Salt-sensitive Rats Research Articles

The p52 isoform of SHC1 is a key driver of breast cancer initiation

BackgroundSHC1 proteins (also called SHCA) exist in three functionally distinct isoforms (p46SHC, p52SHC, and p66SHC) that serve as intracellular adaptors for several key signaling pathways in breast cancer. Despite the broad evidence implicating SHC1 gene products as a central mediator of breast cancer, testing the isoform-specific roles of SHC1 proteins have been inaccessible due to the lack of isoform-specific inhibitors or gene knockout models.MethodsHere, we addressed this issue by generating the first isoform-specific gene knockout models for p52SHC and p66SHC, using germline gene editing in the salt-sensitive rat strain. Compared with the wild-type (WT) rats, we found that genetic ablation of the p52SHC isoform significantly attenuated mammary tumor formation, whereas the p66SHC knockout had no effect. Rats were dosed with 7,12-dimethylbenz(a)anthracene (DMBA) by oral gavage to induce mammary tumors, and progression of tumor development was followed for 15 weeks. At 15 weeks, tumors were excised and analyzed by RNA-seq to determine differences between tumors lacking p66SHC or p52SHC.ResultsCompared with the wild-type (WT) rats, we found that genetic ablation of the p52SHC isoform significantly attenuated mammary tumor formation, whereas the p66SHC knockout had no effect. These data, combined with p52SHC being the predominant isoform that is upregulated in human and rat tumors, provide the first evidence that p52SHC is the oncogenic isoform of Shc1 gene products in breast cancer. Compared with WT tumors, 893 differentially expressed (DE; FDR < 0.05) genes were detected in p52SHC KO tumors compared with only 18 DE genes in the p66SHC KO tumors, further highlighting that p52SHC is the relevant SHC1 isoform in breast cancer. Finally, gene network analysis revealed that p52SHC KO disrupted multiple key pathways that have been previously implicated in breast cancer initiation and progression, including ESR1 and mTORC2/RICTOR.ConclusionCollectively, these data demonstrate the p52SHC isoform is the key driver of DMBA-induced breast cancer while the expression of p66SHC and p46SHC are not enough to compensate.

Protein Kinase C Downregulation Enhanced Extracellular Ca2+-Induced Relaxation of Isolated Mesenteric Arteries from Aged Dahl Salt-Sensitive Rats.

The Ca2+-sensing receptor (CaSR) detects small changes in extracellular calcium (Ca2+ e) concentration ([Ca2+]e) and transduces the signal into modulation of various signaling pathways. Ca2+-induced relaxation of isolated phenylephrine-contracted mesenteric arteries is mediated by the CaSR of the perivascular nerve. Elucidation of the regulatory mechanisms involved in vascular CaSR signaling may provide insights into the physiologic functions of the receptor and identify targets for the development of new treatments for cardiovascular pathologies such as hypertension. Protein kinase Cα (PKCα) is a critical regulator of multiple signaling pathways and can phosphorylate the CaSR leading to receptor desensitization. In this study, we used automated wire myography to investigate the effects of CaSR mutation and small-interfering RNA downregulation of PKCα on CaSR-mediated relaxation of phenylephrine-contracted mesenteric arteries from aged Dahl salt-sensitive (SS) rats on a low-salt diet. The data showed minimal relaxation responses of arteries to Ca2+ e in wild-type (SS) and CaSR mutant (SS-Casrem1Mcwi) rats. Mutation of the CaSR gene had no significant effect on relaxation. PKCα expression was similar in wild-type and mutant rats, and small-interfering RNA downregulation of PKCα and/or inhibition of PKC with the Ca2+-sensitive Gӧ 6976 resulted in a >80% increase in relaxation. Significant differences in EC50 values were observed between treated and untreated controls (P < 0.05 analysis of variance). The results indicate that PKCα plays an important role in the regulation of CaSR-mediated relaxation of mesenteric arteries, and its downregulation or pharmacological inhibition may lead to an increased Ca2+ sensitivity of the receptor and reversal of age-related changes in vascular tone. SIGNIFICANCE STATEMENT: G protein-coupled CaSR signaling leads to the regulation of vascular tone and may, therefore, play a vital role in blood pressure regulation. The receptor has several PKC phosphorylation sites in the C-terminal intracellular tail that mediate desensitization. We have previously shown that activation of the CaSR in neuronal cells leads to PKC phosphorylation, indicating that protein kinase C is an important regulator of CaSR function. Therefore, PKC in the CaSR signaling pathway in mesenteric arteries is a potential target for the development of new therapeutic approaches to treat hypertension and age-related vascular dysfunction. The present studies show that small-interfering RNA downregulation of PKCα and pharmacological inhibition of PKC enhanced CaSR-mediated relaxation of phenylephrine-contracted mesenteric arteries from aged Dahl salt-sensitive rats.

Renal nerves and leukocyte infiltration in the kidney during salt-sensitive hypertension.

Based on previous studies suggesting a role of renal nerves in renal inflammation, the present studies were performed to test the hypothesis that renal nerves mediate renal damage in Dahl salt-sensitive (SS) hypertension by increasing renal leukocyte infiltration. Experiments were performed in Dahl SS rats with bilateral renal denervation (RDN) and bilateral sham operation (n = 10 or 11 per group) and with unilateral RDN and contralateral sham operation (n = 10). After denervation, rats were switched from a low-salt 0.4% NaCl (LS) diet to a high-salt 4% NaCl (HS) diet and maintained on HS diet for 21 days. Bilateral RDN reduced the magnitude of hypertension assessed by radiotelemetry in Dahl SS rats compared with sham-operated rats (mean arterial pressure 140.9 ±4.8 mmHg and 159.7 ± 3.5 mmHg, respectively) and reduced proteinuria at day 21 of HS diet. However, assessment of renal leukocyte infiltration demonstrated no significant effect of bilateral RDN on the number of infiltrating leukocytes (RDN 3.6 ± 0.5 × 106 vs. sham operated 4.3 ± 0.3 × 106 CD45+ cells) or any of the subsets examined by flow cytometry. The unilateral RDN experiment showed no effect of RDN on the renal infiltration of leukocytes (RDN 6.5 ± 0.9 × 106 vs. sham operated 6.1 ± 1.1 × 106 CD45+ cells/kidney) or renal damage in RDN vs. sham-operated kidney after 21 days of HS diet. This work investigated the relationship between renal nerves and renal inflammation during Dahl SS hypertension. Contrary to our hypothesis, the results of this work suggest that immune cell infiltration in the kidney of Dahl SS rats is not mediated by the renal nerves.

Role of the afferent renal nerves in sodium homeostasis and blood pressure regulation in rats.

New Findings What is the central question of this study? What are the differential roles of the mechanosensitive and chemosensitive afferent renal nerves in the reno‐renal reflex that promotes natriuresis, sympathoinhibition and normotension during acute and chronic challenges to sodium homeostasis? What is the main finding and its importance? The mechanosensitive afferent renal nerves contribute to an acute natriuretic sympathoinhibitory reno‐renal reflex that may be integrated within the paraventricular nucleus of the hypothalamus. Critically, the afferent renal nerves are required for the maintenance of salt resistance in Sprague–Dawley and Dahl salt‐resistant rats and attenuate the development of Dahl salt‐sensitive hypertension. These studies tested the hypothesis that in normotensive salt‐resistant rat phenotypes the mechanosensitive afferent renal nerve (ARN) reno‐renal reflex promotes natriuresis, sympathoinhibition and normotension during acute and chronic challenges to fluid and electrolyte homeostasis. Selective ARN ablation was conducted prior to (1) an acute isotonic volume expansion (VE) or 1 m NaCl infusion in Sprague–Dawley (SD) rats and (2) chronic high salt intake in SD, Dahl salt‐resistant (DSR), and Dahl salt‐sensitive (DSS) rats. ARN responsiveness following high salt intake was assessed ex vivo in response to noradrenaline and sodium concentration (SD, DSR and DSS) and via in vivo manipulation of renal pelvic pressure and sodium concentration (SD and DSS). ARN ablation attenuated the natriuretic and sympathoinhibitory responses to an acute VE [peak natriuresis (µeq min−1) sham 52 ± 5 vs. ARN ablation 28 ± 3, P < 0.05], but not a hypertonic saline infusion in SD rats. High salt (HS) intake enhanced ARN reno‐renal reflex‐mediated natriuresis in response to direct increases in renal pelvic pressure (mechanoreceptor stimulus) in vivo and ARN responsiveness to noradrenaline ex vivo in SD, but not DSS, rats. In vivo and ex vivo ARN responsiveness to increased renal pelvic sodium concentration (chemoreceptor stimulus) was unaltered during HS intake. ARN ablation evoked sympathetically mediated salt‐sensitive hypertension in SD rats [MAP (mmHg): sham normal salt 102 ± 2 vs. sham HS 104 ± 2 vs. ARN ablation normal salt 103 ± 2 vs. ARN ablation HS 121 ± 2, P < 0.05] and DSR rats and exacerbated DSS hypertension. The mechanosensitive ARNs mediate an acute sympathoinhibitory natriuretic reflex and counter the development of salt‐sensitive hypertension.

Spontaneous superimposed preeclampsia: chronology and expression unveiled by temporal transcriptomic analysis.

Preeclampsia (PE), a multifactorial pregnancy-specific syndrome accounting for up to 8% of pregnancy complications, is a leading cause of maternal and fetal morbidity and mortality. PE is also associated with long-term risk of hypertension and stroke for both mother and fetus. Currently, the only "cure" is delivery of the baby and placenta, largely because the pathogenesis of PE is not yet fully understood. PE is associated with impaired vascular remodeling at the maternal-fetal interface and placental insufficiency; however, specific factors contributing to this impairment have not been identified. To identify molecular pathways involved in PE, we examined temporal transcriptomic changes occurring within the uterus, uterine implantation sites, and placentae from the Dahl salt-sensitive (Dahl S) rat model of superimposed PE compared with Sprague Dawley (SD) rats. We hypothesized that targeted gene analysis and whole transcriptome analysis would identify genetic factors that contribute to development of the preeclamptic phenotype in the Dahl S rat and unveil novel biomarkers, therapeutic targets, and mechanistic pathways in PE. Quantitative real-time PCR (qRT-PCR) and whole genome microarray analysis were performed on isolated total RNA from uterus (day 0), uterine implantation sites (days 7 and 10), and placenta (days 14 and 20). We found 624, 332, 185, and 366 genes to be differentially expressed between Dahl S (PE) and SD (normal pregnancy) on days 0, 7, 10, and 14, respectively. Our data revealed numerous pathways that may play a role in the pathophysiology of spontaneous superimposed PE and allow for further investigation of novel therapeutic targets and biomarker development.

Guizhi Decoction () Inhibits Cholinergic Transdifferentiation by Regulating Imbalance of NGF and LIF in Salt-Sensitive Hypertensive Heart Failure Rats.

To observe the imbalance of anatomical and functional innervation factors of sympathetic nerves, nerve growth factor (NGF) and leukemia inhibitory factor (LIF), in salt-sensitive hypertensive heart failure rats and to explore the effects of treatment with Guizhi Decoction () on sympathetic remodeling by inhibiting cholinergic transdifferentiation. SS-13BN and Dahl salt-sensitive (DS) rats were divided into 3 groups: SS-13BN group (control group, n=9), DS group (model group, n=9) and GS group (Guizhi Decoction, n=9). After 10 weeks of a high-salt diet, the GS group rats were given Guizhi Decoction and other two groups were given saline at an equal volume as a vehicle. After 4 weeks' intragastric administration, rats were executed to detect the relevant indicators. Echocardiography and plasma n-terminal pro-B type natriuretic peptide (NT-proBNP) levels were used to assess cardiac function. Noradrenaline (NA) levels in the plasma and myocardium were detected to evaluate the sympathetic function. NGF and LIF expression were detected in the myocardium by Western blot or quantitative real-time PCR. Double immunofluorescence or Western blot was used to detect tyrosine hydroxylase (TH), choline acetyltransferase (CHAT) and growth associated protein 43 (GAP43) in order to reflect anatomical and functional changes of sympathetic nerves. DS group had anatomical and functional deterioration of sympathetic nerves in the decompensation period of heart failure compared with SS-13BN group. Compared with the DS group, Guizhi Decoction significantly decreased the expression of LIF mRNA/protein (P<0.01), increased the expression of NGF (P<0.05 or P<0.01), enhanced the levels of TH+/GAP43+ and TH+/CHAT+ positive nerve fibers (P<0.01), and improved the protein expression of TH and GAP43 in left ventricle, but had no effect on CHAT (P>0.05). Guizhi Decoction inhibited inflammatory infiltration and collagen deposition of myocardial injury, increased the content of myocardial NA (P<0.05), reduced the plasma NA level (P<0.01), improved cardiac function (P<0.01), and improved weight and blood pressure to some extent (P<0.05), compared with DS group. Guizhi Decoction could inhibit cholinergic transdifferentiation of sympathetic nerves, improve the anatomical and functional denervation of sympathetic nerves, and delay the progression of decompensated heart failure. The mechanism may be associated with the correction of the imbalance of NGF and LIF.

Inhibitory Effects of Tofogliflozin on Cardiac Hypertrophy in Dahl Salt-Sensitive and Salt-Resistant Rats Fed a High-Fat Diet.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are drugs for diabetes and might prevent heart failure. In this study, we investigated the effects of tofogliflozin, an SGLT2 inhibitor, on cardiac hypertrophy and metabolism in hypertensive rats fed a high-fat diet. Dahl salt-sensitive (DS) rats, hypertensive model rats, and Dahl salt-resistant (DR) rats, non-hypertensive model rats, were fed a high-salt and high-fat diet containing tofogliflozin (0.005%) for 9 weeks to examine the effects of this drug on cardiac hypertrophy and metabolism. Tofogliflozin tended to suppress a rise of the systolic blood pressure, relative to the control, throughout the treatment period in both DR and DS rats, and significantly suppress a rise of the systolic blood pressure, relative to the control, at the 9th week in DS rats. Tofogliflozin reduced cardiac hypertrophy (heart weight/body weight) not only in DS rats but also in DR rats. Histological analysis showed that tofogliflozin significantly decreased cardiomyocyte hypertrophy and perivascular fibrosis in both DS and DR rats. Tofogliflozin significantly decreased the expression levels of genes related to cardiac hypertrophy (encoding for natriuretic peptides A and B and interleukin-6), and to cardiac fibrosis (encoding for transforming growth factor-β1 and collagen type IV), in DS rats. Recent studies have shown that hypertrophied and failing hearts shift to oxidizing ketone bodies as a significant fuel source. We also performed metabolome analysis for ventricular myocardial tissue. Tofogliflozin reduced 3-hydroxybutyrate, a ketone body, and significantly decreased the expression levels of β-hydroxybutyrate dehydrogenase 1 and 3-oxoacid CoA-transferase, which are related to ketone oxidization. In conclusion, tofogliflozin ameliorated cardiac hypertrophy and fibrosis along with reduction of ketone usage in myocardial tissue.

Superoxide increases surface NKCC2 in the rat thick ascending limbs via PKC

The apical Na+-K+-2Cl- cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb (TAL). The free radical superoxide ( ) stimulates TAL NaCl absorption by enhancing NKCC2 activity. In contrast, nitric oxide (NO) scavenges and inhibits NKCC2. NKCC2 activity depends on the number of NKCC2 transporters in the TAL apical membrane and its phosphorylation. We hypothesized that stimulates NKCC2 activity by enhancing apical surface NKCC2 expression. We measured surface NKCC2 expression in rat TALs by surface biotinylation and Western blot analysis. Treatment of TALs with produced by exogenous xanthine oxidase (1 mU/ml) and hypoxanthine (500 µM) stimulated surface NKCC2 expression by ~18 ± 5% (P < 0.05). -stimulated surface NKCC2 expression was blocked by the scavenger tempol (50 µM). Scavenging H2O2 with 100 U/ml catalase did not block the stimulatory effect of xanthine oxidase-hypoxanthine (22 ± 8% increase from control, P < 0.05). Inhibition of endogenous NO production with Nω-nitro-l-arginine methyl ester enhanced surface NKCC2 expression by 21 ± 6% and, when added together with xanthine oxidase-hypoxanthine, increased surface NKCC2 by 41 ± 10% (P < 0.05). Scavenging with superoxide dismutase (300 U/ml) decreased this stimulatory effect by 60% (39 ± 4% to 15 ± 10%, P < 0.05). Protein kinase C inhibition with Gö-6976 (100 nM) blocked -stimulated surface NKCC2 expression (P < 0.05). did not affect NKCC2 phosphorylation at Thr96/101 or its upstream kinases STE20/SPS1-related proline/alanine-rich kinase-oxidative stress-responsive kinase 1. We conclude that increases surface NKCC2 expression by stimulating protein kinase C and that this effect is blunted by endogenous NO. -stimulated apical trafficking of NKCC2 may be involved in the enhanced surface NKCC2 expression observed in Dahl salt-sensitive rats.

High Sodium Intake Impairs Small Artery Vasoreactivity in vivo in Dahl Salt-Sensitive Rats

The effects of high sodium intake on the functionality of resistance arteries have been repeatedly studied in vitro, but no study has focused on salt-sensitive hypertension in vivo. We studied the in vivo reactivity of mesenteric small arteries (MSAs) to vasoactive agents in Dahl salt-sensitive (DS) rats with various sodium diets. Twenty-four male DS rats were randomized into 3 groups: LS (0.3% NaCl diet), NS (0.6% NaCl diet), and HS (8% NaCl diet). After a 12-week intervention, the diameter changes of the MSAs after noradrenaline (NA) and acetylcholine (ACh) exposure were detected by a microscope, and changes in blood perfusion through the MSAs were measured by full-field laser perfusion imaging. HS enhanced the constrictive response of the MSAs to NA and attenuated the relaxing response to ACh. Low sodium intake reduced the response of the MSAs to NA and promoted ACh-induced vasodilatation. HS also aggravated NA-induced blood perfusion reduction and impaired ACh-induced hyperperfusion of the MSAs. Pathologically, HS was associated with arteriolar structural damage and fibrosis of the MSAs. We conclude that sodium intake affects the responsiveness of the MSAs to vasoactive agents in DS rats and might play important roles in modulating blood pressure in hypertensive individuals.

Prenatal Sildenafil Therapy Improves Cardiovascular Function in Fetal Growth Restricted Offspring of Dahl Salt-Sensitive Rats.

Fetal growth restriction (FGR) is associated with increased risk for cardiovascular and renal disorders in later life. Prenatal sildenafil improves birth weight in FGR animal models. Whether sildenafil treatment protects against long-term cardiovascular and renal disease in these offspring is unknown. The aim of this study is to test the hypothesis that prenatal sildenafil ameliorates cardiovascular and renal function in FGR offspring of Dahl salt-sensitive rats. Sildenafil citrate (60 mg/kg per day) or control gel diet (containing 0.3% salt) was administered from gestational day ten until birth. In male and female offspring, the mean arterial pressure was measured by telemetry in 1 subset from week 5 until week twenty. Echocardiographic parameters, glomerular filtration rate, and fractional electrolyte excretion were determined in another subset at week 9. Aortic and mesenteric artery rings were prepared to assess endothelial-dependent (acetylcholine) and -independent (sodium nitroprusside) vasorelaxation (week 10). The rise in mean arterial pressure per week was attenuated in treated versus untreated male offspring. Mesenteric arteries showed an increased endothelium-dependent relaxation and improved endothelium-independent relaxation in treated versus control male offspring. No differences in aortic relaxation, echocardiographic parameters or renal function were observed between groups. Prenatal sildenafil treatment subtly improves cardiovascular but not renal function in the offspring of this FGR rat model. Translationally, in utero treatment could be beneficial for cardiovascular programming in a sex-specific manner; however, caution is warranted since recent human trials have been halted because of potentially deleterious neonatal side effects when treating pregnancies complicated with severe FGR with sildenafil.

Small Amounts of Inorganic Nitrate or Beetroot Provide Substantial Protection From Salt-Induced Increases in Blood Pressure.

To reduce the risk of salt-induced hypertension, medical authorities have emphasized dietary guidelines promoting high intakes of potassium and low intakes of salt that provide molar ratios of potassium to salt of ≥1:1. However, during the past several decades, relatively few people have changed their eating habits sufficiently to reach the recommended dietary goals for salt and potassium. Thus, new strategies that reduce the risk of salt-induced hypertension without requiring major changes in dietary habits would be of considerable medical interest. In the current studies in a widely used model of salt-induced hypertension, the Dahl salt-sensitive rat, we found that supplemental dietary sodium nitrate confers substantial protection from initiation of salt-induced hypertension when the molar ratio of added nitrate to added salt is only ≈1:170. Provision of a low molar ratio of added nitrate to added salt of ≈1:110 by supplementing the diet with beetroot also conferred substantial protection against salt-induced increases in blood pressure. The results suggest that on a molar basis and a weight basis, dietary nitrate may be ≈100× more potent than dietary potassium with respect to providing substantial resistance to the pressor effects of increased salt intake. Given that leafy green and root vegetables contain large amounts of inorganic nitrate, these findings raise the possibility that fortification of salty food products with small amounts of a nitrate-rich vegetable concentrate may provide a simple method for reducing risk for salt-induced hypertension.

Carbon Monoxide Attenuates High Salt-Induced Hypertension While Reducing Pro-inflammatory Cytokines and Oxidative Stress in the Paraventricular Nucleus.

Carbon monoxide (CO) presents anti-inflammatory and antioxidant activities as a new gaseous neuromessenger produced by heme oxygenase-1 (HO-1) in the body. High salt-induced hypertension is relevant to the levels of pro-inflammatory cytokines (PICs) and oxidative stress in the hypothalamic paraventricular nucleus (PVN). We explored whether CO in PVN can attenuate high salt-induced hypertension by regulating PICs or oxidative stress. Male Dahl Salt-Sensitive rats were fed high-salt (8% NaCl) or normal-salt (0.3% NaCl) diet for 4weeks. CORM-2, ZnPP IX, or vehicle was microinjected into bilateral PVN for 6weeks. High-salt diet increased the levels of MAP, plasma norepinephrine (NE), reactive oxygen species (ROS), and the expressions of COX2, IL-1β, IL-6, NOX2, and NOX4 significantly in PVN (p < 0.05), but decreased the expressions of HO-1 and Cu/Zn-SOD in PVN (p < 0.05). Salt increased sympathetic activity as measured by circulating norepinephrine, and increased the ratio of basal RSNA to max RSNA, in part by decreasing max RSNA. PVN microinjection of CORM-2 decreased the levels of MAP, NE, RSNA, ROS and the expressions of COX2, IL-1β, IL-6, NOX2, NOX4 significantly in PVN of hypertensive rat (p < 0.05), but increased the expressions of HO-1 and Cu/Zn-SOD significantly (p < 0.05), which were all opposite to the effects of ZnPP IX microinjected in PVN (p < 0.05). We concluded that exogenous or endogenous CO attenuates high salt-induced hypertension by regulating PICs and oxidative stress in PVN.

Effect of emodin on Ang II/βig-h3 signaling pathway in diabetic nephropathy rats kidney

Salt sensitivity (SS) is associated with increased cardiovascular risk in patients with Type 2 diabetes mellitus (T2-DM) due to an increase in renal oxidation. ω-3 polyunsaturated fatty acids have shown antioxidant effects, but a typical Western diet contains limited content. In particular, ω-3 polyunsaturated fatty acids are able to activate nuclear factor erythroid 2-related factor 2 (Nrf-2) to prevent diabetes mellitus–related complications by mitigating oxidative stress. Therefore, we hypothesized that eicosapentaenoic acid (EPA; ω-3) modulates SS in rats with T2-DM by decreasing renal oxidative stress via Nrf-2 activation and enhancing the antiinflammatory response via interleukin (IL) 6 modulation.Three-month-old male rats (n = 40) were fed with a Normal Na-diet (NNaD) and randomly selected into four groups: Healthy Wistar nondiabetic rats (Wi), diabetic controls (eSS), arachidonic acid-treated eSS (AA; ω-6), and EPA-treated eSS (ω-3). After 1 year, rats were placed in metabolic cages for 7 d and fed a NNaD, followed by a 7-d period with a High Na-diet (HNaD). Systolic blood pressure, body weight, serum IL-6 and reactive oxygen species (ROS) levels were determined at the end of each 7-d period. Glycated hemoglobin (HbA1c), triacylglycerol, creatinine, and cholesterol levels were determined. ROS levels and Nrf-2 expression in kidney lysates were also assayed. Histologic changes were evaluated. A t test or analysis of variance was used for the statistical analysis.After a HNaD, systolic blood pressure increased in both the control eSS and AA groups, but not in the EPA and Wi groups. However, HbA1c levels remained unchanged by the treatments, which suggests that the observed beneficial effect was independent of HbA1c levels. The IL-6 levels were higher in the eSS and AA groups, but remained unaltered in EPA and Wi rats after a HNaD diet. Interestingly, EPA protected against serum ROS in rats fed the HNaD, whereas AA did not. In kidney lysates, ROS decreased significantly in the EPA group compared with the eSS group, and Nrf-2 expression was consistently higher compared with the AA and eSS groups. Diabetic rats presented focal segmental sclerosis, adherence to Bowman capsule, and mild-to-moderate interstitial fibrosis. EPA and AA treatment prevented kidney damage.An adequate ω3-to-ω6 ratio prevents SS in diabetic rats by a mechanism that is independent of glucose metabolism but associated with the prevention of renal oxidative stress generation. These data suggest that EPA antioxidant properties may prevent the development of hypertension or kidney damage.

CACNB2 is associated with aberrant RAS-MAPK signaling in hypertensive Dahl Salt-Sensitive rats

Several independent genome-wide association studies (GWAS) have indicated that calcium (Ca2+) voltage-gated channel auxiliary subunit beta 2 (CACNB2) an L-type Ca2+ channel (LTCC) associated protein has strong association with hypertension. However, the molecular mechanism of CACNB2 and its role in the pathophysiology of hypertension is not clear. To address this knowledge gap, we utilized in vitro and in vivo approaches using HEK293 cells and genetically hypertensive, Dahl Salt-Sensitive (SS) rats. We demonstrated that CACNB2 over-expression in HEK293 cells triggers cell proliferation via an up-regulation of the RAS-MAPK pathway compared to non-transfected cells. These effects were likely independent of LTCC activity as treatment with nifedipine, a well-known LTCC blocker, in CACNB2 overexpressing cells failed to inhibit the RAS-MAPK pathway gene expressions or show an effect on apoptosis marker gene expression. Furthermore, the expression level of CACNB2 was up-regulated in the high salt (HS) diet fed SS rat kidneys compared to low salt diet (LS) fed group. Similar to our in vitro observation the RAS-MAPK mRNA levels were increased in HS fed SS rat kidneys, compared to LS fed group. Collectively, our data suggest that CACNB2 is associated with the increase in RAS-MAPK gene expressions and lead us to speculate that in addition to its role in regulating LTCC α1-subunit trafficking, CACNB2 might lead to aberrant RAS activation, which is one of the key cascade associated with hypertension.

Role of Gut Microbiota and Immunity in the Dietary Modulation of Dahl Salt‐Sensitive Hypertension

Studies from our laboratory have demonstrated that immune cells infiltrate the kidneys and amplify the hypertension and renal damage observed in Dahl Salt‐Sensitive (SS) rats when fed 4.0% high salt (HS). These rats are bred at the Medical College of Wisconsin (SS/MCW) and are maintained on a highly purified, casein‐based diet. Interestingly, SS rats fed a whole grain diet (SS/CRL) or modified wheat gluten diet (SS/WG) have drastically reduced salt‐induced hypertension, albuminuria, and renal inflammation compared to the casein‐fed SS/MCW. With the linkage of the gut microbial composition to many of the risk factors associated with hypertension, the current study sought to define the Dahl SS gut microbiome in the presence of these dietary modulations and to test the hypothesis that gut bacteria directly contribute to the development of hypertension and renal disease. Fecal 16S rRNA sequencing revealed distinct microbiome profiles between SS/MCW, SS/CRL, and SS/WG, with significant shifts in gut microbiome composition due to age, salt, and sex (n=4–6). Furthermore, there were families of salt responsive bacteria that, depending on the diet of the group, became specifically enriched (Ruminococcaceae, Clostridiaceae, Prevotellaceae) or depleted (Lachnospiraceae) after 3 weeks of HS. To assess the direct contribution of gut bacteria to the development of SS hypertension, we treated SS/MCW rats with a broad‐spectrum antibiotic cocktail of bacitracin and streptomycin (Bac/Strep, 2 g/L in drinking water). Bac/Strep treatment resulted in a significant reduction in mean arterial pressure (167.3±5.0 vs 148.9±3.0 mmHg, Vehicle vs Bac/Strep, n=6–8, p&lt;0.001) and albuminuria (221.5±34.3 vs 130.2±16.2 mg/day, p&lt;0.05) compared to vehicletreated rats, suggesting that Bac/Strep treatment eliminated certain gut bacteria responsible for driving the phenotype in Dahl SS rats. As further proof of principle, SS/CRL recipient rats were administered daily fecal microbiota transfer (FMT, 0.1g stool/day) from SS/MCW donor rats starting at 6 weeks of age and throughout the HS period starting at 9 weeks of age. These transfer experiments of the SS/MCW microbiota into SS/CRL rats worsened renal disease progression by increasing both proteinuria (148.0±15.0 vs 209.4±39.8 mg/day, Vehicle vs SS/MCW FMT, n=5, p&lt;0.05) and albuminuria (67.3±6.9 vs 113.7±25.0 mg/day, p&lt;0.01), which also coincided with an increase in systolic arterial pressure (158.6±5.8 vs 177.8±8.9 mmHg, p=0.09). Upon analysis of the immune cells in the kidneys of the SS/MCW FMT, there was a specific increase in the number of CD4+ T helper cells (1.7‐fold, p&lt;0.05) and CD8+ cytotoxic T cells (2.0‐fold, p&lt;0.05), with no differences observed in the number of CD11b/c+ monocytes/macrophages or CD45R+ B cells. While the specific mechanisms linking gut dysbiosis to the SS hypertensive, renal injury, and renal inflammatory phenotype are still under current investigation, our present data excitingly demonstrate that the dietary modulation of gut bacteria are directly contributing to the development and progression of the Dahl SS phenotype.Support or Funding InformationHL116264, HL137748, 15SFRN2391002This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CACNB2 expression is positively associated to the Ras/MAPK pathway in vitro and in an experimental model of hypertension

Several independent genome‐wide association studies highlighted the strong association of voltage‐dependent L‐type calcium channel subunit beta‐2 (CACNB2) gene with hypertension. One such study showed that hypertensive individuals who carry a promoter polymorphism (rs2357928) in CACNB2 exhibited adverse effects during treatment with calcium channel blocker (CCB) ‐ verapamil but responded well to treatment with the β‐blocker‐ atenolol. However, the exact molecular signature of CACNB2 during the pathophysiology of hypertension is currently remains to be explored. In the clinical setting, CCBs are widely used to treat hypertensive individuals, and the inhibition of L‐type Ca2+ channels with CCBs significantly increases the glomerular filtration rate and renal blood flow. Hence, the goal of this present study is to understand the molecular signaling of CACNB2 during hypertension via in vitro and in vivo approaches using HEK293 cells and genetically hypertensive, Dahl Salt‐Sensitive (SS) rats. The over‐expression of CACNB2 in HEK293 cells increased cell proliferation and upregulated the Ras‐MAPK pathway significantly when compared to the controls. These results were consistent with a recent study showing that overexpression of CACNB2, activate Ras/ERK/CREB pathways in vitro. Additionally, the ER stress markers IRE‐1α and PERK were upregulated by CACNB2 over expression compared to the control cells. In contrast, treatment with the L‐type CCB‐ nifidepine in CACNB2 overexpressed cells did not abolish upregulation of Ras‐MAPK and ER stress pathway genes. Similarly, we observed an increase in apoptotic signaling exemplified by the downregulation of Bcl‐2 and upregulation of Caspase 7 and Caspase 9, which were also insensitive to nifidepine treatment. Correspondingly, we investigated whether CACNB2 levels were altered using the SS rats under low salt (0.3% NaCl) and high (2% NaCl) diet fed conditions. We observed higher CACNB2 levels in high salt fed SS rat kidneys compared to SS rats maintained on low salt diet (n=4, p&lt;0.05). In addition, our gene expression studies showed that Mapk1 and Mapk4 levels were upregulated in the kidneys of SS rats under high salt diet (n=4, p&lt;0.05). In summary, our study reveals that CACNB2 significantly associated with the activation of Ras‐MAPK pathway in SS rats. In summary, understanding the regulation of CACNB2 and associated Ras‐MAPK pathway could help in expanding the knowledge of molecular mechanism of hypertension and may help in identifying novel drug targets.Support or Funding InformationFunding support for this study was met from the American Heart Association grant 16SDG27700030 (to S. Kumarasamy) and funding from UT‐COMLS.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolic Insults Drive the Development of Glomerular Sclerosis and Proteinuria in Salt‐Sensitive Hypertensive Nephropathy

Hypertensive nephropathy causes more than one third of end stage renal cases, but its molecular mechanisms are not fully understood. Here, we performed a multi‐layered omic analysis of a well‐established animal model of hypertensive nephropathy, the Dahl salt‐sensitive (SS) rat. Specifically, we performed a time‐course untargeted and targeted metabolomics as well as quantitative phosphoproteomic and proteomic analysis of glomerular and whole kidney cortex. For this experiments three groups of age matched 8 weeks old SS rats were fed either a low salt (0.4% NaCl) or high salt (4% NaCl; 7 and 21 days of HS). Analysis of microalbuminuria, as a marker of kidney injury, revealed that SS rats had a progressive kidney damage following HS treatment (Alb/Cre ratio was 4.6 ± 0.8, 10.7 ± 1.5 and 12.2±2.0, for SS rats fed LS and HS for 7 and 21 days, respectively). Metabolomics, phosphoproteomic and proteomic analyses were performed either in the kidney cortex homogenate or pure fraction of glomeruli. Glomeruli were isolated by consecutive sieving as we previously reported. Metabolomic analysis of the glomeruli revealed a decrease in energy fuels before the onset of proteinuria, including glucose‐6‐phosphate, alpha‐Ketoglutarate, and branched chain amino acids, suggesting a wide‐spread energy depletion. In addition, proinflammatory molecules such as free fatty acids were increased before onset of proteinuria. Notably, these changes did not occur in the kidney cortex where glomeruli fraction was negligeable, suggesting glomerular vulnerability as a key event in hypertensive nephropathy. Phosphoproteomics revealed that podocyte‐specific nutrient‐sensing 5′ AMP‐activated protein kinase (AMPK), as well as regulatory‐associated protein of mTORC1 Raptor, were activated before the onset of structural damage. Structural changes as shown by complement deposition and fibrosis proteins were detectable upon onset of heavy proteinuria. Furthermore, comparative proteomics across glomerular insult models revealed that increased expression of alpha‐Ketoglutarate depleting enzymes is common in glomerular injury. Oxidative protein modifications was also detected in the extracellular matrix of the glomeruli, explaining the energy depletion. In the kidney cortex, reduced uptake of amino acids was observed among other metabolic changes. These data suggest metabolome‐driven insults in the glomeruli of the kidney that proceed the insult in the other parts of nephron. Therefore, this study provides a novel framework for the study of hypertensive nephropathy.Support or Funding InformationSupported by the DFG.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of Nox4 in Angiotensin II‐Mediated Changes in Volume Dynamics and Nitric Oxide Production in Podocytes

Nitric oxide (NO) is a powerful vasodilator that serves important functions in the kidney such as regulation of renal hemodynamics, pressure natriuresis, and diuresis. Significant reduction in NO production has been found to play a role in the pathogenesis of hypertension. Our recent studies revealed that Angiotensin II (Ang II), in addition to causing a release of calcium, stimulates NO production in the glomerular podocytes. This signaling pathway can be also triggered by extracellular application of hydrogen peroxide (H2O2) and is significantly reduced with the development of salt‐sensitive hypertension and kidney injury in Dahl salt‐sensitive (SS) rats (Palygin et al., Nitric Oxide, 2018). Recently, using a knock out of NADPH oxidase 4 (Nox4) in SS rats (SSNox4−/−), one of the primary source of renal reactive oxygen species (ROS) and specifically H2O2 we described the reduction in intracellular calcium activation in podocytes as well as H2O2 production in isolated glomeruli in response to Ang II (Ilatovskaya et al., J Am Soc Nephrol. 2018). Overall, SSNox4−/− rats displayed a protective phenotype with a significant reduction of albuminuria, glomeruli injury and reduction in the elevation of blood pressure under high salt challenge (Cowley et al., Hypertension, 2016). Here we used this model to characterize the effects of alterations in the balance in the relationship between ROS and NO production in podocytes and establish the relation between Nox4 and glomerular/podocyte volume dynamics. The in vivo concentration of H2O2, measured by biosensors amperometry as catalase sensitive current, was significantly reduced in both cortex and medullar parts of the SSNox4−/− rat kidney compared to wild type SS rats, indicating a direct association between Nox4 and the production of renal ROS. We found that acute application of Ang II promoted opposite changes in glomeruli and podocyte volumes, where glomeruli volume decreased, and podocyte volume increased approximately by 18% in SS rats. Overall, SSNox4−/− rats exhibited an increase in total NO production (by 16% to compare with SS), calculated using confocal microscopy as a total integral of NO fluorescent transient (DAF‐FM dye) in the population of podocytes within a freshly isolated glomerulus. In SSNox4−/− rats the elevation in NO together with declining in H2O2 levels directly correlated with an increase of podocyte volume in response to Ang II (13% vs. SS control). We anticipate a rapid podocyte volume change dependent on intracellular NO production and reflecting the ability of these cells to dynamically regulate the permeability of glomeruli filtration barrier. Thus far, we can conclude that in the absence of Nox4 the podocytes shift the ROS production in response to Ang II towards the increase of intracellular NO bioavailability and a decrease of renal H2O2 production. This mechanism may play a key role in the protection of glomeruli form oxidative stress and elevation of blood pressure during the development of salt‐sensitive hypertension.Support or Funding InformationNHLBI R35 HL135749; P01 HL116264; AHA 18PRE34030127; ADA 1‐15‐BS‐172; DK‐020595 Pilot &amp; Feasibility projectThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

α1‐Adrenoceptor Antagonism Attenuates the Development and Progression of Dahl Salt Sensitive Rat Hypertension by Modulating NCC Activity

BackgroundDietary sodium intake plays a significant role in blood pressure (BP) regulation, as increased sodium retention is a well‐established precursor of hypertension risk. In particular, increased dietary sodium intake evokes increased BP in individuals that demonstrate the salt sensitivity of BP and promotes the development of salt sensitive hypertension (SSH). Recent studies suggest that excessive Sympathetic Nervous System (SNS) release of norepinephrine (NE) has the ability to stimulate the sodium chloride cotransporter (NCC) via synergistic α1 and β adrenergic receptor pathways to drive SSH.HypothesisEndogenous SNS release of NE activates an α1‐adrenoceptor pathway to mediate increases in NCC activity that drive the development and maintenance of Dahl rat SSH.MethodsGroups of naïve Dahl Salt Sensitive (DSS) rats, DSS rats receiving a continuous subcutaneous (s.c.) terazosin (10 mg/kg/day), or s.c. propranolol (10 mg/kg/day) infusion were fed a normal salt (NS, 0.6% NaCl) or high salt (HS, 4% NaCl) diet for 21 days respectively. On day 21 basal MAP and NCC activity (peak natriuresis to iv hydrochlorothiazide (HCTZ; 2mg/kg) infusion) were assessed. NCC expression and phosphoNCC levels were assayed via immunoblotting (N=6/gp). Additional groups of DSS rats were fed a 42 day HS diet. On day 21, rats were given s.c. saline/DMSO or s.c. terazosin/DMSO (10 mg/kg/day). On day 42, measurements were taken as described previously (N=4–6/gp).ResultsDSS rats develop SSH when fed a 21 day HS diet and show increased NCC activity compared to rats on a NS diet. NCC expression and phospho levels are similar in both the diets. Use of an α1‐adrenoceptor antagonist, terazosin (confirmed pharmacologically) attenuates SSH and reduces NCC activity and expression as well as phosphoNCC levels in rats on a HS diet. Use of a β‐adrenoceptor antagonist, propranolol fails to attenuate SSH or reduce NCC activity. Significantly, α1 antagonism has a trend to reduce BP and NCC activity in DSS rats with established HTN.ConclusionOur data reveals that SNS release of NE modulates NCC activity via an α1‐adrenoceptor pathway to evoke dietary evoked SSH. Significantly, α1‐adreoceptor antagonism can attenuate the development of SSH by evoking downregulation of NCC activity and expression. In a model of established HTN, α1‐adreoceptor antagonism reduces NCC activity and BP. Taken together, these findings suggest that SNS release of NE activates an α1‐adreoceptor pathway that stimulates NCC activity to drive the development and maintenance of SSH.Support or Funding InformationThis work was funded by NIH R56 AG057687, R01 HL139867, R01HL141406, AHA17GRNT33670023 to RDW, and F31DK116501 to AAF.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Substitution of Casein Dietary Protein with Wheat Gluten Protein Protects Dahl Salt Sensitive Rats from the Development of Maternal Syndrome

Previously our group demonstrated that the maternal diet of the Dahl Salt‐Sensitive (SS) rat, specifically the protein source, during gestation and lactation can impact the severity of salt sensitive hypertension and renal damage of the offspring in adulthood. Offspring born from mothers fed a wheat gluten‐based diet exhibited blunted salt‐sensitive hypertension and renal damage when compared to offspring born from casein fed mothers. Utilizing an embryo transfer approach we determined that regardless of whether an embryo originated from parents fed a casein or a wheat gluten‐based diet, the diet of the surrogate mother that determined the severity of salt‐sensitivity in the offspring. In addition to the maternal diet, the in utero environment has been shown to contribute to the programming of lifelong health of offspring. Dahl SS rats maintained on a casein‐based diet (SSC) develop maternal syndrome (hypertension and proteinuria) compared to virgin controls. Since offspring from mothers fed a wheat gluten based diet were protected from SS hypertension and the Dahl SSC rat develops maternal syndrome, we tested the hypothesis that mothers fed a wheat gluten (SSWG) diet will be protected from the development of maternal syndrome. To test this hypothesis Dahl SSWG and SSC rats were studied on their respective diets, which are identical in every aspect except for the protein source. At 8 weeks of age, females were instrumented with radio‐telemeters for continuous measurement of mean arterial pressure (MAP). After a recovery period, baseline MAP was measured as well as an overnight urine collection for markers of renal damage before females were mated with males of the same age and diet. Virgin littermate controls of the same age and diet for each strain were run in parallel. During the baseline period, there was no difference observed in MAP or protein excretion rate among any of the groups. As previously reported, the SSC mated rats develop an increase in MAP (141±3 vs 129±3 mmHg, p&lt;0.05) and protein excretion rate (98±16 vs 46±5 mg/day, p&lt;0.05) at gestational day 20 (GD) compared to virgin controls. Interestingly, there was a significant reduction in MAP in the SSWG mated rats compared to SSWG virgin controls at GD20 (114±4 vs 126±4 mmHg, respectively, p&lt;0.05). Comparing the SSC and SSWG mated rats at GD20, it is evident that the SSWG mated rats are protected from developing maternal syndrome seen in the SSC mated rats (MAP: 114±4 vs 141±3 mmHg, p&lt;0.001, Proteinuria: 52±12 vs 98±16 mg/day, p&lt;0.01). Additionally, uterine artery resistance index, which is utilized as a surrogate marker for placental perfusion, is significantly greater in the SSC rats compared to the SSWG at GD20 (0.9±0.02 vs 0.5±0.04, p&lt;0.01, n=2/group). While the SSWG rat are protected from the development of maternal syndrome compared to the SSC, the exact mechanisms for this protection is unknown. This study demonstrates how environmental factors such as diet can play a protective role in the development of maternal syndrome with associated effects on the health of the offspring.Support or Funding InformationHL116264, 18POST33990140, 15SFRN2391002This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.