bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased TRF2 binding likely limits telomere uncapping in older human arteries despite age‐related telomere attrition
Authors: Morgan Garrett , Ives Stephen J , Lesniewski Lisa A , Cawthon Richard M , Andtbacka Robert HI , Noyes R Dirk , Richardson Russel S , Donato Anthony J
Published: June 2021

Telomeres are DNA repeats that cap chromosome ends and prevent premature senescence. Capping is maintained by two key regulatory proteins; telomere repeat binding factor 2 (TRF2) and telomerase (TERT). Mean telomere length (mTL) and TERT activity decline with age in various tissues, but little is known regarding arterial tissue. Ninety‐seven human arteries were obtained during outpatient surgeries and mTL (qPCR) as well as telomere localization of TRF2, TERT and phosphorylated H2A.X (p‐H2A.X), a marker of uncapping, were measured (chromatin‐IP). mTL declined progressively with age, as younger adult (YA ≤ 40 yrs) mTL was 22% higher than middle‐age (MA: 41–59 yrs), and MA was 10% higher than older (OA ≥ 60 yrs) mTL (all p ≤ 0.05). TRF2 localization was higher in OA than in YA (p = 0.02), while TERT and p‐H2A.X localization were not different with age (all p ≥ 0.24). Shortest to longest mTL was separated into quartiles (Q1–4) to assess uncapping with mTL attrition. p‐H2A.X and TRF2 localization were higher in Q1 than in Q2–4 (all p ≤ 0.01). OA comprised 48% of Q1 and 9% of Q4, suggesting that age contributes to, but doesn't fully explain, mTL‐related uncapping. These results demonstrate that mTL declines progressively across age‐groups in human arteries despite similar TERT localization, but indicate compensatory increases in TRF2 binding may prevent uncapping in older arteries.

Support: NHLBI‐HL09183, U. Center on Aging
Flow mediated dilation variation based on normotensive rat strain
Authors: Heimlich Jonathan , Pollock David M
Published: June 2021

Previous studies from this lab have suggested that nitric oxide (NO) plays a significant role in the maintenance of arterial pressure in the same strain of rats obtained from different animal vendors. Subsequently, we hypothesized that similar variations in NO and thus, endothelial function, could be found in different strains of rat. Flow mediated dilation (FMD) is traditionally a non‐invasive technique used clinically to assess endothelial function. The hyperemic response elicited using this in vivo technique is thought to be mediated by NO release from the endothelium. The goal of the present study was to determine whether the FMD hyperemic response was different between two normotensive strains from the same supplier, Sprague‐Dawley (SD) and Wistar‐Kyoto rats (WKY) from Charles River (CR). In anesthetized rats, the femoral artery was isolated and occluded for 5 minutes, with femoral blood flow measured continuously before, during and after occlusion by use of an ultrasonic perivascular flow probe. Arterial pressure was measured directly via a catheter inserted into the contralateral femoral artery. The clamp procedure was repeated four times in each rat and then averaged. The average area under the FMD response curve (3 minute duration) for the CR SD rats was 79.0 ± 22.9 mL/min·s (n=6) and 371.0 ± 67.4 mL/min·s (n=5) for the CR WKY rats. These results suggest differences in endothelial function in normotensive rats and could have broad implications for research involving these strains of rat.
Sustained activation of p38 MAPK and MMP2 and 9 exacerbate neointima formation following vascular injury in metabolic syndrome rats
Authors: Weber David S , Jadhav Rashmi , Dodd Tracy , Smith Erika , Bennett Justin R , Rocic Petra
Published: June 2021

Following percutanous transluminal coronary angioplasty (PTCA) restenosis is markedly increased in metabolic syndrome patients. We hypothesized that increased restenosis in the metabolic syndrome was due to enhanced activation of p38 MAPK (p38) and subsequent p38‐dependent activation of MMP2 and 9. Carotid arteries were injured with a Fogarty 2F catheter in a rat model mimicking the human metabolic syndrome (JCR rat) and normal, healthy controls (SD rats) and neointima formation was quantified. p38 phosphorylation and MMP2 and 9 expression were assessed by immunoblotting and MMP2 and 9 activity by zymography in injured and contralateral control carotids at 4, 7, 10 and 14 days post injury. No basal differences were observed between JCR and SD rats. Maximal increase in p38 activation of similar magnitude occurred at 4 days post injury in both rat phenotypes. p38 activation rapidly returned to baseline in SD but remained sustained in JCR rats. MMP2 and 9 expression and activation mirrored that of p38. Thus, while the absolute magnitude of activation in response to vascular injury is similar, our findings suggest that the sustained activation of p38 and MMP2 and 9 in the metabolic syndrome contribute to increased neointima formation and may provide targets for pharmacological therapies devised for prevention of restenosis following PTCA. Support: NIH HL 084159 and HL 093052
Hydrogen sulfide mitigates renovascular matrix pathobiology in hyperhomocysteinemia
Authors: Coley Denise M. , Kundu Sourav , Tyagi Suresh C. , Sen Utpal
Published: June 2021

Homocysteine (Hcy) is a metabolite of methionine and known risk factor for vascular diseases at elevated level (HHcy). In the tissue, Hcy metabolizes to produce hydrogen sulfide (H2S). Although Hcy causes vascular remodeling and recent reports highlights H2S is a physiological gas with multiple beneficial effects, the role of H2S in renovascular remodeling during HHcy is unclear. The goal of this study was to delineate any beneficial role of H2S in Hcy‐induced renovascular remodeling in HHcy. Kidney mesangial cells were treated with or without Hcy and H2S. The groups were: 1) Control, 2) Hcy, 3) H2S, and 4) Hcy + H2S. Protein expression was measured by Western blot. Matrix metalloproteinase (MMP) activity were measured by gelatin zymography, and cells immunostained for confocal microscopy. Results indicated that expression of collagen‐1 & ‐4, MMP‐2, 9,‐13 & ‐14, was increased and TIMP‐1 was decreased in HHcy, whereas H2S normalized expression of these molecules. Activity of MMP‐2 and ‐9 was higher in HHcy and H2S mitigated activity of these two MMPs. Immunostaining revealed that autophagy marker LC3A/B was robustly increased in HHcy, whereas H2S mitigated LC3A/B expression. Western blot analyses indicated that autophagy was mediated through Akt down‐ and/FoxO3 up‐regulation in HHcy, and H2S treatment reversed activation of these signaling molecules. We conclude that H2S play a crucial role in mitigating renovascular pathobiology in HHcy by modulating matrix protein and autophagy through Akt/FoxO3 pathway.
ENaC is active in the ASDN in the absence of mineralocorticoid
Authors: Mironova Elena , Bugaj Vladislav , Stockand James
Published: June 2021

Aldosterone controls extracellular volume and blood pressure by regulating Na+reabsorption via ENaC across epithelial cells of the aldosterone‐sensitive distal nephron (ASDN). Our recent findings suggest that complementary systems function in parallel with aldosterone to modulate ENaC activity, including AVP and purinergic signaling localized to the distal nephron. To explore further the relation of aldosterone with this other regulation and to test the primacy of aldosterone in regulation of ENaC activity, we used mice with bilateral adrenalectomy. The activity of ENaC in the murine ASDN was assayed with patch‐clamp electrophysiology in freshly isolated split‐open tubules. In normal mice, ENaC activity is inversely related to Na+ intake. This relation is thought to be a manifestation, at least in part, of changes in aldosterone levels. Consistent with this, treatment with the mineralocorticoid DOCA reversed the effects of a high Na+ diet returning ENaC activity to normal levels. Unexpectedly, ENaC in the ASDN of Adx mice had robust activity despite the absence of adrenal corticosteroids, as measured with HPLC. Moreover, the response to high salt consumption was similar in both Adx and wild type mice with respect to the number of active channels. Intriguingly, ENaC is more active in Adx mice compared to wild type mice at all dietary Na+ regimens tested. ENaC activity in Adx mice is likely controlled primarily by AVP and/or Ang II responding mostly to changes in plasma tonicity and to a lesser degree volume status. These results demonstrate that in complement to corticosteroids, other hormones play an important role in setting ENaC activity and that ENaC can be present and functional in the ASDN even in the absence of adrenal corticosteroids.
Feedback Inhibition of the epithelial Na+ channel (ENaC): in vitro vs. in vivo
Authors: Patel Ankit Bharat , Frindt Gustavo , Deng Su , Palmer Lawrence G.
Published: June 2021

The mechanism of feedback inhibition of ENaC was investigated in vitro in ENaC‐expressing oocytes and correlated to an in vivo protocol in rats. Two‐electrode voltage clamp (TEVC) and patch clamp was used to measure amiloride‐sensitive currents (INa) in oocytes and split‐open cortical collecting ducts from rats, respectively. Biotin labeling was used to examine cell surface expression of ENaC in both oocytes and rats. 1–1.5 hour incubation in high Na+ of ENaC‐expressing oocytes caused a 74% decrease in INa, but little change in cell‐surface γ ENaC. An overnight incubation in high Na+ caused a 92% decrease in INa and a 73% decrease in cell‐surface γ ENaC. There was no change in cell‐surface expression of β ENaC during either high Na+ incubation. Rats on a low‐Na+ diet with an aldosterone pump had a 70% decrease in INa when resalted with decreases in cell‐surface β and γ ENaC of 60% and 71%, respectively. Resalting with amiloride blocked feedback inhibition but did not significantly change INa compared to resalted rats but increased cell‐surface β and γ ENaC by 123% and 419%, respectively. In oocytes, feedback inhibition causes changes in ENaC activity prior to changes in cell‐surface γ ENaC and no changes in cell‐surface β ENaC. In rats, feedback inhibition during resalting regulates cell‐surface β and γ ENaC exprssion without effecting INa. NIH RO1‐ DK59659 (LGP) and T32‐GM07739 (ABP).
Ankyrin 3 is regulated by aldosterone and mircoRNAs, and alters ENaC‐mediated sodium transport
Authors: Butterworth Michael Bruce , Carananti Maria , Edinger Robert S
Published: June 2021

The role of microRNAs (miRs) in the physiologic regulation of sodium transport has yet to be fully appreciated. miRs are small non‐coding RNAs that selectively target and downregulate mRNAs to inhibit protein expression. Our previous studies indicated that aldosterone altered miR expression to directly modulate Na+ transport via the epithelial sodium channel (ENaC). In silico predicted protein targets were screened in cultured mouse cortical collecting duct (mCCD) cells to test for their involvement in ENaC regulation. One of the candidate miR‐regulated targets was ankyrin 3 (ank3). Ank3 expression increased in mCCD cells stimulated with 50nM aldosterone for 24hrs. By inhibiting the expression of miRs directly we increased ank3 protein levels indicating that ank3 was a miR target. To demonstrate the regulation of ank3 by miRs we transiently transfected the 3’‐untranslated region (UTR) of ank3 linked to a dual luciferase reporter into mCCD cells. Both aldosterone and miRs altered luciferase expression confirming the ank3‐UTR as a miR target. Knockdown of ank3 reduced ENaC short‐circuit currents (53.9 ± 5.13%, n=22) and overexpression of ank3 raised ENaC currents (140.6 ± 9.75%, n=12), independent of aldosterone or miR regulation, thereby linking expression of this protein with Na+ transport. Ank3 represents a novel aldosterone‐induced and miR‐regulated protein involved in altering Na+ transport in the CCD.
Aldosterone and Ang II complementary stimulate ENaC activity during systemic salt restriction
Authors: Mamenko Mykola , Zaika Oleg L , Pochynyuk Oleh
Published: June 2021

Dietary salt intake controls ENaC activity via renin‐angiotensin‐aldosterone system (RAAS) and aldosterone is considered to be a key modulator. However, experimental evidence suggests possible involvement of other RAAS components (such as Ang II) in ENaC regulation additively to aldosterone. Using patch‐clamp electrophysiology in split‐opened distal nephrons, we demonstrate that Ang II, acting via AT1 receptors, acutely stimulates ENaC open probability (Po) and this effect is preserved, when aldosterone levels are clamped high with DOCA. Application of aldosterone does not reproduce the acute stimulatory action of Ang II. Dietary salt restriction (<0.01% Na+ for 7 days) increases both functional ENaC expression (fN) and ENaC Po. Systemic inhibition of mineralocorticoid receptors with spironolactone (7 days) attenuates ENaC activity by decreasing fN, but not Po. Simultaneous treatment with spironolactone and losartan (AT1 receptor antagonist) virtually abolished elevations of ENaC fN and Po by salt restriction. We conclude that Ang II and aldosterone complementary contribute to proper ENaC activation in response to dietary salt restriction, acting primarily on Po and fN, respectively.
Renal proximal tubule ion transporters are discrepantly regulated by parathyroid hormone in acute versus chronic hyperparathyroidism
Authors: Murray Rebecca , Lesousky Nina , Khundmiri Syed J. , Lederer Eleanor D.
Published: June 2021

Under acute experimental conditions, PTH decreases expression of Npt2a (sodium phosphate cotransporter), NHE3 (sodium hydrogen exchanger isoform 3), and Na/K‐ATPase. In contrast, patients with primary hyperparathyroidism (HPTH) frequently have low serum phosphate but infrequently show metabolic acidosis or volume depletion. We hypothesize that acute and chronic PTH stimulation results in differential regulation of proximal tubule (PT) ion transporters. Using a transgenic mouse model that gradually develops HPTH due to parathyroid‐targeted cyclin D1 over‐expression, we examined the expression of Npt2a, Na/K‐ATPase, sodium‐hydrogen exchanger regulatory factor‐1 (NHERF‐1), and the PTH receptor (PTHr). In comparison with control mice, chronic HPTH resulted in sustained decreased expression of Npt2a, whereas expression of Na/K‐ATPase α subunit increased, contrary to what has been shown in acute conditions. The transgenic model showed no discernible difference in the expression of either NHERF‐1 or PTHr when compared to the control mice. We conclude that PTH differentially regulates PT ion transporters in acute versus chronic conditions through as yet unidentified mechanisms. We suggest that PTH regulation of transepithelial sodium transport undergoes desensitization while regulation of phosphate transport does not. Support for this project is provided by VA.
Elucidating the role of a renal proximal tubule‐specific olfactory receptor
Authors: Shepard Blythe D , Cheval Lydie , Doucet Alain , Pluznick Jennifer L
Published: June 2021

Olfactory receptors (ORs) are seven transmembrane domain G protein‐coupled chemosensors that serve as sensors of smell in the nose. Recently, studies have found that ORs and other chemosensors are also expressed outside of their native tissues where they have important functions. We have previously reported that OR signaling plays a role in the kidney, and that Olfr1393 is present in the kidney (PNAS 2009). Using reverse‐transcribed RNA from microdissected renal segments, we found that Olfr1393 is exclusively expressed in all three segments of the proximal tubule (S1, S2 and S3, n=3). Olfr1393, like the majority of ORs, is an “orphan receptor” with no known ligand. To screen for ligands, Olfr1393 must first be expressed on the plasma membrane (PM). However, most ORs are retained in the ER when expressed in HEK293T cells. We have recently cloned this renal OR and found that it can traffic to the PM when coexpressed with Receptor Trafficking Protein. Using a comprehensive screening approach, we have found that Olfr1393 detects cyclic molecules containing a carbonyl group. There are a vast array of physiological chemicals that are derived from this basic unit including bile acids, vitamins and steroid hormones, all of which play important roles in the kidney. We are currently focusing on defining the full complement of ligands for Olfr1393, and understanding the physiological role that this OR plays in the kidney.

Funding NIDDK
Fructose as a modulator of proximal tubule (PT) H+ transport
Authors: Queiroz-Leite Gabriella Duarte , Crajoinas Renato O. , Neri Elida A. , Bezerra Camila N. A. , Girardi Adriana C. C. , Rebouças Nancy A. , Malnic Gerhard
Published: June 2021

High fructose (F) intake adversely affects the kidneys. This study was outlined to investigate F effects on NHE3 activity. Microperfusion showed that 2 and 3mM F stimulate JHCO3‐(nmol/cm2 × s) (2.87 ± 0.19, P < 0.001 and 2.78 ± 0.14, P < 0.01 × 2.08 ± 0.10, control). Action of F on S3226 (NHE3 inhibitor) sensitive‐component showed NHE3 stimulation (1.18 ± 0.07 × 1.81 ± 0.01, P < 0.0001). MAPK pathway is not involved in this activation, as demonstrated by using U1026 and SB203580 (MEK and p38 MAPK inhibitors), whereas 8 br cAMP (cAMP analogue) and forskolin (phosphodiesterase inhibitor) + IBMX (adenylyl cyclase activator) showed that PKA inhibition plays a role in this response. F stimulated JH+ (mM/min) after an acid pulse in LLC‐PK1, a pig PT cell line (11.44 ± 1.09 3mM F × 5.98 ± 0.82 control, P < 0.01). HOE694 (NHE1 inhibitor) and S3226 showed that F increases NHE3, not NHE1 activity. Signaling pathways investigation showed similar results in PT and LLC‐PK1. Cells were treated or not with F for 5, 15 and 30 min and the ATP and cAMP contents were examined. There was a significant decrease in ATP (47, 53 and 37%, respectively) and in cAMP (45 and 31%, respectively) and a not significant reduction at 30 min. Antibody against phosphorylated substrates showed PKA inhibition in F treated cells. Thus, F stimulates NHE3 activity through PKA inhibition, which increases Na+ reabsorption and can contribute to renal injury.

Support: CNPq, FAPESP.
SPAK, OSR1 and Cab39/MO25 form an Interdependent Signaling system which Regulates Thiazide‐Sensitive Salt‐transport, Distal Tubule Mass and Blood Pressure
Authors: Grimm P Richard , Taneja Tarvinder , Liu Jie , Coleman Richard , Chen Yang-Yi , Delpire Eric , Wade James B , Welling Paul A
Published: June 2021

STE20/SPS‐1‐related proline‐alanine‐rich protein kinase (SPAK) and, Oxidative Stress Related Kinase (OSR1), co‐localize at the apical membrane of the Thick ascending Limb (TAL) and Distal Convoluted Tubule (DCT) and both regulate the potassium‐dependent sodium‐chloride co‐transporter, NKCC2, thiazide‐sensitive sodium‐chloride cotransporter, NCC in vitro. Yet genetic ablation of SPAK in mice causes a salt‐wasting nephropathy that is restricted to the DCT, reminiscent of Gitelman's syndrome. Here, we explore why proper DCT function is especially SPAK‐dependent. In the TAL of SPAK−/− mice, OSR1 and Cab39/MO25, a newly described OSR1/SPAK regulatory protein, remain at the apical membrane where they function with a compensatory increase in the AMP‐activated kinase (AMPK) to hyper‐phosphorylate NKCC2. By contrast, the OSR1/SPAK/M025 signal transduction apparatus is completely disrupted in the DCT. OSR1 and MO25 become largely displaced from the thiazide‐sensitive sodium‐chloride cotransporter, NCC, and the apical membrane. OSR1 redistributes to dense punctate structures within the cytoplasm. These changes are paralleled by a dramatic decrease in NCC abundance and phosphorylation. Without SPAK and the proper localization of OSR1 and MO25, phosphorylation‐dependent regulation of NCC by dietary sodium restriction is lost. SPAK−/− mice also exhibit a decrease in the mass of the distal convoluted tubule, exclusive to DCT1. As a result of the interdependent nature of OSR1 and MO25 on SPAK in the DCT, SPAK−/− mice are highly sensitive to dietary salt‐restriction, displaying prolonged negative sodium balance and hypotension.
Stimulation of adenosine A1 receptor inhibits the basolateral 50 pS K channels in the thick ascending limb of rat kidney
Authors: li wennan , Wang Lijun , Kong Shumin , Wu Peng , Wang Mingxiao , Luan Haiyan , Gu Li , Fan Lili , Gu Ruimin
Published: June 2021

We used the patch‐clamp technique to study the effect of stimulation of Adenosine A1 receptor on the basolateral 50 pS K channels in the thick ascending limb (TAL) of rat kidney. 100nM or 200nM Adenosine (ADO) inhibited the basolateral 50 pS K channels. However, 400nM, 800nM and 1μM Adenosine did not decrease the 50 pS K channel activity. Adenosine's effect was mimicked by the specific adenosine A1 receptor agonist Cyclohexyladenosine (CHA). Inhibition of adenosine A1 receptor with 8‐cyclopentyl‐1, 3‐dipropylxanthine (DPCPX), adenosine A1 receptor antagonist, abolished the inhibitory effect of 100nM adenosine. Stimulation of adenosine A1 receptor did not decrease the activity of basolateral 50 pS K channels in the presence of the inhibitor of phospholipase C (PLC), U‐73122 or the inhibitor of protein kinase‐C (PKC), Calphostin‐C, respectively. The mitogen‐activated protein kinase ( MAPK) inhibitors of PD98059 (ERK) or SB202190 (p38) increased the 50 pS K channel activity. PD98059 abolished the inhibitory effect of CHA. CHA decreased the 50 pS K channel activity in the presence of SB202190. These studies show that the inhibitory effect of 100nM adenosine on the basolateral 50 pS K channels, coupled adenosine A1 receptor, is mediated by PLC‐PKC and MAPK (ERK) pathways in the thick ascending limb of the rat kidney.

The work is supported by Chinese National Natural Science Foundation 30770800, 31071017 and 31171110.
The trans‐tubular K gradients (TTKG) reveal long‐term adaptation in mice to Na deficient, high K diets
Authors: Echtenkamp Kari M , Hatcher Lori I , Cornelius Ryan J , Samson Steven C
Published: June 2021

K excretion is dependent on the distal flow rate and the ability to produce a TTKG (lumen [K] to plasma [K]) in the terminal cortical collecting duct (CCD). The TTKG is considered to result from electrogenic Na absorption, which generates the transepithelial potential that drives K secretion. However, Yanomamo Indians secrete K with minimal Na intake. To determine the mechanism of Na‐independent K secretion (NIKS), we measured TTKG when varying dietary K and when adding amiloride (5 μg/day, 2 days) to prevent Na absorption, eplerenone to inhibit aldosterone, or Na deficient diets. The TTKG (mean ±SEM) was 0.48 ±0.02, 9.2 ±0.8, and 26.8 ±0.9 in mice on a low K (0.1%), normal K (0.6%) or high K (5%) diet (7 days), respectively. Compared with high K, the TTKG decreased to 17.7 ±1.1 with high K plus amiloride and to 18.4 ±0.5 with high K plus eplerenone. When treated with a low Na (0.01%) diet the flow was significantly decreased; however the TTKG of 10.7 ±1.2 was not different from control diet. The TTKG value of 27.5 ±2.0 for low Na, high K fed mice was not different from TTKG of high K mice. These results show that mice are able to adapt completely to eliminate K in the near absence of dietary Na; however K secretion remains compromised by pharmacologically preventing Na absorption with amiloride for two days. The reduction in TTKG with high K plus eplerenone, but not with high K, low Na, suggests that NIKS is augmented by aldosterone.
EGF deficiency contributes to the development of salt‐sensitive hypertension via upregulation of ENaC activity
Authors: Pavlov Tengis S. , Levchenko Vladislav , O'Connor Paul M. , Sorokin Andrey , Mattson David L. , Lombard Julian H. , Cowley Allen W. , Staruschenko Alexander
Published: June 2021

Amiloride‐sensitive sodium entry, via the epithelial Na+ channel (ENaC), is the rate‐limiting step for Na+ absorption in the aldosterone sensitive distal nephron (ASDN). Dysfunction and aberrant regulation of ENaC leads to a spectrum of diseases associated with abnormal Na+ handling, including hypertension. The contribution of ENaC appears to be of critical importance in Dahl SS hypertensive rats. Members of the epidermal growth factor (EGF) family play an important role in regulation of transepithelial Na+ transport in the kidney. A combination of electrophysiological, histochemical, molecular and chronic studies in vivo and in vitro was used to investigate how ENaC is regulated by EGF and how changes in this pathway contribute to salt‐induced hypertension in SS rats. As measured by ELISA, EGF concentration in the kidney cortex of SS rats fed a high salt diet was significantly lower compared to rats on a low salt diet. In contrast, ENaC activity was upregulated in SS rats fed a high salt diet. To directly evaluate EGF effect on the development of hypertension and ENaC activity, EGF was intravenously infused and MAP was monitored continuously. Chronic infusion of EGF (2 mg/h; i/v) in SS rats fed a high salt prevented the development of hypertension, increased Na+ excretion and decreased ENaC activity in the ASDN. Furthermore, histological analysis revealed that EGF attenuated renal glomerular and tubular damage.
Hypertension induces proteinuria and renal fibrosis associated with endoplasmic reticulum (ER) stress in the Dahl salt sensitive rat
Authors: Jerome Danielle E. , Yum Victoria , Skelton Meredith M. , Cowley Allen W. , Austin Richard C. , Dickhout Jeffrey G.
Published: June 2021

Hypertension and proteinuria are associated with the progression of chronic kidney disease (CKD). CKD occurs via a process of kidney fibrosis and nephron loss. The Dahl salt sensitive hypertensive rat (Dahl SS) is a model of hypertension caused by high salt, resulting in proteinuria and CKD. We hypothesized that hypertension‐induced proteinuria in the Dahl SS leads to renal fibrosis through the initiation of ER stress and the unfolded protein response (UPR). The UPR is an important modulator in the differentiation of various cell types, including myofibroblasts. We tested this using two models of hypertension, the Dahl SS rat and the spontaneously hypertensive rat (SHR) treated with a high salt (8% NaCl) or maintained on a low salt (0.4% NaCl) diet for 14 days. Kidneys from these animals were processed for histology and immunohistochemistry for markers of both ER stress and fibrosis. In the high salt treated Dahl SS rat, intratubular protein casts were observed. These rats also displayed renal fibrosis as shown by trichrome and α‐smooth‐muscle actin staining, and ER stress, shown by GRP78 and CHOP staining. Contrarily, the SHR developed no significant proteinuria or interstitial fibrosis in response to high salt despite malignant hypertension. These results suggest that hypertension‐induced proteinuria is associated with an ER stress response and renal interstitial fibrosis. Supported by CIHR – OSO‐115895.
Effect of carbon nanotubes on transepithelial resistance in barrier epithelial cells
Authors: Lewis Shanta , Petrache Horia I. , Roark Torri , Witzmann Frank , Blazer-Yost Bonnie L.
Published: June 2021

With the burgeoning of the nanotechnology industry, there is increasing concern regarding the effects of these particles in our environment and food chains and therefore human ingestion and inhalation. Recent research in our laboratory showed that single‐wall and multi‐wall carbon nanotubes (CNTs) decrease the transepithelial resistance of renal principal cells. The effects are manifested at very low concentrations of nanoparticles (0.4 ng/cm2‐ 4 μg/cm2). After 48 hours of CNT exposure, resistances are approximately ½ of the control monolayers, indicating that the barrier function of the tissue is compromised but the cellular monolayer is still intact. To determine the mechanism of action of nanoparticles, we have investigated the interaction of CNTs with model lipid membranes using an ion channel amplifier (planar bilayer Warner workstation). Measurements in 1M KCl solutions indicate that the presence of nanoparticles causes transient disruptions in lipid membranes made of phosphatidylcholine lipids. An ion channel reporter (gramicidin A) is used to quantify the effect of CNT on ion transport. Thus, the CNTs may have a direct effect on the plasma membrane. Funding: NIGMS (RO1GM085218).
Impaired kidney function and anatomy in dietarily‐rescued calcineurin (alpha isoform) knockout mice
Authors: Gooch Jennifer L , Ecelbarger Carolyn , Reddy Ramesh N , Tsukerman Susanna , Madsen Kirsten
Published: June 2021

Previously, we identified a predominant role for the alpha isoform of calcineurin (CnAα) in normal kidney development and function. However, many experiments were limited by failure to thrive and early lethality of CnAα knockout mice. Recently, we reported the rescue of CnAα −/− mice by dietary compensation for defective salivary enzyme secretion. We now provide the first report of kidney anatomy and function in adult knockout mice. The data show that despite comparable body mass, kidney size is reduced and GFR is lower in null mice. In additional, although total glomerular number is unchanged, a significant proportion of glomeruli are abnormal. Examination of the protein expression of major renal sodium transporters, channels, and exchangers reveals modest changes in proximal tubule transporters, but substantial (40–60%) decreases in the aldosterone‐sensitive Na‐Cl cotransporter (NCC), the α‐subunit of the epithelial sodium channel (ENaC), and the 70‐Da band of γ‐ENaC. Null mice also show decreased urinary potassium and hyperkalemia suggesting impaired collecting duct function. These findings are consistent with clinical observations of aldosterone resistance with use of calcineurin inhibitors and provide new experimental evidence that, in addition to glomerular development, CnAα plays a role in sodium and potassium handling by the distal nephron.
Reduced proteoglycans, cystic disease and primary cilia
Authors: Munteanu Maria Cristina , Mansat Robert Silasi , Condac Eduard , Ferencz Beatrix , Lupu Florea , Hinsdale Myron E.
Published: June 2021

Most cells produce proteoglycans. These carbohydrate moieties on these specialized proteins is assembled on specific core protein serines and initiates with a xylose. The enzyme responsible for this is xylosyltransferase and exists in two isoforms. Xylosyltransferase 2 (XylT2) is ubiquitously expressed in many organs suggesting a significant role of XylT2 dependent proteoglycans in those organs. We have shown that XylT2 deficiency causes substantial reduction in liver proteoglycans resulting in cyst development in the biliary tract and in the kidney there is fibrosis and tubule dilation. These findings are similar to those in polycystic kidney disease (PKD). In the liver, biliary epithelial proliferation and fibrosis accompany cyst development. How or if these contribute to cyst formation is unknown, but in other cystic animal models and in patients who suffer from PKD, primary cilia defects occur. Therefore, we determined if primary cilia are abnormal in our cystic model and found that indeed there are cilia abnormalities. These findings provide further evidence illustrating that primary cilia defects can contribute to or may directly cause cystic disease. What is the actual cause of the cilia defects in the Xylt2−/− mice is unknown but is under investigation.
NADPH oxidase mutation protects against skeletal muscle insulin resistance in high fat fed mice
Authors: Stump Craig S. , Chen Guanjie , Diamond-Stanic Maggie K. , Cantoria Mary Jo , Henriksen Erik J.
Published: June 2021

In the present study, we hypothesized that NADPH oxidase‐dependent increases in reactive oxygen species (ROS) associated with diet‐induced obesity impair skeletal muscle insulin action and mitochondrial function. Pathogen free B6(Cg)‐Ncf1m1J/J mice ( Jackson Laboratory), which harbor an inactivating mutation for the p47phox subunit of NADPH oxidase, and C57/BL6J (C57) control mice were maintained on either a high fat diet (HF; 45%) or low fat normal chow diet (LF, 10%) for 10 weeks. Final body weights were significantly greater for mice fed HF compared to LF diets in both C57 (29%) and B6(Cg)‐Ncf1m1J/J (30%) mice. Insulin‐stimulated glucose uptake in soleus muscle (ex vivo) was significantly lower in HF vs. LF fed C57 mice (−41%; p<0.05), but not in HF B6(Cg)‐Ncf1m1J/J mice (−6%; p>0.05), suggesting that NADPH oxidase activation is integral to HF diet‐induced muscle insulin resistance. Hindlimb muscle cytochrome oxidase (COX) activity increased with HF feeding in the B6(Cg)‐Ncf1m1J/J mice (38%; p<0.05), but not in HF fed C57 mice. There was no significant change in muscle citrate synthase activity with HF feeding in either the B6(Cg)‐Ncf1m1J/J or C57 mice. In summary, the adverse effects of HF feeding on skeletal muscle insulin sensitivity and mitochondrial COX activity were abrogated in mice with a p47phox subunit inactivating mutation.

Supported by an Endocrine Society Research Award.
Chronic hyperinsulinemia sensitizes myocytes to hyperglycemia‐induced cell death
Authors: Harmancey Romain , Lubrano Genna , Lam Truong N. , Taegtmeyer Heinrich
Published: June 2021

Recent clinical trials have reported that intensive glucose lowering therapy in type 2 diabetic patients increases death from cardiovascular causes. We therefore investigated whether aggressive insulin therapy sensitizes myocytes to glucose toxicity. Rat L6 myocytes, cultured in low (5.5 mM) glucose conditions, were treated every 24 hours with insulin. After 5 days of treatment, the rates of glucose uptake were measured, and the expression of the glucose transporters GLUT1 and GLUT4 quantified by real‐time PCR and western blotting. Another set of cells was incubated in hyperglycemic (30 mM) conditions, and the activity of the proapoptotic caspase‐3 was measured by spectrophotometry. Chronic insulin treatment increased the rates of glucose uptake compared to non‐treated cells. This increase, which was associated to higher expression levels of both GLUT1 and GLUT4, could not be further potentiated by acute insulin stimulation. In addition, the cells chronically treated with insulin also exhibited an increase in caspase‐3 activity when incubated with high glucose concentrations. In conclusion, simulated chronic hyperinsulinemia upregulates glucose transporters in myocytes, and the ensuing increase in glucose uptake is associated with a sensitization of the cells to hyperglycemia‐induced cell death. This work provides direct evidence for toxic effects of increased glucose metabolism in muscle cells.
Treatment with sulodexide, an endothelial glycocalyx‐mimetic, restores glucose tolerance in high fat diet‐fed mice
Authors: Eskens Bart JM , Vink Hans , van Teeffelen Jurgen WGE
Published: June 2021

We tested if treatment with sulodexide, a glycocalyx‐mimetic, restores glycocalyx dimensions and whole‐body glucose tolerance induced by a high fat diet (HFD) in mice.

C57Bl/6 mice were fed normal chow (n=5) or a HFD for 6 weeks (n=16), and half of these HFD‐fed mice were treated with sulodexide in their drinking water (15mg/100mL) for the final two weeks of the feeding period. At the day of experiment, the microcirculation of the hindlimb muscle was measured with a Sidestream Dark‐field camera under anesthesia, and dynamic variations in RBC column width in all visible microvessels were determined. Further, glucose tolerance was measured by i.p. injection of 1g/kg glucose and determining the area under the curve of blood glucose (AUCgluc).

While median perfused RBC column diameter (± 6 μm) did not change, HFD mice were associated with increased outward movement of RBCs into glycocalyx (2.5 ± 0.3 μm vs 1.6 ± 0.3 μm in control, P<0.05), and this effect was impaired after sulodexide treatment (2.1 ± 0.3 μm; NS vs control). The AUCgluc was also increased in HFD‐fed mice versus control (1005.2 ± 59.8 mmol/L*90min. vs 773.7.1 ± 55.0 mmol/L*90min, P<0.05) and this effect was prevented after sulodexide treatment (780.4 ± 31.8 mmol/L*90min., NS vs control).

In conclusion, two week sulodexide treatment partially restores glycocalyx dimensions and appears to reverse the HFD‐induced decline in whole‐body glucose tolerance in mice.

Supported by DFN 2006.00.027, and CTMM work package 01C‐ 104‐04‐PREDICCT
Association between HOMA‐B and A1C levels in Haitian Americans with type 2 diabetes
Authors: Zarini Gustavo G. , Exebio Joel C. , Podesta Cinthia , Huffman Fatma G.
Published: June 2021

This study examined the association of pancreatic β‐cell function assessed by the homeostasis model assessment (HOMA) and glycosylated hemoglobin (A1C), and determined whether this association varies by gender in Haitian Americans with type 2 diabetes (T2D). The cross‐sectional study included 77 Haitian Americans (males=32 & females=45). Participants were recruited from multiple sources from Miami‐Dade and Broward Counties, Florida. Fasting plasma glucose, A1C, and insulin levels were measured in fasting blood. Analyses included t‐test, chi‐square, Pearson's correlations and linear regression. Males had significantly lower HOMA‐B function (P=.032) and BMI (P=.007) compared to females. Regression analyses showed that after controlling for covariates, the association between HOMA‐B with A1C was only significant for Haitian American males (β= −1.625, SE=−.070, P=.001). The model explained 27.9% of the variability in A1C. A one unit decrease in HOMA‐B increased the A1C levels 1.6%. Assessment of both HOMA‐B and A1C could be a better predictor for diabetes care among Haitian Americans. Funding for this research was provided through an NIH/NIDDK sponsored grant.
MAPK phosphorylation of connexin 43 promotes binding of cyclin E and smooth muscle cell proliferation
Authors: Johnstone Scott Robert , Kroncke Brett M , Straub Adam C , Best Angela K , Dunn Clarence A , Koval Michael , Lampe Paul D , Columbus Linda , Isakson Brant E
Published: June 2021

Mitogen activated protein kinase (MAPK) phosphorylation of connexin 43 (Cx43) is associated with proliferation of vascular smooth muscle cells (VSMC) in atherosclerosis. We aimed to investigate whether MAPK phosphorylation of Cx43 directly regulates VSMC proliferation. Using in vivo models of VSMC proliferation e.g. carotid treatments with platelet‐derived growth factor–BB (PDGF), we identified that MAPK phosphorylated Cx43 interacts with the cell cycle control proteins cyclin E and CDK2. To confirm this in vitro we isolated primary Cx43−/− VSMC and transfected these with constructs for Cx43 containing null phosphorylation (alanine) or phospho‐mimetic (aspartate) mutations of the MAPK serines. Co‐immunoprecipitation and proliferation studies in transfected cells combined with analytical size exclusion studies of purified Cx43 C‐terminus and cyclin E proteins demonstrated that MAPK phosphorylation of Cx43 is critical for its binding with cyclin E and for VSMC proliferation in vitro. Finally, using a novel knock‐in mouse containing Cx43 MAPK alanine mutations (Cx43‐MK4A), we showed that the null‐phosphorylation mutation disrupts interactions between Cx43 and cyclin E and VSMC proliferation in vivo. We conclude that MAPK phosphorylated Cx43 is a novel interacting partner of cyclin E and is required to promote VSMC proliferation.
Probenecid Protects Vascular Contractile Of Thoracic Aorta In Rats Under Hypotonic Condition By Blocking Pannexin‐1
Authors: Li Binyin , Kong Xiangxing , Guan Xiaojing , Xia Qiang
Published: June 2021

Probenecid is a drug proved to act on pannexin‐1(Panx1). This study investigated the involvement of probenecid in the vascular contractile, especially when Panx1 opens under hypotonicity. We first showed the expression of Panx1 on the aortic smooth muscle cell in rats via reverse transcription polymerase chain reaction (RT‐PCR) and immunohistochemistry (Fig. 1). Then the effect of hypotonic condition (201mOsm/L) on vascular contractile of male Sprague‐Dawley rat thoracic aorta with endothelia denuded was measured, with or without adding probenecid. Finally, after incubated in the hypotonic solution with or without probenecid, the efflux adenosine triphosphate (ATP) of smooth muscle was measured. We found that under hypotonic condition, vascular contractile was greatly impaired, while probenecid markedly protected contractility of vascular smooth muscle in hypotonic stress (Fig. 2, * p<0.05, ** p<0.01). And the protection is positively related to concentration of probenecid (Fig. 3, ** p<0.01). Measurement of ATP release showed great increase when aortic rings incubating in hypotonic solution for 15 minutes (Fig. 4, * p<0.05), which was eliminated if probenecid was added in the solution with concentration of 1mM. These results indicated that probenecid protected the vascular contractile, probably via inhibiting Panx‐1 and blocking the efflux of intracellular ATP. This work was supported by grants from the Department of Science and Technology of Zhejiang Province, China (2011C23105).
The CaV1.2 channel C‐terminus fragment is a transcriptional vasodilator
Authors: Bannister John P , Leo Marie Dennis , Naryanan Damodaran , Nair Anitha , Pachuau Judith , Jaggar Jonathan H
Published: June 2021

Arterial myocyte voltage‐dependent L‐type (CaV1.2) channels are essential for physiological control of vascular contractility. The CaV1.2 channel C‐terminus is post‐translationally cleaved, yielding a protein fragment (CCT) that contains a putative nuclear targeting sequence and can act as a transcription factor. Here, we investigated physiological functions of the CCT in myocytes of resistance‐size cerebral arteries. Immunofluorescence using a custom antibody raised to CCT identified CCT localized to both the nucleus and plasma membrane. CCT overexpression reduced CaV1.2 mRNA and protein, and reduced pressure and depolarization‐induced vasoconstriction. Overexpression of a CCT construct lacking the putative nuclear targeting sequence did not alter CaV1.2 mRNA, protein or arterial contractility. These data suggest that the CCT inhibits CaV1.2 transcription and is a cerebral vasodilator. NIH/NHLBI
Comparison of the tolerance on the venous relaxation induced by nitroglycerin and by the new nitrite donor cis‐[Ru(bpy)2(py)(NO2)](PF6)
Authors: Paulo Michele , da Silva Roberto Santana , Bendhack Lusiane Maria
Published: June 2021

Organic nitrates such as nitroglycerin (GNT, glycerol trinitrate), are commonly used in clinical cardiovascular medicine in the acute treatment of angina and severe arterial hypertension. The development of tolerance due to the continuous treatment with high doses leads to a decrease of the magnitude of the vasodilatation induced by GNT. It is a major limitation for the chronic clinical use of GNT. The present study aimed to verify if the new nitrite donor cis‐[Ru(bpy)2(py)(NO2)](PF6) (Rubpy) induces in vitro tolerance in the rat cava vein and to compare this effect with GTN. We have analyzed the maximum relaxing effect (ME) and the potency (EC50) induced by GNT and Rubpy in concentration‐effect curves constructed in vein rings pre‐contracted with endothelin‐1. The experiments were performed on intact endothelial rings following pre‐incubation for 30 min with GTN (EC100: 0.1mmol/L or EC50: 4 μmol/L) or Rubpy (EC100: 0.1μmol/L or EC50: 2 μmol/L). After pre‐incubation with GTN (EC100), the ME was reduced (from 57.5±2.5%, n=4 to 28.1±0.8%, n=4, p<0.001). Pre‐incubation with the EC50 of GTN had no effect. Rubpy induced relaxation in a concentration‐dependent way, that has not been changed by pre‐incubation for 30 min with RuBPY in the EC50 or EC100 (78.6±2.8%, n=4). In conclusion, prior exposure with GTN reduces the relaxation induced by GTN whereas it does not occur with Rubpy. Supported by FAPESP/CNPq.
AMP‐Activated Protein Kinase Reduces Vascular Smooth Muscle Growth by Inhibiting Actin Cytoskeleton Dynamics Necessary for Migration
Authors: Stone Joshua Daniel , Vuncannon Jackson R , Shaver Patti , Fox Jonathan Clay , Tulis David Anthony
Published: June 2021

Abluminal vascular smooth muscle cell (VSMC) migration is a functional underpinning of vasculoproliferative disorders. Upon activation, the dynamic nature of the actin cytoskeleton and rapid focal adhesion turnover provides the structural basis necessary for cell motility. Preliminary data show that activation of the metabolic sensor AMP‐activated protein kinase (AMPK) inhibits migration in rat primary VSMCs. In the current study we tested the hypothesis that AMPK enhances focal adhesion stability and inhibits vasodilator‐stimulated phosphoprotein (VASP) anti‐capping function on microfilament elongation. Treatment of VSMCs with the AMPK activator AICAR (1 mM) significantly (~50%; p<0.05) increased phosphorylated VASP at Thr278, a reported AMPK‐specific site, thus inhibiting microfilament anti‐capping by VASP. This inhibition of VASP was independent of the traditional VASP‐activating kinases PKA and PKG. In addition, AICAR inhibited PDGF‐stimulated autophosphorylation of focal adhesion kinase (FAK) at Ser397, a necessary event for kinase activity and focal adhesion turnover. Inhibition of phospho‐FAK at Ser397 induced focal adhesion stability necessary for cytostasis. These data lend strong support for the notion that AMPK has capacity to promote focal adhesion stability and to inhibit cytoskeletal rearrangement necessary for VSMC migration.
AMP‐Activated Protein Kinase Reduces Vascular Smooth Muscle Growth by Inhibiting TGF‐β/Smad Signaling and MMP Activity
Authors: Stone Joshua Daniel , Vuncannon Jackson R , Shaver Patti , Fox Jonathan Clay , Tulis David Anthony
Published: June 2021

Aberrant vascular smooth muscle cell (VSMC) growth and extracellular matrix synthesis provides mechanistic underpinning for many vascular disorders. One of the major contributors to these phenomena is transforming growth factor‐beta (TGF‐β) and its canonical Smad signaling cascade. Preliminary data suggest that activation of the metabolic sensor AMP‐activated protein kinase (AMPK) has ability to inhibit VSMC growth and neointima formation in the rat carotid artery. In the current study we explored the hypothesis that AMPK inhibits VSMC growth by inhibiting TGF‐β/Smad signaling and matrix turnover by the matrix metalloproteinases (MMPs)‐9. Treatment of VSMCs with the AMPK activator AICAR (1 mM) significantly reduced (~15%; p<0.05) TGF‐β expression in cell lysates after both 24 and 48 hours. AICAR significantly decreased (~15%; p<0.05) stimulatory Smad3 expression and significantly increased inhibitory Smad7 expression (~20%) over a 24 hr period. Furthermore, AICAR significantly decreased (~20%) MMP‐9 activity in 24 hour conditioned media as assessed by gel zymography. AICAR also significantly (p<0.05) inhibited cell proliferation (~40%) after 48 hours. These data support the theory that AMPK inhibits VSM growth by inhibiting the TGF‐β/Smad/MMP signaling cascade and provide solid support for the use of AMPK to halt the VSMC growth and matrix deposition inherent in vascular hypertrophy and stenosis.
Monocytes Induce Apoptosis of Vascular Smooth Muscle Cells via a Monocyte Chemoattractant Protein‐1 (MCP‐1) Mediated Mechanism
Authors: Wang Qiwei , Morgan Stephanie , Liu Bo
Published: June 2021

Excessive apoptosis of smooth muscle cells (SMCs) contributes to pathogenesis of abdominal aortic aneurysm (AAA). We have previously demonstrated in a murine model of AAA that apoptotic SMCs stimulate vascular inflammation through production of MCP‐1. In the current study, we tested the hypothesis that MCP‐1 further induces cell death by enhancing the pro‐apoptotic capability of infiltrating inflammatory cells. Cultured mouse aortic SMCs and a mouse monocyte/macrophage cell line RAW264.7 were used. MCP‐1 (100ng/ml, 24h) primed RAWs elicited significant apoptosis of SMCs (2.21±0.349 fold increase in percentage of Annexin V‐PE+/7AAD− cells compared to untreated SMCs, n=5, p<0.05, ANOVA), while untreated RAWs or MCP‐1 had no effect. The pro‐apoptotic effect required cell‐cell contact or proximity between SMCs and RAWs because conditioned media made by MCP‐1 primed RAWs had minimal effect on apoptosis. In a CaCl2 induced murine aneurysm model, co‐staining with TUNEL and MOMA2 showed spatial correlation of apoptosis and monocyte/macrophage in aortic wall 7 days after AAA induction. In conclusion, our results suggest MCP‐1 may play a central role in pathogenesis of AAA. Produced by apoptotic SMCs and other cell types in injured vessels, MCP‐1 contributes directly to the recruitment of inflammatory cells and indirectly to induce apoptosis of SMCs.

This work was supported by NIH grant R01HL088447‐01A2 (to B. Liu).