bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional microdomain in the cardiac pacemaking in mouse
Authors: Lang Di , Warden Aleah , Kamp Timothy , Glukhov Alexey
Published: November 2023

Background

Heart rhythm imitates from the leading pacemaker called sinoatrial node (SAN). SAN cell contains a number of unique ion channels and transporters responsible for cardiac pacemaking which were prevailingly believed freely mobile in the cell membrane. Recently, it has been reported that Caveolin‐3 (Cav3), a myocyte‐specific scaffolding protein, compartmentalizes these ion channels and transporters within caveolar membranes. These proteins are attributed to a surface “membrane clock” which is spatiotemporally coupled with subcellular calcium handling machinery (i.e. “Ca2+‐clock”) forming a coupled‐clock pacemaker cell system. Such coupling is achieved via extremely complex crosstalk between the two clocks and is hypothesized to rely on functionality of a macro‐molecular pacemaker signaling complex targeted to caveolae.

Methods and Results

In vivo ECG telemetry, ex vivo high‐resolution calcium/voltage optical mapping, and in vitro confocal imaging of [Ca2+]i were performed in wild type (WT, n=17) and conditional cardiac‐specific Cav3 knockout (Cav3−/−, n=14) mice. Cav3−/− mice exhibited SAN pacemaking abnormalities characterized by alternating periods of tachycardia‐bradycardia rhythm originated from distinct anatomical locations, including SAN (during tachycardia) and ectopic foci located within the intercaval region outside of the SAN (during bradycardia). The ectopic foci areas consisted of non‐tubulated myocytes and had prolonged Ca release time and Ca transient duration due to desynchronized sarcoplasmic reticulum Ca release. This was reproduced in non‐tubulated (but not in tubulated) WT atrial myocytes by disruption of caveolae by methyl‐β‐cyclodextrin. To investigate unstable SAN pacemaking, we characterized subsarcolemmal local Ca2+ releases (LCRs) from ryanodine receptors (RyR), a critical component of Na+/Ca2+ exchanger (NCX)‐mediated late diastolic depolarization of SAN action potential (AP). In Cav3−/− SAN myocytes, regular LCRs were recorded; however, a half of them failed to initiate APs resulting in irregular SAN rate. We found significantly prolonged LCR‐to‐AP period (73.9±6.7 ms in WT vs. 130.6±15.0 ms in Cav3−/−, p<0.01) and increased LCR amplitude (normalized value: 0.54±0.046 %/μm in WT vs. 0.90±0.13 %/μm in Cav3−/−, p<0.01). A longer time and a higher driving force needed to activate NCX and boost AP may indicate redistribution of NCX from their canonical location in caveolae to non‐native membrane regions where their functional coupling with RyRs is lost.

Conclusion

Our findings demonstrate that Cav3 plays a crucial role in supporting functional integrity of the SAN by synchronizing calcium‐voltage coupling and regulating rhythmic LCRs. Loss of Cav3 disrupts caveolae‐associated macro‐molecular pacemaker signaling complex which results in functional uncoupling of NCX from RyRs and affects calcium‐voltage synchronization leading to SAN dysfunction.
Ipragliflozin Prevents the Worsening of Ischemic Heart Failure in Diabetic Rats Independent of its Diuretic Effect
Authors: Kamada Kazuhiro , Saku Keita , Kishi Takuya , Akashi Takuya , Takehara Takako , Tsutsui Hiroyuki , Sunagawa Kenji
Published: November 2023

Introduction

Although diabetes mellitus (DM) increases cardiovascular events, no clinical trials had ever shown the benefit of glucose‐lowering therapy on heart failure (HF). Recently, sodium‐glucose cotransporter 2 (SGLT2) inhibitor has shown to lower cardiovascular events and hospitalization for HF in DM (EMPA‐REG OUTCOME). To clarify the beneficial mechanisms of SGLT2‐inhibitor for HF, we investigated the impact of SGLT2‐inhibitor, ipragliflozin, on hemodynamics and cardiac function in a rat model of HF with DM.

Methods

We allocated Sprague‐Dawley rats into streptozotocin (60 mg/kg)‐induced DM or non‐DM groups. We created myocardial infarction (MI) in both groups and administered ipragliflozin (3 mg/kg/day) or vehicle from two weeks after MI. Four weeks after the initiation of treatment, we compared hemodynamic parameters among 4 groups (DM‐MI+ipragliflozin, n=9; DM‐MI+vehicle, n=6; MI+ipragliflozin, n=7; MI+vehicle, n=7).

Results

Daily urine volume nearly quadrupled in DM rats in comparison with non‐DM rats. In DM rats, ipragliflozin significantly lowered blood glucose (309±36 vs. 478±107 mg/dL, p<0.01), prevented the worsening of left ventricular (LV) ejection fraction (−1.4±4.8 vs. −8.8±5.1%, p<0.05), increased max LVdP/dt (6193±667 vs. 5015±456 mmHg/sec, p<0.01) and tended to decrease LV end‐diastolic pressure (24.8±5.3 vs. 30.5±5.7 mmHg, p=0.069). However, ipragliflozin did not affect urine volume. In non‐DM rats, the beneficial effects of ipragliflozin on HF were limited.

Conclusions

Ipragliflozin attenuated the worsening of HF in DM rats, while its beneficial effects were obscured in non‐DM rats. These results indicate that ipragliflozin has direct cardioprotective effects on HF in DM independent of its diuretic effects.
Diaphragm Muscle Function and Antioxidant Levels in Chronic Heart Failure
Authors: Hackfort Bryan T , Lewis Robert , Gao Lie , Zucker Irving H , Schultz Harold D
Published: November 2023

Nearly 5.7 million adults in the United States have chronic heart failure (CHF). CHF is associated with dyspnea and impaired diaphragm function. Shortness of breath in CHF patients may lead to decreased exercise and mobility, further exacerbating skeletal muscle atrophy and the effects of heart failure. Oxidative stress contributes to limb skeletal muscle weakness in heart failure, however the role of oxidative stress in the diaphragm is poorly characterized. Nuclear factor (erythroid derived 2)‐like 2 (Nrf2) is a master regulator of antioxidant enzymes and is decreased in limb skeletal muscles following heart failure. We hypothesize that Nrf2 is decreased and oxidative stress is increased in diaphragm muscle following heart failure resulting in impaired diaphragm muscle function. To test this hypothesis, 8–9 week old, male mice were infarcted by left coronary artery ligation and aged for 10 weeks post MI. Diaphragm function was measured using plethysmography in sham and heart failure conditions. After collection of the diaphragm, protein levels of antioxidant enzymes were measured. Preliminary results (N=2–3) show Nrf2 is not changed (1.1 fold relative to sham) in the diaphragm following CHF. Further, the antioxidant enzymes glutathione peroxidase (2.0 fold), catalase (1.3 fold), and SOD2 (1.9 fold) all tended to increase with CHF compared to sham. Breathing studies showed CHF mice (N=4) tended to have increased respiratory rates (RR = 265 breaths per minute) compared to sham (RR = 208) and decreased tidal volumes. While preliminary, these results suggest that altered breathing dynamics may be associated with altered diaphragm workload in heart failure, with an increase in antioxidant enzymes independent of Nrf2. The increase in antioxidant enzymes in CHF may be related to more oxidative stress from increased workload. Further studies will increase sample size and look at oxidative stress in diaphragm via DHE stain, diaphragm muscle function via muscle tension studies, and mitochondrial respiration.

Support or Funding Information

This work was supported by funds from the National Institutes of Health 5P01HL062222‐17
Increased circadian variations in chemoreflex sensitivity in chronic heart failure
Authors: Lewis Robert , Hackfort Bryan T. , Schultz Harold
Published: November 2023

Respiratory function follows a circadian pattern, and rhythmic changes in carotid body sensitivity to blood gases may contribute to these oscillations in ventilation. Recent studies have demonstrated that the isolated rat carotid body shows enhanced responses to hypoxic and hypercapnic stimulation in the presence of melatonin, a nocturnally released hormone. Carotid body dysfunction is known to occur in several pathological conditions, including chronic heart failure (CHF). CHF is characterized by tonic hyperactivity and exaggerated reactivity of the carotid bodies to hypoxia and hypercapnia. Thus, we've begun to examine circadian rhythms in chemoreflex breathing in CHF to determine if the already heightened hypoxic and hypercapnic responses in CHF are further augmented during the night. Male Sprague‐Dawley rats were divided into either control or CHF groups. Heart failure was induced via coronary ligation. Chemoreflex breathing was assessed during the day and then again 12 hours later during the night. Animals were exposed to room air (baseline), hypoxia (10 % O2) and hypercapnia (5 % CO2). Percent change in respiratory rate from day to night were similar between CHF (17.1 % increase) and control (16.9 % increase) rats when exposed to room air, but hypoxic and hypercapnic challenges induced large circadian differences in CHF (hypoxia: 20.9 % increase; hypercapnia: 23.9 % increase) but not in control (hypoxia: 8.2 % increase; hypercapnia: 13.9 % increase) rats. CHF rats also showed robust night time circadian differences in tidal volume when exposed to room air (31.6 % increase), hypoxia (27.9 % increase), and hypercapnia (43.8 %). In contrast, circadian variations in tidal volume were attenuated in control rats (room air: 11 % increase; hypoxia: 15.6 % increase; hypercapnia: 11.0 % increase). Minute ventilation showed similar differences between CHF and control rats with CHF significantly augmenting circadian variations. CHF rats showed a 53.1 % increase in minute ventilation during the night, and a 52.9 % increase and a 78.2 % increase in response to hypoxia and hypercapnia, respectively. Control rats, in contrast, showed only a 27.7 % increase in minute ventilation to room air, a 25.1 % increase in response to hypoxia, and a 26.9 % increase in response to hypercapnia. Therefore, the data suggest that circadian rhythms in ventilatory responses are amplified in CHF. Future research may directly assess the contribution of the carotid bodies to this phenomenon.
Identification of HuR‐dependent gene expression in cardiac hypertrophy
Authors: Slone Samuel , Anthony Sarah R , Nieman Michelle L , Lorenz John N , Rubinstein Jack , Benoit Joshua B , Tranter Michael
Published: November 2023

HuR is a widely expressed RNA binding protein that interacts with specific AU‐rich domains in target mRNAs and regulates their expression by modulating stability and/or translation. RNA binding proteins such as HuR are becoming recognized as potential mediators of cardiac physiology and pathology but the role of HuR in cardiac hypertrophy is largely unknown. Our results show that HuR undergoes cytoplasmic translocation, indicative of its activation, in hypertrophic cardiac myocytes and is necessary for hypertrophic cell growth. Using an inducible cardiomyocyte‐specific HuR deletion mouse (iCM‐HuR−/−), we have shown that cardiac specific deletion of HuR significantly reduces hypertrophy compared to wild‐type mice at 8 weeks post‐transverse aortic constriction (TAC). More importantly, the iCM‐HuR−/− mice exhibited significantly preserved ejection fraction, reduced chamber dilation, and less fibrosis compared to WT at 8 weeks post‐TAC. To elucidate the downstream target genes of of HuR in the heart and identify potential mechanisms by which HuR deletion reduces pathological cardiac hypertrophy, we used RNA‐seq to identify HuR‐dependent gene expression in the setting of cardiac hypertrophy. Using the NIH DAVID Functional Annotation Tool, our results indicate a significant increase in genes promoting anti‐apoptotic Akt signaling in hearts from iCM‐HuR−/− mice following TAC. However, since HuR binding typically stabilizes target mRNA, we observed a much larger number of significantly down‐regulated mRNA transcripts in iCM‐HuR−/− mice. Among these were genes involved in key cellular growth processes such as muscle cell differentiation, cell proliferation, cell cycle, and positive regulation of transcription. Thus, our results suggest that HuR deletion protects the heart from pathological cardiac hypertrophy by promoting pro‐survival Akt signaling while decreasing the expression of genes involved in cell growth and differentiation.

Support or Funding Information

This work was partially funded by an American Heart Association Scientist Development Grant and NIH R01 HL132111 (MT).
Alterations in the Balance of Cardiomyocyte Glucocorticoid and Mineralocorticoid Receptor Signaling Lead to Heart Failure
Authors: Oakley Robert , Cruz‐Topete Diana , He Bo , Foley Julie , Myers Page , Murphy Elizabeth , Chambon Pierre , Willis Monte , Cidlowski John
Published: November 2023

Stress is associated with adverse cardiac outcomes; however, the mechanisms underlying these effects are poorly understood. Glucocorticoids are stress hormones that regulate cell and tissue homeostasis through two closely related nuclear receptors, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). Cardiomyocytes express both these receptors but little is known concerning their coordinated actions in heart physiology and pathology. Here, we show that patients with failing hearts have a disparity in the relative levels of GR and MR compared to non‐failing donor hearts. To examine the in vivo function of glucocorticoid signaling in the heart, we generated mice with cardiomyocyte‐specific deletion of GR (cardioGRKO), MR (cardioMRKO), or both GR and MR (cardioGRMRdKO). The cardioMRKO mice exhibited normal heart function whereas the cardioGRKO mice spontaneously developed cardiac hypertrophy and left ventricular systolic dysfunction. Surprisingly, the cardioGRMRdKO mice were protected from cardiac disease, despite having many of the same pathological gene changes that were present in the GR deficient hearts. Microarray analysis revealed a set of cardioprotective gene changes that occurred specifically in the cardioGRMRdKO hearts. Re‐installation of MR into the cardioGRMRdKO hearts by adeno‐associated virus gene delivery reversed these cardioprotective gene changes and resulted in cardiac dysfunction. These findings reveal critical gene‐regulatory roles for both GR and MR in the heart and suggest that an imbalance in these two signaling pathways leads to heart disease. Therapies designed to modulate cardiomyocyte glucocorticoid signaling to favor more GR and less MR activity may provide new approaches for treating the failing heart.

Support or Funding Information

NIH
Would healing was improved in mice after myocardial infarction by nature naphthoquinone shikonin
Authors: Cai Ashley , Bedja Djahida , Cai Zheqing
Published: November 2023

Wound healing has an important impact on ventricular remodeling and functional recovery after myocardial infarction (MI). Nature naphthoquinone shikonin has been shown to promote wound healing by reducing reactive oxygen species and inflammation. However, it is still unknown whether shikonin can increase wound healing in infarcted hearts. C57BL6 mice were subjected to MI surgery. Infarcted mice were treated with shikonin by oral gavage, 25 mg/kg once a day for four weeks, or the equal volume of vehicle as controls. At the end of the experiments, echocardiography was performed. Echo study showed that shikonin improved left ventricular fractional shortening and ejection fraction, and that it decreased left ventricular dilation and increased infarcted wall thickening. Moreover, the ratio of heart weight to body weight was decreased in shikonin‐treated group, compared with vehicle group. Masson's trichrome staining demonstrated fibrosis was inhibited in non‐infarcted regions in shikonin‐treated group. Taken together, shikonin promoted wound healing and improved left ventricular functional recovery in infarcted hearts.
Indices of autophagy are unaltered by quercetin consumption in hearts of Mdx/Utrn+/− mice
Authors: Quindry John C , Quindry Tiffany S , Ballmann Christopher G , Selsby Joshua T
Published: November 2023

A significant percentage of Duchenne muscular dystrophy (DMD) is associated with progressive cardiac damage and heart failure. The extent to which autophagy is altered in the dystrophic myocardium with advanced disease is unknown. DMD countermeasures, and prevention of resultant cardiomyopathy, may correct disease‐related autophagic dysfunction. Using mouse models, our lab has demonstrated previously that long‐term consumption of the flavonol quercetin benefits physiologic, biochemical, and histological outcomes in dystrophic hearts. The purpose of this investigation was to determine the extent to which 1) autophagy is dysregulated in dystrophic hearts with advanced disease, and 2) whether indices of autophagic dysfunction are corrected by oral quercetin administration. Hearts harvested from untreated 10‐month old C57, Mdx/Utro+/‐ (heterozygous for a utrophin knockout), and Mdx/Utro+/− treated with a 0.2% quercetin enriched diet from 2–10 months of age. In these hearts we have previously reported widespread dystrophic injury that was largely corrected by the quercetin intervention. Using a western blot approach, dependent measures included relative abundance of key autophagic markers including Atg5, Atg7, beclin‐1, LC3II/I, and p62. Results indicate that Atg5 (p=0.779), Atg7 (p=0.895), beclin‐1 (p=0.251), and p62 (p=0.064) were similar between groups. The LC3II/I ratio was similarly unaffected by 8 months of quercetin treatment, although a strain‐dependent effect (p=0.038) was observed with Mdx/Utrn+/−Q and Mdx/Utrn+/− being 44% and 56% lower than C57, respectively. Based on these preliminary findings, disease‐related injury is independent of changes in autophagic signaling. Moreover, quercetin‐mediated protection appears to be unrealated to alterations to the autophagic process. Additional metrics of autophagic flux and examination using other animal models are needed to confirm the current findings.

Support or Funding Information

Funded by: Duchenne Alliance and its member foundations
Ammonia Promotes Endothelial Cell Survival via the Heme Oxygenase‐1‐Mediated Release of Carbon Monoxide
Authors: Durante William , Liu Xiao‐ming , Peyton Kelly J.
Published: November 2023

Although endothelial cells (ECs) produce substantial amounts of ammonia (NH3) during cell metabolism, the physiologic role of this gas in these cells is not known. In this study, we investigated if NH3 regulates the expression of heme oxygenase‐1 (HO‐1), and if this enzyme influences the biological actions of NH3 on ECs. Exogenously administered NH3, given as ammonium chloride or ammonium hydroxide, or endogenously generated NH3 stimulated HO‐1 protein expression in cultured human and murine ECs. Dietary supplementation of NH3 also increased HO‐1 expression in mouse arteries. The induction of HO‐1 protein by NH3 in ECs was associated with an elevation in HO‐1 mRNA, enhanced production of reactive oxygen species (ROS), and increased expression and activity of the transcription factor Nrf2. NH3 also activated the HO‐1 promoter and this was blocked by mutating the antioxidant responsive elements of the promoter, or by overexpressing dominant‐negative Nrf2. The induction of HO‐1 by NH3 was dependent on ROS formation and prevented by N‐acetyl‐L‐cysteine or rotenone. Significantly, prior treatment of ECs with NH3 inhibited tumor necrosis factor‐α‐mediated cell death. However, silencing HO‐1 expression abrogated the protective action of NH3, and this was reversed by the administration of carbon monoxide (CO) but not bilirubin or iron. In conclusion, this study demonstrates that NH3 stimulates the expression of HO‐1 via the ROS‐Nrf2 pathway, and that the induction of HO‐1 contributes to the cytoprotective action of NH3 by generating CO. Moreover it identifies NH3 as a novel modulator of EC survival and reveals new critical regulatory and functional interactions between signaling gases in the vasculature.

Support or Funding Information

Supported by grants from the NIH (R01HL59976) and AHA (15GRNT25250015).
Neuroprotective Effect of Low Dose Alcohol Consumption Against Transient Focal Cerebral Ischemia
Authors: McCarter Kimberly DeShon , Sun Hong , Li Chun
Published: November 2023

Background

Ischemic stroke, which accounts for 87% of diagnosed strokes, is a leading cause of long‐term disability and mortality worldwide. Chronic alcohol abuse is a risk factor for ischemic stroke and worsens the prognosis of ischemic stroke. On the other hand, increasing evidence suggest that chronic low‐dose alcohol consumption (LAC) reduces the incidence of ischemic stroke and improves the functional outcome of ischemic stroke. Our goal is to delineate the molecular basis whereby LAC alters the incidence and prognosis of ischemic stroke. We hypothesize that LAC protects the brain against its ischemia/reperfusion (I/R) injury by suppressing postischemic inflammation.

Methods

Male Sprague‐Dawley rats were divided into 3 groups, an ethanol group, a wine group, and a control group and fed with ethanol, wine (Castle Rock Pinot Noir), or volume‐matched water via gavage for 8 weeks. Ethanol and wine consumption was 1.4g/kg/day. The animals were then subjected to a 2‐hour middle cerebral artery occlusion (MCAO) and sacrificed at 24 hours of reperfusion after neurological evaluation. Cerebral I/R injury, microglial activation and neutrophil infiltration were evaluated by TTC staining and immunohistochemical staining, respectively. Expression of adhesion molecules were analyzed using western blot analysis. Pro‐inflammatory molecules were measured using ELISA.

Results

Cerebral infarct volume is significantly reduced in ethanol‐ and wine‐fed rats compared to control rats. There is no significant difference in infarct volume between ethanol‐fed and wine‐fed rats. Sensory‐motor functions were significantly improved in ethanol‐fed rats when compared to control rats. Transient focal cerebral ischemia increased neutrophil infiltration, microglial activation and the expression of adhesion molecules such as intercellular adhesion molecule (ICAM), vascular cellular adhesion molecule (VCAM), E‐selectin and P‐selectin. However, the magnitude of microglial activation and ICAM and E‐selectin expression was significantly reduced in ethanol‐fed rats while neutrophil infiltration and the magnitude of the increased expression of P‐selectin were significantly reduced in wine‐fed rats when compared to control rats. VCAM expression after ischemia was significantly increased in ethanol fed rats when compared to control rats.

Conclusions

Our findings suggest that LAC may protect the brain against its I/R injury by suppressing postischemic inflammation.

Support or Funding Information

National Institute of Health Grant; Malcolm Feist PreDoctoral Award (Center for Cardiovascular Diseases, LSU Health Sciences Shreveport)
The Protective Role of NHERF1 in Cisplatin‐Induced Acute Kidney Injury
Authors: Bushau‐Sprinkle Adrienne M , Sherwood Amanda R , Conklin Caryl A , Barati Michelle T , Dupre Tess , Siskind Leah , Gagnon Kenneth B , Khundmiri Syed J , Lederer Eleanor D
Published: November 2023

Acute kidney injury (AKI), an abrupt loss of kidney function which carries a high mortality and confers an increased risk of chronic kidney disease, develops in 30% of patients who receive cisplatin, a widely used and potent chemotherapeutic agent. The sodium hydrogen regulatory factor isoform 1 (NHERF1) is a scaffolding protein that anchors multiple membrane proteins, including the sodium‐phosphate cotransporter (NPT2a) in renal proximal tubules. NHERF1‐deficient proximal tubule cells show slower growth, abnormal transport properties, and aberrant localization of BBM proteins in intracellular compartments. We hypothesize that NHERF1‐deficiency increases susceptibility to AKI through a well‐defined cellular response to conditions of metabolic stress. To test this hypothesis, we used the Opossum kidney (OK) proximal tubule cell line and their NHERF1‐deficient (OKH) cells maintained in minimal essential medium supplemented with 10% FBS and 1% Pen/Strep in humidified 95% O2/5% CO2 incubator. Activated PERK and GRP78 was measured by WB to determine the presence of ER stress. We also used the in vivo model of cisplatin‐induced AKI in C57BL/6 wild type (WT) and NHERF1 knock out (NHERF1‐KO) mice. Male and female WT and NHERF1‐KO mice were treated with vehicle or cisplatin (20 mg/kg dose IP) to induce AKI. After 72 hours, blood samples were collected from these mice to measure blood urea nitrogen (BUN) levels and were sacrificed. The kidneys were analyzed by immunohistochemistry. Hematoxylin and eosin and periodic acid‐schiff staining of the kidney cortex of cisplatin‐treated NHERF1‐KO mice showed greater proximal tubular injury manifested by BBM sloughing, tubular cell detachment and vacuolization. Statistically significant differences were observed in blood urea nitrogen (BUN) levels between cisplatin‐treated WT and NHERF1‐KO male mice (p<0.01) (n= 6/group). NHERF1‐KO mice (n=2/group) treated with cisplatin also demonstrated decreased expression of GRP78, a pro‐survival ER chaperone that is upregulated during ER stress. Female cisplatin‐treated NHERF1‐KO mice (n=2/group) show a decrease in expression of p‐eIF2α/eIF2α total, a translation initiation factor that is phosphorylated in times of ER stress, when compared to male cisplatin‐treated NHERF1‐KO mice. We also observed an increased p‐PERK activity (an eIF2α kinase) in OKH to OK cells. We conclude that NHERF1‐deficiency is associated with an underlying ER stress which in turn may confer enhanced susceptibility to cisplatin‐induced AKI.

Support or Funding Information

Funding for this research was provided by Robley Rex Veterans Affairs Medical Center and University of Louisville School of Medicine (to EDL).
Inhibition of PDE7 Reduces Inflammatory Response Following Renal Ischemia/Reperfusion Injury
Authors: Reis William R. , Sun Michael A. , Kronk Trinity A. , Blount Mitsi A.
Published: November 2023

Ischemia/reperfusion (I/R) injury is an unavoidable consequence after kidney transplantation. I/R affects many regulatory systems to promote pro‐inflammatory responses leading to the progressive interstitial renal fibrosis and declining renal function that can negatively impact long‐term graft outcome. Because elevated cAMP can offset the this harmful, sustained immune response, identification of a targetable protein to increase kidney cAMP levels may provide a viable option to prevent I/R injury before organ recovery by donor pretreatment. Our studies aimed to determine the benefits of raising renal cAMP levels through phosphodiesterase‐7 (PDE7) inhibition for treating the immune response following I/R insult. PDE7 inhibition following TNF‐alpha stimulation of human proximal cells (HK‐2) reduced mRNA expression of cytokines (IL‐2, IL‐6, IL‐12β, IL‐18) and chemokines (CCL2, CCL5) to sham‐treatment levels. Next, age‐matched, male Sprague‐Dawley rats were treated with vehicle or PDE7 inhibitor (TC3.6) 24‐hours before ischemia‐reperfusion (I/R) was induced by inhibiting bilateral renal circulation of for 30 minutes. Perfusion was restored and rats recovered in metabolic cages for 24 hours before sacrificed to collect blood, urine and kidney tissue. Sham surgery was also performed on untreated and TC3.6‐treated rats. I/R injury increased mRNA expression of pro‐inflammatory cytokines and chemokines in whole kidney tissue from vehicle‐treated rats. However, when the rats were treated with TC3.6 prior to I/R injury, cytokine and chemokine mRNA expression was reduced to levels comparable to sham surgery rats. Blood urea nitrogen (BUN) was elevated in vehicle‐treated rats following I/R injury compared to vehicle‐ and TC3.6‐treated rats following sham surgery; however, BUN was reduced in I/R injured TC3.6‐treated rats. In conclusion, PDE7 inhibition prior to I/R injury attenuates pro‐inflammatory responses in the kidney and protects renal function following insult. Our findings provide evidence that PDE7 inhibition may be an effective pretreatment prior to kidney transplant to reduce the harmful effects of I/R and improve graft outcome.
The host engulfment pathway in enteroids is linked to intestinal inflammation following bacterial infection
Authors: Das Soumita , Lim Eileen , Suarez Katherine , Ernst Peter B , Barrett Kim E
Published: November 2023

Intestinal homeostasis is maintained through regulated interactions between epithelial cells, innate immune cells and the microbiota. The immune homeostasis is lost following interaction of the host with enteric pathogens that generate inflammation. Microbes are recognized by specific receptors such as Pattern Recognition Receptors (PRRs). PRRs can bind pathogens as well as commensals, but in most cases inflammation is generated only from pathogenic encounters. Previously, we identified Brain Angiogenesis Inhibitor 1 (BAI1) as a PRR that recognizes bacterial lipopolysaccharide (LPS). BAI1 binds Engulfment and Cell Motility Protein‐1 (ELMO1), which facilitates the engulfment of bacteria by host cells. We have shown that ELMO1 in phagocytes regulates the entry of bacteria and subsequent intestinal inflammation. Cytokine arrays and ELISA showed reduced release of pro‐inflammatory cytokines, including TNF‐α, monocyte chemoattractant protein (MCP‐1) and IL‐1β, from ELMO1‐depleted macrophages. Interestingly, higher expression of ELMO1 in biopsy specimens from inflammatory bowel disease patients correlates with the expression of MCP‐1 and TNF‐a. We developed enteroid‐derived monolayers (EDMs) that mimic the architecture and cell populations of the in‐vivo intestinal epithelium. Here we show that human as well as murine enteroids express ELMO1. We used Salmonella as well as adherent‐invasive E. coli as enteric pathogens. We found a significant reduction of bacterial internalization and pro‐inflammatory cytokines in EDMs generated from ELMO1 knockout mice compared to WT mice. EDMs infected with enteric bacteria generate MCP‐1 in an ELMO1‐dependent manner that attracts monocyte at the site of inflammation. The expression of MCP‐1 is higher in several inflammatory diseases including the atherosclerosis and inflammatory bowel diseases. Therefore, elevated expression of MCP‐1 via ELMO1 may be an early biomarker in inflammatory diseases. Our work also uncovers a central role of ELMO1 in the induction of inflammatory cytokines in non‐phagocytic cells.

Support or Funding Information

UCSD, Academic Senate (Das), NIH‐NIDDK R01 (Das)Clinical and Transitional Research Institute/NIH‐NCATS (Das)
The Effects of Conjugated Linoleic Acid and Berberine Supplementation on Markers of Oxidative Stress in Broiler Chickens
Authors: Dartigue Vincent , Klandorf Hillar , Falkenstein Elizabeth , Barnes Kimberly , Van Dyke Knox
Published: November 2023

Reactive oxidative species (ROS) are oxygen base molecules that are leaked from the electron transport chain during mitochondrial cellular respiration. They are kept in balance by endogenous and exogenous antioxidants, which may include conjugated linoleic acid (CLA) and a plant root extract called berberine. A redox shift towards oxidants causes a state called oxidative stress, which is detrimental if prolonged. A ten week study examined the effect CLA and berberine supplementation on markers of oxidative stress in broiler birds. Broilers (n=60) were divided into six groups; the diet of group four to six included allopurinol (25mg/kg body weight) which induced an oxidative stress state. Pen 1(P1) served as the control, P2 CLA oil (50% substitution), P3 berberine supplementation (20g/kg feed), P4 allopurinol alone, P5 CLA oil + allopurinol and P6 berberine + allopurinol. Body weight, plasma uric acid as a marker of oxidative stress, blood glucose, lipid peroxidation via TBARs assay of serum, and relative gene expression of five endogenous liver antioxidants were measured during the course of the study. The addition of allopurinol to the diet induced an oxidative stress state when compared to the baseline groups as observed by a significant reduction in plasma uric acid. In P5 and P6 treatment we observed on average a 10 fold increase in the relative mRNA expression of superoxide dismutase 2 and 3 as well as glutathione 3 and 7. Notably, CLA increased the expression of uncoupling protein 34 fold compared to control while the addition of allopurinol blocked these changes; in contrast there was a slight increase in the berberine+allopurinol treatment. No significant changes were measured in blood glucose and TBARS. Despite the increase in mRNA expression of the antioxidants genes, the result of the uric acids assay suggest that at the dosages administered CLA and berberine were not effective in reversing the oxidative stress induced by allopurinol.

Support or Funding Information

WVU Hatch
Nicorandil reduces lipoperoxidation in stimulated and non‐stimulated atrophied muscle
Authors: Sanchez‐Perez Alina , Padilla‐Maldonado Paulina , Sanchez‐Duarte Elizabeth , Cortes‐Rojo Christian , Saavedra‐Molina Alfredo , Gomez‐Barroso Mariana , Montoya‐Perez Rocio
Published: November 2023

Muscle atrophy is characterized by a reduction of muscle mass through protein degradation resulting in a reduction of strength development. There is evidence that the opening of the ATP sensitive potassium channels (KATP) causes a protective effect in muscle against deterioration. Thus, the aim of the present study was to explore the effect of nicorandil on lipoperoxidation in stimulated and non‐stimulated atrophied muscle. C57BL/6 male mice 14–16 weeks‐old were used for the experimental basis; these animals were separated in 4 groups: 1) Control, 2) Nicorandil, 3) Atrophy and 4) Atrophy treated with Nicorandil. Atrophy was induced by hindlimb unloading by the tail method. Nicorandil was purveying in 40 mg/kg by intramuscular injection during 14 days. Once finished the treatment, the animals were sacrificed by cervical dislocation and the hindlimb were used lipoperoxidation measurement. In the stimulated muscles, a decrease in membrane damage in the group treated with nicorandil was observed 47.3% (0.157 ± 0.0124 nmol/mg prot), the damage increased to 256.3% (0.85 ± 0.0327 nmol/mg prot) during atrophy; reducing this levels to 128% (0.426 ± 0.0299 nmol/mg prot) in atrophied treated with nicorandil. In non‐stimulated muscles, there was a decrease in membrane damage 56.1% (0.184 ± 0.0.0025 nmol/mg prot) in the group treated with nicorandil, after which the damage increased to 247% (0.81 ± 0.0.0047 nmol/mg prot) during atrophy and decreased to 119.5% (0.392 ± 0.0097 nmol/mg prot). The results suggest that nicorandil decreases lipoperoxidation in stimulated and non‐stimulated soleus muscle during muscle possible through the opening of the ATP sensitive potassium channel.

Support or Funding Information

We thank Universidad Michoacana de San Nicolás de Hidalgo (UMSNH). We acknowledge the partial financing to Coordinación de la Investigación Científica of UMSNH: CCR‐CIC1821440; ASM‐CIC‐2.16; RMP‐CIC 182171.
Chronic Electronic Cigarette Vapor Inhalation Induces Renal Injury and Functional Decline in Female Mice
Authors: Drummond Christopher , Alexander Laura E Crotty , Tian Jiang
Published: November 2023

Clinical studies indicate that combustible cigarette smoke increases renal and cardiac tissue injury progression and functional decline in the setting of chronic kidney disease (CKD). Novel nicotine delivery devices like electronic (e)‐cigarettes are used by over 10% of the population and produce vapor which may also induce renal injury. To establish whether e‐cigarette vapor inhalation induced renal injury in the form of fibrosis and decreased renal injury our current study utilized a nose‐only inhalation exposure system to induce 8 week‐old female mice to inhale e‐cigarette vapor containing 24mg/mL of nicotine suspended in a solution of 50% propylene glycol and 50% vegetable glycerin using the following parameters: 12 seconds of vapor exposure every 60 seconds for 60 minutes five days per week for 1‐, 3‐ and 6‐months. Following e‐cigarette exposure, assessment of renal fibrosis, glomerular filtration rate, and expression of the antifibrotic microRNA miR‐29b‐3p were evaluated. Mice exposed to e‐cigarette vapor suffered a 31% decline in renal tissue expression of miR‐29b‐3p vs air‐exposed controls (p<0.05). Additionally, mRNA targets of miR‐29b‐3p that regulate fibrosis formation or are part of fibrosis were also significantly increased in the kidneys of e‐cigarette exposed mice versus air controls, i.e., Collagen 1A1 (increased 98%; p<0.05); Collagen 3A1 (129% increase; p<0.05); Collagen 4a1 (72% increase; p<0.05); Integrin beta 1 (58% increase; p<0.05); and Fibrillin 1 (100% increase; p<0.05). Additionally, we observed a significant increase in renal fibrosis at the 3‐ and 6‐month time points as assessed by Trichrome staining in these animals. Lastly, following 3‐months of exposure to e‐cigarette vapor renal function was significantly reduced by 21% versus air exposed controls (p=0.017). These data are the first to indicate that e‐cigarettes induce renal fibrosis and functional decline. In addition, these data suggest a novel miR‐29b‐3p mediated mechanism linking e‐cigarette vapor exposure and renal injury and functional decline.

Support or Funding Information

This work was supported by the National Institutes of Health (1F32DK104615‐01 to CAD and HL‐105649 to JT. Additional support was from a Veterans Affairs BLR&D Career Development Award (1IK2BX001313 to LCA), AHA Beginning Grant‐in‐aid (16BGIA27790079 to LCA), and O'Brien Center Daniel O'Connor Memorial Pilot Award to LCA. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
A new view of macula densa cell microanatomy
Authors: Gyarmati Georgina , Moon Ju‐Young , Brison Anne Riquier , Peti‐Peterdi Janos
Published: November 2023

Macula densa (MD) cells are well known as chief regulators of renal and glomerular hemodynamics and renin secretion. However, their new, non‐traditional function as master regulators of renal tissue remodeling is emerging. The present study aimed to explore the microanatomical basis and regulation of the secretory phenotype of MD cells by directly visualizing the morphological features of living MD cells in male and female mice on low, normal, and high salt diet. A new mouse model (MD‐GFP mice) was generated with inducible and conditional MD‐specific, membrane‐targeted expression of the fluorescence reporter EGFP by crossing nNOS‐CreERT2 and mTmG‐floxed mice. MD cells were freshly isolated from digested kidneys of MD‐GFP mice using FACS sorting, and high resolution time‐lapse multiphoton imaging was performed to visualize single, living MD cells. Also, suboptimal tamoxifen induction was performed in MD‐GFP mice, which resulted in the membrane‐targeted expression of EGFP only in a single MD cell per MD plaque and allowed high resolution imaging of the MD ultrastructure on fixed histological sections, including both the plasma and intracellular membranes. MD cells featured very long (~10 μm) single primary cilia at the apical membrane, and a dense network of thin cell processes (up to 20 μm long) at the base of cells projecting into the extraglomerular mesangium. The basal cell processes were more detailed in female mice and in response to low salt diet. Time‐lapse imaging of living MD cells visualized a high number of membrane vesicles trafficking within these cell processes from the cell center to the periphery. These results are consistent with the highly regulated paracrine signaling function and secretory phenotype of MD cells, and suggest the high rate of synthesis and release of novel, yet to be identified secreted MD proteins and their actions on target cell types at the glomerular entrance.

Support or Funding Information

NIH R01 DK64324
Direct Evidence that in Rat the Renal Interstitium Contracts in vivo
Authors: Rodríguez‐Martínez Manuel , López‐Rodríguez Juan Francisco , Flores‐Sandoval Omar , Calvo‐Turrubiartes Miriam Zarahí , LLamazares‐Azuara Lilia
Published: November 2023

Dibutyryl‐cAMP abolishes the RIHP increase in response to renal medullary direct interstitial volume expansion (DIVE = 100 μl in 30 s) with 0.9% saline solution (SS) in hydropenic rats (FASEB J 30:739.9: 2016), suggesting that renal interstitium contracts in vivo. To assess more directly if such an event happens in vivo, we decided to evaluate in hydropenic rats the time course of RIHP before and after either an acute sham (CTR group, n = 9) or an acute (1s) real renal interstitial medullary application (RIMA) of a 5μ bolus of either vehicle (0.9% SS, SS group, n = 9) or 10 μM alpha‐trinositol (anti‐inflammatory drug that increases β1 integrin‐mediated collagen fiber network contraction, A‐T group, n = 9). Left subcapsular RIHP (subcRIHP) and % change of renal outer medullary blood flow (%ΔRoMBF by L‐D) were continuously measured in anesthetized rats with right nephrectomy, acute left renal denervation, hormonal clamp and renal perfusion pressure control (100 mmHg), before (baseline = BL = 30 min) and after sham or real RIMA of 5 μl bolus of either SS or A‐T. There were no differences among groups in body weight, hematocrit nor plasma protein concentration at any time. The table below shows both BL RIHP and ΔRIHP (RIHPafter – RIHPbefore) values (mean ± SEM) obtained at different times points after sham or real RIMA. Three were no differences among groups in BL RIHP. Overall RMMANOVA of ΔRIHP data showed no time effect, but if group (P<0.0004) and time * group interaction effects (P<0.009); 1WRMMANOVA:* P = 0.05, ** P <0.001 vs. Before; 1WANOVA + Tukey test:•P<0.04 vs. SS group, ••P<0.001 vs. CTR group. The %ΔRoMBF showed a similar decrease over time in the three groups.

CONCLUSIONS

The results show that acute RIMA of alfa‐trinositol is able to increase the RIHP in hydropenic rats by a mechanism independent of increased renal medullary blood flow, which reinforces the idea of that, at least in hydropenia, renal interstitium contracts in vivo.

Support or Funding Information

C15‐FAI‐04‐96.66/UASLP

subcRIHP (mmHg)Before RIMA
subcΔRIHP (mmHg)After RIMA

Group
BL
15 min
30 min
45 min
60 min

CTR
3.0 ± 0.1
−0.85 ± 0.22**
−1.11 ± 0.16**
−1.17 ± 0.19**
−1.16 ± 0.17**

SS
3.1 ± 0.03
−0.30 ± 0.20
−0.15 ± 0.36
−0.38 ± 0.56
−0.51 ± 0.55

A‐T
3.1 ± 0.06
0.54 ± 0.25•◆
1.26 ± 0.51*•◆
0.76 ± 0.37◆
0.68 ± 0.38◆
Hydrophobic residues in the Zonula occludens‐1 (ZO‐1) ZU5 domain contribute to essential junctional activity
Authors: King Jonathan M , Thomason Nicole C , White Addison R , Kannen Krista M , Shen Le , Turner Jerrold R
Published: November 2023

Zonula occludens‐1 (ZO‐1) is a cytosolic scaffolding protein involved in tight junction formation and maintenance of barrier function. The carboxy‐terminus contains a unique and poorly characterized ZU5 domain while structural data indicates exposed hydrophobic residues that are putative sites of protein interaction. We hypothesized the ZU5 domain contributes to stabilizing interactions and barrier properties of the tight junction. To determine ZO‐1‐ZU5 domain function, EGFP‐ZO‐1, EGFP‐ZO‐1ΔZU5, and EGFP‐ZO‐1 constructs mutated within the hydrophobic pocket of ZU5 were engineered and stably transfected into low‐resistance MDCK lines. The carboxy‐terminus of ZO‐1 contains an actin binding region (ABR) in close proximity to the ZU5 region that has been found to bind to F‐actin, and we hypothesized that the ZU5 region may have a stabilizing effect on these interactions. Actin protection assays indicated the EGFP‐ZO‐1 provides enhanced protection of perijunctional actin from degradation compared to the EGFP‐ZO‐1ΔZU5. The hydrophobic pocket mutants displayed varying degrees of actin protection. ZO‐1 knockdown cells have an increased paracellular flux, and FITC‐dextran paracellular flux assays in ZO‐1‐ZU5 mutants display enhanced leak pathway permeability. FRAP analyses show that EGFP‐ZO‐1ΔZU5 rapidly recovers and the mobile fraction (Mf) is elevated compared to EGFP‐ZO‐1 suggesting an important role in stabilization. Hydrophobic pocket mutants displayed similar changes suggesting the ZU5 domain participates scaffolding ZO‐1 at the tight junction.

Support or Funding Information

R01DK061931
Autophagy prevents downregulation of AQP1 protein induced by ischemia/reperfusion in cultured IMCD3 cells
Authors: Liu Qiaojuan , Hu Shan , Dong Wei , Liang Xinling , Li Chunling , Wang Weidong
Published: November 2023

Expression of aquaporin‐1 (AQP1) water channels in the kidney are critical for the maintenance of water homeostasis and the urinary concentrating ability. Autophagy is a lysosome‐mediated intracellular catabolic process by which cells remove their damaged organelles for the maintenance of cellular homeostasis. The purpose of current study is to investigate whether activation of autophagy prevents down‐regulation of AQP1 protein induced by in vitro simulated ischemia/reperfusion (I/R) injury in kidney inner medullary cells (IMCD3) (Lanaspa MA, J Biol Chem, 2010). Simulated I/R was induced by immersing the cellular monolayer in mineral oil for two hours followed by oil removal and medium refreshment. I/R induced consistently reduced AQP1 expression and I/R for six hours caused a 45% reduction of AQP1 protein abundance. I/R for six hours induced increased expression of P62/SQSTM1 and decreased ratio of LC3A/B, indicating an inhibition of autophagy flux. The inhibition of autophagy was associated with increased ratio of the serine/threonine kinase pGSK‐3β/GSK‐3β (three‐fold increase compared with controls). The inhibitor of GSK‐3β, TDZD‐8 (1μM), decreased ratio of pGSK‐3β/GSK‐3β and markedly improved autophagy by decreasing P62 (80% of I/R group) and increasing ratio of LC3A/B (1.6‐folds of I/R group). TDZD‐8 also prevented decreased AQP1 expression after I/R. Treatment with necrostatin (4μM), a serine/threonine kinase RIPK1 inhibitor, also decreased pGSK‐3β/GSK‐3β and improved authophagy, which was associated with increased AQP1 expression after I/R group. In conclusion, these data suggest that activation of autophagy in cultured IMCD3 cells prevent downregulation of AQP1 induced by I/R. It is also indicated that autophagy may play a role in the regulation of AQP1 expression in the inner medulla of the kidney under I/R conditions.
Transcranial direct current stimulation transiently increases the blood‐brain barrier solute permeability in rat brain
Authors: Fu Bingmei , Shin Da Wi , Khadka Niranjan , Fan Jie , Bikson Marom
Published: November 2023

Transcranial Direct Current Stimulation (tDCS) is a non‐invasive electrical stimulation technique investigated for a broad range of medical and performance indications. To test the hypothesis that tDCS with proper dosages can achieve non‐invasive, selective and temporary increases in the blood‐brain barrier (BBB) permeability (P), we used laser scanning multiphoton microscopy with 800~850nm excitation wavelength to determine P of the cerebral microvessels in the rat brain 100–200 μm below the pia mater. After 1 mA tDCS treatment for 20 min, sodium fluorescein (MW 376), or FITC‐dextrans 20K and 70K in 1% bovine serum albumin mammalian Ringer was injected into the rat (SD, 250–300g) cerebral circulation via the ipsilateral carotid artery by a syringe pump at a constant rate of ~3 ml/min. P was determined from the rate of dye spreading images surround individual microvessels in the brain tissue. It was found that the relative increase in P is about 8‐fold for small solute, sodium fluorescein, ~35‐fold for both intermediate sized (Dex‐20k) and large (Dex‐70k) solutes, 10 min after 20 min 1 mA tDCS pretreatment. All of the increased permeability returns to the control after 20 min post treatment. Our results demonstrate that tDCS at a proper strength and duration indeed transiently increases the BBB permeability to different sized solutes to different levels. It can be used as a non‐invasive method for delivering therapeutic agents to brain through the BBB.

Support or Funding Information

Supported by NIH R21EB017510‐01.