Integrative Physiology

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    Resistance Breathing and Sympathetic Nerve Activity During Simulated Hemorrhage in Humans
    (2022) Davis, Kenneth; Sprick, Justin; Kay, Victoria; Rickards, Caroline
    Purpose: Resistance breathing amplifies the respiratory pump during inspiration, so may be an effective intervention for treatment of hemorrhagic injuries. In animal studies of actual hemorrhage, and human studies of simulated hemorrhage, resistance breathing protects arterial pressure, and improves tolerance to this stress. Anecdotally, resistance breathing also increases the coupling between sympathetic nerve activity and arterial pressure. The impact of resistance breathing on overall sympathetic nerve activity, however, has not been examined. We tested the hypothesis that resistance breathing increases sympathetic nerve activity during simulated hemorrhage in healthy humans. Methods: Lower body negative pressure (LBNP) was used to simulate hemorrhage in five human subjects (3M, 2F; 29.2 ± 6.8 y). Two experiments were conducted (randomized order): 1) a control condition in which LBNP was applied at 3 mmHg/min until the onset of presyncope, and 2) a resistance breathing condition in which the same LBNP protocol was used, but subjects breathed through a resistance device (set at -7 cm.H2O) during the final stages of the protocol. Arterial pressure and muscle sympathetic nerve activity (MSNA) of the radial nerve were monitored continuously. Bursts frequency (bursts/min) and burst incidence (burst/ 100 heart beats) were used to quantify MSNA. Coupling between diastolic arterial pressure (DAP) and MSNA was assessed by transfer function analysis coherence within the low frequency range (0.04-0.15 Hz). Two-way repeated measures ANOVAs were conducted for assessment of responses in the control and resistance breathing conditions, between baseline and at matched time points late in the LBNP protocol. Results: While LBNP induced increases in both MSNA burst frequency (P=0.003) and burst incidence (P=0.06), there was no effect of resistance breathing on MSNA for either index during LBNP (control, 57.9 ± 25.9 bursts/min vs. resistance breathing, 50.6 ± 21.7 bursts/min, P=0.99; control, 55.6 ± 25.6 b/100 heart beats vs. resistance breathing, 42.3 ± 18.3 b/100 heart beats, P=0.42). Additionally, there was no effect of resistance breathing on DAP (control, 73.2 ± 9.9 mmHg vs. resistance breathing, 72.8 ± 4.4 mmHg; P=0.99), or coherence between MSNA and DAP during LBNP (control, 0.53 ± 0.21 vs. resistance breathing, 0.69 ± 0.17; P=0.46). Conclusion: Contrary to our hypothesis, resistance breathing had no effect on sympathetic nerve activity during LBNP. A limitation of this study is the low sample size (N=5), and high variability of MSNA. Future investigations with a larger sample size are needed to determine if respiratory dynamics can influence the coupling between MSNA and arterial pressure.
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    Effects of Sustained Hypobaric Hypoxia on Amplitude of Forced Hemodynamic Oscillations During Central Hypovolemia
    (2022) Anderson, Garen K.; Rosenberg, Alexander; McKeefer, Haley; Bird, Jordan D.; Pentz, Brandon; Byman, Britta; Jendzjowsky, Nicholas; Wilson, Richard; Day, Trevor; Rickards, Caroline
    Introduction: Forcing oscillations in arterial pressure and cerebral blood flow at 0.1 Hz during simulated hemorrhage protects cerebral oxygenation at both low and high altitude. Arterial pressure oscillations at 0.1 Hz are endogenously driven by rhythmic fluctuations in sympathetic nerve activity. As hypobaric hypoxia increases basal sympathetic activity, we hypothesize that the amplitude of forced oscillations in arterial pressure and cerebral blood flow during simulated hemorrhage will be greater at high altitude compared to low altitude. Methods: 8 healthy human participants (4 M, 24.7 ± 4.1 y; 4 F, 34.3 ± 8.3 y) underwent a hypovolemic oscillatory lower body negative pressure (OLBNP) protocol, where chamber pressure reduced to -60 mmHg then oscillated every 5-s between -30 mmHg and -90 mmHg over 10-min (0.1 Hz). This protocol was performed at both low altitude (LA; Calgary, Alberta, Canada; 1045 m) and high altitude (HA; White Mountain, California, USA; 3800 m). Mean arterial pressure (MAP), mean middle cerebral artery velocity (MCAv), and cerebral tissue oxygenation (ScO2) were recorded continuously. Frequency analysis (via continuous wavelet transform) was used to quantify oscillations in MAP and mean MCAv at ~0.1 Hz. Data were analyzed with linear mixed-models and paired t-tests. All data are represented as mean ± SD. Results: Baseline amplitude of oscillations were similar between HA and LA for MAP (1.9 ± 0.6 mmHg vs. 1.2 ± 0.5 mmHg; P = 0.47) and mean MCAv (0.9 ± 0.4 cm/s vs. 1.1 ± 0.3 cm/s; P = 0.91). Oscillatory amplitudes increased with 0.1 Hz OLBNP and altitude for MAP (ANOVA main effect, OLBNP: P < 0.001, Altitude: P = 0.007) and mean MCAv (ANOVA main effect, OLBNP: P = 0.002, Altitude: P = 0.008). Amplitude of oscillations during OLBNP were greater at HA for both MAP (4.0 ± 2.1 mmHg vs. 2.6 ± 1.4 mmHg, P = 0.05) and mean MCAv (2.4 ± 1.1 cm/s vs. 0.9 ± 0.4 cm/s; P = 0.01). The relative (%Δ) decrease in ScO2 was not different between HA and LA (-0.63 ± 0.92 % vs. -2.56 ± 2.61 %, P = 0.11). Conclusions: Oscillatory amplitudes at 0.1 Hz in both MAP and mean MCAv increased during OLBNP at high altitude. This effect may be due, in part, to the sympathoexcitatory stimulus of hypobaric hypoxia, and does not alter the protection of cerebral tissue oxygenation in this environment.
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    Spatial Transcriptomics Reveal Potential Sex Differences in Supraoptic Nucleus Gene Expression of Adult Rats
    (2022) Nguyen, Dianna H.; Phillips, Nicole; Cunningham, Joseph
    Purpose: The supraoptic nucleus (SON) of the hypothalamus contains magnocellular neurosecretory cells that play a key role in the regulation of fluid and electrolyte homeostasis. Although there are many well-known sexually dimorphic regions of the hypothalamus, little is known about possible sex differences in gene expression in the SON. Our study aims to address this knowledge gap by leveraging spatially-resolved transcriptomics to better visualize gene expression profiles of cells in the SON of male and female rats and gain insight on their physiological functions without sacrificing morphological context. Methods: Visium Spatial Gene Expression (10x Genomics) was used to obtain spatially-resolved gene expression data for the SON of adult male (n=4) and female (n=4) Sprague-Dawley rats. Briefly, each brain was sectioned at 10µm thickness to collect coronal sections (~4x4mm) containing the SON and other brain structures. Each section was then mounted on the capture areas of Visium slides containing probes that bind mRNA. Next, the sections underwent the following workflow: 1) sample staining and imaging, 2) cDNA library preparation, 3) sequencing, and 4) analysis/data visualization. Data were analyzed using 10x Genomics' Space Ranger and Loupe Browser applications and other bioinformatic tools. Results: Gene cluster analysis successfully differentiated myelinated fiber tracts from nuclei and identified several distinct neuronal populations in the coronal brain sections from both male and female rats. From the list of significant genes after performing differential expression analysis on the SON region via Loupe Browser, 22 genes (e.g., Avp and Oxt) were common to both sexes, 24 genes were unique to the females, and no genes were unique to the males. Gene Ontology (GO) Enrichment and pathway analyses revealed GO terms and pathways related to: 1) neurohypophyseal hormone activity, regulation of peptide hormone secretion, and regulation of ion transport for the significant genes common to both males and females and 2) endomembrane system and glycerophospholipid metabolism pathway for the significant genes unique to females, as some examples. Further interrogation of the significant genes with Ingenuity Pathway Analysis showed some overlapping networks and common upstream regulators; however, differences between the male and female groups were also identified in upstream regulators, such as Creb, Pka, and LEPR unique to the males and insulin, Cdkal1, and HIF1A unique to the females. Conclusions: These spatially-resolved transcriptomic data suggest potential sex differences in SON gene expression that may be associated with basic endomembrane structure and function and phospholipid metabolism/signaling. Future spatial transcriptomic studies will investigate changes in SON gene expression that contribute to sex differences in cellular mechanisms involved in body fluid homeostasis and possibly pathophysiology.
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    Hydroxyethyl Starch-Enhanced Flush Initially Minimizes but Ultimately Exacerbates Edema of Machine Perfused Porcine Kidneys
    (2022) Wade, Michael; Ramos, Katherine; Reyad, Ashraf; Williams, Arthur; Yurvati, Albert; Mallet, Robert T.; Horani, Casey
    Hydroxyethyl Starch-Enhanced Flush Initially Minimizes but Ultimately Exacerbates Edema of Machine Perfused Porcine Kidneys. Michael Wade, Katherine Ramos, Casey Horani, Ashraf Reyad, Arthur G. Williams Jr., Albert H. O-Yurvati, Robert T. Mallet. PURPOSE: Improved preservation of explanted kidneys is essential to narrow the supply vs. demand disparity for transplantable human kidneys. Edematous expansion of the explanted organ may make explanted kidneys unsuitable for transplant. This study evaluated the impact of initial flushing of porcine kidneys with a solution containing hydroxyethyl starch (HES), an osmolyte commonly used in preservation solutions for kidney transplantation, on edema during subsequent hypothermic machine perfusion METHODS: This study utilized kidneys from Yorkshire swine due to the anatomical and functional similarities of porcine and human kidneys. Left kidneys were harvested from isoflurane-anesthetized pigs via laparotomy. The renal artery was cannulated and the organ was flushed for 10 min with 400 mL of control Ringer's solution (group A) or with Ringer's solution containing 50 g/L hydroxyethyl starch (group B). Kidneys then underwent hypothermic machine perfusion (2-4°C) for 21-72 hr in a LifePort organ preservation system, with flow rates and resistance recorded throughout perfusion. The kidneys were weighed before and after flush of the organ, weighed again after machine perfusion, and then biopsied for histological analysis of renal cortex and medulla. RESULTS: Group A kidney mass increased by 49 ± 13% (mean ± standard deviation) during initial flush (n =7) and by 52 ± 14% after flush and 72 h machine perfusion (n=8); thus, 95% of the organ expansion occurred during the initial flush. In contrast, Group B kidney mass increased by 19 ± 9% during initial organ flush (n=6), a 60% reduction vs. Group A (P < 0.001), but total weight gain was 83 ± 21% after machine perfusion (n=3). Machine perfusion of 2 of the 3 Group B kidneys failed at 21 hr and 23 hr perfusion, and only 1 was perfused for the entire 72 hours. In both groups, histology revealed preserved tubular and glomerular architecture, but appreciable cellular edema following machine perfusion. Accumulation of debris in the tubular lumina and vacuolization of tubular epithelial cells was evident in Group B, but not Group A. CONCLUSION: In explanted kidneys flushed with colloid-free Ringer's and machine-perfused for 72 h, almost the entire increase in mass occurred during the initial organ flush. As hypothesized, the addition of HES colloid sharply lowered the initial organ expansion, but unexpectedly exacerbated organ expansion during subsequent hypothermic machine perfusion, such that the HES-flushed kidneys gained more mass than the controls flushed without HES. Thus, the potential benefit of including HES in the initial flush solution was lost during machine perfusion. Arguably, the excessive expansion may have contributed to the impaired machine perfusion of these kidneys. Animal Use Protocol: IACUC-2020-0011
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    Norepinephrine innervation of the supraoptic nucleus contributes to dilutional hyponatremia in male BDL rats
    (2022) Aikins, Ato; Little, Joel; Rybalchenko, Nataliya; Cunningham, Joseph
    Purpose: Dilutional hyponatremia is a common complication associated with liver cirrhosis that is linked to inappropriate release of arginine vasopressin (AVP). Elevated plasma AVP causes water retention and hypoosmolality. In the cirrhotic liver, there is increase in resistance to blood flow resulting in portal hypertension, ascites formation, mesenteric vasodilation due to release of vasodilators and increased pooling of blood in the splanchnic circulation. The fluid redistribution decreases central vascular blood volume which is sensed by peripheral baroreceptors and volume receptors located in the aortic arch and heart. This could be relayed to A1 neurons in the caudal ventrolateral medulla (CVLM) and the A2 neurons in the nucleus tractus solitarius (NTS). The A1/A2 neurons stimulate the release of AVP from the supraoptic nucleus (SON). We propose that the A1 and A2 norepinephrine neurons in the hindbrain contribute to the activation of AVP-secreting neurons in the supraoptic nucleus (SON) leading to inappropriate AVP release and dilutional hyponatremia. Method: Anti-DBH saporin [IT-03] (Advanced Targeting Systems), a cytotoxin conjugated to an antibody against DBH was injected to the SON to lesion the norepinephrine innervation of SON including A1/A2 neurons. After two weeks, adult male rats received bile duct ligation surgery (BDL) which was used to model liver cirrhosis. In this model, the common duct that drains bile from the liver to the intestine is cauterized between two ligatures leading to obstructive cholestasis and liver cirrhosis. Four weeks after BDL surgery, rats were anesthetized with inactin (thiobutabarbital sodium salt hydrate; 100 mg/kg, i.p.). Blood samples were taken for plasma copeptin, osmolality, and hematocrit measurements. The rats were then perfused transcardially with 1M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (4% PFA) in 1M PBS. Plasma copeptin concentration was measured as a surrogate marker for AVP using commercially available copeptin ELISA kits. The brains were removed and processed for delta FosB (a marker of chronic activation), dopamine β-hydroxylase (DBH) and AVP immunohistochemistry. Results: Anti-DBH saporin (ADS) injection in the SON of BDL rats significantly decreased the number of cells positive for both delta FosB and DBH in the A1 and A2 cell groups as compared to vehicle injection (A1 neurons, ADS/BDL vs Vehicle/BDL P< 0.001; A2 neurons, ADS/BDL vs Vehicle/BDL P< 0.05). ADS treated BDL rats had fewer SON cells positive for both AVP and delta FosB as compared to vehicle injection (ADS/BDL vs Vehicle/BDL P< 0.05). Reduced colocalization of delta FosB and DBH in A1/A2 neurons was associated with a significantly lower plasma copeptin concentration in BDL rats. (ADS/BDL vs Vehicle/BDL P< 0.05). Similar effects were seen for plasma osmolality and hematocrit (ADS/BDL vs Vehicle/BDL P< 0.05). Conclusion: The result suggests that an increase in cells positive for both delta FosB and DBH in A1/A2 neurons is associated with an increase in plasma AVP and hypoosmolality in male BDL rats. Anti-DBH saporin lesions of SON prevented increases in plasma copeptin and neural activation of A1/A2 and AVP SON neurons associated with BDL.
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    Inhibitor of MyoD family isoform a (I-mfa) regulates contractile function of glomerular mesangial cells and glomerular filtration rate
    (2022) Siebert, Garland; Shotorbani, Parisa Yazdizadeh; Tao, Yu; Chen, Zheng; Ma, Rong
    Purpose: Glomerular filtration is a critical process for maintaining homeostasis of body fluid volume. This process in kidney is regulated by multiple factors inside glomeruli, including the surface area of the glomerular filtration membrane available for filtration. Glomerular mesangial cells (MCs) sit among the networks of glomerular capillaries and regulate glomerular filtration rate (GFR) by changing the surface area of filtration membrane through their contractile function. Inhibitor of MyoD family isoform a (I-mfa) was initially found as a transcription modulator. We recently found that I-mfa was present in MCs. However, the function of this protein in MCs is not known. The aim of this study was to examine if I-mfa regulated contractile response of MCs and GFR. Methods: Experiments were carried out in cultured human MCs and mice. In cultured cells, we examined the contractile function of MCs with and without overexpressing or knocking down I-mfa. I-mfa was overexpressed by transfecting MCs with I-mfa expression plasmids and the no functional I-mfb expression plasmids were used as the control. I-mfa was knocked down using siRNA approach and scramble siRNA was used as the control. MC contraction was evaluated by reduction of planar surface area of the cells in response to Ang II (1 µM) at different time points (10 - 60 min) after treatment. In animal study, we assessed glomerular filtration function in I-mfa+/+ (control) and I-mfa-/- mice (male at age of 12 weeks) using transdermal GFR measurement. Using this approach, we measured GFR by transcutaneous measurement of the elimination kinetics of the fluorescent renal marker FITC-sinistrin under conscious and freely moving conditions. Results: In cultured human MCs, Ang II (1 µM, 60 min) induced ~45% decrease in the planar surface area. This contractile response was significantly enhanced in MCs with I-mfa knocked down, but not in MCs treated with scramble siRNA. Furthermore, the Ang II-stimulated contraction was significantly blunted in MCs with overexpression of I-mfa, but not with overexpression of I-mfb. Consistent with the in vitro experiments, deletion of I-mfa (I-mfa-/-) significantly decreased GFR compared to I-mfa+/+ mice (control) (1351.4 ±51.9 vs. 890 ±166.9, P < 0.05, n= 7 vs. 4, I-mfa+/+ vs. I-mfa-/-). Conclusion: This study suggests that I-mfa inhibits the contractile response of MCs and promotes glomerular filtration function.
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    Sex differences in cerebrovascular dysfunction and hypertension in offspring of hypertensive pregnant rats
    (2022) Smith, Jonna; Powell, Madison; Cromartie, Whitney; Cunningham, Mark; Duncan, Jeremy
    PURPOSE: Offspring from preeclamptic women have a greater risk of hypertension and cerebrovascular dysfunction later in life. One of the major contributors of cerebrovascular dysfunction is impaired cerebral blood flow (CBF) autoregulation. In the USA, 1 in 25 pregnancies are preeclamptic. Preeclampsia, as defined as new-onset hypertension during pregnancy, is an inflammatory condition characterized by elevated interleukin 17 (IL-17), mitochondrial reactive oxygen species (mtROS), mitochondrial dysfunction (mt-Dys), and intrauterine growth restriction (IUGR). Preliminary work from our laboratory demonstrates that offspring from preeclamptic rats have sex differences. Males have hypertension and a greater impairment of CBF autoregulation, while females have no change in blood pressure with impaired CBF autoregulation. The exact mechanisms for the sex differences in cerebrovascular dysfunction and hypertension is unknown and is the focus of this study. We hypothesize that changes in cerebral mt-dys, cerebral mtROS, and circulating IL-17 contributes to the sex differences in hypertension and cerebrovascular dysfunction in male and female offspring from preeclamptic rats. METHODS: In this study, we compared male and female offspring from normal pregnant and preeclamptic Sprague Dawley rats. All offspring were divided into controls (CON) and IUGR by sex. Hypertensive male and non-hypertensive female offspring's with impaired CBF autoregulation were examined at 17 weeks of age for changes in cerebral mitochondrial electron transport chain (ETC) protein complexes, cerebral manganese superoxide dismutase (MnSOD), and circulating IL-17. RESULTS: Female CON (151±5% IU/Protein/male CON) and IUGR (149±8 %IU/Protein/male CON) offspring have increased MnSOD compared to CON (100±7 %IU/Protein/male CON) and IUGR (122±4 %IU/Protein/male CON) males (p< 0.05). No changes in female ETC protein complexes between IUGR and CON. Male IUGR have a decrease in complex II (71±5 vs. 100±8 %IU/Protein/male CON, p< 0.05) and V (57±2 vs. 100±10 %IU/Protein/male CON, p< 0.05) ETC proteins, and elevated IL-17 (944±370 vs. 412±115 pg/mL, ns) compared to CON males. CONCLUSION: Male IUGR offspring have mt-Dys and elevated IL-17, which may contribute to hypertension and a greater impairment in CBF autoregulation. Female IUGR offspring may be protected due to an increase in mitochondrial antioxidants. In summary, studying the dimorphic sex differences in the mechanisms of hypertension and cerebrovascular dysfunction in offspring of preeclamptic women, may improve the offspring's risk of hypertension, cardiovascular disease, stroke, and cognitive dysfunction later in life.
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    Store-operated Ca2+ entry contributed to high glucose- induced podocyte injury
    (2022) Tao, Yu; Chaudhari, Sarika; Shotorbani, Parisa Yazdizadeh; Ma, Rong; Chen, Zheng
    Purpose: Diabetic Nephropathy is one of the major complications of diabetes. Hyperglycemia is a known initiator of diabetes mellitus. Evidence suggests that podocyte injury is associated with diabetic nephropathy onset and progression. However, the mechanisms underlying podocyte injury induced by high glucose (HG) are poorly understood. Store-operated calcium entry (SOCE) is a multifunctional signaling pathway in many cell types. However, its role in podocyte injury in the settings of diabetes is not known. The present study was aimed to determine that enhanced SOCE mediated high glucose (HG)-induced podocyte injury by upregulating calpain activity. Methods: All experiments were performed using cultured human podocytes. Western blot was conducted to estimate Orai1, STIM1, and nephrin protein abundance. Ca2+ imaging was used to analyze SOCE. Confocal microscopy was used to visualize podocyte actin arrangement. Calpain activity was determined by calpain activity assay kits. Results: HG (25mM) treatment significantly increased Orai1, but not STIM1 protein abundance for time periods ranging from 2 to 12 hours. The HG-induced Orai1 response was dose dependent. Ca2+ imaging experiment showed that HG treatment for 12 hours significantly increased SOCE. In addition, HG treatment significantly decreased nephrin (a podocyte marker) protein abundance and resulted in cytoskeleton rearrangement by the formation of cortical F-actin. Both HG responses were significantly blunted by BTP2 (4 µM), a SOCE inhibitor. Furthermore, we found that activation of SOCE by thapsigargin (1 µM) increased calpain activity which was abolished by BTP2. In addition, BTP2 blunted the increased calpain activity induced by HG treatment. Moreover, calpeptin (a calpain inhibitor) attenuated the HG-induced reduction of nephrin protein abundance. Conclusions: The present study suggests that enhanced SOCE contributes to HG-induced podocyte injury by increasing calpain activity.
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    Travel-Induced Stress at Varying Ages Modulates the Pathogenesis of Autoimmunity in Female Lupus Mice
    (2022) Dinh, Viet; Chaudhari, Sarika; Mathis, Keisa W.;
    Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that causes inflammation throughout the body, notably in the kidneys. Inflammatory flares that occur during the pathogenesis of SLE increase morbidity in humans and are associated with stress and environmental factors. Heat shock proteins (HSPs) are chaperone proteins that are elevated in the stressful conditions, especially HSP90 which is increased in subsets of SLE patients. HSP90 is also correlated with interleukin (IL)-6, a proinflammatory cytokine known to stimulate autoimmune processes in many diseases including SLE. The lupus mice used in our lab travel to our university from Bar Harbor, ME. However, it is not known if this travel period induces stress on these mice that significantly affects the pathogenesis of SLE and renal damage. Likewise, it is unknown if the age at which the mice travel also predicts outcomes of the disease. Based on this, we hypothesize that stressors that occur early in life have a greater impact on the pathogenesis of SLE and renal inflammation than stressors that occur during adulthood in lupus-prone mice. We used female NZBWF1 mice - an established model of SLE - that traveled to our university at 6 weeks of age (pre-pubertal stage) or at 19 weeks of age (mature adult stage) to compare the effects of travel at different ages of life. At 30 weeks of age, they were placed in metabolic cages weekly to collect urine samples. Upon reaching 35 weeks of age, a point at which these mice usually develop severe lupus symptoms, we collected blood samples, euthanized the mice, and collected tissues. Urine, plasma, and homogenized right kidneys were analyzed via ELISA to compare various biomarkers, including double-stranded (ds)DNA autoantibodies (a hallmark of SLE), urinary albumin (a marker of renal injury), IL-6, and HSP90. No differences were found in plasma dsDNA autoantibodies between mice that traveled in younger life vs. in adulthood (Young: 6.0e5 ± 8.4e4 vs. Adults: 5.2e5 ± 7.6e4 U/mL; p=0.6930). Likewise, no differences were found in urinary albumin between these groups (Young: 1.2e7 ± 3.2e6 vs. Adults: 1.0e7 ± 8.4e6 ng/mL; p=0.8208). In mice that traveled as adults, IL-6 was significantly higher in plasma (Young: 195.8 ± 87.4 vs. Adults: 826.7 ± 130.0 pg/mL; p=0.0096) and in kidneys (Young: 177.1 ± 60.6 vs. Adults: 798.1 ± 166.5 pg/mL; p=0.0002). Levels of plasma HSP90 were lower in mice that traveled as adults (Young: 152.1 ± 23.4 vs. Adults: 65.70 ± 8.60 ng/mL; p=0.0302). In conclusion, our data indicate that mice subjected to travel-induced stress as adults developed higher levels of plasma and renal IL-6 and lower levels of plasma HSP90 at 35 weeks of age. We will continue to evaluate these outcomes in more mice and further data will expand on these findings, which will act as an important steppingstone for furthering our understanding of how environmental stressors play a role in the progression of autoimmunity well into adulthood.
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    Effect of systemic administration of α7-nicotinic acetylcholine receptor ligands on renal inflammation in young mice with systemic lupus erythematosus
    (2022) Brooks, Calvin D.; Young-Stubbs, Cassandra M.; Shimoura, Caroline; Dinh, Viet; Chaudhari, Sarika; Uteshev, Victor; Mathis, Keisa W.
    Systemic lupus erythematosus (SLE) is an autoimmune disease where renal inflammation contributes to hypertension. The cholinergic anti-inflammatory pathway is a recently described pathway where stimulating the vagus nerve causes release of acetylcholine from choline acetyltransferase (ChAT)+ T-cells in the spleen. This acetylcholine acts on alpha-7 nicotinic acetylcholine receptors (α7nAChR) of immune cells to hault the production of pro-inflammatory cytokines. Our lab has shown stimulation of this pathway at multiple levels lessens autoimmunity, renal inflammation and hypertension in SLE mice. However, our recent attempts to target the α7nAChR directly with a positive allosteric modulator (PAM) in mice with advanced SLE have not yielded similar results. This may be due to decreased parasympathetic tone in these mice in which the PAM is not able to compensate for. The aim of the current study was to determine if activating the α7nAChR in SLE mice at an earlier age, before dampening of parasympathetic tone, prevents the onset of hypertension and renal inflammation. Twelve week old female NZBWF1 mice, which spontaneously develop SLE, and NZW controls were given a partial agonist of the α7nAChR, GTS-21, a PAM, PNU-120596, or vehicle continuously for two weeks via subcutaneous osmotic mini-pump. Mean arterial pressure (MAP) was measured by carotid artery catheter in conscious, freely moving mice at 14 weeks. Mice were then euthanized and blood, spleen and kidneys harvested to allow measurement of plasma double stranded (ds) DNA autoantibodies via ELISA to assess severity of disease. There was no difference in dsDNA autoantibody activity (U/mL) between SLE mice and controls (all data presented as mean±SEM; 76026.3±38901.4 vs. 19617.4±4092.7; p=0.1141). The treatments had no effect on autoantibody activity in SLE mice [76026.3±38901.4 (SLE vehicle) vs. 36951.7±5962.3 (SLE PNU) vs. 56279.7±31381.0 (SLE GTS)] or controls [19617.4±4092.7 (Con vehicle) vs. 17293.2±3384.1 (Con PNU) vs. 16016.2±3059.6 (Con GTS)]. MAP (mmHg) did not differ significantly between young SLE and control mice (143.53±3.26 vs. 128.8±4.95). Additionally, the treatments had no effect on MAP of SLE mice [143.53±3.26 (SLE vehicle) vs. 128.32±10.92 (SLE PNU) vs. 129.56±19.50 (SLE GTS)] or controls [128.8±4.95 (Con vehicle) vs. 127.60±4.43 (Con PNU), vs. 125.65±5.54 (Con GTS)]. Based on these results, we suspect that the disease process has not progressed enough in 14-week-old mice to see differences due to these treatments. Although the changes in the blood pressure and dsDNA antibodies are not significant, we will continue to evaluate renal damage and cytokine profile to determine the effect of these α7nAChR ligands on pathogenesis of SLE. Future studies will aim to modulate α7nAChRs in SLE mice before the onset of disease (~12 weeks of age) through 35 weeks when mice usually experience terminal disease to determine efficacy of early activation of the cholinergic anti-inflammatory pathway in halting the progression of SLE.
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    Optimizing Preservation Solutions for Hypothermic Machine-Perfused Porcine Kidneys
    (2022) Ramos, Katherine; Wade, Michael; Williams, Arthur; Mallet, Robert T.; Yurvati, Albert
    Background: With the mounting incidence of hypertension, type 2 diabetes and other risk factors, end-stage renal disease (ESRD) is increasingly prevalent in the U.S. and worldwide, especially among racial minorities including Hispanics and African Americans. Kidney transplant is the most effective treatment for ESRD, but the supply of transplantable kidneys is inadequate to meet the increasing demand. Before transplantation, kidneys from deceased donors are maintained in a device (LifePort) that pumps ice-cold preservation solution through the organ. Although this hypothermic machine perfusion (HMP) is the most effective kidney preservation method available, kidneys from deceased donors often fail to resume function after transplant, especially when HMP is prolonged. Consequently, any kidneys not transplanted within 30 hours of harvest are discarded. We propose that optimizing the composition of the preservation solution may prevent post-transplant kidney failure and thereby improve kidney transplant outcomes, and potentially extend the useful lifespan of explanted kidneys beyond 30 hours, thereby increasing the pool of available kidneys for transplant. Hypothesis: Hypothermic perfusion of kidneys with solutions containing pyruvate, an energy substrate, anti-oxidant and anti-inflammatory compound, will better maintain perfusion flow and lower vascular resistance, indicating improved stability of the explanted organs. Methods: Kidneys obtained from thirteen anesthetized female Yorkshire pigs were fluid-flushed and then perfused for 72 h with modified, cold (2-4?C) Ringer's solution containing 20 mM glucose, 30 mM mannitol, 50 g/l hydroxyethyl starch ? 20 mM pyruvate in a LifePort organ preservation device while flow and renal vascular resistance were monitored and recorded. Results: Kidneys maintained adequate perfusion for 72 hours despite a 50 ? 4% (mean ? SEM) increase in kidney mass indicating edema. Flows plateaued within the first 6 hours of perfusion and then declined slowly with prolonged perfusion. At 6 h perfusion, flows (ml/min*g) were 0.54 ? 0.06 in kidneys receiving control solution, and 0.61 ? 0.06 in kidneys perfused with pyruvate-enriched solution. At 24 and 72 h, flows were 0.49 ? 0.06 and 0.38 ? 0.04 in the control kidneys, and 0.49 ? 0.03 and 0.37 ? 0.02 in the kidneys perfused with pyruvate enriched solution, respectively. The increases in mass of the control (52?4%) and pyruvate (46?6%) over 72 h perfusion did not differ. Conclusions: Pyruvate augmentation of preservation solution did not affect hypothermic kidney perfusion. The possibilities that pyruvate may affect the kidney's energy and/or antioxidant metabolism without altering organ perfusion or edema, and that measures limiting edema could improve kidney perfusion during HMP, remain to be tested.