bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Use of Precision‐Cut Kidney Slices (PCKS) to Investigate Intracellular Calcium Signaling and Contraction in Small Renal Arteries of Heathy and Diabetic Nephropathy Rats
Authors: Perez‐Zoghbi Jose F , Roghani Ali 
Published: November 2023

Diabetic nephropathy (DN) is a leading cause of mortality and morbidity in diabetic patients. Hyperglycemia‐induced metabolic stimuli and hemodynamic factors including elevated glomerular capillary hydrostatic pressure are the main cause of kidney injury leading to a progressive decline in glomerular filtration rate, proteinuria, and cardiovascular complications. Although control of glycemic and systemic hypertension somewhat improve disease management, a significant number of DN patients are still in need of new and effective therapeutics. Glomerular hydrostatic pressure is regulated locally by changes in vascular resistance of small pre‐ and post‐glomerular arteries. However, the mechanisms that regulate the constriction of these small renal arteries are not fully understood and whether DN causes alterations to these mechanisms is unknown. To investigate contraction and signaling mechanisms in the small renal arteries, we have developed a unique, “living”, precision‐cut kidney slice (PCKS) preparation and employed video phase‐contrast and confocal microscopy to determine dynamic changes in artery cross‐sectional lumen area and in Ca2+ signaling in their vascular smooth muscle cells (VSMC). We found that small renal arteries in SD rat constricted strongly in response to the vasoconstrictors phenylephrine (PE), 5‐hydroxytriptamine (5‐HT), and angiotensin II (Ang‐II). The contractile responses developed fast, were sustained during the presence of the vasoconstrictor, and were quickly reversed upon vasoconstrictor washout. Addition of nitric oxide (NO) donor SNAP to arteries pre‐contracted with 5‐HT generated strong artery dilation that persisted in the presence of SNAP and was reversed after SNAP washout. Vasoconstrictor‐induced artery narrowing was accompanied by the induction of intracellular Ca2+ oscillations in the VSMCs. These Ca2+ oscillations propagated as repetitive Ca2+ waves in individual VSMCs, persisted with a constant frequency in the continuous presence of the vasoconstrictor, and ceased after vasoconstrictor washout. We conclude that the use of PCKS with imaging tools is a novel and promising approach to investigate contraction and cell‐signaling in small renal arteries in health and disease states.Support or Funding InformationAmerican Heart Association
Meloxicam fails to augment the reno‐protective effects of soluble epoxide hydrolase inhibition in streptozotocin‐induced diabetic rats via up‐regulation of cytochrome P450 hydroxylase‐mediated 20‐HETE production
Authors: Katary Mohamed M , Pye Chelsey , Elmarakby Ahmed A. 
Published: November 2023

Previous studies suggest that the inflammatory cyclooxygenase (COX)‐derived prostaglandins and the anti‐inflammatory cytochrome P450 epoxygenase‐derived epoxyeicosatrienoic acids (EETs) play an important role in the regulation of vascular injury. The current study examined whether COX inhibition exacerbates the reno‐protective effects of increased EETs levels, via inhibiting EETs degradation by soluble epoxide hydrolase (sEH) using sEH inhibitor, in type 1 diabetic rats. Twelve week old male Sprague Dawley rats were used to induce diabetes using streptozotocin (50 mg/kg, i.v). Rats were then divided into 5 groups (n=6); control non diabetic, diabetic, diabetic treated with the sEH inhibitor trans‐4‐[4‐(3‐adamantan‐1‐yl‐ureido)‐cyclohexyloxy]‐benzoic acid (t‐AUCB, 10 mg/L in drinking water), diabetic treated with the COX inhibitor meloxicam (5 mg/kg/day in drinking water) and diabetic treated with both inhibitors for two months. Inhibition of sEH reduced markers of renal injury in diabetic rats as it significantly reduced proteinuria and creatinine clearance (P< 0.05). Inhibition of sEH also reduced glomerular permeability to albumin and restored the decrease in glomerular integrin and nephrin expression levels in diabetic rats. Meloxicam treatment failed to reduce renal injury or even to synergize the reno‐protective effects of sEH inhibitor in diabetic rats. Moreover, inhibition of sEH reduced renal collagen deposition and urinary monocyte chemoattractant protein‐1 (MCP‐1) excretion levels together with a reduction in the number of renal TUNEL positive cells in diabetic rats (P< 0.05). Meloxicam did not significantly reduced renal inflammation or apoptosis in diabetic rats or even exacerbate the anti‐inflammatory and anti‐apoptotic effects of sEH inhibition. Expression levels of renal cytochrome P450 hydroxylase (CYP4A) derived 20‐HETE production and the inflammatory 20‐HETE levels were elevated in meloxicam treated diabetic rats. In conclusion, increasing EETs levels in type 1 diabetic rats, using sEH inhibition, reduced renal apoptosis, inflammation and injury and inhibition of COX failed to provide renal protection or to augment the reno‐protective effect of sEH inhibition in diabetic rats, at least in part, via increased CYP4A derived 20‐HETE production.Support or Funding InformationGeorgia Regents University Bridge funding to AAEAmerican Heart Association Scientist development Grant AAE
ER Stress and Mitochondrial ROS Contribute to the Development of Hypertensive‐Diabetic Nephropathy
Authors: Wang Zhen , do Carmo Jussara M. , Hall John E. 
Published: November 2023

Hypertension (HT) is common in diabetes and substantially increases the risk for diabetic nephropathy. Current therapies only slow the progression to end stage renal disease (ESRD) in hypertensive‐diabetic patients rather than halt it. Thus, new insights into the mechanisms of this disease are needed. To investigate the mechanisms by which HT and diabetes interact to promote nephropathy, 6 month‐old male Goto‐Kakizaki (GK) rats, a model of spontaneous type 2 diabetes, were used in this study. HT was induced by aorta coarctation (AC) between the renal arteries to determine the impact of hypertension plus diabetes in the right kidney (above the coarctation) and normal or slightly reduced blood pressure plus diabetes in the left kidney (below the coarctation). Blood pressures (BP) above and below the AC were measured by telemetry and femoral artery catheter, respectively. Glomerular filtration rate (GFR) and urinary albumin excretion (UAE) in left and right kidneys were measured separately by collecting urine from each kidney to evaluate the change of renal function.To assess the potential roles of ER stress and mitochondrial dysfunction in causing kidney injury, GK rats with AC were treated with the ER stress inhibitor, tauroursodeoxycholic acid (TUDCA, 200 mg/kg/day, i.p., 2 to 8 wks after AC), or Mito‐TEMPO, a mitochondrial targeted antioxidant (0.4mg/kg/day, osmotic mini‐pump, 2 to 6 wks after AC) and BP and renal function were examined.After 6 weeks of AC, BP above the AC and urinary albumin excretion (UAE) were significantly increased from baseline (BP: 155±4 vs 108±2 mmHg; UAE: 39.2±14.8 vs 0.9±0.7 mg/24h, n=6). The ER stress marker CHOP was increased 2‐fold in the HT‐diabetic right kidney compared to the normotensive‐diabetic left kidney. After 6 wks of TUDCA treatment, CHOP expression from the right kidney was significantly reduced by 47% compared to untreated rats, and BP above the AC was significantly lower (135±4 vs 155±4 mmHg, n=7). TUDCA treatment also increased GFR (0.9±0.1 vs 0.6±0.1ml/min/g of kidney weight, n=6) and decreased UAE (5.6±1.5 vs 18.4±5.8 mg/min, n=4–6) when comparing right kidneys between treated and untreated GK‐AC rats. Mito‐TEMPO treatment for 4 weeks in GK‐AC rats also attenuated the BP increase after AC compared to untreated rats (8± 3 vs 17± 5 mmHg, n=3–9).These results suggest that ER stress and mitochondrial generated ROS may contribute to kidney injury when HT is superimposed on diabetes. Pharmacological inhibition of ER stress and mitochondrial ROS attenuate increases in BP and prevent kidney injury in hypertensive‐diabetic nephropathy.Support or Funding InformationFunded by NHLBI‐PO1HL51971, AHA 14POST18160019 and NIGMS P20GM10435776
ENaC in Renal Cortical Collecting Duct Principal Cells Is Stimulated by Increasing Intracellular Ca2+ in The Basal Compartment of The Cell Via a Process Involving Src but not CAMK II
Authors: Thai Tiffany L , Yu Ling , Wu Ming Ming , Galarza‐Paez Laura I , Duke Billie Jeanne , Al‐Khalili Otor , Ma Heping , Eaton Douglas C 
Published: November 2023

We have recently shown that intracellular Ca2+ ([Ca2+]i) in renal cortical collecting duct (CCD) principal cells is compartmentalized and that this compartmentalization is maintained by mitochondria which localize in two separate areas beneath the apical and basolateral membranes and sequester Ca2+ to greatly reduce the rate of cytosolic Ca2+ diffusion. When [Ca2+]i is increased in the cytosolic space adjacent to the apical plasma membrane, ENaC is inhibited, but when [Ca2+]i is increased in the cytosolic space adjacent to the basolateral membrane, ENaC activity is stimulated. While the pathways mediating the effects of apical [Ca2+]i are well characterized, the pathways that transduce the basolateral pathways are unknown. We tested which signaling molecules may be mediating these effects. We used a combination of transepithelial current measurements and single channel patch clamping to identify these molecules. Inhibitors of common Ca2+ ‐sensitive signaling molecules were used in combination with either ionomycin or methyl‐thio‐ATP applied basolaterally to increase [Ca2+]i. Since some basolaterally expressed receptor tyrosine kinases stimulate ENaC via activation of Src and since Src is a Ca2+ ‐sensitive protein, we first tested whether basolateral [Ca2+]i stimulates ENaC via a Src‐mediated pathway. Basolaterally applied ionomycin (15 μM) or methyl‐thio‐ATP (50 μM), causes a two‐fold increase in ENaC open probability (N≥6; P<0.05 per group), but has no effect in the presence of the Src inhibitor PP2 (12 μM; N=6; P>0.5). We also considered another Ca2+ ‐dependent that might have stimulated ENaC. Hypotonicity can stimulate ENaC via activation of calmodulin‐dependent protein kinase II (CAMK II). Using concentrations of the CAMKII inhibitor KN93 from 1–100μM, we found no effect of this drug on amiloride‐sensitive transepithelial current induced by basolateral [Ca2+]i. Overall, we conclude that Src mediates the stimulatory effects of basolateral [Ca2+]i on ENaC but that CAMKII likely does not. Whether this occurs via cross talk with receptor tyrosine kinase signaling pathways remains to be seen.Support or Funding InformationNIH R37‐DK037963 to DCE, NIH R01‐DK100582 to HM and AHA 13POST16820072 to TLT
Vitamin E mitigates the decremental effect of paraquat on Substantia Nigra neurons
Authors: Fahim Mohamed Abdelmonem , Nemmar Abderrahim , Shehab Safa , Dhanasekaran Subramanian , Yasin Javed , Shafiullah Mohamed , Hasan Mohamed 
Published: November 2023

BackgroundParaquat (PAR) is a toxic chemical that is widely used as an herbicide in developing countries and has been described as a major suicide agent. Patients with acute PQ poisoning show damage to the lungs, liver, and kidneys, and some also show symptoms of central nervous system toxicity. The hypothesis tested here is that PAR decreased dopamine producing neurons which may be attributed to oxidative stress and therefore the possible effect of vitamin E as an antioxidants to mitigate such loss would be explored.MethodologyForty adult male Wistar rats were divided into 4 groups. Animals received daily intraperitoneal injections for 3 weeks as follows; Group 1 received PAR (10 mg/kg), Group 2 served as controls and received saline, Group 3 received vitamin E (100mg/kg) and Group 4 received PAR (10mg/kg) and vitamin E (100mg/kg). Dopamine concentrations in the ventral mesencephalon were measured by high performance liquid chromatography and the number of dopaminergic neurons in substantia nigra pars compacta was estimated by tyrosine hydroxylase immunohistochemistry.Principal FindingsThe number of tyrosine hydroxylase positive neurons was significantly less in the substantia nigra pars compacta and nigral dopamine level was significantly reduced in PQ treated animals (20.4±3.4 pg/mg) when compared with control animals (55.0±2.4 pg/mg wet tissue). Daily treatment of PQ for three weeks induces selective dopaminergic neuronal loss in the substantia niagra (32.6±2.1 compared with control animals 47.5±4.2 and vitamin E treated animals 46.0±3.3). PAR caused a significant increase in superoxide dismutase (SOD) and catalase (CAT) levels in brain tissue, indicating that PAR could inititate adaptive responses that counterbalance the potatially damaging activity of oxygen radicals induced by PAR exposure. Adininistration of vitamin E in combination with PAR alleviated most of these adverse effects induced by PAR. SOD and CAT activities were increased in a comparable way in both PAR and vitamin E plus PAR groups.ConclusionsIt can be concluded that dopaminergic neurons in substantia niagra is compromised in rats exposed to PAR and that the abnormalities are ameliorated by vitamin E treatment.
Quantitative Ex Vivo Imaging of Tissue O2 Reveals Effects of Inflammation, Mitochondrial Respiration and ROS Production on O2 Levels in Colonic Mucosa
Authors: Zhdanov Alexander V , Okkelman I. A. , Golubeva A. V. , Aviello G. , Melgar S. , Hyland N. P. , Knaus U. G. , Cryan J. F. , Papkovsky D. B. 
Published: November 2023

IntroductionMucosal epithelium is a multifunctional barrier tissue protecting the organism against bacterial invasion from intestinal lumen. Loss of mucosal barrier function leads to the development of intestinal inflammation, which is associated with re‐arrangement of tissue metabolism and oxygenation. However, data on the oxygenation of healthy and inflamed intestinal mucosa are controversial. In healthy gut, mucosal epithelium sustains deep hypoxia with periodic fluctuations of O2 levels; however the borderline between normal and pathological tissue oxygenation is hard to define. O2 availability reciprocally regulates the composition and activity of colonising bacteria, absorption and permeability of the epithelium and gene expression. Hence, knowledge on local O2 levels is of paramount importance. Here, we present quantitative ex vivo imaging of mucosal oxygenation using high‐resolution phosphorescence lifetime imaging microscopy (PLIM) in different models of intestinal dysfunction.Methods10 week old C57BL/6 mice were given 3% dextran sulphate sodium (DSS group, 7 mice) or water (control group, 7 mice) for 4 days, and then euthanized. The distal colon was dissected for phosphorescent staining, O2 imaging and RNA extraction followed by gene expression analysis. Mucosal epithelium was stained ex vivo with a cell‐penetrating O2 sensitive probe, Pt‐Glc and then imaged on a confocal PLIM system equipped with a time‐correlated single photon counting (TCSPC) module (405 nm excitation and 635–675 nm emission).ResultsPt‐Glc brightly stained mostly epithelial cells within the upper 50–60 μm layer of the tissue. In healthy mucosa O2 levels were 20±15 μM, tending to decrease in deeper tissue areas. Four‐day treatment with DSS induced colonic inflammation, as reported by elevated local IL‐6 and mKC gene expression, and significantly increased mucosal oxygenation, suggesting reduced O2 consumption. Treatment with dimethyloxalylglycine (DMOG), known to protect the tissue against DSS‐induced injury, inhibited mitochondrial respiration and increased mucosal O2. This demonstrates the potential of DMOG to relieve metabolic stress imposed by hypoxia on the inflamed colon in vivo. Analysing the impact of non‐mitochondrial O2 consumption, we found that mucosal oxygenation was affected by NADPH oxidase deficiency. The imaging of human colon biopsies is being pursued, but currently the technique will require refinement to allow discrimination between healthy and inflamed tissue samples.ConclusionsPLIM‐TCSPC technique shows high applicability to study oxygenation of the colonic mucosa and sufficient sensitivity to detect changes in O2 levels in different animal and disease models.Support or Funding InformationThis work was supported by Enterprise Ireland (CF/2012/2346) and Science Foundation Ireland (12/RC/2273)
Antitumor activity of Salvianolic acid against Ehrilich solid carcinoma in Swiss albino mice
Authors: Katary Mohamed , Elmarakby Ahmed 
Published: November 2023

Breast cancer is the current leading cause of cancer death in females worldwide. Current chemotherapeutic drugs have many unwanted side effects. Therefore, new anticancer drugs with less side effects are required. Salvianolic acid (Sal), a bioactive compound isolated from the root of Danshen Radix has antioxidant and anti‐inflammatory activities. Ehrlich tumor is undifferentiated malignant neoplasia derived from breast carcinoma in mice. To investigate whether, and how, Sal may subside Ehrilich solid carcinoma (ESC), mice with established (ESC) tumors received I.P injection of Sal (25 mg/kg, daily for 2 weeks) and compared to control group receiving ESC tumor. Treatment with Sal showed a reduction in the tumor volume by 2 fold when compared to tumor control group. Furthermore, biochemical results showed a decreased in plasma level of thiobarbituric acid reactive substances whereas plasma level of reduced glutathione was raised in treated mice. Importantly, Sal was also able to decrease levels of TNF‐α and cyclin d1 in ESC with no changes in tissue Ki‐67 protein. Histopathological examination also suggested a reduction in the proliferative cells and increases in the necrotic cells necrosis in Sal‐treated group. Immunohistochemical results revealed elevation in levels expression of caspase 3, P53 and number of TUNEL‐positive cells and reduction in level of VEGF expression in Sal‐treated mice. Our data suggest that Sal may exhibit antitumor features through anti‐angiogenic and apoptotic effects in addition to immune modulation and antioxidant activities.Support or Funding Informationpersonal funding
Locomotor and Diaphragm Muscle Mitochondrial Oxidative Capacity in Lung‐Targeted VEGF Gene Deleted Mice Exposed to Cigarette Smoke
Authors: Witte Tyler , Wong Fiona , Lee Jasmine , Fine Janelle , Gutierrez Alma , Bigby Timothy , Cannon Daniel , Breen Ellen 
Published: November 2023

Locomotor skeletal muscle function is impaired in patients with chronic obstructive pulmonary disease (COPD). The mechanisms responsible for these skeletal muscle abnormalities are not well understood. We hypothesized that the severity of lung damage following chronic exposure to cigarette smoke (CS) would determine the mitochondrial oxidative capacity of locomotor and respiratory skeletal muscle.Adult VEGFLoxP mice with or without lung‐targeted VEGF gene deletion (5 months of age) were used as a model of progressively compromised static lung mechanics following exposure to 2 × 30 min of daily, nose‐only CS for an additional 4 months. Deletion of the VEGF gene was accomplished by instilling half the mice with AAV‐Cre one month before initiating CS exposure. The remaining mice received a control AAV (LacZ). Further, both groups (Cre and LacZ) were assigned either to CS exposure or room air control (RA). Mitochondrial respiration in permeabilized fiber bundles from the diaphragm and medial gastrocnemius was measured using high‐resolution respirometry.Gastrocnemius ADP‐stimulated O2 consumption with malate and glutamate as substrates was 78±6%, 66±7% and 60±11% lower in CS‐LacZ, RA‐Cre, and CS‐Cre, respectively, than RA‐LacZ (p<0.05). Gastrocnemius electron transport system capacity was 68±5% (p<0.05), 53±11% (p<0.05) and 48±21% (NS) lower in the CS‐LacZ, RA‐Cre, and CS‐Cre, respectively, than RA‐LacZ. Diaphragm ADP‐stimulated O−2 consumption was 56±9% lower in CS‐LacZ fibers than RA‐LacZ (p<0.05). Diaphragm fiber electron transport system capacity was not different between the groups.Mice with a COPD‐like phenotype exhibited impaired locomotor muscle oxidative capacity irrespective of whether the lung damage was caused by cigarette smoke exposure or VEGF gene deletion. In contrast, diaphragm mitochondrial respiration only showed a deficit in ADP‐stimulated O2 consumption with CS exposure. Thus, mitochondrial respiration in locomotor skeletal muscle appears to be less well‐preserved than diaphragm in this model of COPD.Support or Funding InformationDepartment of Veterans Affairs
Purinergic signaling in sepsis induced by a‐hemolysin producing Escherichia coli in mice
Authors: Greve Anne‐Sofie , Praetorius Helle A. , Skals Marianne 
Published: November 2023

Invasive E. coli are phenotypically different from the sub‐strains found in the intestinal flora and frequently produce the virulence factor α‐hemolysin (HlyA). HlyA forms pores in plasma membranes and the lytic process is markedly amplified by ATP‐release directly through the pore and subsequent P2X receptor activation. Interestingly, free plasma hemoglobin contributes significantly to the symptoms during sepsis and is associated with a poorer outcome in both mice and humans. We hypothesize that ATP release and purinergic signaling is a key player in the pathogenesis of sepsis.MethodsSepsis was induced in anaesthetized mice by iv‐injection of HlyA‐producing E. coli or vehicle. Body temperature and blood pressure were measured continuously and time of death was noted during 6 hours observation. For determination of hemolysis, plasma IL‐1β, IL‐6, TNF‐α, KC and thrombin‐antithrombin complexes (TAT), mice were terminated after 2.5 hours.ResultsMice iv‐injected with HlyA‐producing E. coli demonstrated increased body temperature and pulse pressure. The P2X7−/− mice showed a substantial immunosuppression and with markedly lower survival compared to P2X7+/+. The P2X7−/− mice had increased levels of hemolysis, the pro‐inflammatory cytokines IL‐1β and IL‐6 and TAT‐complexes compared to P2X7+/+. Surprisingly, pre‐injection with BBG (50 mg/kg BW), which primarily targets P2X7, but also inhibits P2X1 and P2X4 receptors, reduced the hemolysis, lowered the plasma‐IL‐1b and showed a tendency for prolonged survival of the mice. Thus, it is likely that either P2X1 or P2X4 has an opposing effect on the prognosis of E. coli induced sepsis.ConclusionP2X7 deficient mice are more susceptible to acute gram‐negative sepsis. Since non‐specific inhibition of P2X receptors has a different profile from the P2X7−/− mice, it is important to investigate specific outcome of sepsis in the absence of functional P2X1 and P2X4 receptors.Support or Funding InformationThis study was funded by:The Danish Council for Independent Research, The Lundbeck Foundation and the research centre MEMBRANES