bluuebunny/crossref_metadata_2025 huggingface dataset:
V‐ATPase B1 Subunit Knockout Mice Have A Gender‐Dependent Defect In Urine Concentrating Ability
Authors: Nair Anilkumar , Paunescu Teodor , Bouley Richard , Brown Dennis
Published: June 2021

Men dehydrate more than women during prolonged exercise, but the etiology is unclear. We assessed gender specific water homeostasis in C57BL/6 mice, and found that the basal urine osmolality is higher in female than in male mice (2756±278 vs 2420±185 mOsm/kg: mean±SEM). 12 h water deprivation decreased urinary output by 57 % in females and by 52 % in males (osmolality: 3664±244 vs 3468±318 mOsm/kg). Since β‐intercalated cells (ICs) were shown to play a role in body fluid balance (e. g., Gueutin et al, JCI 2013), we studied the water concentrating ability of mice deficient in the B1 subunit of the V‐ATPase (B1‐/‐ mice). Urinary volume was 10 % higher in B1‐/‐ males and 29 % higher in B1‐/‐ females compared to wild type mice (WT). Unexpectedly, urine output in water‐deprived B1‐/‐ mice decreased by 46 % in females compared to only 17 % in males. Western blot and immunofluorescence staining revealed lower AQP2 expression in B1‐/‐ mice of both sexes compared to WTs. While in dehydrated females AQP2 relocated to the apical membrane region of principal cells (PCs), this effect was much less significant in males, consistent with their weaker concentrating ability. Females have a higher IC/PC ratio than males, and B1‐/‐ females have a higher IC percentage than WTs, whereas B1‐/‐ and WT males do not differ. Our data support the idea that ICs are, via the V‐ATPase, involved in body fluid homeostasis in addition to acid/base balance, and that these effects are gender dependent.
Telemetry‐based Recording of Renal Cortex Oxygenation During Endogenous RAS Activation: Preliminary Observations
Authors: Emans Tonja , Koeners Maarten , Joles Jaap , Krediet C T , Debets Jacques , Janssen Ben
Published: June 2021

Upon feeding of the food additive indole‐3‐carbinol (I3C) the liver of Cyp1A1Ren2 transgenic rats produces high levels of renin which leads to hypertension and tissue damage in the kidney. We hypothesized that endogenous RAS activation in this model would be associated with renal hypoxia. Using a new technology that enables the telemetric recording of intra‐renal tissue oxygen pressure (PO2) in conscious, free roaming animals, we here report preliminary observations on renal cortical PO2 changes during endogenous RAS activation. 1 week after implantation of the electrodes the relative change in renal cortex PO2 during bouts of 3 days of endogenous activation of RAS by feeding an 0.3% or 0.6% I3C containing diet (n=5) were recorded. 3±2 days after start of the 0.6% I3C diet, s.c. bolus infusions of valsartan/losartan (20/30 mg/kg) were administered. During 0.3% and 0.6% I3C feeding, renal PO2 levels fell to 75±7 and 63±13% from baseline values (mean±SD), with a nadir after 25±5 hours. Exogenous blockade of the angiotensin‐II receptor increased oxygen levels within 14±3 min to 98±38% of baseline values (mean±SD). These data suggest that oxygen levels in the renal cortex decrease rapidly during the onset of endogenous RAS hyperactivation. The effect is mediated by AT1‐receptor activation and may precede the known development of systemic hypertension and renal injury in this model.
The Effects of Atorvastatin on the Pogression of Metabolic Syndrome Associated Kidney Renal Disease
Authors: Taylor Lateia , Fizer Brianca , McPherson Kasi , Williams Jan
Published: June 2021

We recently created a 16 base pair frame‐shift deletion in exon 11 of the leptin receptor gene within the genetic background of the Dahl SS rat (SSLepR‐/‐ strain) using Zinc‐finger nucleases that develops many characteristics of metabolic syndrome (MetS) along with progressive kidney disease. The kidneys from the SSLepR‐/‐ strain displayed increased lipid accumulation compared to their control counterparts (SSWT). Lipid lowering drugs such as statins have proven to be renoprotective in various models of kidney disease. Therefore, the purpose of this study was to evaluate the impact of atorvastatin on the progression of renal disease in the SSLepR‐/‐ strain. Atorvastatin was given orally for 28 days at a dose of 10 mg/kg/day in 10 week‐old SSLepR‐/‐ rats with pre‐existing renal injury. Treatment with atorvastatin reduced serum cholesterol levels by 42% over the course of the study. After 14 days, MAP (via tailcuff) and proteinuria increased to 157±10 mmHg and 464±49 mg/day, respectively, in vehicle treated rats. While we did not observe any differences in MAP after 14 days of treatment with atorvastatin (144±8 mmHg), proteinuria decreased by 28% (333±28 mg/day). After 28 days of atorvastatin treatment, MAP and proteinuria was reduced by 18% and 51%, respectively, in comparison to vehicle treated rats. In conclusion, these data suggest that early treatment with atorvastatin delayed the development of severe proteinuria in the SSLepR‐/‐ strain independent of changes in arterial pressure. However, further studies are needed to determine the mechanism of the early beneficial effects of statin therapy on the progression of kidney disease associated with MetS.This research was supported by NIGMS NIH P20GM104357 and AHA 12SDG9440034.
Early Development of Glomerular Injury in Dahl Salt‐Sensitive (SS) Rats with Metabolic Syndrome Independent of Diabetes and Hypertension
Authors: McPherson Kasi , Cornelius Denise , Thomas D'Andrea , Taylor Lateia , Spires Denisha , Johnson Ashley , LaMarca Babbette , Garrett Michael , Williams Jan
Published: June 2021

The incidence of obesity‐induced metabolic syndrome (MetS) has paralleled the increasing rate of renal disease. Recently, we characterized a rodent model of MetS that develops progressive renal injury without developing overt diabetes and hypertension (SSLepR‐/‐ strain). Therefore, the current study tested the hypothesis that alterations in renal hemodynamics and/or inflammation is associated with the early development of renal injury in the SSLepR‐/‐ strain (n=6) compared to SSWT rats (n=6). At 6 weeks of age, we did not observe any differences in mean arterial pressure (MAP, telemetry) between SSWT and SSLepR‐/‐ rats (114±1 and 119±3 mmHg, respectively). Interestingly, proteinuria was significantly elevated at this time period in the SSLepR‐/‐ rats versus SSWT rats (225±38 and 21±2 mg/day, p<0.05, respectively). Similar to proteinuria, significant podocyte injury (podocalyxin excretion) was observed in the SSLepR‐/‐ strain suggesting that glomerular disease develops early (p<0.05). Renal blood flow (RBF) and glomerular filtration rate (GFR) were increased by 40% and 17% (p<0.05), respectively, in the SSLepR‐/‐ rats compared to the SSWT rats indicating renal hyperfiltration. Kidneys from SSLepR‐/‐ rats exhibited increased macrophage infiltration (CD68+cells) vs SSWT rats (30±9% vs 6±4% of gated cells, p<0.05). Overall, these data suggest that the early development of renal injury in the SSLepR‐/‐ strain may be due to either elevations in GFR or macrophage infiltration causing podocyte injury and progressive proteinuria independent of hypertension and diabetes. NIGMS P20GM104357, AHA 12SDG9440034
Sex Differences in the Progression of Diabetes‐associated Cardiac Pathology
Authors: Lum‐Naihe Kelly , Raja Ahmad , Bajwa Jamal , Mahmood Abuzar , Luck Christian , Emter Craig , Pulakat Lakshmi
Published: June 2021

Type 2 Diabetes Mellitus (T2DM) is a major contributor to cardiovascular disease (CVD). We hypothesized that there are significant sex‐related differences in the progression of T2DM‐induced cardiac pathology in pre‐menopausal diabetic female rats and age‐matched diabetic male rats. These differences were assessed in 3 study groups: ZDF male rats (ZDF‐M) and lean female rats (L‐F) on Purina 5008 diet, and ZDF female (ZDF‐F) rats on Research Diets D12468 (to induce T2DM) from the age of 6‐20 weeks. ZDF‐M went from having a ~26 fold higher fasting plasma insulin (INS) than L‐F at 9‐weeks, to <2 fold at 20‐weeks (p<0.05). In contrast, in ZDF‐F, INS was ~ 64 fold higher than L‐F at 15‐weeks and was still ~50 fold higher than L‐F and ~21 fold higher than ZDF‐M at 20‐weeks (p<0.05). Hyperglycemia was lower in ZDF‐F than ZDF‐M at all ages, but ZDF‐F had lower plasma HDL and higher LDL levels and higher % body fat and lower % lean muscle mass (as determined by EchoMRI) compared to ZDF‐M and L‐F (p<0.05). Cardiac echocardiography showed heart rate was significantly different among all three groups with ZDF‐F being greatest and ZDF‐M lowest. However, only ZDF‐M displayed cardiac diastolic dysfunction, indicated by dilatory remodeling, increased isovolumic relaxation time, increased late diastolic filling (A) and decreased E/A and E'/A' ratios. ZDF‐M had the highest 24‐hr urine clearance of Na+, K+, glucose and protein (p<0.05).

Conclusion

T2DM had different effects on ZDF‐F and ZDF‐M. ZDF‐F had a quicker loss of lean muscle mass and HDL despite longer hyperinsulinemic stage of T2DM, whereas ZDF‐M had higher HDL, but worse cardiac dysfunction.

Funding: LSME, UM‐Columbia (LP), NIH 1R01HL118376‐01(LP), R01HL112998 (CAE).
Brain Oxidative Stress in Neural Mechanism of Programmed Hypertension to Maternal High Fructose Diet
Authors: Wu Kay , Tain You‐Lin , Chan Julie
Published: June 2021

Early life exposure to adverse environments can lead to a variety of adult diseases by a process referred to as developmental programming. Maternal high fructose diet (HFD) promotes programmed hypertension in adult offspring, although the underlying mechanism is not fully understood. Brain oxidative stress is pivotal in neural mechanism of hypertension. We tested in this study the hypothesis that brain oxidative stress contributes to programmed hypertension in adult offspring to maternal HFD. In normotensive rats, maternal HFD during gestation and lactation led to insulin resistance, increase in sympathetic activity and hypertension in the 3‐month‐old young offspring. This was associated with augmented neurogenic sympathetic vasomotor tone and high tissue levels of reactive oxygen species (ROS) at the brain stem. Maternal melatonin treatment or simvastatin treatment to the 2‐month‐old offspring significantly ameliorated oxidative stress in the brain stem of the offspring. Simvastatin treatment also abrogated sympathetic overexcitation and prevented the development of programmed hypertension in the 3‐month‐old rats to maternal HFD. These results suggest that brain stem oxidative stress may contribute to sympathoexcitation and programmed hypertension in the offspring to maternal HFD.

This study was supported by grant (CMRPG8C0051) from Kaohsiung Chang Gung Memorial Hospital.
Low birth weight associated renal and vascular impairment
Authors: Ratliff B , Zullo J , Barnett C , Shen M , Rabadi M , Nesi L , Lamba R , Aldumuahdhi W
Published: June 2021

In the United States, 8% of all births are to low birth weight (LBW) neonates, mostly due to maternal malnourishment and preterm delivery. The LBW neonate has a high rate of mortality and morbidity after birth. In experiments, we used a maternal malnourished mouse model to examine the effects of LBW on kidney development and short/long‐term function. Circulating cyto‐/chemokines, renal blood flow, renal developmental gene expression, nephron formation, autophagy, renal function and blood pressure were examined in the LBW offspring. The therapeutic potential of glutamine, methionine and calendula treatment was examined in these animals. LBW male offspring glomerular endowment and survival were reduced by 50%. The LBW neonate experienced attenuated renal blood perfusion by 37%, enhanced circulating proinflammatory cyto‐/chemokines (IL‐1β, IL‐5, IL‐12(p40), IL‐12(p70), IFNγ and GM‐CSF), reduced levels of IL‐10 and VEGF, blunted autophagy, altered expression of critical developmental genes (Wnt9b, Wnt4, Bmp7, FoxD1, Eya1, Sall1 and Fgf8), and a 25% reduction of pre‐nephron structures. Supplementation of methionine and glutamine during nephrogenesis normalized kidney development and function in the LBW neonate. At 2 months post‐birth, LBW mice showed early tubular damage and glomerular abnormalities. At 5 months post‐birth, LBW animals had 10% elevated blood pressure, and elevated serum creatinine and proteinuria. Treatment with calendula improved blood pressure and renal function in the older animals. LBW compromises the kidney and vascular system, enhances circulating proinflammatory cyto‐/chemokines, interferes with normal nephron/glomeruli development and predisposes mice to acute and chronic illness. Effects are improved upon treatment with glutamine, methionine or calendula.
Effect of Prior Use Menopausal Hormone Therapy on Blood Pressure Responses in Women
Authors: Johnson Maja , Kruse Sarah , Harvey Ronee , Joyner Michael , Miller Virginia , Barnes Jill
Published: June 2021

Cerebrovascular reactivity to hypercapnia is higher in women receiving menopausal hormone therapy (MHT) compared with controls, and this may persist after cessation of MHT. We hypothesized that the effect of MHT may be influenced by mean arterial pressure (MAP). Therefore, we examined both middle cerebral artery velocity (MCAv) and MAP responses to stepped hypercapnia in 60 postmenopausal women who were previously randomized to receive oral conjugated equine estrogen (oCEE, n=15), transdermal 17β estradiol (tE2, n=22), or placebo (PLA, n=23) for 4 years. After a 3 year washout period, reactivity was calculated from the slope of the relationship between MCAv or MAP and end‐tidal CO2. MCAv reactivity was higher in the oCEE group compared with the PLA group (1.6±0.2 vs. 1.1±0.1 cm/s/mmHg; oCEE vs. PLA, respectively, p<0.05), but similar between the tE2 and PLA group (1.3±0.2 vs. 1.1±0.1 cm/s/mmHg; tE2 vs. PLA, respectively, p>0.05). There were no significant differences in MAP reactivity (1.0±0.2, 0.6±0.1, 0.7±0.2 mmHg/mmHg of CO2, oCEE vs. tE2 vs. PLA, respectively, p>0.05) among the groups. However, in the combined MHT group, MAP reactivity was positively correlated with MCAv reactivity (r=0.58, p<0.001), such that women with higher MAP responses demonstrate greater MCAv responses to hypercapnia. In conclusion, there were no differences in MAP reactivity 3 years after cessation of MHT. In addition, our results suggest that greater cerebrovascular reactivity in postmenopausal women who received MHT may be driven by augmented blood pressure responses to hypercapnia. Funding: K99 HL 118164, AG 044170 and HL 83947.
Afternoon Spot Urine Osmolality as a Biomarker of Hydration in Free‐living Healthy Adults
Authors: LEMETAIS Guillaume , BOTTIN Jeanne , POUPIN Marie , JIMENEZ Liliana , PERRIER Erica
Published: June 2021

Objective

Previous research has highlighted links between fluid intake (FI), hydration parameters, and disease risk. Specifically, hydration markers in 24h urine samples respond quickly to a change in FI and have been linked to risk of CKD and lithiasis. However, 24h urine collection is burdensome, inconvenient, and time consuming. Spot urine collections would simplify daily hydration monitoring for researchers, clinicians, and individuals. Therefore, we assessed the equivalence of 24h and spot urine osmolality (24h Uosm; Spot Uosm) to determine whether a single spot may replace 24h collection.

Methods

82 healthy adults with low to high habitual FI (50%M; age 23.6±2.9 y; BMI 22.2±1.5 kg/m2) recorded all fluid consumed and collected individual voids over 24h. Uosm was measured on each void and on the pooled 24h sample. Equivalence between Spot and 24h Uosm was evaluated using the Two One‐Sided Test method (α=0.025).

Results

Spot Uosm from afternoon samples (14:00‐16:00; 16:00‐18:00; 18:00‐20:00) was equivalent to 24h Uosm whereas morning (08:00‐10:00; 10:00‐12:00) and midday (12:00‐14:00) spots tended to overestimate 24h Uosm. The mean [95% CI] difference between 24h and Spot Uosm was 287 [209;365] between 08:00‐10:00; 83 [12;154] between 10:00‐12:00; and 107 [58;157] between 12:00‐14:00. From 14:00‐16:00; 16:00‐18:00; and 18:00‐20:00, the mean difference was ‐25 [‐72;22]; 29 [‐35;92] and 12 [‐41;66].

Conclusion

Afternoon Spot Uosm is equivalent to 24h Uosm. Thus, short, well‐timed urine collections may represent a convenient alternative to 24h samples, providing an easy way to monitor daily hydration in real‐life conditions. Afternoon Spot could also serve as an early warning signal to increase FI for maintaining 24h euhydration.
Alterations in Placental Eicosanoid Production Contribute to Impaired Vascular Remodeling in Preeclampsia
Authors: Murphy Sydney , Lee Nicole , Spencer Shauna‐Kay
Published: June 2021

Preeclampsia (PreE) results from an impaired vascular remodeling of the placental spiral arteries. Recent data suggests cytochromeP450 (CYP450) vasoactive eicosanoids in the etiology of the disease. Therefore, we investigated whether alterations in the profile of arachidonic acid (AA) metabolites of the CYP450 pathway contribute to the impaired vascular remodeling in preeclampsia. To this end, we isolated microsomes from placental vessels of normal pregnant (NP) and PreE women and CYP450 enzyme activity was determined by HPLC/mass spectrometry. In addition, the proliferative actions of 20‐HETE, a major CYP450 metabolite were investigated in an in vivo model of trophoblast syncytialisation. Placental vascular microsomes from PreE women displayed a significant increase in 20‐HETE production relative to NP women (0.192±0.04 vs 0.119±0.06 pg/min/mg, p<0.05). 15‐HETE (74±8 vs 43±11 pmol/min/mg), 12‐HETE (70±20 vs 27±7 pmol/min/mg, p<0.05), and total HETE production (287±61 vs 170±44 pmol/min/mg) were also increased in PreE compared to NP. Incubation of BeWo cells with 20‐HETE (10nM; 100nM; 1uM) displayed an increase in cell proliferation, while inhibition of 20‐HETE with 17‐ODYA (10uM) and HET0016 (1nM) displayed a 10% and 75% inhibition. Thus, increases in 20‐HETE production promote, while inhibition of 20‐HETE reduces proliferation of trophoblasts in vitro and may contribute to the altered trophoblast invasion and vascular remodeling in PreE.
The Effects of Sepsis on Osmosensory Neurons Mediating Thirst
Authors: Stare J , Siami S , Trudel E , Prager‐Khoutorsky M , Sharshar T , Bourque C
Published: June 2021

The ability of osmosensory neurons within the hypothalamus to sense changes in blood osmolality is essential for maintaining hydromineral homeostasis. Typically, an increase in blood osmolality excites the osmosensitive neurons of the organum vasculosum of the lamina terminalis (OVLT), which then stimulate neural pathways to induce thirst sensation. Patient studies show that thirst is depressed in sepsis, a deadly disease defined by the systemic inflammatory response to a severe infection, and that this impairment may be due to a deficit in the osmoregulatory pathway. Using the rat cecal‐ligation and puncture (CLP) sepsis model, we tested the hypothesis that sepsis impairs osmotically‐induced thirst and that this is due to changes in osmosensory OVLT neuron properties. Septic rats did indeed drink significantly less under systemic hypertonic conditions (CLP 4.2 ± 0.58mL; controls 16.4 ± 1.8mL; p < 0.001). Electrophysiological recordings of acute in vitro hypothalamic preparations show that septic OVLT neurons are quiescent due to their hyperpolarized state (CLP −47.9 ± 2.4mV; controls −41.0 ± 2.2mV; p = 0.02). Furthermore, they display an impaired increase in firing rate in response to hypertonicity (CLP 0.1 ± 0.05Hz; controls 0.52 ± 0.18Hz; p < 0.01). These results imply that the CLP rat model mimics the human septic condition, and provide a platform to further our understanding of how central osmoregulation and osmotic thirst are perturbed at a cellular and molecular level in sepsis.
Humanized Sickle Cell Disease Mice Display an Increased Sensitivity to α1‐adrenergic Mediated Vasoconstriction
Authors: Fox Brandon , Pollock David , Pollock Jennifer
Published: June 2021

Sickle cell disease (SCD) is the most common inherited blood disorder in the United States. Vascular dysfunction is a common pathway contributing to the diverse pathologies seen in SCD, however, the mechanisms are not fully understood. We utilized a novel mouse globin knockout, human sickle cell gene knockin mouse (βS/βS) and the heterozygous control mouse (βA/βS). The α1 adrenergic and endothelin (ET‐1) pathways are activated in SCD. We hypothesized that thoracic aorta from SCD mice would demonstrate increased sensitivity to α1‐adrenergic mediated vasoconstriction with increased aortic endothelin (ET‐1) expression. Thoracic aortae were isolated from 12‐week‐old male βS/βS or βA/βS mice and mounted on wire myographs (n=3 per group) to assess vasoconstriction. In addition, ET‐1 expression was analyzed using qRT‐PCR (n=5 per group). Cumulative concentration‐response curves were generated for phenylephrine (PE) and KCl to assess vasoconstriction as well as acetylcholine (ACh) and sodium nitroprusside (SNP) for vasodilation. Vasoconstriction to PE was significantly increased in βS/βS mice compared to βA/βS (90±5 vs. 38±7 %Max KCl, p=0.004), without a change in vasodilation between groups. ET‐1 expression was similar in both groups (0.98±0.23 vs. 1.00±0.18 ΔΔCT). Thus, the thoracic aorta of SCD mice display exaggerated α1‐mediated vasoconstriction without a change in vasodilation or ET‐1 expression. These data indicate that SCD enhances vascular α1‐adrenergic mediated signaling in the thoracic aorta, which provides mechanistic insight into a link between stress and vaso‐occlusive crises in SCD patients. Funded by T32 HL007918 to BMF, U01 HL117684 to DMP and JSP, and partially supported by MSTP T32GM008361.
Blockade Of The ETA Receptor In An Animal Model Of HELLP Syndrome
Authors: Wallace Kedra , Spencer Shauna , Kyle Patrick , Moseley Janae , Morris Rachael
Published: June 2021

Women with hypertensive forms of pregnancy such as hemolysis‐elevated liver enzymes‐low platelet‐syndrome have increased circulating levels of endothelin‐1 (ET‐1), however the relationship between hypertension and ET‐1 in this hypertensive disorder of pregnancy has not been studied. Using an animal model of hemolysis elevated liver enzyme low platelet (HELLP) syndrome we sought to determine if along with the hypertension that occurs in this animal model if there was increased activation/dysfunction of ET‐1 and if blockade of the endothelin‐1 receptor‐A (ETA) attenuated hypertension. On gestational day (GD) 12 timed‐pregnant rats were infused with sFlt‐1 and sEng (4.7 and 7µg/kg) via mini‐osmotic pumps for 8 days. A subset of rats were treated with ETA antagonist (ABT‐627, 5mg/kg) for 8 days. Rats with HELLP had significantly increased hypertension (P=0.0001), circulating ET‐1 (P=0.03) and a significant 3.3 and 7.2‐fold increase in pre‐proendothelin (PPET) mRNA placenta and liver expression (P=0.01; P=0.04). Urinary protein:creatinine (PC) ratio was significantly increased in these animals (P=0.0007) and circulating factors from these rats stimulated a significant increase in endothelial cell ET‐1 secretion (P=0.001) in an in vitro assay. Blockade of the ETA receptor significantly decreased hypertension (P=0.001), circulating ET‐1 (P=0.004), placental PPET mRNA expression (P=0.016) and urinary PC ratio (P=0.007) in rats with HELLP. Blockade of the ETA receptor significantly decreased hemolysis (P=0.009), liver enzymes (P=0.006) and significantly increased platelet levels (P=0.03) in HELLP rats. These data support the hypothesis that ET‐1 activation has a critical role in pathophysiology of as HELLP syndrome. KW received IRSP support.
Renal Functional Responses to Angiotensin 1‐7 (Ang1‐7) in the Spontaneously Hypertensive Rat (SHR)
Authors: Barry Elaine , Johns Edward
Published: June 2021

There is evidence that the natriuretic and diuretic actions of Ang1‐7 are dependent on the degree of activation of the renin angiotensin system but it is uncertain whether they are independent of blood pressure. This study examined the effect of intra‐renal infusion of Ang1‐7 on sodium and water excretion in SHR and normotensive Wistar rats, in which both strain's plasma renin and angiotensin II are similar. Twelve week old male Wistar or SHRs (n=7) were anaesthetised and prepared for measurement of mean arterial pressure (MAP), glomerular filtration rate (GFR), urine flow (UV) and fractional sodium excretion (FENa). Clearances, of 20 min, were taken before and during the intra‐renal infusion of saline or Ang1‐7 (3x10‐3M) at 1ml/h. Means±SEM were compared (Wilcoxon matched pairs) and significance taken at P<0.05. Intra‐renal Ang1‐7 infusion did not change MAP or GFR in either SHR or Wistar groups, at 172±9 and 113±6mmHg and 3.4±0.3 and 4.8±0.2ml/min/kg respectively. UV and FENa increased from 57.5±11.1 to 69.3±9.1µl/min/kg and 1.6±0.3 to 2.5±0.3% (P<0.05) in the SHR group and from 96.0±3.9 to 132.1±8.1µl/min/kg and from 1.8±0.1 to 2.3±0.1% (P<0.05) in the Wistar group. These responses were not significantly different across the groups. These findings showed that Ang1‐7 infusion into the kidney caused a diuresis and natriuresis, the magnitude of which was indistinguishable between SHR and normotensive Wistar rats. They indicate that the elevated MAP in the SHR does not contribute to the magnitude of the excretory responses. The data further support the view that the natriuretic and diuretic actions of Ang1‐7 are dependent on the status of the endogenous renin‐angiotensin system but are independent of blood pressure.
Characterization of Atp6ap2 ((Pro)‐Renin Receptor) in Receptor Mediated Endocytosis by the Renal Proximal Tubule
Authors: Figueiredo Marta , Sihn Gabin , Müller Dominik , Nguyen Genevieve , Bader Michael , Wagner Carsten
Published: June 2021

Atp6ap2 may be an accessory subunit of the V‐type H+ ATPase regulating organellar, cellular, and systemic acid‐base homeostasis. Here we examine whether the knock‐down of Atp6ap2 affects renal V‐ATPase function in the proximal tubule. There, V‐ATPases are important for receptor‐mediated endocytosis of low molecular weight proteins from urine via the megalin/cubilin receptors. In order to elucidate the role of Atp6ap2 in this function we used an inducible shRNA Atp6ap2 rat model and a doxycyline‐inducible kidney specific Atp6ap2 KO mouse model. Knock‐down of Atp6ap2 reduced Atp6ap2 expression by approximately 90 %. Atp6ap2 knock‐down animals showed higher proteinuria (albumin and Vitamin D binding protein).To further examine proximal tubular endocytosis, we injected animals with markers for fluid phase endocytosis (FITC‐ dextran, 10 kDa) and a for receptor mediated endocytosis (human transferrin). Immunofluorescence of kidney slices from both animal groups did not show a difference in FITC‐dextran intensity or localization. Furthermore, evaluation of receptor mediated endocytosis of human transferrin, showed in immunofluorescence stainings no differences between the animal groups but the knock‐down animals excreted human transferrin in the urine while control animal did not so. Hence, our data suggests a possible role for ATP6ap2 for proximal tubule function in the kidney; however, the defect in receptor‐mediated endocytosis is mild in rats and mice.

Funded by the Swiss National Science Foundation.
Calpain‐2 Proteolysis of MARCKS is a Negative Feedback Regulator of ENaC
Authors: Montgomery Darrice , Ghazi Zinah , Thai Tiffany , Eaton Douglas , Alli Abdel
Published: June 2021

Proteolytic and hormonal regulation of ENaC activity is important for total body sodium homeostasis and blood pressure control. We recently identified a role for myristoylated alanine‐rich C kinase substrate (MARCKS) in the phosphotidylinositol 4,5‐bisphosphate (PIP2) dependent regulation of ENaC. Like ENaC, MARCKS is regulated by proteases. Calpains are intracellular calcium‐dependent cysteine proteases involved in many physiological functions. The purpose of this study was to examine the ability of calpain‐2 to serve as a negative feedback regulator of ENaC by cleaving MARCKS. Immunoprecipitation and Western blot studies showed the interaction between ENaC and MARCKS in Xenopus 2F3 cells decreased in response to the calcium ionophore A23187, and this effect was attenuated when cells were pretreated with the pharmacological inhibitor of calpain‐2 A6060. A difference in MARCKS expression in sucrose density gradient fractions was observed for cells treated with the calcium ionophore and the calpain‐2 inhibitor compared to untreated cells. Similarly, amiloride‐sensitive transepithelial current across cell monolayers was altered. Taken together, calcium‐dependent calpain‐2 proteolysis of MARCKS prevents constitutive stimulation of ENaC activity at the level of its open probability. This research was supported by a National Institutes of Health grant (1K01DK099617‐01A1).
Short‐term Regulation of Amino Acid Transporters by Amino Acids
Authors: Jando Julia , Jordi Josua , Verrey François
Published: June 2021

To ensure body homeostasis amino acids (AA) are (re)absorbed by epithelial cells of the intestinal mucosa and renal proximal tubules. This sequential transepithelial transport includes an apical uptake followed by a basolateral efflux. The luminal uptake is mediated by different AA transporters but most neutral AAs are transported by the luminal Broad neutral Amino acid Transporter B0AT1 (SLC6A19).

The regulation of this transporter is poorly known; in particular its potential short‐term regulation by its substrates has not been investigated. We hypothesized that similarly to glucose transporters the expression of B0AT1 might be repressed if costs of its synthesis and expression exceed its benefits and thus it would be upregulated by AAs to absorb them efficiently when available. To investigate the possible short‐term regulation of B0AT1 by AAs in vivo, we assessed in rats whether after a fast of 4 or 16 hours, intragastric AA application upregulates B0AT1 expression in intestinal brush border membranes. The time point of analysis after amino acid gavage was determined based on micro Computer Tomography measurements showing when the AA mix reached the small intestine. However, functional analysis of AA absorption in isolated ex vivo intestinal rings showed no significant difference between rats having received a water or an AA gavage. In addition, protein expression levels of B0AT1 in the intestinal brush border analyzed by Western blotting did not reveal a difference between rats having received a water or AA gavage. In conclusion, in the selected experimental conditions, the expression and function of the apical AA transporter B0AT1 was not regulated in the short‐term by the preceding application of its substrate AAs.

Supported by the Swiss National Science Foundation.
Cooperation of Basolateral Epithelial Amino Acid Transporters TAT1 and LAT2 Investigated in a Double Knockout Mouse Model
Authors: Boiadjieva Emilia , Vilches Clara , Bodoy Susanna , Oparija Lalita , Jando Julia , Nunes Virginia , Verrey François , Palacin Manuel
Published: June 2021

Basolateral efflux is a crucial step for amino acid (AA) (re)absorption across small intestine and kidney proximal tubule epithelia mediated by various transporters. There are uniporters that mediate the facilitated diffusion of essential AAs, as does for instance aromatic AA transporter TAT1 (Slc16a10) and also antiporters such as LAT2‐4F2hc (SLC7A8‐SLC3A2) that exchanges neutral AAs. To test the hypothesis that the recycling of aromatic AAs via TAT1 allows the vectorial efflux of other AAs via obligatory exchanger LAT2‐4F2hc, LAT2‐/‐ TAT1‐/‐ double knockout (dKO) mice were generated. These mice have a reduced body weight (~‐17% at 3 months) but no other overt phenotypic alteration. Under normal protein diet, they excrete in the urine, compared to LAT2‐/‐ and TAT1‐/‐ knockout mice, higher amounts of aromatic AAs and of some other AAs that are not substrates of TAT1. The amino aciduria was further increased under high protein diet and involved all proteogenic AAs but some charged ones. Screening the mRNA levels of ~20 AA transporters in the kidney suggested a trend of compensatory changes as the level of the transcript of 5 basolateral AA transporters was increased in dKO mice under high protein diet. Preliminary transport experiments with small intestine gut sacs confirm the alteration of transepithelial transport. These observations support the hypothesis that the basolateral efflux of LAT2 substrates depends on the recycling of aromatic AAs via TAT1.

Supported by Swiss National Science Foundation.
IRBIT Mediates Trafficking and Activation of Na+,K+‐ATPase by Angiotensin II
Authors: He Peijian , Xie Zijian , Yun Chris
Published: June 2021

Na+ reabsorption by the kidney is tightly regulated to maintain Na+, fluid and blood pressure homeostasis. Angiotensin II (Ang II) plays a critical role in Na+ reabsorption in the kidney during health and diseases conditions. Na+,K+‐ATPase on the basolateral membrane is one of the important targets subjected to Ang II regulation. However, the mechanism by which Ang II activates Na+,K+‐ATPase remains poorly understood. We have previously shown that IRBIT (inositol 1,4,5‐triphosphate receptor binding protein released with inositol 1,4,5‐triphosphate) binds and activates Na+/H+ exchanger (NHE3) in response to Ang II. By mass spectrometry analysis, we identified IRBIT as a novel binding protein of Na+,K+‐ATPase, prompting us hypothesize that IRBIT might coordinate transcellular Na+ absorption by regulating apical NHE3 and basolateral Na+,K+‐ATPase. Indeed, overexpression of IRBIT resulted in a greater activation of Na+,K+‐ATPase activity by Ang II in OKP cells, whereas lentiviral shRNA‐mediated knockdown of IRBIT significantly attenuated the stimulatory effect of Ang II. Confocal microscopic analysis showed colocalization of IRBIT and Na+,K+‐ATPase in the basolateral membrane and cytoplasm of OKP cells. Native‐PAGE analysis showed that IRBIT and Na+,K+‐ATPase are co‐expressed in large protein complexes of 350 and 650 kD. Importantly, IRBIT‐Na+,K+‐ATPase association was acutely enhanced by Ang II. Moreover, knockdown of IRBIT abrogated AngII‐induced membrane expression of NKA. In conclusion, IRBIT associates with Na+,K+‐ATPase and mediates Ang II‐induced membrane localization of Na+,K+‐ATPase. (This work was supported by AHA‐SDG grant)
Role of Bile Acids for Regulation of Aquaporins in Rodent Large Intestine
Authors: Yde Jonathan , Moeller Hanne , Lajczak Natalia , O'Dwyer Aoife , Keely Stephen , Fenton Robert
Published: June 2021

Bile acids (BAs) affect water transport in the colon, as evident in Bile acid malabsorption (BAM). In BAM patients, an increased production of BAs or a decrease in the capacity for BA reabsorption in the ileum cause BAs to overflow into the colon, where they lead to diarrhoea through mechanisms that are yet to be determined. Although there is evidence for expression of specific water channels (AQPs) in the colon, results from different studies have been contradictory. A connection between BAs and AQPs has not been described. Here we profiled mRNA and protein levels of AQPs in the colons of rodents and examined the short and long‐term effects of naturally occurring BAs on the expression of AQPs in human colonic T84 and HT29 epithelial cells. In colonic epithelial cells isolated from rats, AQP1, ‐3, ‐4, ‐7 and ‐8 were detected at the mRNA level by RT‐PCR, with AQP1,‐3 and ‐8 expression confirmed at the protein level by immunohistochemistry (IHC). In mouse AQP1, ‐4 and ‐8 mRNA and protein were detected by RT‐PCR and IHC. Compared to the proximal segment, AQP3, ‐4, ‐7, ‐8 mRNAs were expressed at higher levels in the distal half of the rat colon as seen by RT‐qPCR. In T84 and HT‐29 cells, the BA sodium deoxycholate increased AQP3‐mRNA expression in a time‐dependent manner. Furthermore, the BA sodium ursodeoxycholate or the Farnesoid X receptor agonist GW4064 increased AQP8 mRNA in T84 cells. In conclusion, AQPs likely have a spatiotemporal expression profile in the rodent large intestine. Cell line studies suggest that specific BAs may alter AQP expression in the large intestine, and as such AQPs may play a role in BAM syndromes. The study was supported by The Danish Council for Independent Research, MEMBRANES and Gangstedfonden.
Intravital Imaging Reveals Angiotensin II‐induced Endocytosis and Shedding of Plasma Albumin by Podocytes
Authors: Schießl Ina , Kattler Veronika , Hammer Anna , Castrop Hayo
Published: June 2021

We recently showed that angiotensin II (ANGII) acutely increases the albumin filtration in the healthy kidney. Here we used intravital microscopy to assess the effects of ANGII on podocyte function in rats. Acute infusion of 0, 10, 30, 60, and 80 ng/kg/min ANGII enhanced the endocytosis of albumin and resulted in an average of 0, 0, 3.7±2.2, 72.3±18.6 (p=0.0004) and 239.4±34.6 µm3 (p<0.0001), respectively, of albumin‐containing vesicles/glomerulus. This effect was abolished in the presence of the AT1 receptor antagonist losartan. Immunostainings of ANGII‐infused kidneys confirmed the presence of albumin‐containing vesicles in the podocytes, which colocalized with podocin and the multiligand receptor megalin. Furthermore, in the presence of gentamicin, podocyte endocytosis of albumin was markedly reduced to 1.5±1.5 and 22.5±6.3 µm3 during infusion of 60 and 80 ng/kg/min ANGII (p=.0025 and .0001 vs. non‐gentamicin‐treated rats, respectively), suggesting a megalin‐dependent uptake mechanism. ANGII infusion increased the concentration of albumin in the subpodocyte space, a potential source for endocytic protein uptake, and this concentration was further increased in the presence of gentamicin. Some of the endocytic vesicles were acidified, and colocalized with the lysosomal marker lysotracker in vivo, suggesting lysosomal degradation. Another subset of vesicles migrated from the capillary to the apical aspect of the podocyte and was eventually shed into the urinary space. In summary, the transcellular transport of proteins across the podocyte constitutes a new pathway of glomerular protein filtration. ANGII enhances the endocytosis and shedding of plasma albumin by podocytes and this may eventually impair podocyte function.
Silver Nanoparticles Increase Epithelial Sodium Channel (ENaC) Currents in Isolated Sheep Pleura
Authors: Arsenopoulou Zoi , Molyvdas Paschalis , Gourgoulianis Konstantinos , Zarogiannis Sotirios , Hatzoglou Chrissi
Published: June 2021

Objective

Exposure to engineered nanoparticles (NPs) has been associated with lung and pleura pathology such as inflammation, fibrosis and cancer. Silver (Ag) NPs are used in a large number of consumer products and medical devices increasing therefore the possibility of user exposure. The aim of this study was to investigate the possible effects of engineered AgNPs on the function and expression of ENaC on parietal sheep pleura.

Materials and methods

In the present study the short circuit current (ISC) and the transmesothelial resistance (RTM) of sheep parietal pleura was monitored in Ussing chambers after the preincubation of the tissue with spherical AgNPs of 20nm and 60nm in size for 30 min. The effect of the AgNPs on the function of the epithelial sodium channel (ENaC) was investigated by the addition of the inhibitor amiloride (10‐5M) apically. Moreover, the expression of αENaC subunit was analyzed in these samples by Western Blotting in order to assess effects on the protein level.

Results

AgNPs (20 nm; 2μg/ml) increased the amiloride sensitive ISC indicating that ENaC function is enhanced by this treatment. The RTM of the sheep parietal pleura was not altered, suggestive of no effects on the tight junctions and the tissue integrity. Finally, the levels of αENaC expression were similar in all groups. AgNPs of 60nm had no significant effects in any of the parameters.

Conclusions

AgNPs of 20 nm affect sodium permeability of the sheep parietal pleura by increasing ENaC activity. This effect is not due to increased αENaC expression. These data suggest that AgNPs induce changes in the function of pleural ENaC.
Regulation of Intestinal SGLT1 by Catestatin in Hyperleptinemic Type 2 Diabetic Mice
Authors: Dominguez Rieg Jessica , Chirasani Venkat , Köpsell Herrmann , Senapati Sanjib , Mahata Sushil , Rieg Timo
Published: June 2021

We previously showed that intestinal SGLT1 abundance is severely decreased in hyperleptinemic db/db mice, but not leptin‐deficient ob/ob mice. Since the short form of the leptin receptor (LEPRa) is fully functional in db/db mice, we hypothesized that leptin‐induced downregulation of SGLT1 is mediated by LEPRa. Catestatin, a 21‐amino acid fragment of chromogranin A, was shown to alter leptin signaling but the mechanism remains unknown. By conducting molecular dynamics modeling, we identified that the AB‐loop of leptin and catestatin have high structural similarity (>40%). Pairwise sequence alignment showed 29% identical amino acid residues, 19% high in similarity, and 10% low in similarity. A secondary structure prediction identified a high propensity of β‐strands and random coils in the structures of catestatin and leptin's AB‐loop. Protein‐protein docking studies showed that leptin and catestatin exhibit similar modes of binding to the receptor, both targeting the Ig‐like domain of LEPR. Our modeling analyses suggest that in db/db mice with hyperleptinemia, catestatin blocks the biological action of leptin by competing for LEPR. To test this, we treated db/db (n=4) and db/con (n=5) mice with catestatin (5 µg/g bw/day). After 6 days, blood glucose levels rose higher in db/db vs db/con mice (232±51 vs 50±12 Δmg/dl; P<0.05), which corresponded to a restoration of SGLT1 abundance. In summary, when increased leptin signaling via LEPRa in db/db mice is inhibited by catestatin, intestinal SGLT1 expression is restored resulting in overt hyperglycemia.
Comparative structural biology of human and opossum AT1Rs reveals different ARB binding sites
Authors: Chandler Andrew , Kurita Tomomi , Alsamarah Abdelaziz , Kanassatega Rhye‐Samuel , Luo Yun , Andresen Bradley
Published: June 2021

Angiotensin II type 1 receptors (AT1Rs) are involved in numerous physiological processes and AT1R competitive antagonists (ARBs) are used widely as treatments for cardiovascular diseases. However, not all patients, nor species, respond equally to ARBs presumably due to alterations within the ARB binding pocket. Previous studies indicate that the opossum (o) AT1R does not bind, or binds with very low affinity, to ARBs containing a di‐benzyl and five membered ring heterocycle moiety, such as valsartan, due to an altered ARB binding pocket. Thus, the oAT1R can serve as a model of ARB resistance, and an experimentally validated control for elucidating the binding sites of each ARB. AT1R models were prepared and simulated using explicit water solvent and equilibrated for 100 ns. To test the hypothesis that structurally unique ARBs bind to unique sites, each clinically viable ARB was docked to the final eleven configurations of molecular dynamics equilibrated inactive human (h) AT1R and oAT1R. Initial docking of valsartan confirmed the experimental data showing a lack of affinity for the oAT1R (‐0.28 ± 2.72 kcal/mol compared to ‐8.12 ± 0.77 kcal/mol for the hAT1R, n = 10). Surprisingly, ARBs lacking this moiety bound to a different site with reasonable affinity in both oAT1R and hAT1R models (‐6.29 ± 0.44 and ‐7.19 ± 0.63 kcal/mol, respectively). These data suggest that resistance to valsartan, the most commonly prescribed ARB, may not translate to a general ARB resistance. Furthermore, ARBs lacking the di‐benzyl and pentameric ring moiety, such as eprosartan, may be more efficacious in “ARB‐resistant” patients.
Interleukin‐6 Produces Oxidative Stress and Endothelial Dysfunction in the Absence of a Single Endothelial Nitric Oxide Synthase Gene
Authors: Didion Sean
Published: June 2021

The goal of the present study was to test the hypothesis that heterozygous endothelial nitric oxide synthase (eNOS)‐deficiency is associated with oxidative stress and endothelial dysfunction produced by interleukin‐6 (IL‐6). Carotid arteries from eNOS+/+ and eNOS+/‐ mice were incubated with either vehicle or interleukin‐6 (100 or 300 nM) for 22 hrs. IL‐6 produced concentration‐dependent impairment (P<0.05) of responses to acetylcholine in eNOS+/‐ mice. For example, acetylcholine (100 µM) produced 89±1, 60±8, and 35±4% relaxation in arteries from eNOS+/‐ mice treated with vehicle, 100 nM IL‐6, or 300 nM IL‐6, respectively. Basal and NADPH‐stimulated superoxide levels were also selectively increased in vessels from eNOS+/‐ mice incubated with IL‐6. IL‐6 had no effect (P>0.05) on endothelial responses or superoxide levels in eNOS+/+ mice. The effects of IL‐6 were selective for endothelium as responses to nitroprusside were unaltered (P>0.05) by IL‐6 or genotype. Taken together, these findings demonstrate that loss of a single eNOS gene predisposes blood vessels to the negative effects of IL‐6. These findings are an example of eNOS haploinsufficiency and have important implications in disease states associated with reductions in eNOS expression and/or activity in combination with increases in IL‐6, such as aging and obesity. Supported by NIH HL‐089884 and HL‐107632.
SIRT3 Deficiency Impairs Hypoxic Signaling, Reprograms Basal Glycolytic Metabolism and Exacerbates Myocardial Ischemic Injury
Authors: He Xiaochen , Zeng Heng , Chen Jian‐xiong
Published: June 2021

Increased glycolytic metabolism is critical for maintaining homeostasis of cardiomyocytes and endothelial cells (EC) during myocardial ischemia. Sirtuin 3 (SIRT3) is involved in epigenetic regulation of variety of cellular functions. The present study investigated the role of SIRT3 in altering glycolytic metabolism and hypoxia/ischemia responses using SIRT3 knockout (SIRT3 KO) mice and SIRT3 deficient EC. In vitro, exposure of EC to hypoxia up to 24 hours resulted in a gradual increase in HIF‐2α expression in WT and SIRT3 KO‐EC. Interestingly, hypoxia‐induced HIF‐2α expression was less in SIRT3 KO‐EC compared to EC isolated from WT mice. Moreover, hypoxia‐induced VEGF expression was reduced in SIRT3 KO‐EC. Using Seahorse XFe24 analyzer, EC glycolysis and mitochondrial function were further examined. Basal glycolysis, glycolytic reserve and glycolytic capacity were dramatically reduced in Sirt3 KO‐EC compared to WT‐EC. Moreover, Sirt3KO EC exhibited higher oxygen consumption rate than WT‐EC. Intriguingly, treatment with prolyl‐hydroxylase inhibitor‐dimethyloxalylglycine (DMOG) or VEGF rescued the impaired basal glycolytic metabolism in Sirt3 KO‐EC. Treatment with DMOG resulted in a reduction of basal oxygen consumption in WT‐EC, but failed to reduce basal oxygen consumption in Sirt3 KO‐EC. In vivo, SIRT3 KO mice subjected to ischemia exhibited a larger infarct size and impaired cardiac function compared to WT mice. VEGF and glycolytic enzyme PFKFB3 levels were significantly decreased in the heart of SIRT3 KO relative to WT mice. These studies suggest that the loss of SIRT3 aggravates myocardial ischemic injury by altering hypoxic signaling, glycolytic metabolism and increasing oxygen consumption.