Conscious Mice Research Articles

Abstract 38: Loop Diuretic Resistance in a Preclinical Model of Cardiorenal Syndrome 2: Role of Enhanced Distal Convoluted Tubule Sodium Transport

Nearly 60% of heart failure (HF) patients develop a progressive decline in kidney function. Decompensated HF also causes edema, usually treated with loop diuretics, like furosemide, that inhibit the Na-K-2Cl cotransporter NKCC2 in the thick ascending limb (TAL). However, nearly 50% of HF patients do not respond adequately to loop diuretics and become resistant. The mechanisms causing loop diuretic resistance are unclear but may involve TAL-dependent and independent mechanisms. Few have used the mouse as a model of cardiorenal syndrome due to its resistance to renal damage. We hypothesize that mice develop loop diuretic resistance and lower GFR after chronic HF induced by myocardial infarction (MI). MI was induced in C57Bl6J mice through ligation of the left anterior descending coronary artery and compared to a sham surgery group. We assessed heart function by echocardiography and GFR (FITC inulin) in conscious mice. The response to diuretics was studied in metabolic cages. MI mice had a 50% reduction in ejection fraction (EF) one month after MI (MI: 29.94 ± 1.94, Sham: 67.02 ± 1.14%, n= 8, p<0.05). EF was still lower 3 and 6 months after MI. The MI group showed decreased GFR, 6 months after MI (MI: 0.50 ± 0.06, Sham: 0.75±0.05 µl/min, n= 5, p<0.05). Urinary albumin was similar in both groups (n=8). In metabolic cages, a saturating dose of the NKCC2 inhibitor bumetanide (20 mg/Kg), induced diuresis and natriuresis in sham mice (Delta UV: 1.96 ± 0.27 ml/4h, Delta UNa: 117.2 ± 11.02µmols/4h, n= 6) but had an attenuated response in MI mice (Delta UV: 0.88 ± 0.23 ml/4h, Delta UNa: 65.00 ± 4.53 µmols/4h, n= 4, p<0.05 vs sham). To study the distal convoluted tubule (DCT) we measured the response to hydrochlorothiazide (HCTZ). HCTZ (100 mg/Kg) induced a natriuretic response in sham mice (Delta UNa: 75.24±11.18µmols/4h, n= 10) and a higher response in the MI group (Delta UNa: 129.54±11.59µmols/4h, n= 12, p<0.05 vs sham). RNAseq in isolated mTALs from MI and sham mice identified 85 up- and 160 down-regulated genes (n=5, p<0.025). Up-regulated genes involved those in the sphingosine pathway, protein trafficking, PKA signaling (PDE4B, PRKD2) and cell growth/tubule differentiation (FREM2, WFDC1). mRNA expression of TAL NaCl transporters was not affected. We conclude that HF decreases GFR, induces loop diuretic resistance and affects mTAL signaling in mice. Loop diuretic resistance may involve enhanced DCT NaCl reabsorption.

Role of AMPK in Atrial Metabolic Homeostasis and Substrate Preference.

Atrial fibrillation is the most common clinical arrhythmia and may be due in part to metabolic stress. Atrial specific deletion of the master metabolic sensor, AMP-activated protein kinase (AMPK), induces atrial remodeling culminating in atrial fibrillation in mice, implicating AMPK signaling in the maintenance of atrial electrical and structural homeostasis. However, atrial substrate preference for mitochondrial oxidation and the role of AMPK in regulating atrial metabolism are unknown. Here, using LC-MS/MS methodology combined with infusions of [ 13 C 6 ]glucose and [ 13 C 4 ]β-hydroxybutyrate in conscious mice, we demonstrate that conditional deletion of atrial AMPK catalytic subunits shifts mitochondrial atrial metabolism away from fatty acid oxidation and towards pyruvate oxidation. LC-MS/MS-based quantification of acyl-CoAs demonstrated decreased atrial tissue content of long-chain fatty acyl-CoAs. Proteomic analysis revealed a broad downregulation of proteins responsible for fatty acid uptake (LPL, CD36, FABP3), acylation and oxidation. Atrial AMPK deletion reduced expression of atrial PGC1-α and downstream PGC1-α/PPARα/RXR regulated gene transcripts. In contrast, atrial [ 14 C]2-deoxyglucose uptake and GLUT1 expression increased with fasting in mice with AMPK deletion, while the expression of glycolytic enzymes exhibited heterogenous changes. Thus, these results highlight the crucial homeostatic role of AMPK in the atrium, with loss of atrial AMPK leading to downregulation of the PGC1-α/PPARα pathway and broad metabolic reprogramming with a loss of fatty acid oxidation, which may contribute to atrial remodeling and arrhythmia.

The pontine Kölliker-Fuse nucleus is important for reduced post-inspiratory airflow elicited by stimulation of the ventral respiratory parafacial region.

Considering that the retrotrapezoid nucleus/respiratory parafacial region (RTN/pFRG) would be an important center in the central nervous system involved in the maintenance and modulation of respiratory activity, we hypothesized that neurons in this nucleus would also be involved in the postinspiratory phase of the respiratory cycle through a connection with the pontine Kölliker-Fuse (KF) region. Here we performed pharmacogenetic manipulation (AAV-hM3D(Gq)-mCherry or AAV-hM4D(Gi)-mCherry) in VGlut2-cre, Ai6 conscious mice to evaluate breathing parameters through whole body plethysmography under baseline conditions (normoxia: FiO2 = 0.21) or under hypercapnia or hypoxia challenges (FiCO2 = 0.07 or FiO2 = 0.08). Under normoxia, selective stimulation of RTN/pFRG resulted in a smaller increase in VE (1,272 ± 102.5, vs. RTN/pFRG stimulation: 1,878 ± 122.1 ml/kg/min), due to a smaller increase in VT (5.4 ± 0.35, vs. RTN/pFRG stimulation: 7.77 ± 0.21 ml/kg) without changing fR in a condition of KF inhibition. However, inhibition of the VGlut2 neurons in the KF did affect the TE1 produced by selective activation of RTN/pFRG (119.9 ± 2.53, vs. RTN/pFRG stimulation: 104 ± 2.46 ms). Both the hypercapnia and hypoxia ventilatory response were reduced after inhibition of VGlut2-expressing KF neurons. Therefore, consistent with anatomical projections RTN/pFRG neurons regulate lung ventilation by controlling all aspects of breathing, i.e breathing frequency, inspiration, postinspiration and active expiration. All the modulation seems to be dependent on the integrity of the glutamatergic neurons in the KF region.

Chronic N-acetyl cysteine treatment does not improve respiratory system performance in the mdx mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is characterised by respiratory muscle injury, inflammation, fibrosis and weakness, ultimately culminating in respiratory failure. The dystrophin-deficient mouse model of DMD (mdx) shows evidence of respiratory muscle remodelling and dysfunction contributing to impaired respiratory system performance. The antioxidant N-acetylcysteine (NAC) has been shown to exert anti-inflammatory and anti-fibrotic effects leading to improved respiratory muscle performance in a range of animal models of muscle dysfunction, including mdx mice, following short-term administration (2weeks). We sought to build on previous work by exploring the effects of chronic NAC administration (3months) on respiratory system performance in mdx mice. One-month-old male mdx mice were randomised to receive normal drinking water (n=30) or 1% NAC in the drinking water (n=30) for 3months. At 4months of age, we assessed breathing in conscious mice by plethysmography followed by ex vivo assessment of diaphragm force-generating capacity. Additionally, diaphragm histology was performed. In separate studies, in anaesthetised mice, respiratory electromyogram (EMG) activity and inspiratory pressure across a range of behaviours were determined, including assessment of peak inspiratory pressure-generating capacity. NAC treatment did not affect force-generating capacity of the mdx diaphragm. Collagen content and immune cell infiltration were unchanged in mdx + NAC compared with mdx diaphragms. Additionally, there was no significant effect of NAC on breathing, ventilatory responsiveness, inspiratory EMG activity or inspiratory pressure across the range of behaviours from basal conditions to peak system performance. We conclude that chronic NAC treatment has no apparent beneficial effects on respiratory system performance in the mdx mouse model of DMD suggesting limited potential of NAC treatment alone for human DMD.

The Role of Renal Medullary Bilirubin and Biliverdin Reductase in Ang II Induced Hypertension

Previous studies have demonstrated the importance of renal medullary heme oxygenase-1 (HO-1) in blood pressure response to angiotensin II. However, the role of the HO metabolite, bilirubin, in this response remains unknown. The goal of the present study was to test the hypothesis that generation of bilirubin in the renal medulla plays an important role in the regulation of blood pressure in response to angiotensin II. Twenty-week-old male C57Bl/6J mice were implanted with intrarenal medullary interstitial (IRMI) catheters following unilateral nephrectomy. After this time, biliverdin IXα was specifically infused into the kidney (3.6 mg/day) for 3 days prior to implantation with an osmotic minipump delivering angiotensin II (1000 ng/kg/min). Blood pressure was recorded for 3 days, 1 week after minipump infusion, in conscious mice. IRMI infusion of biliverdin significantly decreased blood pressure as compared to mice infused with vehicle (118 ± 4 vs. 158 ± 2 mmHg, p<0.05). To further explore the antihypertensive role of renal medullary bilirubin generation, mice with specific deletion of biliverdin reductase-A (BVRA) in the thick ascending loop of Henle (TALH) were generated by crossing BVRAflox/flox mice with Tamm-Horsfall Cre (THP-Cre) mice. THP-Cre/BVRAflox/flox mice exhibited specific deletion of BVRA in the TALH by co-immunostaining with endogenous THP protein. THP-Cre/BVRAflox/flox mice did not exhibit any differences in baseline blood pressure as compared to BVRAflox/flox mice (108 ± 2 vs. 108 ± 2 mmHg). However, angiotensin-II infusion resulted in significantly higher blood pressure measured in conscious mice 7 days after implantation in THP-Cre/BVRAflox/flox as compared to BVRAflox/flox mice (152 ± 2 vs. 140 ± 3 mmHg, p<0.05). These results demonstrate an important role for renal medullary bilirubin and biliverdin reductase in the blood pressure response to angiotensin II. Strategies which increase renal medullary bilirubin/biliverdin reductase levels could be novel antihypertensive therapies. Future studies will examine the mechanistic link between bilirubin and electrolyte transporters in the kidney, specifically, the Na+-K+-2Cl2 cotransporter (NKCC2), which is a major transporter of electrolyte in the thick ascending limb and is known to play a role in Ang II-induced hypertension. This work was supported by National Institutes of Health grants (RO1-HL088421 to DS). 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.

Macrophage Targeted Dendrimer-Tesaglitazar Therapy for Atherosclerosis

Background: Atherosclerosis is a leading contributor to cardiovascular disease, the number one cause of death globally. Although atherosclerosis is clearly a multicellular disease that also affects endothelial cells as well as smooth muscle cells of arteries, it is increasingly recognized as an inflammatory disease. Our goal is to determine if targeting immune cells implicated in atherosclerosis can halt plaque accumulation and resulting derangement of vascular cells in the aorta. Peroxisome proliferator activated receptors (PPARs) within macrophages present a promising therapeutic target as PPAR-α and PPAR-γ agonists have been shown to improve lipid metabolism and inhibit macrophage foam cell formation. However, due to high doses required and associated side effects, clinical translation was halted. We target anti-atherogenic pathways in immune cells using the novel treatment D-Tesa, a dendrimer conjugate of a dual PPAR-α/γ agonist, Tesaglitazar. Methods: 25-week-old, male, apolipoprotein E knockout (ApoE−/−) mice were fed high fat diet (HFD) for 16 weeks to induce atherosclerosis and randomized into two groups: 1) treatment group: D-Tesa for 6 weeks (20μg/kg twice weekly by oral gavage and 2) untreated group. HFD was continued for the duration of the study. Pulse wave velocity (PWV: Doppler), blood pressure (BP: tail cuff on trained, conscious mice), and body weight were monitored. Dendrimer mediated delivery of drug payload to plaque macrophages was studied using D-Cy5, a fluorescently labeled dendrimer, in an atherosclerotic mouse. Aortas were isolated and used for vasoreactivity studies and western blot analysis. THP-1 cell derived foam cells were used to study D-Tesa’s mechanism of action in vitro. Results: Immunofluorescence staining and confocal microscopy revealed that D-Cy5 localized in macrophages within the atherosclerotic plaque in ApoE−/− mice on HFD, indicating that the dendrimer can deliver drugs specifically into the plaque. D-Tesa slowed plaque progression, preserved endothelial function, and decreased arterial stiffness. BP was similar and unchanged between groups. D-Tesa surprisingly decreased body weight, which was the opposite effect observed from the free drug in clinical trials. Western blotting of aorta revealed that D-Tesa treated mice experienced an upregulation of PPAR-α, PPAR-γ, and ABCA1. The in vitro studies with THP-1 derived foam cells confirmed the upregulation of the cholesterol efflux pathway and anti-inflammatory benefit of D-Tesa. Conclusion: D-Tesa offers a novel therapy to ameliorate multiple damaging consequences of atherosclerosis on the cardiovascular system including improvements in plaque burden, endothelial function, arterial stiffness, and body weight in an ApoE−/− model of atherosclerosis. Excitingly, these effects were seen at 2-15 times lower doses despite continued HFD than those reported in literature with a combination of free Tesa and low-fat diet. Thus, D-Tesa offers great translational relevance because of potential reduction in negative side effects and higher patient benefit due to noted lack of compliance with dietary changes in the clinic. Further research is ongoing to evaluate sex differences, dose response, and long-term effects of this highly promising therapy. This work was supported by a NHLBI grant R01HL148112 01 (L.S.) and the Traystman Endowment (S.K.). 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.

Loop Diuretic Resistance in a Mouse Model of Cardiorenal Syndrome

Nearly 30% of patients with heart failure (HF) develop Cardiorenal syndrome type 2 (CRS2), characterized by renal injury and lower glomerular filtration rate (GFR) secondary to lower cardiac function. Patients with uncompensated heart failure develop edema and are usually treated with a loop diuretic, such as furosemide, that acts in thick ascending limb (TAL) to inhibit NKCC2. However, over 50% of HF patients develop resistance to loop diuretics. The molecular mechanisms underlying the development of diuretic resistance are not clear and may involve decreased NKCC2-mediated NaCl transport, enhanced distal tubule NaCl reabsorption, or decreased accessibility of the diuretic drug to the nephron lumen. Few studies have used the mouse as a model of cardiorenal syndrome after myocardial infarction (MI). We hypothesize that C57Bl6J mice would develop kidney damage several months after myocardial infarction (MI) and resistance to loop diuretics. Heart failure was induced in C57Bl6J mice through ligation of the left anterior descending coronary artery (LAD) and classified as MI group and compared to a sham surgery group (without ligation) as controls. Both groups were assessed for heart function by echocardiography and renal filtration function by measuring GFR (FITC inulin) in conscious mice. The response to diuretics was studied in metabolic cages, 7-8 months after MI. MI mice had a 50% reduction in ejection fraction (EF) 4 weeks after MI compared to sham group (MI: 29.94 ± 1.94, Sham: 67.02 ± 1.14, n= 8 each group, p<0.05). Decreased EF continued to be lower 6 and 12 months after MI. Six months after surgery, the MI group showed decreased GFR compared to Sham (MI: 0.50 ± 0.06, Sham: 0.75±0.05 μl/min, n= 5 MI, n=7 sham, p<0.05). Despite lower GFR, 24h-urinary albumin excretion was similar in both groups (n=8 each group). In metabolic cage studies, a saturating dose of the NKCC2 inhibitor bumetanide (20 mg/Kg), induced diuresis and natriuresis in sham mice (Delta UV: 1.96 ± 0.27 ml/4h, Delta UNa: 117.2 ± 11.02μmols/4h, UCl: 175.65 ± 13.01 μmols/4h, n= 6) but had a smaller effect in MI mice which exhibited an attenuated diuretic response (Delta UV: 0.88 ± 0.23 ml/4h, Delta UNa: 65.00 ± 4.53 μmols/4h, UCl: 102.89 ± 14.08 μmols/4h, n= 4, p<0.05 vs sham), indicative of loop diuretic resistance. To study if bumetanide resistance is secondary to enhanced distal convoluted tubule (DCT) NaCl reabsorption, we measured the diuretic response to the NCC inhibitor hydrochlorothiazide (HCTZ). HCTZ (100 mg/Kg) induced diuresis and natriuresis in sham mice (Delta UNa: 75.24±11.18μmols/4h, UCl: 39.44±10.15 μmols/4h, n= 10) and the diuretic response was slightly higher in the MI group (Delta UNa: 129.54±11.59μmols/4h, UCl: 94.61±11.56 μmols/4h, n= 12, p<0.05 vs sham). We conclude that chronic heart failure in C57Bl6J mice, decreases GFR and induces loop diuretic resistance. Loop diuretic resistance after MI may be secondary to enhanced DCT NaCl transport in mice. This model could be useful to study the mechanisms causing decreased renal function and loop diuretic resistance after MI and pursuing new therapeutic avenues. No funding was received by the authors for conducting this study. 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.

Gut microbes regulate glomerular filtration rate in health and in a model of chronic kidney disease

Gut microbes have been implicated in varied physiological and pathophysiological processes. In this study, we hypothesized that gut microbes modulate glomerular filtration rate (GFR). Gut microbiota was depleted using a modified antibiotic (ABX) treatment protocol with a mixture of 1g/L ampicillin, 1g/L neomycin, and 0.5 g/L vancomycin (modified from Rakoff-Nahoum et al. 2004). C57BL/6J male and female mice (6-weeks-old) were treated with ABX in drinking water for five weeks. Bacteria reduction with ABX treatment was confirmed by qPCR of fecal samples using pan-bacterial primers (Ct value for Females, F: ctrl 13±0.18 vs ABX 21±0.41; Males, M: 13±0.11 vs ABX 20±0.44, n=8). GFR measurements were performed in conscious and unrestrained mice by transcutaneous detection of fluorescent FITC-sinistrin. The GFR was significantly increased in ABX-treated mice (F: ctrl 1190±14 vs ABX 1638±21, p=0.03; M: ctrl 1020±25 vs ABX 1381±29, p=0.005, n=8). To investigate if the increased GFR was due to the absence of gut microbiota, we measured GFR in germ-free (GF) mice (born and raised without gut microbes), conventional mice (Ctrl, born with gut microbes), and conventionalized GF mice (CGF, born without gut microbes but given a fecal slurry containing gut microbes by oral gavage). GFR was increased in GF mice (F: 1853±32, p=0.003; M: 1308±16, p=0.02) versus both conventional (F: 1217±17; M: 1087±18) and CGF (F: 1326±31, p=0.02; M: 1101±10, p=0.03, n=7-8). GFR was similar in Ctrl and CGF mice. To further investigate if ABX can increase GFR in chronic kidney disease (CKD) when GFR is impaired, GFR was measured in adenine-induced CKD. Adenine (in food pellets) and ABX (in drinking water) treatments were begun simultaneously; adenine decreased GFR in females at weeks 4 and 6, and in males at weeks 2, 4 and 6. In females, mice co-treated with ABX and adenine had increased GFR on week 4 (chow: 1298±11 vs adenine: 853±8, p<0.0001; adenine vs adenine+ABX: 1040±9, p=0.04) but not week 6 (chow: 1238±21 vs adenine: 618±9, p=0.0001; adenine vs adenine+ABX: 881±14, p=0.09, n=7-9). Of note, GFR exhibited no difference between chow vs adenine (data not shown) on week 2. In males, ABX increased GFR on week 2 (chow: 1116±31 vs adenine: 913±47, p=0.002; adenine vs adenine+ABX: 1150±49, p=0.0003) but not week 4 (chow: 1154±38 vs adenine: 732±58, p<0.0001; adenine vs adenine+ABX: 877±81, p=0.37) or 6 (chow: 1157±43 vs adenine: 513±51, p<0.0001; adenine vs adenine+ABX: 591±50, p=0.56, n=7-9). To explore the mechanism by which ABX increase GFR we tested the hypothesis that, in the absence of gut microbes, proximal tubular Na+ reabsorption increases, leading to a reduced Na+ delivery to the macula densa and altering tubuloglomerular feedback (TGF) to increase GFR. To test this, we used the sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin (EMPA) to impair Na+ reabsorption and increase Na+ delivery to the macula densa. As seen previously, treatment with ABX increased GFR on week 3 (F: 1430±13, p=0.04; M:1341±9, p=0.01) and week 5 (F: 1857±21, p=0.002, M: 1443±10, p=0.0001) compared to before treatment (F: 1140±10, M: 1090±11, n=8-9). Co-treatment of EMPA and ABX impaired GFR increases on week 3 (F: 1281±18, p=0.9, M: 1201±11, p=0.9) but not week 5 (F: 1650±21, p=0.01; M: 1281±20, p=0.003) when comparing to before the treatment (F: 1199±15; M: 1109±8, n=8-9). In this study, both the absence (GF) and suppression (ABX) of gut microbes increased GFR in healthy females and males. Moreover, ABX partially restored GFR in adenine-induced CKD females and males. These data suggest that gut microbes influence GFR in both health and disease, and that the mechanism may partially involve TGF. AHA Career Development Award (JX) and NIDDK Diabetic Complications Consortium (RRID:SCR_001415m www.diacomp.org, grants DK076169 and DK 115255 (subaward to JLP)) and American Heart Association Established Investigator Award (to JLP). 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.

Galantamine Reduces Blood Pressure and Renal Injury in Female Angiotensin II-induced Hypertensive Mice

Inflammation is a known culprit in the development and progression of hypertension. The autoimmune disease, systemic lupus erythematosus (SLE), is an important model of hypertension in the setting of chronic kidney inflammation. We have shown that administration of an acetylcholinesterase inhibitor, galantamine, attenuates the hypertension, kidney inflammation, and kidney injury in female SLE mice by activating the vagally-mediated cholinergic anti-inflammatory pathway. Although SLE hypertension represents a specific population of patients, we hypothesized that galantamine would have similar anti-inflammatory effects in the angiotensin II (AngII)-induced hypertensive mouse, which more commonly models essential hypertension. Female C57BL/6 mice were implanted with minipumps at 9 weeks of age for subcutaneous delivery of either AngII (1000 ng/kg/min) or saline (controls), and galantamine hydrobromide (4 mg/kg/day) or vehicle, for 21 days. At 12 weeks, carotid catheters were implanted and mean arterial pressure (MAP) was measured in conscious mice for two consecutive days. Mice were then euthanized, and kidneys collected. Markers of kidney inflammation and injury, including interleukin (IL)-6 and kidney injury molecule (KIM)-1, were measured by ELISA and normalized to total kidney protein. MAP (mmHg) was higher in AngII-treated mice compared to controls (174±4 vs. 133±3; n=4-5, p=0.0023). Galantamine lowered MAP in AngII mice (157±8; n=7, p=0.2567) without altering MAP in controls (133±4; n=5, p=0.9999). Renal IL-6 (pg/μg) was not different between AngII and control mice (0.004±0.0001 vs. 0.005±0.0006; p=0.6685). Although galantamine lowered IL-6 in both groups, it was only significant in controls (0.003±0.0002; p=0.0134) and not AngII mice (0.003±0.0003; p=0.4677). Renal cortical KIM-1 (pg/μg) trended higher in AngII mice compared to controls (2.53±0.50 vs 0.63±0.16; p=0.0741). Galantamine lowered KIM-1 in AngII mice (1.18±0.39, p=0.2064), but not controls (1.45 ± 0.59; p=0.6285). In summary, galantamine protected female mice from hypertension and renal injury in a model of AngII hypertension. This suggests that acetylcholinesterase inhibitor therapy may be effective in patients with hypertension. Studies funded by NIH R01HL153703 and K01HL139859 for KWM. 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.

Partial Resistance to Thyroid Hormone-Induced Tachycardia and Cardiac Hypertrophy in Mice Lacking Thyroid Hormone Receptor β.

Background: Thyroid hormones regulate cardiac functions mainly through direct actions in the heart and by binding to the thyroid hormone receptor (TR) isoforms α1 and β. While the role of the most abundantly expressed isoform, TRα1, is widely studied and well characterized, the role of TRβ in regulating heart functions is still poorly understood, primarily due to the accompanying elevation of circulating thyroid hormone in TRβ knockout mice (TRβ-KO). However, their hyperthyroidism is ameliorated at thermoneutrality, which allows studying the role of TRβ without this confounding factor. Methods: Here, we noninvasively monitored heart rate in TRβ-KO mice over several days using radiotelemetry at different housing temperatures (22°C and 30°C) and upon 3,3',5-triiodothyronine (T3) administration in comparison to wild-type animals. Results: TRβ-KO mice displayed normal average heart rate at both 22°C and 30°C with only minor changes in heart rate frequency distribution, which was confirmed by independent electrocardiogram recordings in freely-moving conscious mice. Parasympathetic nerve activity was, however, impaired in TRβ-KO mice at 22°C, and only partly rescued at 30°C. As expected, oral treatment with pharmacological doses of T3 at 30°C led to tachycardia in wild-types, accompanied by broader heart rate frequency distribution and increased heart weight. The TRβ-KO mice, in contrast, showed blunted tachycardia, as well as resistance to changes in heart rate frequency distribution and heart weight. At the molecular level, these observations were paralleled by a blunted cardiac mRNA induction of several important genes, including the pacemaker channels Hcn2 and Hcn4, as well as Kcna7. Conclusions: The phenotyping of TRβ-KO mice conducted at thermoneutrality allows novel insights on the role of TRβ in cardiac functions in the absence of the usual confounding hyperthyroidism. Even though TRβ is expressed at lower levels than TRα1 in the heart, our findings demonstrate an important role for this isoform in the cardiac response to thyroid hormones.

544 Interleukin-6 protects renal dysfunction in mouse models of hypertension and salt-sensitive hypertension

OBJECTIVES/GOALS: We are investigating the role of IL-6 in regulating renal function by measuring mean arterial pressure (MAP), renal plasma flow (RPF) and glomerular filtration rate (GFR) in wild type (WT) and IL-6-knockout (KO) mice in established mouse models of angiotensin II (AII)-dependent- hypertension and -salt-sensitive hypertension. METHODS/STUDY POPULATION: Twelve-week-old male WT and KO mice on the C57BL6 background strain were infused with vehicle (V; saline) or angiotensin II (AII; 200 ng/kg/min) for 12-14 days. Half of the AII-treatment groups were maintained on a high salt (HS; 6% NaCl) diet for the duration of the experiment, while the other half of the AII treatment groups and both vehicle groups were fed normal rat chow. MAP was continuously measured by a fluid filled catheter in conscious mice for the duration of the experiment. RPF and GFR were measured on days 12-14 in anesthetized mice by the para-aminohippurate, and fluorescein isothiocynate-Inulin techniques, respectively. All data were analyzed by 2-way ANOVA; *p<0.05 vs. WT, same treatment; #p<0.05 vs.V, same genotype; ^p<0.05, AII vs. AII+HS, same genotype. RESULTS/ANTICIPATED RESULTS: MAP was 31% lower in KO vs WT mice. AII increased MAP (1.2-fold) in WT but not KO mice. HS diet magnified AII-induced increases in MAP in WT and moderately increased MAP in AII-KO mice: [MAP (mmHg): WT+V, 130±7.0; KO+V, 91.0+4.0*; WT+AII, 153±5.0#; KO+AII, 83.0±4.0*; WT+AII+HS, 150±11#; KO+AII+HS, 93.0±4.0#]. AII infusion reduced RPF in the KO but not WT mice. Addition of HS reduced RPF in WT and exacerbated AII-induced reductions in RPF in KO mice [RPF (ml/min/g): WT+V, 1.82±0.23; KO+V, 1.91+0.40; WT+AII, 3.16±0.75#; KO+AII, 1.65±0.42*; WT+AII+HS, 1.10±0.31#^; KO+AII+HS, 1.13±0.XX#^]. The HS diet reduced GFR in AII-infused KO but not WT mice [GFR (µl/min/g): WT+V, 756±XX; KO+V, 788±XX; WT+AII, 1010±63*#; KO+AII, 756±23*; WT+AII+HS, 1100±150#; KO+AII+HS: 540±210*#^]. DISCUSSION/SIGNIFICANCE: The absence of IL-6 in male mice attenuated AII- and/or AII+HS-induced increases in MAP; however, it exacerbated HS-induced reductions in RPF and GFR. These findings suggest inhibiting IL-6 has therapeutic potential as an antihypertensive but not as a renal protective agent in hypertension and salt-sensitive hypertension disease states.

Hemodynamic Renal Reserve Response in Conscious Normotensive and Hypertensive Mice

Introduction: Renal function may be compromised following recovery from kidney insults. Renal functional reserve (RFR) is a measure of the difference between the kidney’s maximum capacity and its baseline function, which helps identify any areas of the kidney with compromised function. Usually, RFR is evaluated using acute volume expansion (AVE), but this is typically done in anesthetized animals, which may not accurately represent the kidney’s complete functional capacity. In this study, we have introduced a novel method that enables AVE to be conducted in conscious mice. Methods: We have implemented this innovative approach in two animal models representing either intact or impaired renal function, specifically utilizing a lower nephron hypertensive model. Mice were implanted with radio-transmitters for mean artery blood pressure (MAP) monitoring during the experiment. After recovery, half of the mice were induced hypertension by right kidney nephrectomy combined with the ligation of the upper branch of the left kidney. For the AVE, a volume equivalent to 5% of the mouse’s body weight was administered via intravenous (IV) or intraperitoneal bolus injection. Subsequently, the mice were individually housed in cages covered with plastic wrap. Urine was collected every hour for a total of 3 h for the measurement of urine and sodium excretion. Results: The MAPs for all normotensive mice were consistent throughout the AVE, but it increased 5–16 mm Hg in the hypertensive mice upon AVE. Remarkably, conscious mice exhibited a significantly stronger response to IV-administered AVE when compared to anesthetized mice. This response was evident in the increase in urinary flow, which was approximately 170% and 145% higher in conscious normotensive and hypertensive mice, respectively, compared to their respective baselines. In contrast, anesthetized normotensive and hypertensive mice showed only around a 130% and 100% increase in urinary flow, respectively. Additionally, upon AVE, conscious normotensive mice excreted approximately 47% more sodium than conscious hypertensive mice. In contrast, anesthetized normotensive mice excreted only about 30% more sodium than their anesthetized hypertensive counterparts. Conclusion: Performing a kidney stress test with a significant solution load in conscious mice seems to be a superior method for evaluating RFR compared to conducting the test under anesthesia. Assessing kidney clearance while the mice are conscious has the potential to enhance the precision of diagnosing and predicting both acute and chronic kidney diseases.

Wireless ECG Monitoring in Conscious Mice: A Valuable Tool for Investigating Cardiac Function in Myocardial Infarction Research

Wireless ECG Monitoring in Conscious Mice: A Valuable Tool for Investigating Cardiac Function in Myocardial Infarction Research

The effects of sex hormones on the size of intestinal lipoproteins.

Larger intestinal lipoproteins are more likely to be retained longer in the intestinal wall, allowing more time for their fat to be hydrolyzed and subsequently taken up by the abdominal viscera. Since men generally accumulate more abdominal visceral fat than women, we sought to determine if males produce larger intestinal lipoproteins compared to females. Using the conscious lymph fistula mouse model, we discovered that the male mice indeed produced larger intestinal lipoproteins than the female mice when they were intraduodenally infused with lipid emulsion. We then employed our differentiated Caco-2 cell model with semipermeable membrane system to determine the effects of sex hormones on the size of intestinal lipoproteins. Lipoprotein size was quantitatively measured by calculating the ratio of triglycerides (TG)/Apolipoprotein B (ApoB) and by analyzing their transmission electron micrographs. Our studies showed that while there was no dose-dependent effect of estrogen and progesterone, testosterone significantly increased the size of lipoproteins. When these hormones were combined to resemble the physiological concentrations observed in males and the different ovarian cycle phases in premenopausal females, both the male and luteal groups had significantly larger lipoproteins than the ovulatory group; and the male group also had significantly larger lipoproteins than the follicular group. The ovulatory group secreted a significantly lower amount of TG than the male and luteal groups. ApoB was comparable among all these groups. These findings support our hypothesis that, through their testosterone effects, males are more likely to produce larger intestinal lipoproteins. Larger lipoproteins tend to remain longer in the intestinal wall and may facilitate fat uptake preferentially by the abdominal viscera. Our studies may partly explain why men are more prone to accumulating abdominal visceral fat, which is an independent predictor of mortality.

TRPA1 channel mediates methylglyoxal-induced mouse bladder dysfunction.

Introduction: The transient receptor potential ankyrin 1 channel (TRPA1) is expressed in urothelial cells and bladder nerve endings. Hyperglycemia in diabetic individuals induces accumulation of the highly reactive dicarbonyl compound methylglyoxal (MGO), which modulates TRPA1 activity. Long-term oral intake of MGO causes mouse bladder dysfunction. We hypothesized that TRPA1 takes part in the machinery that leads to MGO-induced bladder dysfunction. Therefore, we evaluated TRPA1 expression in the bladder and the effects of 1 h-intravesical infusion of the selective TRPA1 blocker HC-030031 (1nmol/min) on MGO-induced cystometric alterations. Methods: Five-week-old female C57BL/6 mice received 0.5% MGO in their drinking water for 12weeks, whereas control mice received tap water alone. Results: Compared to the control group, the protein levels and immunostaining for the MGO-derived hydroimidazolone isomer MG-H1 was increased in bladders of the MGO group, as observed in urothelium and detrusor smooth muscle. TRPA1 protein expression was significantly higher in bladder tissues of MGO compared to control group with TRPA1 immunostaining both lamina propria and urothelium, but not the detrusor smooth muscle. Void spot assays in conscious mice revealed an overactive bladder phenotype in MGO-treated mice characterized by increased number of voids and reduced volume per void. Filling cystometry in anaesthetized animals revealed an increased voiding frequency, reduced bladder capacity, and reduced voided volume in MGO compared to vehicle group, which were all reversed by HC-030031 infusion. Conclusion: TRPA1 activation is implicated in MGO-induced mouse overactive bladder. TRPA1 blockers may be useful to treat diabetic bladder dysfunction in individuals with high MGO levels.

Vasodilators activate the anion channel TMEM16A in endothelial cells to reduce blood pressure.

Systemic blood pressure is acutely controlled by total peripheral resistance as determined by the diameter of small arteries and arterioles, the contractility of which is regulated by endothelial cells lining the lumen of blood vessels. We investigated the physiological functions of the chloride (Cl-) channel TMEM16A in endothelial cells. TMEM16A channels generated calcium (Ca2+)-activated Cl- currents in endothelial cells from control (TMEM16Afl/fl) mice that were absent in those from mice with tamoxifen-inducible, endothelial cell-specific knockout of TMEM16A (TMEM16A ecKO). TMEM16A currents in endothelial cells were activated by the muscarinic receptor agonist acetylcholine and an agonist of the Ca2+ channel TRPV4, which localized in nanoscale proximity with TMEM16A as assessed by single-molecule localization imaging of endothelial cells. Acetylcholine stimulated TMEM16A currents by activating Ca2+ influx through surface TRPV4 channels without altering the nanoscale properties of TMEM16A and TRPV4 surface clusters or their colocalization. In pressurized arteries, activation of TMEM16A channels in endothelial cells induced by acetylcholine; TRPV4 channel stimulation; or intraluminal ATP, another vasodilator, produced hyperpolarization and dilation. Furthermore, deficiency of TMEM16A channels in endothelial cells resulted in increased systemic blood pressure in conscious mice. These data indicate that vasodilators stimulate TRPV4 channels, leading to Ca2+-dependent activation of nearby TMEM16A channels in endothelial cells to produce arterial hyperpolarization, vasodilation, and reduced blood pressure. Thus, TMEM16A is an anion channel in endothelial cells that regulates arterial contractility and blood pressure.

Abstract 17088: Pharmacological SERCA Activation Shifts Cardiac Fuel Preference Without Impairing Function in Obese Mice

Introduction: Prior studies suggest that restoring Ca 2+ homeostasis by activating SERCA improves liver health and metabolism in a mouse model of metabolic syndrome. We were interested to know if these benefits extended to the heart. Here, we test the impact of pharmacological SERCA activation by CDN1163 on heart metabolism. We modeled data from in vivo 13 C tracer experiments to determine cardiac metabolic fluxes in obese mice at the end of an 8-week CDN1163 treatment period. Flux measurements were combined with echocardiography data to assess the effects of SERCA activation on heart function and metabolism. Methods: All mice in the study were hyperphagic Mc4r -/- mice. We studied three groups of Mc4r -/- mice at 16 weeks of age (n≥6 per group): mice fed a chow diet (Chow) or those switched to a western diet at 8 weeks of age (WD) ± CDN1163 (3 injections per week). Stable and radio-labeled isotopes were infused and blood was sampled from unrestrained, conscious mice through jugular vein and carotid artery catheters, respectively. Fluxes were estimated by fitting a mathematical model of liver and cardiac metabolism to 13 C enrichment measurements of plasma and tissue metabolites taken at the end of the infusion. A separate cohort of mice was used for echocardiography and gene expression analysis. Results: We found that WD increased absolute cardiac glycolytic, anaplerotic, and citric acid cycle (CAC) fluxes (~2-fold; p<0.05). In contrast, CDN1163 treatment decreased these fluxes compared to both Chow and WD (~2-3-fold; p<0.05). Additionally, the pyruvate partitioning between pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) favored PDH with WD feeding (~2-fold), concurring with increased glycolysis. However, in WD+CDN1163 mice, pyruvate partitioning was restored to the Chow phenotype, favoring PC (p<0.05 WD vs. WD+CDN11163), suggesting decreased reliance on glucose for the generation of acetyl CoA for the CAC. These metabolic shifts occurred without changes in cardiac function or compensatory structural remodeling of the left ventricle. Conclusions: CDN1163 treatment changes metabolic fluxes in the hearts of obese mice. These metabolic changes do not impact heart function.

Remote Neuronal Activation Coupled with Automated Blood Sampling to Induce and Measure Circulating Luteinizing Hormone in Mice.

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of numerous inputs and neuronal populations in the modulation of LH release is still unknown. Measuring changes in LH levels in mice is often a challenge since they are easily disrupted by environmental stress. Current techniques to measure LH release and pulsatility require long-term training for mice to adapt to manipulation stress, certain restraint, the presence of the investigator, and working on individual animals, reducing its usefulness for many research questions. This paper presents a technique to remotely activate specific neuronal populations using Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) technology coupled with automated sequential blood sampling in conscious, freely moving, and undisturbed mice. We first describe the stereotaxic surgery protocol to deliver adeno-associated virus (AAV) vectors expressing DREADDs to specific neuronal populations. Next, we describe the protocol for carotid artery and jugular vein cannulation and postsurgical connection to the CULEX automated blood sampling system. Finally, we describe the protocol for clozapine-N-oxide intravenous injection for remote neuronal activation and automated blood collection. This technique allows for programmed automated sampling every 5 min or longer for a given period, coupled with intravenous substance injection at a desired time point or duration. Overall, we found this technique to be a powerful approach for research on neuroendocrine control.

Evidence that methylglyoxal and receptor for advanced glycation end products are implicated in bladder dysfunction of obese diabetic ob/ob mice.

Glycolytic overload in diabetes causes large accumulation of the highly reactive dicarbonyl compound methylglyoxal (MGO) and overproduction of advanced glycation end products (AGEs), which interact with their receptors (RAGE), leading to diabetes-associated macrovascular complications. The bladder is an organ that stays most in contact with dicarbonyl species, but little is known about the importance of the MGO-AGEs-RAGE pathway to diabetes-associated bladder dysfunction. Here, we aimed to investigate the role of the MGO-AGEs-RAGE pathway in bladder dysfunction of diabetic male and female ob/ob mice compared with wild-type (WT) lean mice. Diabetic ob/ob mice were treated with the AGE breaker alagebrium (ALT-711, 1 mg/kg) for 8 wk in drinking water. Compared with WT animals, male and female ob/ob mice showed marked hyperglycemia and insulin resistance, whereas fluid intake remained unaltered. Levels of total AGEs, MGO-derived hydroimidazolone 1, and RAGE in bladder tissues, as well as fluorescent AGEs in serum, were significantly elevated in ob/ob mice of either sex. Collagen content was also markedly elevated in the bladders of ob/ob mice. Void spot assays in filter paper in conscious mice revealed significant increases in total void volume and volume per void in ob/ob mice with no alterations of spot number. Treatment with ALT-711 significantly reduced the levels of MGO, AGEs, RAGE, and collagen content in ob/ob mice. In addition, ALT-711 treatment normalized the volume per void and increased the number of spots in ob/ob mice. Activation of AGEs-RAGE pathways by MGO in the bladder wall may contribute to the pathogenesis of diabetes-associated bladder dysfunction.NEW & NOTEWORTHY The involvement of methylglyoxal (MGO) and advanced glycation end products (AGEs) in bladder dysfunction of diabetic ob/ob mice treated with the AGE breaker ALT-711 was investigated here. Diabetic mice exhibited high levels of MGO, AGEs, receptor for AGEs (RAGE), and collagen in serum and/or bladder tissues along with increased volume per void, all of which were reduced by ALT-711. Activation of the MGO-AGEs-RAGE pathway in the bladder wall contributes to the pathogenesis of diabetes-associated bladder dysfunction.

Characterization of the Cardiac Structure and Function of Conscious D2.B10-Dmdmdx/J (D2-mdx) mice from 16-17 to 24-25 Weeks of Age.

Duchenne muscular dystrophy (DMD) is the most common form of muscle degenerative hereditary disease. Muscular replacement by fibrosis and calcification are the principal causes of progressive and severe musculoskeletal, respiratory, and cardiac dysfunction. To date, the D2.B10-Dmdmdx/J (D2-mdx) model is proposed as the closest to DMD, but the results are controversial. In this study, the cardiac structure and function was characterized in D2-mdx mice from 16-17 up to 24-25 weeks of age. Echocardiographic assessment in conscious mice, gross pathology, and histological and cardiac biomarker analyses were performed. At 16-17 weeks of age, D2-mdx mice presented mild left ventricular function impairment and increased pulmonary vascular resistance. Cardiac fibrosis was more extended in the right ventricle, principally on the epicardium. In 24-25-week-old D2-mdx mice, functional and structural alterations increased but with large individual variation. High-sensitivity cardiac Troponin T, but not N-terminal pro-atrial natriuretic peptide, plasma levels were increased. In conclusion, left ventricle remodeling was mild to moderate in both young and adult mice. We confirmed that right ventricle epicardial fibrosis is the most outstanding finding in D2-mdx mice. Further long-term studies are needed to evaluate whether this mouse model can also be considered a model of DMD cardiomyopathy.