Overnight Urine Collections Research Articles

The effect of chronic unpredictable stress on renal mitochondrial function in female mice

Background: Chronic stress can induce high blood pressure and lead to decreased renal function by mechanisms that remain unclear. The exposure of rodents to chronic unpredictable stress (CUS) is a translational model used for investigating the physiological impact of stress. The CUS protocol consists of unpredictable and random stressors that mimic real life exposure to stress. While stress is known to impact mitochondrial function, little is understood about the direct impact of chronic stress on renal mitochondrial respiration. Moreover, while studies have shown a link between estrogen and mitochondria through estrogen receptors, the relation between stress induced renal mitochondrial dysfunction and estrous cycle is unknown. Hypothesis: Exposing female mice to CUS will increase blood pressure and cause decreased renal mitochondrial respiration. Methods: We exposed adult female C57BL/6 mice to CUS for 28 days. Before and after stress, all control (CON, n=8) and CUS (n=10) mice were placed in metabolic cages for overnight urine collection. Urine albumin was measured by dipstick assay. On day 28, mice were assessed for blood pressure measurement via tail cuff. 24 hours later, mice were euthanized, and vaginal lavage was performed. The left kidney was removed, transected into thirds, and the poles were placed in relaxing buffer. Kidney mitochondrial oxygen consumption was assessed using high-resolution respirometry. Results: Data were stratified by phases of the estrus cycle. Neither blood pressure nor urinary albumin was significantly altered by CUS in stressed females. Females in estrus and proestrus exposed to CUS (n=5) exhibited a decrease of 20.8% ± 5.1 (n=5, p=0.03) in renal mitochondrial maximal uncoupled respiration compared to non-stressed CON. Renal mitochondrial respiration in diestrus was similar between CUS and CON. Conclusion: Results from this study reveal a link between the female estrus cycle, stress, and mitochondrial function. This work was supported by NIH grants P20GM109091 (F.H.), R01HL130972-01A1 (F.G.S.), R01HL5949 (F.G.S.), RO1DK132948 (C.W. & F.P.) and R01 MH129798 (SKW); VA grants: VISN7 RDA (F.H.), Merit awards: BX000168-10A1 (F.G.S.), BX005320 (F.G.S.), and BX002604 (M.J.R.). 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.

Abstract 028: Toll-Like Receptor 4 Promotes Renal Immune Cell Infiltration And The Development Of Renal Damage During Dahl Salt-Sensitive Hypertension

Previous work in our laboratory has implicated a significant role for the immune system in potentiating the hypertensive and kidney damage phenotypes which occur in the Dahl Salt-Sensitive (SS) rat. Toll-like receptors (TLRs), usually expressed on macrophages, play a key role in the innate immune system. A number of TLRs have been implicated in cardiovascular disease and chronic kidney disease with particular interest in TLR4, which specifically binds gram-negative lipopolysaccharide or endogenous molecules produced as a result of tissue injury. We previously performed a transcriptomic analysis which demonstrated TLR4 upregulation in renal medullary tissue of Dahl SS rats fed a high salt (4.0% NaCl) diet. The present study tests the hypothesis that TLR4 influences high salt diet-induced hypertension and renal damage in the Dahl SS rat. Using CRISPR/Cas9 technology, a mutation was generated in the TLR4 gene on the Dahl SS background, creating a 1-bp frameshift deletion resulting in a premature stop codon. SS TLR4-/- males and parental Dahl SS males (SS TLR4+/+ ), maintained on a 0.4% NaCl chow, underwent carotid artery telemetry implantation at 7 weeks of age. After recovery and baseline mean arterial pressure (MAP) recordings (day 0: 125.6 ± 0.5 vs 124.2 ± 2 mmHg), animals were switched to a 4.0% NaCl chow for 3 weeks. Overnight urine collections occurred once during the low salt period and weekly throughout the high salt challenge. Kidneys were flushed prior to euthanasia. Although SS TLR4-/- MAP was not different from SS TLR4+/+ during the high salt challenge (day 21: 176.8 ± 14 mmHg, n=2 vs 172.3 ± 18 mmHg, n=4), high salt day 21 urinary albumin excretion was significantly reduced in SS TLR4-/- (272.7 ± 65 mg/day, n=5, p<0.01) compared to SS TLR4+/+ (427.5 ± 67 mg/day, n=7), indicating reduced renal damage in TLR4 absence. Furthermore, flow cytometric analysis of isolated renal immune cells indicated a 33% reduction in CD45+ total leukocytes (5.0 ± 1.2 vs 3.4 ± 0.9 x10 6 cells) and a 31% reduction in CD11bc+ monocytes/macrophages (3.9 ± 0.9 vs 2.6 ± 0.7 x10 6 cells) in SS TLR4-/- (n=5) vs SS TLR4+/+ (n=7) kidneys. Together, these data indicate that TLR4 may drive the immune response during the induction and progression of salt-sensitive hypertension and renal disease.

Urinary Concentrations of Triclosan, Bisphenol A, and Brominated Flame Retardants and the Association of Triclosan with Demographic Characteristics and Body Fatness among Women with Newly Diagnosed Breast Cancer.

Background: Triclosan, bisphenol A (BPA), and brominated flame retardants are environmental estrogenic endocrine-disrupting compounds that may influence the prognosis of breast cancer. We examined the urinary concentrations of these compounds and their associations with demographic characteristics and body fatness in a population of women with newly diagnosed breast cancer. Methods: Overnight urine collection and anthropometric measures were obtained from 302 participants. Triclosan, BPA, tetrabromobisphenol A (TBBPA), and tetrabromobenzoic acid (TBBA) concentrations were determined using ultra-performance liquid chromatography–tandem mass spectrometry. Regression analyses were conducted to examine associations of urinary compound concentration with age, menopause, race, ethnicity, educational level, estrogen receptor status, body size, and body composition. Results: Triclosan, BPA, and TBBA were detected in urine samples from 98.3%, 6.0%, and 0.3% of patients, respectively; TBBPA was undetectable. Among patients with quantifiable values, the geometric mean concentrations were 20.74 µg/L (27.04 µg/g creatinine) for triclosan and 0.82 µg/L (1.08 µg/g creatinine) for BPA. Body mass index ≥ 30 vs. <25 kg/m2 was associated with lower creatinine-corrected urinary concentrations of triclosan (−40.00, 95% confidence interval [CI] = −77.19 to −2.81; p = 0.0351). The observed association was predominantly in postmenopausal women (−66.57; 95% CI: −109.18% to −23.96%). Consistent results were found for associations between triclosan levels and fat mass variables. Conclusion: In this study population, women with newly diagnosed breast cancer had triclosan exposure. Assessments of the implications of urinary concentrations of triclosan for women should consider body fatness and menopausal status.

Evaluation of CYP1A2 activity: Relationship between the endogenous urinary 6-hydroxymelatonin to melatonin ratio and paraxanthine to caffeine ratio in dried blood spots.

The suitability of the endogenous 6‐hydroxymelatonin/melatonin urinary metabolic ratio as a surrogate for the paraxanthine/caffeine ratio to predict cytochrome P450 1A2 (CYP1A2) activity was assessed in this study. Twelve healthy volunteers completed four study sessions spread over 1 month (including overnight urine collection with first morning voids collected separately). Except for the third session, volunteers were asked to abstain from methylxanthine‐containing beverages and foods at least 24 h before urine collection. At the end of urine collection, subjects were given a caffeinated beverage and capillary blood samples were collected 2 h after the drink administration. A significant linear relationship between the 6‐hydroxymelatonin/melatonin ratios from 12‐h urine samples and first morning voids was observed (R 2 = 0.876, p < 0.0001). In contrast to the paraxanthine/caffeine ratio, consumption of methylxanthine‐containing beverages during session three did not significantly influence the 6‐hydroxymelatonin/melatonin ratios compared with the other sessions requiring abstinence from caffeine. A larger intra‐ and interindividual variability in the 6‐hydroxymelatonin/melatonin ratios compared with the paraxanthine/caffeine ratio was also observed. A very weak correlation was observed between the paraxanthine/caffeine ratio and both of the endogenous 6‐hydroxymelatonin/melatonin ratios (Pearson r < 0.35, p < 0.05). All these results question whether this endogenous metric could adequately reflect CYP1A2 activity or substitute for the probe caffeine. Additional studies with larger study samples are needed to examine this endogenous metric in more details.

Relationship of the Intake of Water and Other Beverages With Renal Endpoints: Cross-Sectional and Longitudinal Data—Observational, Population-Based Study

The relationship of water intake with kidney function in the population is uncertain. This study investigated cross-sectionally and longitudinally the relationship of the intake of water and other beverages with kidney function within an adult Italian population sample. In 4,554 Gubbio Study examinees (54.4% women, age 18-95years), data collection at baseline included demographics, anthropometry, questionnaires on habitual intakes of water and other beverages (non-water fluids), a timed overnight urine collection, estimated glomerular filtration rate (eGFR), decreased eGFR (<60mL/minute/1.73m2), and other variables including urinary markers of diet. At 15-year follow-up, the incidence of renal/kidney replacement therapy, the eGFR change from baseline, and the incidence of decreased eGFR were used as indices of kidney function change over time. In multivariable analyses, higher water intake is independently related to higher urine flow (beta=0.163, P<.001), lower urine osmolality (beta=0.184, P<.001), lower eGFR (beta=0.030, P=.002), and higher prevalence of decreased eGFR (logisticcoefficient±standard error=1.13±0.32, P<.001). Water intake did not relate to kidney function change over time. Intake of non-water fluids did not independently relate to urinary indices nor to kidney function. In the general population, water intake relates cross-sectionally to urine flow, urine concentration, and kidney function but it does not relate to kidney function change over time. The intake of other beverages does not relate to urinary indices or kidney function. Results do not support a role of water intake in kidney function decline over time in the population.

Abstract MP21: Suppression Of Reactive Oxygen Species Protects The Dahl Salt-sensitive Rat From Developing Maternal Syndrome

The Dahl Salt-Sensitive (SS) rat maintained on a low salt diet exhibits a differential response to pregnancy; approximately half develop maternal syndrome (hypertension and proteinuria) while the other half are protected. The exact mechanisms of maternal syndrome are unclear, but it is proposed that reactive oxygen species (ROS) are involved. Previous studies from our laboratory demonstrated the importance of NADPH oxidase (NOX) 2-derived ROS produced by immune cells in the development of salt-sensitive hypertension and renal disease. Given the role of NOX2-derived ROS in the Dahl SS rat, we tested the hypothesis that genetic deletion of the p67phox subunit of NOX2 (SS Ncf2-/- ) will result in the protection from developing maternal syndrome. To test this hypothesis, 8 week old female Dahl SS and SS Ncf2-/- were instrumented with radio-telemeters for continuous measurement of mean arterial pressure (MAP). After a recovery period, baseline MAP was measured as well as an overnight urine collection for markers of renal damage before females were mated with males of the same strain and age. Virgin litter-mate controls were run in parallel. As expected, MAP and protein excretion levels were comparable at baseline between virgin and mated SS Ncf2-/- rats. These parameters remained comparable at the late pregnancy time point (108±3 vs 112±1mmHg, 9±3 vs 16±5mg/day, p &gt;0.05). In contrast to the SS preeclamptic pregnancy, the SS Ncf2-/- mated rats are protected from the development of maternal syndrome (141±8 vs 112±1mmHg, p &lt;0.001; and 98±16 vs 16±5mg/day, p &lt;0.001; in the SS vs SS Ncf2-/- , respectively). This protection is accompanied with a significant reduction in uterine artery resistance index which is a marker of impaired blood flow, in the SS Ncf2-/- (0.85±0.03 vs 0.59±0.02, p &lt;0.01). Despite these phenotypic differences, the number of infiltrating immune cells into kidneys or placentas do not differ between these two groups. These results suggest that NOX2-derived ROS contribute to the pathogenesis of maternal syndrome in the Dahl SS rat, and further investigations are necessary to clarify the source of the ROS within key organs such as the kidney and placenta.

Dose-dependent detoxication of the airborne pollutant benzene in a randomized trial of broccoli sprout beverage in Qidong, China

Airborne pollutants have collectively been classified as a known human carcinogen and, more broadly, affect the health of hundreds of millions of people worldwide. Benzene is a frequent component of air pollution, and strategies to protect individuals against unavoidable exposure to this and other airborne carcinogens could improve the public's health. Earlier clinical trials in Qidong, China, demonstrated efficacy in enhancing the detoxication of benzene using a broccoli sprout beverage. A randomized, placebo-controlled, multidose trial of a broccoli sprout beverage was designed to determine the lowest effective concentration that enhances benzene detoxication adjudged by enhanced excretion of the urinary biomarker, S-phenylmercapturic acid (SPMA). Following informed consent, 170 subjects were randomly assigned in 5 blocks of 34 each to drink either a placebo beverage (n=55) or 1 of 3 graded concentrations of a broccoli sprout beverage [full (n=25), one-half (n=35), and one-fifth (n=55)] for 10 consecutive days. Concentrations of SPMA arising through induced benzene conjugation with glutathione were quantified by MS in sequential 12-h overnight urine collections during the intervention. MS was also used to quantify urinary sulforaphane metabolites in each dosing regimen that resulted in a median 24-h urinary output of 24.6, 10.3, and 4.3 µmol, respectively, confirming a dose-dependent de-escalation of the inducing principle within the beverage. A statistically significant increase in benzene mercapturic acids in urine was found for the high-dose group (+63.2%) during the 10-d period. The one-half dose (+11.3%) and one-fifth dose groups (-6.4%) were not significantly different from placebo controls. An intervention with a broccoli sprout beverage enhanced the detoxication of benzene, an important airborne pollutant, when dosed at a concentration evoking a urinary elimination of ∼25 µmol sulforaphane metabolites per day, and it portends a practical and frugal population-based strategy to attenuate associated long-term health risks of air pollution. This trial was registered at clinicaltrials.gov as NCT02656420.

Liposome Delivery Enhances Clodronate Nephrotoxicity in Dahl SS Hypertension and Renal Injury

Studies in the Dahl Salt‐Sensitive (SS) rat have shown that infiltrating renal CD3+ T cells and ED‐1+ macrophages are involved in the development of SS hypertension (HTN) and renal damage. We have previously demonstrated the importance of infiltrating cells by pharmacological immunosuppression and genetic deletion of adaptive immune cells, but the role of innate immunity in SS HTN is still unclear. It is practically more difficult to assess the contribution of the innate immune system macrophage to this pathology due its vital role in development and in bone and liver physiology. A proposed method of macrophage depletion is via liposome‐encapsulated Clodronate which has shown mixed results, from attenuation of Ang‐II induced HTN, to inducing salt‐sensitive HTN. This study tests the hypothesis that Clodronate‐Liposome treatment will attenuate Dahl SS HTN and renal damage through macrophage depletion. To do so, animals were administered either Clodronate‐Liposomes (Clod‐Lipo) or PBS‐Liposomes (PBS‐Lipo) as a vehicle control. A third group of animals were treated with Clodronate alone (Clod) to assess direct effects of the drug. 7‐week‐old male Dahl SS rats were implanted with radiotelemeters and after recovery and baseline pressure recordings on a low‐salt (0.4% NaCl) diet, animals received a tail vein injection of either PBS‐Lipo (equivalent volume), Clod‐Lipo (50mg/kg), or Clod (50mg/kg). Booster injections (25mg/kg) were administered every 7 days thereafter. Three days after the initial injection, all animals were switched to a high‐salt (4.0% NaCl) diet. Animals were followed for 21 days of the high‐salt diet with weekly overnight urine collections. Clod treatment alone did not alter circulating or splenic levels of macrophages unlike Clod‐Lipo treatment which depleted macrophages by about 50%. Contrary to our hypothesis, by the end of the study, Clod‐Lipo treated animals developed an exacerbated SS response in respect to blood pressure (Clod‐Lipo 178.7±7.5mmhg, PBS‐Lipo 157.2±3.9mmhg, p&lt;0.01 Two‐way RM‐ANOVA, n=8/7) and albuminuria (Clod‐Lipo 403.6±32.8mg/day, PBS‐Lipo 180.6±22.4mg/day, p&lt;0.001 Two‐way RM‐ANOVA, n=11/7). This exacerbation in the Clod‐Lipo group was accompanied by an increase in infiltration of adaptive T and B cells despite a depletion of circulating macrophages and a reduction in infiltrating macrophages. The third group of animals receiving Clod alone demonstrated a similar, though delayed, exacerbation in the SS increase in blood pressure (181.6±6.2mmhg p&lt;0.05 Two‐way RM‐ANOVA, n=4). In addition, Clod treated animals showed elevated albuminuria (259.2±48.6mg/day, p&lt;0.05 Two‐way RM‐ANOVA, n=5) though not to the same extent as Clod‐Lipo treated animals. These SS phenotypic changes in Clod treated animals were independent of changes to circulating, splenic, and infiltrating renal immune cells. This study demonstrates Clodronate's direct nephrotoxic effects similar to those of other bisphosphonates. Furthermore, liposome delivery of Clodronate further exacerbated the SS renal damage and lymphocyte infiltration, indicating the potential adverse effects of this macrophage depletion strategy.Support or Funding InformationHL‐116264, HL‐137748, and 18PRE3400038This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of the MCP‐1/CCR2 Axis in the Development of Dahl Salt‐Sensitive (SS) Hypertension and Renal Damage

Studies examining mechanisms of SS hypertension have implicated the infiltration of immune cells into the kidneys which contribute to elevated blood pressure and associated renal damage. However, the signaling pathways by which immune cells traffic to the kidneys remain poorly understood. The present set of studies were performed to test the hypothesis that the MCP‐1/CCR2 pathway contributes to the development of hypertension and renal damage in Dahl‐SS rats fed a high salt diet by modulating renal immune cell infiltration. Monocyte Chemoattractant Protein‐1 (MCP‐1) was nominated from a transcriptomic study of kidneys from Dahl‐SS rats fed either a Low Salt (LS‐0.4% NaCl) or a High Salt (HS‐4.0% NaCl) diet. Of all the chemokines examined, MCP‐1 was one of only two significantly upregulated in kidneys following HS diet. As measured by ELISA, initial experiments demonstrated increased urinary excretion of MCP‐1 in Dahl‐SS rats switched from a LS to a HS diet within seven days of HS (11.2±1.3 vs 1.6±0.4 μg/24hr at LS baseline, n=4/group), while there was no difference in serum. To test the functional role of MCP‐1 in SS hypertension, seven‐week‐old male Dahl‐SS rats maintained on a LS diet were instrumented with radio‐telemeters for continuous measurement of blood pressure. After 1 week of recovery, LS baseline blood pressure was measured for 1 week and then the rats were switched to HS and randomly divided into two groups (n=6/group) to receive daily intraperitoneal shots of either CCR2 (MCP1 receptor) antagonist (RS102895 at 5mg/kg/day in DMSO) or DMSO vehicle for 3 weeks. Overnight urine collections were performed at baseline and then weekly until the conclusion of the study. While LS baseline mean arterial pressure (MAP) was similar in both groups (124.7±2.2 vs 124.1±1.7 mmHg in vehicle group), the rats receiving CCR2 antagonist had significantly blunted hypertension after 3 weeks of HS diet compared to the vehicle group (146.3±4.1 vs 160.1±7.3 mmHg respectively), p‐value&lt;0.001 by two‐way ANOVA. This protection was mirrored in the urinary albumin excretion rate where both groups had comparable LS baseline levels (12.6±4.4 vs 16.3±4 mg/24hr in vehicle group), but the CCR2 antagonist group had significantly lower albumin excretion rate after 3 weeks of HS diet compared to vehicle (42.2±8.3 vs 94.4±16.3 mg/24hr respectively) p‐value=0.026. When examining renal immune cell infiltration, we observed a nonsignificant reduction in infiltration of total leukocytes in the drug treated rats when compared to vehicle treated rats (2.1±0.3 vs 3.3±0.6 X10^6 CD45+ cells/kidney p=0.141). The drop was primarily due to a decrease in CD11b/c+ cells of the monocyte/macrophage lineage. These studies are the first to demonstrate an important role of the MCP‐1/CCR2 pathway in the development of hypertension, renal damage and renal immune cell infiltration in the Dahl‐SS rat. These studies prompt further investigation into this mechanism and the specific subsets of immune cells targeted. Follow‐up studies are ongoing to assess effect of blocking this pathway only in the kidneys to determine renal specific effects.Support or Funding InformationSupported by HL137748, HL116264 and AHA 15SFRN2391002.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Substitution of Casein Dietary Protein with Wheat Gluten Protein Protects Dahl Salt Sensitive Rats from the Development of Maternal Syndrome

Previously our group demonstrated that the maternal diet of the Dahl Salt‐Sensitive (SS) rat, specifically the protein source, during gestation and lactation can impact the severity of salt sensitive hypertension and renal damage of the offspring in adulthood. Offspring born from mothers fed a wheat gluten‐based diet exhibited blunted salt‐sensitive hypertension and renal damage when compared to offspring born from casein fed mothers. Utilizing an embryo transfer approach we determined that regardless of whether an embryo originated from parents fed a casein or a wheat gluten‐based diet, the diet of the surrogate mother that determined the severity of salt‐sensitivity in the offspring. In addition to the maternal diet, the in utero environment has been shown to contribute to the programming of lifelong health of offspring. Dahl SS rats maintained on a casein‐based diet (SSC) develop maternal syndrome (hypertension and proteinuria) compared to virgin controls. Since offspring from mothers fed a wheat gluten based diet were protected from SS hypertension and the Dahl SSC rat develops maternal syndrome, we tested the hypothesis that mothers fed a wheat gluten (SSWG) diet will be protected from the development of maternal syndrome. To test this hypothesis Dahl SSWG and SSC rats were studied on their respective diets, which are identical in every aspect except for the protein source. At 8 weeks of age, females were instrumented with radio‐telemeters for continuous measurement of mean arterial pressure (MAP). After a recovery period, baseline MAP was measured as well as an overnight urine collection for markers of renal damage before females were mated with males of the same age and diet. Virgin littermate controls of the same age and diet for each strain were run in parallel. During the baseline period, there was no difference observed in MAP or protein excretion rate among any of the groups. As previously reported, the SSC mated rats develop an increase in MAP (141±3 vs 129±3 mmHg, p&lt;0.05) and protein excretion rate (98±16 vs 46±5 mg/day, p&lt;0.05) at gestational day 20 (GD) compared to virgin controls. Interestingly, there was a significant reduction in MAP in the SSWG mated rats compared to SSWG virgin controls at GD20 (114±4 vs 126±4 mmHg, respectively, p&lt;0.05). Comparing the SSC and SSWG mated rats at GD20, it is evident that the SSWG mated rats are protected from developing maternal syndrome seen in the SSC mated rats (MAP: 114±4 vs 141±3 mmHg, p&lt;0.001, Proteinuria: 52±12 vs 98±16 mg/day, p&lt;0.01). Additionally, uterine artery resistance index, which is utilized as a surrogate marker for placental perfusion, is significantly greater in the SSC rats compared to the SSWG at GD20 (0.9±0.02 vs 0.5±0.04, p&lt;0.01, n=2/group). While the SSWG rat are protected from the development of maternal syndrome compared to the SSC, the exact mechanisms for this protection is unknown. This study demonstrates how environmental factors such as diet can play a protective role in the development of maternal syndrome with associated effects on the health of the offspring.Support or Funding InformationHL116264, 18POST33990140, 15SFRN2391002This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CD14 as a Novel Negative Modulator of Immune System‐Dependent Renal Damage and Salt‐Sensitive Hypertension

Hypertension affects one‐third of Americans of which approximately half are termed salt‐sensitive. Previous work in our laboratory has implicated a significant role for the immune system in potentiating the hypertensive and kidney damage phenotypes which occur in the Dahl Salt‐Sensitive (SS) rat. A previously performed RNA‐Seq based transcriptomic experiment on medullary tissue from SS rats fed either a low (0.4% NaCl) or high salt (4.0% NaCl) diet demonstrated an upregulation of molecules involved in the innate immune response including Cluster of Differentiation 14 (CD14). CD14 has been associated with hypertension in various human and animal studies, but mechanistic studies have not been previously performed. The present study tests the hypothesis that CD14 influences the extent of high‐salt diet induced renal damage and hypertension in the Dahl SS rat, by modulating activation of the innate immune system. Using CRISPR‐Cas9 technology, we generated a targeted mutation in the CD14 gene on the Dahl SS rat background, creating a 7‐bp frameshift deletion of bases 248–254 resulting in a predicted premature stop codon (SSCD14−/−). As CD14 is involved in macrophage activation, Interleukin‐1β production, a stereotypical pro‐inflammatory activation response by macrophages, was assessed. SSCD14−/− peritoneal macrophages demonstrated an increased ability to produce IL‐1β (307.8±31.3pg/ml) in response to Lipopolysaccharide + ATP stimulation compared to SSCD14+/+ littermates (142.9±31.5pg/ml, p=0.0099). We then assessed salt‐sensitive hypertension and renal damage phenotypes in these animals. SSCD14+/+ and SSCD14−/− female littermates, maintained on 0.4% NaCl chow, underwent a carotid artery telemetry implantation surgery at 7 weeks of age. After recovery and six days of baseline mean arterial pressure (MAP) recording (day 0: MAP SSCD14+/+=117.2±1.5mmhg, SSCD14−/−=132±11.0mmhg) animals were switched to a 4.0% NaCl diet for three weeks. Overnight urine collections occurred during the low salt period and throughout the high salt diet period. At 21 days, SSCD14−/− had a significantly greater MAP (172.0±11.4mmhg) than SSCD14+/+ animals (148.2±3.5mmhg), p&lt;0.001. Urinary albumin excretion, though not different at baseline (SSCD14+/+=5.6±0.7, SSCD14−/−=21.9±10.5, p&gt;0.05), was significantly greater in SSCD14−/− compared to SSCD14+/+ littermates at days 14 (SSCD14+/+=47.4±8.7, SSCD14−/−=139.8±51.7mg/day, p=0.011) and 21 (SSCD14+/+=79.8±15.9, SSCD14−/−=164.8±36.4mg/day, p=0.017) of the high salt diet. Together these data indicate that CD14 may modulate the full extent of the immune response in the macrophage during the induction and potentiation of salt‐sensitive hypertension and renal disease.Support or Funding InformationSupported by DK96859, R24HL114474, and HL116264.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enhanced vasculotoxic metal excretion in post-myocardial infarction patients following a single edetate disodium-based infusion

Toxic metals have been associated with cardiovascular mortality and morbidity. We have hypothesized that enhanced excretion of vasculotoxic metals might explain the positive results of the Trial to Assess Chelation Therapy (TACT). The purpose of this study was to determine whether a single infusion of the edetate disodium- based infusion used in TACT led to enhanced excretion of toxic metals known to be associated with cardiovascular events. MethodsTwenty six patients (post-MI, age > 50 years, serum creatinine ≤ 2.0 mg/dL) were enrolled in this open-label study. Urinary levels of 20 toxic metals normalized to urinary creatinine concentrations were measured at baseline in overnight urine collections, for 6 h following a placebo infusion of 500 mL normal saline and 1.2% dextrose, and for 6 h following a 3 g edetate disodium-based infusion. Self-reported metal exposure, smoking status, food frequency, occupational history, drinking water source, housing and hobbies were collected at baseline by a metal exposure questionnaire. ResultsThe mean age was 65 years (range 51–81 years). All patients were male. 50% had diabetes mellitus and 58% were former smokers. Mean (SD) serum creatinine was 0.95 (0.31) mg/dL. Toxic metals were detected in the baseline urine of >80% of patients. After placebo infusion there were no significant changes in total urinary metal levels. After edetate infusion, total urinary metal level increased by 71% compared to baseline (1500 vs. 2580 µg/g creatinine; P<0.0001). The effect of edetate was particularly large for lead (3835% increase) and cadmium (633% increase). ConclusionsEdetate disodium-based infusions markedly enhanced the urinary excretion of lead and cadmium, toxic metals with established epidemiologic evidence and mechanisms linking them to coronary and vascular events.

Daytime cold exposure and salt intake based on nocturnal urinary sodium excretion: A cross-sectional analysis of the HEIJO-KYO study

Increased cardiovascular incidence in winter is partly explained by higher blood pressure due to cold exposure. Although higher salt intake induced by cold exposure has been reported in mice, the association remains unclear in humans. To investigate the association between salt intake and cold exposure in winter, a cross-sectional study was conducted among 860 elderly subjects (mean±standard deviation: 72.0±7.1years).We determined ambient temperature at every 10min according to indoor temperature measured in the subjects' home, outdoor temperature, and self-administered diary logging time spent outdoors. Salt intake was estimated by nocturnal sodium excretion rate of overnight urine collection. A 1°C lower daytime ambient temperature was significantly associated with a higher urinary sodium excretion rate by 0.07mmol/h in the subsequent night independent of age, sex, body weight, alcohol intake, calcium channel blocker use, diabetes, household income, estimated glomerular filtration rate, daytime physical activity (p=0.02). After further adjustment for outdoor temperature and day length, the lowest tertile groups of ambient daytime temperature (10.1±2.3°C) showed the nocturnal urinary sodium excretion rate was higher by 14.2% (7.62 vs. 6.54mmol/h) compared with the highest tertile group (19.3±1.8°C). Higher sodium excretion rate was associated with higher nighttime ambulatory blood pressure (p<0.01) and its lower nocturnal dipping (p<0.01). Significant association between higher salt intake and daytime cold exposure partly explain the mechanism of higher blood pressure in winter, and suggest that a reduction of cold exposure might be effective to decrease salt intake.

Natriuresis and blood pressure reduction in hypertensive patients with diabetes mellitus: the NESTOR study

The antihypertensive effect of indapamide has never been clearly understood, particularly in hypertensive patients with diabetes mellitus. A total of 565 patients were randomly selected to receive either indapamide 1.5 mg or enalapril 10 mg daily for 12 months. Brachial blood pressure (BP) and plasma and urinary electrolytes were measured at baseline and at the end of follow–up. Sodium and potassium levels and excretion rates were measured in overnight urine collections. After 12 months' treatment, similar significant reductions were observed in systolic and diastolic BP and pulse pressure levels in both treatment arms (P < .001). However, age, body mass index, diabetes duration, and plasma sodium reductions were shown to be major, independent factors influencing BP reduction with indapamide, but not with enalapril. Regression coefficients were positive for age and plasma sodium reductions (P ≤ .009) but negative for body mass index and diabetes duration (P ≤ .008). Similar findings were observed for pulse pressure. These results were more notable in elderly patients, did not differ regardless of whether BP reduction was measured in absolute or percent values, and were associated with increased sodium and potassium excretion rates.Indapamide is more effective than enalapril at reducing BP in elderly diabetic hypertensives with marked sodium retention.

Glomerular Hyperfiltration and Renal Disease Progression in Type 2 Diabetes

OBJECTIVETo describe the prevalence and determinants of hyperfiltration (glomerular filtration rate [GFR] ≥120 mL/min/1.73 m2), GFR decline, and nephropathy onset or progression in type 2 diabetic patients with normo- or microalbuminuria.RESEARCH DESIGN AND METHODSWe longitudinally studied 600 hypertensive type 2 diabetic patients with albuminuria <200 μg/min and who were retrieved from two randomized trials testing the renal effect of trandolapril and delapril. Target blood pressure (BP) was <120/80 mmHg, and HbA1c was <7%. GFR, albuminuria, and glucose disposal rate (GDR) were centrally measured by iohexol plasma clearance, nephelometry in three consecutive overnight urine collections, and hyperinsulinemic euglycemic clamp, respectively.RESULTSOver a median (range) follow-up of 4.0 (1.7–8.1) years, GFR declined by 3.37 (5.71–1.31) mL/min/1.73 m2 per year. GFR change was bimodal over time: a larger reduction at 6 months significantly predicted slower subsequent decline (coefficient: −0.0054; SE: 0.0009), particularly among hyperfiltering patients. A total of 90 subjects (15%) were hyperfiltering at inclusion, and 11 of 47 (23.4%) patients with persistent hyperfiltration progressed to micro- or macroalbuminuria versus 53 (10.6%) of the 502 who had their hyperfiltration ameliorated at 6 months or were nonhyperfiltering since inclusion (hazard ratio 2.16 [95% CI 1.13–4.14]). Amelioration of hyperfiltration was independent of baseline characteristics or ACE inhibition. It was significantly associated with improved BP and metabolic control, amelioration of GDR, and slower long-term GFR decline on follow-up.CONCLUSIONSDespite intensified treatment, patients with type 2 diabetes have a fast GFR decline. Hyperfiltration affects a subgroup of patients and may contribute to renal function loss and nephropathy onset or progression. Whether amelioration of hyperfiltration is renoprotective is worth investigating.

Cardiac and sympathetic activation are reduced in children with Down syndrome and sleep disordered breathing.

Sleep disordered breathing (SDB) occurs at an increased incidence in children with Down Syndrome (DS) compared to the general pediatric population. We hypothesized that, compared with typically developing (TD) children with SDB, children with DS have a reduced cardiovascular response with delayed reoxygenation after obstructive respiratory events, and reduced sympathetic drive, providing a potential explanation for their increased risk of pulmonary hypertension. Beat-by-beat heart rate (HR) was analyzed over the course of obstructive events (pre, early, late, post-event) and compared between groups. Also compared were the time for oxygen resaturation post-event and overnight urinary catecholamines. Pediatric sleep laboratory. Sixty-four children aged 2-17 y referred for investigation of SDB (32 DS; 32 TD) matched for age and obstructive apnea/hypopnea index. Children underwent overnight polysomnography with overnight urine collection. Compared to TD children, those with DS had significantly reduced HR changes post-event during NREM (DS: 21.4% ± 1.8%, TD: 26.6% ± 1.6%, change from late to post-event, P < 0.05). The time to resaturation post-event was significantly increased in the DS group (P < 0.05 for both NREM and REM sleep). Children with DS had significantly reduced overnight urinary noradrenaline (P < 0.01), adrenaline (P < 0.05) and dopamine levels (P < 0.01) compared with TD children. Children with DS and SDB exhibit a compromised acute cardio-respiratory response and dampened sympathetic response to SDB compared with TD children with SDB. These data may reflect autonomic dysfunction in children with DS that may place them at increased risk for cardiovascular complications such as pulmonary hypertension.

Protein intake and yoga influence whole body protein turnover in middle‐aged women

This study characterized effects of protein intake on whole body protein turnover (WBPTO) between middle‐aged (50–70 y) women who practice yoga (YOGA) and healthy sedentary controls (CON). WBPTO (15N glycine method) was determined at baseline (BASE) and following 2 wk of diet interventions (T2W) where protein intakes approximated the RDA (RDA; .8 g/kg) or 1.6 g/kg (HP). 15N ammonia enrichment was determined for an overnight urine collection at BASE and T2W. Protein and energy intakes were estimated from 7 d diet records. Data represent mean ± SE and level of significance was P&lt;0.05. Age, weight and BMI were similar for YOGA (n=4, 54.8±1.4 y, 62.0±5.3 kg, BMI=24.4 ± .9) and CON (n=8, 54±1.5 y, 66.3± 2.6 kg, BMI = 25.6 ± .7). Q was lower for YOGA vs CON (Q: .62 ± .05 vs .75 ± .04) while PB (2.86 ± .29 vs 3.07 ± 47), PS (3.33 ± .33 vs 4.47 ± .52) did not differ. Q increased with HP for YOGA (61 ± .11 vs 1.1 ± .19) and CON (72 ± .03 vs 1.03±.10). The change (T2W‐BASE) in Q was greater for HP for YOGA (.48 ± .08 vs .04 ± .03) vs CON (.32 ± .08 vs ‐.30±.16). PS increased for HP for YOGA (3.14 ± .56 vs 5.29 ± .58) and CON (3.81 ± .18 vs 5.68 ± .62) from BASE to T2W. PB increased for CON – HP (2.79 ± .64 vs 5.88 ± 0.72). At T2W, PS was greater for HP for CON (5.68 ± .62 vs 3.74 ± .07). Increased consumption of protein affected WBPTO in middle‐aged women. Research is warranted to delineate the protein related metabolic response to protein intake in active and sedentary middle‐aged women.Grant Funding Source : Dairy Research Institute

Does cellular aging relate to patterns of allostasis?: An e`xamination of basal and stress reactive HPA axis activity and telomere length

Long-term exposure to stress and its physiological mediators, in particular cortisol, may lead to impaired telomere maintenance. In this study, we examine if greater cortisol responses to an acute stressor and/or dysregulated patterns of daily cortisol secretion are associated with shorter telomere length. Twenty-three postmenopausal women comprising caregivers for dementia partners (n=14) and age- and BMI-matched non-caregivers provided home sampling of cortisol–saliva samples at waking, 30min after waking, and bedtime, and a 12-hour overnight urine collection. They were also exposed to an acute laboratory stressor throughout which they provided saliva samples. Peripheral blood mononuclear cells were isolated from a fasting blood sample and assayed for telomere length. As hypothesized, greater cortisol responses to the acute stressor were associated with shorter telomeres, as were higher overnight urinary free cortisol levels and flatter daytime cortisol slopes. While robust physiological responses to acute stress serve important functions, the long-term consequences of frequent high stress reactivity may include accelerated telomere shortening.

Heart rate variability, overnight urinary norepinephrine, and plasma cholesterol in apparently healthy human adults

ObjectiveThe aim of this study was to assess the relationship between autonomic nervous system activity as indexed by measures of heart rate variability and overnight urinary norepinephrine, and plasma cholesterol levels in a large sample of working adults. MethodsThe study population comprised 611 apparently healthy employees of an airplane manufacturing plant in Southern Germany. Heart rate variability was calculated as beat-to-beat intervals over the course of one 24-hour weekday measured with an ambulatory ECG recorder. Overnight urine collection and blood samples were also obtained. ResultsWe found an inverse association between indices of vagally-mediated heart rate variability and plasma levels of total cholesterol, low density lipoprotein (LDL), and the ratio of LDL to high density lipoprotein (HDL) that remained significant in multivariate models after controlling for relevant covariates including norepinephrine. Urinary norepinephrine was not significantly related to any measure of cholesterol in multivariate models. ConclusionsWe report here for the first time, in a large sample of healthy human adults, evidence supporting the hypothesis of a clinically relevant inverse relationship between measures of plasma cholesterol and vagally-mediated heart rate variability after controlling for sympathetic nervous system activity. This suggests an important role for the vagal control of plasma cholesterol levels in cardiovascular disease.

Tolerance of Bacteriuria After Urinary Diversion Is Linked to Antimicrobial Peptide Activity

To compare the cationic antimicrobial peptide gene expression profiles and urinary cationic antimicrobial activities of patients after urinary diversion according to their urinary tract infection (UTI) status. Ileal conduit urinary diversion joins the bacterial-tolerant ileal epithelium and intolerant urothelium. After this procedure, one quarter of patients develop repeated symptomatic UTIs. Such development might reflect the altered innate immune mechanisms centered on epithelial expression and urinary activity of cationic antimicrobial peptides, such as defensins. Ileal and ureteral biopsy specimens from ileal conduit subjects with (n = 18) and without (n = 18) recurrent symptomatic UTIs were assessed for cationic antimicrobial peptide gene expression using quantitative reverse transcriptase polymerase chain reaction. Overnight urine collections were analyzed for antimicrobial activity against a laboratory Escherichia coli strain, and infecting organisms were isolated from individual subjects. Overall, the ureteral epithelium showed increased expression of human α-defensin 5 and decreased expression of the human β-defensin 1 after urinary diversion (P < .05). No significant changes were seen for the ileal epithelium. The expression levels of both defensins also did not differ significantly according to UTI status. Urinary cationic activity against infecting bacterial isolates from the individual subjects was significantly greater in those with symptomatic UTI (P < .001), and the activities against the laboratory E. coli strain were similar. The changes in the human β-defensin 1 and human α-defensin 5 expression profiles and the link between symptomatic infection and high urinary antimicrobial activity suggest that innate mechanisms play significant roles in balancing bacterial tolerance and killing after ileal conduit urinary diversion. Future work needs to determine whether these changes can be therapeutically modulated to benefit the patients.