bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucose Promotes Metabolic Reprogramming of Male Rat Hearts in the Early Stress Response Following Hypothermic Ischemic Arrest and Reperfusion
Authors: Bux Ahmed , Vasquez Hernan , Taegtmeyer Heinrich , Harmancey Romain
Published: November 2023

The mechanisms by which hyperglycemia increases mortality in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) are not well defined. The present study aimed to elucidate the effects of glucose on early cardiac remodeling following cardiac arrest and reperfusion. Isolated hearts of male Sprague Dawley rats were cold‐arrested and subjected to 60 minutes normothermic reperfusion in the working mode with Krebs‐Henseleit buffer supplemented with glucose (25 mM) or mannitol (25 mM; osmotic control). Alterations of gene expression in the left ventricle were then determined by microarray analysis. Compared to non‐perfused hearts, perfused hearts displayed a more than twofold increased expression for 71 genes (mannitol group) and 103 genes (glucose group) connected to inflammation, apoptosis, and remodeling. There was a significant overlap between the transcriptional changes found in the perfused rat hearts and gene alterations previously reported in the right atrium (p < 2.34E‐16) and left ventricle (p < 4.83E‐46) of patients who underwent cardiac surgery with CBP. A rank‐rank hypergeometric overlap analysis of the genes differentially expressed in perfused hearts demonstrated that the transcriptional remodeling occurred independently from the presence of glucose. However, compared to hearts perfused with mannitol, glucose further increased the expression of early stress response genes and master regulators of metabolic reprogramming Myc (+ 56%), Nr4a1 (+ 30%), and Nr4a2 (+ 76%). This was accompanied by the increased expression of enzymes and membrane transporters controlling metabolic flux in pathways associated with the activation of immune cells and fibroblasts, including glycolysis (HK2, SLC2A3), glutamine catabolism (SLC1A5, GLS), fatty acid biosynthesis (OLAH), polyamine biosynthesis (ARG1, SAT1), and hexosamine biosynthesis (UAP1, GFPT1). Interestingly, glucose failed to enhance the expression of these transcription factors and enzymes in the heart of rats rendered insulin resistant by high‐sucrose feeding. This study demonstrates that the isolated working rat heart accurately reproduces the stress response associated with cardiac surgery with CPB. The results also suggest that glucose promotes the activation of resident cardiac immune cells and stromal cells in the stressed heart by enhancing their metabolic reprogramming. Lastly, we demonstrate that insulin resistance blocks the metabolic reprogramming induced by glucose in the isolated heart.

Support or Funding Information

Supported by grants K99/R00 HL112952 (to R.H.) and R01 HL061483 (to H.T.) from the National Institutes of Health
Na+,HCO3−‐cotransporter NBCn1 promotes Filopodia Generation, Smooth Muscle Cell Migration And Conduit Artery Remodeling
Authors: Aalkjaer Christian , Boedtkjer Ebbe , Bentzon Jacob Fog , Dam Vibeke Secher
Published: November 2023

We tested the role of cellular acid‐base transport in vascular smooth muscle cell (VSM) proliferation and migration and in structural remodeling of conduit arteries.

We used VSM from primary explants of mouse carotid arteries and aorta. In primary explants, pHi gradients exist along the axis of VSMC migration. In the presence of CO2/HCO3−, but not in its absence, knockout or pharmacological inhibition of the Na,HCO3‐cotransporter NBCn1 abolishes the pHi gradients, reduces filopodia generation and lowers the initial rate of VSMC migration after wound infliction. Interventions to reduce buffer mobility (omission of CO2/HCO3− or inhibition of carbonic anhydrase) reestablish pHi gradients and migration velocity in NBCn1 knockout mice. In vivo, carotid arteries undergo hypertrophic inward remodeling in response to ligation but the increase in media area and thickness and the reduction in lumen diameter are attenuated in arteries from NBCn1 knockout compared to wild type mice.

With CO2/HCO3− present, NBCn1‐mediated HCO3− uptake promotes filopodia generation and migration. Knockout of NBCn1 inhibits hypertrophic inward remodeling of carotid arteries. We propose that acid‐base transport, particularly via NBCn1, is a promising target for treatment of occlusive artery disease.

Support or Funding Information

Danish Research Council, Danish Heart Foundation
High Affinity Bent β2 Integrin Binds Ligand in Cis and Regulates Inflammation
Authors: Fan Zhichao , McArdle Sara , Mikulski Zbigniew , Gutierrez Edgar , Ginsberg Mark , Groisman Alex , Ley Klaus
Published: November 2023

Integrins are bidirectional signaling molecules that are bent at rest. Upon cell activation, integrins can extend (E+) and acquire a high affinity conformation with an “open” headpiece (H+). The canonical “switchblade” model of integrin activation proposes that the E+ conformation precedes H+, and the two are thought to be structurally linked. Only the E+H+ conformation can mediate cell adhesion by binding to ligand in trans. Here, by using high‐resolution quantitative dynamic footprinting (qDF) microscopy combined with a homogenous conformation‐reporter binding assay, we show that a substantial fraction of β2 integrins on human neutrophils acquires an unexpected E−H+ conformation not previously seen on primary cells. We show that E−H+ integrin is functional because it binds its ligand intercellular adhesion molecule 1 (ICAM‐1) in cis and significantly inhibits neutrophil adhesion under flow, which is demonstrated by Förster resonance energy transfer assay, qDF imaging and microfluidic neutrophil adhesion assay and intravital cremaster imaging on neutrophil adaptive transfered mice. These findings suggest that integrin extension does not necessarily precede high affinity and the two are regulated independently.

Support or Funding Information

NIH funding HL078784
Endothelial Cell Secreted MIF Regulates Pericyte Contractility to Decrease Barrier Function
Authors: Pellowe Amanda , Hou Yue , Harris Mariah , Liu Rebecca , Pober Jordan S , Gonzalez Anjelica
Published: November 2023

Microvascular endothelial cells (EC) and pericytes (PC) regulate neutrophil (PMN) transmigration through a variety of mechanisms, including adhesion molecule expression and contractility. Specifically, the contraction of endothelial cells creates F‐actin docking structures rich in intercellular adhesion molecule 1 (ICAM‐1) expression and generates gaps between cells that allow for PMN transmigration. Recent evidence has alternatively suggested that PC relaxation, rather than contractility, results in the loss of focal adhesions, disassembly of stress fibers, and subsequent increase in PMN transmigration. Both in vivo and in vitro experimental systems of composite EC/PC microvascular constructs demonstrate that PC are less permissive to neutrophil transmigration in the absence of EC, suggesting that communication between EC and PC may regulate PC contractility. Here, we demonstrate that tumor necrosis factor alpha (TNFa) induced EC secretion of the pro‐inflammatory cytokine macrophage migration inhibitory factor (MIF), which is accompanied by a TNFa induced upregulation of the MIF receptor (CD74) in PC, significantly decreases PC contractility. We confirm that TNFa mediated transmigration is differentially regulated by contractility. EC contractile ability is required to facilitate PMN transmigration through the formation of F‐actin docking sites and ICAM‐1 localization. In contrast, inhibition of PC contractility increases PMN transmigration via mechanisms that are not related to the loss of ICAM‐1 localization and F‐actin disassembly, but rather the loss of focal adhesion plaques, the loss of tension fibers and the formation of PC junctional gaps. We have confirmed that EC secreted MIF signaling results in the loss of PC focal adhesion plaques and significantly decreases PC contractility. This study suggests for the first time that EC paracrine signaling can mediate PC regulated PMN transmigration, and specifically that EC to PC MIF signaling may be an important factor for reducing PC barrier function during inflammation.

Support or Funding Information

Dubinsky Initiative
A BIOPOLYMER‐STABILIZED VEGF CHIMERA REVERSES ANGIOGENIC IMBALANCE IN THE REDUCED UTERINE PERFUSION PRESSURE MODEL OF PREECLAMPSIA
Authors: LOGUE OMAR CHRISTOPHER , Mahdi Fakrhi , George Eric Matthew , Bidwell Gene Lee
Published: November 2023

Preeclampsia (PE) is a hypertensive disorder that complicates approximately 5–8% of all pregnancies. The unknown etiology of PE originates from defective spiral artery remodeling at the maternal‐placental interface. The resulting hypoxia creates placental ischemia from which the anti‐angiogenic factor soluble fms‐like tyrosine kinase‐1 (sFlt‐1) is released. sFlt‐1 sequesters VEGF from binding to fully functional VEGF receptors, leading to endothelial dysfunction and maternal hypertension. The rodent model of PE, reduced uterine perfusion pressure (RUPP), exhibits the sFlt‐1 pathophysiology observed in human PE. To restore endothelial function impaired by excessive placental sFlt‐1 production, we have constructed a VEGF chimera fused to an Elastin‐like Polypeptide (ELP) carrier with increased in vivo half‐life and stability. We observe that ELP‐VEGF exhibits potency equal to that of free VEGF in stimulating tube formation in human umbilical vein vascular endothelial cells (HUVEC) cells exposed to serum from pregnant RUPP Sprague‐Dawley rats. Moreover, intraperitoneal administration of ELP‐VEGF at 5 mg/kg/day ameliorates hypertension in timed pregnant RUPP rats. Pharmacokinetic and biodistribution studies of this therapeutic dose demonstrate nearly complete fetal exclusion. These data indicate that ELP‐delivered VEGF has the potential to preserve endothelial function by counteracting the anti‐angiogenic factors circulating in the maternal plasma while preventing fetal exposure to VEGF supplementation.

Support or Funding Information

AHA 13SDG16490006 to Bidwell

NHLBI 1R01HL121527 to Bidwell

NHLBI 1R00HL116774 to George

Ruth L. Kirschstein INRSA 5 T32 HL 105324‐5 to Logue
Decreased Uterine Vascularization and Uterine Arteriolar Expansive Remodeling by Matrix Metalloproteinases in Hypertensive Pregnancy
Authors: He Hong , Lin Chen , Ren Zongli , Zhu Minglin , Khalil Raouf A
Published: November 2023

Normal pregnancy is associated with extensive remodeling of spiral arteries in order to maintain nutrients and blood supply to the growing fetus. Inadequate placentation and invasion of the uterine wall could lead to uteroplacental ischemia and hypertensive pregnancy (HTN‐Preg); however, the extent of uterine vascularization and the mechanisms involved are unclear. We hypothesized that HTN‐Preg is associated with changes in uterine vascularization and arterial remodeling by matrix metalloproteinases (MMPs). Blood pressure (BP) and fetal parameters were assessed on day‐19 in normal pregnant rats (Preg) and pregnant rats with reduced uterine perfusion pressure (RUPP), and the uteri were isolated for measurement of tissue vascularity and MMPs expression/activity. BP was greater and pups number and weight, and uterus weight were reduced in RUPP vs. Preg rats (BP 128.2±1.7 vs. 91.60±2.8mmHg; pups number 8.8±1.1 vs. 13.00±0.7; pup weight 1.48±0.059 vs. 2.02±0.02g; uterus weight 3.82±0.37 vs. 4.92±0.27g). Histological analysis of uterine tissue sections showed reduced total number and whole area of uterine arterioles in RUPP vs. Preg rats (number 5.75±0.95 vs. 11.50±0.87; area 0.05±0.01 vs. 0.12±0.02mm2). Immunohistochemistry with the endothelial cell marker CD31 showed localization in the uterine arterial intima and confirmed reduction in the number and size of uterine arterioles in RUPP vs. Preg rats. Further analysis of the uterine arterial wall showed reduction in the area of all three layers of the uterine arterial wall in RUPP vs. Preg rats (intima 0.015±0.003 vs. 0.031±0.005; media 0.012±0.002 vs. 0.038±0.005; adventitia 0.021±0.003 vs. 0.048±0.007mm2). Western blots and gelatin zymography showed a decrease in expression and activity of MMP‐2 and ‐9 in uterus and uterine arteries of RUPP vs. Preg rats. Immunohistochemistry showed decreased total amount of MMP‐2 and ‐9 in the uterus, and reduction in MMP distribution in the intima and media of uterine arteries of RUPP vs. Preg rats. Thus, in HTN‐Preg rats there is reduction in the number and size of uterine arteries and decreased expression and activity of MMP‐2 and ‐9. These results suggest that reduced uterine vascularization and uterine arteriolar expansive remodeling by MMPs could be a factor in the uteroplacental ischemia associated with HTN‐Preg and preeclampsia.

Support or Funding Information

This work was supported by grants from National Heart, Lung, and Blood Institute (HL‐65998,HL‐111775).
Perinatal Resveratrol Supplementation to Spontaneously Hypertensive Rat Dams Mitigates the Development of Hypertension in Adult Offspring
Authors: Bourque Stephane L , Care Alison , Sung Miranda , Panahi Sareh , Gragasin Ferrante , Davidge Sandra T , Dyck Jason R
Published: November 2023

Background

The spontaneously hypertensive rat (SHR) is a model of essential hypertension in which high blood pressure (BP) develops after 7 wks of age. Altered vascular resistance profiles and renal hemodynamics are evident by 3 wks of age, implicating these early functional changes as a cause, rather than a consequence, of hypertension in this model. As such, the developmental period before weaning may constitute a critical time for therapeutic intervention.

Objective

This study was undertaken to determine whether perinatal maternal resveratrol (Resv)—a phytoalexin known to confer cardiovascular protection—could prevent the development of hypertension and improve vascular function in adult SHR offspring.

Methods

SHR dams were fed either a control or a Resv‐supplemented diet (4g/kg diet) from gestational day 0.5 until postnatal day 21. Indwelling catheters were used to assess BP and vascular function in vivo; wire myography was used to assess vascular reactivity ex vivo.

Results

Perinatal Resv supplementation in dams had no effect on fetal body weights, albeit continued maternal treatment postnatally resulted in growth restriction in offspring by PD21; growth restriction was no longer evident after 5 wk of age. Maternal perinatal Resv supplementation prevented the onset of hypertension in adult offspring (−18mHg, P=0.007), and L‐NAME treatment normalized these BP differences, suggesting improved NO bioavailability underlies the BP reductions in the SHR+Resv group. In vivo and ex vivo, vascular responses to methylcholine were not different between treatment groups, but prior treatment with L‐NAME attenuated the vasodilation in SHR, but not SHR+Resv adult offspring, suggesting a shift towards NO‐independent vascular control mechanisms in the treated group. Finally, bioconversion of the inactive precursor bET‐1 to active ET‐1 in isolated mesenteric arteries was reduced in SHR+Resv offspring (−28%; P<0.05), and this difference could be normalized by L‐NAME treatment.

Conclusion

Perinatal Resv mitigated the development of hypertension and causes persistent alterations in vascular responsiveness in SHRs.

Support or Funding Information

This work is funded by grants from the Canadian Institutes of Health Research and the Women and Children's Health Research Institute through the generous contributions of the Stollery Children's Hospital Foundation and the Royal Alexandra Hospital Foundation. AC was supported by fellowships from the National Health and Medical Research Council of Australia, Alberta Innovates ‐ Health Solutions, and the Heart and Stroke Foundation of Canada; SD is a Canada Research Chair in Maternal and Perinatal Cardiovascular Health.
Cardiometabolic Risk Factors in the Mouse are Shaped by Metabolic State of the Mother During Pregnancy
Authors: Thompson Jennifer Anne , Mintz James
Published: November 2023

Objective

To determine if gestational diabetes (GDM) programs an aberrant cardiometabolic phenotype in the offspring.

Methods

At 14 weeks of age and after assessment of pre‐conception metabolic status, female wild type (Wt) and female mice heterozygous for the leptin receptor mutation (Hetdb) were mated with Wt males. To confirm development of GDM, intra‐peritoneal glucose tolerance tests (IPGTT) were performed on Gd17 (term = Gd19) in Wt and Hetdb pregnant females. Maternal body composition was determined by NMR spectroscopy. One group of pregnant females was sacrificed on Gd18 for collection of maternal blood; the second group delivered spontaneously. Only Wt offspring were examined. At 3‐weeks (weaning), 3‐months and 6‐months of age, offspring were evaluated for body composition using NMR. At 6‐months, fasting plasma levels of lipids were determined by Colorimetric assay.

Results

Prior to conception, regulation of blood glucose appears normal in Hetdb females compared to Wt controls, yet non‐pregnant Hetdb females displayed increased adiposity and reductions in lean body mass relative to age‐matched, non‐pregnant controls (p < 0.001). On Gd17 there was an increase in fasting blood glucose, insulin and triglyceride levels, and reduction in glucose clearance in pregnant Hetdb vs. Wt females (all p < 0.05). At 3‐weeks of age, % body fat was increased in male (p < 0.001) and female (p < 0.001) Wt offspring born from a Hetdb dam (Male: Wt dam 6.6 ± 0.4 %, Hetdb dam 10.8 ± 0.8 %; Female: Wt dam 7.0 ± 0.37 %, Hetdb dam 10.4 ± 0.7 %). At 3‐months and 6‐months, adiposity remained higher in offspring born to Hetdb dams (p < 0.05). At 6‐months, fasting plasma triglyceride levels were increased 2‐fold in Wt offspring of Hetdb vs. Wt pregnancies; non‐esterified fatty acids (p < 0.01) and cholesterol (p < 0.05) levels were increased by 54% and 68%, respectively, in Wt offspring of Hetdb pregnancy.

Conclusions

The Hetdb female has high pre‐pregnancy adiposity and spontaneously develops glucose intolerance during pregnancy and thus is a valuable model for the study of GDM. Similar to human GDM, the Hetdb model programs an abnormal metabolic phenotype in the offspring.

Support or Funding Information

JAT supported by AHA 15POST25730006JAT
Increased hepatic lipogenic gene expression correlates with enhanced central and ectopic adiposity in female C57BL/6 mice exposed to maternal separation
Authors: Murphy Margaret , Cohn Dianne , Wills Caleb , Gilbert Lucas , Powell David , Loria Analia
Published: November 2023

We previously reported that maternal separation (MatSep), a model of early life stress, in combination with high fat (HF) diet leads to exaggerated weight gain; however, female mice present with significantly greater levels of adiposity compared to male mice. The objective of this study was to investigate the sex‐specific differences within MatSep mice in driving fat tissue expansion by measuring 1) food intake and energy expenditure (EE), 2) body fat distribution using MRI, 3) ectopic lipid deposition, 4) systemic and local production of CORT, and 5) liver and white adipose tissue (WAT) gene expression in response to HF diet. MatSep was performed in male and female C57Bl/6 mice with early weaning at PND17 and mice were weaned onto a HF diet for 16 weeks. Normally reared mice served as controls (C). A group of mice (n=6–10) were placed into TSE metabolic chambers to obtain net food intake and EE normalized to lean body mass. In female mice, food intake was significantly higher in MatSep compared to C mice (slope: 0.522±0.59 vs −2.67±1.79, p<0.05) with no differences observed between groups in male mice. MatSep had no effect on EE in both male and female mice. MRI analysis revealed elevated levels of visceral fat in MatSep female mice compared to C (1550.7 ± 233.5 vs 904.1 ± 104.5 mm3 p=0.08, n=4). In addition, oil Red O staining within the liver and kidney revealed an elevated accumulation of neutral lipids suggesting that MatSep increases both central and ectopic adiposity. In response to HF diet, plasma levels of CORT were not different between MatSep and C in male (145.8 ± 38.6 vs. 111.04 ± 35 ng/ml, respectively) and female mice (193.8 ±36.3 vs. 176.5 ± 31 ng/ml, respectively, 2‐way ANOVA: NS, n=6). RT‐PCR analysis revealed that MatSep upregulated 11β‐HSD1 in subcutaneous WAT in both genders; however, only female MatSep mice showed upregulation of 11β‐HSD1 in liver (3.71±0.61 vs 0.82±0.37 2^ddCt, p<0.05, n=6–8) suggesting an elevated local CORT regeneration. In addition, we found a significant upregulation of several hepatic lipogenic genes including PPARγ, ACC‐1, FAS, SCD‐1, and CREB in MatSep females compared to C. No differences between MatSep and C were found in hepatic lipogenic genes from male mice. Interestingly, obese MatSep female mice showed increased ER β expression compared to C in gonadal WAT (113.91±22.63 vs. 1.41±0.43 2^ddCt, p<0.05) and SQ WAT (56.34±11.19 vs 1.05±0.23 2^ddCt, p<0.05) which may have diabetogenic effects. No differences in ER β expression were observed in male mice. We propose that excessive glucocorticoid signaling at the tissue level may contribute to the exacerbated lipogenesis and fat expansion in female mice exposed to MatSep, suggesting a potential mechanism by which female mice exposed to early life stress develop metabolic dysfunction.

Support or Funding Information

NIH R00 HL111354 COBRE P20 GM103527‐06
Pulmonary and Systemic Vascular Responses in Rats Exposed to Perinatal Hyperoxia
Authors: Greiner Tyler J. , Rotella Diane , Bates Melissa L.
Published: November 2023

In the last 25 years, advances in perinatal therapy have dramatically increased the survival of very premature infants. Still, because the lungs of these babies are underdeveloped, they frequently require supplemental oxygen. The overall objective of our work was to investigate the impact of perinatal supplemental oxygen on pulmonary and systemic vascular reactivity. We hypothesized that exposure to oxygen leads to autonomic imbalance that promotes heightened hypoxic pulmonary vasoconstriction and the baroreflex in response to hypo‐ and hypertensive stress. To test our hypothesis, we used a well‐described rat model of 80% oxygen exposure for 14 days immediately after birth and comparable controls exposed to room air (21%). When they reached adulthood, two to three months post‐exposure, the change in pulmonary vascular resistance during 10 minutes of exposure to 12% oxygen was measured. We found that pulmonary vascular resistance was higher during the first two minutes of hypoxia in rats exposed to perinatal oxygen. This heightened vasoconstrictor response could be eliminated by stimulating the vagus nerve. The baroreflex was also evaluated using the Modified Oxford Technique during exposure to 21%, 12% and 100% oxygen. Our studies revealed that the baroreflex response to sodium nitroprusside was reduced during hypoxia in female rats that had been exposed to 80%. We speculate that they have higher sympathetic tone during hypoxia, limiting their ability to respond to acute hypotension. Together, our studies suggest that rats exposed to perinatal oxygen have long‐term autonomic abnormalities that persist into adulthood, may contribute to pulmonary hypertension and poor systemic blood pressure regulation, and that these effects may be different in male and female subjects.
Enhanced Dietary Fructose Rapidly Induces Salt‐Sensitive Hypertension in Rats
Authors: Gordish Kevin L , Ortiz Pablo A , Garvin Jeffrey L , Beierwaltes William H
Published: November 2023

Fructose consumption is associated with increased sodium reabsorption, increased formation of reactive oxygen species, and the development of hypertension. In normal rats, 20% dietary fructose supplementation induces hypertension within 8–12 weeks. We hypothesized a diet combining 20% fructose supplemented with high salt (4% NaCl) would induce abnormal sodium retention, increased oxidative stress, and accelerate the development of elevated blood pressure. Rats weighing 200–225 g were pre‐trained for 2 weeks for non‐invasive tail cuff blood pressure measurements and then placed in metabolic caging. Rats were pair‐fed for 2 weeks with one of 4 different diets: 1) control group (0.4% NaCl), 2) high salt chow group (4% NaCl), 3) 20% fructose group (in the drinking water), and 4) 20% fructose plus high salt group (n's = 9,5,9,18). Blood pressure, urinary sodium excretion and cumulative sodium balance were measured over 2 weeks and urinary 8‐Isoprostane excretion, as a marker for reactive oxygen species, during the final two days. High salt chow replaced normal chow at the beginning of the second week. By the end of the protocol, systolic blood pressure was unchanged in control group (121±2 to 122±2 mmHg) as well as the high salt group (122±2 to 122±1 mmHg). 20% fructose alone had no effect on blood pressure over the 2 weeks (121±1 to 125±1 mmHg). However, blood pressure significantly increased in the 20% fructose plus high salt group (125 to 140 mmHg p<0.001). The increased blood pressure was concomitant with increased positive cumulative sodium balance. During the final week mean urinary sodium excretion in the high salt and 20% fructose plus high salt groups were 5–6 fold higher than normal salt controls (p<0.001): 1.13±0.07, 7.67±0.31, 1.20±0.10, 5.33±0.21 μmol/24 hrs, respectively. Despite similar consumption, urinary sodium excretion in the 20% fructose plus high salt group was significantly lower than the high salt group (p<0.0001). Urinary 8‐Isoprostane excretion was significantly higher in the high salt, 20% fructose, and 20% fructose plus high salt groups compared to control: 16.4±1.3, 29.5±2.5, 32.2±3.9, 31.2±2.5 ng/24 hrs, (p<0.001). Our results suggests enhanced fructose and salt consumption increases sodium retention and oxidative stress. High salt can induce elevations in blood pressure in 20% fructose‐fed rats within just one week. Increased blood pressure appears to associate with increased sodium retention and positive cumulative sodium balance. Overall, fructose combined with high salt in the diet contributes to the accelerated development of salt‐sensitive hypertension.

Support or Funding Information

National Institutes of Health Grant 5P01HL090550‐05
Hypoxia precedes proteinuria in mice with diabetic nephropathy
Authors: Franzen Stephanie , Pihl Liselotte , Gustafsson Håkan , Palm Fredrik
Published: November 2023

Kidney hypoxia has been suggested to be a unifying mechanism for the progression of diabetic nephropathy and chronic kidney disease. Though, for hypoxia to be the actual causing mechanism it needs to be present before other functional alterations. Proteinuria is a commonly used biomarker for kidney damage and therefore, we investigated if hypoxia can be seen as early as proteinuria in mice with diabetic nephropathy.

Kidney cortex oxygen tensions were analyzed using a minimally invasive electron paramagnetic resonance oximetry technique with implanted oxygen sensing probes. Measurements were made before and up to 15 days after the induction of type‐1 diabetes in male mice and compared to corresponding controls. After 16 days, proteinuria and conscious glomerular filtration rates were analyzed in order to investigate the progressive relationship between kidney hypoxia and disease progression.

Diabetic mice developed pronounced intrarenal hypoxia three days after the induction of diabetes, which maintained throughout the study. On day 16, diabetic mice had increased glomerular filtration rate but normal proteinuria.

In conclusion, kidney hypoxia precedes proteinuria and could therefore be considered a promising biomarker for the progression if kidney damage.

Support or Funding Information

All experiments was conducted according to the FASEB Statement of Principles. The study was supported and funded by the Swedish Research Council, the Swedish Diabetes Foundation and the Swedish Heart Lung Foundation.
Angiotensin receptor blockade and glucagon like peptide‐1 receptor activation ameliorates NADPH oxidase 4‐associated oxidative injury in insulin‐resistant rats
Authors: Escobedo Benny , Rodriguez Ruben , Nishiyama Akira , Moreno Meagan , Ortiz Rudy
Published: November 2023

Diabetic nephropathy is associated with oxidative stress and increased renal NADPH oxidase 4 protein expression. Glucagon‐like peptide‐1 receptor (GLP‐1r) activation decreases glomerular NOX 4 expression and albumin excretion in streptozotocin‐induced diabetic rats. Angiotensin receptor type 1 (AT1) blockade improves renal oxidative injury via downregulation of NOX 4 and reducing urinary albumin excretion. To test the hypothesis that the combination of GLP‐1r activation and AT1 blockade decreases oxidative stress and subsequent kidney damage, we measured renal NOX 4 protein expression and albumin excretion in five rat groups: 1) untreated, lean LETO (n=7), 2) untreated, obese OLETF (n=9), 3) OLETF + angiotensin receptor blocker (ARB; 10 mg olmesartan /kg/d; n=9), 4) OLETF + GLP‐1 mimetic (Exe; 10 ug exenatide/kg/d; n=7), and 5) OLETF + ARB + exenatide (combo; n=6). Albumin excretion increased in OLETF compared to LETO; whereas, ARB and Exe decreased it, and combo treatment decreased it further. Renal NOX 4 protein expression increased 23% in OLETF compared to LETO. Exe and ARB decreased NOX 4 protein expression in OLETF rats by 18% and 14% respectively, while the combo treatment decreased it further by 24%. These data suggest that AT1 blockade and GLP‐1r activation ameliorate oxidative renal injury, highlighting the impact of the activation of these receptors in the pathogenesis of diabetes‐associated renal impairments.
Chronic Renal Hypoxia Leads to Injury and Dysfunction: Role of HIF1alpha
Authors: Welch William J , Araujo Magali , Hosszu Adam , Luo Zaiming , Solis Glenn
Published: November 2023

Renal hypoxia precedes renal injury and impaired GFR in multiple renal disease models and in hypertension. Further, in imaging studies in humans, renal hypoxia correlates with hypertension and reduced renal function. We propose that renal injury that accompanies hypertension is mediated by events stimulated by hypoxia and responses linked to HIF1a. To test these effects, we developed a pilot study to generate reproducible renal hypoxia in rats by treatment with 2,4‐dinitrophenol (DNP, 30 mg/kg/day). DNP is a mitochondrial uncoupling agent that acts as a proton ionophore, which reduces production of ATP, yet still consumes O2. Renal cortical and medullary pO2 levels were lower in DNP‐treated rats compared to vehicle (Cortex pO2: 34±4 vs 44±4 mmHg; Medulla pO2: 22±3 ve 31±3 mmHg, p<0.001, n=6). Blood pO2 was unchanged by DNP treatment. Expressions of HIF1a and the pro‐oxidant enzyme, NADPH oxidase‐2 (NOX‐2) were increased in DNP‐treated rats. Conversely the antioxidant enzyme, superoxide dismutase‐2 (SOD‐2), was not changed. Further, the renal injury markers TGFb and NGAL were higher in DNP‐treated rats. MAP, basal GFR and RBF were unchanged in response to DNP‐generated renal hypoxia. Separate DNP‐fed rats were treated with direct left renal injections of siRNA directed to HIF1a, 48 hours prior to measuring renal pO2. Expression of HIF1a was reduced by 66±3% in the left kidneys treated with siRNA in DNP‐treated rats and was 32% lower than HIF1a in Veh‐treated rats. Expression of HIF1a in the right kidney was not altered by siRNA injection in the left kidney. Knockdown of HIF1a further reduced pO2 compared to DNP‐treated rats (Cort pO2 = −4±2 mmHg; Med pO2 = −8±1 mmHg, p<0.01), suggesting HIF1a protects the kidney from greater renal hypoxia. Further, expression of renal injury markers was higher in HIF1a reduced kidneys. We conclude that chronic renal hypoxia induces renal injury via oxidant pathways and stimulates HIF1a, which is only partially protective. DNP‐induced renal hypoxia is a viable model to test the long‐term effects of hypertension on renal oxygen metabolism.

Support or Funding Information

HL089583; HL068686
Prasugrel Suppresses Development of Lithium‐induced Nephrogenic Diabetes Insipidus in Mice
Authors: Kishore Bellamkonda K , Brandes Anna U , Carlson Noel G , Zhang Yue
Published: November 2023

Previously we localized ADP‐activated P2Y12 receptor in rodent kidney and showed that its blockade by clopidogrel bisulfate (CLPD) attenuates lithium(Li)‐induced nephrogenic diabetes insipidus (NDI) (Zhang et al, JASN ePub 2015; Zhang et al, Puriner Signal ePub 2015). In order to explore whether such attenuation is possible by the use of other drugs that are known to block P2Y12 receptor, we evaluated the effect of administration of prasugrel (PRSG) on Li‐induced NDI in mice. Similar to CLPD, PRSG belongs to thienopyridine class of ADP receptors inhibitors, and is a prodrug metabolized in the liver into its active form, which irreversibly binds to P2Y12 receptor. Groups of age‐matched adult B6D2 mice (n = 5/group) were fed either regular rodent chow (controls, CNT) or rodent chow with added LiCl (LI; 40 mmol/kg chow) or PRSG in drinking water (PRG; 10 mg/kg bw/day) or a combination of Li and PRSG for 14 days. Twenty‐four hour water consumption and urine output were determined on day 0 and day 14, and the mice were humanely euthanized. Blood and kidney tissues were collected and analyzed. Administration of PRSG completely suppressed Li‐induced NDI as assessed by water intake, urine output and urine osmolality. Semi‐quantitative immunoblotting revealed that administration of PRSG significantly prevented Li‐induced decrease in AQP2 protein abundance in renal cortex (3.4‐fold higher) and medulla (3.2‐fold higher). Serum Li, Na, and K levels and osmolality were not affected by the administration of PRG. Similar to CLPD, PRSG administration had no effect on Li‐induced increase in urinary Na excretion. Taken together, our data suggest that pharmacological inhibition of P2Y12 receptor by thienopyridine group of drugs may potentially offer therapeutic benefits in Li‐induced NDI.

Support or Funding Information

Dept. of Veterans Affairs
Endothelin‐1 mediates glomerular and tubular injury in sickle cell mice
Authors: Kasztan Malgorzata , Sun Chiao‐Wang , Townes Tim M. , Pollock David M.
Published: November 2023

Sickle nephropathy (SN) is associated with structural and functional abnormalities in kidneys that lead to albuminuria/proteinuria, a major mortality risk factor in sickle cell disease (SCD). Elevated urinary endothelin‐1 (ET‐1) excretion reported in SCD patients correlates with microalbuminuria, suggesting a pathophysiological link between ET‐1 and development and progression of SN. The aim of the study was to determine whether ET‐1 contributes to glomerular and tubular injury in SCD and if ETA receptors blockade ameliorates these damages. Experiments utilized 14 week old humanized sickle cell mice (HbSS) and genetic controls (HbAA). Ambrisentan (ETA antagonist), A‐182086 (ETA/B antagonist) or vehicle was administrated via drinking water (10mg/kg/day) for 10 (beginning at weaning) or 2 weeks (beginning at 12 week of age). Glomeruli were isolated for direct permeability measurements as a volume response to an oncopressive medium generated by changing concentrations of albumin. Markers of glomerular and tubular injury were measured in 24h urine samples. Administration of ambrisentan for 2 weeks significantly reduced elevated Palb (0.22±0.03 vs. 0.48±0.05), urinary albumin (20.2±3.9 vs. 68.5±20.7μg/24h) as well as markers of tubular injury excretion: KIM‐1(171.3± 13.4 vs. 347±53 pg/24h) and NAG (3.07±1.9 vs. 15.9±1.5 U/L) in HbSS mice. Treatment with the dual ETA/B receptor antagonist had similar effect on Palb, however there was no effect on the excretion of the markers of glomerular and tubular injury. Administration of ambrisentan for 10 weeks significantly reduced elevated Palb (0.14±0.03 vs. 0.52±0.04), markers of glomerular injury: albumin (17.2±4.6 vs. 38.7±5.8μg/24h) and protein (0.74± 0.17 vs. 3.23± 0.4mg/24h), as well as markers of tubular injury excretion: KIM‐1(96.1± 21.6 vs. 373±111.5pg/24h) and NAG (5.8±2.1 vs. 14.5±1.2 U/L) in HbSS mice. Treatment with the dual ETA/B receptor antagonist had similar effect on Palb, however there was no effect on the excretion of the markers of glomerular and tubular injury. Neither antagonist altered any of the measured parameters in HbAA mice. These data support the hypothesis that ET‐1 contributes to the development and progression of sickle cell nephropathy and support the use of chronic ETA antagonism as a prospective treatment for sickle cell nephropathy.

Support or Funding Information

This work was supported by the program project grant on The Role of Endothelin‐1 in Sickle Cell Disease (U01 HL117684).
Gene Set Enrichment Analysis of the Renal Cortex of Fructose Fed Rats
Authors: Gonzalez‐Vicente Agustin , Hopfer Ulrich , Garvin Jeffrey L
Published: November 2023

Rats consuming 20% of their calories as fructose or a high‐salt diet are able to maintain blood pressure within physiological range. However when animals are giving high salt in addition to 20% fructose, their blood pressure rises; i.e. they exhibit salt‐sensitive hypertension. Fructose‐induced salt‐sensitive hypertension is likely important to more than 30 million Americans because they consume 20% or more of their calories as fructose and the average American diet also contains excessive salt. The kidney is important in blood pressure regulation because it is the primary regulator of salt and volume homeostasis. Therefore changes in renal function are required to initiate salt‐sensitive increases in blood pressure. More than 70% of the salt filtered by the glomerulus is reabsorbed in cortical segments of the nephron. Therefore, we measured the change in transcription in the renal cortex induced by dietary fructose in the presence and absence of excess of salt. We hypothesize that a diet containing moderate amounts of fructose predisposes to salt‐sensitive hypertension by affecting pathways that regulate Na reabsorption in the renal cortex. To test our hypothesis, we perform Gene Set Enrichment Analysis (GSEA) of the renal cortex of rats consuming either tap water or a 20% fructose solution for 1 week. We used the Affymetrix RATgene2 microchip, and compared the results using BAMarray software. From a total of 220231 probes mapping within exons; 2207 were upregulated and 2047 downregulated by the diet. After filtering the results to correct for multiples probes mapping within the same gene, we found that 1346 genes were upregulated and 1033 genes were downregulated by fructose. The gene list was load into the Database for Annotation, Visualization and Integrated Discovery (DAVID) and the affected pathways visualized in the Kyoto Encyclopedia of Genes and Genomes. We found that 24 major pathways were affected by fructose. Some of these pathways such as signaling cascades involving MAPK, Calcium, and PIP3, as well as some genes mediating aldosterone signaling are important in salt handling by the kidney, and might be responsible for the fructose induced salt‐sensitivity.
The ubiquitin ligase Ubr4 controls disassembly and stability of mechanosensitive podocin/MEC‐2 ion channel supercomplexes
Authors: Rinschen Markus M , Benzing Thomas
Published: November 2023

The kidney expressed PHB‐domain protein podocin maintains the renal filtration barrier. Podocin and its C. elegans orthologue MEC‐2 have emerged as key components of mechanosensitive membrane protein signalling complexes. Whereas podocin resides at a specialized cell junction at the kidney filtration barrier, MEC‐2 is found in neurons required for touch sensitivity. Here we show that the ubiquitin ligase Ubr4 binds to and colocalizes with podocin in kidney podocytes and regulates podocin stability. We also find that this process is conserved in C. elegans and responsible for the degradation of mislocalized MEC‐2 multimers. Surprisingly, degradation depends on Ubr4‐dependent ubiquitylation of very specific lysine residues in the PHB‐domain that are conserved in evolution. Molecular dynamics simulations revealed that ubiquitylation of these sites does not only target podocin/MEC‐2 for proteasomal degradation but may also affect stability and disassembly of the multimeric complex. We suggest that ubiquitylation of specific lysine residues may represent an evolutionarily conserved principle in proteostasis of podocin/MEC‐2 supercomplexes.

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DFG
Mass spectrometric imaging of metabolites in kidney tissues from rats treated with furosemide
Authors: Choi Hyo‐Jung , Park Eui‐Jung , Kim Dasom , Lee Yu‐Jung , Kwon Tae‐Hwan
Published: November 2023

In the kidney, metabolic processes are different among the cortex (COR), outer medulla (OM), and inner medulla (IM). Using a novel technology of the matrix‐assisted laser desorption/ionization (MALDI) and imaging mass spectrometry (IMS), we examined the regional differences of the metabolites in the kidneys of furosemide‐infused rats. Osmotic minipumps were implanted in male Sprague‐Dawley rats to deliver 12 mg/d/rat of furosemide. Vehicle‐treated‐ (n = 14) and furosemide‐treated‐ (furosemide rats, n = 15) rats in metabolic cages received a fixed amount of rat chow (15 g/220 g bw/day/rat) with free access to water intake for 6 days. At day 6, higher urine output (32 ± 4 vs. 9 ± 1 ml/day) and lower urine osmolality (546 ± 44 vs. 1,677 ± 104 mOsm/KgH2O) were observed in furosemide rats. Extracts of COR, OM, and IM were analyzed by UPLC/Q‐TOF‐MS, where multivariate analysis revealed significant differences between the two groups. Several metabolites, including acetyl carnitine, betaine, carnitine, choline, and glycerophosphorylcholine (GPC), were significantly changed. The changes of metabolites were further identified by MALDI‐TOF/TOF and IMS. Importantly, their spatial distribution and relative quantitation in the kidneys were analyzed by IMS. Choline compounds were increased in COR and OM, but decreased in IM from furosemide rats. Carnitine compounds were increased in COR and IM, whereas carnitine and acetyl carnitine were decreased in OM. Betaine and GPC were decreased in OM and IM. Taken together, MALDI‐TOF/TOF and IMS successfully provide the spatial distribution and relative quantitation of metabolites in the kidney.

Support or Funding Information

This study was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning, Korea (2013R1A1A2007266, and 2014R1A5A2009242).
High Fructose Concentrations Could Cause Changes in Intracellular Signaling Pathways and transepithelial electrical resistance in Proximal Tubule Cells
Authors: Matsumoto Larissa Emi , Yokota Rodrigo , de Oliveira Leite Ana Paula , Jara Zaira Palomino , Casarini Dulce Elena
Published: November 2023

In the western lifestyle is observed the excessive consumption of industrialized foods composed with high fructose. This sugar is an important contributor of the Metabolic Syndrome (MS). MS is a set of pathologies that includes cardiovascular and renal diseases, like diabetes, hypertension, and obesity. Recent studies showed that hypertension in MS is induced by high fructose consumption and it is associated with direct changes of Renin‐Angiotensin System (RAS) balance. This system is present locally in several tissues. In the kidney, the proximal tubule (PT) that is the major region of sodium and water re‐absorption expresses all of the RAS components. In high fructose ingestion, inflammation has been observed in this region, but it is unclear how Ang II contributes to fructose inflammatory process, as well as which signaling pathways are involved in this process. We hypothesized that high fructose intake can increase Ang II concentration, which results in activation of proinflammatory second messengers as mitogen‐activated protein kinases, including extracellular‐signal‐regulated kinases (ERK 1/2), as well as modified the transepithelial electrical resistance (TEER) consequently impacting sodium and water transport. To verify the ERK phosphorilation pattern, it was used immortalized rat proximal tubule cells (IRPTC) treated with high fructose concentrations, RAS antagonists (angiotensin II type 1 receptor (AT1R) antagonist: losartan, angiotensin II type 2 receptor (AT2R) antagonist: PD123319, and angiotensin‐(1–7) antagonist: D‐Ala), and angiotensin‐converting enzyme inhibitor. To verified TEER changes it was used IRPTC treated with fructose, fructose + NaCl, and NaCl alone. Our results indicate that the group treated with fructose together with D‐Ala and PD123319 enhanced phosphorilated ERK 1/2 expression. The TEER results showed that voltage in fructose and fructose + NaCl treated cells were significantly decreased compared to the negative (without any treatment) and positive (NaCl) control. In conclusion, the enhanced phosphorilation of ERK via AT2R and MAS antagonism could indicate; first, modulation of angiotensins levels, second that there is anti‐inflammatory response activated; and third, this response could be increase to compensate the inflammatory process fructose‐dependent. The drastic voltage reduction could be involved in modulation of sodium transporter such as NHE3, among others and modulation of AQP‐1, main water channel in the PT.

Support or Funding Information

Supported by Capes
Subunit‐Specific Role of the Epithelial Na+ Channel Thumb Domain
Authors: SHENG SHAOHU , CHEN JINGXIN , TOLINO MICHAEL A. , KLEYMAN THOMAS R.
Published: November 2023

Members of the epithelial Na+ channel/degenerin (ENaC/DEG) family of ion channels have large extracellular domains with common structural features, including a β‐strand central core embraced by helical peripheral domains (referred to as finger, thumb and knuckle). We hypothesized that the peripheral helical domains serve as functional modules that facilitate interactions between ENaC and extracellular regulatory factors. In this study, we examined the properties of channels where the thumb domain was deleted from a specific subunit. We found that channels lacking the γ subunit thumb domain retained activity when expressed in Xenopus oocytes, but channels lacking the α or β subunit thumb domain had greatly reduced activity. Surface expression of channels lacking the thumb domain of any subunit was significantly lower than wild‐type. Channels with a γ subunit thumb domain deletion exhibited a loss of the Na+ self‐inhibition response, suggesting a high channel open probability in the presence of a high extracellular Na+ concentration. We conclude that the presence of the thumb domain in each ENaC subunit is required for optimal surface ENaC expression in oocytes. Furthermore, the presence of the γ thumb domain is required for the inhibitory response to extracellular Na+ (Na+ self‐inhibition).

Support or Funding Information

Supported by NIH ES014701, DK051391 and P30 DK079307
Simulation of Cl− secretion in epithelial tissues
Authors: Marunaka Yoshinori , Sasamoto Kouhei , Niisato Naomi , Taruno Akiyuki
Published: November 2023

Transcellular Cl− secretion is mediated by two steps; 1) the entry step of Cl− into the cytosolic space from the basolateral space across the basolateral membrane by Cl− transporters, such as Na+‐K+‐2Cl− cotransporter, and 2) the releasing step of Cl− from the cytosolic space into the luminal (air) space across the apical membrane via Cl− channels. Various experimental techniques have characterized transcellular Cl− secretion: e.g., short‐circuit current techniques and patch clamp techniques provide us useful information on transepithelial ion movements. Many investigators have tried to clarify roles of Cl− channels and transporters located at the apical and basolateral membranes in transcellular Cl− secretion. However, we have little information how Cl− channels/transporters contribute to transcellular Cl− secretion and are regulated by various stimuli such as Ca2+ and cAMP. In the present study, we tried to simulate transcellular Cl− secretion using mathematical models combined with electrophysiological measurements, providing information on contribution of Cl− channels/transporters to transcellular Cl− secretion, activity of electro‐neutral ion transporters and how Cl− channels/transporters are regulated.