bluuebunny/crossref_metadata_2025 huggingface dataset:
Sexual dimorphism in mesenteric arterial function of a novel rat model of type 2 diabetes, UC Davis‐Type 2 Diabetes Mellitus (UCD‐T2DM)
Authors: Shaligram Sonali , Akther Farjana , Karimi Mujibullah D , Graham James L , Stanhope Kimber J , Havel Peter , Rahimian Roshanak
Published: November 2023

This is the first study to characterize the vascular functions in a well characterized and validated polygenic rat model of type 2 diabetes (UC Davis‐Type 2 Diabetes Mellitus, UCD‐T2DM), exhibiting etiology more similar to clinical T2DM in humans than other rodent models. The objectives of this study were to investigate whether 1) the endothelium‐dependent and ‐independent vasodilation in mesenteric arteries (MA) are impaired, 2) sex differences exist in the development of abnormal vascular responses, and 3) the relative contributions of endothelium derived relaxing factors in modulating vascular reactivity of MA is altered in UCD‐T2DM Rats.

Endothelium‐dependent vasodilation to acetylcholine (ACh, 10−8 to 10−5 M) in pre‐contracted arteries with phenylephrine (PE, 2 μM) was measured before and after pre‐treatment with indomethacin (cyclooxygenase inhibitor, 10 μM), L‐NAME (nitric oxide synthase inhibitor, 200 μM), or barium chloride (KIR blocker, 100 μM) plus ouabain (Na+‐K+‐ATPase inhibitor, 10 μM). Vascular responses to sodium nitroprusside (SNP) (endothelium‐independent vasodilator, 10−9 to 10−5 M) and contractile agents such as PE (10−7 to 3×10−5 M) or endothelin‐1 (ET‐1, 10−10 to 10−7 M) were also determined.

Both ACh‐ and SNP‐induced relaxation were significantly impaired in MA of diabetic rats, regardless of sex. However, the extent of impairment was greater in female diabetic rats compared to male diabetic animals. Diabetes also significantly increased sensitivity to PE or ET‐1 in females compared to males. Finally, the relative importance of endothelium‐derived hyperpolarizing factor (EDHF) to vascular regulation was lost in diabetic rats, regardless of sex.

These data, for the first time, show that the endothelial function in MA of UCD‐T2DM Rats is impaired in both sexes, possibly due to the loss of EDHF, initially a major vasodilatory factor. Furthermore, we showed a greater predisposition of MA to injury in diabetic female rats, in part due to a higher sensitivity of female arteries to contractile agents.
Sex Differences in the Gene Expression Profile of LOX‐1 in Mouse Tissues
Authors: Kim Seong Chul , Hamblin Milton H.
Published: November 2023

Lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1) is recognized as an important cardiovascular cell scavenger receptor for oxidized low‐density lipoproteins (oxLDL). LOX‐1 activation mediates several oxLDL‐stimulated intracellular signaling pathways and tissue processes that lead to oxidative stress, inflammatory cell infiltration in the vessel wall, endothelial dysfunction, and cardiovascular pathophysiology. In the current study, we analyzed the gene expression profiles of LOX‐1 in the aortas, hearts, liver, lungs, and kidneys of male and female C57BL/6J mice. Significant sex differences were observed in LOX‐1 mRNA expression. LOX‐1 mRNA levels were higher in male mouse aortas and hearts compared with female tissues. Conversely, LOX‐1 gene expression was elevated in female versus male kidneys. In mouse liver and lungs, we found LOX‐1 mRNA levels were comparable in males and females. These results clearly suggest that significant sex differences exist in the gene expression profiles of LOX‐1 in mouse tissues, and given the data supporting the tissue‐specific differences in LOX‐1 gene expression between males and females, we speculate these studies will provide new insights into the cellular mechanisms directly responsible for the critical, sex‐linked roles of LOX‐1 in both vascular dysfunction and other disease complications.

Support or Funding Information

(Supported by 5K12HD043451).
The Chronic Effect of Tubular pH on TGF
Authors: Wei Jin , Wang Shaohui , Wang Lei , Zhang Gensheng , Zhang Jie , Yip Kay‐Pong , Liu Ruisheng
Published: November 2023

Tubuloglomerular feedback (TGF) is an important mechanism for regulation of glomerular filtration rate (GFR), sodium excretion and blood pressure. Nitric oxide synthase 1 (NOS1) is the primary isoform of NOS expressed in the macula densa and the major splice variant is NOS1β. Nitric oxide (NO) generated by the macula densa blunts TGF response. Elevation of intracellular pH at macula densa activates NOS1 during TGF. However, whether chronic changes of renal pH have any effect on TGF response in vivo has not been clarified yet. We hypothesized in present study that elevation of renal pH inhibits TGF response by increasing NOS1β expression in the macula densa. C57BL/6J mice were divided into 3 groups treated with 0.5% NaCl, 0.72% NaHCO3 (same mEq of Na+ as in NaCl) or 0.46% NH4Cl (same mEq of Cl− as in NaCl), respectively, for two weeks. Urine pH was significantly increased to 7.8±0.5 in NaHCO3 treated group and decreased to 6.8±0.3 in NH4Cl treated group, compared with the control NaCl group (7.3±0.3). TGF was assessed in vivo by measuring the change in proximal tubular stop flow pressure (ΔPsf) induced by an increase of perfusion rate in late proximal tubules from 0 to 40nl/min. TGF was significantly blunted to 4.2±0.5 mmHg in NaHCO3 treated group but significantly enhanced to 7.4±1.1 mmHg in NH4Cl treated group, compared with the mice fed NaCl (6.0±0.7mmHg,). GFR in conscious mice were measured by plasma FITC‐inulin clearance after a single bolus injection of FITC‐inulin. There was no significantly difference among three groups, which was 237±13.7 μl/min in NaHCO3 group, 219±17.2 μl/min in NH4Cl group and 241±12.8 μl/min in NaCl group. The expression of NOS1β in MD, measured by Western Blot was significantly increased with a more than 5 folds higher in NaHCO3 treated group than that in NaCl treated group. On the contrary, NOS1β level was significantly decreased in NH4Cl group with about 1/3 value of that in NaCl group. To further determine the role of NOS1β in the alteration of TGF by renal pH, we repeated the experiments in MD specific NOS1 KO (MD‐NOS1‐KO) mice, which were developed by our laboratory. Although urine pH in the MD‐NOS1‐KO mice was similar to the wild type mice in 3 treated goups (7.9±0.8, 6.6±0.5, and 7.4±0.2), the difference in TGF among 3 groups were diminished. The TGF response was 7.5±0.7mmHg in NaHCO3 group, 8.5±1.6 mmHg in NH4Cl group, and 7.9±1.2 mmHg in NaCl group. In conclusion, chronic elevation of tubular pH by NaHCO3 treatment increases the expression of NOS1β in MD and blunts TGF; on the contrary, acid tubular solutes decrease the NOS1β level in the MD and enhance TGF. Therefore, NOS1β in MD modulated by renal pH by NaHCO3 treatment or dietary acid‐base load could be a potential target for prevention and treatment for hypertension and chronic kidney disease.
Components of the Innate Immune System Induce an Increase in Mitochondrial Membrane Potential, Reactive Oxygen Species and Necrotic Cells in Renal Medulla
Authors: Robertson Kirsti , Wildman Scott , Peppiatt‐Wildman Claire
Published: November 2023

The innate immune system is thought to play a role in the pathogenesis of kidney diseases, such as acute kidney injury (AKI), and yet the precise mechanisms involved are unknown [1]. Mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS) and necrosis are thought to be involved in the injury process of AKI [2] and dysregulation of pericyte function has been associated with ischemic injury, AKI and progression to chronic kidney disease [3].

Here we use a rat live kidney slice model [4] in combination with multiphoton microscopy, to investigate whether various components of the innate immune system alter ΔΨm and/or induce production of mitochondrial ROS and cell death in the renal medulla, to assess their effect on cell function. Kidney slices were loaded with the appropriate fluorescent dye; tetramethylrhodamine methyl ester (TMRM), mitoSOX and propidium iodide (PI), respectively. Tissue was then superfused with complement component 5a (C5a), interleukin‐33 (IL‐33) and tumour necrosis factor alpha (TNF‐α), and changes in fluorescent signal were recorded via imaging techniques.

C5a evoked a significant increase (34.73% ± 4.56%, 40.87% ± 8.34%, 34.40% ± 7.12%) in TMRM signal in endothelial (E), pericyte (P) and tubule cells (T), respectively, indicating membrane hyperpolarisation. Similar increases in TMRM signal were measured in response to IL‐33 (E: 26.25% ± 2.68%, P: 29.27% ± 8.82, T: 20.79% ± 2.61%) and TNF‐α (E: 44.72% ± 3.72%, P: 52.64% ± 4.29%, T: 54.75% ± 6.14%). C5a also evoked an increase (24.96% ± 5.87%, 31.41% ± 7.70%, 50.14% ± 7.69%) in mitoSOX signal in E, P and T cells, respectively, indicating an increase in mitochondrial ROS, associated with membrane hyperpolarization. Both C5a and TNF‐α evoked increases in mitoSOX fluorescence (E: 44.59% ± 8.59%, P: 30.92% ± 9.07%, T: 26.25% ± 3.37% and E: 61.10% ± 9.72%, P: 51.51% ± 12.08%, T: 70.41% ± 9.25% respectively). Furthermore, C5a also evoked a significant increase (22.74% ± 4.80%) in propidium iodide fluorescent cells across the tissue slice, as did C5a (15.58% ± 3.81%) and TNF‐α (17.02% ± 4.78%) when compared to PSS control experiments (1.58% ± 0.35%), indicating an increase in necrotic cells.

We conclude that the innate immune system components C5a, IL‐33 and TNF‐α act at medullary endothelial, pericyte and tubule cells causing membrane hyperpolarisation, and increases in mitochondrial ROS resulting in an increase in necrotic cells. These initial observations provide preliminary evidence that suggests C5a, IL‐33 and TNF‐α may mediate some of the pathophysiological changes thought to underlie AKI.

Support or Funding Information

Kidney Research UK
Prostamide‐Mediated Protective Action of Anandamide on Nlrp3 Inflammasome Activation in Glomeruli of Mice with Hyperhomocysteinemia
Authors: Xia Min , Li Guangbi , Conley Sabena M , Meng Nan , Boini Krishna M , Ritter Joseph K , Li Pin‐Lan
Published: November 2023

Recent studies have shown anandamide (AEA), endogenous cannabinoids, and its major renal cyclooxygenase‐2 (Cox‐2) metabolite, prostamide E2 (PGE2‐EA), attenuate Hcys‐induced Nlrp3 inflammasome activation and injury in podocytes. The present study was designed to further test whether in vivo treatment of AEA suppresses Nlrp3 inflammasome activation and thereby prevents glomerular injury in mice with hHcys. Uninephrectomized C57BL/6J wild type mice were fed either normal chow (ND) or a folate free (FF) diet for 4 weeks to produce hHcys. By confocal microscopy we demonstrated that hHcys increased the colocalization of inflammasome proteins Nlrp3 with ASC or with caspase‐1 in glomeruli compared to them in ND fed mice. Correspondingly, IL‐1β production in these glomeruli of mice with hHcys was markedly increased. In vivo administration of AEA substantially inhibited Nlrp3 inflammasome formation and IL‐1β increases in glomeruli of hHcys mice. However, if these mice received Cox‐2 inhibitor, celecoxib, the inhibitory effect of AEA on Nlrp3 inflammasome activation was significantly reduced in glomeruli of mice with hHcys. Correspondingly, hHcys‐induced decrease in podocin and increase of desmin in glomeruli were also reversed by treatment of AEA. Proteinuria and glomerular sclerotic alterations as shown by increases in glomerular damage index (GDI) in mice with hHcys were also attenuated by AEA (GDI decreased from 1.5 in FF mice back to 0.5 in AEA treated hHcys mice). Although celecoxib itself had no effect on hHcys‐induced podocyte dysfunction and glomerular injury, it blocked the protective action of AEA. In Nlrp3 KO mice, hHcys failed to induce Nlrp3 inflammasome activation and glomerular injury and AEA also had no effect on glomerular inflammasomes and glomerular injury in these mice. From these results, we conclude that AEA protects glomeruli from hHcys‐induced Nlrp3 inflammasome activation and consequent glomerular injury through its metabolites such as prostamides via Cox‐2 pathway.

Support or Funding Information

Supported by NIH grants HL057244, HL075316, HL091464 and HL122937
Adiponectin Protects Mouse Podocytes from Acid Ceramidase Inhibition‐Induced Injury
Authors: Koka Saisudha , Xia Min , Conley Sabena M , Gehr Todd W , Li Ningjun , Li Pin‐Lan , Boini Krishna M
Published: November 2023

Our recent studies have demonstrated that acid ceramidase (AC) contributes to the regulation of podocyte function and that the defect of AC gene expression triggers podocyte injury ultimately resulting in obesity‐associated end‐stage renal disease. However, it remains unknown how the inhibition of AC leads to podocyte dysfunction and injury. The current study addressed whether adipokine, adiponectin antagonizes AC inhibition‐induced damage in cultured mouse podocytes. Western blot analysis showed that adiponectin treatment dose dependently increased the acid ceramidase expression in podocytes. Confocal and RT‐PCR analyses showed that AC inhibitor, D‐NMAPPD decreased the podocin expression compared to control cells. However, prior treatment with adiponectin attenuated the D‐NMAPPD‐induced podocin decrease. Furthermore, adiponectin treatment was found to preserve podocyte morphology by maintaining the distinct arrangement of F‐actin fibers normally lost in response to D‐NMAPPD. It also prevented podocyte dysfunction by restoring D‐NMAPD‐induced suppression of VEGF production and secretion. By electromagnetic spin resonance, AC inhibitor was shown to increase superoxide production 1.8 folds compared to control podocytes, which was inhibited by adiponectin. Functionally, AC inhibitor, D‐NMAPPD treatment significantly increased the cell permeability in podocytes compared to control cells (Control: 1 ± 0.1 vs. D‐NMAPPD: 1.6 ± 0.1), and adiponectin attenuated the D‐NMAPPD‐induced increases in cell permeability. In conclusion, our observations suggest that adiponectin may exert an antagonistic action on acid ceramidase inhibition‐induced podocyte dysfunction and injury.

Support or Funding Information

Supported by NIH grants DK104031 and DK54927.
Visualizing Regional Kidney Blood Flow in the Mouse
Authors: Cabrales Pedro , Jani Vivek , Hu Vincent J
Published: November 2023

A study of the perfusion and circulation of blood in large organs is an important interest in study for analyzing effects of various physiological conditions on organ function. The spatial distribution of blood flow in the kidney is a phenotype of interest, because derangements in blood flow may precede detectable changes in organ function. However, quantifying the regional distribution of blood flow within organs of mice is challenging because of the small organ volume and the high resolution required to observe spatial differences in flow. Traditional microsphere methods in which the numbers of microspheres per region are indirectly estimated from counts or extracted fluorescence are limited to whole organ. In order to study perfusion, a traditional microsphere method of analysis was modified to obtain spatial and temporal resolution. 15‐μm fluorescent microspheres were administered via cardiac puncture at different time points. Then, the kidneys divided in three regions: two medullar regions and one cortical region. The sections were analyzed and spatial coordinates for each color microspheres were obtained. Mathematical analysis is location and density were used to estimate local perfusion as a function of time. This method of analysis at normal physiological conditions and after infusion of nephro toxic acellular hemoglobin, in order determine reliability and sensitivity of the method. This method can be advantageous in quantitatively defining renal perfusion for normal and diseased physiological conditions, as well as applied to other tissues.

Support or Funding Information

NIH grants from the Heart Lung and Blood Institute, P01‐HL110900, R01‐HL52684, and R56‐HL123015.
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 Information

American Heart Association
NOS1β in the macula densa contributes to the sex differences in decline of GFR with aging
Authors: Zhang Jie , Wang Lei , Wang Shaohui , Wei Jin , Zhang Gensheng , Yip Kay‐Pong , Liu Ruisheng
Published: November 2023

Kidney function indicated by glomerular filtration rate (GFR) decline with aging. The descent of GFR begins at age about 40, and accelerates after age 60 to 70, but females have a much slower decline in GFR compared with males. The mechanisms underlying the decline in GFR with aging and the sex differences have not been clarified yet. In present study, we hypothesized that the expression of NOS1β in macula densa (MD) decreases with aging, which enhances tubuloglomerular feedback (TGF) responsiveness and contributes to declines of GFR. Females are protected by a lower rate of decrease in NOS1β expression compared with age‐matched males, resulting in the relatively retarded decline in kidney function with aging. First, we measured NOS1β protein level in both young mice (3 months) and aged mice (18 months) by Western Blot. The expression of NOS1β was reduced by 83±7.9% in aged male mice, but only reduced by 27±14.3% in aged females. TGF, as indicated by the change in proximal tubular stop flow pressure, was 6.1±0.8mmHg in young male mice and significantly enhanced to 8.7±1.2mmHg in aged males, but there was no significant changes in females with aging (5.9±1.2mmHg vs 6.8±1.6mmHg). GFR in conscious mice were measured by plasma FITC‐inulin clearance after a single bolus injection of FITC‐inulin. GFR was reduced by 36% in aged male mice, which was from 236±9.7 μl/min in young male mice to 150±7.6 μl/min in aged male mice, but only reduced by 17% in aged female mice, which was from 182±9.2 μl/min in young to 151±13.5 μl/min in aged females. Histology with PAS staining showed obvious glomerulosclerosis in aged mice. But the injury in aged male mice was much more severe than that in age matched females, with a higher glomerular injury score (1.4±0.5 vs 0.8±0.4). To further determine the role of MD NOS1β in the decline of GFR and sex differences with aging, we repeated the experiments in MD specific NOS1 knockout (MD‐NOS1KO) mice, which were developed by our laboratory. GFR was decreased more rapidly by 47% in aged male KO mice and by 39% in aged female KO mice, but the sex difference was diminished in the KO mice. Glomerular injury is more severe in both male and female MD‐NOS1 KO mice with a higher injury score (2.1±0.6, 1.7±0.5) than age matched wild type mice (1.4±0.5, 0.8±0.4). There was also no significant sex difference in MD‐NOS1KO mice. In conclusion, NOS1β in MD protects against decline of GFR with aging by modulating TGF response. The decreased expression of NOS1β in MD contributes to the decline of GFR in aged male mice. The relatively higher MD NOS1β level in female mice delays the decrease of GFR with aging.
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 Information

Georgia 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 Information

Funded 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 Information

NIH 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

Background

Paraquat (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.

Methodology

Forty 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 Findings

The 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.

Conclusions

It 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

Introduction

Mucosal 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.

Methods

10 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).

Results

Pt‐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.

Conclusions

PLIM‐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 Information

This 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 Information

personal 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 Information

Department 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.

Methods

Sepsis 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.

Results

Mice 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.

Conclusion

P2X7 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 Information

This study was funded by:

The Danish Council for Independent Research, The Lundbeck Foundation and the research centre MEMBRANES
Age‐Related Changes in Macrophage Proliferative Capacity in Response to Chronic Inflammation and Inhaled Ozone
Authors: Liu Ziping , Groves Angela , Golden Thea , Laskin Debra , Gow Andrew
Published: November 2023

Rationale

Resident pulmonary macrophages originate from embryonic progenitor cells. In response to injury resident macrophages can be depleted and are replaced from the finite progenitor pool or from blood monocytes. We previously reported that mice lacking surfactant protein‐D (SftpD−/−) display chronic inflammation and are hypersensitive to ozone‐induced lung injury.

Objective

In these studies we examined whether constitutive inflammation in SftpD−/− mice results in a loss of the resident progenitor pool, leading to increased recruitment from the vasculature in response to ozone‐induced injury.

Methods

C57BL/6J (WT) and SftpD−/− mice (8, 27, and 80 wk) were exposed to air or ozone (0.8 ppm, 3 h)r. After 3 days, lung sections were prepared and stained for Ki‐67 (a marker of proliferative capacity), CD11b and Cd11c (surface markers of pulmonary residency or recruitment) and scored on a scale of 0 to 3.

Results

Reduced expression of Ki‐67 was observed in lungs of 80‐week mice relative to 27 and 8 week mice (p<0.05); this effect was greater in SftpD−/− mice which showed upregulation of Ki‐67 at 8 weeks of age. Across all ages Ki‐67 staining was greater in SftpD−/− mice than in WT, although these differences were lowest at 27 weeks. These results correlate with previously observed increases in inflammatory staining in Sftpd−/− mice. A significant interaction between age and ozone responsiveness was noted in SftpD−/− mice. At 8 weeks of age ozone exposure increased Ki‐67 staining, while at 80 weeks of age this response was reversed. Consistently for both Ki‐67 staining and leukocyte infiltration, the 27‐week old mice were the least responsive. Qualitatively, leukocyte recruitment in the SftpD−/− mice resulted in congregation into granuloma‐like structures. The granuloma structures stained positively for Ki‐67 in their center within 8‐week‐old mice, but this expression was lost with age. Spearman correlation for CD11b and CD11c confirmed that proliferating cells within the lung are of pulmonary origin. Of note Ki‐67 staining was not observed in the large foamy cells previously identified in this model as expressing the antioxidant enzyme HO‐1

Discussion

These results support the hypothesis that with age there is a loss of proliferative capacity within the macrophages of the lung in both WT and SPD−/− mice. In the background of chronic inflammation, ozone exposure leads to increased proliferation and activation of resident macrophages in young mice. However, in 80‐week‐old mice ozone exposure results in loss of resident proliferative inflammatory cells and recruitment of acutely activated myeloid cells. This indicates that in the context of inflammation/injury there is a greater reliance on recruited cells. This may represent a significant mechanism within senescence related alterations in pulmonary inflammatory response.

Support or Funding Information

(NIH HL086621, HL096426, ES004738, AR055073, ES005022)
Mitigation of Hypoxic‐Ischemic Damage in Newborn Rats by Misoprostol, a Prostaglandin E2 Receptor Agonist
Authors: Martens Matthew , Blaney Caitlin , Archiblad Alyssa , Nguyen Lucas , Rampitsch Christof , Gordon Joseph , Ivanco Tammy , Diehl‐Jones William L
Published: November 2023

Hypoxic‐Ischemic (HI) episodes occur in approximately 50% of preterm infants and result in significant morbidity and mortality. HI activates the nuclear transcription factor HIF1α, which in turn up‐regulates a variety of antioxidant response element‐driven genes including Bnip3, whose protein product has a pivotal role in hypoxia‐induced apoptosis and/or necrosis. Increased Bnip3 expression has been implicated in necrotizing enterocolistis (NEC), an inflammatory bowel disease of premature infants. Previously our laboratory has shown that Misoprostol, a prostaglandin E2 receptor agonist, mitigates the effects of Bnip3 in an enterocyte cell model. We hypothesized that misoprostol regulates intestinal Bnip3 activity in a rat model of HI. Dams and their pups were exposed to environmental hypoxia (10% O2) or normoxia (21% O2) from post‐natal days (PND) 3–10. Experimental animals were injected subcutaneously with 0.1mg/kg Misoprostol each day between PND 3–10, while control animals received saline injections over the same period. PND10 pups were sacrificed, and transverse colon regions were isolated and analyzed for Bnip3 protein expression via western blotting. HI induced a 17‐fold increase in Bnip3 protein expression (p<0.001); in contrast, Misoprostol induced a 72% decrease in protein expression (p<0.05) in hypoxic rats. To explore this effect mechanistically, we examined mitochondrial permeability transition pore (mPTP) opening in HEK293 cells. As expected, Bnip3 transfection increased mPTP opening, whereas Misoprostol inhibited mPTP opening in cells transfected with Bnip3. Furthermore, cotransfection with both Bnip3 and protein kinase A (PKA) mitigated Bnip3‐induced cell death in 3T3 fibroblasts. Finally, we present data showing that PKA phosphorylates Bnip3 protein. We suggest that misoprostol may attenuate hypoxia‐induced cell death via cAMP/PKA signaling pathways.

Support or Funding Information

Athabasca University

Natural Sciences and Engineering Research Council of Canada

Children's Hospital Research Institute of Manitoba