bluuebunny/crossref_metadata_2025 huggingface dataset:
Acute Effects of Conjugate Equine Estrogen and Selective Estrogen Receptor‐α Agonist PPT on Postischemic Pial Arteriole Response in Rats
Authors: Zeynalov Emil , Ma Li , Rezvani Niloofar , Littleton-Kearney Marguerite T
Published: June 2021

Study Objective

We explored the effects of acute administration of conjugated equine estrogens (CEE), on postischemic pial arteriole dilatory capacity. Additionally, we determined determined if the beneficial effects of estrogen are mediated through estrogen receptor‐α (ER‐α) using the specific a specific agonist, propyl pyrazole triol (PPT).

Methods

Ovariectomized (OVX) female or male rats were anesthetized, and after placement of a closed cranial window over the parietal cortex, rats were subjected to 15‐min reversible forebrain ischemia, followed by 2 hr. reperfusion. OVX rats received intravenous CEE (1 mg/kg) or vehicle at the onset of reperfusion. Separate cohorts of OVX female or male rats were treated with the ER‐α agonist PPT intraperitoneally (2 mg/kg) 3 hours prior to ischemia. Pial artery dilatory capacity was tested before and after ischemia.

Results

Administration of CEE improved vasodilatory response from −1.7 ± 4.43% to 15.31± 6.63% (p=0.0002). PPT augmented postischemic vasodilatory response in females from −2.63 ± 1.42% to 23.57 ± 11.3% (p= 0.005), and in male rats from −0.38 ± 2.88% to 30.84 ± 11.66% (p=0.007).

Conclusion

Acute administration of CEE or PPT restores postischemic pial arteriole vasodilatory reactivity to Ach. These findings suggest that the effects acute estrogen treatment after ischemic cerebral injury may be modulated via ER‐α.
Cerebral Vascular Blood Flow Changes During ‘Brain Freeze’
Authors: Blatt Melissa Mary , Falvo Michael , Jasien Jessica , Deegan Brian , Laighin Gearóid Ó , Serrador Jorge
Published: June 2021

‘Brain freeze’ commonly occurs when ice cold food or liquid is rapidly consumed. It's mechanism remains unclear, however rapid cooling of the brain through the upper palate may cause local cerebral blood flow changes. The purpose of this study was to examine whether ‘brain freeze’ induces the hypothesized cerebrovascular changes.

Using transcranial Doppler, we evaluated cerebral blood flow velocity (CBFV) in the middle (MCA) and anterior (ACA) cerebral arteries in 13 healthy adults while consuming ice and ambient water. Subjects drank ice water through a straw against the palate until pain developed. CBFV, heart rate, and blood pressure were analyzed, before pain, during pain and after pain.

Consumption of ice water produced a tendency towards increased cerebral flow velocity in the ACA (P=0.078) but not the MCA (P=0.24). Ice water also resulted in greater cerebrovascular resistance during the experimentally induced “brain freeze” when compared to following ambient water consumption.

Our results support a vascular mechanism for brain freeze. Ice water consumption resulted in a significantly greater cerebrovascular resistance as compared to that during ambient water consumption. However, the fact that cerebral flow increased during pain along with increases in blood pressure may suggest autoregulation was not as effective. Supported by NASA and NIH.
Age‐ and estrogen‐dependent changes in cerebrovascular reactivity are mediated by constrictor prostanoids and superoxide
Authors: Deer Rachel R , Perkins Lisa M , Stallone John N
Published: June 2021

The mechanisms underlying the conflicting effects of age and estrogen on cerebrovascular reactivity were examined in the middle cerebral artery (MCA) of mature multigravid (MA) female (F), older reproductively senescent (RS)‐F, and mature male (MM) rats. Ovary‐intact, ovariectomized (OvX) and OvX+estrogen‐replaced (OvX+ER) F were studied. Vasopressin (VP, 10−12–10−7M) reactivity was compared in pressurized MCAs pretreated with indomethacin (Indo, 10μM), NS398 (NS, 10μM), or SC560 (SC, 1μM). In MA‐F, Indo had no effect on VP response, whereas in MM, COX‐1(NS) or COX‐2 (SC) inhibition attenuated maximal reactivity by 25–26%. In contrast, in RS‐F, Indo reduced reactivity by 65%. The effects of oxidative stress on VP reactivity were examined in tempol (TPL, 1mM) treated MCA. In MA‐F, reactivity to VP was 20% lower in OvX+ER than in OvX. TPL reduced reactivity by 40% in OvX but had no effect in OvX+ER. Reactivity to VP in RS‐F was similar in OvX and OvX+ER. TPL attenuated reactivity by 30% in OvX+ER, but had no effect in OvX. These data suggest that: 1) constrictor prostanoids enhance vasoconstrictor responses to VP in MM and in older RS‐F but not in younger MA‐F; 2) estrogen reduces oxidative stress‐induced vasoconstriction in younger MA‐F, but enhances it in older RS‐F. (NIH: HL‐080402)
ANG (1–7) reduces ANG II‐induced insulin resistance by increasing Akt phosphorylation via a Mas receptor‐dependent mechanism in rat skeletal muscle
Authors: Henriksen Erik John , Prasannarong Mujalin , Santos Fernando Reyes
Published: June 2021

Angiotensin II (ANG II) induces hypertension via the ANG II type 1 receptor, while angiotensin (1–7) (ANG (1–7)), a peptide derived primarily from ANG II, elicits an antihypertensive effect via the Mas receptor. Although a critical role of ANG II in the etiology of skeletal muscle insulin resistance is well documented, the role of the ANG (1–7)/Mas receptor axis in this context is poorly understood. Therefore, we determined whether ANG (1–7) is effective in ameliorating the negative effects of ANG II on insulin‐stimulated insulin signaling and glucose transport in isolated soleus muscle strips from lean Zucker rats. ANG II (500 nM for 2 hr) alone decreased insulin‐stimulated glucose transport by 45% (p<0.05). In the presence of 500–1000 nM ANG (1–7), insulin‐stimulated glucose transport in muscle exposed to ANG II improved by ~25% (p<0.05). Moreover, this ANG (1–7)‐mediated improvement in glucose transport was associated with increased Akt Ser467 phosphorylation (47%, p<0.05). The positive effects of ANG (1–7) on insulin‐stimulated glucose transport and Akt phosphorylation were completely blunted in presence of 1000 nM A779, a selective Mas receptor antagonist. The present study demonstrates that ANG (1–7), via a Mas receptor‐dependent mechanism, can partially prevent the inhibitory effect of ANG II on glucose transport in isolated mammalian skeletal muscle, associated with enhanced Akt phosphorylation.
SUR2a gene is upregulated in skeletal muscle insulin resistance of obese rats: possible correlation with GLUT4 gene
Authors: Marques Milano Felipe dos Santos Ferreira , Mori Rosana Cristina Tieko , Okamoto Maristela Mitiko , Machado Ubiratan Fabres
Published: June 2021

Objective

The aim of this study was to investigate whether SUR2A (sulfonylurea receptor type 2A, ABCC9) MEF2a (myocyte enhancer factor‐2a) and HIF1a (Hypoxia‐inducible factor‐1) can influence glucose uptake in oxidative muscle soleus by regulating GLUT4 expression.

Methods

Insulin resistance was induced in male Wistar rats by inducing obesity with monosodium glutamate (MSG). At the age 3 months, half control and MSG‐treated animals started receiving 0.1 mg/kg/day of glimepiride in the drinking water, and distributed in 3 experimental groups: control rats (C), MSG‐obese rats (O) and MSG‐obese rats treated with glimepiride (OG). Animals were submitted to insulin tolerance test (ITT) and samples of soleus muscle were excised for quantification of gene expression by Real Time RT‐PCR and GLUT4 protein.

Results

OG rats presented a 51% increase in SUR2A mRNA content in comparison to O and C rats (p<0.001). O rats showed 16% increase in GLUT4 mRNA content (p<0.01 vs. C), without any increase in GLUT4 protein. After glimepiride treatment, OG rats increased GLUT4 mRNA (21%, p<0.01 vs. C) and protein (131%, p<0.01 vs. C; and 73%, p<0.05 vs. O). No significant differences (p>0.05) in mRNA expression of MEF2a and HIF1a were found.

Conclusion

We conclude that the glimepiride increased the GLUT4 expression in soleus of the insulin resistant animals which could be associated to SUR2A genes expression.
Moderate increases in dietary fat impair microvascular but not myocyte actions of insulin in skeletal muscle of Sprague Dawley rats
Authors: Richards Stephen Michael , Premilovac Dino , Ng Helena , Bradley Eloise A , Rattigan Stephen , Keske Michelle A
Published: June 2021

Enhanced microvascular perfusion in skeletal muscle is important for insulin‐mediated glucose disposal. We have previously demonstrated that high fat‐fed rats (36% fat wt/wt, 4 weeks) have impaired insulin‐stimulated microvascular perfusion and reduced insulin‐stimulated muscle glucose uptake (IMGU). We hypothesised that moderate increases in dietary fat might impair microvascular responses without altering myocyte insulin sensitivity. Rats were fed 5% (5%FD) or 9% (9%FD) fat (wt/wt) diets. After 4 weeks anesthetized rats were subjected to either a hyperinsulinemic euglycemic clamp, and microvascular perfusion measured from hindleg disappearance of 1‐methylxanthine, or pump‐perfusion of the isolated rat hindlimb, which allows myocyte insulin responses to be measured in the absence of altered perfusion. Body weight, epididymal fat pad weight, and fasting plasma glucose were not altered by the diets. Fasting plasma insulin concentrations were moderately elevated, and plasma free fatty acid concentrations were significantly increased in 9%FD. Glucose infusion rate, IMGU and microvascular perfusion during the clamp were significantly impaired in 9%FD. In contrast, IMGU in the isolated, perfused hindlimb was unaltered by diet. These data show that increased dietary fat impairs microvascular insulin responses before those of skeletal myocytes, but still leads to muscle insulin resistance in vivo.
Myostatin Deletion Improves Insulin Resistance and Vascular Function in Obese Mice
Authors: Qiu Shuiqing , Mintz James , Salet Christina , de Chantemele Eric Belin , Stepp David W
Published: June 2021

Insulin resistance in tissues such as muscle and fat is an underlying cause of obesity‐induced vascular dysfunction. Myostatin, secreted by skeletal muscle, has been associated with muscle metabolism and lowering myostatin may improve insulin sensitivity.

Hypothesis

Increasing muscle mass via myostatin deletion in obesity improves whole‐body metabolism and vascular function. A dual knockout (KO) approach was used including dual heterozygous lean mice, lean myostatin KO, db/db and db/db mice with and without myostatin KO. Myostatin KO increased gastrocnemius and gluteal muscle mass (37–52% in lean mice, 83% in obese mice). Myostatin KO improved HbA1c (6.33±0.50 vs. 10.00±1.54) in db/db mice, and fasting glucose in both lean and db/db mice, while myostatin KO had no effect on lipid metabolism. Glucose tolerance test results showed that db/db mice were insulin resistant and that myostatin KO improved insulin sensitivity in db/db mice. Obese db/db mice demonstrated an impaired microvascular vasodilation to acetylcholine that was improved by KO of myostatin. Adrenergic constriction was similar in all groups. Improvement of vasodilator capacity by myostatin KO was not attributed to vascular sensitivity to nitric oxide or changes in vascular structure and mechanics. Taken together, these data suggested that myostatin deletion in obese mice improved insulin resistance and endothelium dependent vasodilation.
Antenatal Maternal Protein Deprivation and Sexually Dimorphic Programming of the Pancreatic Renin‐Angiotensin System
Authors: Goyal Ravi , Wong Christine , Longo Lawrence D.
Published: June 2021

Alterations in the local pancreatic renin‐angiotensin system (RAS) may play a role in the underlying mechanisms of antenatal maternal malnutrition‐induced type 2 diabetes mellitus (T2DM). In the present study, we tested the hypothesis that antenatal maternal protein deprivation (AMPD) leads to alterations in the local pancreatic RAS, with associated hyperglycemia in the adult progeny. Mice dams were fed either control or 50% protein restricted diet starting 1 week before conception and maintained during complete gestation. From 3‐week old control and AMPD mice offspring (n= 5), we isolated pancreas to examine the RAS mRNA and protein expression. We also examined birth weight, catch‐up growth, insulin, and blood glucose levels of control and AMPD mice offspring. Our results demonstrate low birth weight and sexually dimorphic programming of the pancreatic RAS, with development of hyperglycemia in the mice offspring as a consequence of AMPD during pregnancy. Only female offspring from the AMPD group developed hyperglycemia with no significant difference serum insulin concentration. We conclude that sexually dimorphic programming of the pancreatic RAS expression is associated with AMPD diet‐mediated development of hyperglycemia.
Regulation of Nox4 by Calcineurin in the Diabetic Kidney
Authors: Williams Clintoria R. , Reddy Ramesh N. , Gooch Jennifer L.
Published: June 2021

Diabetic nephropathy is a leading cause of morbidity and mortality and is due to the detrimental effects of hyperglycemia. Hypertrophy, an early response to hyperglycemia, may contribute to pathological changes in the kidney. Previously, we found that calcineurin (Cnβ) is required for diabetic hypertrophy. Other groups have reported a role for the Nox4 isoform of NADPH oxidase in diabetic nephropathy. Therefore, we examined if Cnβ and Nox4 act in a common pathway to induce hypertrophy in vitro and in vivo. First, we examined the effect of high glucose (HG) on Nox4 expression in primary kidney tubule epithelial cells (TECs). In wild‐type (WT) TECs, HG stimulated up‐regulation of Nox4 mRNA and protein expression. In contrast, HG failed to induce mRNA and Nox4 protein expression in Cnβ−/− TECs. Next, we examined regulation of Nox4 expression by hyperglycemia in vivo using WT and Cnβ−/− mice. In diabetic WT mice, whole kidney hypertrophy was induced and Nox4 expression was increased in the kidney cortex compared to control WT mice. In contrast, kidney hypertrophy was reduced in diabetic Cnβ−/− mice. Likewise, Nox4 expression was not increased in the kidney cortex of diabetic Cnβ−/− mice. These data show that Cnβ is required for HG‐mediated Nox4 up‐regulation in vitro and in vivo and establish Cnβ as a new player in Nox4 regulation in the diabetic kidney.

Funding: VA MERIT and PECASE (JLG), NIDDK T32‐DK00756 (CRW)
Salt‐deficient diet does not attenuate the development of slowly progressive ANG II‐dependent hypertension in Cyp1a1‐Ren2 transgenic rats
Authors: Collins Amy E , Howard Catherine G , Mitchell Kenneth D
Published: June 2021

The present study was performed to determine the effects of dietary salt restriction on conscious systolic blood pressure (SBP), and 24‐hour urine flow and urinary sodium and protein excretion in Cyp1a1‐Ren2 rats with slowly progressive ANG‐II dependent hypertension. Adult male Cyp1a1‐Ren2 rats (n=5–6/group) were fed either a normal salt (0.6%) or salt‐deficient diet (<0.01%) containing indole‐3‐carbinol (I3C; 0.15%, wt/wt) to induce slowly progressive ANG II‐dependent hypertension. Both normal salt and salt‐deficient groups fed an I3C‐containing diet showed increases in SBP by day 4 of treatment (134±3 to 162±3 and 147±4 to 160±3 mmHg, respectively, P<0.001) and reached similar SBP levels by day 16 (207±8 vs. 194±7 mmHg, NS). Induced rats on salt‐deficient diet exhibited markedly reduced 24‐hour urinary sodium excretion (74.4±15.8 to 0.20±0.07 mEq/day, P<0.001) and urine output (10.2±1.3 vs. 18.8±3.5 mL/day, P<0.05. Urinary protein excretion increased similarly in response to I3C administration regardless of dietary salt intake (7.5±0.8 to 25.2±4.4, and 6.6±0.3 to 35.7±5.7 mg/d, respectively, P<0.05). These findings demonstrate that dietary salt restriction does not attenuate the development of slowly progressive ANG II‐dependent hypertension or the associated proteinuria in Cyp1a1‐Ren2 transgenic rats.

Support: NHLBI, Tulane COBRE in Hypertension and Renal Biology.
Tumor necrosis factor alpha activates RhoA and decreases NO synthase type 3 expression in rat thick ascending limbs
Authors: Ramseyer Vanesa Daniela , Hong Nancy , Garvin Jeffrey Lawrence
Published: June 2021

NO produced by NO synthase type 3 (NOS3) decreases thick ascending limb (THAL) NaCl reabsorption and induces natriuresis. Tumor necrosis factor alpha (TNF) stimulates RhoA GTPase and Rho kinase (ROCK) and decreases NOS3 expression in endothelial cells. Therefore, we hypothesized that TNF decreases NOS3 expression via Rho/ROCK in rat THALs. We measured: 1) NOS3 expression by Western blot and 2) RhoA activity by ELISA. Treatment of THAL primary cultures with 1 nM TNF for 24 hrs decreased NOS3 expression by 54 ± 4% (n= 6, p<0.001). Treatment with TNF for 10 min increased RhoA activity from 0.520 ± 0.038 OD to 0.816 ± 0.090 (Δ= 60±23%, p<0.03). Blockade of RhoA with 0.05 ug/mL C3 exoenzyme inhibited TNF‐induced reductions in NOS3 expression by 30 ± 9% (n=7, p<0.02) while C3 exoenzyme alone did not change NOS3 expression. Blockade of ROCK with 10 μM H‐1152 blunted TNF‐mediated reductions in NOS3 expression by 66 ± 15 % (n=6 p<0.001) while H‐1152 alone did not affect NOS3 expression. Simultaneous blockade of RhoA and ROCK were not additive (inhibition of TNF's effect = 56 ± 19 % n=5, p<0.05). We conclude that: 1) TNF decreases NOS3 expression in THALs; and 2) this effect is partially mediated by Rho/ROCK. Blockade of Rho/ROCK may improve renal function in hypertension by preventing TNF‐induced decreases in NOS3 expression.

Sources of funding: NIH HL 028982 to J.L.G. and AHA 11PRE7510005 to V.D.R
Characterization of glucose and SGLT mediated effect on NHE3 in renal proximal tubule
Authors: Pessoa Thaissa Dantas , Malnic Gerhard
Published: June 2021

We previously reported that proximal tubule perfusion with glucose 5mM stimulates Na+/H+ exchanger isoform 3 (NHE3) activity via p38MAPKinase pathway. Perfusions with higher glucose concentrations (up to 40 mM) were seen to inhibit NHE3 in comparison to a nominally glucose free solution. However, the role of SGLT1 and/or SGLT2 or glucose metabolism in these distinct effects on NHE3 has not been determined so far. We therefore performed microperfusion experiments with sugar free (CTRL), glucose 5mM (G5) and glucose 40mM (G40) plus 2mM Phloridzin (Plz, a SGLT inhibitor). Plz inhibited CTRL (46,2%) and G5 (60,7%) NHE3 activity but had no additional inhibitory effect on G40 perfusion, showing that SGLT activity is necessary for NHE3 function. In addition, the substitution of glucose by galactose (transported only by SGLT1) completely inhibited the G5 and G40 effect, suggesting that only SGLT2 activity or glucose metabolism modulates NHE3 activity. The substitution of glucose by Methyl α‐D‐glucopyranoside (non‐metabolizable glucose) abolished the G5 but not G40 effect, showing that G5 stimulates NHE3 through glucose metabolism, while G40 did not. We concluded that SGLT activity is necessary for NHE3 function, SGLT2 mediates the effects of both low and high glucose on NHE3 activity and the glucose metabolism is involved with the stimulatory effect of G5 on the transporter. Supported by FAPESP and CNPq
Inhibition of KV7 channels causes rat renal vasoconstriction in vivo
Authors: Sorensen Charlotte Mehlin , Holstein-Rathlou Niels-Henrik , Salomonsson Max
Published: June 2021

We investigated the possible role of KV7 (KCNQ)‐channels in the control of rat renal vascular hemodynamics. We also investigated whether this family of K+ channels participated in the acetylcholine (ACh) mediated renal vasodilation.

We measured renal blood flow (RBF) in vivo in Sprague Dawley rats using an ultrasonic flow probe. Basal RBF was 6.0 ± 0.38 ml/min (n=5). Intrarenal infusion of XE991 (a potent blocker of KV7.1‐7.5) at rates of 0.6 nmol/min and 6 nmol/min did not affect RBF (n=5). However, intrarenal infusion of XE991 for 1.5 min at rates of 15 nmol/min and 30 nmol/min (estimated plasma concentrations ~2.5 μM and 5 μM, respectively) significantly decreased RBF by 2.2 ± 0.6 % and 3.1 ± 0.8 %, respectively (n=5). After 4 min XE991 infusion the corresponding RBF decreases were 3.5 ± 0.8 % and 4.6 ± 0.8 %, respectively (n=5). Intrarenal ACh infusion at a rate of 3 nmol/min (estimated plasma concentration ~ 0.5 μM) increased RBF by 26 ± 2.8 % (n=3). Pretreatment with XE991 (30 nmol/min) for 1.5 min did not affect the response to ACh as RBF increased by 28 ± 2.9 % (n=3). In summary, we show that KV7 channels have a potential role in the regulation of renal hemodynamics. Furthermore, we find no indication that these channels play a role in ACh induced renal vasodilation. This study is supported by the Danish National Research Foundation, the Danish Heart Foundation, and the Lundbeck Foundation.
Autophagy Maturation Controlled by CD38‐Lysosome Signaling in Glomerular Podocytes of Mice
Authors: xiong jing , Xia Min , Xu Ming , Li Xiao-Xue , Abais Justine , Boini Krishna , Li Pin-Lan
Published: June 2021

Podocytes are well differentiated glomerular epithelial cells, and their structural and functional integrity are mainly maintained by a self‐defense or adaptive mechanism, autophagy. However, it remains unknown how podocyte autophagy is regulated to maintain the integrity of glomerular structure and function. The present study tested whether CD38‐mediated signaling, an important regulatory pathway of lysosome function, plays a critical role in the regulation of podocyte autophagy. Western blot analysis showed that rapamycin stimulation significantly increased the LC3‐II and beclin expression with or without prior treatment of nicotinamide, an inhibitor of CD38 or bafilomycin, a lysosome function inhibitor. Laser flow cytometry demonstrated that treatment of podocytes with nicotinamide enhanced the autophagosome (AP) accumulation (2.34±0.19 vs 1.63±0.21 of control), but decreased the formation of autophagolysosomes (1.12±0.18 vs 1.65±0.14 of control) under rapamycin stimulation. Using CD38 shRNA, we confirmed that CD38 gene silencing had similar effects to those observed by using pharmacological inhibition of CD38 activity or lysosome function, as analyzed both Western blot analysis and cytometry. To explore the possibility that CD38 may control podocyte autophagy through its regulation of lysosome function, we directly investigated the fusion of APs with lysosomes in living podocytes by co‐transfection of GFP‐LC3B and RFP‐Lamp1 expression vectors to express both proteins that were located to APs and lysosome, respectively. It was found that a colocalization of GFP‐LC3B and RFP‐Lamp1 upon stimulation of rapamycin became obvious in transfected podocytes, which could be substantially blocked by nicotinamide, CD38 shRNA, and bafilomycin. In conclusion, CD38 is a critical molecule that controls lysosome function and thereby determine autophagy maturation in podocytes (supported by NIH grants HL‐091464 and DK54927).
Renal function in T‐type voltage‐gated calcium channel knock‐out mice
Authors: Hansen Pernille B.L. , Andersen Henrik , Cardel Majken , Shin Hee-Sup , Skott Ole , Jensen Boye L. , Bie Peter
Published: June 2021

T‐type voltage‐gated calcium channels (Cav) are expressed in renal resistance vessels and collecting duct and calcium channels play an essential role in regulation of renal blood flow and glomerular filtration rate. The objective of the present study was to investigate renal function in two T‐type calcium channel knock‐out (KO) mice strains (Cav3.1 and Cav3.2). Continuous recordings of blood pressure (MAP), heart rate and inulin clearance (GFR) were performed in Wt and Cav3.1 and Cav3.2 KO mice using indwelling catheters. The night heart rate was significant lower in Cav3.1 KO compared to Wt with no difference in MAP. GFR was significantly higher in Cav3.2 KO compared to Wt. GFR in Cav3.1 KO were indistinguishable from Wt. No significant difference was observed in water intake, diuresis and electrolyte excretion measured before and after 24 hour water deprivation comparing Wt and Cav3.1 KO mice however a significant increase in sodium excretion rate was observed in both strains after water deprivation. We tested whether the high GFR in Cav3.2 KO was related to an increased contractility of the efferent arteriole in isolated perfused efferent arterioles. However depolarization and endothelin‐1 induced significant and similar constriction in Wt, Cav3.1 and Cav3.2 KO mice. In conclusion, lack of Cav3.1 is associated with a decreased heart rate whereas lack of Cav3.2 T‐type channels is associated with an increased GFR.

Research Support: Lundbeck Foundation, The Danish Medical Research Council, The Danish Heart Foundation
Chronic direct renin inhibition with aliskiren normalizes blood pressure and improves renal hemodynamics in Cyp1a1‐Ren2 transgenic rats with ANG II‐dependent malignant hypertension
Authors: Howard Catherine G , Mitchell Kenneth D
Published: June 2021

The present study determined if direct renin inhibition with aliskiren can normalize blood pressure and improve renal hemodynamics in Cyp1a1‐Ren2 transgenic rats with ANG II‐dependent malignant hypertension. Adult male Cyp1a1‐Ren2 rats (n=6) received 0.3% indole‐3‐carbinol (I3C) in the diet to induce ANG II‐dependent malignant hypertension [systolic blood pressure (SBP): 137±2 to 222±5 mmHg, P<0.001)]. Subsequent chronic renin inhibition with aliskiren (60 mg/kg/d, sc) during continued I3C administration normalized SBP within one day (102±3 mmHg, P<0.001) and maintained SBP at normotensive levels for 10 days (135±2 mmHg, P<0.001). Pentobarbital sodium anesthetized rats treated chronically with aliskiren exhibited lower MAP (138±3 vs. 187±6 mmHg, P<0.001) and renal vascular resistance (19.4±0.8 vs. 39.8±2.3 mmHg/ml/min.g, P<0.001), and increased renal plasma flow (3.8±0.2 vs. 2.4±0.1 ml/min.g, P<0.001) compared to untreated hypertensive rats (n=6). Glomerular filtration rates were comparable in the two groups of rats. The present findings demonstrate that chronic renin inhibition with aliskiren is highly effective in normalizing the elevated arterial blood pressure and in improving renal hemodynamics in ANG II‐dependent malignant hypertension.

Support: NHLBI, Tulane COBRE in Hypertension and Renal Biology, Novartis Pharma, AHA Greater Southeast Affiliate Predoctoral Fellowship.
Imaging intracellular calcium signals in intact kidney tissue
Authors: Crawford Carol , Hall Andrew M , Peppiatt-Wildman Claire M
Published: June 2021

Calcium (Ca2+) is an intracellular secondary messenger with multiple important physiological roles; disturbances in intracellular Ca2+ homeostasis have been implicated in the pathogenesis of various kidney diseases. We have recently demonstrated that multiphoton microscopy can be used to image a range of intracellular signals in ex vivo rat kidney preparations such as tissue slices [1, 2] and isolated perfused organs [3].

Here, we demonstrate that the tissue slice model can be used to load established Ca2+ indicators into both tubular and endothelial cells in the kidney. Kidney slices (200 μm thick) were obtained from adult, male sprague dawley rat kidneys (following cervical dislocation) and maintained in a physiological saline solution, bubbled with 95% O2/5% CO2. Loading of exogenous Ca2+ sensitive dyes (Fluo4, Rhod‐2, Fura Red) was achieved ex vivo by using a re‐circulating perfusion system. Confocal microscopy was used to image Ca2+ loading in the renal medulla, and to record changes in signal in response to physiological stimuli, i.e. ATP, Angiotensin II.

We believe that the application of these imaging techniques have much potential to enhance understanding of the roles of intracellular Ca2+ signalling in renal physiology and pathophysiology.

Research supported by the Medical Research Council
Effects of nitrosonifedipine, a photodegradation product of nifedipine, on diabetic nephropathy in type II diabetic mice
Authors: Sakurada Takumi , Ishizawa Keisuke , Fujii Shoko , Izawa-Ishizawa Yuki , Imanishi Masaki , Nuno Asami , Kihira Yoshitaka , Ikeda Yasumasa , Tomita Shuhei , Tsuchiya Koichiro , Tamaki Toshiaki
Published: June 2021

As the number of the patients with diabetic nephropathy (DN) has been increasing worldwide despite current treatment, more patients have received dialysis treatment. Oxidative stress and endothelial damage are involved in the development and progression of DN. Recently, we have demonstrated that Nitrosonifedipine (NO‐NIF), a photolytic compound of nifedipine, possesses radical scavenging activity and improves endothelial cell damage induced by tumor necrosis factor‐α (TNF‐α). Therefore, in this study, we investigated the effect of NO‐NIF on DN by using KKAy mice developing type II diabetes. In NO‐NIF treated KKAy mice, the urinary protein and albumin were significantly decreased compared with non‐treated KKAy mice. The pathological analysis showed expanded glomerular mesangium in KKAy mice, which was suppressed by NO‐NIF administration. NO‐NIF decreased urinary 8‐hydroxy‐20‐deoxyguanosine and reactive oxygen species detected by dihydroethidium staining in kidney. The mRNA expression of TNF‐α in kidney was also inhibited by NO‐NIF. However, NO‐NIF had no effect on body weight, kidney weight, adipocyte hypertrophy, blood glucose levels, insulin resistance, and systolic blood pressure. We conclude that NO‐NIF protects diabetic renal dysfunction via the attenuation of oxidative stress, and it might be a potential drug for the prevention of DN.
Role of Different Reactive Oxygen Species in Homocysteine‐induced NALP3 Inflammasome Activation in Mouse Podocytes
Authors: Abais Justine , Boini Krishna , Xia Min , Li Pin-Lan
Published: June 2021

Our recent studies have demonstrated the importance of NADPH oxidase‐mediated NALP3 inflammasome activation in both cultured podocytes and glomeruli of mice with hyperhomocysteinemia (hHcys). However, it remains unknown whether NADPH oxidase‐derived superoxide (O2•−) or other reactive oxygen species (ROS) are responsible for this activation. The present study tested the role of four common endogenous ROS – O2•−, hydrogen peroxide (H2O2), peroxynitrite (ONOO−), and hydroxyl radical (OH) in NALP3 inflammasome activation by using their selective scavengers or enzyme inhibitors. Confocal microscopy showed that dismutation of O2•− by 4‐hydroxy‐2,2,6,6‐tetramethyl‐piperidinyloxy (TEMPOL) markedly reduced Hcysinduced colocalization of inflammasome protein NALP3 with ASC by 60.4% and NALP3 with caspase‐1 by 64.1% in podocytes. Size exclusion chromatography also demonstrated that TEMPOL inhibited Hcys‐induced NALP3 inflammasome formation by preventing ASC recruitment to higher molecular weight fractions. Scavenging of ONOO− by uric acid also prevented Hcys‐induced aggregation of NALP3 inflammasome proteins. Furthermore, TEMPOL and uric acid treatment were able to inhibit Hcys‐induced caspase‐1 activation, respectively by 40.5% and 39.2%, as well as IL‐1β production by 46.8% and 39.4%. By electromagnetic spin resonance spectrometry, Hcys was shown to increase superoxide production by 2.2 folds compared to control podocytes, which was inhibited by 71.5% by TEMPOL and 62.2% by uric acid. However, H2O2 decomposer, catalase and hydroxyl radical scavenger, tetramethylthiourea (TMTU) had no inhibitory effects on Hcys‐induced NALP3 inflammasome protein aggregation and increases in caspase‐1 activity and IL‐1β production. These results indicate that O2•− and ONOO−, but not H2O2 or OH play an important role in mediating Hcys‐induced NALP3 inflammasome activation in podocytes (supported by NIH grants HL057244 and DK54927).
Moderate hyperoxia treatment increases glutathione levels during direct LPS‐induced lung injury in mice
Authors: Britt Rodney D , Velten Markus , Rogers Lynette K
Published: June 2021

Acute lung injury (ALI) is a major health threat. Supplemental oxygen therapy is often used to maintain blood oxygenation levels, however, its impact during ALI remains poorly understood. We tested the hypothesis that hyperoxia exposure following administration of LPS would increase inflammation, impair lung function, and alter glutathione (GSH) levels in adult mice. C3H/HeN male mice (10–12 wks) were treated with vehicle (PBS) or 0.375 μg/g E. coli LPS. After 12 hours, mice were exposed to room air (RA) or 60% O2 for 6 days. Cytokine and chemokine expression was measured by ELISA in BALF. Pulmonary function tests were performed with a Flexivent. GSH and GSSG levels were measured in lung tissue homogenates. On day 1, TNF‐á, IL‐6, RAGE, and MCP‐1 were significantly increased in LPS/RA and LPS/O2 exposed mice compared to PBS/RA or PBS/O2 exposed mice. KC and MIP‐2 were significantly increased in LPS/O2 mice only. Central airway resistance was increased on day 3 in LPS/O2 compared to PBS/RA mice. At day 6, LPS/O2 mice had higher GSH and GSSG levels compared to other groups. Our data indicate that LPS/O2 uniquely increases expression of chemokines, increases airway resistance, and increases GSH and GSSG levels. We speculate that LPS and hyperoxia act synergistically and that increases in GSH levels may be needed to cope with increased oxidation during development of LPS‐induced ALI in adult mice.
Apurinic/apyrimidinic endonuclease 1 (APE1) inhibits oxidative stress in gastric epithelial cells during H. pylori infection through binding and inhibition of Rac1
Authors: Chattopadhyay Ranajoy , Das Soumita , Hall Emily , Ablack Amber , Ernst Peter , Crowe Sheila
Published: June 2021

Background

We have shown that H. pylori (Hp) infection of gastric epithelial cells (GEC) increases reactive oxygen species (ROS) and induces APE1 expression. Rac1 and NAPDH oxidase contribute to Hp‐mediated oxidative stress. Thus, we hypothesized that APE1 regulates Rac1 and NADPH oxidase (Nox1).

Methods

After Hp infection, superoxide was measured by luminol oxidation in stable APE1 deficient AGS cells (shRNA) and wild‐type (WT) cells. Diphenylene iodonium (DPI) and NSC23766 were used to inhibit NADPH oxidase and Rac1 respectively. Rac1 activity was assessed by GST‐PBD pulldown assay. Rac1‐APE1 interaction was measured by co‐immunoprecipitation and confocal microscopy. Nox1 and APE1 mRNA levels in patient gastric tissues were measured by RT‐ PCR.

Results

Increased ROS, Rac1 activity and Nox1 were measured in shRNA cells compared to WT after Hp infection. APE1 overexpression decreased Hp induced Rac1 activation as well as Nox1 expression. N‐terminal Lys mutation of APE1 reduced the interaction with Rac1. Mutant APE1 overexpression resulted in increased ROS accumulation and Nox1 expression after Hp infection. Both Nox1 and APE1 mRNA levels were elevated in GEC from Hp infected patient tissues.

Conclusions

APE1 acts as a negative regulator of ROS accumulation due to Hp infection by inhibiting Rac1 and Nox1. (Research Support, R01DK061769‐09).
Effect of Some Antioxidant Vitamins on Some Enzymes of Ocular Tissues Exposed to Potassium Bromate
Authors: Osagie Victor Ehigie , Okolie Paulinus Ngozi
Published: June 2021

The effects of potassium bromate on ocular tissues (lens, retina and cornea) and subsequent treatment with antioxidant vitamins (A, C and E) on New Zealand white rabbits were studied for a period of eight weeks. Three groups of eight rabbits each were used. One test group (B) was administered 60mg KBrO3/kg body weight/day. The other test group, BV, was in addition to KBrO3 given 200,000IU vit. A/kg body weight/day, 1000IU vit. E/kg body weight/day and vit. C 40mg/kg body weight/day. The control group (N) received only normal saline. The study considered the levels of malondialdehyde (MDA), catalase and superoxide dismutase (SOD) in the ocular tissues. The results show that the lenticular SOD activity of the B group was significantly higher (p<0.05) than the control while the BV recorded an insignificant (p>0.05) increase over the control. The B and BV groups recorded significant increases in corneal SOD activity over the control. The BV group showed significant increase (p<0.05) in catalase activity when compared with N and B groups of all the tissues. The retinal MDA concentration recorded a significant increase (p<0.05) when the B group is compared with the control but no significant difference (p>0.05) between the BV and N. The corneal MDA level did not record any significant change (p>0.05) but the lenticular MDA level recorded a significant increase (p<0.05). This preliminary study indicates that potassium bromate adversely affects ocular tissues, and that antioxidant vitamins may play a role in mitigating these effects.
Alteration of GSSG/GSH Ratio Modulates Expression of Phase 2 Detoxifying Enzymes through Nrf2 Signaling Pathway
Authors: Kim Jong-Sang , Han Jung-Hwa , Seo Jiyeon , Lim Ji Sun , Jung Chaelim , Park In-Sil , Kang Hye-ryung , Kim Seong Soon , Kim Hyo Jung , Yoon Jung Han Park
Published: June 2021

Glutathione (GSH), a coenzyme of glutathione peroxidase, is involved in protection of cells from oxidative stress through termination of oxidants with concomitant oxidation of GSH to GSSG. The ratio of GSH/GSSG is viewed as the major indicator of the cellular redox status. We investigated whether intracelluar level of GSH or GSH/GSSG ratio plays an important role in Nrf2‐mediated regulation of phase 2 detoxifying enzymes such as NAD(P)H:quinone oixdoreductase (QR). In order to change the ratio of GSH/GSSG, we attempted to deplete NADPH, a cofactor of glutathione reductase, by inhibiting pentose phosphate pathway in mouse hepatoma cell (Hepa1c1c7) and measured the phase 2 enzyme expression. We also examined whether some known phase 2 enzyme inducers such as tert‐butylhydroquinone (tBHQ), sulforaphane, delphinidin, genistein and glyceollins exert their effect by modulating GSSG/GSH ratio. Our data suggest that perturbation of intracellular GSSG/GSH ratio affects the expression of phase 2 detoxifying and antioxidant enzymes maybe through Nrf2 signaling pathway.
M‐cadherin Modulates Phosphorylation of Beta‐catenin Nterminus and Promotes Myogenic Differentiation in a TCF/LEF‐Independent Manner
Authors: Wang Yan , Alway Stephen E.
Published: June 2021

β‐catenin is an essential molecule in both cadherin‐mediated cell‐cell adhesion and canonical Wnt signaling. The phosphorylation of β‐catenin by Glycogen Synthase Kinase‐3β (GSK‐3β) at residues serine31/37/threonine41 of N‐terminus regulates its stability and its role in canonical Wnt signaling. Recent work from our lab found that M‐cadherin‐mediated signaling protects myoblasts against apoptosis and promotes myogenic differentiation by suppressing GSK‐3β activation. In this study, we further investigated the possibility that the N‐terminal phosphorylation of β‐catenin is regulated by M‐cadherin and that this regulation mediates M‐cadherin's role in promoting myogenic differentiation. We demonstrated that knockdown of M‐cadherin expression by RNA interference (RNAi) in C2C12 myoblasts significantly enhances the phosphorylation of β‐catenin at ser33/37/thr41 and decreased the protein abundance of ser37/thr41‐unphosphorylated active β‐catenin. This effect can be relieved by GSK‐3β inhibition via lithium chloride (LiCl) treatment. Furthermore, M‐cadherin RNAi promotes LiCl or Wnt‐3a‐induced increase in TCF/LEF transcription activity but significantly blunts the myogenic induction by LiCl or Wnt‐3a treatment. Forced expression of phosphorylation‐resistant mutated β‐catenin (S33Y‐β‐catenin) alone failed to enhance myogenic differentiation but partially rescued the myogenic and apoptotic phenotype of myoblasts caused by M‐cadherin RNAi. These data show that M‐cadherin‐mediated signaling attenuates β‐catenin phosphorylation by GSK‐ 3β and this regulation has a positive effect on canonical/Wnt‐induced myogenic differentiation independent of TCF/LEF transcription activity. Our findings provide a novel mechanism by which M‐cadherin mediates myogenic differentiation.
Endoplasmic reticulum stress transducer OASIS promotes terminal differentiation of goblet cells in large intestine
Authors: Asada Rie , Saito Atsushi , Imaizumi Kazunori
Published: June 2021

OASIS is a transmembrane transcription factor which is localized at endoplasmic reticulum, and is cleaved at membrane region in response to ER stress. OASIS is expressed in bones, intestine and astrocyte in central nervous system. Among these, the most intense signals are detected in large intestine. In situ hybridization showed OASIS strongly expressed in pre‐goblet cells at crypt base of large intestine. In 3‐weeks‐old Oasis−/− mice, the expression of Mucin2, a mature goblet cell marker, was lower, and that of Trefoil factor 3, a pre‐goblet cell marker, was higher than in those of wild‐type mice. Electron microscope analysis showed abnormal morphology of mucus vesicles in the goblet cells of Oasis−/− mice, indicating that OASIS is essential for normal formation of mucus vesicle. We further examined the roles of OASIS in goblet cell differentiation using LS174T colon cancer cells. The expression of OASIS was up‐regulated, and the cleavage of OASIS at the membrane region was accelerated during differentiation of LS174T to mature goblet cells. Knockdown of OASIS transcript resulted in disturbance of goblet cell terminal differentiation. Taken together, OASIS could play crucial roles in accelerating terminal differentiation of goblet cells in large intestine involving formation of mucus vesicles.
Functional Expression of MRP4 in the Ciliary Epithelium of the Eye
Authors: Pelis Ryan M , Coca-Prados Miguel
Published: June 2021

The multidrug‐resistance associated protein 4 (MRP4) is an efflux pump that transports organic anions, including para‐aminohippurate. Transepithelial transport of para‐aminohippurate by the ciliary epithelium was previously demonstrated (Pelis and Delamere, The FASEB Journal 23, Abstract #797.9, 2009). This led to the hypothesis that MRP4 contributes to organic anion transport by the ciliary epithelium. MRP4 expression was examined using reverse transcriptase PCR, Western blotting and functional assays. MRP4 mRNA and protein were detected in the freshly dissected human ciliary body and in a simian virus transformed cell line derived from human ciliary body epithelial cells (SVHCE cells). MRP4 protein levels (relative to total protein) in the native tissue and cultured cells were comparable. In SVHCE cells, the intracellular accumulation of the fluorescent MRP4 substrate bimane glutathione increased in a dose‐dependent manner in the presence of the MRP4 inhibitors, probenecid and indomethacin. These data indicate that MRP4 contributes to organic anion transport by the human ciliary epithelium. The functional expression of MRP4 in SVHCE cells makes them a potentially useful system for examining MRP4‐mediated organic anion transport by ciliary epithelia. Supported by NIH.
CFTR‐dependent HCO3‐ defect, cause of viscous mucus in distal intestinal obstruction syndrome (DIOS)?
Authors: Yang Ning , Quinton Paul M.
Published: June 2021

Background & Aim

Distal intestinal obstruction syndrome (DIOS) is a condition unique to Cystic Fibrosis, which is due to the accumulation of abnormally viscous mucus in the intestinal tract. However, the pathogenesis of DIOS has not been determined. This study is to test our hypothesis that the CFTR‐dependent HCO3− defect is a cause of more viscous mucus in DIOS.

Methods

Segments of distal small intestine (ileum) and distal colon from CL57B1/6 WT mice were incubated with stimulants in vitro in either NaCl (no HCO3−) or NaHCO3 (with HCO3−) Ringers solution. Mucus samples were collected from the incubated intestines and analyzed by a spot tracking method for mucus viscosity. pH‐stat and Ussing chamber techniques were used to measure HCO3− secretion.

Results

After stimulating with 5‐HT (10 μM) and PGE2 (1 μM), we found that the mucus viscosity from both ileal and distal colonic sacs incubated with HCO3− Ringer solution was significantly lower than the mucus from sacs incubated in Ringer solution without HCO3−, indicating that HCO3− secretion decreased the mucus viscosity in both the mouse ileum and distal colon. However, we found that CFTR inhibitor, glyH‐101 (20 μM) significantly inhibited 5‐HT and PGE2‐induced HCO3− secretion only in the ileum, not in the distal colon, suggesting that HCO3− secretion in the ileum is CFTR‐dependent.

The CFTR‐dependent HCO3−defect in the distal small intestine is probably a cause of the more viscous mucus in DIOS.
Role of BKCa channels in regulating uterine vascular contractility: effect of high altitude hypoxia
Authors: zhu ronghui , Xiao DaLiao , Zhang Lubo
Published: June 2021

BKCa channels play a key role in the increased uterine artery blood flow in pregnancy. The present study tested whether chronic hypoxia attenuates pregnancy‐mediated enhanced BKCa channel activities and increases PKC‐mediated uterine vascular tone. Uterine arteries were isolated from nonpregnant (NPUA) and nearterm pregnant (PUA) sheep maintained at sea level (~300 m) or high altitude (3801 m) for 110 days. In contrast to previous studies in normoxic sheep, PKC activator PDBu‐induced contractions were increased in PUA, which was not significantly different from that of NPUA in hypoxic sheep. BKCa channel blocker TEA increased PDBu‐induced contractions in NPUA but not PUA. However, TEA increased norpinephrine (NE)‐induced contractions in both NPUA and PUA. Both BKCa channel opener, NS1619 and SKCa channel opener, NS309 caused minimal relaxations (~20%) of NPUA and PUA pre‐contracted by NE in the present or absence of endothelial cells. In addition, Western blot showed a decrease in the β1, but not α, subunit of BKCa channels in hypoxic PUA as compared with normaxic PUA. The results suggest that chronic hypoxia selectively attenuates basal uterine vascular BKCa channel activity, but not BKCa channel activation in pregnant animals, which may contribute to enhanced uterine vascular tone observed in sheep during pregnancy at high altitude hypoxia. (Supported in part by NIH grant HD31226)
ANTI‐OBESITY EFFECT OF SHK‐186, A K+ CHANNEL BLOCKER
Authors: Upadhyay Sanjeev K , Mahan Kristin Eckel , Mirbolooki MohammedReza , Tjong Indra W , Tjong Indra W , Mukharjee Jogeshwar , Iadonato Shawn P , Sassoni-Corsi Paolo , Wang Ping H , Chandy K George
Published: June 2021

We describe the anti‐obesity effects of ShK‐186, a Kv1.3‐selective peptide inhibitor. C57BL/J mice fed a diet containing high fat (21%) diet and fructose solution (60%) were administered ShK‐186 (500 μg/kg) or vehicle on alternate days by subcutaneous injection. ShK‐186‐treatment caused a dose‐dependent reduction in weight gain over the 45‐day trial, the maximal weight difference being 30%. ShK‐186‐treatment reduced plasma levels of free fatty acids without affecting cholesterol, HDL, LDL or glucose. CT scan revealed significant reduction in volume of white adipose tissue in ShK‐186‐treated mice compared to controls. However, histology revealed no difference in cell size and fat content of white fat cells. In metabolic cage experiments, ShK‐186‐treatment increased cumulative intake of food and fluid compared to vehicle‐treated mice, especially during the light‐cycle. Furthermore, their respiratory exchange ratio and energy expenditure were also higher. In PET experiments, ShK‐186 treatment increased uptake of 18‐fluoro‐deoxyglucose (18FDG) by brown adipose tissue (BAT) and skeletal muscle compared to vehicle‐controls; pre‐administration of the beta‐adrenoceptor blocker propranolol (5mg/kg) inhibited 18FDG uptake by BAT but not skeletal muscle. ShK‐186's anti‐obesity effect appears to be mediated by activation of BAT and/or skeletal muscle, two important thermogenic tissues in the body