bluuebunny/crossref_metadata_2025 huggingface dataset:
The Cardioprotective Effects of the Mitochondrial Fission Inhibitor, P110, on Myocardial Ischemia/Reperfusion (MI/R) Injury
Authors: Vu Jonathan , Chen Qian , Remarcke Ryan , Benjamin Israel , Stutzman Devon , Patel Harsh , Chau William , Patel Samir , Peters Colin , Smith Edward , Pang Clifford , Barsotti Robert , Young Lindon
Published: June 2021

Mitochondrial dynamics are altered in favor of mitochondrial fission during MI/R. Mitochondrial fission is associated with shortening of mitochondria, decreased ATP production, and increased reactive oxygen species during MI/R, and is thought to promote cardiomyocyte loss. Therefore, inhibition of mitochondrial fission may be a new strategy to salvage injured cardiac myocytes and limit infarct size. To test this hypothesis, the cardioprotective effects of a novel mitochondrial fission inhibitor, P110 (MW=2427 g/mol), a cell permeable peptide, that selectively inhibits the interaction between dynamin related protein 1 and fission protein 1, were determined in isolated perfused rat hearts subjected to I (30 min)/R (45 min). P110 (1 µM) given for 10 min before ischemia and for 20 min post‐reperfusion, significantly restored left ventricular developed pressure (LVDP) and the peak of the first derivative of left ventricular pressure (dP/dtmax) to 77 ± 9% and 58 ± 7% of baseline values at 45 min post‐reperfusion, respectively, as compared to control I/R hearts (both p<0.01, n=6). The LVDP and dP/dtmax of control I/R hearts (n=8) only recovered to 34 ± 8% and 29 ± 4% of baseline values at 45 min post‐reperfusion, respectively. P110 also significantly reduced infarct size to 20 ± 3% compared to 41 ± 4% in control I/R hearts (p<0.01). The preliminary results suggest that inhibition of mitochondrial fission during MI/R improves post‐reperfused cardiac contractile function and reduces infarct size.
The Activation of Matrix Metalloproteinease‐2 by Mitochondrially‐Generated Reactive Oxygen/Nitrogen Species
Authors: Hughes Bryan , Schulz Richard
Published: June 2021

Matrix metalloproteinase‐2 (MMP‐2), along with other MMPs, has been implicated in heart injury and disease. These pathologies are characterized by both acute and chronic oxidative stress. In vitro, low concentrations of reactive oxygen/nitrogen species (RONS) can directly activate MMP‐2 via post‐translational modifications. However, this has yet to be demonstrated in a living system. We investigated whether endogenous RONS produced by pharmacological inhibition of mitochondrial ubiquinol‐cytochrome c reductase with antimycin could increase MMP‐2 activity in cardiomyocyte and non‐cardiomyocyte cell lines. We exposed neonatal rat cardiomyocytes (NRVMs), H9c2 cardiomyoblasts and HT1080 fibrosarcoma cells to antimycin (0.01‐1, 0.3‐3.2 and 1‐100 µM for NRVMs, H9c2 and HT1080 respectively) in serum‐free media for short (0.5 h) or long (6 h) intervals to measure changes in MMP‐2 activity in cell lysates and conditioned media by gelatin zymography. The antimycin concentrations used did not affect viability or cell growth, but increased oxidative stress in a dose‐ and time‐dependent fashion as shown by decreased aconitase activity. Antimycin decreased lysate MMP‐2 activity in NRVMs, but increased it in H9c2 cells, despite comparable decreases in aconitase activity. HT1080 cells also exhibited increased MMP‐2 activities, but only at the lowest concentrations of antimycin used. The activity of secreted MMP‐2 was not consistently affected by antimycin treatment in any cell line. We conclude that intracellular MMP‐2 activity can be affected by endogenous oxidative stress in cultured cells, dependent upon cell type. This provides evidence that RONS could modulate MMP‐2 activity in vivo, independent of gene expression.
Support: CIHR
N‐Acetyl Cysteine Attenuates Hypoxia Induced Sympathoexcitation in Human Subjects
Authors: Jouett Noah , Moralez Gilbert , Raven Peter , Watenpaugh Don , Smith Michael
Published: June 2021

This investigation tested the hypothesis that central and peripheral reactive oxygen species (ROS) mediate hypoxia induced sympathoexcitation, which is a central feature of the Obstructive Sleep Apnea‐Hypopnea Syndrome (OSAS). 10 healthy human subjects were recruited. One hour prior to experimentation, each subject randomly ingested either 70 mg.kg‐1 of N‐Acetyl Cysteine (NAC, n=5) or vehicle placebo (n=5). ECG, BP, muscle sympathetic nerve activity (MSNA) and plasma ROS and catecholamines were measured. Subjects underwent a 20 minute intermittent hypoxia training (IHT) protocol consisting of cyclical end‐expiratory apneas with 100% N2. Venous blood was analyzed pre/post IHT for ROS by electron paramagnetic spectroscopy and for catecholamines by ELISA. In the placebo group, MSNA was increased between pre vs. post IHT (P=0.01). In the NAC group, however, MSNA was unchanged (P=0.26). NAC reduced the percent change (% Δ) of ROS observed from pre‐ vs. post‐IHT compared to placebo (P=0.02). The % Δ of ROS was directly related to increasing MSNA (R2=0.83, p=0.01). The % Δ of norepinephrine was lower in the NAC vs. placebo group after IHT (P=0.05), whereas the % Δ of epinephrine was unchanged (P=0.40). These data indicate that NAC reduces central sympathetic outflow in response to IHT, and thereby could reduce cardiovascular risk in OSAS patients.
Doxorubicin‐induced Activation of Intracellular Matrix Metalloproteinase‐2 by Oxidative Stress
Authors: Chan Brandon , Hughes Bryan , Schulz Richard
Published: June 2021

Matrix metalloproteinase‐2 (MMP‐2) activity is involved in several heart pathologies resulting from increased oxidative stress including ischemia‐reperfusion injury. In addition to its roles in extracellular matrix remodeling, MMP‐2 is also an intracellular protease with specific intracellular substrates. MMP‐2 mRNA and protein levels are elevated in cancer patients treated with doxorubicin (DXR). Though effective in chemotherapy, DXR's dose‐limiting cardiotoxicity may cause heart failure by activating intracellular MMP‐2. Human fibrosarcoma cells (HT1080 cells) and neonatal rat ventricular myocytes (NRVM) were treated with 0.01‐1 μM DXR for 0.5 and 2 h at 37°C. DXR treatment had no effect on cell viability. MMP‐2 activity and protein levels were measured by gelatin zymography and immunoblotting, respectively. Oxidative stress was measured by changes in mitochondrial aconitase activity. To visualize changes in intracellular MMP‐2 activity in real time, we will use a genetically encoded, fluorescence resonance energy transfer (FRET)‐based biosensor. In HT1080 cells 0.1‐1 μM DXR stimulated a 1.6 fold increase in intracellular MMP‐2 activity after 2 h without changing its protein level. DXR had no effect on MMP‐2 activity in NRVM. The FRET‐based MMP‐2 biosensor detected MMP but not other intracellular proteases, such as calpains and caspases, in vitro. In human cancer cells, acute treatment with DXR activates intracellular MMP‐2. The biosensors are now being expressed in cells to directly study intracellular MMP‐2 activity. The FRET‐based biosensor will allow us to better understand how oxidative stress stimuli affects intracellular MMP‐2 activity and its specific substrates.
Hyperacetylation of SOD2 Mediates Transporting Its Cytosolic Precursor into Mitochondrial Matrix in the Murine Heart of SOD2‐tg
Authors: Chen Yeong‐Renn , Zhang Liwen , Chen Chwen‐Lih , Kang Patrick
Published: June 2021

SOD2 is the primary enzyme that neutralizes •O2‐ to H2O2 in mitochondria. Cardiac‐specific SOD2 overexpression (SOD2‐tg mouse) induces supernormal function and physiological hypertrophy in mouse heart. However, the stress imposed by SOD2 overexpression also resulted in protein aggregation of SOD2 and SOD2 hyperacetylation marked in mitochondria, which is associated with the phenotype of hypertrophy. We studied SOD2 acetylation from SOD2‐tg murine heart. We found acetylation of mature SOD2 and its precursor were detected in the mitochondrial matrix and cytosol by immunoblotting using the acetyllysine antibody. LC/MS/MS analysis indicated that K68, K122, K130, and K202 of mature SOD2 in matrix were acetylated. LC/MS/MS analysis further revealed that K68, K75, K89, K114, K122, K130, K132, K134, K154, and K202 of cytosolic SOD2 precursor were acetylated except residues of K194, K202, K221 and K222 at the C‐terminus. The results indicated that transport of SOD2 precursor into mitochondrial matrix resulted in Sirt 3‐mediated deacetylation at the residues of K75, K89, K114, K132, K134, K154; and site‐specific acetylation at K202. In vitro acetylation of mature SOD2 with acetic anhydride deaggregated pentameric SOD2, restored the profile of hyperacetylation observed in the cytosolic SOD2 precursor, and subsequently decreased the enzymatic activity of SOD2 as measured by EPR. The results concludes protein hyperacetylation of SOD2 inactivates the cytosolic precursor, and mediates transporting SOD2 into mitochondrial matrix, subsequently activating mature SOD2 via Sirt 3‐mediated deacetylation.
Assisted Reproductive Technologies‐Induced Premature Vascular Ageing Persists and Evolves into Arterial Hypertension in Adolescents
Authors: Rexhaj Emrush , von Arx Robert , Cerny David , Soria Rodrigo , Bouillet Elisa , Sartori Claudio , Scherrer Urs , Rimoldi Stefano
Published: June 2021

Assisted reproductive technologies (ART) induce vascular dysfunction and premature vascular ageing in young apparently healthy children and mice (Scherrer et al. Circulation 2012; Rexhaj et al. JCI 2013). In adult ART mice, premature vascular ageing translates into arterial hypertension. Given the young age of the human ART population, the evolution and long‐term consequences of ART induced vascular alterations are unknown. We speculated that vascular alterations persist in ART adolescents and that arterial hypertension represents the first clinically relevant end‐point of this problem in humans. We, therefore, 5 years after the initial assessment, reassessed vascular function (flow mediated dilation, FMD, pulse wave velocity, PWV, and carotid intima media thickness, IMT) in 54 ART young adults (16.4,y) and 43 controls (17.6,y) together with 24h ambulatory blood pressure measurements.

The main new findings were 2‐fold: First, premature vascular ageing persisted in ART adolescents, as evidenced by decreased FMD (P<.01), and increased PWV (P=.039) and IMT (P<.01), compared to controls. Endothelial dysfunction and arterial stiffness were comparable to the one observed 5 years ago, whereas IMT had increased in ART subjects (P<.01). Second and most importantly, 24h blood pressure was markedly higher in ART than in control subjects (119.8±1.3 vs. 115.7±1.1, P=.02, vs. ctrl).

These findings provide the first evidence that ART‐induced premature vascular ageing persists in adolescents and translates into arterial hypertension. These data further underscore the potential of ART to increase cardiovascular risk in this exponentially growing population.
Effects of Palmitate on Mitochondrial Number and PGC1α Splicing in Primary Human Myocytes
Authors: Devarshi Prasad , Taylor Erin , Jones Aarin , Henagan Tara
Published: June 2021

PGC1α is an epigenetically regulated transcriptional coactivator that promotes mitochondrial biogenesis. Long chain fatty acids (LCFA), such as palmitate, decrease PGC1α expression in association with decreased mitochondrial function and increased insulin resistance in primary myocytes. Several splice variants of PGC1α have been identified and may compensate for the loss of full length PGC1α; yet, it is unclear if these variants are epigenetically regulated or altered by LCFA treatment in skeletal muscle. Here, we determined the effects of 48h of palmitate treatment on known PGC1α splice variant mRNA levels in cultured and differentiated primary human myocytes (Life Technologies). Genomic DNA and total RNA were isolated and mitochondrial number and PGC1α splice variants' expression, respectively, were determined by real time‐PCR. Palmitate treatment tended to increase mitochondrial number (P = 0.14), despite a significant decrease in the combined expression of all PGC1α splice variants (P < 0.05). The decrease in combined PGC1α splice variant expression appeared to be due to specific decreases in the unspliced PGC1α and A4 variants, although these were not significantly decreased on their own. In contrast, we observed a significant increase in the A2 splice variant (P < 0.05) and a trend towards an increase in N‐truncated PGC1α (P = 0.11). Palmitate had no significant effect on other PGC1α splice variants. N‐truncated PGC1α has been shown to compensate for loss of unspliced PGC1α. We propose that palmitate‐induced upregulation of A2 and N‐truncated PGC1α may be sufficient to acutely increase mitochondrial number, leading to compensatory increases in the cellular capacity for beta oxidation of LCFA.
RELATION BETWEEN ALDOSE REDUCTASE ENZYME AND THE SIGNAL PATHWAY OF PROTEIN KINASE C IN THE IN VITRO DIABETIC RETINOPATHY MODEL
Authors: Das Evcimen Net , Sarikaya Mutlu , Yazihan Nuray
Published: June 2021

Diabetic retinopathy is an important microangiopathy which effects the pre‐capillary arterioles, capillaries and venules of retina. More than 90 % of all diabetic patients have retinopathy within which causes serious health problems. Several molecular mechanisms have been described to explain the retinal, neural and vascular disorders caused by diabetes. Formation of advanced glycation products, oxidative stress, hexosamine pathway, PKC pathway and polyol pathways are among of these mechanisms. Although it has been known that the PKC signaling pathway and the aldose reductase related polyol pathway are interacting with each other, it is not clearly known whether inhibition or activation of aldose reductase in human retinal cells effects the PKC level and activity, cell proliferation and apoptosis under hyperglycemia and oxidative conditions. In this study, the interaction between AR and PKC in ARPE‐19 cells have been investigated. The relationship between oxidative stress, PKC and AR is very important for the development of diabetic complications such as retinopathy. Our studies have shown that these three mechanisms are closely related to each other. In this context, the AR enzyme can be considered as tissue dependent and its inhibition for the benefit/harm of the cell must be evaluated carefully. In conclusion, AR and PKC pathways are closely related to each other in in‐vitro diabetic retinopathy model. The relation between AR and PKC signaling pathways under hyperglycemia and oxidative stress conditions plays an important role in the control of various cellular pathways.
Renoprotective Effects of N‐acetyl‐seryl‐aspartyl‐lysyl‐proline (Ac‐SDKP) in Dahl Salt‐Sensitive Rats
Authors: Worou Morel , D'Ambrosio M , Liao T‐D , Nakagawa P , Peterson E , Janic B , Rhaleb N‐E , Carretero Oscar
Published: June 2021

Ac‐SDKP is a natural tetrapeptide, hydrolyzed by angiotensin‐converting enzyme, with anti‐inflammatory and anti‐fibrotic properties. The effect of Ac‐SDKP on Salt‐Sensitive (SS) hypertension is unknown; here, we hypothesized that Ac‐SDKP prevents renal inflammation, fibrosis, glomerulosclerosis and albuminuria in Dahl SS rats on high salt diet, without affecting the blood pressure. Dahl SS rats and their consomic controls, SS‐13BN were fed either 0.23% NaCl (low salt, LS) or 4% NaCl (high salt, HS) diet and infused with vehicle or Ac‐SDKP at either low or high dose (800 or 1600 µg/kg/day, respectively) subcutaneously via osmotic minipump for 6 weeks. Animals were divided into the following groups: LS + vehicle, HS + vehicle, HS + low AcSDKP, and HS + high AcSDKP.

HS increased systolic blood pressure (SBP) in SS rats (HS + vehicle 186±5 vs. LS + vehicle 141±3 mmHg), but not in SS‐13BN rats (HS+vehicle 140±3 vs. LS+vehicle 140±2 mmHg). Ac‐SDKP did not affect SBP. In both strains, Ac‐SDKP prevented HS‐induced renal macrophage and T cell infiltration, interstitial fibrosis and glomeruloslerosis. In SS‐13BN rats, low dose of Ac‐SDKP were able to prevent albuminuria (marker of renal function) induced by HS (LS+vehicle 7±1 vs. HS + vehicle 44±8 vs. HS + low Ac‐SDKP 24±3.18 mg/24h), while in SS rats only high Ac‐SDKP dose had the same effect.

In summary, at low and high doses, Ac‐SDKP prevented renal damages in SS rats on HS diet without affecting the blood pressure. But, only at high dose, Ac‐SDKP prevented HS‐induced albuminuria in this strain. On the other hand, in consomic controls, both doses of Ac‐SDKP prevented HS‐induced renal damages and albuminuria.

Supported by NIH PPG grant P01HL028982
Calcineurin homologous protein 1 interferes with calcineurin‐dependent regulation of the renal Na‐K‐2Cl‐cotransporter
Authors: Boldt Christin , Paliege Alexander , Jankowski Vera , Wakabayashi Shigeo , Hisamitsu Takashi , Bachmann Sebastian , Mutig Kerim
Published: June 2021

The Na‐K‐2Cl‐cotransporter (NKCC2) of kidney thick ascending limb (TAL) is crucial for renal salt handling. Its activity tightly regulated by phosphorylation. Our previous data suggested that calcineurin is involved in dephosphorylation and thus, deactivation of NKCC2. Further results suggested calcineurin activity may be modulated by members of the calcineurin homologous protein family (CHP). Among these, CHP1 has been implicated in the regulation of several membrane proteins including ion transporters. The role of CHP1 in calcineurin‐dependent regulation of NKCC2 activity has therefore been studied.

CHP1 was co‐localized with NKCC2 in rat TAL by immunofluorescence. Both products co‐immunoprecipitated, suggesting their interaction. GST pull down assays with recombinant N‐terminal NKCC2 mutants, mimicking its (de)phosphorylation at functionally relevant residues (T96, T101, T114, and S126), suggested competitive interactions of CHP1 and calcineurin with phosphorylated NKCC2. Overexpression of CHP1 in cultured TAL cells substantially increased the phosphorylation of endogenous NKCC2 at baseline and upon low‐chloride stimulation. Acute stimulation of NKCC2 by administration of desmopressin in rats facilitated NKCC2 interaction with CHP1 while attenuating its interaction with calcineurin.

In sum, our data provide evidence that CHP1 facilitates NKCC2 phosphorylation, likely by interfering with calcineurin function in TAL.
Renal Specific Upregulation of Mitochondrial Genes in a Translational Primate Model of Spontaneous Hypertension
Authors: Goleva Slavina , Rhoads Megan , Osborn Jeffrey
Published: June 2021

Mitochondrial gene expression in African Green Monkeys (Chlorocebus aethiops sabaeus common name, vervet) may importantly influence renal function and long‐term blood pressure control. Vervets share over 90% of their genome with humans and exhibit heritable, spontaneous hypertension.We hypothesized that altered renal mitochondrial gene expression in hypertensive vervets may be associated with arterial hypertension. Vervets were phenotyped as normotensive (NT, systolic blood pressure (SBP) <120 mmHg) or hypertensive (HT, SBP > 140 mmHg). RNA was extracted from kidney cortex and liver tissues from 18 HT (mean SBP 166±7 mmHg) and 18 NT animals (mean SBP 98±3 mmHg) and gene expression determined using semi‐quantitative RT‐PCR. In renal cortical tissue of HT vervets, COX 3 (Complex IV) and Cyt B (Complex III) were upregulated by 1.6‐fold (p=0.03) and 1.4‐fold (p=0.05) respectively, while NADH4 (Complex I) and ATP8 (Complex V) expression were unchanged. In contrast, liver COX 3 expression was decreased in HT monkeys (1.9‐fold; p=0.04), 1.6‐fold for ATP8 (p=0.03), and 2.0‐fold for CYTB (p=0.01). Gene expression of nuclear encoded subunits in the oxidative phosphorylation chain SDH (Complex II) and COX4 (Complex IV) were not different in both kidneys and liver of HT vs. NT vervets. Selected mitochondrial genes are upregulated in renal cortex of hypertensive vervets and may be associated with excess reactive oxygen species. Mitochondrial gene downregulation in the liver may result from the systemic hypertension.

(Supported by APS, Biomedical Sciences Research Group, LLC, Telabio, Inc and Behavioral Sciences Foundation)
Nitric Oxide and Superoxide Significantly Affect Medullary Oxygenation and Urinary Output
Authors: Fry Brendan , Edwards Aurelie , Layton Anita
Published: June 2021

The goal of this study was to investigate the reciprocal interactions among oxygen (O2), nitric oxide (NO), and superoxide (O2‐), and their effects on medullary oxygenation and urinary output. To accomplish that goal, we developed a detailed mathematical model of solute transport in the renal medulla of the rat kidney. The model represents the radial organization of the renal tubules and vessels, which centers around the vascular bundles in the outer medulla and around clusters of collecting ducts in the inner medulla. Model simulations yield significant radial gradients in interstitial fluid oxygen tension (PO2) and NO and O2‐ concentration in the outer medulla and upper inner medulla. Due to the nonlinear interactions among O2, NO, and O2‐, the effects of NO and O2‐ on sodium transport, osmolality, and medullary oxygenation cannot be gleaned by considering each solute's effect in isolation. Model simulations further suggest that a sufficiently large reduction in the efficiency of oxygen utilization for tubular transport may be the key contributing factor – more so than oxidative stress – to hypertension‐induced medullary hypoxia. This research was supported in part by NIH grant DK‐89066.
Role of NOX4 in Superoxide Production Stimulated by H+ Efflux in mTAL of SS Rat
Authors: Zheleznova Nadezhda , O'Connor Paul , Cowley Allen
Published: June 2021

Renal medullary thick ascending limb (mTAL) tubular segments produce superoxide mostly derived from NADPH oxidases (NOXs).Superoxide production is augmented in Dahl salt‐sensitive (SS) rats and contributes to the development of hypertension in these animals. We have shown that the voltage‐gated proton channel Hv1 contributes to superoxide production in the mTAL of the SS rat during proton efflux however, as Hv1 itself does not produce superoxide, the molecular source of superoxide radicals remains unknown. We hypothesize that NOX4 might be involved in Hv1‐dependent superoxide production during proton efflux in mTAL. To test this, mTAL superoxide production (ΔEth/ΔDHE ratio) was compared between control SS and mutant SSNox4‐/‐rats (where Nox4 gene was knocked out in SS genetic background using zinc finger nuclease (ZFN) technique) in response to cellular acidification (using an NH4Cl loading/removal; 20 mmol/L). It was found that increase in superoxide production after acidification in microdissected mTAL from SS rats was blunted nearly 30% in mTAL isolated from SSNox4‐/‐ mutant rats. We conclude that Hv1 dependent O2−· production following mTAL cellular acidification is partially dependent on the presence of NADPH oxidase 4 (NOX4) although NOX4 may not be the source of O2−· production (HL116264)
Acute IP Furosemide Increases Medullary PO2 in The Diabetic Rat Kidney
Authors: o neill julie , Fasching Angelica , Patinha Daniela , Pihl Liselotte , Franzen Stephanie , Palm Fredrik
Published: June 2021

We recently observed medullary hypoxia during SGLT (sodium glucose linked transport) inhibition in the diabetic rat kidney. Underlying mechanisms are unclear but may reflect compensatory increases in sodium reabsorption in distal nephron segments, such as the medullary thick ascending limb (mTAL), where sodium transport is less efficient. To test this hypothesis, we investigated the impact of NKCC (sodium/potassium/chloride) co‐transporter inhibition on renal oxygen metabolism in the diabetic rat kidney.

Diabetes was induced by intraperitoneal (IP) STZ (50mg/kg) injection in Sprague Dawley rats (n=11), 2 weeks prior to experimentation. Animals were surgically prepared for intravenous infusion of [3H]Inulin and [14C]PAH, the IP delivery of furosemide (NKCC inhibitor; 3mg/Kg) and the measurement of mean arterial pressure (MAP), left kidney function and medullary (M) PO2 (Clark electrode). Data± SEM was analyzed using a two‐way RM ANOVA. Baseline MAP, transported sodium (TNa), total kidney oxygen consumption (QO2), and MPO2 was 107±4 mmHg, 189±23 µmol/min, 23±3 µmol/min and 26±2 mmHg, respectively. IP furosemide, had no effect on MAP, reduced TNa and QO2 to 58±7 µmol/min and 8±2 µmol/min respectively and increased MPO2 to 36±2 mmHg (all P<0.05).

Sodium transport via NKCC is clearly a major consumer of oxygen in the diabetic rat kidney. Moreover, the furosemide‐induced increase in MPO2 is in vast contrast to the reduction in medullary PO2, previously observed during SGLT inhibition. These data provide evidence that medullary hypoxia during SGLT inhibition in the diabetic rat kidney results from the diversion of TNa to the mTAL where sodium transport is less efficient.
Histone Deacetylase‐1 is Critical for Flow‐mediated Nitric Oxide Production by the Inner Medullary Collecting Duct
Authors: Hyndman Kelly , Pollock Jennifer
Published: June 2021

Collecting duct (CD) nitric oxide synthase‐1 (NOS1, nNOS) is critical for maintaining fluid‐electrolyte balance when eating a high sodium diet. During high sodium feeding, there is increased water intake to maintain hydration resulting in an increased urine flow/shear stress (SS) against the epithelium and increased NO production. The mechanism of the flow‐mediated increase in NO is undetermined. We recently reported that histone deacetylase enzyme‐1 (HDAC1) regulated endothelial‐derived NO. We hypothesized that HDAC1 regulates flow‐stimulated inner medullary (IM) NO production. We determined that HDAC1 and NOS1 form a protein‐protein complex in mIMCD3 cells and mouse IM tissue. mIMCD3 cells were subjected to a normal flow (NF, SS= 3 dynes/cm2 for 1 h) or high flow (HF, 30 dynes/cm2 for 1h) to mimic urine flow on normal or high salt diets. Cells were either pretreated with vehicle (0.1% DMSO), MS275 (300 nM) to inhibit HDAC1, MS275 (8 μM) to inhibit Class 1 HDACs, or TSA (500 nM) to inhibit Class 1/2 HDACs. Vehicle treated cells had a flow‐dependent increase in nitrite production (static = 278±35, NF = 515±124, HF = 803±140 pmol/mg pr/h, P<0.01). HDAC1 inhibition (300 nM MS275) attenuated the flow‐mediated increase in nitrite in both the NF and HF (113±44 and 223±51 pmol/mg pr/h, respectively) but not static nitrite production. Whereas, inhibition of multiple Class 1/2 HDAC isoforms did not inhibit static or flow‐mediated nitrite production. These data suggest that HDAC1 is a positive regulator of NOS1, while other class 1 and/or class 2 HDAC isoforms may negatively regulate IMCD NO production. In conclusion, HDAC1 is a novel NOS1 interacting protein and critical for IMCD NO production.
Identification of Phosphate Regulated Transcripts in Ileum and Renal Proximal Tubules
Authors: Knöpfel Thomas , Kampik Nicole , Hernando Nati , Rubio Aliaga Isabel , Biber Jürg , Wagner Carsten
Published: June 2021

Phosphate (Pi) is a vital mineral for every cell in order to maintain energy metabolism, cell signaling and structure. The kidney and intestine are two major organs in maintaining Pi homeostasis by their role in absorbing Pi from the diet and by the reabsorption of filtered Pi. Apical uptake of Pi into intestinal and proximal tubular epithelial cells is well characterized, whereas basolateral efflux mechanisms are poorly understood. The amount of Na‐Pi cotransporters (members of the Slc34 family) expressed at the apical membrane is regulated by changes in dietary Pi intake through mechanisms, which are not fully understood. The aim of this project is to identify transcripts in ileum and renal proximal tubules regulated by dietary Pi. Identified genes may be involved in the transcellular transport of Pi like the unknown basolateral transport pathway or contribute to adaption of apical Pi uptake and metabolism. C57BL/6 male mice (8 weeks old) were fed a high (1.2%) or low (0.1%) Pi diet for 3 days. Epithelial scrapings were prepared from ileum and micro‐dissected renal proximal tubules were collected. Renal and intestinal adaption was confirmed by qPCR for known Pi regulated transcripts. RNA was then analysed using the RNA sequencing service provided by the Functional Genomics Center Zurich. Comparison of the transcriptome of low and high dietary Pi conditions revealed 2029 genes to be differently (at least 2 fold changes) expressed in ileum and 327 in renal proximal tubules. Regulation of selected transcripts was confirmed by qPCR. Pathway analysis is currently being performed and selected candidates for basolateral Pi transporters will be expressed in Xenopus laevis oocytes.

Funded by the Swiss National Science Foundation.
Renal Localization and Characterization of Monocarboxylate Transporter Family Member Slc16a14
Authors: Knöpfel Thomas , Atanassoff Alexander , Kampik Nicole , Hernando Nati , Wagner Carsten , Biber Jürg
Published: June 2021

Monocarboxylate transporter 14 (MCT14) is an orphan member of the solute carrier family 16 for monocarboxylates, thyroid hormone and aromatic amino acids. The kidney shows the highest abundance of MCT14 mRNA, however, the renal localization, function and regulation are poorly understood. The aim was to localize MCT14 in murine kidney and address the question of its possible function. To this end an antibody was raised against a peptide located at the extracellular side of MCT14 and its specifity was confirmed by peptide protection and heterologous expression of MCT14 in Xenopus laevis oocytes.

Colocalization experiments with markers for different nephron segments indicate that MCT14 is localized to the apical membrane of the thick ascending limb of the loop of Henle. Members of the Slc16 family showed changes in protein and mRNA abundance in kidney due to metabolic acidosis. MCT14 itself showed an upregulation under acidotic conditions in both mRNA and protein levels, but the expression pattern in the kidney was not altered.

Uptake experiments in Xenopus laevis oocytes expressing MCT14 suggest that MCT14 does not transport lactate or pyruvate unlike the classical monocarboxylate transporters of the Slc16 family. To identify the substrate of MCT14 we used a mass spectrometry based approach in order to cover many substrates. Therefore Xenopus laevis oocytes expressing MCT14 and corresponding negative controls were incubated in mouse serum. Intracellular metabolites were then analyzed by mass spectrometry in the Functional Genomics Center Zurich. Major substrate candidates identified by mass spectrometry are currently being examined to confirm whether they are substrates of MCT14.

Funded by the Swiss National Science Foundation.
CFTR and SLC5A8 Associate in Thyroid Epithelial Cells
Authors: Fong Peying , Li Yonghai , Ganta Suhasini , Chen Grace
Published: June 2021

The cystic fibrosis transmembrane conductance regulator, CFTR, is expressed at the apical membranes of many epithelial cells, where it mediates cAMP‐stimulated anion secretion. In thyroid, CFTR may indirectly and/or directly furnish iodide for hormone synthesis. CFTR functionally interacts with many membrane transporters via PSD‐Discs large homologous‐ZO‐1 (PDZ) domain‐containing scaffolding proteins, thereby forming macromolecular transport complexes. SLC5A8 is an electrogenic, sodium‐coupled monocarboxylate transporter that also is expressed at thyrocyte apical membranes. CFTR and SLC5A8 contain the identical PDZ domain interaction motif; preliminary studies utilizing overexpression systems indicate a role for PDZ adaptor proteins in linking CFTR and SLC5A8. The present studies use the Fisher rat thyroid cell line, FRTL‐5, to test the hypothesis that PDZ proteins facilitate CFTR/SLC5A8 interactions in an endogenously expressing system. Expression of CFTR (~150 kDa), SLC5A8 (~68 kDa) and NHERF‐1 (~50 kDa) in total FRTL‐5 lysates was confirmed by immunoblot analysis using anti‐CFTR (M3A7 and Cystic Fibrosis Foundation Therapeutics (CFFT) 660), anti‐SLC5A8 (Abcam) and anti‐NHERF‐1 (Alomone) antibodies. Anti‐SLC5A8‐immunoprecipitated (IPd) fractions of FRTL‐5 lysates reacted with both CFFT 660 and anti‐NHERF‐1. Fractions IPd using anti‐CFTR (M3A7) were probed with anti‐SLC5A8 and anti‐NHERF‐1. M3A7‐IPd fractions showed positive reactivity with anti‐SLC5A8, but NHERF‐1 was not detected. These data suggest that although CFTR and SLC5A8 associate in FRTL‐5 cells, the participation of NHERF‐1 is not required.
Support: NIH/NIGMS Award 1R15GM101674‐01A1 (to PF); Office of the Director, NIH Award Number T35OD010979.
RanBPM Likely Mediates Sodium Chloride Cotransporter (NCC) Regulation through a WNK4‐ERK 1/2 Signaling Pathway
Authors: Sun Michael , Caroti Courtney , Feng Xiuyan , Zhou Bao , Cai Hui
Published: June 2021

Ran‐Binding protein M (RanBPM) is a nucleoplasmic protein. A recent study has shown that RanBPM is an inhibitor of ERK 1/2 signaling pathway by regulating c‐Raf stability. WNK is a serine/threonine kinase. Mutations in WNK1 and WNK4 cause pseudohypoaldosteronism type II (PHA II), characterized by hypertension, hyperkalemia and metabolic acidosis. Our previous study has shown that WNK4 inhibits NCC by activating ERK 1/2 signaling pathway. To determine whether WNK4 inhibits NCC through RanBPM‐dependent ERK 1/2 signaling pathway, we first investigated interaction between WNK4 and RanBPM using yeast‐two hybrid screening technique. We found that WNK4 interacted with RanBPM. RanBPM was also shown to prevent ERK 1/2 phosphorylation in response to EGF stimulation. When mouse distal convoluted cells (mDCT) were transfected with myc‐RanBPM, ERK 1/2 phosphorylation was decreased while NCC protein expression increased. When mDCT cells were transfected with siRNA RanBPM, ERK 1/2 phosphorylation was increased. These data suggest that 1) WNK4 interacts with RanBPM which is an inhibitor of ERK 1/2 phosphorylation; 2) RanBPM mediates NCC regulation likely through ERK1/2 signaling pathway.
Comparison of protein expression in kidney tubular apical and basolateral membranes in young and old rats
Authors: Khundmiri Syed , Murray Rebecca , LeBlanc Amanda , Sen Utpal , Lederer Eleanor , Weinman Edward , Jose Pedro , Barati Michelle , Merchant Michael
Published: June 2021

Na‐H Exchanger Regulatory Factor Isoform 1 (NHERF1) is critical for the expression of epithelial signaling complexes. We previously demonstrated loss of NHERF1 expression in kidney apical (BBM) and basolateral (BLM) membranes from old (22 m, O) compared to young (4 m, Y) rats, but the effect of NHERF1 loss on expression of kidney epithelial transporters is unknown. We hypothesize that loss of NHERF1 expression in aging will result in differential expression of ion transporting proteins in the BBM and BLM. To address this hypothesis, we compared protein expression and phosphorylation in BBM and BLM from Y and O female F344 rat kidneys using proteomic analysis. We identified 1331 differentially expressed proteins of which 18 were differentially phosphorylated in BBM and BLM from O animals as compared to Y animals. NHERF1 expression significantly decreased in BBM and BLM from O animals. Phosphoproteomic analysis identified phosphorylation of Ser276 on NHERF1 in membranes from Y animals. An additional phosphorylation at Ser285 on NHERF1 was identified in O animals. Expression of NKA a1, NpT2c, NHE3, NBCe1, g‐glutamyl peptidase, and ezrin were significantly decreased in membranes from old animals as compared to young animals. These data suggest that age‐related alterations in ion homeostasis may result from changes in transporter expression and from loss of normal hormonal regulatory pathways. Supported by NIH.
Role of insulin and insulin‐like‐growth factor (IGF) receptors in renal proximal tubule (PT) phosphorus handling
Authors: Doh Kwame , Li Lijun , Ecelbarger Carolyn
Published: June 2021

The renal PT is involved in the control of phosphorus (P) homeostasis via regulated reabsorption by the sodium phosphate cotransporter (NaPi‐2 or Slc34a1). Previous work suggests NaPi‐2 activity is increased by insulin and/or IGF; however, the mechanisms remain obscure. Our aim was to determine whether PT‐cell‐select knockout (KO) of both the insulin receptor (IR) and the IGF1 receptor (IGFR) would impact NaPi‐2 regulation and phosphorus homeostasis. Mice with both IR and IGF1R genes floxed were crossed for 2 generations with mice with Cre‐recombinase driven by the PT‐select γ‐glutamyl transferase promoter. No differences due to genotype were observed in food intake, body weight, or growth rate in either male (M) or female (F) mice. Similarly, 24‐hour urine volumes and P excretion did not differ. In contrast, plasma P levels were significantly lower in adult KO (6‐9 months) as compared to wild‐type (WT) mice (mg/dl): 12.9 ± 1.2 (WTM), 16.5 ± 2.3 (WTF), 10.2 ± 0.3 (KOM), 8.9 ± 1.3 (KOF), p = 0.014, 2‐way ANOVA for genotype, with no significant effect of sex. Western blotting of whole kidney homogenates revealed no differences due to genotype or sex for the major (84 kDa) band of NaPi‐2; however, a higher molecular weight band on the NaPi‐2 blots (~250 kDa) was markedly reduced in the KO, as well as in female mice. Band densities were (% WTM): 100 ± 7 (WTM), 32 ± 11 (WTF), 16 ± 9 (KOM), 1.6 ± 0.5 (KOF), p <0.001 for genotype and sex. We speculate that this band may represent NaPi‐2 bound to regulatory proteins. Our data support a role for insulin and/or IGF1 receptor‐mediated action in the regulation of renal P reabsorption. Additional studies will be needed to determine the role and cellular actions of each individual receptor.
Gastric iodide secretion; possible role in antimicrobial oxidant hypoiodous acid production
Authors: Akiba Yasutada , Kaji Izumi , Said Hyder , Narimatsu Kazuyuki , Kaunitz Jonathan
Published: June 2021

Ingested iodide is absorbed in the small intestine, followed by uptake into the thyroid follicles, whereas iodide in the systemic circulation is secreted via the salivary glands and the stomach in an entero‐gastric loop. Since the gastric mucosa generates luminal H2O2 via dual oxidase, and hypoiodous acid (HOI) is an antimicrobial oxidant with strong antifungal and antiviral activity, we hypothesized that gastric iodide secretion (GIS) might be related to the production of luminal HOI in the stomach.

We measured GIS in the perfused stomach in vivo using flow‐through iodide sensitive electrode. Sodium iodide (NaI) was given IV or intraduodenally (Id). Transmucosal iodide movement was measured in an Ussing chamber.

Immunostaining localized the Na+/I‐ symporter (NIS) to the basolateral membranes in fundic surface cells and pyloric gland cells. Mucosal NIS mRNA expression was highest in the fundus and antrum to levels similar to that in the thyroid. Bolus iv injection of NaI dose‐dependently increased luminal iodide output in the stomach, reduced by pretreatment with the NIS inhibitor ITB5. Id NaI gradually increased GIS, paralleled by an increase of HOI output into the gastric lumen. Antral > fundic iodide secretion rates were present, enhanced by the presence of luminal amiloride or serosal forskolin, suggesting that amiloride 'unmasks' electrogenic epithelial Na+ uptake via the basolateral NIS, and apical iodide secretion is related to cAMP‐mediated anion secretion.

These results suggest that GIS may affect host defenses via luminal HOI production, complementing antimicrobial gastric acid secretion.

Supported by VA Merit Review, NIH R01 DK54221
OCTN1‐Mediated Ergothioneine Uptake Protects Lung Epithelial Cells From Tobacco Smoke‐Induced Damage
Authors: Ehrhardt Carsten , Nickel Sabrina , Selo Mohammed Ali , Clerkin Caoimhe , Salomon Johanna , Talbot Brian , Walsh John
Published: June 2021

Smoking is a major contributing factor for chronic obstructive pulmonary disease (COPD). Tobacco smoke exposure causes oxidative stress which results in alveolar wall destruction, mucus hyper secretion, inflammation and defective tissue repair. Ergothioneine (ET), an amino acid transported by OCTN1, has been shown to exhibit antioxidant and cytoprotective capacities in vitro. Hence, the objective of this study is to understand the role of OCTN1 and the effect of ET on human alveolar cells when subjected to tobacco smoke‐induced oxidative stress.

Confocal microscopy and Western blot was used to determine the presence of OCTN1 in A549 cells and freshly isolated human alveolar epithelial type II (ATII) and type I‐like (ATI‐like) cells in primary culture. A549 cells were cultured in medium containing 1 mM of ET and its intracellular accumulation was measured by LC‐MS/MS over a 3‐week period. A549 cells were also was exposed to various concentrations of cigarette smoke extract (CSE). Prior to the experiment, cells were either cultured for 1 week in the presence or absence of 1 mM ET. An MTT assay was used to assess cell viability for the difference exposures to CSE.

Immunoblot and CLSM showed OCTN1 expression in A549, ATII and ATI‐like cells. The uptake of ET by A549 epithelial cells was saturated after approximately 7 days of culture. Western blot data supported the CLSM results and also indicated that OCTN1 levels were increased in the presence of ET. The MTT assay showed that cells pre‐treated with ET were less affected by the cytotoxic effects of CSE.

These preliminary data indicate that OCTN1 might play a role in the pathogenesis of tobacco smoke‐induced COPD by regulating ergothioneine transport.
Towards New Insights in Epithelial Ion Channels Regulation by the ER Luminal Chaperone ERp29
Authors: Bikard Yann , Suaud Laurence , Grumbach Yael , Litvak Jonathan , Rubenstein Ronald
Published: June 2021

ERp29 is an endoplasmic reticulum (ER) 29 kD thioredoxin‐homologous protein that displays chaperone‐like properties. We have previously demonstrated that ERp29 promotes CFTR biogenesis. As the biogenesis of CFTR and ENaC share similar features, we here tested the hypothesis that ERp29 would also regulate ENaC biogenesis and functional expression, as well as further probed ERp29 mechanism. Overexpression of wt ERp29 increased the abundance of the active form of γ‐ENaC, as well as ENaC functional expression. In contrast, ERp29 overexpression of a mutant ERp29 lacking its single Cysteine (C157S ERp29) decreased ENaC functional expression. These observations were not associated with altered expression of β‐ENaC at the apical surface, suggesting that ERp29 may modulate ENaC open probability at the apical surface. ERp29 overexpression promoted the interaction of both ENaC and CFTR with the coat complex II ER exit machinery, whereas C157S ERp29 overexpression decreased this interaction. These data suggest a model where ERp29 may promote ENaC cleavage by directing ENaC to the Golgi. Finally, we also tested the hypothesis that ERp29 subcellular localization is a crucial determinant for its action. We therefore designed a mutant ERp29 containing a KDEL retention motif (ERp29 KDEL) that should be better retained in the ER, and two mutants that would better escape the ER, by mutation and deletion (ERp29 KDEV and ΔKEEL, respectively). Together our findings suggest a key role for ERp29 in the biogenesis of ENaC and CFTR as well as emphasize ERp29 mechanism.
LMTK2, a Novel Target in Prostate Cancer Therapy
Authors: Shah Kalpit , Bradbury Neil
Published: June 2021

Androgens and their receptors play essential roles in the development and progression of prostate cancer, which claims around 28,000 lives every year. Kinase activity is shown to be responsible for activating androgen receptor in castrate resistant prostate cancer, the stage most associated with mortality. Several Genome Wide Analysis Studies (GWAS) has shown that a decrease in mRNA expression of Lemur Tyrosine Kinase 2 (LMTK2) is associated with development of prostate cancer. In agreement with GWAS studies, we show that LMTK2 protein expression is significantly lower in prostate adenocarcinomas in comparison to normal prostate tissue. Furthermore, our study shows that manipulating LMTK2 levels in HEK293 cells and the prostate adenocarcinoma cell line, LNCaP, markedly affects the expression of androgen dependent genes. In addition, loss of LMTK2, as seen in prostate cancer, leads to an increase in cell proliferation and enhanced tumor‐forming capacity of LNCaP cells. We found that at least one reason for increased androgen response following the loss of LMTK2 is a marked increased in the half‐life of androgen receptors. In addition, in vivo Proximity Ligation Assays and Co‐immunoprecipiation assay shows that the androgen receptor and LMTK2 are binding partners in LNCaP cells. Using a combination of biochemical, molecular and cellular approaches, our work defines a novel cellular pathway for androgen receptor signaling in castrate resistant prostate cancer; pharmacologic manipulation of such regulation will provide innovative and more effective therapies for the treatment of patients with castrate resistant prostate cancer.
Mimicking the Diabetic State in the Non‐Diabetic β‐cell to Elucidate Critical Pathways in β‐cell Dysfunction
Authors: Brar Harpreet , Neuman Joshua , Brill Allison , Cadena Mark , Kimple Michelle
Published: June 2021

EP3, a receptor for prostaglandin E2 (PGE2), plays a key role in the β‐cell dysfunction of diabetes. We demonstrated EP3 expression and PGE2 production are increased in islets isolated from diabetic mice and humans, and targeting this pathway in vitro restores proper β‐cell function. Yet, the mechanisms involved in the pathogenesis of β‐cell dysfunction upstream and downstream of EP3 remain elusive. The INS‐1 832/3 cell line has high EP3 expression, just as in diabetic islets. Interestingly, even though EP3 expression is high, PGE2 production is not; thus, an EP3 antagonist has no effect on β‐cell function. We hypothesized one or more of the multitude of insults β‐cells encounter in the diabetic state are necessary to stimulate PGE2 production and resulting β‐cell dysfunction. We mimicked diet by enriching INS‐1 cells with arachidonic acid (AA), an ω‐6 polyunsaturated fatty acid and PGE2 precursor, and inflammation by IL‐1β treatment. This combination significantly increased PGE2 production, blunting insulin secretion. Further, β‐cell function could be restored by an EP3 antagonist. In islets isolated from non‐diabetic mice and humans, AA and IL‐1β had a strong effect on PGE2 synthetic enzyme expression, but EP3 itself was relatively unchanged, and EP3‐mediated β‐cell dysfunction minimal. We hypothesized that high glucose or free fatty acids are responsible for the dysfunctional up‐regulation of β‐cell EP3 in vivo. Finally, we demonstrate the relevance of targeting this pathway in vivo, as mice lacking the EP3 effector, Gαz, specifically in the β‐cell are protected from the β‐cell dysfunction induced by inflammation.
Knockdown of the 110kDa Subunit of OGT Dysregulates Cardiac Remodeling in Response to Pressure Overload
Authors: Medford Heidi , Miller Lindsey , Dieter Brad , Johnson Emily , Marsh Susan
Published: June 2021

Hypertension causes pathological cardiac hypertrophy and heart failure, but the mechanisms underlying this process are poorly understood. The attachment of O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) to serine/threonine amino acids by O‐GlcNAc transferase (OGT) is increased in cardiovascular pathology; OGT is singly encoded with three splice variants (110, 103, and 78 kDa). Hypertension incites pathological cardiac remodeling via aberrant calcium‐signaling and induction of the pro‐hypertrophic fetal cardiac gene program (FCGP). Activity of cardiac specific calcium‐bound calmodulin kinase II (CaMKIIδ) and histone deacetylase 4 (HDAC4) is altered by phosphorylation and O‐GlcNAcylation, and interactions between these proteins regulate cardiac hypertrophy. We used a cardiomyocyte‐specific OGT knockdown (OGTKD) mouse lacking 110kDa OGT, along with transaortic constriction (TAC), to investigate the effects of OGTKD and pressure overload (PO) on HDAC4 phosphorylation by CaMKIIδ. OGTKD mice exhibited extremely low postoperative survival, and HDAC4 and CaMKIIδ reacted oppositely to OGTKD by increasing and decreasing, respectively. OGTKD mice exhibited pathological hypertrophy independent of surgery, were cachexic, and OGTKD‐TAC mice weighed less than OGTKD‐sham mice (p=0.05). Calcium signaling was altered in OGTKD‐TAC hearts, and the FCGP was induced in OGTKD and TAC (p=0.05). These data suggest that 110kDa OGT is essential to prevent pathological cardiac remodeling and also for the cardiac stress response to PO.