Control Male Mice Research Articles

Expression of human sPRR in renal collecting duct cells increases blood pressure in female mice independently of its circulating levels

Elevated circulating sPRR has been associated with essential hypertension, obesity-related hypertension, heart failure and chronic kidney disease in humans. However, the exact contribution of tissue-specific human sPRR in blood pressure control remains to be investigated. Therefore, we evaluated the effect of human sPRR on circulating sPRR and blood pressure control when expressed in mouse kidney, adipose tissue or liver from both male and female mice.Human sPRR-Myc-tag transgenic mice were bred with mice expressing either Hoxb7/Cre, Adiponectin/Cre or Albumin/Cre to selectively express human sPRR (HsPRR) in the collecting duct of the kidney (Kid-HsPRR), adipose tissue (Adi-HsPRR) or liver (Liv-HsPRR), respectively. The presence of human sPRR-Myc-tag (28 KDa) was validated in each mouse model by detecting the presence of C-myc. HsPRR and control (WT) male and female mice were fed a control diet throughout the study (n=8-12/group). At 6 months, baseline and acute blood pressure responses were assessed by radio-telemetry.Tissue expression of sPRR increased in kidney-derived HsPRR (M: 1.0±0.2 vs. 1.6±0.3 AU, F: 1.0±0.1 vs. 1.7±0.2 AU p<0.05), adipose-derived HsPRR (M: 1.0±0.3 vs. 4.7±1.5 AU, F: 1.0±0.2 vs. 3.2±1.4 AU p<0.05) and liver-derived HsPRR mice (M: 1.0±0.2 vs. 9.7±0.8 AU, F: 1.0±0.1 vs. 8.7±0.9 AU p<0.05) but only liver-derived HsPRR mice elevated circulating sPRR (M: 7.0±2.4 vs. 125.0±7.9 ng/ml, F: 2.2±0.4 vs. 93.9±13.4 ng/ml p<0.05). Human sPRR increased blood pressure only in female mice expressing HsPRR in kidney (F: 111±2 vs. 121±4 mmHg p<0.05). Also, only female mice expressing HsPRR in kidney displayed a blunted acute response to losartan injection (M: -30.1±3.3 vs. -22.1±3.7, F: -16.3±2.4 vs. 14.1±5.8 Δ mmHg p<0.05) and Ang II-induced pressor response in acute (M: 28.9±5.1 vs. 47.6±4.4 mmHg p<0.05, F: 35.2±7.2 vs. 26.9±7.5 mmHg) and chronic fashion (M: 33.9±3.6 vs. 30.7±5.0, F: 35.1±6.8 vs.17.6±3.2 Δ mmHg, p<0.05).Overall, our data show that increased intrarenal human sPRR expression contributes to increase blood pressure in a sex-specific manner. Taken together, increased HsPRR in kidney is associated with hypertension and impaired response to AT1R blockade and AngII-induced pressor response suggesting a dysfunctional AT1R-mediated signaling that could be due to ligand competition. These studies were supported by grants from the American Heart Association (13SDG17230008), the National Institute of General Medical Sciences (P30 GM127211), the Center for Clinical and Translational Sciences (UL1TR000117) and the National Heart Lung and Blood Institute (R01HL142969-01) 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.

Sex differences in the development of coronary microvascular diseases in type-2 diabetes: possible role of gap junction protein connexin-40

Type-2 diabetes (T2D) increases the risk of cardiovascular diseases 2-4 folds in both sexes compared to non-diabetic individuals. However, more than 50% of female patients who experience chest pain have coronary microvascular disease (CMD) instead of obstructive CAD. On the other hand, 20% of men with chest pain have CMD. CMD is characterized by reduced coronary flow reserve, and coronary flow is regulated by the change in capillary density and vessel diameter. Gap junction protein connexin-40 (Cx40) plays a significant role in coronary microvascular function by controlling endothelium-dependent relaxation (EDR), cardiac endothelial cell (CEC) migration and capillary density. We hypothesized that T2D differentially affects the CX-40 expression and function in male and female coronary artery (CA) and causes CMD. The objective of this study is to identify the molecular mechanisms underlining the development of T2D-induced CMD in male and female mice.T2D mice were generated by a combination of high-fat diet feeding and a low-dose of streptozotocin (75 mg/kg, i.p.). The oral glucose tolerance was conducted at 4-, 8-, 12-, and 16 weeks after diabetes induction. Coronary flow velocity reserve (CFVR) was used to assess coronary microvascular function in mice. Coronary endothelial function was determined by 1) capillary density measurement in the left ventricle and 2) isometric tension experiment in small CAs (3rd order). CECs from male and female mice were isolated to conduct molecular biological experiments.There was no significant difference in CFVR and capillary density between control male and female mice. However, T2D mice of both sexes showed significant differences in the properties of EDR, CFVR and capillary density. T2D male mice exhibited a significant attenuation of EDR accompanied by impaired endothelium-derived nitric oxide(EDNO)-dependent relaxation and endothelium-dependent hyperpolarization (EDH)-mediated relaxation. On the contrary, there is no difference in EDR between control and diabetic female mice. Capillary density was reduced in T2D mice of both sexes compared to the respective controls. Our RNA sequencing data in CECs revealed that Cx40 is one of the genes which contributes to diabetes-mediated CMD. Systemic Cx40-KO male mice showed reduced EDR, EDH-mediated relaxation and capillary density compared to wild type control. T2D reduced the expression of Cx40 and overexpression of Cx40 restored EDR in CAs and increased capillary density in T2D male mice.T2D impaired coronary microvascular function in both sexes. However, the cause of reduced CFVR was different between diabetic male and female mice. In T2D females, capillary rarefaction was a primary cause of reduced CFVR. In contrast, attenuated EDNO- and EDH-mediated relaxation, reduced capillary density and downregulation of Cx40 contributed to decreased CFVR in T2D male mice. Future studies are required to establish the role of Cx40 in the development of CMD in T2D females. Supported by NIH R01HL142214 and DOD W81XWH2110472 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.

Small Extracellular Vesicles From Spared Nerve Injury Model and Sham Control Mice Differentially Regulate Gene Expression in Primary Microglia

Nerve injury outcomes might be predicted by examining small extracellular vesicles (sEVs) in circulation, as their biomolecular cargo facilitates cellular communication and can alter transcriptional state and behavior of recipient cells. We found that sEVs from the serum of spared nerve injury (SNI) model male mice had 7 differentially expressed miRNAs compared to sEVs from sham-operated control mice 4 weeks postsurgery. We investigated how these sEVs alter transcription in primary cortical microglia, a crucial mediator of neuropathic pain, using RNA sequencing. While the uptake of sEVs from both SNI model and sham groups changed gene expression in microglia compared to PBS treatment, sEVs from the sham group induced a more drastic change, particularly in genes involved in immune response. This was recapitulated by increased levels of pro-inflammatory cytokines and chemokines in microglia incubated with sEVs from sham control compared to sEVs from SNI model, naïve mice, or PBS. However, treating microglia with sEVs from female mice showed that serum sEVs derived from female SNI mice but not from female sham mice induced a more pronounced microglial secretion of pro-inflammatory mediators. Our data demonstrate that the molecular changes induced by sham surgery injuring skin and muscles are reflected in circulating sEVs in male mice 4 weeks later. Thus, when using sEVs from sham mice as control in comparative mechanistic studies after nerve injury, sex of mice should be taken into consideration. PerspectiveMicroglial uptake of sEVs from male sham control mice induces higher pro-inflammatory responses compared to SNI sEVs but the reverse was observed upon treatment with sEVs from female mice. Wound healing may have a long-term impact on sEVs in male mice and should be considered for comparative studies using sEVs.

Skeletal muscle single fiber force production declines early in juvenile male mice with chronic kidney disease.

Children with chronic kidney disease (CKD) frequently exhibit delayed physical development and reduced physical performance, presumably due to skeletal muscle dysfunction. However, the cellular and molecular basis of skeletal muscle impairment in juvenile CKD remains poorly understood. Cellular (single fiber) and molecular (myosin-actin interactions and myofilament properties) function was examined ex vivo in slow (soleus) and fast (extensor digitorum longus) contracting muscles of juvenile male (6 weeks old) CKD and control mice. CKD was induced by 0.2% adenine diet for 3 weeks starting at 3 weeks of age. Specific tension (maximal isometric force divided by cross-sectional area) was reduced in larger myosin heavy chain (MHC) I and IIA fibers and in all IIB fibers in juvenile male mice with CKD due to fewer strongly bound myosin-actin cross-bridges. Fiber cross-sectional area in juvenile CKD mice was unchanged in MHC I and IIB fibers and increased in MHC IIA fibers, compared to controls. CKD slowed cross-bridge kinetics (slower rate of myosin force production and longer myosin attachment time, ton ) in MHC IIA fibers, and accelerated kinetics (shorter ton ) in MHC IIB fibers, which may indicate fiber type dependent shifts in contractile velocity in juvenile CKD. Overall, our findings show that single fiber myopathy is an early event during juvenile CKD, manifesting prior to the development of cellular atrophy as reduced force generation due to fewer strongly bound myosin heads. These results warrant clinical translation and call for early interventions to preserve physical function in children with CKD.

Evidence of sex differences in cancer-related cardiac complications in mouse models of pancreatic and liver cancer.

Abnormal heart rate variability (HRV) is commonly observed in cancer patients who have undergone targeted therapy and/or surgery, yet the effects of cancer itself on cardiac function remain underexplored. Specifically, there is limited knowledge about sex-specific manifestations of HRV in cancer patients. Transgenic mouse models are widely used to study different types of cancer. Here, we aimed to investigate the sex-specific effects of cancer on cardiac function using transgenic mouse models of pancreatic and liver cancers. This study used male and female transgenic mice with cancer and wild-type controls. Cardiac function was assessed by recording electrocardiograms in conscious mice. RR intervals were detected to determine HRV using time and frequency domain analyses. Histological analysis with Masson's trichrome staining was performed to determine structural changes. In females, increased HRV was observed in both pancreatic and liver cancer-bearing mice. In contrast, in males, increased HRV was observed only in the liver cancer group. Male pancreatic cancer mice demonstrated autonomic balance shift showing an increase in parasympathetic to sympathetic tone. The heart rate (HR) was higher in control and liver cancer male mice groups than in females. Histological analysis did not show significant sex differences but suggested a higher degree of remodeling in liver cancer mice than in control, specifically in the right atrium and left ventricle. This study revealed sex differences in cancer's HR modulation. Specifically, female cancer mice had lower median HR and higher HRV. These findings indicate that sex must be considered when using HRV as a cancer biomarker.

Exposure to Doxorubicin Modulates the Cardiac Response to Isoproterenol in Male and Female Mice.

Sex is a salient risk factor in the development of doxorubicin-induced cardiotoxicity. Sex differences in the heart's ability to respond to hypertrophic stimuli in doxorubicin-exposed animals have not been reported. We identified the sexual dimorphic effects of isoproterenol in mice pre-exposed to doxorubicin. Male and female intact or gonadectomized C57BL/6N mice underwent five weekly intraperitoneal injections of 4 mg/kg doxorubicin followed by a five-week recovery period. Fourteen days of subcutaneous isoproterenol injections (10 mg/kg/day) were administered after the recovery period. Echocardiography was used to assess heart function one and five weeks after the last doxorubicin injection and on the fourteenth day of isoproterenol treatment. Thereafter, mice were euthanized, and the hearts were weighed and processed for histopathology and gene expression analysis. Doxorubicin did not produce overt cardiac dysfunction in male or female mice before starting isoproterenol treatment. The chronotropic response to a single isoproterenol injection was blunted by doxorubicin, but the inotropic response was maintained in both males and females. Pre-exposure to doxorubicin caused cardiac atrophy in both control and isoproterenol-treated male mice but not in female mice. Counterintuitively, pre-exposure to doxorubicin abrogated isoproterenol-induced cardiac fibrosis. However, there were no sex differences in the expression of markers of pathological hypertrophy, fibrosis, or inflammation. Gonadectomy did not reverse the sexually dimorphic effects of doxorubicin. Additionally, pre-exposure to doxorubicin abrogated the hypertrophic response to isoproterenol in castrated male mice but not in ovariectomized female mice. Therefore, pre-exposure to doxorubicin caused male-specific cardiac atrophy that persisted after isoproterenol treatment, which could not be prevented by gonadectomy.

Low energy availability reduces bone mass and gonadal function in male mice

In women, the female athlete triad, marked by low energy availability, functional hypothalamic amenorrhea and osteoporosis, is a recognized risk for stress fractures. Stress injuries also occur in men, but by contrast risks and mechanisms underlying them are less characterized. 5week-old wild-type male mice were fed ad libitum (ad) or subjected to 60% food restriction (FR) for five weeks. In both groups, some mice were allowed access to an exercise wheel in cages to allow voluntary wheel running (ex) and/or treated with active vitamin D analogues. Mice were sacrificed and analyzed at 10weeks of age. Male FR mice exhibited significantly reduced testicle weight, serum testosterone levels and bone mass. Such bone losses in FR male mice were enhanced by exercise. Histological analysis revealed that both bone-resorbing and -forming activities were significantly reduced in FR or FR plus exercise (FR + ex) mice, mimicking a state of low bone turnover. Significantly reduced bone mass in FR or FR + ex male mice was significantly rescued by treatment with active vitamin D analogues, with significant restoration of osteoblastic activities. Serum levels of insulin-like growth factor I (IGF-I), which is critical for bone remodeling, were significantly lower in FR versus control male mice. Low energy availability puts men at risk for stress injuries as well, and low energy availability is upstream of gonadal dysfunction and osteoporosis in males. Active vitamin D analogues could serve as therapeutic or preventive options for stress injuries in men.

Stress-induced plasticity of a CRH/GABA projection disrupts reward behaviors in mice

Disrupted operations of the reward circuit underlie major emotional disorders, including depression, which commonly arise following early life stress / adversity (ELA). However, how ELA enduringly impacts reward circuit functions remains unclear. We characterize a stress-sensitive projection connecting basolateral amygdala (BLA) and nucleus accumbens (NAc) that co-expresses GABA and the stress-reactive neuropeptide corticotropin-releasing hormone (CRH). We identify a crucial role for this projection in executing disrupted reward behaviors provoked by ELA: chemogenetic and optogenetic stimulation of the projection in control male mice suppresses several reward behaviors, recapitulating deficits resulting from ELA and demonstrating the pathway’s contributions to normal reward behaviors. In adult ELA mice, inhibiting–but not stimulating–the projection, restores typical reward behaviors yet has little effect in controls, indicating ELA-induced maladaptive plasticity of this reward-circuit component. Thus, we discover a stress-sensitive, reward inhibiting BLA → NAc projection with unique molecular features, which may provide intervention targets for disabling mental illnesses.

Machado Joseph disease severity is linked with gut microbiota alterations in transgenic mice

Emerging evidence suggests the presence of bidirectional interactions between the central nervous system and gut microbiota that may contribute to the pathogenesis of neurodegenerative diseases. However, the potential role of gut microbes in forms of spinocerebellar ataxia, such as the fatal neurodegenerative disease Machado Joseph disease (MJD), remains unexplored. Here, we examined whether gut microbiota alterations may be an early disease phenotype of MJD. We profiled the gut microbiota of male and female transgenic MJD mice (CMVMJD135) expressing human ATXN3 with expanded CAG repeats (133–143 CAG) at pre-symptomatic, symptomatic and well-established stages of the disease (7, 11 and 15 weeks of age, respectively). We compared these profiles with the gut microbiota of male and female wild-type (WT) littermate control mice at same ages. Correlation network analyses were employed to explore the relevance of microbiota changes to disease progression. The results demontrated distinct sex-dependent effects in disease development whereby male MJD mice displayed earlier motor impairments than female MJD mice. The gut microbiota community structure and composition also demonstrated sex-specific differences between MJD and WT mice. In both male and female MJD mice, the shifts in the microbiota were present by 7 weeks, before the onset of any symptoms. These pre-symptomatic microbial changes correlated with the severity of neurological impairments present at later stages of the disease. Previous efforts towards developing treatments for MJD have failed to yield meaningful outcomes. Our study reports a novel relationship between the gut microbiota and MJD development and severity. Elucidating how gut microbes are involved in MJD pathogenesis may offer new and efficacious treatment strategies for this currently untreatable disease.

Sex Difference in Capillary Reperfusion After Transient Middle Cerebral Artery Occlusion in Diabetic Mice.

Type 2 diabetes (DM2) exacerbates stroke injury, reduces efficacy of endovascular therapy, and worsens long-term functional outcome. Sex differences exist in stroke incidence, response to therapy, poststroke microvascular dysfunction, and functional recovery. In this study, we tested the hypotheses that poor outcome after stroke in the setting of DM2 is linked to impaired microvascular tissue reperfusion and that male and female DM2 mice exhibit different microvascular reperfusion response after transient middle cerebral artery occlusion (MCAO). Transient MCAO was induced for 60 minutes using an intraluminal filament in young adult DM2 and nondiabetic control male and female mice. Capillary flux in deep cortical layers was assessed using optical coherence tomography-based optical microangiography (OMAG), and associated regional brain infarct size was evaluated by hematoxylin and eosin staining. Compared to baseline, MCAO reduced absolute capillary red blood cell flux by 84% at 24 hours post-MCAO in male DM2 (P<0.001) but not male control mice. When normalized to pre-MCAO baseline, red blood cell flux 24 hours after stroke was 64% lower in male DM2 mice than male nondiabetic controls (P<0.01). In females, MCAO decreased capillary flux by 48% at 24 hours post-MCAO compared with baseline in DM2 (P<0.05) but not in control mice. Red blood cell flux of female DM2 mice did not differ from that of nondiabetic controls either before or 24 hours after MCAO. Furthermore, normalized capillary flux 24 hours after MCAO failed to differ between female DM2 mice and nondiabetic controls. Concomitantly, male but not female DM2 mice experienced 25% larger infarct in caudate-putamen versus respective nondiabetic controls (P<0.05). DM2 impairs capillary perfusion and exacerbates ischemic deep brain injury in male but not female young adult mice. Premenopausal females appear to be protected against DM2-related capillary dysfunction and brain injury.

Abstract WP240: TRPM2 Channel Deletion In Neurons Reduces Injury Following Ischemic Stroke And Improves Post-stroke Cognitive Function

Background: Emerging evidence implicates post-stroke cognitive impairment as a major contributor to long-term disability. Therefore, optimal therapeutic targets reduce acute ischemic injury and enhance post-stroke brain function. Strong data demonstrates that a novel TRPM2 channel antagonist (tat-M2NX) provides neuroprotection and improves synaptic function, thereby reducing post-stroke cognitive impairment (PSCI). Hypothesis: Knockout of neuron-specific TRPM2 channel expression reduces infarct volume and enhances functional recovery following MCAO Methods: Transient MCAO (60 min) was performed on adult (8-10 week) male and female TRPM2 neuron-specific KO (TRPM2fl/fl, CaMKII Cre) and TRPM2 floxed controls (TRPM2fl/fl)Hemispheric infarct volume analyzed from MRI (T2) images 3 days post-injury by a blinded investigator. Extracellular field recordings of CA1 neurons were performed in acute hippocampal slices prepared 7 days after recovery from MCAO to analyze synaptic placticity (LTP). Results: We observed that neuronal-specific TRPM2 channel knockout reduces acute ischemic injury (infarct volume) in male animals, while having minimal effect on female mice. Consistent with the hypothesis that neuronal TRPM2 channels contribute to ischemia-induced synaptic dysfunction, recordings obtained in brain slices from neuronal TRPM2 channel KO mice (TRPM2fl/fl-CaMKII CRE) mice 7 days after recovery from 60 min MCAo exhibited intact hippocampal plasticity compared to control TRPM2fl/fl mice not expressing CRE. Male sham control mice exhibit robust LTP of 163±10.4% (n=3) compared to 115±5.6% (n=4) in TRPM2fl/fl mice after MCAO. Neuronal KO exhibited 175±9.8% (n=2). Similarly, female mice had control LTP of 170±6.8% (n=4) compared to 125±6.8% (n=3) in TRPM2fl/fl mice after MCAO. Neuronal KO exhibited 208±7.8% (n=5, p&lt;0.05 ANOVA compared to TRPM2fl/fl MCAO). Conclusion: Our data highlight that TRPM2 channels expressed in neurons contribute to both acute injury following transient ischemic stroke and subacute/chronic functional recovery.

Duality of Nrf2 in iron-overload cardiomyopathy.

Cardiomyopathy deeply affects quality of life and mortality of patients with b-thalassemia or with transfusion-dependent myelodysplastic syndromes. Recently, a link between Nrf2 activity and iron metabolism has been reported in liver ironoverload murine models. Here, we studied C57B6 mice as healthy control and nuclear erythroid factor-2 knockout (Nrf2-/-) male mice aged 4 and 12 months. Eleven-month-old wild-type and Nrf2-/- mice were fed with either standard diet or a diet containing 2.5% carbonyl-iron (iron overload [IO]) for 4 weeks. We show that Nrf2-/- mice develop an age-dependent cardiomyopathy, characterized by severe oxidation, degradation of SERCA2A and iron accumulation. This was associated with local hepcidin expression and increased serum non-transferrin-bound iron, which promotes maladaptive cardiac remodeling and interstitial fibrosis related to overactivation of the TGF-b pathway. When mice were exposed to IO diet, the absence of Nrf2 was paradoxically protective against further heart iron accumulation. Indeed, the combination of prolonged oxidation and the burst induced by IO diet resulted in activation of the unfolded protein response (UPR) system, which in turn promotes hepcidin expression independently from heart iron accumulation. In the heart of Hbbth3/+ mice, a model of b-thalassemia intermedia, despite the activation of Nrf2 pathway, we found severe protein oxidation, activation of UPR system and cardiac fibrosis independently from heart iron content. We describe the dual role of Nrf2 when aging is combined with IO and its novel interrelation with UPR system to ensure cell survival. We open a new perspective for early and intense treatment of cardiomyopathy in patients with b-thalassemia before the appearance of heart iron accumulation.

Abcc8 (sulfonylurea receptor-1) knockout mice exhibit reduced axonal injury, cytotoxic edema and cognitive dysfunction vs. wild-type in a cecal ligation and puncture model of sepsis

Sepsis-associated brain injury (SABI) is characterized by an acute deterioration of mental status resulting in cognitive impairment and acquisition of new and persistent functional limitations in sepsis survivors. Previously, we reported that septic mice had evidence of axonal injury, robust microglial activation, and cytotoxic edema in the cerebral cortex, thalamus, and hippocampus in the absence of blood–brain barrier disruption. A key conceptual advance in the field was identification of sulfonylurea receptor 1 (SUR1), a member of the adenosine triphosphate (ATP)-binding cassette protein superfamily, that associates with the transient receptor potential melastatin 4 (TRPM4) cation channel to play a crucial role in cerebral edema development. Therefore, we hypothesized that knockout (KO) of Abcc8 (Sur1 gene) is associated with a decrease in microglial activation, cerebral edema, and improved neurobehavioral outcomes in a murine cecal ligation and puncture (CLP) model of sepsis. Sepsis was induced in 4–6-week-old Abcc8 KO and wild-type (WT) littermate control male mice by CLP. We used immunohistochemistry to define neuropathology and microglial activation along with parallel studies using magnetic resonance imaging, focusing on cerebral edema on days 1 and 4 after CLP. Abcc8 KO mice exhibited a decrease in axonal injury and cytotoxic edema vs. WT on day 1. Abcc8 KO mice also had decreased microglial activation in the cerebral cortex vs. WT. These findings were associated with improved spatial memory on days 7–8 after CLP. Our study challenges a key concept in sepsis and suggests that brain injury may not occur merely as an extension of systemic inflammation. We advance the field further and demonstrate that deletion of the SUR1 gene ameliorates CNS pathobiology in sepsis including edema, axonal injury, neuroinflammation, and behavioral deficits. Benefits conferred by Abcc8 KO in the murine CLP model warrant studies of pharmacological Abcc8 inhibition as a new potential therapeutic strategy for SABI.

PS-B01-11: GENERATION OF TRANSGENIC MICE WITH PROXIMAL TUBULE-SPECIFIC OVEREXPRESSION OF ATRAP AND ANALYSIS OF PROXIMAL TUBULE ATRAP IN BLOOD PRESSURE REGULATION

Objective: Proximal tubule angiotensin II type 1 receptor (AT1R) is involved in blood pressure (BP) regulation (Li XC, et al. Hypertension, 77:1285-1298, 2021). AT1 receptor-associated protein (ATRAP) interacts with AT1R and promotes constitutive internalization of AT1R, to inhibit pathological activation of its downstream signaling (Tamura K, et al. Hypertens Res, 45:32–39, 2022). Endogenous ATRAP is abundantly expressed along kidney tubules in the kidney, and our previous investigations using systemic ATRAP knockout and distal tubule-dominant ATRAP transgenic mice revealed that ATRAP suppressed Ang II-induced hypertension mainly through a distal tubule-mediated mechanism (Wakui H, et al. Hypertension, 61:1203–10, 2013; Ohsawa M, et al. Kidney Int, 86:570–81, 2014). On the other hand, proximal tubule-specific ATRAP knockout mice exhibited similar baseline BP and pressor response to Ang II compared with wild-type mice (Kinguchi S, et al. J Am Heart Assoc, 8(8):e012395, 2019). The present study was performed to investigate the effects of enhancement of proximal tubule ATRAP on Ang II-mediated hypertension, using proximal tubule-specific ATRAP transgenic mice. Design and method: [Experiment 1] Using mouse ATRAP cDNA linked to the type II sodium-dependent phosphate cotransporters (NPT2) promoter, we generated transgenic mice (NPT2-Tg mice) with proximal tubule-specific overexpression of ATRAP. ATRAP mRNA expression in the proximal tubule was quantified by a laser capture microdissection and quantitative reverse transcription-polymerase chain reaction analysis. [Experiment 2] Male NPT2-Tg and wild-type littermate control (LC) mice were divided into Ang II (1000 ng/kg/min) and vehicle groups. BP was measured using a tail-cuff method before and 14 days after treatment, and then mice were sacrificed and their organs were collected. Results: [Experiment 1] 5 of 12 lines of ATRAP Tg mice exhibited kidney overexpression of the ATRAP-transgene in comparison with LC mice. Kidney ATRAP expression at the protein level revealed the highest expression level of ATRAP in lines 23, approximately 10-fold higher compared to LC mice. Therefore, we chose line 23 (NPT2-Tg23) for further analysis. ATRAP mRNA expression was increased approximately 9-fold in the proximal tubules of NPT2-Tg23 mice compared with LC mice. On the other hand, ATRAP mRNA expression in the distal tubules was comparable between NPT2-Tg23 and LC mice. [Experiment 2] We are currently conducting the experiments and will present our findings at this conference. Conclusions: We succeeded in generating transgenic mice with proximal tubule-specific overexpression of ATRAP. We plan to present the results of the impact of enhancement of proximal tubular ATRAP on Ang II-dependent hypertension at this conference.

1235: ABBCC8 (SULFONYLUREA RECEPTOR 1) KO CONFERS NEUROPROTECTION IN SEPSIS-ASSOCIATED BRAIN INJURY

Introduction: Sepsis-Associated Brain Injury (SABI) Is Characterized by an Acute Deterioration of Mental Status Resulting in Cognitive Impairment and Acquisition of New and Persistent Functional Limitations in Sepsis Survivors. Previously, We Reported That Septic Mice Had Evidence of Axonal Injury, Robust Microglial Activation, and Cytotoxic Edema in the Cerebral Cortex, Thalamus, and Hippocampus in the Absence of Blood-Brain Barrier Disruption. In Central Nervous System Injury, Sulfonylurea Receptor 1 (SUR1), a Member of the Adenosine Triphosphate (ATP)-Binding Cassette Protein Superfamily, Associates With the Transient Receptor Potential Melastatin 4 (TRPM4) Cation Channel to Play a Crucial Role in Cerebral Edema Development. Therefore, We Hypothesized That Knockout (KO) of Abcc8 (Sur1 Gene) Is Associated With a Decrease in Microglial Activation, Cerebral Edema, and Improved Neurobehavioral Outcomes in a Murine Cecal Ligation and Puncture (CLP) Model of Sepsis. Methods: Sepsis Was Induced in 4-6-Week-Old Abcc8 KO and Wild-Type (WT) Littermate Control Male Mice by CLP. We Used Immunohistochemistry to Define Microglial Activation Along With Parallel Studies Using Magnetic Resonance Imaging, Focusing on Cerebral Edema on Days 1 and 4 Days After CLP. Results: Abcc8 KO Mice Exhibited a Decrease in Axonal Injury and Cytotoxic Edema vs. WT on Day 1. Abcc8 KO Mice Also Had Decreased Microglial Activation in the Cerebral Cortex vs WT. These Findings Were Associated With Cognitive Improvement on Days 7–8 After CLP. Conclusions: Our Study Challenges a Key Concept in Sepsis and Suggests That Brain Injury Does Not Merely Happen as an Extension of Systemic Inflammation, but Upregulation of the SUR1-TRPM4 Channel in the Brain Directly Contributes to the CNS Pathobiology Previously Demonstrated. Benefits Conferred by Abcc8 KO in the Murine CLP Model Warrant Studies of Pharmacological Abcc8 Inhibition as a New Potential Therapeutic Strategy for SABI.

Inhibition of the LRRC8A channel promotes microglia/macrophage phagocytosis and improves outcomes after intracerebral hemorrhagic stroke.

Promoting microglial/macrophage (M/Mφ) phagocytosis accelerates hematoma clearance and improves the prognosis of intracerebral hemorrhagic stroke (ICH). Cation channels such as Piezo1 modulate bacterial clearance by regulating M/Mφ. Whether LRRC8A, an anion channel, affects M/Mφ erythrophagocytosis and functional recovery after ICH was investigated here. We found that LRRC8A is highly expressed on M/Mφ in the perihematomal region of ICH mice. Conditional knockout of Lrrc8a in M/Mφ or treatment with an LRRC8A channel blocker accelerated hematoma clearance, reduced neuronal death, and improved functional recovery after ICH. Mechanistically, the LRRC8A channel inhibition promoted M/Mφ phagocytosis by activating AMP-activated protein kinase (AMPK), thereby inducing nuclear translocation of nuclear factor-erythroid 2 related factor 2 (Nrf2) and increasing Cd36 transcription. Our findings illuminate the regulation of M/Mφ phagocytosis by the LRRC8A channel via the AMPK-Nrf2-CD36 pathway after ICH, suggesting that LRRC8A is a potential target for hematoma clearance in ICH treatment.

Preclinical toxicity assessment of anionic nanoliposomes

AimTo evaluate the in vivo toxicity of the anionic nanoliposome formulation containing [hydrogenated soy phosphatidylcholine (HSPC)] and [1,2-distearoyl-sn-glycero-3- phosphoglycerol (DSPG)]. MethodsThe anionic nanoliposome formulation was prepared by the lipid film hydration method. To assess the toxicity of anionic nanoliposomes, male and female albino mice were weakly treated with intravenous injection of the formulation (100 μmol/kg) for four weeks. The toxicity study was performed by the subacute protocol, four weeks after the last injection. To this end, the plasma levels of lipid indexes, urea, creatinine, AST, ALT, ALP, and fasting blood glucose (FBG) were measured. To evaluate histopathological alterations, the tissues of the vital organs including the heart, liver, kidneys, spleen, and brain were studied using hematoxylin & eosin (H&E) staining. ResultsThe results showed nonsignificant changes in total cholesterol, LDL-C, HDL-C, creatinine, urea, AST, ALP, and ALT in the liposome-treated mice when compared with control mice. However, plasma levels of triglycerides were significantly decreased (by 64.5 ± 15.3 mg/dL, p = 0.001) and (by 58.75 ± 15.3 mg/dL, p = 0.002) in the liposome-treated male and female mice, respectively, when compared with corresponding control mice. The FBG level was significantly increased by154 ± 20 mg/dL, p = 0.001 in the liposome-treated male mice when compared with the control male mice. The PAB level was significantly decreased by 12 ± 4.2 HK, p = 0.03 in the liposome-treated male mice when compared with the control male mice. Histological examination of vital organs indicated no significant differences in tissue damage between the liposome-treated and control mice. ConclusionThe findings of the present study indicated that DSPG-containing nanoliposome formulation exerted no significant adverse effects on the function of vital organs and blood levels of biochemical biomarkers in healthy mice. However, further investigations are needed to find a safe dose of DSPG liposomes concerning the risk of diabetes.

Neuroinflammation Underlies the Development of Social Stress Induced Cognitive Deficit in Sickle Cell Disease

Cognitive deficits have been observed in children and adults with sickle cell disease (SCD), with and without cerebral infarcts. Risk factors include systemic inflammation, neuroinflammation, and severe anemia, and sociological risk factors such as parental education level and income. Additionally, there is a reported relationship between plasma cytokine levels and cognitive function in children with SCD, suggesting a link between inflammation and cognitive deficit in SCD. This study aims to examine the mechanism underlying the development of cognitive deficit in SCD with exposure to social stress, by using the repeat social defeat (RSD) paradigm in sickle cell mice. We hypothesized that neuroinflammation is a major underlying mechanism mediating the relationship between social stress and development of cognitive deficit in SCD. RSD was done by introducing a male intruder mouse (an aggressor) into an established cage of 3, 6 months old male sickle cell or control mice, every day, for two hours, for six consecutive days. Age and sex-matched control (sickle cell and non-sickle cell mice) cages were set up but without aggressor mice. On the seventh day, mice were tested for cognitive/behavioral deficit using novel object recognition (NOR) and fear conditioning (FC) test paradigms. Except for the aggressors, all mice used were Townes humanized sickle cell (SS) and control (AA) mice. To test the hypothesis that neuroinflammation is an underlying mechanism, a second cohort of SS and AA nice were randomly assigned to oral (in drinking water) minocycline treatment (90mg/kg) or placebo (drinking water). Mice within treatment arms were randomly assigned to RSD exposure or no RSD exposure. Minocycline treatment was started one day prior to the day of commencing RSD and terminated on the same day as the final RSD session and the dose was kept constant by adjusting the amount administered daily, using the water consumption from the previous day. Cognitive/behavioral testing was performed as before, starting the next day after day 6 of RSD and day 7 of treatment. Mice were sacrificed 1-2 days after completion of behavioral testing and brains were extracted for histological analysis. Cellular evidence of neuroinflammation in the hippocampus/dentate gyrus was determined using immunohistochemistry to quantify peripheral immune cell infiltrates (CD45+ (bone marrow-derived microglia), CD3+ (T-cell density), B220+ (B-cell density), and Iba1+ (activated microglia). We used Golgi-Cox impregnation to quantify spine density and maturity in cortical and hippocampal pyramidal neurons. There were no differences between 6 months old AA and SS with regards to percent preference on NOR test (53.1 ± 4.4% vs. 56.1 ± 5.6%). However, SS mice exposed to RSD, had a lower percent preference (36.1 ± 6.7%, p = 0.06) for the novel object compared to SS mice not exposed to RSD (56.1 ± 5.6%), indicating the presence of a memory deficit in SS mice exposed to RSD. The SS mice exposed to RSD had worse performance on contextual FC test (p = 0.001), indicated by a lower percent freezing compared with SS mice not exposed to RSD. Additionally, SS mice exposed to RSD also had a higher density of activated microglia (260.2 ± 3.7 vs. 233.6 ± 4.0; p ≤ 0.0001) and bone marrow derived microglia (125.4 ± 6.2 vs. 93.7 ± 5.0; p = 0.0004) in the dentate gyrus, compared to SS mice not exposed to RSD, suggesting increased neuroinflammation after RSD exposure. There were no differences in B or T cell density. SS mice treated with minocycline during RSD exposure showed improvement in memory based on NOR (67.1 ± 4.5% vs 36.1 ± 6.7%, p = 0.0007) and contextual FC (p = 0.01) tests compared to SS mice exposed to RSD and not treated with minocycline. The treated mice had fewer activated microglia (168.4 ± 6.7 vs. 260.2 ± 3.7; p ≤ 0.0001) and bone marrow derived microglia (31.79 ± 2.2 vs. 125.4 ± 6.2; p≤0.0001) in the dentate gyrus compared to the non-treated SS mice exposed to RSD. Additionally, the treated SS mice exposed to RSD also showed increases in neuron progenitor stem cells density improvement in neurogenesis and a decrease in astrogliosis in granular zone of the dentate gyrus, compared to the SS mice exposed to RSD without minocycline treatment. We demonstrate for the first time that neuroinflammation is one of the underlying mechanisms of social stress related cognitive impairment in mice and may be applicable to children with SCD and might be treatable using anti-neuroinflammatory drugs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Palmitoylethanolamide Attenuates Pain-like Behavior in Factor VIII Deficient Mice

Coagulation factor VIII (FVIII) deficiency gives rise to hemophilia A, which is characterized by spontaneous joint bleeding. Uncontrolled bleeding drives inflammation and release of noxious and neuropathic factors, accompanied by joint degeneration, arthropathy, joint/chronic pain and impairment in physical activity. Thus, an unmet need is to develop strategies to target pain and improve physical activity in hemophilia. Chronic joint pain observed in osteoarthritis (OA) shares features of inflammation and hemarthropathy with hemophilia joint pain. Palmitoylethanolamide (PEA) is being evaluated for joint pain in OA in an interventional clinical trial (NCT05406726), which builds upon a previous double-blind, placebo-controlled evaluation suggesting its safety, tolerability, and efficacy in alleviating OA joint pain. PEA is an endogenous lipid mediator that interacts with the endogenous cannabinoid system to alleviate chronic pain, inflammation and signs of neuropathy without the intoxicating effects of cannabinoids. We reported that PEA reduced chronic pain in a mouse model of sickle cell disease (Argueta et al., Blood 2021), which presents with release of cell free heme, consequent inflammation and tissue damage similar to hemophilia. Due to its ability to reduce clinical pain outcomes and its beneficial effects on inflammation, osteoclastogenesis, and neuropathy, PEA may provide substantial benefits as a novel treatment for pain in hemophilia. To test this hypothesis, we utilized FVIII-knockout (KO) mice, which recapitulate features of human hemophilia A including spontaneous bleeding and pain (Cooke et al., J Thromb Haemost 2019), to evaluate the impact of daily PEA treatment on nociceptive and nocifensive behaviors. We treated 12 wks old male control (B6129SF2/J) and FVIII-KO (B6;129S-F8tm1Kaz/J) mice (Jackson Laboratory, Bar Harbor, ME) intraperitoneally with 20 mg/kg/d PEA (Cayman Chemical, Ann Arbor, MI) for 7 days followed by 7 day withdrawal. Mice were weighed and examined for mechanical hyperalgesia by application of Von Frey monofilaments to the plantar surface of the hindpaws (10 repetitions per paw) and weight bearing on pressure-sensitive plates (Incapacitance Tester, Ugo Basile, Gemonio, Italy) at baseline (BL), then after PEA treatment at 1, 4, and 24 hours, days 3 and 7; and after discontinuation of treatment on days 10 and 14. We also measured spontaneous nocifensive behaviors (locomotion, rearing, and grooming) over a 2-minute period in homecage (Generic USB webcam; Streampix 7, Norpix, Montreal, Canada) at BL, days 7 and 14. PEA treatment reduced mechanical hyperalgesia (~60%, P<0.001) in FVIII-KO mice after 1 hour, which continued to improve over 24 hours and was maintained for 7 days of treatment. Following withdrawal, mechanical hyperalgesia was no longer alleviated. These data suggest increased sensory pain in FVIII-KO mice, and that PEA has an analgesic effect. PEA treatment improved stance instability in weight bearing test of FVIII-KO mice (~66%, P<0.05) after 4 hours, which continued to improve over treatment period and persisted after withdrawal, up to day 10. However, improved weight bearing regressed by day 14. Improved weight bearing even after withdrawal of the PEA for 3 days suggests a disease modifying effect perhaps via reduction in joint inflammation, modulation of arthropathy and/other mechanisms. Non-evoked, spontaneous nocifensive behaviors show associations (Pearson correlation, P<0.05) with weight bearing at BL, days 7 and 14 in FVIII-KO mice. Thus, nocifensive behaviors may be a less invasive, indirect measure of weight bearing and joint pain. Body mass was not significantly affected by treatment and subsequent withdrawal. We provide the first evidence that PEA may reduce mechanical hyperalgesia and improve weight bearing in a translational model of hemophilia A. Several clinical evaluations of PEA indicate its safety, even at relatively large doses, and its efficacy in reducing inflammation and pain, which include joint pain and damage observed in OA. Our observations and the availability of PEA as a dietary supplement underscore the need for clinical and preclinical evaluation of PEA for hemophilia pain.

LBSUN132 Adipocyte-specific Ablation Of Fgf23 Attenuates Diet-induced Obesity In Female Mice

Abstract Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that regulates phosphate homeostasis and vitamin D metabolism. In addition to its central role in mineral balance, recent findings suggested that FGF23 is associated with chronic metabolic conditions including cardiovascular disease, diabetes mellitus, and obesity. Circulating FGF23 levels are elevated in obese individuals, and they are correlated with body fat mass and abdominal obesity. Moreover, consumption of high fat diet (HFD) increases serum FGF23 levels. However, the actions of FGF23 in adipocytes and its role in lipid metabolism have not been demonstrated. To understand the role of FGF23 in adipocytes, particularly in obesity, we generated adipocyte-specific Fgf23 null mice using Cre recombinase under the control of the mouse adiponectin (Adipoq) promoter. Both male and female control (Fgf23flox/flox) and knockout (KO, AdipoqCreFgf23flox/flox) mice were fed either HFD (60%kcal fat) or normal fat diet (NFD) at 8 weeks of age for 24 weeks. Body weight measurements were taken every week and percentage of body fat was determined after longitudinally during 24 weeks of diet interventation using in vivo by an EchoMRI scannermicroCT. Serum, adipose tissue, and liver were collected upon sacrifice. After 24 weeks of HFD, male control and KO mice had comparable gain in body weight (BW) and body fat percentage. However, female KO mice had significantly lower body weight and percentage of body fat. Moreover, serum total cholesterol levels were reduced in female KO mice. Triglyceride (TG) content in the liver was significantly reduced in female KO mice, and it corresponded with downregulation of lipid droplet protein Perilipin and fatty acid transporter CD36 mRNA expression in the liver. Transcript levels of hepatic PPARγ, which is positively correlated with fat accumulation in obesity, were also suppressed in female KO mice compared to controls. Obesity is associated with low-grade chronic inflammation, and we found that hepatic mRNA expression of the inflammatory cytokine TNFα was diminished in female KO mice, suggesting that adipocytic Fgf23 modulates lipid accumulation and inflammation. In summary, our results demonstrate for the first time that adipocyte-specific ablation of Fgf23 alleviates attenuates diet-induced obesity and regulates lipid metabolism in a sex-specific manner. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.