bluuebunny/crossref_metadata_2025 huggingface dataset:
Radiation‐induced Afferent Arteriolar Endothelial Dysfunction Involves Decreased Epoxygenase Metabolites
Authors: Sharma Amit , Khan Md. Abdul Hye , Fish Brian , Cohen Eric P. , Imig John D.
Published: November 2023

Chronic kidney disease (CKD) occurs in 15% of hematopoietic stem cell transplant (HSCT) patients, and as been clearly linked to irradiation at the time of the HSCT. CKD due to radiation also occurs after radionuclide therapy for cancer. There is a 6–8 week latent period after irradiation that leads to the development of proteinuria, azotemia, and hypertension in rats. We have previously demonstrated that impaired endothelial‐dependent responses to acetylcholine occur at 3 and 6 weeks that occur prior to the development of proteinuria, azotemia, and hypertension in rats. The current study tested the hypothesis that decreased endothelial‐derived relaxing factors contribute to the impaired afferent arteriolar responses to acetylcholine in rats exposed to total body irradiation (TBI). Male WAG/RijCmcr rats were subjected to TBI (11 Gy) and afferent arteriolar responses to acetylcholine using the juxtamedullary nephron technique were determined at three weeks. Systolic blood pressure (118 ± 5 vs. 121 ± 6 mmHg) and BUN (15.2 ± 1.3 vs. 15.7 ± 0.7 mg/dl) were not different between control and TBI groups. Afferent arteriolar diameters averaged 21.4 ± 0.9 ±m (n=25) in controls and 21.5 ± 0.8 ±m (n=25) in TBI rats. During exposure to acetylcholine (0.01. 0.1, 1, and 10 ±mol/L), afferent diameter increased by 8±2%, 18±3%, 27±2% and 31±3% in control rats (n=7), and the acetylcholine response was impaired in TBI and averaged 3±2%, 9±3%, 12±3%, and 16±3% (n=7, p<0.05). The nitric oxide synthase (NOS) inhibitor, L‐NAME, reduced the afferent arteriolar acetylcholine response by 50% in controls and 90% (p<0.05) in 3‐week TBI rats. Cyclooxygenase (COX) inhibition with indomethacin did not significantly alter the acetylcholine response in the presence or absence of L‐NAME. Epoxygenase (EPOX) inhibition with MS‐PPOH significantly decreased the acetylcholine responses (35%) in controls but did not significantly alter the acetylcholine responses (4%) in TBI rats. Combined inhibition of NOS, COX, and EPOX pathways eliminated the afferent arteriolar response to acetylcholine in control and TBI rats. Renal microvascular protein expression of CYP2C, COX2, and NOS revealed decreased EPOX expression of CYP2C23 (±45±6%) and CYP2C11 (±12±4%) at 3 weeks TBI. COX‐1, COX‐2, and eNOS renal microvascular protein expression was not altered at 3 weeks TBI. Taken together, these results indicate that the impaired afferent arteriolar endothelial‐dependent acetylcholine responses involve decreased EPOX activity that precedes the development of proteinuria, azotemia, and hypertension in irradiated rats.
Increased Urinary Exosomal Kidney Injury Molecule‐1 in Patients with Type 2 Diabetes
Authors: George Jasmine , Meng Yuan‐Xiang , Huang Ming‐Bo , Morgan‐Billingslea Jan , Garba Halima , Bond Vincent , Zhao Xueying
Published: November 2023

Diabetic nephropathy (DN), a leading cause of end‐stage renal disease, occurs in 25%–40% of patients with diabetes, highlighting the importance of early identification and management of DN. Urinary exosomes are 40–100 nm vesicles released by epithelial cells along the nephron and they may serve as biomarkers of renal dysfunction and structural injury. Kidney injury molecule‐1 (KIM‐1) is expressed on the apical membrane of dedifferentiated proximal tubule cells and its ectodomain can be cleaved and released into the urine in both rodents and humans with proteinuric kidney diseases. In this study, we investigated urinary exosomal KIM‐1 in type 2 diabetic patients to confirm its role as a non‐invasive biomarker for predicting kidney injury and dysfunction. Exosomes were prepared from spot urine samples of healthy (n=11), prediabetic (n=12) and type 2 diabetic patients (n=10) using serial ultracentrifugation. Nanoparticle tracking analysis (NTA) was applied to quantify the size and concentration of urinary exosomes. The majority of the captured particles had a size ranging from 20 nm to 100 nm with a peak size of 78±14 nm in healthy subjects, 67±10 nm in prediabetic, and 49±7 nm in diabetic patients with or without albuminuria. Urinary exosomes were significantly increased in diabetic patients (16.2±2.7 ×1010 particles/mg creatinine) compared to healthy donors (7.4±0.9 ×1010 particles/mg creatinine) or prediabetic patients (8.8±1.5 ×1010 particles/mg creatinine). As expected, soluble KIM‐1 was significantly increased in the urine of diabetic patients with microalbuminuria. Interestingly, Western blot also detected an increase in urinary exosomal KIM‐1 in the diabetic patients without microalbuminuria. In conclusion, type 2 diabetes is associated with an increase in urinary exosomes and exosomal KIM‐1 may represent a novel candidate biomarker in diabetic renal complication.

Support or Funding Information

This work was supported by the NIH SDK096441, 8G12MD007602 and 8U54MD007588.
Increased I‐mfa in Mesangial Cells Contributes to Increased Abundance of Fibronectin Protein in Diabetic Kidney
Authors: Chaudhari Sarika , Li Weizu , Wang Yanxia , Ma Rong
Published: November 2023

Accumulation of extracellular matrix (ECM) proteins in glomerulus is one of early features of diabetic nephropathy. Glomerular mesangial cell (MC) is the major source of ECM. Inhibitor of Myogenic Family isoform a (I‐mfa) is a cytosolic protein interacting with components of the Wnt/β‐catenin pathway and of store‐operated Ca2+ signaling pathway, both of which are involved in the development of diabetic nephropathy. The present study was carried out to determine if I‐mfa in MCs promoted expression of fibronectin protein, a major component of ECM using in vitro and in vivo settings. In cultured human MCs, high glucose treatment (25 mM) significantly increased I‐mfa protein expression in time and dose dependent manners. Importantly, abundance of I‐mfa protein markedly increased in the renal cortex and glomerulus in streptozotocin‐induced diabetic mice. Furthermore, overexpression of I‐mfa increased fibronectin expression at protein level and knockdown of I‐mfa decreased fibronectin expression at mRNA level in human MCs. We then generated diabetes in I‐mfa−/− and wild type control mice by multiple injections of low dose streptozotocin. As expected, diabetes greatly increased fibronectin protein expression in renal cortex in wild type mice. However, this fibronectin response was significantly reduced in I‐mfa deficient diabetic mice. Consistently, histological examination showed a decrease in mesangial expansion in I‐mfa deficient diabetic mice compared to wilt type control mice. Taken together, our data indicate that diabetes increased I‐mfa expression in MCs, which stimulates fibronectin protein expression in glomerulus. Therefore, I‐mfa may be a potential therapeutic target for diabetic nephropathy.

Support or Funding Information

UNTHSC Institutional Seed Grant, UNTHSC Doctoral student Bridge Grant, Sigma Xi Grant‐in ‐Aids Research Grant.
Renal effects of glucagon‐like peptide‐1 receptor knockout in Akita‐diabetic mice
Authors: Song Panai , Fu Yiling , Qiu Hongyu , Onishi Akira , Drucker Daniel J , Vallon Volker
Published: November 2023

Background

Glucagon‐like peptide‐1 (GLP‐1) is a gut‐derived hormone that enhances glucose‐dependent insulin release and has anti‐oxidative and protective effects mediated through GLP‐1 receptor (GLP1R) in various tissues. Here, we determined whether the GLP1R has a pathophysiologic role in the diabetic kidney.

Methods

GLP1R‐KO mice (C57BL/6J) were backcrossed to DBA2 background for at least 4 generations and crossed with heterozygous Ins2Akita mice, a genetic model of type 1 diabetes, to generate 4 experimental groups; i) WT (n=7), ii) GLP1R‐KO (n=14), iii) diabetic WT (n=6), and iv) diabetic GLP1R‐KO (n=10). Body weight, blood glucose, urine glucose and albumin to creatinine ratios, GFR (by plasma elimination kinetics of FITC‐sinistrin in conscious mice), and kidney weight were determined at 16 weeks of age.

Results

In non‐diabetic mice, no significant differences were observed between GLP1R‐KO and WT for bodyweight (26.1±1.0 vs 26±1.3 g), blood glucose (127±4 vs 112±5 mg/dl), urine glucose (2.5±0.3 vs 3.6±0.8 umol/mg), albuminuria (39±7 vs 37±19 μg/mg), and GFR (18±1 vs 22±2 μl/min/g), while kidney weight tended to be reduced in GLP1R‐KO (7.8±0.2 vs 8.5±0.3 mg/g; P=0.064). In GLP1R‐KO and WT mice, diabetes increased to similar levels: blood glucose (515±21 vs 501±32 mg/dl), urine glucose (3504±426 vs 5515±864 umol/mg), albuminuria (117±41 vs 102±22 ug/mg), food and fluid intake (not shown), and GFR (28±2 vs 33±3 ul/min/g), associated with a similar reduction in body weight (23.1±0.9 vs 23.7±0.6 g). While diabetes increased kidney weight in GLP1R‐KO and WT to similar absolute levels (11.8±0.8 vs 10.3±0.4 mg/g), the diabetes‐induced fractional increase was enhanced in GLP1R‐KO vs WT (increase of 51 vs 21%).

Conclusion

1) On a predominant DBA2 genetic background, type 1 Akita diabetes increased blood glucose levels, GFR, kidney to body weight ratios, and albuminuria. 2) The effects of diabetes on blood glucose, GFR and albuminuria occurred independent of the GLP1R, whereas the absence of the GLP1R appeared to facilitate the diabetes‐induced increase in kidney to body weight ratios.

Support or Funding Information

National Institutes of Health (R01DK56248, P30DK079337) and AstraZeneca
The Impact of Elevations of Renal Perfusion Pressure and Hyperglycemia on MMP‐2 Expression
Authors: McPherson Kasi Christine , Womack Judson , Taylor Lateia , Fizer Brianca , Green DesTenee , Spires Denisha , Robinson Joihn , Williams Jan M
Published: November 2023

The early stages of diabetic nephropathy (DN) are characterized by elevations in glomerular filtration rate (GFR) or renal perfusion pressure (RPP) that initiates the development of renal injury. Matrix metalloproteases (MMPs) have been shown to participate in renal fibrosis during DN. However, whether increases in GFR or hyperglycemia stimulate MMPs during DN remains to be determined. The current study determined the impact of elevations in RPP and/or hyperglycemia on MMP activity in Dahl salt‐sensitive (SS) rats. Experiments were performed on 6‐week old SS rats treated with either vehicle or streptozotocin (STZ, 50 mg/kg i.p.) to induce diabetes. After the induction of diabetes, both groups underwent surgery to place a silastic cuff on the aorta between the left and right kidneys to cause an increase in RPP to the right kidney by 10–20 mmHg: left kidney‐low RPP (110±3 mmHg) and right kidney‐high RPP (130±6 mmHg). After 3 weeks, blood glucose levels were markedly increased in the STZ‐treated rats versus vehicle rats (482±52 vs. 120±18 mg/dL, respectively). GFR was significantly elevated in response to both hyperglycemia and elevations in RPP compared to either hyperglycemia or elevated RPP alone. In comparison to the low RPP kidneys from the vehicle‐treated rats, proteinuria doubled in response to elevations in RPP alone (32±2 vs 16±8 μg/min) and increased by 4‐fold in response to both elevations in RPP and hyperglycemia (147±50 vs 16±8 μg/min, respectively). Renal MMP‐2 expression was 80% higher in response to elevations in RPP alone versus low RPP kidneys from vehicle rats. However, the effects of either hyperglycemia alone or in combination with increases in RPP on MMP‐2 expression were similar. Thus, these data suggest that the effects of elevations in RPP have a larger impact on renal MMP‐2 expression compared to either hyperglycemia alone or in combination with elevations in RPP.

Support or Funding Information

This research was supported by NIGMS NIH P20GM104357 and AHA 12SDG9440034.
Oxygen metabolic competition in the lactic acidotic diabetic kidney: A point of no return?
Authors: Laustsen Christoffer , Lipsø Kasper , Østergaard Jakob Appel , Mose Per , Flyvbjerg Allan , Pedersen Michael , Palm Fredrik , Ardenkjaer‐Larsen Jan Henrik
Published: November 2023

Diabetic nephropathy is directly related to renal hypoxia, with an increased mitochondrial uncoupling and increased energy demand to maintain normal renal function. Lowering the oxygen content in inspired air has shown to worsen the prognostic outcome of diabetic patients independent of glycemic control. We therefore tested the hypothesis that acutely altered renal oxygen availability alters metabolic pathways related to cellular energy production.

Methods and materials

Hyperpolarized 13C‐MRI and blood oxygenation level‐dependent (BOLD) 1H‐MRI were used to investigate the renal metabolic changes following acute changes in oxygen content in the inspired air in control and streptozotocin‐induced insulinopenic rats with and without insulin treatment.

Results

The severe lactic acidosis phenotype of diabetes, show a negative sensitivity towards oxygen changes, with a 12% and 16% reduction in the lactate‐to‐total carbon ratio change in both treated and untreated animals, however with no response differences between the treated and untreated group.

Discussion

The negative response to changes in oxygen availability can be the result of an increased lactate pool size masking the Pasteur effect, thus highlighting a key event in the development of kidney disease. The inability of insulin to normalize the lactate‐to‐pyruvate conversion in the present study indicates maintained elevated metabolic flux in these kidneys.

Support or Funding Information

The study was supported by The Simon Spies Foundation and the Danish National Research Foundation. Sascha Gude and Line Nielsen are acknowledged for their laboratory assistance.
(Pro)Renin Receptor Mediates Antidiuretic Action of Vasopressin/Prostaglandin EP4 Subtype
Authors: Yang Tianxin , Lu Xiaohan , Peng Kexin , Wang Fei
Published: November 2023

Within the kidney, (pro)renin receptor (PRR) is predominantly expressed in the collecting duct (CD), particularly in intercalated cells and is regulated by the EP4 subtype. Here we investigated the function of CD PRR during antidiuresis and defined its relationship with the EP4 subtype as well as vasopressin (VP). Water deprivation (WD) elevated renal PRR expression and urinary soluble PRR (sPRR) excretion in rats. Intrarenal infusion of a PRR decoy peptide PRO20 to rats partially attenuated the decrease in urine volume and the increase in urine osmolality in response to 48‐h WD, in parallel with suppressed aquaporin‐2 expression; a similar effect was seen with EP4 antagonism. The mice lacking PRR in the CD exhibited increased urine volume and decreased urine osmolality at basal condition and exhibited impaired urine concentrating capability after WD, accompanied with severe volume loss and a dangerous level of plasma hyperosmolality. In primary culture of rat inner medullary collecting duct (IMCD) cells, exposure to 10 nM prorenin for 24 h remarkably induced AQP2 protein expression, which was blunted by a PRR decoy inhibitor PRO20 and PRR siRNA. VP treatment for 24 h similarly induced aquaporin‐2 (AQP2) expression, accompanied with increased release of PGE2 and sPRR; the AQP2 upregulation was blunted by antagonism of EP4 or PRR. Overall, we have defined a VP/PGE2/EP4/PRR/AQP2 pathway that appears to play an important role in regulation of urine concentrating capability.

Support or Funding Information

This work was supported by National Institutes of Health Grants DK104072 and DK094956, VA Merit Review from the Department of Veterans Affairs, and National Natural Science Foundation of China Grant No. 91439205 and No. 31330037. T. Yang is a Research Career Scientist in the Department of Veterans Affairs.
Lack of adenylyl cyclase 6 induces alkalosis and enhances urinary acidification
Authors: Poulsen Søren Brandt , Fenton Robert A , Rieg Timo
Published: November 2023

Adenylyl cyclase isoform 6 (AC6) is expressed along the entire nephron, catalyzes the synthesis of cyclic adenosine monophosphate (cAMP) and contributes to the regulation of various aspects of renal transport. Recent in vitro and ex vivo studies identified that cAMP regulates Cl−/HCO3− exchanger (pendrin) trafficking to the apical plasma membrane of type B intercalated cells. However, the isoforms of AC involved in the regulation of urinary pH are not known. To gain insight in this regard, we studied AC6 knockout (AC6−/−) and littermate wild‐type (WT) mice. As a model of metabolic alkalosis and to test for a defect in urinary alkalinization, mice were supplied sodium‐bicarbonate in their drinking water for 8 days. Baseline urinary pH was significantly lower in AC6−/− mice (AC6−/−: 6.5 ± 0.2; WT: 7.7 ± 0.2, P < 0.05), which was associated with a more alkaline blood pH (AC6−/−: 7.44 ± 0.01; WT: 7.41 ± 0.01, P < 0.05). While blood pH was unaffected in both genotypes after bicarbonate administration compared to baseline conditions, urinary pH was increased in AC6−/− mice during bicarbonate administration, and pendrin abundance was higher compared to WT mice (AC6−/−: 225 ± 16%; WT: 100 ± 6%, P < 0.001). Immunohistochemistry showed a comparable subcellular pendrin distribution and number of pendrin‐positive cells/mm2 cortex in AC6−/− compared to WT mice (AC6−/−: 121.2 ± 8.7; WT: 108.3 ± 6.6, NS), thus possibly indicating intact pendrin regulation. Proton pump (H+‐ATPase B1 subunit) abundance was higher in AC6−/− mice both under baseline conditions (AC6−/−: 175 ± 25 %; WT: 100 ± 7%, P < 0.05) and after bicarbonate administration (AC6−/−: 196 ± 16%; WT: 100 ± 15%, P < 0.01). Furthermore, the number of H+‐ATPase B1 subunit‐positive cells/mm2 was higher after bicarbonate administration both in the cortex/outer stripe outer medulla (AC6−/−: 192.8 ± 10.5; WT: 161.0 ± 9.3, P < 0.05) and in the inner stripe outer medulla (AC6−/−: 312.6 ± 23.5; WT: 224.1 ± 11.3, P < 0.01). In conclusion, our results suggest that lack of AC6 induces mild metabolic alkalosis and enhances urinary acidification under baseline conditions. Stable blood pH after alkaline loading in AC6−/− may require a higher pendrin and H+‐ATPase B1 subunit abundance.

Support or Funding Information

This work was supported by the O'Brien Center for Acute Kidney Injury Research Grant P30DK079337, Diabetes Endocrinology Research Center P30DK063491, American Heart Association 15BGIA22410018, Satellite Healthcare (a not‐for‐profit renal care provider), Department of Veterans Affairs Merit Review Award, and Center on Genetics of Transport and Epithelial Biology at University of Cincinnati. Further funding is provided by the Lundbeck Foundation, the Danish Medical Research Council, and the Novo Nordisk Foundation.
The Sex‐Specific Adaptation of the Mitochondrial Lon protease in the model organism, Drosophila Melanogaster
Authors: Pomatto Laura Corrales‐Diaz , Shen Brenda , Carney Caroline , Tower John , Davies Kelvin J. A.
Published: November 2023

Due to the maternal inheritance of mitochondria, many of the mitochondrial genes and targeted proteins are optimized in a sex‐dependent manner, favoring females. In turn, this generates a biological power‐play between the sexes. One example is the mitochondrial stress response protein, the Lon protease. Our work in the model organism Drosophila melanogaster, has uncovered a sexual difference in Lon expression. A major hallmark of this sexual dimorphism is the ability of females, pre‐treated with hydrogen peroxide to adapt (measured by increased survival time) compared to non‐pretreated females, when subsequently exposed to a lethal dose of hydrogen peroxide. In contrast males, regardless if they are pre‐treated or only challenged, show no adaptation. As well, the removal of Lon from females prevents adaptation, with no impact upon males. In addition, Lon protein expression and activity is only inducible in a sex‐dependent manner. Based on our findings, we attribute these differences to the unique Lon protein banding variation that we have identified in males and females.

Support or Funding Information

National Science Foundation Graduate Research Fellowship; NIH/NIEHS RO1ES003598 (PI Davies, K.J.A.)
Increased O‐GlcNAcylation Attenuates Autophagic Flux, Induces Mitochondrial Dysfunction and Leads to Accumulation of Alpha‐Synuclein in Neurons
Authors: Wani Willayat Yousuf , Benavides Gloria , Ahmad Israr , Shacka John , Chacko Balu , Chatham John C , Paterson Andrew J , Darley‐Usmar Victor M , Zhang Jianhua
Published: November 2023

Alpha‐synuclein accumulation and mitochondrial dysfunction are prominent in Parkinson's disease (PD). To determine how autophagy, the cellular protein homeostasis and mitochondrial quality control process is regulated by a nutrient sensing post‐translational modification pathway, the O‐linked attachment of β‐N‐acetyl‐glucosamine on protein S/T residues (O‐GlcNAcylation), we first performed western blot analyses of lysates from postmortem temporal cortex of controls and PD patients, and found significantly increased O‐GlcNAcylation levels in PD brains compared to control. Whether increased O‐GlcNAcylation affects neuronal function and survival was then tested in rat primary cortical neurons. We found that thiamet G, a highly selective inhibitor of the O‐GlcNAcase (OGA) that removes O‐GlcNAc modification, significantly increased protein O‐GlcNAcylation, decreased autophagic flux, decreased mitochondrial complex IV activities, increased accumulation of mitochondrial peroxynitrite, and led to an increased α‐synuclein accumulation. To confirm our results in vivo, we assessed cortical synaptosomal characteristics in wildtype and dnOGA transgenic (CaMKIIα‐tTA::Tet‐dnOGA, dominant negative OGA with a catalytic site deletion) mice. We found that dnOGA synaptosomes exhibited increased O‐GlcNAcylation, decreased mitochondrial function, increased α‐synuclein, and accumulation of autophagosomes. Taken together we proposed that a significant increase of O‐GlcNAcylation levels in PD brains may contribute to impairment of neuronal bioenergetics and attenuation of autophagic activities and accumulation of α‐synuclein.

Support or Funding Information

UAB School of Medicine Multi‐Investigator Pilot Grant and NIH R01‐NS064090 (JZ)
S‐nitrosylation of Mitochondrial Proteins Preserves Skeletal Muscle Function From Ischemia‐Reperfusion‐Induced Injury
Authors: Wilson Rebecca June , Zhang Mei , Murphy Michael P. , Yan Zhen
Published: November 2023

Ischemia‐reperfusion (IR) injury in skeletal muscle is a common complication in many diseases and clinical settings that leads to loss of muscle mass and function. Unfortunately, there are currently no preemptive treatments to reduce IR injury in skeletal muscle. Generation of mitochondrial reactive oxygen species (ROS) during rapid re‐establishment of oxidative phosphorylation in mitochondria upon reperfusion has been identified as a major mediator of IR injury in skeletal muscle. We hypothesized that reversible suppression of mitochondrial respiration by covalently linking nitric oxide moieties to protein thiols (S‐nitrosylation or SNO), which is known to inhibit enzyme activities, may mitigate IR injury to skeletal muscle. We subjected C57BL/6 mice to 1 hour of unilateral ischemia by application of a rubber‐band tourniquet or to a sham procedure (Con). Five minutes prior to tourniquet removal, we performed intramuscular injection of an endogenous s‐nitrosothiol (S‐nitrosoglutathione, GSNO group), a mitochondria‐specific S‐nitrosothiol (MitoSNO1 group) or the respective vehicles (IR group). We assessed muscle weight and maximal specific force production 14 days following IR injury. The IR group had significantly less muscle force than Con mice (~50% reduction), while GSNO injection led to a moderate, but not statistically significant improvement of muscle force compared to the IR group (~10% improvement). Treatment with MitoSNO1 resulted in a complete recovery of muscle force. Gastrocnemius and tibialis anterior muscle masses were significantly reduced by ~40% and ~20%, respectively, in the IR and GSNO groups, but preserved in the MitoSNO1 group. Taken together, targeted S‐nitrosylation of mitochondrial proteins profoundly protects skeletal muscle against IR injury‐induced loss in muscle mass and function. Therefore, mitochondria targeted interventions may provide an efficacious means for protection of skeletal muscle against IR injury.

Support or Funding Information

AHA Predoctoral Award #14PRE20380254
PKD Translocation to the Outer Mitochondrial Membrane Induces Mitochondrial Fragmentation and Cell Death via DLP1 Phosphorylation in Cardiomyocytes
Authors: Jhun Bong Sook , O‐Uchi Jin , Mishra Jyotsna , Xu Xiaole , Hurst Stephen , Mende Ulrike , Sheu Shey‐Shing
Published: November 2023

Heart failure (HF) occurs in response to various types of stimulus including Gq‐protein coupled receptor (GqPCR) stimulation. GqPCR‐mediated mitochondrial dysfunction is frequently observed in HF animal models, but the molecular mechanism remains unclear. Recently, Protein kinase D (PKD) located at GqPCR downstream has been recognized as a key signaling nodal point affecting various cardiac functions. Therefore, we hypothesize that PKD acts as a mitochondrial signaling component and induces mitochondrial dysfunctions under GqPCR‐mediated HF. Using the outer mitochondrial membrane (OMM)‐targeted CFP (mt‐CFP), we found that the value of the Förster resonance energy transfer (FRET) between mt‐CFP and YFP‐tagged PKD significantly increases upon GqPCR stimulation, indicating PKD translocation from cytosol to the OMM. Next, using the FRET‐based OMM‐targeted PKD‐activity sensor, we observed that PKD is activated at the OMM after GqPCR stimulation. Furthermore, we found that GqPCR‐mediated PKD activation at the OMM induced mitochondrial fragmentation, increased reactive oxygen species generation and mitochondrial permeability transition pore opening, followed by caspase‐3 activation in cardiomyocytes. These morphological and functional alterations in cardiac mitochondria were mediated via PKD‐dependent phosphorylation of Dynamin‐Like Protein 1 (DLP1) at S637. Importantly, PKD‐dependent DLP1 phosphorylation concurrent with abnormal mitochondrial morphology and apoptotic signaling were also observed in ventricular tissue from transgenic mice with cardiac‐specific overexpression of constitutively active Gaq. In summary, we conclude that GqPCR stimulation induces PKD translocation to the OMM and induces mitochondrial fragmentation and dysfunction, which likely contributes to cardiomyocyte dysfunction during HF.

Support or Funding Information

This work was partly supported by American Heart Association (AHA) grant (14BGIA18830032 to J.O.‐U.), Medical Research Grant from W.W. Smith Charitable Trust (H1403 to J.O.‐U.) and NIH grants (2R01HL093671 and 1R01HL122124 to SSS).
Denervation alters micro‐RNA expression profiles of microvesicles released by cultured muscle fibers
Authors: Hamidi Sayyed , De Gasperi Rita , Harlow Lauren M. , Ksiezak‐Reding Hana , Bauman William A. , Cardozo Christopher P.
Published: November 2023

OBJECTIVES

Skeletal muscle releases a wide variety of substances that signal to distant tissues to integrate physiologic responses to physical activity or immobilization. Microvesicles have been the focus of intense study for their ability to signal from cell to cell. We tested whether skeletal muscle fibers produce microvesicles and examined their microRNA composition.

METHODS

These studies were conducted using fibers isolated from gastrocnemius muscle from mice at 7 days after sciatic nerve transection or sham surgery. Skeletal muscle fibers were maintained in culture for 48 hours and the released microvesicles collected by ultracentrifugation. The microvesicles were characterized by electron microscopy and labeled with PHK67 to evaluate their uptake by C2C12 cells. The microRNA expression profiles of fibers and corresponding microvesicles were analyzed by Nanostrings microRNA microarrays.

RESULTS

Skeletal muscle fibers maintained in culture for 48 hours released microvesicles which, by electron microscopy, were ~100 nm in diameter, had a bilamellar membrane, and were labeled by anti‐CD63. Vesicles labeled with PHK67, a fluorescent dye taken up by vesicle membranes, were avidly taken up by C2C12 cells. We observed high‐levels of microRNA expression in sham fibers for let7‐d, miR‐1, miR‐133a, miR‐125‐5p and miR‐22. In microvesicles, there was high‐level expression of miR‐1, 133a, 133b, and 378. When comparing miR expression in microvesicles from denervated versus sham muscle fibers, miR 1, 133a, and 145 were downregulated while miR‐206 was greatly up‐regulated. A similar though less dramatic pattern of changes was observed when comparing denervated and sham fibers. These studies reveal that skeletal muscle fibers release microvesicles that contain microRNA, the relative expression levels of these microRNAs is similar to that observed in skeletal muscle, these vesicles can be transferred to other cells, and paralysis resulting from nerve transection alters the microRNA expression profiles in vesicles in a manner similar to the parent muscle fibers.

CONCLUSIONS

These findings have important implications for understanding the mechanisms by which skeletal muscle fibers signal locally to adjacent muscle cells, as well other more distant cell types, including those found in bone.

Support or Funding Information

NA
Chronic alcohol ingestion induces alveolar macrophage oxidative stress and phagocytic dysfunction by down‐regulating NADPH oxidase‐related microRNAs
Authors: Yeligar Samantha M. , Harris Frank L. , Hart C. Michael , Brown Lou Ann S.
Published: November 2023

Objective

Compared to non‐alcoholics, chronic alcohol abusers are 2–4 times as likely to develop respiratory infections and acute respiratory distress syndrome (ARDS). Alcohol abuse increases susceptibility to lung infections in alcoholics through enhanced oxidative stress and impaired alveolar macrophage (AM) phagocytic function. NADPH oxidases (Nox) are primary sources of oxidative stress in AMs, and the Nox isoforms, Nox1 and Nox2, are required for Nox4 expression and activity. We hypothesize that alcohol attenuates Nox1‐ and Nox2‐associated microRNAs (miRs) to up‐regulate Nox1, Nox2, Nox4, oxidative stress, and phagocytic dysfunction.

Methods

AMs were obtained from the bronchoalveolar lavage (BAL) fluid of C57BL/6J mice fed ± ethanol (20% w/v) in the drinking water for 12 wks. In parallel, MH‐S cells, a mouse AM cell line, were transfected ± 50 nM miR mimics and treated ± 0.08% ethanol for 3 d. Levels of Nox1‐related miRs (miR‐1264 and ‐1982), which bind to the Nox1 3′UTR to decrease Nox1 levels, and Nox2‐related miRs (miR‐103 and ‐107), which bind to the Nox2 3′UTR to decrease Nox2 levels, were assessed by qRT‐PCR. Nox1, Nox2, and Nox4 mRNA and protein expression were measured by qRT‐PCR and western blot. Oxidative stress was measured with DCFH‐DA and Amplex Red assays. AM function was evaluated by phagocytosis assay (S. aureus internalization).

Results

In vivo and in vitro studies demonstrated that ethanol: 1) decreased AM levels of Nox1‐related miR‐1264 and Nox2‐related miR‐107; 2) increased Nox1, Nox2, and Nox4 mRNA and protein expression; 3) enhanced oxidative stress; and 4) impaired phagocytic capacity. miR mimics reversed these ethanol‐induced AM derangements.

Conclusions

Chronic alcohol ingestion enhances AM Nox expression, oxidative stress, and phagocytic dysfunction by down‐regulating Nox‐related miRs. Our studies suggest that targeting Nox‐related miR expression may attenuate alcohol‐induced AM derangements and decrease susceptibility to respiratory infections in patients with a history of alcohol use disorders.

Support or Funding Information

Research Funding Source: AHA SDG 13SDG13930003 (SMY), NIAAA 1K99AA021803 (SMY), Emory Alcohol & Lung Biology Center 1P50AA135757 (LAB & CMH), and Merit Review Funding from the Atlanta VA Medical Center (CMH).
Interplay Between Nutrition and Environment in Altering the Gut Microbiome and Susceptibility to Type 2 Diabetes
Authors: Schaid Mike , Neuman Joshua , Wisinski Jackie , Reuter Austin , Laundre Erin , Fenske Rachel , Kimple Michelle
Published: November 2023

It has recently been established that changes in the gut microbiotic profile are influential in development of obesity and type 2 diabetes mellitus (T2DM). Homozygosity for the leptinOb mutation in the BTBR mouse strain induces hyperphagia resulting in obesity, insulin resistance, and ultimately severe T2DM with full penetrance by 10 weeks of age. The BTBR ob/ob mouse is a strong, well‐accepted pre‐clinical model of T2DM: no one has ever reported conditions in which BTBR ob/ob mice do not become diabetic. We found that BTBR ob/ob mice housed in a specific “dirty” environment are completely protected from developing T2DM by 10 weeks of age. Studies out to 30 weeks indicate a relative protection that is durable. The protected phenotype directly correlates with improved liver metabolism, beta‐cell function, and glucose homeostasis as compared to littermate BTBR ob/ob mice continuously housed in our breeding colony, in which all rodent housing, bedding, and food is sterile. This is in the face of the severe insulin resistance and obesity expected with this strong genetic model of T2DM. To begin to elucidate mechanisms, we began a study in both rodent housing facilities testing the impact of diet nutrition, diet sterility, and environment on the protective phenotype, with the hypothesis that the gut microbiome was being altered in the “dirty” environment either by the nutritional composition of the diet or resident bacteria in the environment, which then colonize the chow. Preliminary data shows significant differences between the gut microbiome of protected vs. susceptible mice, with altered ratios of bacteroides to firmicutes, as well as other species that are all but absent in the susceptible mice. We are now correlating species with the plasma metabolome, peptide hormones, markers of inflammation and pancreatic islets function, followed by fecal transplant experiments, in order to show causality.
Pulsed Ultrasound Stimulation Prevents Bacterial Lipopolysaccharide Induced Muscle Wasting and p38 MAPK Phosphorylation in Mouse C2C12 Skeletal Myotubes
Authors: Ueno Mizuki , Maeshige Noriaki , Hirayama Yusuke , Nakanishi Ryosuke , Yoshikawa Madoka , Fujino Hidemi
Published: November 2023

Cachexia is a multifactorial syndrome characterized by involuntary weight loss due to skeletal muscle wasting and fat depletion. In cachectic state, inflammatory cytokines increase systemically, and following p38 MAPK phosphorylation leads to muscle protein degradation. While pulsed ultrasound stimulation can provide the mechanical stimulation to the target tissue, and has been reported to show anti‐inflammatory effects. Therefore, the preventive effect of pulsed ultrasound stimulation on muscle protein degradation induced by lipopolysaccharide (LPS) using C2C12 was investigated in this study. Pulsed ultrasound stimulation was performed immediately before LPS treatment. The pulsed ultrasound signal consisted of a series of 3 MHz, 20% burst rate, and was delivered at an intensity of 0.5 W/cm2 for 30 sec. LPS treatment resulted in the increase of p38 MAPK phosphorylation and myofibril protein degradation. In addition, the treatment induced the decrease of cell viability and diameter in C2C12 myotubes. Meanwhile, pulsed ultrasound stimulation prevented myofibril protein degradation induced by LPS in C2C12 myotubes, and inhibited the phosphorylation of p38 MAPK. These results indicate that mechanical stimuli with pulsed ultrasound could be a preventive intervention against cachectic muscle atrophy.
Microglial Inflammatory Responses to Systemic Immune Challenge are Attenuated over the Course of Chronic Intermittent Hypoxia Exposure
Authors: Ouellette Jonathan N. , Smith Stephanie M.C. , Watters Jyoti J.
Published: November 2023

Intermittent hypoxia (IH) is a hallmark of sleep disordered breathing that causes significant neuronal death, in part, via elevated levels of neuroinflammatory molecules. We previously identified microglia as one cellular source of these pro‐inflammatory molecules in the CNS within 1–2 weeks of IH exposure. However, microglial responses during IH to a concomitant inflammatory challenge are not yet known, an important consideration given that sleep disordered breathing and systemic inflammation are common co‐morbidities. Toll‐like receptor 4 (TLR4) is an innate immune receptor whose activation mediates inflammation in response to gram‐negative bacterial lipopolysaccharide (LPS) and endogenous molecules released from injured/dying cells prevalent in the CNS during IH. We found that TLR4 activation was necessary for microglial pro‐inflammatory responses to IH in vitro, and that TLR4 mRNA and activated cell surface protein levels increase and remain elevated in microglia for at least 28 days of IH in vivo, suggesting that microglia in the brains of IH‐exposed mice may be primed for greater responsiveness to subsequent inflammatory challenges. To test this hypothesis, we evaluated inflammatory gene expression in immunomagnetically‐isolated microglia from adult male C57Bl/6 mice that received 7–14 days of either normoxia (room air) or IH (60–90 second intervals of 6.5–10.5% O2 for 8–12 hours) followed by intraperitoneal injection of LPS (1 mg/kg; 14 hours) or vehicle (HBSS). Surprisingly, we found that LPS‐stimulated microglial pro‐inflammatory gene expression (COX‐2, IL‐1β, TNFα and CCL3) was significantly attenuated in IH‐exposed mice, whereas microglial expression of anti‐inflammatory/trophic factors (IFNβ and BDNF) was increased by IH alone (and unaltered by LPS in either O2 condition). Studies to investigate microglial responses to other forms of systemic inflammation during IH are ongoing. These data suggest that microglial responses are not exaggerated during IH, despite chronically elevated TLR4 levels. Further, they support the idea that microglia undergo a phenotypic transition during chronic exposure to IH that dampens their ability to respond to inflammatory stimuli, perhaps as an adaptive or compensatory response to protect the CNS against continued IH insult.

Support or Funding Information

Supported by R01 HL111598 and R01 NS085226
Investigating the Role of Mitochondrial Fission in Cardiac Myocyte Hypoxia/Re‐oxygenation‐Induced Cell Death
Authors: Pratt Amina Malika , Heine Erin , Chen Qian , Barsotti Robert , Young Lindon
Published: November 2023

Currently there is no effective pharmacological intervention to attenuate myocardial ischemia reperfusion (IR) injury. Understanding the mechanisms that govern IR injury would benefit myocardial infarction, coronary bypass and organ transplant patients. Recent studies suggest that cardiac mitochondria undergo excessive fragmentation during IR, a process that is called fission. Increased mitochondrial fission may lead to increased cell death during IR. Mitochondrial fission has been shown to be mediated by association of dynamin related protein‐1(Drp1), a GTPase that translocates from the cytosol to interact with outer mitochondrial membrane proteins, fission protein 1 and mitochondrial fission factor, to induce fission. We hypothesize that attenuating mitochondrial fission during IR may prove to be an effective strategy to mitigate IR injury. To test this hypothesis, we used a pharmacological inhibitor of Drp‐1 GTPase activity, Mdivi‐1(MW=353 g/mol). We subjected murine cardiac myocytes (HL‐1 cells) to 12 hours of hypoxia in hypoxic buffer (pH 6.8, no glucose or pyruvate) and then 1 hour of re‐oxygenation in normoxic media (pH 7.4), in the presence/absence of Mdivi‐1 (5–25μM). We predicted that HL‐1 cells subjected to simulated IR (SIR) would exhibit increased cell death in relation to their normoxic control counterparts, and that Mdivi‐1 treated cells would have attenuated SIR‐induced cell death. Cell death was determined by trypan blue (0.3%) staining. Preliminary data shows that control SIR cells (n=4) exhibited 25% cell death, by contrast Mdivi‐1 treated cells only showed 7% (5μM, n=4) and 11% (25μM n=4) cell death. Normoxic untreated (n=3) and Mdivi‐1 treated cells (5 and 25 μM; n=3) showed only 2% cell death in all study groups. The data suggests that inhibiting Drp‐1 may mitigate cardiac cell death during SIR. Future studies will determine the role of Drp‐1 in the regulation of mitochondrial dynamics during SIR.

Support or Funding Information

This study was supported by the Department of Bio‐Medical Sciences, Division of Research and the Center for Chronic Disorders of Aging at Philadelphia College of Osteopathic Medicine
P2X receptor inhibition protects THP‐1 monocytes against cytolysis by α‐hemolysin from Escherichia Coli and leukotoxin from Aggregatibacter actinomycetemcomitans
Authors: Fagerberg Steen K , Skals Marianne G , Praetorius Helle A
Published: November 2023

The Repeats in Toxins (RTX) α‐hemolysin (HlyA) from E. coli and leukotoxin A (LtxA) from A. actinomycetemcomitans are important virulence factors for ascending urinary tract infections and aggressive forms of periodontitis respectively. Upon insertion into the plasma membrane ATP is released through the toxin pore and is as signaling molecule essential for the HlyA‐ and LtxA‐induced erythrocyte damage. The immediate effects after ATP release are a massive erythrocyte shrinkage and phosphatidylserine exposure in the outer leaflet of the plasma membrane. These events are eminent for the erythrocytes to be recognized and engulfed by the monocyte cell line THP‐1. Here, we investigate how these toxins and ATP, affect the survival and function of THP‐1 monocytes.

By measurements of [Ca2+]i, we demonstrate that the THP‐1 express functional P2X (P2X1, P2X4 and P2X7) and P2Y2 receptor. We show that desensitization of either P2X1 by either low (0.001 μM) or more pronounced of P2X4/P2X7 by high concentrations of ATP (0.1 to 1 mM) increases the survival rate of THP‐1 cells attacked by either HlyA or LtxA in a calcein release assay. This protection is also found by specific blockage of the ATP sensitive receptors P2X1 (100 μM NF449) or P2X7 (100 μM oxATP or A804589). By erythrocyte phagocytosis assay, we determined that low dose HlyA (EC25) preconditioning is increasing the function of erythrocyte clearance at the place of injury.

These findings suggest a new target for protecting monocytes during pore‐former toxemia – both pharmacologically through P2X blockers or receptor desenzitisation. Both mechanisms may be relevant for improving monocyte survival and function in patients suffering from infection with RTX producing bacteria.

Support or Funding Information

Funded by the Danish Council for Independent Research, MEMRANES and Lundbeck Foundation
Umbilical cord stromal cell (UCSC) exosomes protect tubular epithelial cells from gentamicin injury
Authors: Borges Fernanda Teixeira , Novaes Antonio , Schor Nestor
Published: November 2023

Bone Marrow Mesenchymal Stromal Cells (MSCs) and Umbilical Cord Stromal Cells (UCSC) protect the kidney against nephrotoxic insults. This effect could be paracrine, possibly mediated by extracellular vesicles. Exosomes (EXOs) are extracellular vesicles that transfer mRNA and microRNA to the recipient cells, mediating the communication between them. Mir 126 mediates the protective effect of stem cell microvesicles. The goal of the present study was to analyze the effects of EXOs from MSCs and UCSC on proximal tubular epithelial cells from rats (RPTC) exposed to gentamicin (genta). UCSC and MSCs were maintained in culture conditions and every 3 days the culture medium was collected. EXOs were extracted through filtration in the 0.1μm filter, followed by ultracentrifugation (100,000g during 2h). Mir 126 from stromal cells and EXOs were extracted and analyzed by real time‐PCR. RPTC cells were exposed to gentamicin (2mM) in the presence or absence of exosomes (50 μg/ml), DNA damage and proliferation was analyzed by H2Ax and BrDU immunofluorescence, respectively. The expression of p21 and c‐myc was analyzed in RPTC through western blot. Mir 126 expression was increased in exosomes in comparison to stromal cells. It was increased in BM‐MSC exosomes (2.52 ± 0.12 AU) in comparison to UCSC exosomes (1.2 ± 0.2 AU). The treatment with UCSC‐EXO, but not with MSC‐EXO, stimulated the proliferation and decreased DNA damage induced by gentamicin in tubular cells. MSC‐EXO increased proliferation and decreased DNA damage when mir126 was depleted from exosomes with 126 antimir. RPTC cells treated with UCSC‐EXO increased the tumor suppressor protein p21 and increased in cell cycle progress protein c‐myc. MSC‐EXO did not show the same effect. Our results suggest that stromal cell exosomes carry mir 126 and this microRNA could be related to the paracrine protective effect of umbilical stromal cell exosome.

Support or Funding Information

Supported by FAPESP, CNPq, CAPES and FOR
Pharmacological activation of LPA receptors regulates erythro‐megakaryocytic differentiation in myeloid lineage
Authors: Chiang Jui‐Chung , Lin Kuan‐Hung , Ho Ya‐Shuan , Yao Chao‐Ling , Lee Hsinyu
Published: November 2023

Myeloid cell differentiation is one of the crucial process of hematopoiesis. It has been revealed that lysophosphatidic acid (LPA), through activating G‐protein‐coupled receptors of LPA1–6, may contribute to the regulation of this process. However, the underlie mechanisms in vivo remains unclear. Here, we aim to investigate the effects of LPA and its corresponding receptors, LPA2 and LPA3, during erythropoiesis/megakaryopoiesis. Two chemical drugs, LPA2 agonist GRI977143 and LPA3 agonist 1‐oleoyl‐2‐O‐methyl‐rac‐glycerophosphothionate (OMPT) were injected in BALB/c mice. Blood samples were collected and analyzed to determine the quantity of differentiation‐stage‐specific erythro‐megakaryocytic progenitors in spleen/bone marrow and terminally differentiated cells in circulatory blood, respectively. Our results revealed that the activation of LPA2 inhibit erythro‐megakaryocytic differentiation, whereas activation of LPA3 may promote erythroid development and decrease megakaryocyte generation in vivo. These result implied that LPA2 may dominate and inhibit the erythro‐megakaryocytic differentiation in the early stage of myeloid lineage. In contrast, LPA3 mainly regulates and promotes erythroid differentiation except for megakaryocyte. Taken together, our findings showing a remarkable difference between LPA receptor activation on regulating hematopoiesis in vivo. Moreover, the detailed impacts of LPA2/3 on different stages of myeloid stem cells during erythropoiesis/megakaryopoiesis will be elucidated in the future.