDS symptoms in rats by increasing 11β-HSD1 expression and reductase activity, which regulates the HPA axis.

THE IMPACT OF HOP EXTRACT ACTIVE COMPONENTS ON METABOLIC DISTURBANCES IN ESTROGEN DEFICIENT FEMALE RATS WITH PREDIABETES

A number of studies have shown a greater cardiovascular risk in women with type 2 diabetes compared to men. The latter may be due to more excessive manifestations of metabolic disorders in women, in particular postmenopausal, that develop even at the stage of prediabetes. Since the use of hormone replacement therapy in women after menopause had adverse results on the progression of cardiovascular events, an alternative method of reducing cardiovascular risk in postmenopausal women with prediabetes may be the use of phytoestrogens, which possess estrogen-like activity without negative side effects specific to exogenous estrogens. The aim of our study was to evaluate the effect of hop extract on metabolic disturbances in female rats with prediabetes under estrogen deficiency. Materials and methods. The study was conducted on three-month-old intact and ovariectomized Wistar rats with metabolic syndrome (MS) induced by high calorie diet (15% lard, 25% sucrose, 1% bile salts, 59% standard feed) for eight weeks. The test substances were administered intragastrically once per day in a dose of 20 μg/kg or 200 μg/kg b.w. in recalculation on 8-prenylnaringenin for hop extract and 200 μg/kg b.w. in recalculation on estradiol for the reference drug, from the fifth week after induction of metabolic syndrome. Results. It was revealed that administration of hop extract in a dose of 200 μg/kg, similar to estradiol, led to improvement in glucose tolerance and decrease in insulin resistance (p˂0.05) in estrogen deficient rats with MS. In contrast to estradiol, hop extract had no effect on the mass of uterus in ovariectomized animals. The use of hop extract only in the dose of 200 μg/kg, similar to estradiol, was accompanied by a significant decrease in body weight gain and visceral fat, as well as normalization of the total lipids content in serum of experimental animals (p˂0.05). It is of note, that hop extract resulted in decrease of hypertriglyceridemia (p˂0.05), but did not affect the level of total cholesterol, while estradiol reduced the concentration of total cholesterol (p˂0.05), and had no effect on the level of triglycerides. Hop extract in a dose of 200 μg/kg, as well as estradiol, led to a decrease in the intensity of lipid peroxidation induced by MS under estrogen deficiency, as evidenced by reduced concentration of conjugated dienes in serum (p˂0.05). At the same time, the total antioxidant activity of serum in these animals remained elevated compared to intact group. In rats with MS and hypoestrogenia, hop extract in the highest dose and estradiol resulted in complete normalization (p˂0.05) of the decreased level of NO stable metabolites in serum and the reduced activity of NO-synthase in heart. Conclusions. The revealed cardioprotective effect of hop extract that is realized due to the improved lipid profile and reduced insulin resistance, visceral obesity, oxidative stress and endothelial dysfunction in estrogen deficient female rats with MS indicates the prospects of its use for prevention and treatment of cardiovascular complications in postmenopausal women with prediabetes.

Research on Experimental Hypertension in Prague (1966-2009).

The study of ontogenetic aspects of water and electrolyte metabolism performed in the Institute of Physiology (Czechoslovak Academy of Sciences) led to the research on the increased susceptibility of immature rats to salt-dependent forms of hypertension since 1966. Hemodynamic studies in developing rats paved the way to the evaluation of hemodynamic mechanisms during the development of genetic hypertension in SHR. A particular attention was focused on altered renal function and kidney damage in both salt and genetic hypertension with a special respect to renin-angiotensin system. Renal damage associated with hypertension progression was in the center of interest of several research groups in Prague. The alterations in ion transport, cell calcium handling and membrane structure as well as their relationship to abnormal lipid metabolism were studied in a close cooperation with laboratories in Munich, Glasgow, Montreal and Paris. The role of NO and oxidative stress in various forms of hypertension was a subject of a joint research with our Slovak colleagues focused mainly on NO-deficient hypertension elicited by chronic L-NAME administration. Finally, we adopted a method enabling us to evaluate the balance of vasoconstrictor and vasodilator mechanisms in BP maintenance. Using this method we demonstrated sympathetic hyperactivity and relative NO deficiency in rats with either salt-dependent or genetic hypertension. At the end of the first decennium of this century we were ready to modify our traditional approach towards modern trends in the research of experimental hypertension. Keywords: Salt-dependent hypertension o Genetic hypertension o Body fluids o Hemodynamics o Ion transport o Cell membrane structure and function o Renal function o Renin-angiotensin systems.

Experimental Vitamin D Deficiency in Rats: Clinical Chemistry, Histopathological, and Immunological Evaluation.

Vitamin D deficiency is a significant global health concern. Experimental models are essential to elucidate the biochemical, histopathological, and immunological consequences of this deficiency. This study established a vitamin D deficiency rat model to mimic insufficient vitamin D intake and examine the resulting health impacts, particularly on liver, kidney, and immune functions. Sprague-Dawley male rats were randomly assigned to two groups. The control group received a standard rodent diet, while the experimental group was fed a modified diet with reduced vitamin D for three months. Analyses included serum vitamin D levels, clinical chemistry, renal and liver histopathology, and blood immunophenotyping and cytokine analysis for both the control (n=7) and experimental (n=7) groups. Serum vitamin D 25-OH levels were threefold lower in the experimental group (p < 0.001), indicating the induction of vitamin D deficiency. No significant differences in weight gain were observed between the groups. All clinical chemistry parameters remained within reference ranges. However, the experimental group showed significant declines in triglycerides (TG, p=0.0441), alkaline phosphatase (ALP, p=0.0021), and alanine aminotransferase (ALT, p=0.0002). Histopathology revealed normal liver and kidney architecture in the control group, while the experimental group exhibited hepatic cord deterioration, severe vacuolization in the liver, and edema and dilatation in the renal cortex tubular epithelium. Immunophenotyping analysis of lymphocyte subsets and assessment of serum cytokines did not reveal any differences between the two groups. A vitamin D deficiency model without complications such as obesity, parathyroid issues, or mortality was established in rats. This method could be applied in specific disease experimental models.

Abstract Tu132: Estrogen modulation of ethanol-evoked cardiac oxidative stress and dysfunction: Role of circadian clock period-2 and ferroptosis in estrogen deficient rats

Background: Alcohol triggers exacerbated cardiovascular effects in females, compared to males, in estrogen (E2) dependent manner. E2 provides cardiac protection in premenopausal women and models of menopause, yet the interplay between the circadian clock proteins and E2 in the female heart, particularly in the presence of ethanol (EtOH) remains unclear. Aim: This study aims to determine if the suppression of the circadian clock protein Period 2 (Per2) and associated redox enzymes contribute to E2-mediated exacerbation of the EtOH-induced cardiac ferroptosis, and dysfunction. Methods: Two groups of female Sprague-Dawley rats (n=6-8) were subjected to bilateral ovariectomy (OVX) and fed with EtOH (5% in liquid diet) in the presence of E2 or its vehicle for 8 weeks. Measurements of cardiac function using radiotelemetry and echocardiography along with ex vivo biochemical/molecular studies. Results: Compared to vehicle treated OVX+EtOH group, the E2-treated OVX+EtOH group exhibited: (1) reduced (p&lt;0.05) body weight gain and fat mass; (2) higher (p&lt;0.05) telemetry-measured blood pressure and heart rate; (3) reductions (p&lt;0.05) in echocardiography derived contractility indices, ejection fraction and fractional shortening. In the same group, ex vivo molecular analysis demonstrated: (1) lower cardiac Per2 and suppressed (p&lt;0.05) redox enzymes; (2) increased (p&lt;0.05) ROS level and glutathione depletion (p&lt;0.05), leading to the degradation of glutathione peroxidase 4 (GPX4); (3) heme oxygenase-1 upregulation leading to iron overload (p&lt;0.05). These ferroptosis-related effects resulted in increased lipid peroxidation (p&lt;0.05) compared to findings in OVX+EtOH rats in the absence of E2. Conclusion: This study is the first to suggest that abrogation of the E2-mediated Per2 upregulation unraveled cardiac ferroptosis and contributes to the detrimental cardiac effects of ethanol in E2-replete rats. These novel findings underscore the potential risk for myocardial ferroptosis and dysfunction in menopausal women who are consuming ethanol while on E2 replacement therapy.

Zinc Deficiency Causes Glomerulosclerosis and Renal Interstitial Fibrosis Through Oxidative Stress and Increased Lactate Metabolism in Rats.

Chronic kidney disease (CKD) is a highly prevalent condition characterized by renal fibrosis as its ultimate manifestation. Zinc deficiency is closely associated with CKD, evidenced by its link to renal fibrosis.Recently, local lactic acidosis has beendemonstrated topromote renal fibrosis. Under zinc-deficient conditions,mitochondrial functioniscompromised and abnormal lactate metabolism might be inducedpotentially. However, it remains unclear whether zinc deficiency leads to renal fibrosisthrough local lactic acidosis. Zinc deficiency rat models were successfully established by feeding zinc-deficient diet. Western blot, qPCR, IHC, and other experiments were employed to investigate the key markers and molecular mechanisms of glomerulosclerosis and renal interstitial fibrosis. Our resultsindicate that zinc deficiency reduces specific markers of podocytes (podocalyxin, WT1, and nephrin) andactivates the Wnt3a/β-catenin pathway, a key pathway in podocyte injury. Concurrently, glomerulosclerosis is indicated by increased urinary microalbumin and serum creatinine levels along with histological alteration observed through PAS and Masson staining in zinc-deficient rats. Furthermore, various degrees of upregulation for several markers of interstitial fibrosis including α-SMA, FN1 and collagen III are also revealed. These findings were further confirmed by Masson staining and IHC. Additionally, alterations in four markers in the EMT process, N-cadherin, E-cadherin, Vimentin, and snail, were consistent with expectations. We then confirmed the activation of the non-canonical TGF-β1 pathway known as the PI3K/AKT/mTOR pathway. An elevation in renal ROS levels accompanied by increased mitochondrial marker cytochrome C expression as well as an elevated NADH/NAD + ratio is also observed within the kidneys. Furthermore, the activity of both MMP/TIMP system and fibrinolytic system was abnormally enhanced under zinc deficiency conditions. Finally, we find zinc supplementation could significantly ameliorate relevant pathological alterations induced by zinc deficiency. These results collectively point that zinc deficiency causes podocyte damage ultimately resulting in glomerulosclerosis via accumulation of ROS and induces interstitial fibrosis via lactic acidosis.

TSPO in pancreatic beta cells and its possible involvement in type 2 diabetes

Amyloidosis forms a large family of pathologies associated with amyloid deposit generated by the formation of amyloid fibrils or plaques. The amyloidogenic proteins and peptides involved in these processes are targeted against almost all organs. In brain they are associated with neurodegenerative disease, and the Translocator Protein (TSPO), overexpressed in these inflammatory conditions, is one of the target for the diagnostic. Moreover, TSPO ligands have been described as promising therapeutic drugs for neurodegenerative diseases. Type 2 diabetes, another amyloidosis, is due to a beta cell mass decrease that has been linked to hIAPP (human islet amyloid polypeptide) fibril formation, leading to the reduction of insulin production. In the present study, in a first approach, we link overexpression of TSPO and inflammation in potentially prediabetic patients. In a second approach, we observed that TSPO deficient rats have higher level of insulin secretion in basal conditions and more IAPP fibrils formation compared with wild type animals. In a third approach, we show that diabetogenic conditions also increase TSPO overexpression and IAPP fibril formation in rat beta pancreatic cell line (INS-1E). These data open the way for further studies in the field of type 2 diabetes treatment or prevention.

Treatment with mannose oligosaccharides reverses the intestinal injury in the acetylsalicylic acid-treated rat model

Mannose oligosaccharide (MOS) has been shown to promote animal growth, maintain intestinal health, and activate the intestinal immune system. However, the question of whether MOS can stimulate the immune system and alleviate acetylsalicylic acid (ASA)-induced gut damage remains unresolved. The purpose of this study was to investigate the impact of MOS pretreatment on the immunological and anti-inflammatory capabilities of rats with ASA-induced intestinal injury. Thirty-six male Sprague-Dawley rats were divided into 6 groups and were fed with 0 (negative control), 100, 300, 600, and 800 mg/kg·Body weight (BW) of MOS for 3 weeks. From day 8, rats were fed with 200 mg/kg BW of ASA for 14 days to induce intestinal injury. The growth performance, viscera index, serum and intestinal immunity, intestinal inflammation and morphology of ASA-induced intestinal injury rats with or without MOS administration were investigated. In MOS deficient rats, oral treatment of ASA causes severe intestine damage and immunological dysfunction. In a rat model, 600 mg/kg BW MOS can lower the expression of inflammatory markers and effectively increase liver index, serum interleukin-2 (IL-2), lysozyme contents, intestinal secretory immunoglobulin A (sIgA) and mucus volume, intestinal villus height, crypt depth and villus height/crypt depth in comparison to the ASA group. These results imply that providing rats with MOS at the appropriate dosage can significantly improve their immune system and successfully shield the intestines from ASA damage. MOS is therefore expected to be a promising gut immunopotentiator for enhancing intestinal health in animals.

Effects of pre- versus post-conceptional iron and n-3 fatty acid supplementation of deficient rat dams on neurodevelopmental outcomes in the offspring

This abstract was awarded the Student Competition Prize.Periconception is the period spanning the weeks directly before and after conception and is a crucial window for optimising neurodevelopment in offspring(1-4). Iron and omega-3 fatty acids (n-3 FAs) are two vital nutrients for the development of the nervous system however, little research has been conducted into their importance during the periconceptional window(5-8). This study investigated whether supplementing iron and n-3 FA deficient (ID and n-3 FAD) rat dams before conception compared to after conception results in different neurodevelopmental outcomes in offspring. We hypothesised that initiating iron and n-3 FA supplementation in deficient dams after conception would not be as efficacious in preventing impaired offspring neurodevelopment induced by double- deficiency, compared to initiating supplementation before conception.Female rats consuming an ID and n-3 FAD diet were randomly allocated to receive iron and DHA/EPA supplementation either 10 days before (Pre-Fe+DHA/EPA) or 10 days after conception (Post-Fe+DHA/EPA). Dams and offspring (Pre-: n = 24; Post-: n = 26) were subsequently maintained on supplemented diets throughout the experiment. Between postnatal days 31–41, cognitive and behavioural tests were conducted on offspring. Offspring were euthanised between postnatal day 42–45 and n-3 FAs, iron and monoamine concentrations were measured in the hippocampus, striatum and frontal cortex. All outcomes were compared to offspring who were either iron and n-3 FA deficient (ID+n-3 FAD: n = 24) or sufficient (Control+Fe+DHA/EPA: n = 22). One-way ANCOVA, with sex as a covariate, was used to determine between-group differences and two-way ANOVA was used to explore diet-sex interactions.There were no differences in brain iron or n-3 FA levels between Pre- and Post-Fe+DHA/EPA offspring (P &gt; 0.05). Female Post-Fe+DHA/EPA offspring had greater norepinephrine concentrations in the frontal cortex (Pre-: 3.21 ± 0.57 ng/mg vs Post-: 2.50 ± 0.55 ng/mg; P = 0.014) and consumed less sucrose in the sucrose preference test (Pre-: 96.16 ± 1.73%; Post-: 90.15 ± 1.66%; P = 0.010) compared to Pre-Fe+DHA/EPA offspring. Female Post-Fe+DHA/EPA offspring also had significantly lower liver iron concentrations compared to female Pre-Fe+DHA/EPA offspring (Pre-: 537 ± 47.13 μg/L vs Post-: 310 ± 45.28 μg/L; P = 0.034). There were no other significant differences in monoamine concentrations or behavioural tests.Our results indicate that supplementing ID and n-3 FAD mothers both before and after conception is efficacious in preventing neurodevelopmental deficits associated with deficiency. However, ID and n-3 FAD during the periconceptional period may alter reward-based learning in female offspring(9-11). Additionally, optimising iron provision during periconception may have important implications for the prevention of postnatal ID-anaemia, particular during early infancy(12).

Material basis of kidney Yang deficiency rats before and after salt processing of Dipsacus asper based on spectrum-effect relationship

This paper aims to study the spectrum-effect relationship between the fingerprints before and after salt processing of Dipsacus asper and the efficacy of warming and tonifying kidney Yang and find the main active components against kidney Yang deficiency before and after salt processing of D. asper, so as to provide the basis for clarifying the effect of salt processing on kidney Yang deficiency. The HPLC fingerprint before and after salt processing of D. asper was established by the HPLC-DAD. 15 common peaks were obtained, and 11 components were identified. The content changes of various components in rat serum were detected, and the difference in efficacy before and after salt processing was compared. The results of pharmacological experiments showed that salt processing of D. asper could enhance the kidney index. At the same dose, there was a significant difference between the raw D. asper and D. asper after salt processing groups. Compared with the model group, the contents of ACTH, cAMP, CORT, E_2, GH, Na~+-K~+-ATPase, T, and T4 in the serum of rats in the administration group increased to a certain extent, and the contents of cGMP and TNF-α decreased to a certain extent. Among them, there were significant differences in the above indexes in the serum of rats in the high-dose group of raw D. asper, middle-dose group of D. asper after salt processing, high-dose group of D. asper after salt processing, and the positive drug group. The overall results showed that D. asper after salt processing was more effective than raw D. asper in preventing kidney yang deficiency. The efficacy of D. asper was evaluated by grey correlation analysis, entropy method, and Pearson correlation analysis, and the components of D. asper after salt processing against kidney yang deficiency were screened out. According to the results of correlation degree ranking, the components with increased ranking before and after salt processing of D. asper were loganin, chlorogenic acid, dipsacoside A, asperosaponin Ⅵ, caffeic acid, and isochlorogenic acid B. It was preliminarily speculated that these compounds may be the potential pharmacodynamic components for the treatment of kidney yang deficiency before and after salt processing of D. asper. The changing components before and after the salt processing of D. asper were determined, which proved that D. asper after salt processing was superior to D. asper in the treatment of kidney yang deficiency. The spectrum-effect relationship between the efficacy of D. asper before and after salt processing and the treatment of kidney yang deficiency was established, which laid a foundation for the subsequent study on the pharmacodynamic components and molecular mechanism of salt processing of D. asper.

Recovery of optimal vitamin D improves responsiveness to phosphodiesterase-5 inhibitors therapy in pulmonary arterial hypertension

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): R.A. has received funding from the European Union’s Horizon 2020 research and innovation programme under Marie-Curie grant agreement Nº 847635. Introduction Vitamin D (vitD) deficiency is highly prevalent in patients with pulmonary arterial hypertension (PAH). Moreover, PAH-patients with lower levels of vitD have worse prognosis. We demonstrated that in animals with PAH and severe deficit of vitD, restoring vitD levels to an optimal range partially improves some pathophysiological features of PAH. Also, recent evidence suggests that vitD deficiency may cause insufficient response to phosphodiesterase-5 inhibitors (PDE5i), such as sildenafil, in some patients with PAH. Thus, in this study, we hypothesize that the recovery of optimal vitD levels in experimental PAH might help to improve responsiveness to PDE5i therapy. Methods Male Wistar rats were fed a vitD-free diet for five weeks and then received a single dose of Su5416 (20 mg/Kg) and were exposed to vitD-free diet and chronic hypoxia (10% O2) for three weeks to induce PAH. Following this, vitD deficient rats with PAH were housed in room air and divided into two groups: (a) daily tadalafil therapy (oral;10mg/kg) + continued vitD-free diet (n=9) or (b) daily tadalafil therapy (oral;10mg/kg) + single oral dose of 50,000 IU/Kg of vitD plus standard diet (n=9) for four weeks. Animals were then used for exercise capacity evaluation, invasive haemodynamic, pulmonary vascular analysis, and sample collection. Evolution of cardiac (dys)function was assessed by echocardiography. Results Recovering optimal vitD levels improved pulmonary endothelial function, measured by an increase in the endothelium-dependent vasodilator response to acetylcholine. It also increased the vasodilator response to sildenafil. Moreover, pulmonary small artery (&amp;lt;55 µm) remodeling was decreased in vitD-restored group, as measured by a decline in mean lumen/total ratio and mean medial thickness. In a morphometric analysis, vitD treatment attenuated increases in both right ventricle (RV) and right atrial hypertrophy, as well as the fulton index. VitD supplementation also improved the exercise capacity with increases seen in distance run, evaluated by endurance tests in a treadmill set. RV catheterization showed that vitD did not decreased the RV dysfunction, evaluated by end-systolic and end-diastolic pressure. Similar to the RV, no alterations were observed in pulmonary artery pressures. However, echocardiographic analysis revealed an improved pulmonary flow in the vitD-restored group, where pulmonary artery acceleration time normalized to ejection time ratio and pulmonary artery velocity-time integral were increased. It also improved tricuspid annular plane systolic excursion (TAPSE). Conclusion Altogether, these data suggest that in animals with PAH treated with tadalafil, restoring vitD levels to an optimal range improves some pathophysiological features of PAH. Therefore, in addition to recovery of optimal vitD status being indicated to restore calcium homeostasis in those PAH patients with severe deficiency, it might help to improve responsiveness to PDE5i.

Estrogen deficiency in rats alters the expression and localization of redox proteins in the bone and bone marrow

Imbalances in bone formation/bone resorption ratio can occur under diverse conditions and diseases leading to an alteration of bone mineral density and strength. One of the most dramatic changes altering bone’s microarchitecture occurs under estrogen deficient conditions in humans and in other mammals. For instance, estrogen deficiency during menopause in women induces the formation and accumulation of reactive oxygen species (ROS) and a dysregulation of redox signaling. The bone and bone marrow are especially susceptible to the effects of oxidative stress and ROS. In tissues like the bone, cells are equipped with important defense systems to maintain homeostasis and to survive. Redox proteins of the thioredoxin family represent the most important protective system in cells that act against ROS and repair their intracellular effects. Redox proteins of the thioredoxin family comprise thioredoxins (Trxs), glutaredoxins (Grxs), and peroxiredoxins (Prxs). From all these protein classes, Prxs are the only ones that work as classical ROS scavengers being able to reduce hydrogen peroxide (H2O2), peroxynitrites and organic peroxides. Prxs are localized in different cellular compartments such as mitochondria (Prx3), cytosol and nucleus (Prx1 and Prx2), endoplasmic reticulum (Prx4), and peroxisomes (Prx5). We hypothesized that changes in estrogen levels may alter the expression and localization of Prxs in the bone and bone marrow. In this study, we analyzed the cellular localization and expression patterns of Prxs in the bone and bone marrow of rats as well as the effects of estrogens on their expression levels. We compared sham, ovariectomized, as well as ovariectomized/estrogen-replaced female rats of the same age. Western blot and immunohistochemistry revealed that all peroxiredoxins were abundantly expressed in different cell types of the bone and bone marrow such as osteocytes, chondrocytes, osteoclasts, bone lining cells, osteoblasts, and hematopoietic precursor cells. After ovariectomy, redox protein levels were significantly reduced in most cell types of the bone and bone marrow compartment. In addition to this, estrogen deficient animals showed increased levels of Prx1 in bone marrow supernatant suggesting a major redox dysregulation in this compartment. Estrogen replacement restored the levels of most redox proteins in the bone and bone marrow compartment and reduced the amount of extracellular Prx1. Based on these results, we conclude that estrogen deficiency negatively affects the ability of bone and bone marrow cells to maintain redox proteins at physiological levels. These results also reveal the widespread effects that are mediated by estrogen affecting different protein systems and tissues in the body. This work was funded by a Seed Discovery Grant (grant #3670920201102 to JG) from Long Island University. 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.

Profound Sex Differences in the Kidney Expression of Mitochondria-Related Genes in Endothelin-1 Receptor B (ETB) Deficient Rats Fed a High Salt Diet

Elevated levels of endothelin-1 (ET-1), overactivation of the ETA receptor and oxidative stress are critical in the development of hypertensive kidney disease. Females are more resistant than males to kidney damage; however, it is unclear if overactivation of the ETA receptor disrupts mitochondrial homeostasis similarly in both sexes. With these studies we aimed to determine if overactivation of the ET-1/ETA axis has a differential effect on mitochondrial homeostasis in the male and female kidney during high salt consumption. Male and female ETB deficient (ETB def) and transgenic (TG) control rats were placed on a high salt diet (4.0% NaCl) for 3 weeks. Kidneys were collected and the expression of 84 mitochondria-related genes in renal cortex and outer medulla was assessed with qRT-PCR arrays. In response to high salt, male ETB def rats displayed cortical overexpression of 13 genes when compared to male TG controls, while only 7 genes were upregulated in cortex of female ETB def rats compared to female TG controls. Genes upregulated in male ETB def rats are involved in apoptosis, mitochondrial biogenesis and transport, energy metabolism and ROS handling (selected genes, male ETB def vs. TG controls, n=3/group: Ucp1 (19-fold increase, p=0.03), Bnip3 and Scl25a21 (6-fold, p=0.003 and p=0.004, respectively), and Mfn1 (5-fold, p=0.03). Genes upregulated in females are involved in apoptosis, ROS handling and mitochondrial transport (selected examples, female ETB def vs. TG control, n=3/group: 2-fold increase in Sod1 (p=0.03), Akt1 (p=0.003) and Grpel1 (p=0.019). No common genes were changed between the sexes. When comparisons were made in the outer medulla, we found that deficiency of the ETB receptor in males resulted in a ≥30% reduction in the expression of 29 genes involved in apoptosis, mitochondrial biogenesis and transport, energy metabolism, ROS handling and other mitochondrial functions (p&lt;0.05). On the other hand, the expression of 8 genes involved in mitochondrial transport and apoptosis was reduced by ≥80% in the female ETB def rat outer medulla vs. TG controls, while 3 genes involved in mitochondrial transport and energy metabolism had a 2-fold overexpression (p&lt;0.05). Altogether, our results demonstrate that salt-sensitive hypertension affects mitochondrial homeostasis differentially in distinct regions of the kidney, and that the male and female kidney respond to salt in a disparate manner: with overactivation of the ET-1/ETA axis resulting in greater dysregulation of mitochondria-related genes in the male versus the female kidney during a high salt diet. Funded by UAB-UCSD O’Brien Center Summer Research Pathway for Students P30 DK079337 and KURE R25 DK115353 to BSM, NIGMS UAB CORD BBRT to GJC, and NIH K01HL145324 and Sapphire North America Return to Science Research grant to CDM. 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.

Linking Altered Nephrogenesis with Hypertension using Single Nuclei RNA Sequencing of Fetal Kidney at E15.5, E16.5 and E17.5 in a Novel Nephron Deficient Rat Model

This study sought to elucidate molecular mechanisms linking altered nephrogenesis in the HSRA rat, a novel model of nephron deficiency, to the development of hypertension and chronic kidney disease (CKD) later in life. The HSRA model gives rise to offspring born with a single kidney (-S) 50-75% of the time. The single kidneys from HSRA-S animals exhibit reduced nephron endowment compared to either kidney from two-kidney offspring (-C). We hypothesize that early molecular factors during development contribute to the observed reduction in nephron number in -S animals. Timed breeding and embryonic kidney isolation from -S and -C animals were performed at gestational days (GD) 15.5, 16.5, or 17.5. Single nuclei RNA sequencing (snRNA-seq) was conducted using n=3 -S kidneys and n=3 -C kidneys for each timepoint. The library was prepared using a 10x Chromium Single Cell 3’ kit, followed by sequencing on an Illumina NextSeq2000. The average number of cells captured per sample was 16,997, yielding an average of 25,335 reads/cell, with a median of 1,512 genes/cell identified. Bioinformatic analysis using Cell Ranger and Seurat (v4) revealed various renal progenitor cell types including metanephric mesenchymal (MM) and ureteric bud cells, alongside tubular, stromal, and glomerular endothelial progenitors. Podocytes emerged by GD17.5, originating from the MM cell population, as evidenced by their extension from this cluster. Global differential expression analysis revealed minimal transcriptional differences between -S and -C fetal kidneys at GD 16.5 and 17.5. However, a down-regulation of ribosomal genes was observed on GD15.5 in the -S group, suggesting premature cellular differentiation contributing to the diminished nephron formation. Markedly, cell type-specific gene expression differences were observed at GD 16.5 and 17.5, highlighting the superior resolution that snRNAseq provides compared to bulk RNA sequencing methods. We are currently performing trajectory analysis using Monocle3 to investigate cellular fate pathways and uncover genes involved during nephron progenitor cell differentiation. These preliminary findings shed light on the cellular and molecular landscape during critical developmental stages in the kidney of the HSRA rat that could link developmental factors affecting nephron number to hypertension and CKD pathologies later in life. This research was generously supported by the Graduate School of Health Sciences at UMMC. MRG is supported by NIH R01HL137673. The UMMC Molecular and Genomics Facility is supported, in part, by funds from the NIGMS, including Molecular Center of Health and Disease-COBRE (P20GM144041-Garrett), Mississippi INBRE (P20GM103476-Flynt), and Cardiorenal and Metabolic Diseases-COBRE (P20GM104357-Hall). 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.

Post-weaning social isolation modifies neonatal anoxia-induced changes in energy metabolism and growth of rats.

Neonatal oxygen deficiency in rats may disturb growth and long-term metabolic homeostasis. In order to facilitate metabolic evaluation, the subjects are usually housed individually. However, social isolation associated with individually housed conditions alters animal behavior, which may influence the experimental results. This study investigated the effects of social isolation on neonatal anoxia-induced changes in growth and energy metabolism. Male and female Wistar rats were exposed, on postnatal day 2 (P2), to either 25-min of anoxia or control treatment. From P27 onward, part of the subjects of each group was isolated in standard cages, and the remaining subjects were housed in groups. At P34 or P95, the subjects were fasted for 18 h, refeed for 1h, and then perfused 30 min later. Glycemia, leptin, insulin, and morphology of the pancreas were evaluated at both ages. For subjects perfused at P95, body weight and food intake were recorded up to P90, and the brain was collected for Fos and NeuN immunohistochemistry. Results showed that male rats exposed to neonatal anoxia and social isolation exhibited increased body weight gain despite the lack of changes in food intake. In addition, social isolation (1) decreased post-fasting weight loss and post-fasting food intake and (2) increased glycemia, insulin, and leptin levels of male and female rats exposed to anoxia and control treatments, both at P35 and P95. Furthermore, although at P35, anoxia increased insulin levels of males, it decreased the area of the β-positive cells in the pancreas of females. At P95, anoxia increased post-prandial weight loss of males, post-fasting food intake, insulin, and leptin, and decreased Fos expression in the arcuate nucleus (ARC) of males and females. Hyperphagia was associated with possible resistance to leptin and insulin, suspected by the high circulating levels of these hormones and poor neuronal activation of ARC. This study demonstrated that continuous social isolation from weaning modifies, in a differentiated way, the long-term energy metabolism and growth of male and female Wistar rats exposed to neonatal anoxia or even control treatments. Therefore, social isolation should be considered as a factor that negatively influences experimental results and the outcomes of the neonatal injury. These results should also be taken into account in clinical procedures, since the used model simulates the preterm babies' conditions and some therapeutic approaches require isolation.

Annexin A1 conveys neuroprotective function via inhibiting oxidative stress in diffuse axonal injury of rats.

Diffuse axonal injury (DAI) is a critical pathological facet of traumatic brain injury (TBI). Oxidative stress plays a significant role in the progress of DAI. Annexin A1 (AnxA1) has been demonstrated to benefit from recovery of neurofunctional outcomes after TBI. However, whether AnxA1 exhibits neuronal protective function by modulating oxidative stress in DAI remains unknown. Expression of AnxA1 was evaluated via real-time PCR and western blotting in rat brainstem after DAI. The neurological effect of AnxA1 following DAI through quantification of modified neurologic severity score (mNSS) was compared between wild-type and AnxA1-knockout rats. Brain edema and neuronal apoptosis, as well as expression of oxidative factors and inflammatory cytokines, were analyzed between wild-type and AnxA1 deficiency rats after DAI. Furthermore, mNSS, oxidative and inflammatory cytokines were assayed after timely administration of recombinant AnxA1 for DAI rats. In the brainstem of DAI, the expression of AnxA1 remarkably increased. Ablation of AnxA1 increased the mNSS score and brain water content of rats after DAI. Neuron apoptosis in the brainstem after DAI was exaggerated by AnxA1 deficiency. In addition, AnxA1 deficiency significantly upregulated the level of oxidative and inflammatory factors in the brainstem of DAI rats. Moreover, mNSS decreased by AnxA1 treatment in rats following DAI. Expression of oxidative and inflammatory molecules in rat brainstem subjected to DAI inhibited by AnxA1 administration. AnxA1 exhibited neuronal protective function in the progression of DAI mainly dependent on suppressing oxidative stress and inflammation.

Study on the intervention effect of Epimedium before and after suet-oil-processed on kidney yang deficiency rats based on intestinal flora and fecal metabolomics

Epimedium is a Chinese herbal medicine commonly used in clinical practice to reinforce yang. Previous studies have shown that Epimedium fried with suet oil based has the best effect on warming kidney and promoting yang. Evidence suggests a relationship between kidney yang deficiency syndrome (KYDS) and metabolic disorders of the intestinal microflora. However, the specific interaction between KYDS and the intestinal microbiome, as well as the internal regulatory mechanism of the KYDS intestinal microbiome regulated by Epimedium fried with suet oil, remain unclear. The purpose of this study was to investigate the regulatory effects of different processed products of Epimedium on intestinal microflora and metabolites in rats with kidney yang deficiency, and to reveal the processing mechanism of Epimedium fried with suet oil warming kidney and helping yang. 16S rRNA and LC-MS/MS technology were used to detect fecal samples. Combined with multivariate statistical analysis, differential intestinal flora and metabolites were screened. Then the content of differential bacteria was then quantified using quantitative real-time fluorescence PCR. Furthermore, the correlation between differential bacterial flora and metabolites was analyzed using Spearman's method. The study found that the composition of intestinal flora in rats with kidney yang deficiency changed compared to healthy rats. Epimedium fried with suet oil could increase the levels of beneficial bacteria, while significantly reducing the levels of harmful bacteria. Real-time quantitative PCR results were consistent with 16S rRNA gene sequencing analysis. Fecal metabolomics revealed that KYDS was associated with 30 different metabolites, involving metabolic pathways steroid hormone biosynthesis etc. Moreover, differential bacteria were closely correlated with potential biomarkers. Epimedium could improve metabolic disorders associated with KYDS by acting on the intestinal flora, with Epimedium fried with suet oil demonstrating the most effective regulatory effect. Its potential mechanism may involve the regulation of abnormal metabolism and the impact on the diversity and structure of the intestinal flora.

Insights into Q-markers of honey-fried licorice in treating spleen deficiency based on substance and energy metabolism regulation

BackgroundHoney-fried Licorice (HFL) is a dosage form of Glycyrrhizae Radix et Rhizome processed with honey, which has been recorded to exhibit better efficacy in tonifying the spleen compared to the raw product. In contrast, different processing methods of Glycyrrhizae Radix et Rhizome exhibit different efficacies and applications, but their current quality control index components remain consistent. PurposeBased on the discovery and research strategy of traditional Chinese medicine decoction piece quality marker (Q-marker), this study aimed to conduct a multidimensional integration of constituents absorbed into the body and metabolomics based on the tonifying spleen and stomach effects of HFL to effectively identify the Q-marker of HFL. MethodsIn this study, a spleen deficiency rat model was established using the "exhausted swimming + poor diet" method to investigate the pharmacodynamics of tonifying the spleen and stomach by HFL. The constituents absorbed into blood was conducted using UPLC-Q-TOF/MS, correlation analysis between metabolomics and constituents absorbed into blood recognized the Q-Marker of HFL. ResultsThe pharmacodynamic data demonstrated that HFL exhibited a significant regulatory effect on the disordered levels of PP, trypsin, chymase, PL, α-Glu, MTL, GAS, VIP, IL-2, IFN-γ, and IgA in the spleen deficiency model. Furthermore, HFL was found to improve the pathological changes in the spleen and intestine in the spleen deficiency model, highlighting its significant "tonifying spleen and stomach" effect. In the serum containing HFL, a total of 17 constituents were identified as being absorbed into the blood. Among these, 11 were prototypical components, while 6 were metabolites. Metabolomics data revealed that 9 differentially expressed metabolic markers were observed. Furthermore, the analysis of endogenous metabolic markers indicated that 10 components exhibited significant correlations with these biomarkers. ConclusionThe effect of "tonifying spleen and stomach" of HFL is closely related to the regulation of the material and energy metabolism pathway. The Q-Marker of HFL is glycyrrhizic acid and 18β-glycyrrhetinic acid as the main control standards and liquiritin, isoliquiritin, liquiritin, isoliquiritin, isolicorice flavonol, licorice chalcone C and Formononetin were used as auxiliary standards.

Nitric oxide-induced lipophagic defects contribute to testosterone deficiency in rats with spinal cord injury.

Males with acute spinal cord injury (SCI) frequently exhibit testosterone deficiency and reproductive dysfunction. While such incidence rates are high in chronic patients, the underlying mechanisms remain elusive. Herein, we generated a rat SCI model, which recapitulated complications in human males, including low testosterone levels and spermatogenic disorders. Proteomics analyses showed that the differentially expressed proteins were mostly enriched in lipid metabolism and steroid metabolism and biosynthesis. In SCI rats, we observed that testicular nitric oxide (NO) levels were elevated and lipid droplet-autophagosome co-localization in testicular interstitial cells was decreased. We hypothesized that NO impaired lipophagy in Leydig cells (LCs) to disrupt testosterone biosynthesis and spermatogenesis. As postulated, exogenous NO donor (S-nitroso-N-acetylpenicillamine (SNAP)) treatment markedly raised NO levels and disturbed lipophagy via the AMPK/mTOR/ULK1 pathway, and ultimately impaired testosterone production in mouse LCs. However, such alterations were not fully observed when cells were treated with an endogenous NO donor (L-arginine), suggesting that mouse LCs were devoid of an endogenous NO-production system. Alternatively, activated (M1) macrophages were predominant NO sources, as inducible NO synthase inhibition attenuated lipophagic defects and testosterone insufficiency in LCs in a macrophage-LC co-culture system. In scavenging NO (2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO)) we effectively restored lipophagy and testosterone levels both in vitro and in vivo, and importantly, spermatogenesis in vivo. Autophagy activation by LYN-1604 also promoted lipid degradation and testosterone synthesis. In summary, we showed that NO-disrupted-lipophagy caused testosterone deficiency following SCI, and NO clearance or autophagy activation could be effective in preventing reproductive dysfunction in males with SCI.