bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chronic Insulin‐clamp Causes Postprandial Diuresis in Sprague Dawley Rats
Authors: Irsik Debra L. , Alaisami Rabei , Washington Ashley R. , Brands Michael W. 
Published: November 2023

Understanding the physiologic role of insulin in salt and volume homeostasis is important for interpreting its contribution to disease states such as diabetes or metabolic syndrome. This study tested the hypothesis that the rise in plasma insulin that occurs postprandially is required to minimize renal sodium (Na+) and volume losses. Rats were instrumented with chronic artery and vein catheters, housed in metabolic cages, and connected to Instech hydraulic swivels. Na+ intake was clamped chronically at normal by feeding Na+ deficient diet and infusing saline 24 hr/day. Measurements of 24 hr Na+ and volume excretions and 4‐ and 8‐hr postprandial Na+ and volume excretions were made under control conditions (CTL) and during insulin‐clamped conditions (I‐C) to prevent the postprandial insulin surge. The chronic I‐C was accomplished by administering STZ and subsequently infusing insulin 24 hr/day to clamp insulin at normal levels that maintained normal fasting blood glucose. Twenty‐four hr urine volume and Na+ excretion were greater in the I‐C vs. CTL rats (33±2 vs. 17±1 ml/day and 3.8±0.1 vs. 2.9±0.2 mmol/day, respectively; n=5–9; p<0.05) despite lower fasting blood glucose levels (93±9 vs. 129±5 mg/dl). Postprandial measurements were made after feeding a calorie‐dense gel to rats that had been fasted overnight. Postprandial urine volume was greater in I‐C rats after 4 hrs (5.5±1.6 vs. 2.7±0.3 ml, n=4–8; p<0.05) and 8 hrs (7.0 vs. 2.8 ml), and postprandial GFR was lower in the I‐C rats (1.8±0.4 vs. 2.2±0.4 ml/min), consistent with decreased tubular reabsorption. Postprandial blood glucose increased more in the I‐C rats (307±80 vs. 141±11 mg/dl, n=4–8; p<0.05). These data suggest that the postprandial surge in insulin is necessary to prevent renal salt and volume losses, because rats that were unable to increase insulin after a meal exhibited increased diuresis. Future studies will be needed to quantify the relative roles of insulin‐mediated glucose disposal vs. insulin‐mediated renal tubular Na+ reabsorption.Support or Funding InformationHL056259 and HL056259‐S1
High Salt Activation of Collecting Duct‐Derived Nitric Oxide Synthase Suppresses the Renin‐Angiotensin‐Aldosterone (RAAS) System
Authors: Pollock Jennifer S , Pollock David M , Dugas Courtney , Hyndman Kelly A 
Published: November 2023

We reported that collecting duct nitric oxide synthase‐1 knockout (CDNOS1KO) mice display a salt‐sensitive blood pressure phenotype with reduced Na excretion in response to a high salt (HS) diet. To further delineate the relationship of CD NOS1 activation and Na homeostasis, we designed experiments to test the hypothesis that HS induced activation of CD NOS1 functions to suppress the RAAS. Adult male CDNOS1KO and flox control mice were placed on normal (NS) or HS diet for 7 days (NS: 0.4% NaCl, HS: 4% NaCl). NO production in isolated CD preparations from the CDNOS1KO mice was significantly reduced compared to control mice on NS (0.5±0.09 vs 1.0±0.03 pmol/mg pr/h, p< 0.05) and HS (1.5±0.3 vs 2.8±0.2 pmol/mg pr/h, p<0.05). HS induced urinary NOx excretion was also significantly reduced in CDNOS1KO mice by >50% compared to control. HS induced significantly higher plasma Na levels in CDNOS1KO mice (+8 mEq/L; p<0.05) compared to control mice. Plasma K levels were similar in NS and HS in both genotypes. HS significantly reduced plasma aldosterone (532.6±93.0 vs 55.6±10.4 pg/ml) and urinary aldosterone excretion in the control mice (4.2±0.7 vs 1.7±0.4 ng/day), however this reduction was abolished in CDNOS1KO mice (402.0±68 vs 276.5±55 pg/ml; 4.1±0.8 vs 3.7±0.7 ng/day). Plasma renin concentration was similar in both genotypes with HS intake yet blood pressure was significantly higher in CDNOS1KO mice, thus CDNOS1KO mice display an in appropriately high level of plasma renin with HS. Furthermore, renal renin expression was enhanced in CDNOS1KO mice compared to controls. These data demonstrate that loss of CD NOS1 facilitates sustained elevations in plasma Na levels as well as plasma and intra‐renal renin that further enhances activation of aldosterone. These findings indicate that CD NO, especially NOS1‐derived, is vital for systemic Na homeostasis under HS conditions.Support or Funding InformationNIH P01HL95499
Median Preoptic AT1a Receptor Knockdown Does Not Diminish Drinking in Response to Peripheral Angiotensin II Administration
Authors: Shell Brent , Farmer Gef , Emeh Robert , Little Joel , Cunningham Tom 
Published: November 2023

Thirst is an essential mechanism for organisms to maintain body fluid homeostasis and is therefore tightly regulated. Angiotensin II (Ang II) has long been known to stimulate thirst by both by its action peripherally as a circulating hormone, and centrally acting as a peptide neurotransmitter. Peripherally circulating Ang II can elicit thirst by stimulating the Subfornical Organ (SFO), which in turn stimulates other hypothalamic nuclei regulating body fluid homeostasis such as the Median Preoptic Nucleus (MnPO) presumably through Ang II signaling. Central Ang II administered via intracerebroventricular (ICV) infusion also induces thirst by directly stimulating regions such as the MnPO. Previous studies have shown that lesions of the MnPO block drinking responses to peripheral or ICV Ang II while lesions of the SFO decrease drinking stimulated by peripheral Ang II but not ICV Ang II. Based on these observations, it has been hypothesized that Ang II receptors in the MnPO are critical for drinking responses to centrally or peripherally administered Ang II. We tested this hypothesis by microinjecting an adeno‐associated virus with short hairpin RNA matched to the Angiotensin Type 1a receptor (AT1aR) into the MnPO and testing the rats for drinking responses elicited by peripheral or centrally administered Ang II. Sprague Dawley rats were separated into subcutaneous (SC) and ICV Ang II administration groups. The SC group was pretested for their drinking response to 2 mg/kg Ang II, and those animals that drank in response to the SC Ang II administration were utilized in the study. All animals were injected with the shAt1aR virus or a control virus on Day 0, and were allowed to recover before drinking tests on day 14 and day 18. ICV animals were microinjected with the viruses on Day 0 and instrumented with a chronic lateral ventricle cannula. These animals were tested for drinking responses to 2 ng Ang II in 1ul aCSF 14 and 18 days later. Unexpectedly, we found that knockdown of AT1aR in the MnPO did not reduce drinking in response to SC Ang II (p>.05), but did significantly reduce drinking in response to ICV Ang II (P<.05). These results suggest that AT1aRs in MnPO are necessary for drinking responses following ICV Ang II but not SC Ang II. It is possible that drinking responses to peripheral Ang II involves co‐transmitters, such as glutamate, in the MnPO that produce drinking in the absence of AT1aRs or that other Ang II receptors mediate this response.Support or Funding InformationR01 HL119458P01 HL088052
Blood Pressure in Old Female SHR is not Salt Sensitive and Tempol Fails to Decrease Blood Pressure during High Salt Intake
Authors: Dalmasso Carolina , Maranon Rodrigo O. , Patil Chetan N. , Reckelhoff Jane F. 
Published: November 2023

Oxidative stress contributes to the development and maintenance of hypertension in male rodents; however studies in females have rarely shown a reduction in blood pressure (BP) with antioxidants. Tempol, a superoxide dismutase mimetic, decreases BP in young male spontaneously hypertensive rats (SHR), but fails to reduce BP in either young or old female SHR, despite the fact that females have similar or higher levels of oxidative stress markers. In contrast, recent experiments from our laboratory have shown that Tempol decreases BP in old female SHR treated chronically with Acetazolamide. Acetazolamide is a carbonic anhydrase inhibitor that increases sodium delivery to the distal nephron and may increase distal oxidative stress. Thus the data suggest that oxidative stress‐mediated BP control is dependent on increased sodium delivery to the distal nephron. The aim of the present work was to test the hypothesis that tempol will decrease blood pressure in old female SHR chronically given a high salt intake. Female SHR (20 months of age) were divided into two groups (n=7/group): Control + Tempol (C+T), and High Salt Diet + Tempol (HSD+T). After baseline mean arterial pressure (MAP; telemetry; 5 days), rats received low salt diet (NaCl 0.3%; C+T) or high salt diet (NaCl 8%; HSD+T). On day 22, rats in both groups (C+T and HSD+T) were given tempol (30 mg/kg/d) in the drinking water for 21 days. Baseline MAP was similar between groups (C+T: 174±7; HSD+T: 168±5 mm Hg, p=NS), and MAP was not salt sensitive (C+T: 178±5; HSD+T: 178±4 mm Hg, p=NS). Tempol treatment had no effect on MAP in either group of female SHR (C+T: 179±5; HSD+T: 178±5 mm Hg). Because tempol in acetazolamide treated female SHR reduces MAP, whereas tempol has no effect on MAP in females on high salt diet, the data suggest that in hypertensive female rats, tempol reduces MAP only when proximal sodium reabsorption is blocked.Support or Funding InformationSupported by NIH‐R01HL66072, PO1HL51971 (JFR), 14POST18640015 (ROM).
Flow‐Induced Activation of NO Production is Calcium‐ and HDAC1‐Dependent in Inner Medullary Collecting Duct Cells
Authors: Sedaka Randee S. , Hyndman Kelly A. , Pollock Jennifer S. 
Published: November 2023

High salt intake mediates an increase in sodium delivery and flow within renal tubules, especially the inner‐medullary collecting duct (IMCD). We previously reported that isolated IMCD from mice on high salt diet produce significantly more NO compared to mice on normal salt diet without a change in NO synthase (NOS) expression suggesting that high salt and/or flow increases NO production through increased NOS activity. NOS activity is activated by intracellular calcium signaling in other cell types, thus we hypothesized that flow increases NO production through increased intracellular calcium. Mouse IMCD cells were grown to confluency on glass slides, then placed under static conditions or high flow (10 dynes/cm2) for 1 hour at 37°C in the absence or presence of calcium‐free media or BAPTA. NO production was analyzed by HPLC. NO production increased in response to flow >5‐fold (static: 653.8±165.9, flow: 3357.3±1024.7 pmol/mg pr/hr; n=4–9/group, p<0.001). Flow‐induced NO production was abolished in the absence of calcium (BAPTA: 609.6±152.5 pmol/mg pr/hr; calcium‐free media: 1440.0±907.0 pmol/mg pr/hr; n= 4–9/group, p<0.001). Previous reports from the literature show that histone deacetylase (HDAC) 1 is activated with intracellular calcium and is flow‐responsive. Thus, we further hypothesized that flow‐mediated increases in NO production occur via HDAC1. HDAC1‐specific inhibition with MS‐275 (300 nM) significantly decreased NO production in response to flow (static: 215.8±20.6, flow + vehicle: 1619.2±44.1 pmol/mg pr/hr, flow + MS‐275: 381.9±77.6 pmol/mg pr/hr; n=5–10/group, p<0.0001). HDAC1 expression was unchanged with flow (static: 2.86±0.9, flow + vehicle: 2.91±0.3, flow+MS‐275: 3.26±0.6 RDU; n=3/group, p>0.05). Additionally, preliminary HDAC1‐specific immunofluorescence staining of post‐flow IMCD cells suggests the presence of HDAC1 in both the nuclear and cytosolic compartments. Therefore, our data suggest that NO production in response to flow is calcium‐ and HDAC1‐dependent. Future studies will elucidate HDAC1 expression and activity in nuclear and cytosolic cell fractions as well as HDAC1 translocation utilizing immunofluorescence staining.Support or Funding InformationSupported by NIH T32 HL007918 to RSS and P01 HL95499 to JSP.
Radiofrequency Renal Denervation Decreases Inflammation in Kidney Medulla in Spontaneously Hypertensive Rats (SHR)
Authors: Gao Juan , Xiao Liang , Polhemus David J , Smart Frank , Mernaugh Raymond , Goodchild Traci T , Lefer David J , Harrison David G , Kapusta Daniel R 
Published: November 2023

We have shown that radio frequency ablation (RF‐ABL) of the renal arteries decreases blood pressure and sympathetic activity in hypertensive SHR. Recently, it has been shown that in angiotensin II‐induced hypertensive mice, surgical renal denervation reduces renal inflammatory cell infiltration, fibrosis, albuminuria and nephrinuria independent of blood pressure. The purpose of this study was to determine if RF‐ABL affects regional localization of inflammatory markers in kidneys of spontaneously hypertensive rats (SHR). SHR (n=8 /group) received bilateral RF‐ABL or Sham‐ABL (Biosense Webster Stockert 70 generator and RF‐probe). After 4wks, kidneys were collected, cortex and medulla separated, and processed for either flow cytometric determination of infiltrating tissue numbers of B cells and T cells (n=8/group) or for measurement of tissue cytokine levels (Bio‐Plex; n=10/group) including Interleukin 2 (IL‐2), IL‐5, IL‐6, IL‐7, IL‐10, IL‐17, Interferon gamma (INF‐γ), Granulocyte Monocyte Colony Stimulating Factor (GM‐CSF), Macrophage Inflammatory Protein‐3a (MIP‐3a), and Tumor Necrosis Factor alpha (TNF‐α). RF‐ABL significantly decreased B cells (56% of Sham‐ABL), CD4+ T cells (55% of Sham‐ABL) and CD8+ T cells (61% of Sham‐ABL) in the medulla, but had no effect on the numbers of B and T cells in the cortex. At 4 weeks following RF‐ABL, tissue levels of IL‐2, IL‐6, IL‐7, IL‐10, IL‐17, INF‐γ, MIP‐3a, and TNF‐α were decreased in the medulla as compared to Sham‐ABL. These findings demonstrate that RF‐ABL is associated with a preferential reduction of inflammatory markers in the kidney medulla, which may contribute to blood pressure independent effects of RF‐ABL in hypertensive rats.Support or Funding InformationAHA 14POST20450173 to JG; Biosense Webster IIS‐175 to DRK & FS; P30GM106392 to DRK. 1R01 HL092141, 1R01 HL093579, 1U24 HL 094373, and 1P20 HL113452 to DJL.
Sex‐Specific Cell Death Response Following Renal Ischemia‐Reperfusion in Spontaneously Hypertensive Rats (SHR)
Authors: Crislip G. Ryan , Sullivan Jennifer C. 
Published: November 2023

Males develop a greater extent of ischemia‐reperfusion (IR) induced injury than females. Recent studies have shown that renal IR injury is primarily mediated by necrosis in male mice, and pilot studies in our lab indicate a sex difference in renal cell death in SHR with females having more apoptotic cell death than males under control conditions. Based on the potential protective role of apoptosis vs. necrosis, the goal of this study was to test the hypothesis that males exhibit greater necrotic cell death following IR while female SHR favor apoptotic cell death. 13 week old male and female SHR were studied: control and 45 minute warm bilateral renal ischemia followed by 24 hours reperfusion (N=6–9). Renal injury was assessed by measuring plasma creatinine (PCr) and histological analysis. Kidneys were harvested to measure cell death via TUNEL assay, PCR Arrays, and Western blot analysis. IR increased PCr in both sexes (M: 0.34±0.04 vs. 3.07±0.5; F: 0.41±0.06 vs. 2.81±0.6), although kidneys from males exhibited a greater extent of red blood cell clogging than females when stained slides were scored (M: 1.0±0.0 vs. 2.4±0.2; F: 1.1±0.1 vs. 1.8±0.3). IR increased renal apoptotic cell death in both sexes, although females tended to have greater apoptotic cell death than males (M: 1.6±0.6 vs 12.0±3.9; F: 5.0±1.0 vs 18.3±3.9 cells per area; effect of treatment: p=0.01; effect of sex: p=0.1). RIP3 activates the formation of a pro‐necrotic complex with RIP1 during the cell death process. RIP3 mRNA increased 24 hours post‐IR in both sexes, although the increase only reached significance in males (M: p=0.01; F: p=0.09; n=4). RIP1 mRNA decreased only in females (M: p=0.8; F: p=0.03; n=4), however, the protein expression decreased in both sexes (M: 0.27±0.02 vs. 0.15±0.03; F: 0.31±0.01 vs. 0.20±0.05; effect of treatment: p=0.003; effect of sex: p=0.1). These data support a sex‐specific cell death response following renal IR with greater necrotic cell death in males and greater apoptotic cell death in females. A sex difference in cell death may underlie the sex difference observed in renal IR induced injury.Support or Funding InformationAmerican Heart Association Predoctoral Fellowship; AHA Award Number: 15PRE25850008 (PI: Sullivan)
Exposure to Cigarette Smoke Worsens Chronic Kidney Disease Induced Cardiac Hypertrophy and Fibrosis
Authors: Drummond Christopher A , Hernandez Dawn Alita , Haller Steven T , Liu Jiang , Shapiro Joseph I , Cooper Christopher J , Tian Jiang 
Published: November 2023

IntroductionAlthough smoking accelerates renal and cardiac disease progression, the mechanisms by which it does so are unclear. Previously our lab has found that platelet activation can lead to increased renal injury. As smoking is a well‐known cause of platelet activation we postulated that smoking would accelerate renal disease progression as well as the associated cardiomyopathy in experimental renal failure.MethodsWe used male Sprague‐Dawley rats which were divided into two surgical groups one receiving sham (control) operations the other receiving the 5/6th partial nephrectomy (PNx) operation, a model of Chronic Kidney Disease (CKD). Each surgical group of animals were further split into two groups, one receiving exposure to side stream smoke from five 3R4F combustible cigarettes, delivered over 4 hours per day, 5 days per week for 4 weeks and non‐exposed controls. Blood pressure via tail cuff, renal and cardiac fibrosis, proteinuria, cardiac ejection fraction and Western blots to evaluate CD40 expression in kidney were analyzed 4 weeks following the start of the procedure.ResultsPNx increases systolic blood pressure compared to sham controls (177±16 vs 128±6 mmHg, p<0.05). Cigarette smoke exposure also increased blood pressure in Sham operated animals (142±3 mmHg, p<0.05) and with PNx (191±10 mmHg, p<0.05). PNx alone increased cardiac and renal fibrosis by 2.7‐ and 8.7 fold, respectively (p<0.01) versus sham‐operated animals. Cigarette smoking worsened cardiac and renal fibrosis (4.4‐ and 9.4‐fold, p<0.01) in Sham operated animals. Additionally, smoking significantly increased cardiac and renal fibrosis in PNx‐operated animals, resulting in 2.5‐ and 2.4‐fold higher fibrosis.Proteinuria increased significantly with PNx alone vs shams (p<0.05). Smoke exposure doubled proteinuria in PNx (p<0.05), a similar trend was not observed in sham animals exposed to smoke vs those that were not. Ejection fraction as determined by echocardiography and cardiac hypertrophy as measured by heart weight to body weight ratio increased significantly in PNx operated animals versus shams (p<0.01) and smoking induced further significant increases in ejection fraction and cardiac hypertrophy vs non‐exposed animals (p<0.01). Finally, western blot analysis revealed that PNx and smoking both individually increased kidney expression of the pro‐inflammatory molecule CD40 and when combined synergized to increase CD40 protein expression over that observed for either treatment alone.ConclusionsCombustible cigarette smoking significantly worsens progression of experimental cardiac and renal fibrosis and hypertrophy.Support or Funding InformationThis work was supported by NIH grant R01 HL‐105649 and F32 DK‐104615
Transferrin Receptor Levels are increased by Interleukin 1β in a Human Kidney Proximal Tubular Cell line
Authors: Bonnemaison Mathilde L , Boesen Erika I 
Published: November 2023

BackgroundIron is required for several essential cellular processes such as DNA synthesis and energy metabolism, but when present in excess can cause cellular injury. Although kidney iron levels and urinary excretion of iron and iron transport proteins such as transferrin are increased in patients with kidney disease, it remains unclear how iron is handled by the kidneys during inflammation. We hypothesize that inflammation increases renal tubular iron uptake mechanisms, which in turn may promote cellular injury.Methods and ResultsTo understand how iron is handled by proximal tubule cells during inflammation, we turned to HK‐2 cells, a human proximal tubular cell line, and investigated the levels and distribution of transferrin receptor and divalent metal transporter 1 (DMT‐1) in response to interleukin 1β, a pro‐inflammatory cytokine. Transferrin receptor allows the uptake of iron after binding and internalization of iron‐loaded transferrin while DMT‐1 carries divalent metals like iron, zinc and copper inside the cells. Our data with HK‐2 cells indicated that transferrin receptor levels doubled upon treatment with 0.1 ng/mL interleukin 1β for 24 hours (P<0.005 by one‐way ANOVA, followed by Dunnett's test). Immunofluorescent staining of HK‐2 cells showed no apparent change in transferrin receptor distribution. In contrast, DMT‐1 levels in HK‐2 cells remained unchanged upon treatment with interleukin 1β for 24 hours at concentrations as high as 50 ng/mL. Increased intracellular levels of iron may lead to mitochondrial oxidative stress, which in turn may impact ATP production. After treating HK‐2 cells with 1 ng/mL interleukin 1β for 24 hours, intracellular ATP levels were measured and indicated no significant difference with untreated cells.ConclusionsThese data suggest that under inflammatory conditions, the uptake mechanism for the key iron transport protein transferrin is increased in the proximal tubule. This could have important implications in the setting of glomerular injury, which permits greater amounts of transferrin to be filtered, resulting in increased exposure of proximal tubular cells to transferrin and thus iron uptake.