Salt-sensitive Rats Research Articles

Fumarase Overexpression Abolishes Hypertension Attributable to eNOS Haploinsufficiency in Dahl Salt‐Sensitive Rats

BackgroundHuman blood pressure salt sensitivity is associated with changes in urinary metabolites related to fumarase (Fh) and nitric oxide (NO) metabolism, and fumarase promotes NO production through a malate‐aspartate‐arginine pathway.ObjectiveWe examined the role of the fumarase‐NO pathway in the development of hypertension in the genetic background of the Dahl salt‐sensitive (SS) rat, a model of human salt‐sensitive hypertension and renal injury used widely.Methods and ResultsSS rats with heterozygous mutation of endothelial NO synthase (eNOS or Nos3) (SS‐Nos3+/−) were bred with SS rats with a hemizygous Fh transgene. SS‐Nos3+/− rats without the Fh transgene (SS‐Nos3+/−/Fh0/0) developed substantial hypertension with a mean arterial pressure (MAP) of 138.5 ± 3.8 mmHg on a 0.4% NaCl diet, and 183.0 ± 3.7 mmHg after 14 days on a 4% NaCl diet. MAP decreased remarkably to 123.1 ± 1.4 mmHg on 0.4% NaCl, and 143.3 ± 1.5 mmHg on 4% NaCl in SS‐Nos3+/− rats with a Fh transgene (SS‐Nos3+/−/Fh0/1), and proteinuria, renal fibrosis, and tubular casts were attenuated in SS‐Nos3+/−/Fh0/1 rats compared to SS‐Nos3+/−/Fh0/0 rats. eNOS protein abundance decreased in rats with the Nos3 heterozygous mutation, which was not influenced by Fh overexpression. However, the decrease in NO metabolite in the renal outer medulla of SS‐Nos3+/−/Fh0/0 rats was reversed in SS‐Nos3+/−/Fh0/1 rats. Levels of L‐arginine, but not the other 12 amino acids analyzed, were significantly higher in SS‐Nos3+/−/Fh0/1 rats than in SS‐Nos3+/+/Fh0/0 rats.ConclusionsFumarase has potent effects in restoring NO production in renal outer medulla and blunting the development of hypertension attributable to eNOS haploinsufficiency.Support or Funding InformationThis work was supported by US National Institutes of Health grant HL116264‐5244 (M.L.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Postpartum Changes in Microglia Density and Activation in a Rat Model of Superimposed Preeclampsia

Microglia are the primary immune cells of the central nervous system, surveying the microenvironment and responding to cell injury or tissue damage. The Dahl Salt Sensitive (Dahl‐S) rat model of superimposed preeclampsia (PE) has preexisting hypertension and chronic kidney disease and exhibits characteristic symptoms of PE during pregnancy. Preexisting cardiovascular and renal disease increase the risk of superimposed PE, which further increases risk of maternal complications. Recently, we showed that the rat model of placental ischemia (induced preeclampsia) has increased posterior cortical microglia activation 2 months postpartum. However, whether superimposed PE exacerbates postpartum microglia activation is not known. Because the Dahl‐S rat has increased peripheral inflammation (increased circulating TNFα) during pregnancy and exacerbated hypertension and proteinuria with subsequent pregnancies, we hypothesized that the Dahl‐S model has increased microglia activation after two pregnancies. Dahl‐S rats were randomly assigned to virgin (DSV) or postpartum (DSP) groups (n=3 per group). Postpartum rats were mated twice (3 and 5 months of age) and brains were collected at 8 months of age. Posterior cerebrum was cryo‐sectioned and immuno‐stained using ionized calcium binding adapter molecule (Iba1). Microglia density and morphology were analyzed using ImageJ in 3 images per region and averaged per rat. There was no difference in microglia density (number of Iba1+ microglia per image) in the hippocampus (10±2 in DSV vs. 9±1 in DSP) or cortex (7±1 in DSV vs. 7±2 in DSP). DSP rats had higher proportion of hippocampal Type 3 (78% vs. 62% in DSV) and fewer Type 4 (5% vs. 22% in DSV) microglia (p<0.05). DSP had fewer hippocampal amoeboid (activated) microglia and higher cortical ramified (surveying) microglia compared to DSV (Figure). Taken together, history of superimposed preeclampsia symptoms in DSP rats does not induce exacerbated microglia activation suggesting that pregnancy is either protective to the brain or that following damage, microglia fail to activate, contributing to incomplete repair processes. These hypotheses will be tested in future studies. Future studies will also determine cortical and hippocampal cytokine levels and astrocyte activation in the postpartum Dahl‐S rats.Support or Funding InformationNIH R00HL129192, R25HL121042, 5T32HL105324‐09, R01HL134711This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pre‐transcriptional fibrotic factor alterations do not contribute to high fat diet associated renal fibrosis in Dahl salt sensitive male rats

We previously reported that high fat diet (HFD) feeding for 24 weeks promotes hypertension similarly in male and female Dahl salt‐sensitive rats. However, HFD fed males exhibited more severe renal inflammation, fibrosis and injury than females. The underlying mechanisms responsible for sex differences in HFD associated renal fibrosis/injury are not fully understood. It is well established that extracellular matrix (ECM) remodeling is the hallmark of hypertension associated renal fibrosis. Proteins involved in ECM remodeling are key mediators in the fibrogenic process. Therefore, we determined if HFD increases ECM remodeling resulting in renal fibrosis and if sex differences of ECM remodeling in response to HFD contribute to hypertension‐associated renal fibrosis/injury in males. Renal cortex was collected from male and female rats fed a control diet (CD, 10% kcal fat) or a high fat diet (60% kcal fat) for 24 weeks. mRNA expression of fibrosis markers in the renal cortex was assessed, which included transforming growth factor (TGF), smooth muscle actin (SMA), cysteine‐rich angiogenic inducer 61 (CYRG1), connective tissue growth factor (CTGF), collagen (COL1A1, COL3A1, COL1A2), and matrix metalloproteinases (MMP9, MMP14). We found that only MMP9 mRNA levels were significantly elevated in HFD fed females compared to CD fed females. However, MM9 mRNA expression was similar in HFD fed and CD fed males. HFD feeding did not change expression of the other fibrosis markers in males or females. We next determined if elevated MMP9 mRNA levels in HFD females were translated into increased total MMP9 protein and/or increased MMP9 activity. MMP9 activity was determined by zymograph assay. We found that the total MMP9 protein expression and the activity were similar in HFD fed males and females. Our studies suggest that it is unlikely that HFD causes pre‐transcriptional alterations in the fibrosis signaling pathway. HFD associated post‐transcriptional alterations may contribute to renal fibrosis in males, but the underlying mechanisms are still undetermined. In addition, the role of ECM remodeling in HFD associated renal fibrosis needs to be assessed prior to the onset of renal fibrosis.Support or Funding InformationSupported by NHLBI 2P01HL070687 for G.D.F, J.J.G. and H.XThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Curcumin Does Not Attenuate the Preeclamptic Phenotype in the Dahl Salt‐Sensitive Rat

Preeclampsia (PE) is associated with new onset of hypertension and proteinuria and affects 2–8% of pregnancies worldwide. It is a leading cause of maternal and fetal morbidity and mortality with no current therapy to manage the disease. While its etiology is yet to be elucidated, immune system activation of various inflammatory pathways is reported to be a contributing factor. Toll‐like receptor (TLR) activation by damage‐associated molecular patterns (DAMPS) released following poor placentation and oxidative stress has been linked to the development of PE and TLR4 expression is increased in the placenta of preeclamptic women. We performed a whole transcriptomic analysis on placental tissues from gestation day (GD) 14 rats using Affymetrix Rat 2.0ST GeneChip and noted significant dysregulation in multiple genes during pregnancy in the Dahl salt sensitive (S) rat, a model of spontaneous superimposed preeclampsia, compared to the normal pregnant control Sprague Dawley (SD) rat. These genes included upregulation of TLR4 in the placenta of Dahl S rats (1.9 fold, p<0.05), which was subsequently confirmed via quantitative real‐time PCR (2.9 fold, p<0.05). To further investigate this pathway, the goal of this study was to assess the effect of TLR4 inhibition in pregnant Dahl S rats. Curcumin, the anti‐oxidant and anti‐inflammatory active ingredient in the spice turmeric, has been reported to inhibit TLR4 activation, and reduced blood pressure in rodent models of LPS‐induced preeclampsia. We hypothesized that curcumin would inhibit TLR4 activation and reduce blood pressure and uterine artery resistance index during pregnancy in the Dahl S rat. Starting on gestational day (GD) 10, 17 weeks old rats were divided into two groups: a treated group (N=7) that orally received dough containing 400mg/kg of curcumin daily and a control group (N=6) that received the vehicle dough through GD20. On GD18, Uterine Artery Resistance Index (UARI) was measured via Doppler ultrasound. On GD20, terminal blood pressure measurements were taken, and pups and placentas harvested and measured. Curcumin had no effect on UARI (0.78 vs. 0.82, p=0.4) or mean arterial pressure (146 ± 5 vs. 133 ± 5 mmHg, p=0.08). Fetal outcome assessed by litter size (10 ± 1 vs. 11 ± 1 pups); pup weight (2.23 ± 0.08 vs. 2.30 ± 0.07 g) and pup length (29.44 ± 0.31 vs. 29.6 ± 0.46 cm) was similar between groups. These results suggest that administration of curcumin does not alleviate the preeclamptic phenotype in the Dahl S rat.Support or Funding InformationR01HL1347111R01HL137673This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bioinformatics analysis for heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) represents approximately half of heart failure but little is known about the underlying mechanisms. Dahl salt‐sensitive (Dahl‐SS) rats were fed a high‐salt diet (8% NaCl) from 7 weeks of age to induce HFpEF and controls were fed a normal‐salt diet (0.3% NaCl). Echocardiograms and electrocardiography (ECG) was performed to assess cardiac function. Diastolic dysfunction and preserved ejection fraction along with signs of heart failure was showed and QTc interval on ECG was prolonged (p<0.001) at 14 weeks of age. Hearts were harvested for gene array and label‐free quantitative proteomics. Bioinformatics analysis using DAVID website, GSEA software and BinGO plugin in Cytoscape environment. GO analysis showed microtubule binding and calcium dependent phospholipid binding protein were significant downregulated while oxidative stress response protein were upregulated (p<0.05, q<0.05). GSEA results indicated that gap junction, focal adhesion and actin cytoskeleton regulation gene sets were concentrated in upregulated transcripts (p<0.01, q<0.25) while cardiac muscle contraction and oxidative phosphorylation pathway were downregulated in proteomics (p<0.001, q<0.001). Western blot and PCR experiments showed significant changes in the expression of Dynamin‐1, Integrin‐α9 and MFF, which had important functions including cell stiffness regulation. Our study provides valuable information on the molecular functions and regulatory network of HFpEF, as well as new perspectives on the relationship between diastolic dysfunction and calcium stimulate, cytoskeleton stiffness and oxidative stress.Support or Funding InformationShenzhen Science and Technology Innovation Institution (China) Grant JCYJ20160427170536358 (Y Huo)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Contribution of the Renal Lymphatic Circulation to the Development of Salt‐Sensitive Hypertension

More than one billion people worldwide suffer from hypertension, which is a leading cause of stroke and cardiovascular disease. According to the National Heart, Lung, and Blood Institute (NHLBI) Working Group, an estimated 40% to 85% of hypertensive patients exhibit salt‐sensitivity, which increases the risk for cardiovascular events and mortality. Salt‐sensitive hypertension is known to be caused by an imbalance between sodium retention and excretion in the kidney. However, the mechanisms that enable the kidney to deliver excessive re‐absorbed salt and volume back to the systemic circulation remain obscure. Interestingly, recent studies indicate that the peripheral lymphatics act as a “salt sink” to store excessive salt and volume, thereby maintaining sodium homeostasis to regulate blood pressure. This finding led us to consider whether renal lymphatics contribute to excessive sodium retention in the kidney during the development of salt‐sensitive hypertension. To test this hypothesis, we initially evaluated lymph‐angiogenesis in the kidneys of rats with or without salt‐sensitive hypertension. Real‐time PCR, immunohistochemistry and Western blots revealed increased expression of the lymphatic‐specific marker VEGFR3 in the kidneys of Dahl salt‐sensitive (DSS) rats fed a high‐salt diet compared to either DSS rats fed regular chow or Dahl salt‐resistant (DSR) rats with or without high‐salt intake. To explore the impact of renal lymph‐angiogenesis on blood pressure, we surgically ligated the renal collecting lymph vessel (LV) in DSS rats. Ligation of the renal LV in DSS rats attenuated the increase in systolic blood pressure caused by a high‐salt diet. Notably, a growing body of evidence has implicated macrophages in lymph‐angiogenesis. Indeed, we also observed that a high‐salt diet promoted renal infiltration of macrophages, which stimulated renal lymph‐angiogenesis via upregulating VEGFR3 and its ligand VEGFC. Collectively, our data suggest that in response to a high‐salt diet, kidney‐infiltrated macrophages stimulate renal lymph‐angiogenesis, which appears to support the development of salt‐sensitive hypertension.Support or Funding InformationSupported by NIH grants T32 GM106999 (BRG) and R21 CA187325‐01A1 (NJR), and a Medical Research Endowment Award (SM) from the Univ. of Arkansas for Medical Sciences.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Anti‐hypertensive Treatment Lisinopril on Anxiety‐like Behavior in Adult Hypertensive Dahl Salt‐Sensitive Rats

BackgroundIndependent of a high salt intake, Dahl Salt‐Sensitive (DSS) rats demonstrate an age‐associated increase in blood pressure (BP) accompanied by cognitive decline. We sought to explore the effect of anti‐hypertensive treatment, Lisinopril, which lowers tissue angiotensin II levels, on BP and behavior in adult DSS rats. We hypothesized, that Lisinopril, in addition to BP decrease, will affect the cognitive status of adult DSS.MethodsSixteen 6‐month‐old male DSS rats were kept on normal salt diet for the duration of the study; eight of them were continuously treated with 250 mg/L Lisinopril administered through their water for 2 months (15 mg/kg/day), and eight DSS rats remained on regular water as a control. Systolic BP (SBP) was measured in conscious male DSS rats via tail‐cuff plethysmography, and open field test (OFT) was performed to assess locomotor and anxiety‐like behavior at baseline prior to treatment and repeated 2 months after treatment began. Statistical regression analyses were performed using Prizm 7: 1‐way and 2‐way ANOVA, T‐test when applicable. The data are presented as mean ± standard deviation.ResultsPrior to treatment, all DSS rats displayed hypertension and BP elevation with age (SBP: 170 ± 11 vs. 142 ± 5 mmHg, 6‐mo vs. 3‐mo, n=16; P<0.01). After 2 months, control untreated animals showed no difference in SBP and were hypertensive (170 ± 15 vs. 168 ± 13 mmHg, 8‐mo vs. 6‐mo, n=8; n/s). Rats treated with Lisinopril for 2 months showed a dramatically decreased SBP compared to their baseline prior to treatment (119 ± 9 vs. 171 ± 11 mmHg, P<0.0001, n=8; Fig. 1), verifying the efficacy of the drug. In OFT, treatment with Lisinopril increased exploration of the center of the field compared to the control group (160 ± 45 vs. 91 ± 55 % of distance traveled in center to baseline, p=0.0166, n=8/group; Fig. 2) without affecting total distance traveled, indicating a reduction in anxiety‐like behavior. Furthermore, there was a significant negative correlation between SBP and increased activity in the center of the field (Pearson R=−0.58, R2=0.34, P=0.018; Fig. 3), suggesting anxiolytic effects of BP lowering medication.ConclusionLowering of BP is associated with reduced anxiety‐like behavior in DSS, which indicate the connection of cardiovascular disorder and cognitive function in adult hypertensive DSS. Whether Lisinopril has an additional direct effect on brain functions in DSS rats will be further studied.Support or Funding InformationSupported by NIH/NIA Intramural Research ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ALMS1 deletion in rats causes hyperleptinemia, progressive obesity, and renal damage

Genome‐wide association studies identified single nucleotide polymorphisms in the Alström syndrome 1 (ALMS1) gene linked to chronic kidney disease (CKD). ALMS1 has also been associated to obesity and insulin resistance. Inactivating mutations in the ALMS1 gene cause Alström syndrome in humans, characterized by early onset obesity, insulin resistance and renal dysfunction. We recently found that ALMS1 is expressed in renal tubules and its deletion from Dahl salt sensitive rats induced hypertension, in part by enhancing renal NaCl reabsorption. We also observed that ALMS1 KO rats developed progressive obesity as they aged which was evident by 11 weeks of age. In mice, mutations in ALMS1 cause obesity and hyperleptinemia, yet it is not known whether higher leptin levels are secondary to obesity. The effect of deleting ALMS1 in glomerular filtration is not known. We hypothesized that ALMS1 deletion causes hyperleptinemia before an increase in body weight (BW) and also induces decreased renal function in rats. To test this we studied homozygous ALMS1 knockout (KO) and littermates wild‐type Dahl SS (WT) rats at different time points. We found age‐dependent effects. ALMS1 KO rats developed a progressive increase in BW. At 7 weeks BW was not different between strains but was significantly higher by 11 weeks (ALMS1: 377±18 vs. WT: 319±18 g, p<0.05) or 16 weeks (ALMS1: 442±21 vs. WT: 366±22 g, p<0.05). We then measured plasma leptin prior to the development of obesity (7 week old) and at 16 weeks. Interestingly leptin was already higher at 6–7 weeks (ALMS1: 3530±300 vs. WT: 172±16 pg/ml, p<0.05) and was higher at 18 weeks of age (ALMS1: 39517 ± 3843 vs. WT: 1954 ± 171 pg/ml, p<0.05). We also found ALMS1 rats were hyperphagic (ALMS1: 19±1 vs. WT: 27±2 g/day, p<0.05). Since the ALMS1 gene is associated to lower renal function in humans, we measured GFR in conscious rats, calculated from inulin clearance. We found that GFR was not different by 10 weeks of age (ALMS1: 2.8±0.1 vs. WT: 3.1±0.2 ml/min, p=0.28) but was severely reduced by 50% at 18 weeks of age (ALMS1: 2.3±0.4 vs. WT: 5.0±1.1 ml/min, p<0.05). This difference was maximized after correction by BW, thus we elected to show net filtration rates. To further asses renal damage we measured urinary albumin/creatinine ratio. Urinary albumin/creatinine ratio was similar in 7 week‐old rats (ALMS1: 0.85±0.07 vs. WT: 0.98±0.12, p=0.12) but was higher in 15 week‐old ALMS1 KO (ALMS1: 28.74±6.28 vs. WT: 3.20±1.38, p=0.07) indicating progressive renal damage. We concluded that deletion of ALMS1 caused primary hyperleptinemia, progressive obesity and gradual renal dysfunction. This is the first study indicating a role of ALMS1 in leptin metabolism prior to the development of obesity. Our findings establish a link between the ALMS1, obesity and progressive renal dysfunction. Our data point to ALMS1 as a potentially novel gene involved in leptin biology/function and renal disease.Support or Funding InformationFunding: RO1 DK105818A1, Henry Ford Fund.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pairing Electrophysiology and Single Cell RNA Sequencing to Identify Mechanisms of Cellular pH/CO2 Sensitivity in Respiratory Chemoreceptor Neurons

Respiratory chemoreceptors are specialized neurons that are excited by hypercapnic acidosis (HA) and encode brain pH and/or CO2 levels to drive the appropriate changes in breathing. A subset of serotonin‐producing (5‐HT) neurons develop an apparent intrinsic chemosensitivity to HA after ~P12, but the molecular mechanisms driving their cellular pH/CO2 sensitivity remains unknown. Given that Sudden Infant Death Syndrome (SIDS) in humans has been linked to developmental 5‐HT system defects and potential alterations in ventilatory chemoreflexes, we aimed to develop a new technique to identify transcriptional differences among pH‐sensitive and ‐insensitive 5‐HT neurons in order to ultimately define the genetic determinants of cellular pH/CO2 sensitivity. Acute brainstem slices (200 uM) were prepared from 18–23 day‐old transgenic Dahl Salt Sensitive rats that express eGFP in all 5‐HT neurons (SSeGFP). Firing rates were measured via cell‐attached patch in eGFP+ 5‐HT neurons superfused with an artificial CSF (aCSF) containing inhibitors for synaptic blockade (10 mM CNQX, 50 mM D‐AP5, 20 mM Gabazine) while cycling between control (5% CO2 bal. O2; pH= 7.36; 5 min) and HA (15% CO2 bal. O2; pH= 7.10; 10 min) conditions. Once the cellular phenotype was established for pHsensitive (n=6 cells) or ‐insensitive (n=5) cells, a second electrode filled with small amounts (<1ul) of intracellular solution spiked with RNAse inhibitor was used to isolate the cellular contents for subsequent single cell RNA Sequencing (scRNA‐Seq). Specificity of cellular isolation was confirmed by high levels of expression of several serotonin and neuronal genes, and a lack of glial gene expression by sc‐qRT‐PCR (n=7) or sc‐RNAseq. The scRNA‐Seq read summary indicated an average of ~45M reads, a quality score of ~33.5, and ~80% mapping rate per sample, from which >10,000 known genes were quantified. However, small sample sizes prevented obtaining statistical significance for differential expression (Q > 0.05). Using R software packages, unsupervised analyses (hierarchical clustering and principal component analyses) failed to clearly separate samples by phenotype, and a power analysis based on FPKM values from the 11 samples suggested sample sizes of 20–60 cells/group are required. However, a supervised analysis (pre‐defining experimental groups) using Support Vector Machine (SVM) learning coupled with Recursive Feature Elimination (i.e. creates a model with all genes and recursively reduces the number of genes to identify the fewest genes to explain the predefined phenotype) revealed a candidate molecular signature for CO2 sensitive 5‐HT neurons. Overall, we demonstrated we can obtain transcriptomic data from electrophysiologically‐characterized 5‐HT neurons at the single cell‐level, and analyses of these pilot data suggest unsupervised and supervised machine learning approaches may identify unique genetic profiles in pH‐sensitive 5‐HT neurons. These data provide the field of respiratory physiology with preliminary markers of 5‐HT neuron CO2/pH sensitivity and validate that this approach may be used to address questions pertaining to genetic determinants of a given cellular phenotype.Support or Funding InformationNIH R01 HL12358 04 & Parker B. Francis FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sympathetic Neurotransmission in Resistance Mesenteric Arteries in Obesity‐Related Hypertension

Hyperactivity of sympathetic nerves has an important role in the pathogenesis of obesity‐related hypertension. The mechanisms linking obesity to hypertension are unclear. Small mesenteric arteries (MA, <300 mm dia.) are resistance blood vessels that are densely innervated by sympathetic nerves. Nerve varicosities release norepinephrine (NE) and ATP which cause arterial constriction. Impaired control of NE and/or ATP release and clearance would cause hypertension. Alterations in sympathetic nerves supplying MAs contribute to hypertension in several animal models. This study examined whether alterations of sympathetic neurotransmission in the resistance MAs contribute to high blood pressure in high fat‐fed (HF) Dahl salt‐sensitive rats. Sympathetic nerves supplying MAs maintained in vitro were stimulated focally. Electrically‐evoked NE and ATP release were detected by using amperometry with carbon fiber electrodes, and intracellular recording of excitatory junction potentials (EJPs) from arterial smooth muscle cells, respectively. Data were obtained from male and female rats at 10, 17, and 24 weeks of control and HF diet. Peak amplitudes of NE oxidation current and EJPs were assessed. NE oxidation current from all groups were blocked by a Na+ channel blocker, tetrodotoxin (TTX, 300 nM), suggesting that NE release in neurogenic. EJPs from all groups were abolished by TTX, and PPADS (10 μM), a P2X receptor antagonist, indicating that EJPs were neurogenic and purinergic. Frequency (1–30 Hz, 50 pulses) response curves using amperometric measurement of NE release showed similar responses in MAs from control and HF fed male and female rats. EJP frequency (0.5–10Hz, 5 pulses) response curves were also similar in MA from control and HF fed male and female rats. We investigated prejunctional α2‐adrenergic receptor function using the α2 receptor agonist (UK14,304) and antagonist (yohimbine). UK14,304 (0.0001–10 μM) inhibited NE and ATP release equally well and in a concentration‐dependent manner in MAs from control and HF rats. In the presence of 1 μM yohimbine, NE and ATP release was increased equally in MA from all rats. We conclude that adrenergic and purinergic neurotransmission by periarterial sympathetic nerves are not affected in HF Dahl salt sensitive rats. Elevated blood pressure in this animal model is not dependent on altered sympathetic neurotransmission to the mesenteric vasculature.Support or Funding InformationNIH 2P01HL070687 (For JJG), and Post‐doctoral training scholarship, Faculty of Medicine, Thammasat University, Thailand (For SS).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of Formyl Peptide Receptor Precedes the Onset of Hypertension in Dahl Salt Sensitive Rats: Effects of Microbiota and Salt

AimsChronic activation of the immune system contributes to kidney injury and hypertension. Mitochondria carry hallmarks of their bacterial ancestry and thus have emerged as a significant source of inflammatogenic damage‐associated molecular patterns. One of these hallmarks is that they still use formylated peptides (NFPs) as an initiator of protein synthesis. NFPs activate formyl peptide receptor (FPR), a G‐protein coupled receptor, and leads to receptor internalization and desensitization. We have observed that mitochondrial NFPs are elevated in the circulation of spontaneously hypertensive rats (SHR) and FPR blockage decreases blood pressure in this strain. Also, we and others observed that FPR downregulates upon activation in immune and non‐immune cells. Given that cell death and gut dysbiosis are present in salt sensitive hypertension, we hypothesized that Dahl sensitive rats would have decreased FPR expression, and that the decrease could be attributed to increased plasma levels of circulating mitochondria and/or bacteria NFPs from the cell death and leaky gut, respectively, and contributes to hypertension. Sex differences were also investigated.MethodsMale and Female Dahl Sensitive (S) and Resistant (R) rats (6‐week old) were given a low (0.3% NaCl) or high salt diet (2% NaCl) for 38 days. Rats received normal drinking water or water supplemented with neomycin (0.5 g/l, GIBCO) for 3 weeks. Kidney samples were taken from all rats and used for mRNA extraction and purification. The mRNA was made into cDNA to test gene expression for FPR. Blood was collected to measure mitochondrial NFPs. T‐test *p<0.05; n= 8–11 for Dahl S and R; n=7 for Dahl + neomycin.Results and ConclusionFPR expression was downregulated 4‐fold from male and female Dahl S when compared to Dahl R (AU: FPR mRNA: Dahl R 4.5 ± 1.3 vs. Dahl S: 0.7 ± 0.2*) (Figure 1). This phenomenon was independent of salt and sex differences. Antibiotic treatment partially restored FPR expression from Dahl S animals when compared to Dahl S treated with neomycin (AU FPR mRNA: Dahl S 4.5 ± 1.3 vs. Dahl S‐neomycin treated: 2.74 ± 0.99*). ND6, a mitochondrial protein, is 1.7‐fold higher in plasma from Dahl S compared to Dahl R. Unexpected, salt diet did not change this parameter. We have observed for the first time that FPR is downregulated in Dahl animals independent of salt probably due to overstimulation of this receptor. Therefore, FPR activation due to increased levels of bacterial (NFPs) could be associated with the development of renal injury at an early age independently of salt and elevations in arterial pressure.Support or Funding InformationAmerican Heart Association (18POST34060003) and National Institutes of Health (K99GM118885 and R01HL143082)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Kir5.1‐Mediated Changes in Renin‐Angiotensin‐Aldosterone System Balance in Salt Sensitive Hypertension

Kir5.1 (encoded by Kcnj16 gene) is a member of the inwardly rectifying K+ channel (Kir) family which is expressed in the kidney where it plays an essential role in the maintenance of electrolyte balance and blood pressure control. More specifically, Kir5.1 is highly expressed in the distal convoluted tubule and collecting ducts, also known as the aldosterone‐sensitive distal nephron (ASDN), where it forms a functional channel with Kir4.1. Our previous studies revealed that a knockout of Kcnj16 in the Dahl salt‐sensitive rat (SSKcnj16−/−) results in decreased blood pressure, salt wasting tubulopathy, and hypokalemia (Palygin et al., JCI Insight, 2017). Furthermore, when fed a high salt diet (HS; 4% NaCl), hypokalemia was exacerbated and resulted in mortality of SSKcnj16−/− rats within a few days. Importantly, dietary potassium supplementation or ENaC inhibition with benzamil treatment prevented salt‐induced death. However, specific mechanisms pertaining to cardiorenal abnormalities in SSKcnj16−/− rats and the interaction of Kir4.1/Kir5.1‐mediated potassium transport with the renin‐angiotensin‐aldosterone system (RAAS, a major hormone system that controls fluid and electrolyte balance in ASDN) remains unclear. The main objective of this study was to assess the contribution of RAAS, and specifically aldosterone, in the phenotype observed in SSKcnj16−/− rats. We used mass spectrometry‐based quantification of individual RAAS components to examine whether RAAS hormones were altered in SSKcnj16−/− rats. We found that SSKcnj16−/− rats have drastically elevated serum aldosterone (1878 ± 303 pM for SSKcnj16−/− rats vs 183 ± 35 pM for SS control rats) which becomes significantly attenuated preceding mortality upon introduction of a high salt diet (394 ± 82 pM). All angiotensin derivatives were similarly elevated in SSKcnj16−/− rats, as were RAAS hormone ratios as surrogate measures of angiotensin‐converting enzyme (ACE) activity (Ang II/Ang I), plasma renin activity (PRA, Ang I+Ang II), and primary aldosteronism (AA2‐Ratio). No significant differences in circulating RAAS hormones between male and female rats were observed. Furthermore, blocking aldosterone action with the administration of the potassium‐sparing diuretic, spironolactone (50 mg/kg IP), which is a potent mineralocorticoid receptor antagonist, did not prevent mortalities from a HS diet. Moreover, spironolactone caused 100% mortality within 48 hrs in SSKcnj16−/− (N = 17) but not SS rats during low salt treatment. Blood electrolyte analysis after 24 hrs treatment with spironolactone did not indicate a decline in serum potassium, which was the cause of mortality due to HS intake. Despite this, dietary potassium supplementation (0.36% to 1.41% dietary K+) prevented mortalities due to spironolactone administration in SSKcnj16−/− rats (N = 6). Our data suggest that there is a direct correlation between Kir4.1/Kir5.1‐mediated potassium transport and RAAS function. Our current studies further reveal that hypokalemia due to Kir5.1 deletion results in compensatory RAAS elevation and that supplementation of the diet with high K+ protects from mortality resulting from aldosteronemediated mechanisms.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

β‐Hydroxybutyrate (βHOB) Increases Nitric Oxide Synthase Activity in Resistance Arteries from Dahl Salt‐sensitive Rats

The deleterious link between overconsumption of dietary salt and hypertension is unequivocal. This dogma has recently expanded with the report that a high salt diet lowers the circulating levels of the ketone metabolite, beta‐hydroxybutyrate (βHOB), in Dahl salt‐sensitive rats (Dahl S‐HS). While stimulators of ketogenesis are well known for improving cardiovascular health (e.g., exercise and caloric restriction), less is known about negative‐regulation of ketone body metabolism and how it may impact hypertension and vascular aging. Therefore, we hypothesized that raising the circulating levels of βHOB would improve endothelium‐dependent relaxation in mesenteric resistance arteries (MRA) of Dahl S‐HS. To test this hypothesis, 1,3‐butanediol (20% v/v), which is a precursor of βHOB, was supplemented in the drinking water of 5 week old, male Dahl S‐HS for 8 weeks. Subsequently MRA function was analyzed using wire myographs. Although relaxation to endothelium‐dependent vasodilator acetylcholine (ACh) was not different [%Relaxation, Dahl S‐low salt (LS): 40±11 vs. Dahl S‐HS: 52±11 vs. Dahl S‐HS+βHOB: 44±11, p>0.05], the mechanism of relaxation was different between the groups. Specifically, while nitric oxide synthase inhibitor (L‐NAME; 100 μM) completely prevented relaxation in Dahl S‐LS and Dahl S‐HS+βHOB groups, MRA from Dahl S‐HS still partially relaxed in the presence of L‐NAME (%Relaxation, Dahl S‐LS: 1±4 vs. Dahl S‐HS: 33±5 vs. Dahl S‐HS+βHOB: 6±4, p<0.05; Figure 1A–C). These data suggest that MRA from Dahl S‐HS relax through nitric oxide‐independent mechanisms and that βHOB treatment restores nitric oxide synthase activity, despite consumption of a high salt diet. To test whether 1,3‐butanediol treatment had direct effects on arterial function, concentrations‐response curves were performed to βHOB and 1,3‐butanediol in MRA from naïve 5 week old, male Dahl S‐LS rats. We observed that βHOB and 1,3‐butanediol caused triphasic‐quadriphasic responses after pre‐contraction to phenylephrine (PE; Figure 2A–B). Specifically, we observed that low concentrations (<3 nM) of βHOB caused pronounced vasodilation and this was inhibited by both L‐NAME and tetraethylammonium (TEA; potassium channel inhibitor; both, p<0.05; Figure 3A–B). Intermediate concentrations of βHOB (3 nM–100 nM) caused contraction and this tended to be inhibited by indomethacin (cyclooxygenase inhibitor; p=0.08; Figure 3C). Finally, high concentrations (100 nM–30 μM) of βHOB caused relaxation again and this was predominately mediated by nitric oxide (p<0.05; Figure 3A). Overall, these data suggest that βHOB stimulates the production of endothelium‐derived factors and low concentrations of βHOB could be a novel therapy against hypertension‐associated vascular aging by increasing nitric oxide synthesis.Support or Funding InformationAmerican Heart Association (18POST34060003) and National Institutes of Health (K99GM118885 and R01HL143082).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Liposome Delivery Enhances Clodronate Nephrotoxicity in Dahl SS Hypertension and Renal Injury

Studies in the Dahl Salt‐Sensitive (SS) rat have shown that infiltrating renal CD3+ T cells and ED‐1+ macrophages are involved in the development of SS hypertension (HTN) and renal damage. We have previously demonstrated the importance of infiltrating cells by pharmacological immunosuppression and genetic deletion of adaptive immune cells, but the role of innate immunity in SS HTN is still unclear. It is practically more difficult to assess the contribution of the innate immune system macrophage to this pathology due its vital role in development and in bone and liver physiology. A proposed method of macrophage depletion is via liposome‐encapsulated Clodronate which has shown mixed results, from attenuation of Ang‐II induced HTN, to inducing salt‐sensitive HTN. This study tests the hypothesis that Clodronate‐Liposome treatment will attenuate Dahl SS HTN and renal damage through macrophage depletion. To do so, animals were administered either Clodronate‐Liposomes (Clod‐Lipo) or PBS‐Liposomes (PBS‐Lipo) as a vehicle control. A third group of animals were treated with Clodronate alone (Clod) to assess direct effects of the drug. 7‐week‐old male Dahl SS rats were implanted with radiotelemeters and after recovery and baseline pressure recordings on a low‐salt (0.4% NaCl) diet, animals received a tail vein injection of either PBS‐Lipo (equivalent volume), Clod‐Lipo (50mg/kg), or Clod (50mg/kg). Booster injections (25mg/kg) were administered every 7 days thereafter. Three days after the initial injection, all animals were switched to a high‐salt (4.0% NaCl) diet. Animals were followed for 21 days of the high‐salt diet with weekly overnight urine collections. Clod treatment alone did not alter circulating or splenic levels of macrophages unlike Clod‐Lipo treatment which depleted macrophages by about 50%. Contrary to our hypothesis, by the end of the study, Clod‐Lipo treated animals developed an exacerbated SS response in respect to blood pressure (Clod‐Lipo 178.7±7.5mmhg, PBS‐Lipo 157.2±3.9mmhg, p<0.01 Two‐way RM‐ANOVA, n=8/7) and albuminuria (Clod‐Lipo 403.6±32.8mg/day, PBS‐Lipo 180.6±22.4mg/day, p<0.001 Two‐way RM‐ANOVA, n=11/7). This exacerbation in the Clod‐Lipo group was accompanied by an increase in infiltration of adaptive T and B cells despite a depletion of circulating macrophages and a reduction in infiltrating macrophages. The third group of animals receiving Clod alone demonstrated a similar, though delayed, exacerbation in the SS increase in blood pressure (181.6±6.2mmhg p<0.05 Two‐way RM‐ANOVA, n=4). In addition, Clod treated animals showed elevated albuminuria (259.2±48.6mg/day, p<0.05 Two‐way RM‐ANOVA, n=5) though not to the same extent as Clod‐Lipo treated animals. These SS phenotypic changes in Clod treated animals were independent of changes to circulating, splenic, and infiltrating renal immune cells. This study demonstrates Clodronate's direct nephrotoxic effects similar to those of other bisphosphonates. Furthermore, liposome delivery of Clodronate further exacerbated the SS renal damage and lymphocyte infiltration, indicating the potential adverse effects of this macrophage depletion strategy.Support or Funding InformationHL‐116264, HL‐137748, and 18PRE3400038This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Disruption of mesothelial connections to the spleen inhibits splenic anti‐inflammatory responses and protects from the development of insulin resistance in rats

We have recently reported that stimulation of gastric acid secretion with NaHCO3 promotes an anti‐inflammatory response in both rats and healthy human subjects. Importantly, disruption of ‘neuronal like’ mesothelial cell connections to spleen abolished this response. The Dahl salt‐sensitive (SS) rat model develops insulin resistance when fed a high salt diet. As low grade inflammation is thought to promote insulin resistance, we hypothesized that ‘Ingestion of NaHCO3 would prevent the development of insulin resistance in Dahl SS rats and that this protection would be lost following surgical disruption of mesothelial connections to the spleen’.9 week old male Dahl salt‐sensitive rats were anesthetized with isoflurane, an abdominal incision performed and either; mesothelial connections to the spleen physically disrupted (DISRUPTED) or; the spleen left untouched (SHAM). Following 1 wk of recovery from surgery, all rats were placed on an 8% high salt (HS) diet for 3 weeks. Upon starting the HS diet, the drinking water was replaced with either 0.1M NaHCO3 (n=12 sham, n=10 disrupted) or equimolar NaCl solutions (n=11 sham, n=9 disrupted). An insulin tolerance test as performed at day 17 of HS feeding. Following 21 days of HS the spleen was harvested for flow cytometry analysis (n=6 each group).Ingestion of NaHCO3 in SHAM rats resulted in significantly fewer splenic pro‐inflammatory M1 macrophages when compared to SHAM rats drinking NaCl (4.8±0.3 vs 7±0.5% splenocytes, ANOVA p=0.01, respectively) and this difference was abolished following mesothelial disruption (NaHCO3 + DISRUPTED =7.2±0.5%, p=N.S). Insulin administration resulted in a decrease in blood glucose in all groups, however the decrease in blood glucose was significantly greater in the DISRUPTED + NaHCO3 group when compared to all other groups (p=0.02). The % of splenic cells identified as myeloid dendritic cells mirrored the development of insulin resistance, with myeloid dendritic cells being significantly lower (p=0.02) only in the DISRUPTED + NaHCO3 group.In agreement with our previous studies, ingestion of NaHCO3 promoted an anti‐inflammatory response in the spleen which was abolished by surgical disruption of splenic mesothelial connections. Protection from the development of insulin resistance however, did not correlate with this anti‐inflammatory effect. Rather, the degree of insulin resistance was mirrored by differences in splenic myeloid dendritic cells. These data are important as they demonstrate that, despite promoting an inflammatory phenotype, disruption of mesothelial connections to the spleen can contribute to protection from the development of insulin resistance. As both NaHCO3 ingestion and vagal nerve stimulation promote gastric acid secretion, and the anti‐inflammatory response to both stimuli can be inhibited by a nicotinic receptor antagonist, we speculate that these mesothelial connections mediate the anti‐inflammatory response to efferent vagal stimulation. Our data indicate however, that multiple signals are received by the spleen and that these act in combination to modulate immune responses resulting in varied functional effects.Support or Funding InformationDK099548, P01HL134604This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mitochondria ROS in podocytes of freshly isolated glomeruli during salt‐sensitive hypertension

The hallmark of salt‐sensitive (SS) hypertension is an early onset of proteinuria, which results from loss of epithelial cells of the glomerular filtration barrier – podocytes. Several cell culture studies show that mitochondria dysfunction can contribute to podocyte apoptosis, thus leading to proteinuria. During hypertension, mitochondria in the kidney cells are known to lose cristae membranes, reduce their size and have reduced matrix density, leading to decreased ATP levels and increased reactive oxygen species (ROS). The goal of this study was to develop the method to monitor mitochondrial damage in the podocytes and test the role of mitochondria in podocyte dysfunction during SS.Hypertension was induced in Dahl SS rats by a high salt diet challenge (HS, 4% NaCl, 21 days); control group was maintained on the low salt diet (0.4% NaCl, LS). At the end of the protocol blood pressure was measured using the tail cuff, kidney flush via abdominal aorta was performed, tissues were collected, glomeruli were isolated by differential sieving and then loaded with TMRM (mitochondria membrane potential marker) to visualize podocytic mitochondria, or MitoPY1 (a mitochondria‐targeting ROS indicator), and imaged with a Zeiss 880 confocal microscope. Blood electrolyte levels were measured using a CareLyte Electrolyte ISE Analyzer. Kidney samples were stained using Masson's trichrome in order to characterize damage. Using a BD CARV II Confocal Imager, fifty glomeruli from each sample were blindly scored on a scale of 0–4.Systolic blood pressure was elevated in the HS diet fed rats (171 ± 9 vs 148 ± 6 mmHg in HS vs LS diet fed groups, respectively, n=11 per group); a trend for renal hypertrophy was noted in the HS group compared to controls. No differences in plasma Na+, Cl− or K+ were observed. Histological scoring revealed that HS diet fed rats had exacerbated glomeruli damage compared to the LS group (3.1 ± 0.2 vs 1.9 ± 0.1, p = 0.006). Using an uncoupling agent FCCP we confirmed in the TMRM‐loaded samples that the signals we are recording are generated specifically by mitochondria since upon FCCP application we observed fast (within 40 ± 12 s) quenching of TMRM fluorescence. In MitoPY1‐stained glomeruli we detected higher baseline mitochondrial ROS level, as well as more fragmented mitochondria in the podocytes of the HS diet, fed group. In addition, we revealed a differential response to an acute application of 20 μM H2O2 between the LS vs HS groups. Future directions of the study involve assessing mitochondria biogenesis, performing a detailed structural analysis of mitochondria using electron microscopy, and testing a panel of mitochondrial damage markers in qPCR in the setting of SS hypertension. Further work will be devoted to discovering molecular pathways contributing to altered mitochondria structure and function in podocytes in SS hypertension.Support or Funding InformationSupported by NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A dipeptidyl peptidase-IV inhibitor improves diastolic dysfunction in Dahl salt-sensitive rats

To date, there is no established treatment for heart failure with preserved ejection fraction (HFpEF). Dipeptidyl peptidase-IV (DPP-IV) inhibitors reportedly have improved not only diabetes mellitus but also heart failure with systolic dysfunction in experimental models. We investigated the effects of a DPP-IV inhibitor on HFpEF in rats. Dahl salt-sensitive rats were fed either high-salt (high-salt diet (HSD): 8% NaCl) or low-salt diets (0.3% NaCl) from 6.5 weeks of age. They were then treated with or without a DPP-IV inhibitor, vildagliptin (10 mg/kg/day, orally), from 11 weeks of age for 9 weeks and analyzed at the age of 20 weeks. HSD rats mimicked the pathophysiology of HFpEF. There were no differences in heart rate, blood pressure, left ventricular (LV) systolic function, or the extent of LV hypertrophy between HSD rats with or without vildagliptin. However, vildagliptin decreased LV end-diastolic pressure, the most reliable hemodynamic parameter of HFpEF in HSD rats. Vildagliptin also decreased the LV distensibility index, a sensitive marker of LV diastolic function in HSD rats. Vildagliptin decreased the expression of collagen genes in HSD hearts and attenuated LV interstitial fibrosis (HSD with vehicle and vildagliptin, 2.9% vs. 1.9%; P < 0.05). Furthermore, vildagliptin administration reduced both plasma renin activity and aldosterone concentrations in HSD rats. A DPP-IV inhibitor, vildagliptin, improved the severity of LV fibrosis, and thus, diastolic dysfunction of HFpEF in Dahl salt-sensitive hypertensive rats. DPP-IV inhibitors are promising medicines for treatment of HFpEF in patients with diabetes mellitus.

Therapeutic Suppression of mTOR (Mammalian Target of Rapamycin) Signaling Prevents and Reverses Salt-Induced Hypertension and Kidney Injury in Dahl Salt-Sensitive Rats.

mTOR (mammalian target of rapamycin) signaling has emerged as a key regulator in a wide range of cellular processes ranging from cell proliferation, immune responses, and electrolyte homeostasis. mTOR consists of 2 distinct protein complexes, mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2) with distinct downstream signaling events. mTORC1 has been implicated in pathological conditions, such as cancer and type 2 diabetes mellitus in humans, and inhibition of this pathway with rapamycin has been shown to attenuate salt-induced hypertension in Dahl salt-sensitive rats. Several studies have found that the mTORC2 pathway is involved in the regulation of renal tubular sodium and potassium transport, but its role in hypertension has remained largely unexplored. In the present study, we, therefore, determined the effect of mTORC2 inhibition with compound PP242 on salt-induced hypertension and renal injury in salt-sensitive rats. We found that PP242 not only completely prevented but also reversed salt-induced hypertension and kidney injury in salt-sensitive rats. PP242 exhibited potent natriuretic actions, and chronic administration tended to produce a negative Na+ balance even during high-salt feeding. The results indicate that mTORC2 and the related downstream associated pathways play an important role in regulation of sodium balance and arterial pressure regulation in salt-sensitive rats. Therapeutic suppression of the mTORC2 pathway represents a novel pathway for the potential treatment of hypertension.

CT-1 (Cardiotrophin-1)-Gal-3 (Galectin-3) Axis in Cardiac Fibrosis and Inflammation.

Myocardial fibrosis is a main contributor to the development of heart failure (HF). CT-1 (cardiotrophin-1) and Gal-3 (galectin-3) are increased in HF and associated with myocardial fibrosis. The aim of this study is to analyze whether CT-1 regulates Gal-3. Proteomic analysis revealed that Gal-3 was upregulated by CT-1 in human cardiac fibroblasts in parallel with other profibrotic and proinflammatory markers. CT-1 upregulation of Gal-3 was mediated by ERK (extracellular signal-regulated kinase) 1/2 and Stat-3 (signal transducer and activator of transcription 3) pathways. Male Wistar rats and B6CBAF1 mice treated with CT-1 (20 µg/kg per day) presented higher cardiac Gal-3 levels and myocardial fibrosis. In CT-1-treated rats, direct correlations were found between cardiac CT-1 and Gal-3 levels, as well as between Gal-3 and perivascular fibrosis. Gal-3 genetic disruption in human cardiac fibroblasts and pharmacological Gal-3 inhibition in mice prevented the profibrotic and proinflammatory effects of CT-1. Dahl salt-sensitive hypertensive rats with diastolic dysfunction showed increased cardiac CT-1 and Gal-3 expression together with cardiac fibrosis and inflammation. CT-1 and Gal-3 directly correlated with myocardial fibrosis. In HF patients, myocardial and plasma CT-1 and Gal-3 were increased and directly correlated. In addition, HF patients with high CT-1 and Gal-3 plasma levels presented an increased risk of cardiovascular death. Our data suggest that CT-1 upregulates Gal-3 which, in turn, mediates the proinflammatory and profibrotic myocardial effects of CT-1. The elevation of both molecules in HF patients identifies a subgroup of patients with a higher risk of cardiovascular mortality. The CT-1/Gal-3 axis emerges as a candidate therapeutic target and a potential prognostic biomarker in HF.

Superimposed Preeclampsia Exacerbates Postpartum Renal Injury Despite Lack of Long-Term Blood Pressure Difference in the Dahl Salt-Sensitive Rat.

Preeclampsia results in increased susceptibility to hypertension and chronic kidney disease postpartum; however, the mechanisms responsible for disease progression in these women remain unknown. The purpose of this study was to test the hypothesis that 2 mechanisms contribute to the link between the maternal syndrome of preeclampsia and the increased postpartum risk of cardiovascular and renal disease: (1) increased T cells in the kidney and (2) a decreased NO:ET-1 (endothelin-1) ratio. Dahl S rats (a previously characterized model of preeclampsia superimposed on chronic hypertension) who experienced 2 pregnancies and virgin littermate controls were studied at 6 months of age. Mean arterial pressure was measured via telemetry, and renal injury was assessed through both histological analysis and measurement of urinary markers including nephrin, podocalyxin, and KIM-1 (kidney injury marker 1). Contributing mechanisms were assessed through flow cytometric analysis of renal T cells, quantification of plasma TNF-α (tumor necrosis factor-α) and IL-10 (interleukin-10), and quantification of urinary concentrations of NO metabolites and ET-1. Although prior pregnancy did not exacerbate the hypertension at 6 months, this group showed greater renal injury compared with virgin littermates. Flow cytometric analyses revealed an increase in renal T cells after pregnancy, and cytokine analysis revealed a systemic proinflammatory shift. Finally, the NO:ET-1 ratio was reduced. These results demonstrate that the link between the maternal syndrome of superimposed preeclampsia and postpartum risk of chronic kidney disease could involve both immune system activation and dysregulation of the NO:ET-1 balance.