bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of MicroRNA‐429 Expression Mediates Angiotensin II‐induced Kidney Damages in Rats
Authors: Wang Zhengchao , Zhu Qing , Wang Weili , Li Pin‐Lan , Hu Junping , Li Ningjun
Published: June 2021

MicroRNA (miR) 429 has been shown to inhibit epithelial‐to‐mesenchymal transition (EMT) and promote mesenchymal‐to‐epithelial transition (MET). The present study determined whether miR429 participated in angiotensin (ANG) II‐induced transdifferentiation and consequent fibrogenesis in renal cells. In NRK‐52E cells, a rat proximal tubular cell line, incubation of ANG II (1 nM) for 24 h significantly reduced the level of mir429 by 60% and meanwhile increased the protein levels of mechansymal markers α‐smooth muscle actin (α‐SMA) and fibroblast‐specific protein (FSP)‐1 by 3 fold and decreased epithelial marker E‐cadherin by 60%. In cells transfected with plasmids expressing mir429, ANG II‐induced changes in the above markers were abolished. Male Sprague‐Dawley rats were treated with chronic infusion of vehicle, ANG II (200 ng/kg/min) or ANG II + intrarenal transfection of lentivirus expressing mir429 for 12 days. The levels of mir429 in the kidneys were reduced by 75% in ANG II‐treated rats. The protein levels of α‐SMA, FSP‐1 and collagen I/III were much higher and E‐cadherin lower in the kidneys in ANG II‐treated rats than that in control. These changes were reversed in ANG II + mir429‐treated rats. The urinary albumin were 0.9±0.36 mg/Kg/24h in control, 2.8±0.28 mg/Kg/24h in ANG II‐treated and 1.2± 0.27 mg/Kg/24h in ANG II+mir429‐treated rats, respectively, at the end of experiment. There was no difference in the increases of blood pressure between ANG II‐ and ANG II+mir429‐ treated rats. These data suggested that ANG II produces transdifferentiation and fibrogenesis in renal cells via inhibiting mir429 expression and that miR429 protects against ANG II‐induced kidney damage. (Grant supports: NIH HL89563 and HL106042)
Chronic fish oil supplementation partially reverted renal alterations observed in mice fed with a high fat diet
Authors: Fernandez Ricardo , Pequito Danielle , Coelho Isabela
Published: June 2021

The objective of this study was evaluate the effect of chronic fish oil (FO) supplementation on renal morphology and function of mice fed with a high fat (HF) diet. Six weeks male mice were divided in two groups and fed during eight weeks with regular rodent chow (S), or HF diet. After this, they were supplemented orally for 30 days with FO, 1g/kg body weight. Renal function parameters, urinary excretion of TXB2, PGE2 and albumin, immunolocalization of vimentin, desmin were measured. The weight gain of HF group was higher, almost 2 times, than S group. HF group present glucose intolerance, an effect that was not modified by FO. Plasma insulin levels increased significantly in HF. Creatinine clearance was lower in HF (0.81±0.09 mL/min/kg of body wt, n=31) if compared to S (1.28±0.22, n=20) (p<0.05), independent of FO supplementation. High‐fat diet induced a significant albuminuria (expressed as urine albumin/creatinine ratio): 35.65±7.32ng/mg, n=7 in HF vs 11.06±2.16 in S (p=0.01). FO supplementation reduced in 40% albumin excretion in HF (21.33±1.33). Urinary TXB2 excretion was reduced in 58% by high‐fat diet and FO supplementation was not able to reverse this modification. Urinary excretion of PGE2 was similar among groups. The high‐fat diet was able to increase in almost 100% the concentration of TNF‐a in renal tissue; an effect completely reverted by FO supplementation in tubular cells.The intrarenal expression of vimentin was significantly higher in the interstitium and tubules of HF group, an effect reverted by FO supplementation. Desmin expression in glomerulus edge increased in obese animals, but was not modified by FO supplementation. In summary, FO supplementation partially reverted renal alterations induced by HF diet.
Reactive Oxygen Species and NFkB Increase Peritoneal Filtration and Generate Ascites in Nephrotic Syndrome
Authors: Doucet Alain , Brideau Gaelle , Vogt Bruno , Udwan Khalil
Published: June 2021

Clinical and experimental observations suggest that nephrotic ascites is not accounted for by hypoalbuminemia but by changes of the capillary endothelium permeability. We investigated the mechanism of these changes in puromycin aminonucleoside (PAN) nephrotic rats. The peritoneal filtration coefficient (Kf) was estimated by the slope of the regression line connecting the trans‐peritoneal gradient of osmotic pressure which was varied experimentally to the trans‐peritoneal water flux measured by osmotic dilution.

Kf doubled and expression of aquaporin 1 (AQP1) increased in peritoneum of PAN rats. PAN rats displayed peritoneal oxidative stress attested by ROS accumulation and dysregulation of NADPH oxidase and ROS detoxifying enzymes expression. The ROS scavenger N‐acetyl cysteine (NAC) prevented the increase in Kf and in AQP1 expression. It also reduced by half the volume of ascites without changing renal sodium handling, albuminuria or hypoalbuminemia. PAN rats peritoneum also displayed activation of NFkB attested by increased nuclear labeling with anti‐p50 antibody. Activation of NFkB was blunted in NAC‐treated PAN rats. JSH‐23, an inhibitor of NFkB transcriptional activity, mimicked all the effects of NAC.

These results show that nephrotic syndrome is associated with increased filtration coefficient of the peritoneal barrier which stems in part from increased expression of AQP1. These changes are triggered by oxidative stress and subsequent activation of NFkB. They account for at least half of increased filtration rate, the remaining stemming possibly from decreased plasma oncotic pressure.
Utilizing a Type 1 Diabetic Nephropathy Model Developed on the Basis of Streptozotocin‐Treated Dahl SS Rats for the Studies of Calcium Handling in the Podocytes
Authors: Lowing Andrea , Ilatovskaya Daria , Palygin Oleg , Pavlov Tengis , Levchenko Vladislav , Staruschenko Alexander
Published: June 2021

Podocytes play a key role in the development of nephropathy: podocyte depletion results in reduced GFR and progressive glomerulosclerosis. Circulating factors can activate calcium influx in these cells triggering processes that lead to podocyte loss. This study was aimed at developing a proper model of diabetic nephropathy (DN) on the basis of the Dahl Salt‐Sensitive (SS) rat that could be further used for assessing calcium signaling in podocytes. Type I diabetes was induced in 6 week old SS rats by an injection of 75 mg/kg streptozotocin (STZ) which caused an elevation of blood glucose up to 700 mg/dL; this was then decreased to 350 mg/dL by an insulin pellet implant. Rats were monitored throughout the next 6 or 12 weeks; urine analyses revealed progressive proteinuria, whereas sodium and potassium fractional excretion levels were normal in both control and diabetic groups (0.3 and 14 for fractional excretion of Na and K, respectively). However, 6 weeks after the STZ injection was not sufficient to develop histological features characteristic in DN, whereas the 12 week treatment resulted in typical DN kidney damage. Urinary ELISA showed a 50‐fold increase in nephrin excretion in the 12 week hyperglycemic animals, which is indicative of podocyte depletion and foot processes effacement. At the end of the experiment rat glomeruli were isolated and successfully used for ratiometric confocal measurements with fluorescent dyes or patch‐clamp measurements. In conclusion, this rat model shows the features of diabetic kidney injury and is a good basis for the assessment of calcium signaling and glomerular filtration barrier function during type 1 DN.
The Effect of Fetal Hypoxia on Programing Cardiovascular Function
Authors: Prestipino Louise , Polson Jaimie , Brolin Elisabeth , Ritchie Helen
Published: June 2021

Chronic prenatal hypoxia is associated with intrauterine growth retardation and programs hypertension in the offspring. However, previous methods for inducing fetal hypoxia used either maternal hypoxia or placental insufficiency, both of which may confound the interpretation because of side effects. We have developed a model in which hypoxia is restricted to the fetus. Dofetilide (Dof) is a class III antiarrhythmic agent that specifically blocks the IKr channel. This channel is expressed in the rat fetus but not adult and its blockade induces fetal bradycardia. In this study, we confirmed the effects of Dof on the fetal heart rate (HR) and determined its effects on the adult offspring. Dams were dosed with Dof (2.5 mg/kg) or saline at gestational days E11‐E14. In the first series of experiments dams were sacrificed after two hours and 8 embryos were collected from each rat and placed in culture for 15 min. Embryos were then videoed for HR analysis, the results confirming that Dof induced a 35‐45% drop in HR in Dof embryos compared to controls. In the second series of experiments, litters were born and at 3‐4 months of age, blood pressure (BP), HR and spontaneous baroreflex gain (sBRG) were recorded using radiotelemetry. At 3 days of age, Dof pups were 6% lighter than control rats, and remained ~8% lighter at 3‐4 months. Mean BP was ~10 mmHg higher in the adult Dof rat than controls during both the night (active phase) and day (night: controls, 81.7±1.8 mmHg; Dof, 91.8±2.3 mmHg, P<0.01). There were no differences in HR or sBRG. Our results show that Dof produces a large fall in HR in the fetus at E11‐E14, producing small birth weight pups and programing for hypertension in later life. We found no evidence of altered baroreflex function.
Early Life Stress (ELS) Induces Dysregulation of Pro‐ and Anti‐Inflammatory Gene Expression in Renal Cortex of Adult Rats in Response to Chronic Angiotensin II
Authors: Obi Ijeoma , Loria Analia , Ho Dao , De Miguel Carmen , Pollock Jennifer
Published: June 2021

ELS increases cardiovascular disease risk and chronic inflammation in human adults. We previously showed that maternal separation (MatSep), an ELS rat model, induces exaggerated blood pressure and renal cortical T‐cell infiltration in response to chronic AngII. We hypothesized that MatSep induces dysregulation of pro‐ and anti‐inflammatory gene expression in response to AngII infusion in adult rats. Expression of pro‐ and anti‐inflammatory genes was measured by PCR array or qRT‐PCR in circulating T cells, renal vessels, and renal cortices from control and MatSep rats. We found that at baseline MatSep significantly (p<0.05) induces CCL2, IL‐2R‐g, CD40 ligand, and TNFR1 genes in circulating T cells; CX3CL1, NFkB1 and TGFb in renal vessels; and caspase3, Flt3, IL‐15, Pik3r1, and Prlr in renal cortex when compared to control rats. In response to AngII infusion, renal cortices from control rats showed significant up‐regulation of mRNA expression of Cd1d1 (baseline vs. AngII, fold‐change, p<0.05: 1.0+0.04 vs. 1.9+0.30), Flt3 (1.0+0.30 vs. 1.9+ 0.20), and IL‐11 (1.0+0.30 vs. 3.5+0.60). In addition, control rats down‐regulated expression of interleukin 12‐b (1.0+0.30 vs. 0.2+0.10) and AT1 receptor (1.0+0.09 vs. 0.3+0.08), while MatSep rats showed an increase in AT1 receptor (1.1+0.20 vs. 2.4+0.40) in response to chronic AngII infusion. These data suggest that ELS induces sensitivity to chronic AngII infusion in adult rats via differential transcriptional responses. Funded by K99/R00HL111354 to ASL, F32HL116145 to DHH, T32DK007545 to CDM, and P01HL69999 to JSP.
Glucocorticoids and the diurnal rhythm of the thiazide‐sensitive NaCl cotransporter
Authors: Ivy Jessica , Aldujaili Emad , Flatman Peter , Bailey Matthew
Published: June 2021

Reabsorbing ~7% of the sodium load, the distal convoluted tubule plays key roles in blood pressure (BP) homeostasis. Here, Na‐Cl co‐transport (NCC) is the major route for apical sodium entry making thiazide diuretics, NCC inhibitors, a mainstay hypertension treatment. Predictive adaptions of sodium excretory rhythms are supported by an intrinsic renal clock, which regulates transporter activity according to physiological need. Peripheral clocks can be influenced by glucocorticoids, which also have a circadian rhythm and we therefore hypothesized that the diurnal rhythm of NCC was regulated by glucocorticoids. C57BL6 mice were kept on a 12h light cycle with subjective day starting at 7am local time. Urine was collected in 12h periods and kidneys harvested at 1am (night) and 1pm (day). Slc12a3 (NCC encoding gene) mRNA and NCC protein abundance were similar between night and day but phosphorylation at threonine 53 was significantly higher at night compared to the day. Both plasma and urinary corticosterone levels were elevated at night. Glucocorticoid inducible leucine zipper (GILZ) and serum and glucocorticoid inducible kinase (SGK1) transcripts were upregulated at 1am compared to 1pm. Chronic corticosterone infusion flattened the corticosterone rhythm at intermediate levels. The diurnal rhythm of T53 phosphorylation was dampened in these mice but not in vehicle‐treated mice. These data indicate that glucocorticoids contribute to the regulation of a diurnal rhythm of NCC phosphorylation. This may be clinically relevant particularly in the pathogenesis of hypertension in conditions associated with elevated glucocorticoid levels such as metabolic syndrome and chronic stress.
Impaired baroreflex response correlates with reduced conduit vessel contractility in female maternally separated (MS) rats and reveals α1D adrenergic receptor (AR) dysfunction
Authors: Murphy Margaret , Evans Logan , Mahanes Timothy , Loria Analia
Published: June 2021

Previously, we have shown that male MS rats display impaired baroreflex function and chronic blood pressure control but normal acute reactivity to α1 adrenergic stimulation. This study tests in female (F) MS rats: 1) MAP and HR response to phenylephrine (PE), 2) ex vivo vascular reactivity in aorta and 3) aortic contractile protein expression. MS was applied 3 hr/day from postnatal day 2‐14. Normally reared rats served as controls (C). Adult F rats (n=4) were anesthetized. Saline solution or PE (0.3 μg/μl, 2 min) was administrated i.v. Hemodynamic data was collected using an arterial catheter. PE infusion was performed before and after a bolus of mecamylamine (MECA, 0.1 mg/kg, i.v). HR/MAP slope was greater in MS compared to C rats (‐1.9±0.2 vs. ‐0.9± 0.1 bpm.mmHg‐1, p<0.05). MECA‐induced drop in blood pressure was not different between groups. Maximal PE‐induced MAP response was exaggerated in MS compared with C rats (p<0.05), showing no changes in HR between groups. PE‐induced reactivity in aorta was attenuated in MS rats (68±6 vs. 96±8 % KCl, p<0.05, n=6). ACh‐induced relaxation was impaired in aorta from MS rats (59±7 vs. 81±3 % relax, p<0.05). α1A AR protein expression was similar between groups, but α1D AR was increased in MS compared to C rats (0.4±0.1 vs. 1.3±0.2 AU, p<0.05, n=6). Contractile proteins RhoA, MLC2 and ROCKII were similar between groups. In MS rats, impaired contractile function in conduit vessels suggests an inadequate subcellular localization of the α1D AR, that may impact the myogenic tone and the baroreflex function as well. Due to the lack of vascular response, PE‐induced central effects need further investigation. Funding: R00 HL111354
Dynamin‐2 is a negative regulator of NOS1β in the collecting duct
Authors: Arguello Alexandra , Hyndman Kelly , Morsing Sofia , Pollock Jennifer
Published: June 2021

Nitric oxide synthase 1 (NOS1)/NO pathway in the inner medullary collecting duct (IMCD) promotes natriuresis. Rat IMCD express NOS1α and NOS1β splice variants, while mouse and human IMCD expresses predominantly NOS1β. We reported dynamin 2 (DNM2) activates NO production via direct interaction with NOS1α; however it is undetermined if DNM2 also activates NOS1β. Accordingly, we hypothesized that DNM2 is an activator of NOS1β. To test this hypothesis, we used COS7 cells, which lack endogenous NOS, and mIMCD‐3 immortalized cells that express NOS1β. By immunoprecipitation and western blot, we confirmed that NOS1β interacts with DNM2. Nitrite production (an index of NO production) was assessed by HPLC, and we determined that overexpression of DNM2 in mIMCD‐3 cells resulted in a significant reduction in nitrite production (323 ± 25 pmol/mg pr/h vs. 1734 ± 35, N = 3‐4, P = 0.016). Using a variety of pharmacological inhibitors and control agents targeting different domains of DNM2, we determined that inhibition of DNM2 resulted in a significant increase in mIMCD‐3 nitrite production (ranged from 1410 ± 419 to 1910±105 pmol/mg pr/h) compared to controls (ranged from 665 ± 57 to 839 ± 120 pmol/mg pr/h; n = 4, P < 0.05). Finally, we confirmed our pharmacological experiments by decreasing DNM2 expression with siRNA. DNM2 siRNA resulted in a 95% reduction in mIMCD‐3 DNM2 expression and produced a significant increase in nitrite production (scramble siRNA 112 ± 15 vs DNM2 siRNA 249 ± 21 pmol/ mg pr/ h). In conclusion, these data suggest that DNM2 is a novel negative regulator of NOS1β.
Vasopressin Increases Adrenal Blood Flow in a Pig Model of Hemorrhagic Shock
Authors: Uyehara Catherine , Wetstein Paul , Ichimura Wayne , Murata Lee‐Ann , Sato Aileen , Hernandez Claudia , Kajiura Lauren
Published: June 2021

Vasopressin (VP) is effective in restoring mean arterial pressure (MAP) in catecholamine‐resistant shock and seems to augment adrenergic vasopressor action. We thus examined whether VP may influence adrenal function early on in hemorrhage. Using a pig model of severe rapid hemorrhage (30 ml/kg blood loss in 20 minutes), we compared the effect of fluid resuscitation (NS; normal saline equal to no more than 2 times the shed volume, n=9) in combination with VP (30 ng/kg/min, n=7), phenylephrine (PE; 5 mg/kg/min, n=6), or norepinephrine (NE; 0.1 mg/kg/min, n=7) against fluid resuscitation alone. Hemorrhage caused a 50% drop in MAP (74±2 to 36±1 mm Hg, p<0.05) and decreased cardiac output (CO; from 3.5±0.1 to 2.0±0.1 L/min, p<0.05). VP returned MAP to baseline (72±4 mm Hg), versus partial restoration with the other treatments (50±6, 53±4, and 53±3 mm Hg, for NS, PE, and NE, respectively). CO was restored with fluid resuscitation in all groups. Interestingly, adrenal blood flow doubled with the endogenous response to hemorrhage. VP administration further increased adrenal blood flow to 3.6 + 0.9 times baseline. PE, NE or NS did not exhibit this enhanced augmentation. Cortisol, aldosterone, adrenocorticotropic hormone, epinephrine and norepinephrine levels all increased in response to hemorrhage and returned to baseline with correction of blood pressure. There was no evidence of adrenal insufficiency in this acute phase of shock. VP‐induced increase in adrenal blood flow during hemorrhage may help prevent development of adrenal insufficiency as a long term complication of severe hemorrhagic shock.
Increased Renal Potassium Secretion Associated with Increased Vasopressin Prevents Hyperkalemia in a Rat Model of Hemorrhagic Shock
Authors: Lee Debby , Hernandez Claudia , Murata Lee‐Ann , Sato Aileen , Uyehara Catherine
Published: June 2021

Trauma is the leading cause of death in young adults worldwide, and up to 50% of these deaths involve hemorrhage. Hyperkalemia is associated with non‐survival of severe hemorrhage. During shock, urine flow is dramatically decreased, and thus, in order to maintain electrolyte balance, renal handling of solute secretion must be altered. In this study, we tested the hypothesis that the kidneys respond to hemorrhage by increasing potassium (K) secretion to prevent hyperkalemia. We compared renal handling of K and sodium (Na) in conscious, chronically‐catheterized rats (n=15) before, during, and after hemorrhage (2 ml shed blood/100 g body weight, 25% hematocrit decrease). One hour after hemorrhage, rats were resuscitated with normal saline. With shock, urine flow decreased from 14 ± 2 to 4 ± 1 ul/min/100g BW, and osmotic clearance also concomitantly decreased (34 ± 3 to 15 ± 2 ul/min/100g, p<0.05). There was a slight increase in plasma K (3.94 ± 0.06 to 4.34 ± 0.07 mEq/L, p<0.05) during hemorrhage. Endogenous plasma vasopressin (VP) levels increased (5.3 ± 1.2 to 26.8 ± 8.4 pg/ml, p<0.05), followed by an increase in renal trans‐tubular K gradient (11 ± 1 to 14 ± 1, p<0.05). Urine K levels increased (111 ± 13 to 191 ± 10 mEq/L, p<0.05) whereas urine Na levels did not. Results suggest that a relative increase in renal tubular K secretion mediated by VP plays an important role in the regulation of K homeostasis during hemorrhage. This increase in K secretion may protect against hyperkalemia in hemorrhagic shock.
Sex Differences in the Diurnal Natriuretic Response to a Salt Load in Rats Lacking a Functional ETB Receptor
Authors: Johnston Jermaine , Mason Jourdan , Jin Chunhua , Speed Joshua , Pollock David
Published: June 2021

Male rats lacking the ETB receptor (ETB def) exhibit salt‐sensitive hypertension due to impaired renal sodium handling. Given that females generally have a reduced incidence of salt‐sensitive hypertension compared to males, we hypothesized that female ETB def rats are better able to excrete a salt load compared to males. ETB def and genetic control rats of both sexes were implanted with telemetry transmitters to monitor mean arterial pressure (MAP) and given at least a week to recover. Following several days of baseline urine collection in 12 hr light/dark increments, rats were given a single 900µEq Na salt load (NaCl) by oral gavage at the beginning of their active (7pm‐7am, dark) or inactive (7am‐7pm, light) period. Urine was again collected in 12 hr intervals for several more days. We previously showed that ETB def males excrete significantly less salt during the initial 12 hr following an acute salt load whether given at the beginning of the active or inactive period compared to genetic controls, an effect that was more pronounced when the salt load was given prior to the inactive period. ETB def females excreted significantly more salt vs. ETB def males following the acute salt load given during the beginning of the active period (673 ± 111 vs. 320 ± 98µEq Na/12hr, P<0.05) and again showed an improved natriuresis vs. ETB def males following a salt load given at the start of the inactive period (183 ± 60 vs. 50 ± 91µEq Na/12hr). There was no significant effect of the salt loading on MAP in any group, although both male and female ETB def rats had significantly elevated MAP compared to controls. These data show that despite the loss of the ETB receptor, females excrete an acute salt load more efficiently than males.
The Renal Aldosterone‐Endothelin Feedback System (RAES) Controls Total Na Balance and Blood Pressure
Authors: Welch Amanda , Lynch I Jeanette , Gumz Michelle , Cain Brian , Wingo Charles
Published: June 2021

Aldosterone increases blood pressure (BP) by stimulating sodium (Na) reabsorption in the collecting duct (CD) and, by negative feedback, stimulates CD endothelin‐1 (ET‐1) that acts to inhibit Na and water reabsorption. We tested the hypothesis that a renal aldosterone‐endothelin feedback system (RAES) regulates Na balance and BP by comparing the effect of a high NaCl diet (2% gelled diet with saline to drink) and mineralocorticoid stimulation in wild‐type mice (WT) and CD‐specific ET‐1 knockout mice (KO). Metabolic balance and radiotelemetric BP were measured before and after treatment with desoxycorticosterone pivalate (DOCP, 0.07mg/g, IM). KO mice consumed more HS diet and saline and had greater urine output than WT. Before DOCP, the KO mice, but not WT, increased fluid balance, body weight (BW), and BP. WT mice did not increase fluid balance, BW, or BP until after DOCP treatment. DOCP further increased systolic BP in the KO. After DOCP, KO mice exhibited greater positive electrolyte and fluid balance and BW than WT. Unlike WT, KO mice failed to respond acutely to DOCP treatment with transient positive Na balance and subsequent return to neutral Na balance (mineralocorticoid escape). Thus, the absence of RAES impairs renal response to Na loading, which results in abnormal fluid and electrolyte handling when challenged with a HS diet and DOCP treatment. We conclude that 1) CD expressed ET‐1 functions as a principal mechanism for mineralocorticoid escape; 2) disruption of RAES prevents renal compensation to, and escape from, the chronic effects of mineralocorticoids on electrolyte balance.
Epithelial Sodium Channel (ENaC) Tagged with Bungarotoxin Binding Site to Study Channel Plasma Membrane Expression and Trafficking
Authors: Gonzalez‐Montelongo Rafaela , Alvarez de la Rosa Diego , Giraldez Teresa
Published: June 2021

Transepithelial sodium reabsorption through ENaC plays a key role in electrolyte and extracellular volume homeostasis and regulation of blood pressure. ENaC is a heterotrimeric ion channel formed by α, β and γ subunits. Channel activity is tightly regulated by many local and circulating factors, which in turn control channel cell surface expression (N) and open probability (Po). ENaC upregulation resulting from decreased endocytosis or enhanced delivery to the membrane have been described. However, there are several technical challenges in the study of ENaC trafficking, including low levels of plasma membrane expression. The aim of this study was to develop a new tool to directly study the dynamics of ENaC trafficking to and from the plasma membrane. To that end we inserted an α‐bungarotoxin (BTX) binding site (BBS) in the extracellular domain of the a subunit and used fluorescently labeled BTX to study ENaC plasma membrane expression and trafficking in Xenopus laevis oocytes. Mutant αBBSβγ channels do not shown any functional abnormality in the absence or presence of the toxin. Confocal imaging of Alexa Fluor 488 (AF488)‐labeled BTX showed specific labeling in plasma membrane of oocytes expressing αBBSβγ channels. ENaC retrieval was detected by imaging AF488‐BTX labeled oocytes at different time points whereas ENaC insertion rate was detected by blocking αBBSβγ‐expressing oocytes with unlabeled BTX and then staining with AF488‐BTX at different times. Our results suggest that tagged αBBS subunits provide a useful tool to study plasma membrane ENaC abundance and trafficking to and from the membrane.
CRISPR/Cas mediated genome editing reveals new insights into the WNK‐SPAK/OSR1 network
Authors: Roy Ankita , Goodman Joshua , Pittman Gabrielle , Begum Gulnaz , Donnelly Bridget , Sun Dandan , Subramanya Arohan
Published: June 2021

Sodium‐coupled SLC12 cation chloride cotransporters play important roles in cell volume and chloride homeostasis, epithelial fluid secretion, and renal tubular salt reabsorption. These cotransporters are phosphorylated and activated indirectly by With‐No‐Lysine (WNK) kinases through their downstream effector kinases, SPAK and OSR1. Multiple WNKs can coexist within a single cell type, although their relative contributions to SPAK/OSR1 activation and salt transport remain incompletely understood. Here, we outline a simple method to selectively knock out WNK expression from mammalian cells using single guide RNA (sgRNA) targeted CRISPR/Cas9 endonucleases. Genetically and biochemically validated WNK1 knockout HEK293T cells exhibited low baseline WNK4 and SPAK/OSR1 activity. This was associated with increased expression of KLHL3/CUL3, suggesting that WNK1 regulates WNK4 stability through their cognate E3 ubiquitin ligase. WNK1 KO cells also failed to trigger regulatory volume increase (RVI) during hypertonic stress, proving that WNK1 is necessary for cell volume control. Currently, we are adapting this technology to generate WNK4 and WNK3 KOs. To optimize sgRNA efficiency, we used a surrogate reporter‐based assay that triggers eGFP fluorescence upon target site editing, permitting enrichment of edited cells by FACS. This technique dramatically enhanced detection of genomic modification by T7E1 mismatch screening. Collectively, our data illustrate how this new and powerful technology can be used to dissect and analyze complex signaling networks. Support: NIDDK, VA
ADF/COF Have Different Effects on Intestinal Brush Border Structure and NHE3 Activity
Authors: Gao Lucy , Sarker Rafiquel , Donowitz Mark
Published: June 2021

The Na‐H exchanger 3 (NHE3) protein complex accounts for most Na absorption at the intestinal brush border. NHE3 expression/activity is acutely regulated by endocytosis/exocytosis. Cofilin‐1 (COF) and actin depolymerizing factor (ADF) are highly conserved isoforms of the cofilin family of actin severing proteins. COF/ADF are ubiquitously expressed though ADF has a stronger presence in intestinal tissue. We hypothesized that COF and/or ADF are necessary for basal and regulated NHE3 activity by remodeling the terminal web actin structure to allow endocytosis/exocytosis. COF and ADF knockdown (KD) cells were engineered with lentivirus/puromycin in Caco‐2 cells, a polarized intestinal Na‐absorptive cell line. COF‐KD reduced COF by 67±5% (p蠄0.01) without significantly altering ADF, while ADF‐KD cells reduced ADF by 60±7% (p蠄0.01) without altering COF. By transmission electron microscopy, COF‐KD cells had straighter and a greater number of microvilli (MV) than control and ADF‐KD cells had more bent MV. By immunofluorescence, neither COF nor ADF had a predominantly MV distribution. NHE3 activity was quantified by measuring pH change (BCECF/fluorometry) under basal and inhibited (forskolin (FSK), 2.5 uM) conditions. COF‐KD had 35±12% less activity than control cells (p蠄.05). ADF‐KD had 34±12% greater NHE3 activity than control cells (p蠄.05). FSK inhibited NHE3 similarly in all three conditions (approximately 49%).

Conclusions

1. Using KD studies we conclude that COF normally increases and ADF decreases basal NHE3 activity, but neither are involved in cAMP induced inhibition of NHE3. 2. COF normally reduces apical microvillar number while ADF is necessary for microvillar rigidity. The lack of ADF/COF in MV suggest that they affect the MV through intermediate proteins.
Abnormal Calcium Metabolism in the Living Human Inner Ear Disorders
Authors: Tanioka Hisaya , Kaga Kimitaka
Published: June 2021

Aims

1) What kinds of metabolic abnormalities were caused by the inner ear disorders such as presbycusis, sudden deafness, tinnitus, BPPV, and Meniere's disease? 2) To investigate an imaging method of abnormal calcium oxide concentrated in the inner ear used by a conventional spiral CT.

Subjects

6 normal ears of adolescence, 6 normal ears of elderlies, 3 ears of sudden deafness, 3 ears of BPPV, 6 ears of tinnitus, and 3 ears of Meniere's disease. Every cases were undergone follow‐up exams after medical treatments.

Methods

3D virtual histology of the inner ear used by the volume rendering technique was reconstructed by a conventional temporal HR‐CT. For the imaging study of calcium oxide in the desiccant, quantitative analysis based on 3D volume rendering technique used by a spiral thin section CT.

image

Results

6 normal adolescent ears showed no image of calcium oxide. Meniere's disease, tinnitus originating from the ear, sudden deafness, and BPPV and aging changes revealed abnormal deposited of calcium oxide at the cochlea and saccule. For tinnitus originating from the ear, the calcium oxide deposited in the cochlea. Otherwise, tinnitus from brain did not deposit in the inner ear. Aging change showed calcium oxide deposited in the cochlea and vestibule in correlation with their aging.

image

Conclusion

All inner ear disorders and aging changes showed abnormal images of calcium oxide like figure 2. But, tinnitus originating from brain showed no calcium oxide deposited in the cochlea. These facts implicated that inner ear disorders and aging change were caused by unbalance metabolism between calcium ion and oxygen. But we cannot deny the existence of calcium peroxide (CaO2) that was produced by free radical action.
Exercise Training Reduces AngII and Increases Ang‐(1‐7) Concentration in Cardiac Muscle of CHF Rats
Authors: Gomes‐Santos Igor , Fernandes Tiago , Couto Gisele , Ferreira‐Filho Julio , Salemi Vera , Fernandes Fernanda , Casarini Dulce , Brum Patricia , Rossoni Luciana , Oliveira Edilamar , Negrao Carlos
Published: June 2021

ACE‐AngII axis of the renin angiotensin‐system is increased in the heart of chronic heart failure (CHF) and contributes to its pathophysiology. Recent studies show that ACE2‐Ang‐(1‐7) counteracts the ACE‐AngII effects. However, the cardiac balance between ACE‐AngII/ACE2‐Ang‐(1‐7) in CHF remains unknown. We studied: 1) The cardiac ACE‐AngII/ACE2‐Ang‐(1‐7) balance in CHF; and 2) The effects of exercise training (ExT, treadmill running, 60min/d, 5 d/wk, 60% VO2max for 8wk) on ACE‐AngII/ACE2‐Ang‐(1‐7) balance in the heart of CHF rats. Male Wistar rats underwent coronary artery ligation for CHF induction or sham surgery (C). Four weeks later, they were divided into four groups: 1) C (untrained), 2) C+ExT, 3) CHF and 4) CHF+ExT. ExT. CHF or ExT did not change cardiac ACE activity (fluorimetry, uF/min/mg). In contrast, CHF increased cardiac AngII concentration (HPLC, 139.1±22.6 and 96.8±8.1 pmol/mg) and ExT reduced it (70.4±9.7 pmol/mg, P=0.01). Unexpectedly, CHF provoked an increase in ACE2 activity. ExT increased ACE2 activity, even further in the CHF+ExT rats. CHF caused no changes in Ang‐(1‐7). ExT increased cardiac Ang‐(1‐7) concentration in both C and CHF rats, which resulted in an augmented Ang‐(1‐7)/AngII ratio. We conclude that CHF provokes an imbalance in cardiac ACE‐AngII/ACE2‐Ang‐(1‐7) toward ACE‐AngII axis. ExT counteracts this imbalance by increasing cardiac ACE2‐Ang‐(1‐7) axis. Sponsored by FAPESP, CNPq and CAPES.
The Percent Composition of Carbon, Oxygen and Nitrogen on a Nitrocellulose Filter after Exposure to Vaping or Smoking
Authors: Palazzolo Dominic , Crow Andrew , Johnson Robert
Published: June 2021

Electronic cigarettes are at the center of much controversy concerning their safety relative to conventional cigarettes. This study investigates the effect vaping electronic cigarettes, compared to smoking conventional cigarettes, has on the percentages of carbon (C), oxygen (O) and nitrogen (N) that makeup a nitrocellulose filter (13 mm diameter, 5µm pore size), and serves as a basic index of the relative impact vaping and smoking have on the respiratory tract. A peristaltic pump (400 ml/min) is used to impact air (n=4), vape (n=4) or smoke (n=4) onto nitrocellulose filters housed inside an in‐line chamber. These filters were exposed to air, vape or smoke during 45 cycles of a 5 second puff followed by a 10 second rest period. Virgin filters not exposed to air, vape or smoke served as control (n=4). The percentages of C, O and N for each exposed filter were determined using scanning electron microscopy with X‐ray microanalysis. The percentage of C in control filters and filters exposed to air, vape and smoke were 46.3 ± 0.3%, 46.6 ± 0.5%, 45.3 ± 0.2% and 73.5 ± 0.3%, respectively. The percentage of O in control filters and filters exposed to air, vape and smoke were 39.8 ± 0.3%, 39.7 ± 0.6%, 44.5 ± 0.3% and 15.9 ± 0.3%, respectively. The percentage of N in control filters and filters exposed to air, vape and smoke were 9.2 ± 0.2%, 9.2 ± 0.4%%, 9.1 ± 0.1% and 8.2 ± 0.3%, respectively. This study indicates that smoking, but not vaping, significantly (p < 0.001) increases the C to O ratio on the nitrocellulose filter, implying that smokers may be more at risk for development of inflammatory or other respiratory pathology.

This work is supported by a Debusk College of Osteopathic Medicine internal grant.
Bile Acid and ATP Signaling in Exocrine Pancreatic Cells
Authors: Kowal Justyna , Haanes Kristian , Christensen Nynne , Novak Ivana
Published: June 2021

Bile acids (BAs) have a multitude of effects in many cells, some of which are mediated by specific receptors such as the TGR5 receptor (Schaap et al., 2014 Nat. Rev. Gastroenterol. Hepatol. 11, 55‐67). Pancreatic cells could be exposed to systemic BAs, as well as to intra‐ductal BAs from bile reflux. They could affect secretory function and/or integrity of pancreatic ducts and acini. Extracellular ATP and purinergic signaling have similar regulatory targets as BAs in pancreas (Novak et al., 2013 Front Physiol 4, 380). The aim of the study was to investigate the interplay between ATP and BA signalling in exocrine pancreas.

Using live‐cell detection of extracellular ATP, we show that chenodeoxycholic acid (CDCA) caused fast and concentration‐dependent ATP release from acinar cells, AR42J (614 ± 79 nM; n = 5) and duct cell line, Capan‐1 (848 ± 16 nM; n = 6). In duct monolayers, ATP was released mainly across the luminal membrane via vesicular and non‐vesicular secretory pathways. Duct cells were not depleted of intracellular ATP (ATPi), but acinar cells lost some ATPi, as detected by several methods including ATP sensor AT1.03YEMK and Magnesium Green. In duct cells, CDCA caused an increase in the intracellular Ca2+ [Ca2+]i (177 ± 21 nM, n = 5), which was significantly inhibited by a mix of antagonists of purinergic receptors (90 ± 9 nM, n = 5). The TGR5 receptor is expressed mainly on luminal membrane of duct epithelia and it can stimulate the Na+/Ca2+ exchanger, protecting cells against high [Ca2+]i.

In conclusion, the CDCA effect can be mediated by ATP release and purinergic signalling. We propose that purinergic signalling must be taken into consideration when interpreting the multifaceted effects of BAs.

Support: The Danish Council for Independent Research/Natural Sciences (0602‐01527B; 4002‐00162).
Probing unexplored neutrophil GPCR signaling pathways to discover novel anti‐bacterial targets
Authors: Corriden Ross , Flores Roxana , Nizet Victor , Insel Paul
Published: June 2021

Polymorphonuclear neutrophils (PMNs), the primary phagocytic leukocytes in the blood, play a central role in the innate immune response to endovascular pathogens such as Staphylococcus aureus (SA). However, excessive PMN activation, such as in septic shock or traumatic injury, can severely damage tissues, including in the cardiovascular system, and is associated with high mortality rates (30‐50%). Enhanced understanding of signaling, in particular by GPCRs that regulate PMN function, may reveal new therapeutic approaches. We thus used Taqman GPCR arrays to define the basal expression of GPCRs (which represented ~190 receptors) by human PMNs and after 3 hr incubation with methicillin‐resistant SA (MRSA). MRSA‐incubated PMNs had altered expression of numerous Gi‐, Gs‐ and Gq‐coupled GPCRs. Antagonists of one newly recognized GPCR, GPR30/GPER, inhibited the formation of DNA‐based neutrophil extracellular traps (NETs) but not phagocytosis or reactive oxygen species production by PMNs. The potential to selectively inhibit NETs, which have been implicated as important mediators of vascular inflammation, while not blocking other responses to pathogens, suggests that PMN‐expressed GPCRs, including newly recognized GPCRs, such as GPR30/GPER are novel targets for the therapy of endovascular pathogens.
Differential Expression of Wnt‐pathway in Late Fetal Ovine Heart and Lung Development
Authors: Gupta Nikita , Krauss Ramona , Smolich Joseph , Cheung Michael , Pepe Salvatore
Published: June 2021

Wnt signalling pathways critical to early cardiogenesis have not been extensively studied in sheep. The activity of Wnt signalling during the rapid heart growth occurring between late‐fetal and early newborn development is not known but may be important given that a right ventricular (RV) functional dominance occurs in utero, but a left ventricular (LV) dominance after birth. Thus our aims were to measure cardiopulmonary changes in 7 key proteins (Wnt3a, Wnt5a, Dvl‐1, phospho‐GSK3β, cdc42, ROCK2 and TCF7L2) and genes (Wnt4 and Wnt7b) in the Wnt signalling pathway in fetal and neonatal sheep. Lung, ventricular and atrial samples from pre‐term (age=128‐129 days; term=147 days), near‐term fetal (age=139‐141 days), and neonatal lambs (age= 5‐7 days post birth) were processed for western immunoblots and qRT‐PCR assays. Four main developmental patterns were evident. In the first pattern, the relative ventricular and atrial expression of Wnt3a, Dvl‐1, phospho‐GSK3β increased during fetal development (P<0.05), but decreased after birth (P<0.05). In the second pattern, fetal and newborn Wnt5a and ROCK2 expression was relatively constant, but with RV expression 2‐3 fold higher (P<0.025 – P<0.001). cdc42 expression decreased in both the ventricular and atrial samples. TCF7L2 expression decreased progressively in both ventricles from preterm to neonatal stages, but increased in the atria. Gene expression of Wnt4 and Wnt7b were greater in atrial samples than ventricular samples. Wnt3a, cdc42 and ROCK2 expression decreased in the lungs post birth but increased for Wnt5a and Dvl‐1. These data show that distinct patterns in Wnt signalling occur during ovine fetal growth, with evidence of differential expression between ventricles and atria.
Porosome: Involvement of Hsp90 in its Assembly and Function
Authors: Jena Bhanu , Rajagopal Amulya , Kulkarni Sanjana , Lewis Kenneth , Chen Xuequn , Taatjes Douglas
Published: June 2021

Cup‐shaped lipoprotein structures at the cell plasma membrane called 'porosomes' are involved in the regulated fractional release of intravesicular contents from cells during secretion. Porosomes enable secretion from cells with exquisite precision, involving membrane fusion and secretory vesicle volume regulation. Porosomes from a number of secretory cells including neurons have been isolated, their composition determined, and they have been functionally reconstituted it in artificial lipid membrane. The dynamics and high‐resolution structure of porosomes in several cell types have been determined using a variety of approaches, including atomic force microscopy, electron microscopy, and X‐ray solution scattering. In the current study, the proteome of the immunoisolated porosome complex in mouse insulinoma Min6 cells was determined using mass spectrometry, demonstrating among other proteins, the presence of 30 core proteins including the heat shock protein Hsp90. Half maximal inhibition of Hsp90 using the specific inhibitor 17‐Demethoxy‐17‐(2‐prophenylamino)geldanamycin, results in the loss of proteins, including the calcium‐transporting ATPase type 2C and the potassium channel subfamily K member 2 from the Min6 porosome. This loss of porosome proteins is reflected in the inhibition of glucose stimulated insulin release from Min6 cells exposed to the Hsp90 specific inhibitor. Results from the study implicate Hsp90 in the assembly and function of the insulin‐secreting porosome.
Cyclic Stretch Induces Fibronectin Expression via c‐Src‐ and STAT3‐ Dependent Pathways in Renal Epithelial Cells
Authors: Alexander Larry
Published: June 2021

Extracellular matrix (ECM) proteins, especially fibronectin, are critical pathological characteristics of renal fibrosis and contribute to the progression of chronic renal disease. Recent studies showed that mechanical stretch is associated with the development of renal fibrosis in a murine model of unilateral ureteral obstruction (UUO). However, the molecular events responsible for stretch‐induced renal fibrosis, following UUO, have yet to be determined. In this study, we investigated the mechanism by which mechanical stretch, an in vitro model for chronic obstructive nephropathy, regulates fibronectin expression in cultured human proximal tubular epithelium (HK‐2) cells. Mechanical stretch up‐regulated fibronectin expression and activated STAT3 in a time‐dependent manner. Stretch‐induced fibronectin were suppressed by a STAT3 inhibitor, S3I‐201 and by small interfering RNA (siRNA) targeting human STAT3 (STAT3 siRNA). Similarly, fibronectin expression and STAT3 activation induced by mechanical stretch was suppressed by the Src family kinase inhibitor PP2 and by transfection of HK‐2 cells with a dominant‐negative mutant of c‐Src (DN‐Src), whereas PP3, an inactive analogue of PP2, had no significant effect. This work demonstrates, for the first time, a role for c‐Src and STAT3 signaling pathways in stretch‐induced renal fibrosis.
Effect of Gradient Ionizing Radiation on ROS Formation in Human Breast Cancer Cells
Authors: Zuo Li , Rong Yi , Roberts William , Jin Feng , He Feng , Zhang Dongqing
Published: June 2021

The application of ionizing radiation (IR) in cancer radiotherapy can induce collective cellular damages via non‐targeted effects, in which neighboring cells are affected by irradiated cells. This so‐called bystander effect (BE) remains an area of interest. Since reactive oxygen species (ROS) play a vital role in biological systems, ROS induced by IR may contribute to BE. However, this mechanism has not been fully elucidated in carcinomas such as human breast cancer cells (MCF‐7). Hereby, we hypothesized that the application of gradient radiation (GR) can reduce the damage to neighboring healthy cells, compared to uniform radiation (UR). We studied radiation dose responses by evaluating ROS generation in MCF‐7 exposed to UR (5 Gy) or GR (8‐2 Gy). Using dihydroethidium (DHE)/ethidium (ET), a fluorescent probe for intracellular superoxide (O2•–, one major ROS), we measured ROS as an indication of cellular oxidative stress. Our results have shown that at 48 hours after irradiation, there is no difference in intracellular ROS formation in GR vs. UR treatment. Antioxidant Tiron treatment eliminated fluorescence, confirming the presence of intracellular ROS. Our project suggests a potential advantage of GR over UR to reduce the damage to neighboring healthy cells via a BE.
Can hyperglycemia‐induced endothelial dysfunction be reversed by regulating the fate of superoxide?
Authors: Patel Hemang , Chen Juan , Kavdia Mahendra
Published: June 2021

Increased superoxide (O₂‾) levels and diminished nitric oxide (NO) are hallmarks of hyperglycemia‐induced endothelial dysfunction. As per the literature increased O₂‾ levels rapidly reacts away with NO to reduce its bioavailability, which sets the stage for endothelial dysfunction in hyperglycemia. With that perspective several therapeutic strategies were tried out to improve NO levels through neutralizing O₂‾ to manage endothelial dysfunction. However, the limited outcomes from these strategies have raised concerns on current understanding of the interplay that drives the fate of O₂‾ to regulate the functionality of endothelial cells. Thus, we investigated the effects of regulating fate of O₂‾ on endothelial function during hyperglycemia. Using systems biology approach, we monitored the interplay amongst superoxide (O₂‾), nitric oxide (NO), and hydrogen peroxide (H₂O₂) during superoxide dismutase (SOD) and catalase treatments in hyperglycemia treated HUVEC. Hyperglycemia significantly increased O₂‾ and H₂O₂levels and decreased NO levels in endothelial cells. However, the treatments with exogenous SOD decreased O₂‾ levels and significantly increased NO levels in hyperglycemic HUVEC. Surprisingly, treatment with catalase also inflicted the similar changes. Our results suggest that manipulating either O₂‾ or H₂O₂ levels can improve NO levels during hyperglycemia‐induced endothelial dysfunction. These results also suggest that production of O₂‾ and H₂O₂ is tightly regulated in hyperglycemia‐induced endothelial dysfunction. Thus, manipulating the fate of O₂‾ may hold a key to reverse the endothelial dysfunction.