bluuebunny/crossref_metadata_2025 huggingface dataset:
Cardiomyocytes Release IFN‐β in Response to Hypoxia: A Previously Unrecognized Cardiomyocyte to Fibroblast Signaling Pathway
Authors: Bangeranye Catherine , Wang Ping , Chatterjeee Prodyot K , Predmore Jamie , McNeil Franklin , Metz Christine , Miller Edmund , Day Sharlene , Powell Saul R
Published: November 2023

We have observed a persistent increase in the expression and activity of the immunoproteasome (iProt) in post‐ischemic heart (post‐MI). The objective of this study was to identify the pathway and mediator(s) responsible for iProt upregulation. Using RT‐PCR, ifng, tnfa, and ifnb1 mRNAs were assayed. There were no changes in either ifng or tnfa but ifnb1 was elevated ~ 17‐fold (P<0.01) in the heart 4 days post‐MI. Exposure of a mixture of rat neonatal cardiomyocytes/fibroblasts (CM/CFs) to IFN‐β from 1U/ml up to100u/ml for 24h resulted in a variable response with an 85% (P<0.05) increase in iProt at 100 u/ml as measured using the ProCISE assay. Since the CM/CF preparation typically contains about 20% cardiac fibroblasts, (confirmed by vimentin staining), isolated CFs were exposed to IFN‐β for 24hs which resulted in a robust concentration‐dependent increase in iProt that was almost 3‐fold (P<0.01) at 100 u/ml. An enriched CM (eCM) preparation in which CFs were limited to ≤5% was prepared and these had the lowest iProt response to 10u/ml IFN‐β (eCM, +27% ± 5%; CF, +104 ± 15%; CM/CF, +52 ± 18%). To determine a potential source of IFN‐β in the post‐MI heart, CFs, CM/CFs and eCMs were exposed to 1% O2 for 22h to simulate ischemia. This resulted in +2 ±1%, +31 ± 8% (P<0.05), −16 ± 5% changes in iProt, respectively, suggesting that the CMs were the source of the IFN‐β. RT‐PCR analysis revealed that ifnb1 transcription in the control and in hypoxic eCMs was increased 2.75‐fold (P<0.01) and 3.3‐fold (P<0.01) respectively when compared to hypoxic CM/CFs. To probe a potential function of IFN‐β, CM/CFs were pretreated with 100 u/ml IFN‐β 24h prior to exposure to hypoxia (1% O2) for 22h. Cardiomyocyte injury was determined using protein array to measure release of cardiac injury markers, cTNI, cTNT and FABP3, into the media. All of the markers were significantly increased at the end of the hypoxic challenge in cells that were not pre‐treated with IFN‐β as compared to controls. In the absence of hypoxia, IFN‐β had no effect on the levels of these markers. However, when IFN‐β pre‐treated CM/CFs were exposed to hypoxia there was a significant reduction (P<0.05) in all three markers as compared to hypoxic CM/CFs alone. These results suggest that in response to hypoxia CMs release IFN‐β which then stimulates CF to upregulate an iProt that serves to protect CMs. Overall, these studies suggest the existence of a previously unrecognized signaling pathway in the heart involving CM IFN‐β signaling to the cardiac fibroblast.

Support or Funding Information

This study was supported in part by an AHA ‐ Founders Affiliate Grant in Aid and a grant from Onyx Pharmaceuticals, Inc., an Amgen subsidiary.
The Effect of PAK1 Knockout on the Efficacy of Metoclopramide in a Murine Model of Postoperative Ileus
Authors: Peery Travis , Rivas Orlyn , Bhattarai Deepa , Uray Karen
Published: November 2023

Postoperative ileus (POI) is characterized as a decrease in gastrointestinal motility following a surgical procedure. POI is one of the most common postoperative complications in the United States and presents a considerable cost in both financial terms and patient morbidity. Metoclopramide is a prokinetic agent which antagonizes dopamine receptors in the enteric nervous system (ENS) and is currently employed to treat POI. Reviews of the literature have shown metoclopramide to be ineffective in the treatment of POI. We have shown that intestinal smooth muscle is dysfunctional in animal models of POI, which may account for the lack of response to prokinetics such as metoclopramide. We have also shown that PAK negatively regulation myosin light chain phosphorylation in a POI model and PAK inhibition can improve smooth muscle contractility. Based on these data, we hypothesize that the response to metoclopramide will be improved in PAK1 knockout mice compared to wildtype mice after gut manipulation to induce POI. PAK1 homozygous knockout strains and C57BL/6 wildtype mice of both sexes were used. Mice of both genotypes were divided into four groups: 1) sham surgery and vehicle; 2) POI and vehicle; 3) sham surgery and 0.1mg/kg metoclopramide; 4) POI and 0.1mg/kg metoclopramide. Sections of whole ileum were harvested 24 hours post‐surgery and suspended in 37°C buffered Krebs solution while muscle contractility was measured. Amplitude, frequency, tone, and integral were calculated and a carbachol dose response curve was generated in the organ bath. As expected, a significant decrease in baseline integral and contraction amplitude, and agonist induced contractility was observed in the POI groups compared to the sham surgery groups. Contractility and responsiveness to carbachol improved in PAK1 knockout; however, response to metoclopramide was not significantly different between PAK1 knockout and wildtype mice. We conclude that PAK1 inhibition partially attenuates the development of POI but does not improve the response to metoclopramide.
Interleukin‐19 Can Simultaneously Reduce Atherosclerosis and Increase Perfusion in Hind Limb Ligated LDLr−/− Mice
Authors: Ray Mitali , Gabunia Khatuna , Kelemen Sheri , Kako Farah , Richards James , Autieri Michael V.
Published: November 2023

Peripheral arterial disease is often a co‐morbidity with hypercholesterolemia and atherosclerosis. We have previously characterized Interleukin‐19 (IL‐19) as an anti‐inflammatory, Th2 cytokine. Past studies in our lab have determined that IL‐19 can decrease atherosclerosis in LDLr−/− mice and increase angiogenesis in the hind limb ischemia (HLI) model, suggesting that IL‐19 has both anti‐atherosclerotic and pro‐angiogenic effects.

Objective

The purpose of this study was to determine whether IL‐19 can induce angiogenesis within the background of hyperlipidemia and atherosclerosis and potentially reduce atherosclerosis and induce angiogenesis simultaneously.

Methods and Results

We utilized three different, yet complementary platforms to test this hypothesis. We fed LDLr−/− mice high fat diet (HFD) for 12 weeks, then performed HLI surgery and continued HFD and treatment with rIL‐19 (10ng/g/day) or PBS intraperitoneal (IP) injections for an additional 4 weeks. Doppler imaging revealed increased perfusion in mice treated with IL‐19 when compared to PBS controls. Additional en face evaluation of the aortic arches of these mice demonstrated a trend toward decreased plaque burden in the IL‐19 treated mice, though it was not statistically significant. These data suggest that IL‐19 is capable of promoting angiogenesis, even in a hyperlipidemic environment. We used this same experimental model, but instead treated LDLr−/− mice with IL‐19 (10ng/g/day) or PBS by daily IP injection throughout the initial 12 weeks of HFD, in addition to four weeks following HLI surgery. Doppler imaging demonstrated increased perfusion in IL‐19 treated mice when compared to PBS controls, and en face evaluation showed decreased plaque burden in the IL‐19 treated mice, supporting our hypothesis that IL‐19 is capable of simultaneously decreasing atherosclerosis and promoting angiogenesis in the background of hyperlipidemia. Finally, we hypothesized that lack of IL‐19 would both reduce perfusion and exacerbate atherosclerosis. We generated a double knockout mouse model (dKO) by crossing the LDLr−/− with an IL‐19−/− mouse. We placed dKO and LDLr−/− mice on HFD for 12 weeks, completed HLI and performed Doppler imaging for 4 weeks. Preliminary results suggest dKO mice demonstrate both decreased perfusion and increased plaque burden.

Conclusions

The current dogma referred to as the Janus phenomenon suggests that pro‐angiogenic cytokines are also pro‐atherosclerotic. Collectively, these data suggest IL‐19 is the only cytokine to date that challenges this theory, demonstrating both pro‐angiogenic and anti‐atherosclerotic effects.

Support or Funding Information

This work was supported by grants HL115575 and HL117724 from the National Heart Lung, and Blood Institute of the National Institutes of Health, and Grant 13GRNT1685003 from the American Heart Association to MVA.
Stimulation of Mitochondrial Fragmentation and Mitophagy by TNFα in Microvascular Endothelial Cells
Authors: Kalogeris Theodore , Korthuis Ronald
Published: November 2023

The inflammatory cytokine, TNFα, decreases mitochondrial mass in cultured microvascular endothelial cells. We previously showed that this effect might be explained by an inhibitory effect of TNFα on eNOS‐dependent expression of PGC‐1α. However, other investigators have reported that TNFα stimulates mitophagy, and a possible mitophagic effect of TNFα was not ruled out in our own studies. In order to more fully understand how TNFα affects endothelial mitochondrial function, we have begun to examine the effect of this cytokine on measures of mitochondrial network integrity and mitophagy in human dermal microvascular endothelial cells (HMEC‐1). Since fragmentation of mitochondrial networks via mitochondrial fission has been shown to be necessary for subsequent mitophagy, we examined the time‐dependent effect of TNFα on mitochondrial network integrity using fluorescence microscopy of cells loaded with the mitochondrial‐specific dye, mitotracker green (MTG). Compared with control cells, TNF treatment increased mitochondrial fragmentation by 12% and 20% at 24 and 48 h, respectively. This was associated with a similar time‐dependent increase in mitochondrial PINK‐1, and recruitment of both drp‐1 and parkin, suggesting increases in both mitochondrial fission and mitophagy progression. We also observed an increase in the cellular ratio of LC3II/LC3I, a marker of autophagasome formation. Finally, TNFα‐induced decrease in mitochondrial mass was significantly attenuated in cells co‐incubated with mitophagy inhibitors, 3‐methyladenine or m‐divi. Taken together with our previous results, these findings suggest that TNFα may exert negative effects on endothelial mitochondrial capacity by simultaneously blocking organellar biogenesis and stimulating mitophagic destruction.

Support or Funding Information

Supported by AA014945 & HL095486.
Inhibition of Epidermal Growth Factor Receptor Tyrosine Kinase and Endoplasmic Reticulum Stress Protects Myocardial from Ischemia ‐ Reperfusion Injury in Type 2 Diabetic Mice
Authors: Mali Vishal R. , Ali Maha , Haddox Samuel , Matrougui Khalid , Belmadani Souad
Published: November 2023

Background

We previously reported that epidermal growth factor receptor tyrosine kinase (EGFRtk) activity and endoplasmic reticulum (ER) stress are enhanced in type 2 diabetic mice and cause vascular dysfunction. In the present study, we determined the in vivo contribution of EGFRtk and ER stress in acute myocardial infarction in type 2 diabetic (db−/db−) mice.

Methods and Results

We treated type 2 diabetic mice (db−/db−) mice with EGFRtk inhibitor (AG1478, 10 mg/kg/day) or ER stress inhibitor (tauroursodeoxycholic acid, TUDCA, 150 mg/kg per day) for two weeks. After the treatment, mice were subjected to myocardial ischemia for 40 minutes and followed by reperfusion for 24 hours. The treatment with AG1478 and TUDCA reduced infarct size illustrating a cardioprotection effect of inhibiting EGFRtk signaling or ER stress against ischemia‐reperfusion injury in type 2 diabetic mice. To determine the signaling mechanism responsible for the observed cardioprotection, we determined the level of ER stress proteins Chop and BIP expression. The AG1478 or TUDCA significantly decreased the expression of BIP and Chop in diabetic mice compared to untreated db−/db− mice. Also, AG1478 and TUDCA reduced cardiomyocyte apoptosis, p38, and ERK1/2 MAP‐Kinases, and increased the activity of the pro‐survival signaling cascade Akt. Immunohistochemistry staining showed that EGFRtk inhibition and TUDCA treatment lowered macrophages and neutrophil infiltration after I/R injury in diabetic hearts compared to untreated mice.

Conclusion

Overall, we showed for the first time that inhibition of EGFRtk and ER stress protect diabetic mice against ischemia‐reperfusion injury by activating the reperfusion injury survival pathway and inhibiting apoptotic signaling and inflammation. These findings suggest the importance of targeting ER stress and EGFRtk as a potential therapy for heart complication in type 2 diabetes.

Support or Funding Information

This study was supported by EVMS Research Enhance Grant
Sympathetic Modulation Decreases in Rats with Renovascular Hypertension Treated with Chronic Administration of Catalase Inhibitor
Authors: Lauar Mariana Ruiz , De Luca Laurival Antonio , De Paula Patricia Maria , Colombari Débora S A , Colombari Eduardo , Andrade Carina A F , Menani José Vanderlei
Published: November 2023

Previous studies have shown that the increase of endogenous hydrogen peroxide (H2O2) with the administration of the catalase inhibitor 3‐amino‐1,2,4‐triazol (ATZ) intravenously acutely reduced mean arterial pressure (MAP) and the pressor response induced by angiotensin II intracerebroventricularly in renal hypertensive rats. Recent data have also shown that chronic subcutaneous (sc) administration of ATZ reduced MAP in 2‐kidneys, 1‐clip (2K1C) hypertensive rats. In the present study, we analyzed the autonomic modulation of cardiovascular function in 2K1C hypertensive rats treated with chronic sc injection of ATZ. Male Holtzman rats (initial weight 150–180 g, n=7–11/group) received a silver clip around the left renal artery to generate 2K1C hypertension. After 6 weeks of the surgery, rats were treated with ATZ or saline for 7 days before the recording of arterial pressure and the analysis of systolic blood pressure (SBP) and pulse interval (PI) variability using power spectral analysis. Baseline MAP was increased in 2K1C rats compared to sham (207 ± 9, vs. 100 ± 2 mmHg, respectively). ATZ (600 mg/kg/day) in 2K1C rats reduced MAP (162 ± 10 vs. saline 209 ± 5 mmHg), the low frequency (LF) of SBP (3.3 ± 0.5, vs. saline 7.6±1.5 mmHg2) and LF of PI (14.6±3, vs. saline: 31±3 normalized units). ATZ also increased high frequency (HF) of PI (85±3, vs. saline: 68±3 normalized units) and improved the sympathovagal balance calculated for LF/HF ratio of PI variability (0.2±0.05, vs. saline: 0.52±0.09). The results suggest that the treatment with ATZ reduces MAP in 2K1C hypertensive rats by reducing the modulation of sympathetic activity.

Support or Funding Information

FAPESP, CNPq and PROPE/FUNDUNESP.
Sympathetic Vasomotor Activation To Different Targets In A Model Of Hypertension Induced By Nitric Oxide Synthase Blockade
Authors: Zambrano Lysien Ivania , Pontes Roberto Braz , Garcia Michelle Louvaes , Nishi Erika Emy , Campos Ruy R. , Bergamaschi Cassia T.
Published: November 2023

High blood pressure is a global public health problem. Several factors are involved in the generation and maintenance of hypertension, such as increased sympathetic nerve activity (SNA), although controversies have been reported. Our aims were to evaluate sympathetic activation to two targets, the kidneys (rSNA) and other visceral bed, the splanchnic (sSNA), in animals with hypertension induced by nitric oxide synthase blockade (L‐NAME). Wistar rats were treated with L‐NAME (20mg/kg/day, once a day, for 8 days). L‐NAME‐treated group presented increased blood pressure (154±13 mmHg, N=16), compared with control group (102±2 mmHg, N= 10). Both rSNA (156±37.4 spikes/s, N=16) and sSNA (145±42.7 spike/s, N=16) were higher in L‐NAME‐treated group, compared with control group (111±20.6, N=10 and 63±20.6 spikes/s, N=10, respectively). Taken altogether, our data show that the hypertension induced by nitric oxide synthase blockade is characterized by sympathoexcitation to more than one bed and that can be involved in the generation and/or maintenance of high blood pressure.

Support or Funding Information

CAPES, CNPq, Universidad Nacional de Honduras (UNAH).
Positive and negative inotropic effects of left ventricular mechano‐energetics in hypothermic and hyperthermic rat hearts
Authors: Obata Koji , Morita Hironobu , Takaki Miyako
Published: November 2023

We investigated the effects of cardiac hypothermia or hyperthermia, including capsaicin (a TRPV1 agonist) on LV myocardial mechano‐energetics using the excised, cross‐circulated rat heart model. We analyzed the left ventricular (LV) end‐systolic pressure‐volume relation (ESPVR) and the linear relation between the myocardial oxygen consumption per beat (VO2) and systolic pressure‐volume area (PVA; a total mechanical energy per beat) in isovolumically contracting rat hearts during hypothermia (32°C), normothermia (37°C), and hyperthermia (42°C) under 300‐beats per minute (bpm) pacing. LV ESPVR shifted upward in cardiac hypothermia compared with that in cardiac normothermia, whereas it shifted downward in cardiac hyperthermic and capsaicin‐treated hearts. The hypothemic, normothermic and hyperthermic VO2‐PVA relations were superimposable, however, each data point of VO2‐PVA at each LV volume at 32°C shifted right‐upward from that at 42°C on the VO2‐PVA relation line. In capsaicin treated‐hearts, the slope of VO2‐PVA relation was not also significantly different. However, the VO2 intercept was decreased in capsaicin treated‐hearts differently from that in hyperthermic‐hearts. Decreasing cardiac temperature increased the excitation‐contraction (E‐C) coupling VO2 for Ca2+ handling, and decreased the basal metabolism and oxygen cost of LV contractility. The results indicate that cardiac temperature plays an important role on control ling cardiac mechano‐energetics. In addition, capsaicin decreased the LV contractility like hyperthermia due to the down‐regulation of the calcium handling in E‐C coupling. We conclude that the thermic intervention modulated cardiac inotropism by changing Ca2+ handling and cross‐bridge cycling in rat hearts.
Kv1 Channels are Critical Regulators of Coronary Blood Flow in Swine
Authors: Goodwill Adam G , Noblet Jillian N , Sassoon Daniel J , Fu Lijuan , Schepers Luke , Sturek Joshua , Tune Johnathan D , Dick Gregory M
Published: November 2023

Despite numerous investigations, the mechanisms regulating the relationship between coronary vascular flow and myocardial oxygen consumption remain poorly understood. Recent attention has been given to voltage gated potassium channels (Kv), as data suggest that they mediate redox‐sensitive coronary vasodilation. Through this mechanism, myocardial production of H2O2 increases coronary blood flow, providing a direct mechanistic linkage. The purpose of this study was to establish the role of Kv1 channels in the porcine coronary circulation through blockade of Kv1 channels with the selective channel blocker, correolide. Kv1 transcripts were present in porcine coronary arteries, with KCNA5 (Kv1.5) being the most abundant (p<0.001). Immunohistochemical analyses demonstrated expression of Kv1.5 protein in the vascular smooth muscle layer of both porcine and human coronary arteries. Whole cell electrophysiology demonstrated significant correolide‐sensitive current in coronary smooth muscle. In vivo studies involved direct intracoronary infusion of correolide into the pressure‐clamped left anterior descending artery of healthy swine while simultaneously measuring coronary blood flow. Correolide had no effect on heart rate or systemic pressure development at any tested dose (coronary plasma concentration 0.3–3 μM). Correolide reduced coronary blood flow in a dose‐dependent manner relative to vehicle control (ΔFlow, Vehicle +0.19 ± 0.08 vs. Correolide −0.12 ± 0.03 mL/min/g; p< 0.01). To determine the role of Kv1 channels in coronary metabolic vasodilation, swine received systemic infusion of the sympathomimetic compound dobutamine (0.3–10 μg/kg/min) in the presence or absence of correolide (3 μM). Correolide significantly reduced the coronary flow response to increasing myocardial demand across the range of dobutamine concentrations (~20%; p<0.001). Coronary artery occlusions (15 s) elicited reactive hyperemia and correolide reduced the flow volume repayment by approximately 50% (p=0.02). Taken together, these data support a role for Kv1 channels in modulating baseline coronary vascular tone, in regulating responses to ischemia, and in balancing myocardial oxygen delivery with metabolism.

Support or Funding Information

This study was supported by U01HL118738 (D. Beard and G. Kassab, PI) and R01HL11760 (J. Tune and K. Mather, PI). Dr. Goodwill was supported by American Heart Association 13POST1681001813 (A. Goodwill, PI). Ms. Noblet and Mr. Sassoon were supported by TL1 TR000162 (A. Shekhar, PI) from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award.
Attenuated Response of hCD39 Overexpressing Transgenic Mice to High‐fat Diet‐induced Obesity and Glucose Intolerance
Authors: Brandes Anna U , Zhang Yue , Robson Simon C , Kishore Bellamkonda K
Published: November 2023

CD39 (ENTPD1) is an ecto‐nucleotidase that scavenges extracellular nucleotides, and thus terminates P2 receptor signaling. Previously we reported that genetic deletion ATP/UTP‐activated P2Y2 receptor confers significant protection against high‐fat diet (HFD)‐induced obesity and insulin resistance (IR). In order to understand the role of extracellular nucleotides in diet‐induced obesity and IR, we subjected wild type C57/Bl6 (WT) and syngeneic transgenic (TG) mice overexpressing human CD39 (hCD39) to either regular diet (10% of calories as fat; n = 5/genotype) or HFD (60% calories as fat; n = 7/genotype) feeding for 18 weeks with free access to drinking water and food. Body weights (BW), food and water consumption and urine output were measured every two weeks. Glucose tolerance test (GTT) was performed during 15th week. At the end of the experimental period all mice were humanely euthanized. Both genotypes showed significant increases in BW over time, and reached comparable mean BW toward the end of the study. However, until week 14, the slope of gain in BW was higher in WT vs. TG mice. No significant differences were found in daily food intake between the two groups. And there were no signs of steatorrhea, indicating no malabsorption of fat. Significant increases in the amount of brown and white adipose tissues were recorded in both groups, although the increases were not comparable. Liver and kidney weights, when adjusted to BW, are modestly, but significantly higher in HFD‐fed TG mice vs. WT mice. Glucose tolerance was significantly better in HFD‐fed TG vs. WT mice. Our results demonstrate that scavenging of extracellular nucleotides has the potential to delay the onset of diet‐induced obesity and glucose intolerance, and open potential avenues for the development of new therapies for obesity and insulin resistance.

Support or Funding Information

Dept. of Veterans Affairs (BKK) & PRIA Foundation (SCR)
Contribution of prolyl hydroxyl binding proteins (PHD) to MYD88/NFkB pathways in Nitric Oxide deficiency‐induced hypertensive renal injury
Authors: Choi Myung , Oyekan Adebayo
Published: November 2023

Oxidative stress and associated inflammation, features of hypertension and kidney disease, involve many mediators that include Nrf2, a transcription factor, the primary cellular defense against oxidative stress, MYD88, an adaptor protein implicated in NFkB‐mediated inflammation. PHD, oxygen sensor in hypoxia is intricately linked to NO. This study evaluated the role played by PHD and its association with Nrf2, MYD88 and NFKB in hypertension‐induced renal injury.

Adult rats were placed on LNNA, inhibitor of NO synthase and/or DMOG, an inhibitor of PHD. Blood pressure (BP) was determined on Days 1, 4, 7, and 14 and 24‐hour urine collected for protein excretion. Kidneys were collected to determine expression of PHD1 and PHD2, Nrf2, p65, and MYD88 in nuclear fractions. KIM‐1 expression, index of tubular damage, was assessed in whole kidneys. Plasma TNFa and IL‐1b levels, markers of inflammation, were measured by ELISA. Renal generation of oxidant species was evaluated as xanthine (3,5,10 mg/kg)‐induced generation of H2O2. LNNA, inhibitor of NO synthase increased BP (35%, P<0.05), kidney weight (15%, P<0.05), protein excretion (3.5‐fold, P<0.05) and KIM‐1 expression (3.8‐fold, P<0.05). DMOG blunted the increases. LNNA also reduced PHD2 (20%) but not PHD1 expression or Nrf2 expression. There was increased nuclear expression of p65 (34%, P<0.05) and MYD88 (32%, P<0.05) and increased TNFa and IL‐1b (P<0.05) in LNNA‐treated rats. DMOG blunted these effects. Renal production of H2O2 by xanthine was greater (P<0.05) in LNNA‐treated rats. These data suggest a role for PHD2 in hypertensive renal injury and inflammation induced by NO deficiency. The accompanying changes in MYD88/p65 expression and cytokine production confirm the ongoing inflammatory process. However, despite the increase in production of reactive oxygen species, Nrf2 does not appear to play a part in NO deficiency‐induced cardiovascular effect.

Support or Funding Information

National Institutes of Health grant 5G12 MD007605
Novel Differences in Renal Gene Expression in a Diet Induced Obesity Model of Diabetic Nephropathy
Authors: Kuhns Victoria L Halperin , Pluznick Jennifer L
Published: November 2023

Diabetic nephropathy (DN) is a serious and common complication of diabetes mellitus. Our objective was to identify novel genes differentially regulated in the early stages of DN using an unbiased approach. To this end, male C57bl/6 mice were fed high fat diet (HFD, 60% kcal fat, n=5) or matched control diet (CD, 10% kcal fat, n=5) for 9 weeks starting at 6 weeks of age to induce metabolic syndrome and DN. Renal cortex RNA was isolated from all samples, and RNA Seq was performed on 4 samples from each diet. We identified 1134 differentially expressed genes; of the 22 genes with the greatest fold increase or decrease, only 3 (Sorcs1, Lipg, Slc7a12) have been previously been studied in a diabetic context. We then utilized a Taqman real‐time (RT) PCR array to confirm our initial findings. The array included the 9 most upregulated and 11 most downregulated genes from the RNA Seq data, with an additional 11 genes altered by RNA Seq and of interest to our group (primarily sensory receptors and G proteins). These arrays (31 genes + 18S control) were used to screen additional tissue samples (renal medulla, liver, heart, n=3 CD, n=3 HFD), and an expanded cohort of renal cortex samples (n=5 CD, n=5 HFD). All 9 of the genes significantly upregulated with HFD by RNA Seq were also upregulated in the cortex samples by RT‐PCR (Atp12a, Ccl28, Ctxn3, Cyp2b10, Lhx2, Popdc3, Ptpn5, Sorcs1, Synpr; p<0.05). However, of the remaining 22 genes (downregulated + other genes of interest) only 2 were confirmed in cortex by RT‐PCR (Gsta2, Slc7a12; p<0.05). There were 6 genes for which changes in the renal medulla mirrored changes seen in the renal cortex (upregulation of Ctxn3; downregulation of Bhmt, Cesg1, Gm10639, Gsta2, Slc7a12; p<0.05). As compared to the changes in expression found in renal cortex by HFD diet vs. CD, only 2 genes changed in the same direction in liver (downregulation of Lipg, Slc7a12; p<0.05) with 1 additional gene changing in the opposite direction (upregulation of Scd1; p<0.05). In heart, none of the genes changed in the same direction, while 6 genes changed in opposite directions (downregulation of Cyp2b10, Ccl28, Synpr; upregulation of Gm10639, Lipg, Scnn1g; p<0.05). These data demonstrate that our RNA Seq data regarding upregulation were more reproducible than those regarding downregulation, and that changes are largely kidney specific. Preliminary studies using samples from CD and HFD female mice (on diet for 12 weeks from weaning) indicate that the changes seen in our initial cohort are likely male‐specific, in agreement with the fact that female mice fail to develop metabolic syndrome on this diet. This implies that renal changes are downstream of metabolic changes, and not non‐specific changes due to an alteration in diet. Thus, we have identified novel renal genes differentially regulated in DN, and these genes should be studied to elucidate their role in DN pathogenesis.
Proximal Tubular Cell Angiotensinogen Upregulation by High Glucose is Mediated by Reactive Oxygen Species and not Dependent on AT1 Receptor Activation
Authors: Cypress Michael , Satou Ryousuke , Hopfer Ulrich , Navar Luis Gabriel
Published: November 2023

Enhanced proximal tubular angiotensinogen (AGT) levels have been shown in diabetes mellitus, which can be a key factor in intrarenal RAS activation. AGT is upregulated in proximal tubular cells (PTC) by high glucose (HG), but the detailed mechanisms are unclear. Sodium‐glucose cotransporter 2 (SGLT2) is expressed in PTC and is responsible for glucose reabsorption and consequent ROS generation. Therefore, changes in SGLT2 levels may alter the regulation of AGT in PTC under diabetic conditions. The goal of this study was to determine the effects of HG‐induced ROS and angiotensin II type 1 receptor (AT1R) activation, which also generates ROS, on AGT augmentation by HG in PTC. Immortalized mouse PTC derived from the early proximal tubular segment were treated with 5 mM (normal), 15 mM, or 25 mM D‐glucose for up to 24 hours. D‐Mannitol was used as an osmotic control. PTC were treated with sodium pyruvate (1 mM) to determine if enhanced glycolysis stimulates AGT. Moreover, 2.5 mM tempol, an antioxidant, was used to evaluate if HG‐induced AGT augmentation is ROS‐dependent. To determine the role of AT1R activation in AGT upregulation by HG, 1 mM olmesartan, an AT1R blocker, was used. Furthermore, PTC were treated with 10−10–10−7 M angiotensin II (Ang II) and/or HG. In addition, the effect of HG and Ang II on SGLT2 expression was investigated. AGT and SGLT2 protein levels were quantified by Western blot analysis, and AGT mRNA was measured by qRT‐PCR. AGT protein levels were increased by 15 mM (4.4±0.2‐fold compared to control) and 25 mM (4.6±0.2‐fold) glucose. AGT mRNA augmentation was also observed (31.1±3.5‐fold) by HG. On the other hand, AGT levels were not increased by mannitol, indicating that elevated osmolality does not change AGT levels. Pyruvate stimulated AGT expression (10.7±1.0‐fold) under normal glucose conditions, supporting a role for glycolysis in AGT augmentation. Pretreatment with tempol attenuated AGT augmentation in HG‐treated cells (77% suppression compared to HG without tempol), suggesting that ROS mediates HG‐induced AGT upregulation. AT1R blockade and Ang II did not alter AGT responses to HG. Moreover, SGLT2 protein levels were unchanged by HG or Ang II. These findings indicate that AGT upregulation by HG occurs independently of AT1R activation, but is mediated by glycolysis and ROS in PTC. In addition, SGLT2 upregulation is not required for HG‐induced AGT augmentation.

Support or Funding Information

This study was supported by the National Institute of General Medical Sciences IDeA Program (COBRE, P30GM103337) and the American Heart Association Predoctoral Fellowship (15PRE25090156).
Altered electrolyte homeostasis in mice with collecting‐duct‐principal‐cell select knockout of the mammalian‐target‐of‐rapamycin (mTOR)
Authors: Ecelbarger Carolyn M. , Byrd Marcus , Tiwari Swasti , Li Lijun
Published: November 2023

The mammalian‐target‐of‐rapamycin (mTOR) is a central sensor of energy status in many cell types. One of its primary regulators is insulin. In cell culture, mTOR has been demonstrated to play a role in insulin activation of the epithelial sodium channel (ENaC). To assess the role of mTOR on electrolyte homeostasis by the collecting duct (CD), we generated mice with CD‐principal‐cell select mTOR knockout (KO) by Cre‐lox recombination using the aquaporin‐2 promoter to drive Cre‐recombinase. No differences in body or kidney weights between KO and wild‐type (WT) littermates were observed. Adaptation to a low‐sodium (Na+) diet was assessed in young, male mice. As compared to WT, KO mice (n = 8/genotype) fed the low Na+ diet for one‐week had significantly lower plasma potassium (K+) levels (mM): 4.97 ± 0.24 (wild‐type, WT) versus 3.89 ± 0.14 (KO), p < 0.005, with no significant differences in plasma chloride (Cl−) or Na+. Likewise, no significant differences were found between genotypes for urine excretion of Na+, K+, or Cl− on days 1–3 of the low‐Na+ diet; however, there was a strong trend for a relatively higher urine K‐to‐Cl ratio on day 2 in the KO: 1.64 ± 0.18 (WT) versus 2.26 ± 0.24 (KO), p = 0.053 suggesting impaired homeostatic balance between K+ and Na+ in the KO. A benzamil‐sensitivity test was applied to KO and WT mice, as an assessment of basal ENaC activity, by administering 4.3 mg/kg·bw benzamil, intraperitoneally, and collecting urine for 4 hours. KO mice had significantly reduced urine Cl− concentrations (mM), as well as, absolute excretion of both Na+ and Cl− (mmol/4 hours), as compared to the WT, suggesting reduced basal ENaC activity. However, their urinary ratio of Na‐to‐Cl was higher: 1.37± 0.07 (WT) versus 1.71± 0.11 (KO), p = 0.023, supporting independent and distinct regulation of these two electrolytes by the mTOR pathway. Overall, these findings support a role for mTOR in the modulation of CD electrolyte homeostasis by ENaC and related but distinct Cl− and K+ transporters and channels.

Support or Funding Information

Supported by Georgetown University CTSA award and internal funding.
Activation of Kir4.1 is essential for switching the function of DCT2 to DCT1 by Stimulating NCC and inhibiting ROMK/ENaC during dietary K restriction
Authors: Wang Mingxiao , Su Xiao‐Tong , Wu Peng , Lin Dao‐Hong , McCormick James A. , Ellison David E. , Wang Wen‐Hui
Published: November 2023

Kir.4.1 is an inwardly‐rectifying K channel expressed in the basolateral membrane of distal convoluted tubule (DCT). Previous study demonstrated that Kir4.1 activity determines the expression of NCC. In the present study we test the hypothesis that Kir4.1 plays a key role in suppressing K secretion of the DCT during K‐restriction through inhibiting ROMK and ENaC, and stimulating NCC. The experiments were performed in WT and in inducible kidney specific Kcnj10 knockout mice (Ks‐Kcnj10 KO) using pax8+/Cre+/Kcnj10flox/flox mice treated with doxycycline. The whole‐cell recording shows that low K (LK) intake significantly increases the Ba2+‐sensitive K currents in early DCT (DCT1). Since we did not detect ROMK channel activity in DCT1, this indicates that LK intake stimulates the basolateral K conductance. This notion is also supported by the observation that LK intake not only increases the probability of finding Kir4.1 in the DCT but also channel activity (NPo) detected with single channel recording. The basolateral K conductance in the DCT1 is largely abolished in KS‐Kcnj10 KO mice. Moreover, LK intake fails to increase the basolateral K currents in DCT1, suggesting that LK intake‐induced increase in the basolateral K conductance is due to the activation of Kir.4.1. We confirmed the previous finding that LK intake increases the expression of NCC and phosphorylated NCC (p‐NCC). However, the LK intake induced stimulation of NCC and p‐NCC is completely absent in KS‐Kcnj10 KO mice. Patch‐clamp experiments detected the TPNQ‐sensitive ROMK currents and amiloride‐sensitive ENaC in the split‐open DCT2/CNT of WT and KS‐Kcnj10 KO mice. Moreover, ROMK and ENaC currents are higher in KS‐Kcnj10 KO mice than that of WT. LK intake significantly inhibits ROMK and ENaC channel activity in the WT mice but this effect is blunted in the DCT2/CNT of KS‐Kcnj10 KO mice. We conclude that LK intake stimulates the basolateral Kir4.1 activity and that the activation of Kir4.1 is essential for stimulating NCC and inhibiting ROMK/ENaC in the DCT during K restriction. It is possible that Kir.4.1 plays a key role in converting the function of DCT2 to DCT1 thereby inhibiting K secretion in the DCT2 during hypokalemia.
Intermittent and Sustained Hypoxia Exacerbate the Neonatal Microglial Inflammatory Response to TLR4 Ligands
Authors: Kiernan Elizabeth A. , Gardner Alyssa , Langenstroer Elizabeth A. , Watters Jyoti J.
Published: November 2023

Chronic intermittent hypoxia (IH) is a hallmark of sleep disordered breathing, a condition that occurs in approximately 2–4% of the pediatric population, and in over 50% of premature infants. IH is characterized by increased reactive oxygen species formation, peripheral and CNS inflammation, neuronal death and neurocognitive deficits. However, although IH alone does not promote neuroinflammation in the neonate to the extent it does in adults, few studies have investigated the possibility that IH may exacerbate the neonatal CNS response to systemic inflammation, a common clinical co‐morbidity, especially in premature infants. In neonatal rats, we tested the hypothesis that IH will prime both the peripheral and central immune system leading to potentiation of inflammation. To test this hypothesis, we exposed P1 rat pups to one night of IH (2 min cycles of 10.5% O2). The following morning the pups were challenged with an intraperitoneal injection of the toll‐like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) for 3 hrs. CD11b+ microglia and macrophages were harvested from the brain and spleen, respectively, and iNOS and IL‐1 beta gene expression was analyzed. Preliminary results demonstrated that IH exposure prior to LPS injection increased microglial (but not spleen macrophage) iNOS mRNA levels compared to LPS alone. These findings were confirmed in vitro by exposing murine N9 and primary microglia to sustained hypoxia (1% O2 for 16 hrs) followed by application of multiple TLR4 ligands (LPS, Hsp60, HMGB1) for 3 hrs. Hypoxia potentiated TLR4‐dependent iNOS and IL‐1b mRNA levels, changes that were consistent with increased transcriptional activation marks at the iNOS gene promoter (trimethylation of histone H3 lysine 4; H3K4me3). Future studies will investigate if similar epigenetic modifications also occur in vivo. These data suggest differential responses of peripheral and CNS macrophage populations to the priming effects of hypoxia, and indicate that the CNS may be more vulnerable. By resolving mechanisms whereby hypoxia primes microglial inflammation, novel therapeutic targets can be identified that may be beneficial to an already susceptible pediatric population.

Support or Funding Information

This work was supported by NIH R01 HL111598 and NS085226
Counter‐regulation of the HIF‐dependent adaptive response to hypoxia by hypercapnia
Authors: Taylor Cormac , Selfridge Andrew , Cavadas Miguel , Scholz Carsten , Cambpell Eric , Welch Lynn , Lecuonah Emilia , Colgan Sean , Barrett Kim , Sporn Peter , Sznajder Jacob , Cummins Eoin
Published: November 2023

Molecular O2 and CO2 are the primary substrate and product of oxidative metabolism respectively. Hypoxia (low O2) and hypercapnia (high CO2) are co‐incidental features of the microenvironment in a diverse range of pathophysiologic states including cancer, respiratory disease and inflammation. Hypoxia inducible factor (HIF) is the master transcriptional regulator of the response to low oxygen. Despite the close relationship between O2 and CO2 levels, the potential impact of CO2 on HIF remains unclear. Recent studies have demonstrated the ability of CO2 to regulate gene expression. The purpose of this work was to investigate the effect of hypercapnia on the HIF pathway.

HIF‐2α protein and the HIF targets erythropoietin and carbonic anhydrase IX were decreased by hypercapnia in vivo. Hypercapnia suppressed HIF‐1α and HIF‐2α protein stabilization in a variety of cell lines of diverse origin. High CO2 diminished the activity of HIF‐dependent genes. CO2 did not impact the canonical, oxygen‐dependent HIF degradation pathway. Hypercapnia caused intracellular acidosis, and levels of HIF‐1/2α protein were lower when intracellular pH (pHi) was reduced by means other than CO2. Low pHi is required for optimal lysosomal degradation and a lysosomal inhibitor antagonised the ability of hypercapnia to reduce HIF‐1/2α.

In conclusion, we have demonstrated a novel CO2‐dependent suppression of HIF signalling. As HIF regulates the transcription of a host of genes that have angiogenic, glycolytic and inflammatory functions, manipulating HIF with CO2 could be exploited for therapeutic gain.

Support or Funding Information

This work was supported by a grant from Science Foundation Ireland (11/PI/1005)
Can Augmented Sympathetic Activity in Rosacea be Tracked by Cutaneous Flowmotion?
Authors: Wilson Thad E. , Eaton Emily , Toma Kumika , Jurovcik Andrew , Sammons Dawn , Metzler‐Wilson Kristen
Published: November 2023

Recent supraorbital microneurography evidence indicates that sympathetic outflow to the face is augmented in rosacea (Metzler‐Wilson & Toma, et al, J Neurophysiol, 2015). Our aim was to identify if non‐invasive assessment of skin blood flow (SkBF; laser‐Doppler flowmetry) can be used to track facial sympathetic outflow differences between subjects with and without rosacea and during symptom‐triggering events. To this end, we assessed spectral power (fast Fourier transform) in the sympathetic (low frequency (LF); 0.02–0.06 Hz) and vascular endothelial (very‐low frequency (VLF); 0.009–0.02 Hz) frequency ranges in forehead skin during systemic heating (50° C water delivered through a high density tube‐lined suit to increase mean skin and internal temperature). We hypothesized that compared to healthy age‐sex matched controls (n=9), Individuals with rosacea (n=9) would demonstrate greater changes in LF, but not VLF ranges. SkBF increased to a greater extent during heat stress in rosacea compared to controls (451±40 vs. 352±33% increase from baseline; p=0.04). Heat stress also increased spectral power within both the VLF and LF ranges in both groups (change from baseline to heating in VLF: 0.17±0.16 vs. 0.67±0.32 au; LF: 1.17±0.62 vs. 0.39±0.36 au; rosacea and control, respectively), but significant group difference were not observed (VLF: p=0.21; LF: p=0.16). High between‐subject variance existed in patients and controls. Thus, until better variability controls among subjects during symptom‐triggering events are developed, cutaneous flowmotion does not appear to be a viable surrogate for skin sympathetic nerve activity measurements in this population.

Support or Funding Information

National Rosacea Society
ATP Maintains the Ability to Blunt α1‐Adrenergic Vasoconstriction During Combined Blockade of Nitric Oxide, Prostaglandins, Na+/K+‐ATPase, and KIR Channels
Authors: Hearon Christopher M , Richards Jennifer C , Luckasen Gary J , Larson Dennis G , Joyner Michael J , Dinenno Frank A
Published: November 2023

The vasoactive properties of exogenous adenosine triphosphate (ATP) are unique because it has the dual ability to induce robust vasodilation as well as blunt sympathetic vasoconstriction, similar to what is observed in contracting human skeletal muscle. ATP‐mediated vasodilation and exercise hyperemia are dependent, in part, on activation of inwardly rectifying potassium (KIR) channels and subsequent smooth muscle cell hyperpolarization, with a modest reliance on the production of nitric oxide (NO) and prostaglandins (PGs). However, recent studies from our laboratory indicate that these pathways are not obligatory for contracting skeletal muscle to blunt α1‐adrenergic vasoconstriction. Given the specific role of KIR channels in ATP‐mediated vasodilation, the primary purpose of this study was to determine whether the ability of ATP to blunt α1‐adrenergic vasoconstriction is dependent upon KIR channel activity in humans. A secondary purpose was to determine whether the ability of ATP to blunt α1‐adrenergic vasoconstriction is dependent upon activation of KIR channels and Na+/K+‐ATPase, as well as the synthesis of NO and PGs. In a total of 13 young subjects we measured forearm blood flow (Doppler ultrasound) and calculated changes in vascular conductance (FVC) to local intra‐arterial infusion of phenylephrine (PE; α1‐agonist) during infusion of ATP or a control vasodilator(s) (Protocol 1 (n=7): adenosine (ADO), and sodium nitroprusside (SNP); Protocol 2 (n=6): ADO). Vasomotor responses were assessed before and after blockade of signaling pathways that contribute to ATP‐mediated vasodilation (Protocol 1: KIR channels (barium chloride); Protocol 2: combined blockade of KIR channels, Na+/K+‐ATPase (ouabain), NO (L‐NMMA), PGs (Ketorolac)). Protocol 1: ATP significantly attenuated PE‐mediated vasoconstriction relative to both SNP and ADO (Δ FVC: ATP: −16 ± 2; ADO: −38 ± 6; SNP: −59 ± 6%; P<0.05 vs ADO and SNP). Blockade of KIR channels significantly attenuated ATP mediated vasodilation ~40% (P<0.05 vs. control), but had no effect on the ability of ATP to blunt PE‐ mediated vasoconstriction (Δ FVC: −6 ± 5%; P>0.05 vs. control). Protocol 2: Despite combined blockade of KIR, Na+/K+‐ATPase, NO, and PGs resulting in profound attenuation of ATP‐mediated vasodilation (−~60%; P<0.01 vs. control), ATP was still able to blunt PE‐mediated vasoconstriction (Δ FVC: −4 ± 14%; P=0.50 vs. control). Together these studies highlight that while NO, PGs, and vascular smooth muscle hyperpolarization (via KIR channel activation) are contributors to the normal vasodilatory response to exercise and exogenous ATP, these pathways are not obligatory for exercise or ATP to blunt α1‐adrenergic vasoconstriction in humans.

Support or Funding Information

Support: HL119337, HL095573