NaCl Chow Research Articles

Abstract 21: The Detrimental Effects of a Comorbidity of Increased Blood Pressure Variability and High Salt Diet on Cardiovascular and Neurovascular Outcomes

Recent studies have identified increased blood pressure variability (BPV) as a novel risk factor for cognitive decline, independently from absolute BP values. However, the underlying mechanisms are poorly defined. In addition, whether a high salt diet (HSD) contributes to BPV and exacerbates cognitive decline is not known. We tested the hypothesis that vascular damage resulting from blood pressure fluctuations in BPV disrupts critical mechanisms regulating cerebral blood flow (CBF), impairing perfusion and leading to neurovascular dysfunction and cognitive decline. We established a murine BPV model and evaluated the impact of BPV on systemic BP (baroreceptor reflex) and CBF regulation (myogenic and functional hyperemia (FH) responses). Mice were implanted with a cranial window for in-vivo two-photon (2P) imaging. A programmable pump infused 6-8 pulses/day of angiotensin II ((Ang II 18 µg/day) (BPV) or saline (Control) for 25 days, and cardiovascular variables were measured via DSI telemetry. Ang II pulse infusions evoked transient BP increases (Δ43±4 mmHg, MAP; P<0.01,n=8); however, did not induce hypertension in the BPV group. BPV mice displayed increased BPV index (P=0.04,n=12) and cognitive decline (novel object recognition test (P=0.01,n=10)). Telemetry data showed a decrease in bradycardic responses following chronic BPV (P=0.03, n=8). Data from 2P studies showed enhanced myogenic response in parenchymal arterioles of BPV vs Controls (P<0.01,n=9-15 runs) when BP transitions from low to high BP. We observed a blunted FH in BPV vs Control during low BP (P<0.01,n=6-7). During high BP, FH was pronounced in Controls (P<0.01,n=6-7); however, pressure-induced vascular responses were abolished in BPV mice (P<0.01,n=6-7). In separate groups of mice, the Control and BPV protocol was repeated with all mice receiving a high-salt diet (HS; 4% NaCl chow+1% NaCl drinking water: Control+HS or BPV+HS). There were no changes in 24-hr MAP in Control+HS (P>0.85, n=5). Whereas BPV+HS mice developed sustained hypertension (increased 24-hr MAP; P<0.01, n=4). These data demonstrate that BPV alters mechanisms regulating CBF and BP, contributing to cognitive decline and that HSD may exacerbate the detrimental effects of BPV on BP regulating mechanisms. Ongoing studies are examining the comorbid effects of BPV+HS on the cerebrovasculature and direct CBF changes in BPV and BPV+HS mice.

Excessive sodium chloride ingestion promotes inflammation and kidney fibrosis in aging mice.

In aging kidneys, a decline of function resulting from extracellular matrix (ECM) deposition and organ fibrosis is regarded as "physiological." Whether a direct link between high salt intake and fibrosis in aging kidney exists autonomously from arterial hypertension is unclear. This study explores kidney intrinsic changes (inflammation, ECM derangement) induced by a high-salt diet (HSD) in a murine model lacking arterial hypertension. The contribution of cold shock Y-box binding protein (YB-1) as a key orchestrator of organ fibrosis to the observed differences is determined by comparison with a knockout strain (Ybx1ΔRosaERT+TX). Comparisons of tissue from mice fed with normal-salt diet (NSD, standard chow) or high-salt diet (HSD, 4% NaCl in chow; 1% NaCl in water) for up to 16 mo revealed that with HSD tubular cell numbers decrease and tubulointerstitial scarring [periodic acid-Schiff (PAS), Masson's trichrome, Sirius red staining] prevails. In Ybx1ΔRosaERT+TX animals tubular cell damage, a loss of cell contacts with profound tubulointerstitial alterations, and tubular cell senescence was seen. A distinct tubulointerstitial distribution of fibrinogen, collagen type VI, and tenascin-C was detected under HSD, transcriptome analyses determined patterns of matrisome regulation. Temporal increase of immune cell infiltration was seen under HSD of wild type, but not Ybx1ΔRosaERT+TX animals. In vitro Ybx1ΔRosaERT+TX bone marrow-derived macrophages exhibited a defect in polarization (IL-4/IL-13) and abrogated response to sodium chloride. Taken together, HSD promotes progressive kidney fibrosis with premature cell aging, ECM deposition, and immune cell recruitment that is exacerbated in Ybx1ΔRosaERT+TX animals.NEW & NOTEWORTHY Short-term experimental studies link excessive sodium ingestion with extracellular matrix accumulation and inflammatory cell recruitment, yet long-term data are scarce. Our findings with a high-salt diet over 16 mo in aging mice pinpoints to a decisive tipping point after 12 mo with tubular stress response, skewed matrisome transcriptome, and immune cell infiltration. Cell senescence was aggravated in knockout animals for cold shock Y-box binding protein (YB-1), suggesting a novel protective protein function.

Fructose-induced salt-sensitive blood pressure differentially affects sympathetically mediated aortic stiffness in male and female Sprague-Dawley rats.

Hypertension is the leading risk factor for major adverse cardiovascular events (MACE). Aortic stiffness and sympathoexcitation are robust predictors of MACE. Combined high fructose and sodium intake increases arterial pressure, aortic stiffness, renin, and sympathetic nerve activity in male rats. We hypothesized that activation of the renin angiotensin system (RAS) and/or the sympathetic system mediates aortic stiffness in rats with fructose-induced salt-sensitive blood pressure. Male and female Sprague-Dawley rats ingested 20% fructose or 20% glucose in drinking water with 0.4% NaCl chow for 1 week. Then, fructose-fed rats were switched to 4% NaCl chow (Fru + HS); glucose-fed rats remained on 0.4% NaCl chow (Glu + NS, controls for caloric intake). After 2 weeks, mean arterial pressure (MAP) and aortic pulsed wave velocity (PWV) were evaluated at baseline or after acute intravenous vehicle, clonidine, enalapril, losartan, or hydrochlorothiazide. Baseline global longitudinal strain (GLS) was also assessed. MAP and PWV were greater in male Fru + HS versus Glu + NS male rats (p < 0.05 and p < 0.001, respectively). PWV was similar between the female groups. Despite similarly reduced MAP after clonidine, PWV decreased in Fru + HS versus Glu + NS male rats (p < 0.01). Clonidine induced similar decreases in MAP and PWV in females on either diet. GLS was lower in Fru + HS versus Glu + NS male rats and either of the female groups. Thus, acute sympathoinhibition improved aortic compliance in male rats with fructose salt-sensitive blood pressure. Female rats retained aortic compliance regardless of diet. Acute RAS inhibition exerted no significant effects. Male rats on fructose high salt diet displayed an early deficit in myocardial function. Taken together, these findings suggest that adult female rats are protected from the impact of fructose and high salt diet on blood pressure, aortic stiffness, and early left ventricular dysfunction compared with male rats.

PS-BPB10-1: INVOLVEMENT OF RENAL TUBULAR NFAT5 IN THE REGULATION OF BLOOD PRESSURE AND BODY FLUID VOLUME

Objective: Nuclear factor of activated T-cells 5 (NFAT5) is an osmoresponsive transcription factor that activates osmoprotective genes to protect cells against hypertonicity. Renal tubular cell-specific NFAT5 knockout (KO) mice have been shown to exhibit polyuria, hypernatremia, low urinary sodium excretion, and increased expression of the epithelial sodium channel (ENaC) in the kidney. In the present study, regulation of blood pressure (BP) as well as that of body fluid volume were examined in the wild type (WT) mice and NFAT5 KO mice. Methods: The BP of KO mice and WT mice was measured using radiotelemetry method. The mice were fed with regular salt diet (RSD, 0.3% NaCl chow) or high salt diet (HSD, 8% NaCl chow) followed by the treatment with amiloride, an ENaC blocker. To assess body fluid volume, the plasma renin activity (PRA) and the plasma aldosterone concentration (PAC) were measured at the condition of RSD and HSD, together with plasma arginine vasopressin (AVP) and hematocrit (Ht) levels. Results: Compared to WT mice, the mean BP was significantly higher in KO mice both at the condition with RSD and that with HSD in dark period, but not in light period. Treatment with amiloride significantly decreased the mean BP in KO mice with HSD to the level of WT mice with HSD in dark period. PRA, PAC, and AVP levels were not significantly different between in WT and KO mice with RSD. HSD significantly decreased PRA and PAC and tended to increase the AVP level both in WT and KO mice. The Ht level was significantly lower in KO mice than in WT mice regardless of RSD or HSD (WT with RSD, 42.6 ± 0.5%; WT with HSD, 42.8 ± 0.6%; KO with RSD, 38.3 ± 0.4%; KO with HSD, 39.0 ± 0.4%, n = 4). Conclusions: The results suggest that the renal tubular cell-specific NFAT5 KO mice exhibit excess body fluid retention induced by sodium reabsorption through ENaC, leading to salt-sensitive hypertension. Such phenotypes seem to be independent of the systemic renin-angiotensin-aldosterone and AVP systems. Renal tubular NFAT5 could integrate urine concentration and sodium reabsorption in the kidney, thus preventing salt-dependent hypertension.

Abstract 028: Toll-Like Receptor 4 Promotes Renal Immune Cell Infiltration And The Development Of Renal Damage During Dahl Salt-Sensitive Hypertension

Previous work in our laboratory has implicated a significant role for the immune system in potentiating the hypertensive and kidney damage phenotypes which occur in the Dahl Salt-Sensitive (SS) rat. Toll-like receptors (TLRs), usually expressed on macrophages, play a key role in the innate immune system. A number of TLRs have been implicated in cardiovascular disease and chronic kidney disease with particular interest in TLR4, which specifically binds gram-negative lipopolysaccharide or endogenous molecules produced as a result of tissue injury. We previously performed a transcriptomic analysis which demonstrated TLR4 upregulation in renal medullary tissue of Dahl SS rats fed a high salt (4.0% NaCl) diet. The present study tests the hypothesis that TLR4 influences high salt diet-induced hypertension and renal damage in the Dahl SS rat. Using CRISPR/Cas9 technology, a mutation was generated in the TLR4 gene on the Dahl SS background, creating a 1-bp frameshift deletion resulting in a premature stop codon. SS TLR4-/- males and parental Dahl SS males (SS TLR4+/+ ), maintained on a 0.4% NaCl chow, underwent carotid artery telemetry implantation at 7 weeks of age. After recovery and baseline mean arterial pressure (MAP) recordings (day 0: 125.6 ± 0.5 vs 124.2 ± 2 mmHg), animals were switched to a 4.0% NaCl chow for 3 weeks. Overnight urine collections occurred once during the low salt period and weekly throughout the high salt challenge. Kidneys were flushed prior to euthanasia. Although SS TLR4-/- MAP was not different from SS TLR4+/+ during the high salt challenge (day 21: 176.8 ± 14 mmHg, n=2 vs 172.3 ± 18 mmHg, n=4), high salt day 21 urinary albumin excretion was significantly reduced in SS TLR4-/- (272.7 ± 65 mg/day, n=5, p&lt;0.01) compared to SS TLR4+/+ (427.5 ± 67 mg/day, n=7), indicating reduced renal damage in TLR4 absence. Furthermore, flow cytometric analysis of isolated renal immune cells indicated a 33% reduction in CD45+ total leukocytes (5.0 ± 1.2 vs 3.4 ± 0.9 x10 6 cells) and a 31% reduction in CD11bc+ monocytes/macrophages (3.9 ± 0.9 vs 2.6 ± 0.7 x10 6 cells) in SS TLR4-/- (n=5) vs SS TLR4+/+ (n=7) kidneys. Together, these data indicate that TLR4 may drive the immune response during the induction and progression of salt-sensitive hypertension and renal disease.

High salt diet-induced proximal tubular phenotypic changes and sodium-glucose cotransporter-2 expression are coordinated by cold shock Y-box binding protein-1.

High salt diet (HSD) is a hallmark of blood pressure elevations, weight gain and diabetes onset in the metabolic syndrome. In kidney, compensatory mechanisms are activated to balance salt turnover and maintain homeostasis. Data on the long-term effects of HSD with respect to tubular cell functions and kidney architecture that exclude confounding indirect blood pressure effects are scarce. Additionally we focus on cold shock Y-box binding protein-1 as a tubular cell protective factor. A HSD model (4% NaCl in chow; 1% NaCl in water) was compared to normal salt diet (NSD, standard chow) over 16 months using wild type mice and an inducible conditional whole body knockout for cold shock Y-box binding protein-1 (BL6J/N, Ybx1). HSD induced no difference in blood pressure over 16 months, comparing NSD/HSD and Ybx1 wild type/knockout. Nevertheless, marked phenotypic changes were detected. Glucosuria and subnephrotic albuminuria ensued in wild type animals under HSD, which subsided in Ybx1-deficient animals. At the same time megalin receptors were upregulated. The sodium-glucose cotransporter-2 (SGLT2) was completely downregulated in wild type HSD animals that developed glucosuria. In Ybx1 knockouts, expression of AQP1 and SGLT2 was maintained under HSD; proximal tubular widening and glomerular tubularization developed. Concurrently, amino aciduria of neutral and hydrophobic amino acids was seen. In vitro translation confirmed that YB-1 translationally represses Sglt2 transcripts. Our data reveal profound effects of HSD primarily within glomeruli and proximal tubular segments. YB-1 is regulated by HSD and orchestrates HSD-dependent changes; notably, sets reabsorption thresholds for amino acids, proteins and glucose.

Abstract 46: Vascular Endothelium-derived Endothelin 1 Promotes Salt-induced Kidney Inflammation In Male But Not Female Mice

Endothelin 1 (ET-1), potent vasoactive and pro-inflammatory peptide, plays a criticalrole in hypertension and end-organ damage. The inflammatory response to high saltintake displays sex differences; however, if a sex difference exists in vascular ET-1inducing salt-induced kidney inflammation is unknown. We aimed to assess the role ofendothelium-derived ET-1 in promoting kidney inflammation in male and female micefed high salt. Aged-matched male and female vascular endothelial cell ET-1 knockout(VEET KO) and control floxed ET-1 (VEET fl/fl ) mice were fed 4.0% NaCl chow (HSD).After 3 weeks of HSD, kidney myeloid and lymphoid cell populations and their functionalstatus were examined by flow cytometry. In response to HSD, male VEET fl/fl mice hadsignificantly higher kidney TH17 cell numbers than female VEET fl/fl mice (IL-17A + CD4 + cell absolute count, male vs. female: 229.6±27.7 vs. 84.9±17.2, p=0.0003, n=6-10/group), indicating a substantial sex-dependent difference in kidney TH17 cellabundance. Lack of endothelium-derived ET-1 resulted in 72% reduction in renal TH17cell number in males (VEET fl/fl vs. VEET KO: 229.6±27.7 vs 64.0±64, p=0.0002), but notin females (p&gt;0.05). Frequency of kidney IL-17A + CD4 + cells was not different betweengenotypes in females or between male and female VEET fl/fl mice. However, male VEETKO mice displayed 57% less renal IL-17A + CD4 + cells than male VEET fl/fl (respectively,0.6±0.071 vs. 1.4±0.2, p=0.0055), and 59% reduction compared to female VEET KOmice (1.4±0.2, p=0.0052). Assessment of the effects of vascular ET-1 on the innateimmune response showed that activated kidney resident macrophages(F4/80 hi CD11b + CD64 + ) were increased in male vs. female VEET fl/fl (MFI, respectively,2080±105 vs. 1672±25, p=0.0002). Lack of endothelium-derived ET-1 significantlyreduced CD64 abundance in males (p=0.012), but not females (p&gt;0.05), whencompared to VEET fl/fl . In conclusion, endothelium-derived ET-1 is critical in promotingrenal innate and adaptive inflammatory responses in males in response to high salt, andimportant male-female differences exist in the manner by which vascular ET-1 regulateskidney inflammation in this setting. Funded by NIH F31 HL151264-0 to PAM,P01HL136267 to JSP, and K01HL145324 to CDM.

Do Counter‐Current Exchange and Glycosaminoglycan Buffering Contribute in Salt Storage in Skin of Rats?

We have previously shown that there is an osmolality and urea gradient from epidermis to dermis and subcutis. This gradient may be a result of the proposed counter-current mechanism for electrolyte homeostasis in skin. Our aims were to test whether there are gradients in salt concentration in skin generated by counter-current exchange and whether glycosaminoglycans (GAGs) can store sodium making it ‘inactive’ in relation to fluid exchange and thereby act as buffers during high salt intake. Sprague-Dawley rats were given low salt diet (LSD, <0.1% NaCl in chow and tap water), high salt diet (HSD, 8% NaCl in chow and 1% saline to drink), and DOCA-salt diet (50mg/week deoxycorticosterone acetate, 150mg/21 days and 1% saline to drink). Rats were randomly assigned to either LSD (n=15), HSD (n=7) or DOCA salt (n=8) diet for measurements of extracellular volume (ECV) in tangential sequential skin sections. Two weeks into the diet regimen the rats were anesthetized, neprectomized and given the extracellular tracer 51Cr-EDTA i.v. that was allowed 120 min circulation time. Directly thereafter, shaved back skin was further processed by cutting sequential sections of 40 µm (epidermis) and 100 µm (dermis and subcutis) thickness in a cryostat taking care to avoid evaporation. The skin sections were weighed and counted in a gamma counter and the extracellular volume found as the plasma equivalent space of 51Cr-EDTA. After counting, the samples were dried until constant weight, then eluted in ultrapure water for seven days. The eluate was analyzed in an ion chromatograph to determine sodium and potassium content in each layer. Sulfated GAGs were determined in whole skin, dermis and subcutis using Blyscan sGAG colorimetric assay to determine sGAG content. When relating sodium content to ECV we found that there was a gradient from epidermis to dermis, and that this gradient was more pronounced in the DOCA rats. The LSD group had a mean sodium of 332.7±194.3 mM in epidermis and 118.3±20.15 mM in the following layer at 140±mM, with corresponding concentrations of 219.2±52 mM and 121.1±41.4 mM in HSD, and 757.8±426.1 mM and 123.7±11.45 mM in DOCA. There was no difference in sulfated GAGs in skin between LSD, HSD and the DOCA salt group in any of the skin regions. Our results suggest that there may be a gradient of salt in the skin with a higher concentration in ECV in epidermis supporting the notion of a counter-current mechanism. However, our data do not support that an increased concentration of sulfated GAGs contribute to salt buffering during high salt conditions.

High-salt diet decreases mechanical thresholds in mice that is mediated by a CCR2-dependent mechanism

BackgroundThough it is well-known that a high-salt diet (HSD) is associated with many chronic diseases, the effects of long-term high-salt intake on physiological functions and homeostasis remain elusive. In this study, we investigated whether and how an HSD affects mouse nociceptive thresholds, and myeloid cell trafficking and activation.MethodsHealthy C57BL/6 male and female mice were fed an HSD (containing 4% NaCl in chow and 1% NaCl in water) from the time of weaning for 3 to 4 months. Circulating monocytes, nerve macrophages, spinal microglia, and associated inflammatory responses were scrutinized using flow cytometry, immunohistochemistry, and quantitative real-time polymerase chain reaction (qPCR) approaches. Mouse pain sensitivity to mechanical stimuli was monitored with von Frey tests along the experimental duration.ResultsMice on an HSD have reduced mechanical thresholds. They feel more pain than those on a normal diet (ND), e.g., regular laboratory chow (0.3% NaCl in chow). An HSD induced not only a remarkable expansion of circulating monocytes, CCR2+Ly6Chi inflammatory monocytes in particular, but also an accumulation of CD11b+F4/80+ macrophages in the peripheral nerves and an activation of Iba-1+ spinal microglia. Replacing an HSD with a ND was unable to reverse the HSD-induced mechanical hypersensitivity or rescue the altered immune responses. However, treating HSD-fed mice with a chemokine receptor CCR2 antagonist effectively normalized the pain thresholds and immune cell profile in the periphery and spinal cord. An HSD failed to alter pain thresholds and myeloid cell activation in CCR2-deficient mice. Spinal microglial activation is required for HSD-induced mechanical hypersensitivity in male, but not in female mice.ConclusionOverall, this study provides evidence that an HSD has a long-term impact on physiological function. CCR2-mediated cellular response, including myeloid cell trafficking and associated inflammation, plays pivotal roles in salt-dietary modulation of pain sensitivity.

High Sodium Diet – Role in Renal Sympathodepression

Afferent renal nerve pathways are likely involved in the development of salt sensitive hypertension. We recently reported that intrarenal NaCl elicited a long‐lasting sympatho‐depression via a neuro‐humoral TRPV1 dependent and tachykinin mediated renal afferent nerve pathway. We now wanted to test the hypothesis that high sodium intake impairs this afferent sympatho‐depressory mechanism.Respective groups were put on tap water, 0.9 % saline for drinking or chow containing 8% NaCl. Cultured dorsal root ganglion neurons (DRG Th11‐L2) of rats with renal afferents were investigated in current clamp mode to assess action potential generation during current injection. Rats were equipped with femoral catheters for blood pressure (BP) &amp; heart rate (HR) assessment, drug application, a renal arterial catheter for intrarenal administration (IRA) of NaCl boil (10 % NaCl, 10 μl) or Capsaicin (CAP 3.3, 6.6, 10, 33*10‐7 M, 10 μl) and a bipolar electrode for renal sympathetic nerve activity (RSNA) recordings; eventually an intravenous (iv) bolus of the NK1‐receptor blocker RP67580 (10*10‐3M, 15 μl) was administered. Results are mean±SEM.In neurons from rats on high salt diet, but not on 0.9 % saline or tap water, the relation of tonic highly active neurons to less active neurons shifted towards less active units (62% tonic neurons in a control group vs. 40% on high salt diet and 63% on saline, significant, p&lt;0.05, z‐test, mean +/−SEM). However, cultured renal neurons from rats on 0.9% saline or on high salt diet exhibited increased action potential production upon stimulation (Controls 13,3+/11,03 APs/600ms vs. saline 19,8+/−2,33 APs/600ms vs. high salt diet 22,2+/−4,54 APs/600ms, significant, &lt;0.05, t−test, mean+/−SEM). 10% NaCl boli IRA induced decreases of RSNA from baseline 4.1±0.6 μV*sec to 2.2±0.8 μV*sec (10% NaCl, p&lt;0.05) impaired in rats on 8% NaCl chow. (Suppressed RSNA unmasked in all groups by an i.v. NK1‐inhibitor).In rats on high salt diet highly active tonic neurons with renal afferents in vitro decreased at the expense of less active phasic neurons in spite of tonic neurons producing more action potentials upon stimulation. The sodium inducible long‐lasting sympatho‐depression via a neuro‐humoral tachykinin mediated afferent renal nerve pathway got eventually impaired.

High-Salt Intake Reduces Apomorphine-Induced Penile Erection and Increases Neurally Mediated Contractile Responses of the Cavernosal Smooth Muscle in Rats.

This study was designed to evaluate whether overconsumption of NaCl, a well-known risk factor for hypertension, leads to erectile dysfunction in rodents. Male Wistar rats received regular chow (control group) or 4% NaCl chow for 24 weeks and were subjected to blood pressure measurement and apomorphine-induced erection. Moreover, cavernosal strips from both the control and 4% NaCl groups were evaluated in organ baths. Animals subjected to 4% NaCl chow did not develop hypertension but presented a significant reduction in the total number of erections following apomorphine administration as compared with the control group. The addition of high KCl or phenylephrine resulted in similar contractile responses in the corpus cavernosal strips from both the control and 4% NaCl groups. However, electrical field stimulation-induced contraction was significantly enhanced in cavernosal strips from animals exposed to 4% NaCl. Incubation of Y-27632, but not of atropine and Nω-nitro-l-arginine methyl ester (L-NAME), entirely prevented the potentiation of the contractile responses evoked by electrical stimulation. The enhanced contractile responses evoked by electrical stimulation found in the high-salt group were also avoided in the absence of extracellular calcium. Concentration-response curves of CaCl2 revealed augmented contractility in response to extracellular calcium in cavernosal strips from the 4% NaCl-treated rats, compared with control samples. A high-salt diet alone rendered the animals less responsive to apomorphine-induced penile erection and enhanced neurally mediated contractile responses in the corpus cavernosum, a clear indication that overconsumption of sodium can lead to erectile dysfunction even without the development of hypertension.

MODEST ARTERIAL REMODELING IN MALE AND FEMALE FIBULIN-5 KNOCK-OUT MICE IN RESPONSE TO INDUCED HYPERTENSION

Objective: Aging and hypertension often go hand-in-hand, which makes it difficult to distinguish their biomechanical effects on arteries in clinical studies. While human aortic aging is characterized by reduced elastin (half-life∼70 years), mice age to a mere 2 years and most elastin remains intact. Knock-out of the elastin-associated glycoprotein fibulin-5 (Fbln5−/−) in mice yields a biomechanical phenotype that resembles human aging. Design and method: Adult male and female Fbln5+/+ and Fbln5−/− mice were studied under three conditions: untreated, after 14-day angiotensin-II infusion (AngII) at 490 ng/kg/min, or after 13 weeks on an 8%-NaCl chow with 3 g/L of L-NAME in the drinking water (L-NAME-NaCl). After tail-cuff blood pressure (BP) measurement and euthanasia, descending thoracic (DTA) and infrarenal abdominal (IAA) aortas were dissected and placed within a computer-controlled biaxial testing device. Pressure-diameter tests were performed at 95%, 100%, and 105% of the in vivo axial stretch and axial force-length tests at 10, 60, 100, and 140 mmHg. Data were fit using a nonlinear constitutive model. Results: Neither AngII nor L-NAME-NaCl changed wall thickness or axial stretch in the males (figure), hence circumferential stress and material stiffness increased due to BP elevation (e.g., significant in IAA, trend in DTA with AngII). This finding indicated a lack of adaptive remodeling. Stored strain energy was significantly lower in Fbln5−/− than Fbln5+/+ mice, but was not significantly influenced by AngII or L-NAME-NaCl. Pulse wave velocity, a measure of structural stiffness, increased due to AngII and L-NAME-NaCl in several groups, which was also BP-mediated. Females showed similar trends as males (not shown), except for strain energy, which increased with AngII independent of BP.Conclusions: The present findings for AngII infusion were similar to prior results for the IAA, but very different for the DTA when compared to the 2.62-fold increase in wall thickness and significant decrease in axial stretch in wildtype mice. The prior dramatic changes depended in large part on inflammation. We suspect that the modest arterial remodeling observed herein in response to induced hypertension may have been due more to the genetic background strain of our mice (C57BL/6x129SvEv) than fibulin-5 deficiency.

The Impact of High Salt Diet on the Nociceptive Pain Thresholds and Functional Phenotype of Myeloid Cells

Poster Introduction/Aim: Though several studies have shown that high salt diet (HSD) is related to the development of chronic diseases, the effect of long-term high-salt intake on the immune system and pain behavior remains elusive. Here, we aimed to investigate whether and how HSD affected mouse pain thresholds and innate immunity, especially myeloid cells.Methods: Healthy C57BL/6 male and female mice were fed with HSD (containing 4% NaCl in chow and 1% NaCl in water) right from weaning period (3-week after birth) for 1–3 months.Results: Behavioral analysis demonstrated that HSD-fed mice were hypersensitive to mechanical stimuli. Moreover, HSD induced an expansion of circulating monocytes, especially the proinflammatory monocyte (Ly6Chigh, CCR2+) subsets. HSD also increased the density of Iba-1+ microglia and the number of FcγII/III receptor (CD16/32) expressing cells in the lumbar spinal cord. Prolonged HSD feeding, meanwhile, resulted in increased infiltration of monocyte to the peripheral nerves, contributing to the monocyte-derived macrophage (CD11b+, F4/80+) population. Finally, shifting the HSD back to the regular laboratory chow (with 0.3% NaCl) cannot reverse the HSD-induced mechanical hypersensitivity myeloid cell expansion and polarization towards the inflammatory subsets in the periphery. However, treating HSD mice with CCR2 antagonist effectively normalized the pain threshold and myeloid cell profile in the peripheral blood and nerves.Discussion/Conclusions: Long term HSD feeding decreased mouse mechanical thresholds, in parallel with an expansion and polarization of myeloid cells towards inflammatory status. Such changes were not reversible when HSD-fed mice back to normal diet. However, blocking CCR2 signaling successfully attenuated HSD-induced hypersensitivity.

Abstract 107: Neurons in the Organum Vasculosum of the Lamina Terminalis Sense NaCl and Angiotensin II to Regulate Sympathetic Nerve Activity and Arterial Blood Pressure

The organum vasculosum of the lamina terminalis (OVLT) contains specialized neurons that sense changes in extracellular NaCl concentrations to regulate sympathetic nerve activity (SNA) and arterial blood pressure (ABP). However, OVLT neurons also abundantly express angiotensin II (AngII)-type I receptors. Therefore, we performed a series of in vitro and in vivo studies in adult male Sprague-Dawley rats to determine whether OVLT neurons sense both NaCl and AngII to impact SNA and ABP. First, in vitro whole-cell recordings revealed the majority of OVLT neurons (66%, 23/35) showed an increased discharge frequency to both +7.5mM NaCl (1.1±0.2 to 2.2±0.3Hz, P&lt;0.001) and 100pM AngII (1.3±0.3 to 2.2±0.3Hz, P&lt;0.001). Second, in vivo single-unit recordings demonstrate the majority of OVLT neurons (61%, 11/18) displayed an increase in cell discharge to intracarotid injection of 0.5M NaCl (3.6±0.6 to 9.0±1.6Hz, P&lt;0.01) and 200ng AngII (3.2±0.7 to 9.2±1.9Hz, P&lt;0.01). Third, optogenetic inhibition of OVLT neurons (rAAV2-CaMKII-eNpHR3.0-mCherry, 5x10 12 particles/mL, 561nm, 10mW) attenuated thirst responses to IV infusion of 2M NaCl (1.25mL/30min; Laser OFF: 6.1±0.5mL vs ON: 2.0±0.7mL, P&lt;0.05) and AngII (Laser OFF 7.2±0.7mL vs ON: 2.2±1.2mL; n=5 per group, P&lt;0.05). Fourth, OVLT injection (20nL) of 0.5M NaCl or 10pmol AngII increased SNA (121±6% or 117±5%, n=4/group; P&lt;0.05 respectively) and mean ABP (7±1 or 9±2mmHg, n=4/group; P&lt;0.05 respectively). To determine whether chronic activation of OVLT neurons produces hypertension, rats received an OVLT injection of rAAV2-hSyn-HM3D(Gq)-mCherry (3x10 12 particles/mL, 50nL) and given access to 0.1% NaCl chow and 0.9% NaCl drinking solution. After 4 weeks, daily administration of clozapine-N-oxide to the drinking solution (3mg/kg/day) significantly increased 24-h fluid intake (baseline: 60±10mL vs Day 7: 178±29mL; n=6, P&lt;0.01) and 24-h mean ABP (baseline: 97±2mmHg vs Day 7: 108±1 mmHg; n=6, P&lt;0.05). Ganglionic blockade with hexamethonium (30mg/kg, ip) produced a greater fall in mean ABP at Day 7 (baseline: -38±6 mmHg vs Day 7: -53±5 mmHg, P&lt;0.05). These findings suggest OVLT neurons sense both NaCl and AngII to regulate body fluid homeostasis and SNA to produce a sympathetically-mediated hypertension.

High Salt Diet May Stimulate Fructose Uptake in Brain Neurons and Contribute to Neuronal Apoptosis

Clinical studies have been leaning toward the role that the central nervous system (CNS) is one of the main controls of blood pressure regulation such as the onset of fructose‐induced hypertension. The CNS is by far the most energy demanding and consuming organ in our body and it can metabolize fructose but that has consequences. However, the connection between fructose and hypertension within the CNS is still lacking in definition. Here we tested the hypothesis that a high fructose diet contributes to hypertension via increase neuronal apoptosis. Eight‐week‐old male Sprague Dawley (SD) rats were divided into 4 groups and were fed a normal salt (NS: 0.4% NaCI chow), high salt (HS: 4% NaCl chow), NS diet with fructose (F: 20% fructose drinking water), and high salt plus fructose (HS+F: 4%NaCl chow and 20% drinking water) diet, respectively, for 3 consecutive weeks. Their blood pressure was measured prior and post each diet treatment using the non‐invasive tail‐cuff technologies. All rats were then euthanized, then their blood and cerebrospinal fluid (CSF) was withdrawn for fructose measurements. HS+F rats showed an increase in mean arterial blood pressure (MAP) 126 mmHg compared to control 90 mmHg (p&lt;0.0001). There is no significant MAP alterations in HS or F alone groups compared to NS group. Fructose measurement demonstrated that the serum fructose concentration is significantly increased in the F group (359 μM), and HS+F (409 μM) compared to control NS group (253 μM), while no significant alterations were observed in serum fructose in HS alone group (229 μM). In contrast, CSF fructose concentration is dramatically decreased in F (230 μM) and HS+F (231 μM) groups compared to NS control group (290 μM). In addition, immunostaining showed that HS+F group significantly promoted immunoreactivity of GLUT5, a fructose transporter in the brain paraventricular nucleus (PVN). Furthermore, incubation of brain neurons isolated from neonatal SD rats with 1 mM fructose for 6 hours stimulated neuronal apoptosis (27%) compared to the control (20%, P&lt;0.05). In summary, this study demonstrated that HS+F diet decreases CSF fructose concentration, increases PVN GLUT5 expression, and results in hypertension in normal SD rats. These results suggest that HS facilitates brain neurons to uptake fructose into neuronal cells, resulting in an increase in intracellular fructose, which in turn, stimulate cell apoptosis. Increased neuronal apoptosis may contribute to hypertension.Support or Funding InformationFunding support: R15‐HL122952, Qinghui Chen; R15‐HL129213, Zhiying Shan; Michigan Tech and portage health Foundation REF, Zhiying ShanThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CD14 as a Novel Negative Modulator of Immune System‐Dependent Renal Damage and Salt‐Sensitive Hypertension

Hypertension affects one‐third of Americans of which approximately half are termed salt‐sensitive. Previous work in our laboratory has implicated a significant role for the immune system in potentiating the hypertensive and kidney damage phenotypes which occur in the Dahl Salt‐Sensitive (SS) rat. A previously performed RNA‐Seq based transcriptomic experiment on medullary tissue from SS rats fed either a low (0.4% NaCl) or high salt (4.0% NaCl) diet demonstrated an upregulation of molecules involved in the innate immune response including Cluster of Differentiation 14 (CD14). CD14 has been associated with hypertension in various human and animal studies, but mechanistic studies have not been previously performed. The present study tests the hypothesis that CD14 influences the extent of high‐salt diet induced renal damage and hypertension in the Dahl SS rat, by modulating activation of the innate immune system. Using CRISPR‐Cas9 technology, we generated a targeted mutation in the CD14 gene on the Dahl SS rat background, creating a 7‐bp frameshift deletion of bases 248–254 resulting in a predicted premature stop codon (SSCD14−/−). As CD14 is involved in macrophage activation, Interleukin‐1β production, a stereotypical pro‐inflammatory activation response by macrophages, was assessed. SSCD14−/− peritoneal macrophages demonstrated an increased ability to produce IL‐1β (307.8±31.3pg/ml) in response to Lipopolysaccharide + ATP stimulation compared to SSCD14+/+ littermates (142.9±31.5pg/ml, p=0.0099). We then assessed salt‐sensitive hypertension and renal damage phenotypes in these animals. SSCD14+/+ and SSCD14−/− female littermates, maintained on 0.4% NaCl chow, underwent a carotid artery telemetry implantation surgery at 7 weeks of age. After recovery and six days of baseline mean arterial pressure (MAP) recording (day 0: MAP SSCD14+/+=117.2±1.5mmhg, SSCD14−/−=132±11.0mmhg) animals were switched to a 4.0% NaCl diet for three weeks. Overnight urine collections occurred during the low salt period and throughout the high salt diet period. At 21 days, SSCD14−/− had a significantly greater MAP (172.0±11.4mmhg) than SSCD14+/+ animals (148.2±3.5mmhg), p&lt;0.001. Urinary albumin excretion, though not different at baseline (SSCD14+/+=5.6±0.7, SSCD14−/−=21.9±10.5, p&gt;0.05), was significantly greater in SSCD14−/− compared to SSCD14+/+ littermates at days 14 (SSCD14+/+=47.4±8.7, SSCD14−/−=139.8±51.7mg/day, p=0.011) and 21 (SSCD14+/+=79.8±15.9, SSCD14−/−=164.8±36.4mg/day, p=0.017) of the high salt diet. Together these data indicate that CD14 may modulate the full extent of the immune response in the macrophage during the induction and potentiation of salt‐sensitive hypertension and renal disease.Support or Funding InformationSupported by DK96859, R24HL114474, and HL116264.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Stimulation of Intestinal Cl- Secretion Through CFTR by Caffeine Intake in Salt-Sensitive Hypertensive Rats

Background/Aims: High salt consumption is a major risk factor for hypertension, and sodium homeostasis is regulated by both intestinal sodium absorption and urinary sodium excretion. Chronic caffeine intake has been reported to attenuate salt-sensitive hypertension by promoting urinary sodium excretion; however, its exact role in intestinal sodium absorption remains unknown. Here, we investigated whether and how chronic caffeine consumption antagonizes salt-sensitive hypertension by inhibiting intestinal sodium absorption. Methods: Dahl salt-sensitive rats were fed 8% NaCl chow and 0.1% caffeine in their drinking water for 15 days. The blood pressure and fecal sodium content were measured. The effect of caffeine on the movement of Cl- in enterocyte cells was determined with the Ussing chamber assay. Results: Rats that were treated with caffeine displayed significantly lower mean blood pressure and higher fecal sodium content than the controls. Consistent with these findings, caffeine intake decreased fluid absorption by the intestine in the fluid perfusion experiment. Further, the results from the Ussing chamber assay indicated that caffeine promoted Cl- secretion through enterocyte apical cystic fibrosis transmembrane conductance regulator (CFTR), and thus inhibited sodium absorption. Moreover, depletion of cAMP or inhibition of CFTR completely abolished the effect of caffeine on Cl- secretion. Conclusion: The results indicate that chronic caffeine consumption reduces sodium absorption by promoting CFTR-mediated Cl- secretion in the intestine, which contributes to the anti-hypertensive effect of caffeine in salt-sensitive rats.

Teneligliptin Prevents Cardiomyocyte Hypertrophy, Fibrosis, and Development of Hypertensive Heart Failure in Dahl Salt-Sensitive Rats

BackgroundWe investigated the effects of the dipeptidyl peptidase 4 inhibitor teneligliptin on cardiac function and hemodynamics during heart failure in hypertensive model rats. Methods and ResultsFifty-five male Dahl salt-sensitive rats were divided into 4 groups: control group (0.3% NaCl chow; n = 13), hypertension (HT) group (8% NaCl chow; n = 20), HT–early TNL group (8% NaCl chow and teneligliptin from 6 weeks; n = 10), and HT–late TNL group (8% NaCl chow and teneligliptin from 10 weeks; n = 12). Hemodynamic measurement and tissue analyses were performed at 18 weeks. In all of the HT groups, systolic blood pressures were similarly elevated (P = .66) and heart weights similarly increased (P = .36) with and without TNL administration. LV end-diastolic dimension was significantly enlarged only in the HT–early TNL group compared with the control group (P = .025). Histologic analysis showed less fibrosis (P = .008) and cardiomyocyte widths (P = .009) in the HT–early TNL group compared with the HT group. On hemodynamic analysis, only the HT group showed significant LV end-diastolic pressure elevation (P = .049) and lung congestion (P < .001) compared with the control group. ConclusionsThese results suggest that teneligliptin prevents concentric LV hypertrophy, fibrosis, and development of congestive heart failure in Dahl salt-sensitive rats. Teneligliptin may inhibit pressure-overload hypertrophic adaption and result in LV eccentric hypertrophy with reduced LV ejection fraction.

Chronic Renal Medullary Infusion of Interleukin‐1 Receptor Antagonist Delays the Onset of Salt Sensitive Hypertension in Uninephrectomized Dahl SS Rats

The Dahl SS rat is a well‐established model of salt‐sensitive hypertension and renal disease with the immune system contributing in the pathology of the disease. Recent work from our laboratory demonstrated that in response to a high salt challenge, in addition to increases in blood pressure and urinary albumin excretion, there is an upregulation of the Nod‐like receptor protein‐3 (NLRP3) inflammasome in the renal medulla. This upregulation of the NLRP3 inflammasome leads to a subsequent increase in interleukin 1 beta (IL‐1β) production. This proinflammatory cytokine has been shown to be elevated in hypertension in humans making it a potential therapeutic target for salt sensitive hypertension. Therefore, we tested the hypothesis that chronic renal medullary infusion of IL‐1 receptor antagonist (IL‐1RA) would attenuate the development of salt sensitive hypertension in the Dahl SS rat. Animals underwent a right uninephrectomy at 7 weeks of age to avoid compensatory changes in blood pressure from the contralateral kidney. After a week of recovery, the left femoral artery was cannulated to measure mean arterial pressure (MAP) while a chronically implanted medullary interstitial catheter was placed in the left kidney for continuous infusion IL‐1RA (600μg/day, n=5) or vehicle (0.9% NaCl saline, n=6). MAP was measured during a 4‐day period while animals were maintained on 0.4% NaCl chow then switched to a high salt (4.0% NaCl) challenge for a 10‐day. MAP did not differ between saline and IL‐1RA treated animals during the 0.4% diet period (127±3 mmHg vs 121±4 mmHg, P&gt;0.05). However, following a 10‐day challenge of 4.0% NaCl diet, MAP was significantly reduced in the IL‐1RA treated rats compared to the saline‐treated rats (157±9 mmHg vs 176±5 mmHg, P&lt;0.05). Despite seeing a reduction in MAP, urinary albumin (118±18 vs 126±10 mg/day) and protein (284±28 vs 277±19 mg/day) excretion in IL‐1RA treated did not differ from saline‐treated rats. Furthermore, when medullary IL‐1β levels were measured by ELISA, there was no difference between the IL‐1RA treated rats compared to the saline‐treated rats (76±25 pg/mL vs. 83±11 pg/mL). In contrast to the effects of intarenal infusion, systemic administration of the same dose of IL‐1RA did not alter salt‐sensitive hypertension compared to vehicle‐treated control SS (132±8mmHg vs 141±mmHg, n=5–6/group), demonstrating that the effects observed with medullary interstitial infusion are due to intrarenal effects. These data indicate that blockade of the IL‐1 receptor in the renal medulla attenuates salt‐sensitive hypertension in the Dahl SS rat. Further studies are required to understand the mechanism by which renal medullary inhibition of IL‐1β results in reduction in blood pressure in response to a high salt diet.Support or Funding InformationSupported by DK96859, HL116264, and 15SFRN2391002.

PP.12.13] DIFFERENT EFFECTS OF ACUTE HIGH SALT INTAKE ON ANTIOXIDATIVE GENES EXPRESSION IN CEREBRAL BLOOD VESSELS IN YOUNG AND MATURE SPRAGUE-DAWLEY RATS

Objective: The aim of this study was to assess the level of antioxidative genes expression in Sprague-Dawley rats at different ages and question whether high salt (HS) intake leads to the same changes regardless of the animal age. Design and method: 4- and 11-weeks old healthy male rats (N = 3–6) were divided to low salt (LS) group fed 0.4% NaCl chow and HS group fed 4% NaCl chow for 1 week. After diet protocol rats were anesthetized with ketamin-chloride (75 mg/kg) and midazolam (2.5 mg/kg) and sacrificed. All surface cerebral blood vessels were isolated and collected for mRNA gene expression analysis: SOD isoforms (Cu/ZnSOD, MnSOD, ecSOD) and glutathione peroxidase 4 (GPx4)) using real-time quantitative PCR (rtPCR; BioRad CFX96). Results are presented as mean ± SEM (p < 0.05 was considered significant). All experimental procedures conformed to the European Guidelines for the Care and Use of Laboratory Animals (directive 86/609) and were approved by the local Ethical Committee. Results: mRNA expression of SOD isoforms (MnSOD (4wLS 0.188 ± 0.012 vs. 11wLS 0.952 ± 0.078, p < 0.001) and ecSOD (4wLS 0.240 ± 0.009 vs. 11wLS 0.609 ± 0.083, p = 0.002)) was significantly higher, while expression of GPx4 (4wLS 1.299 ± 0.20 vs. 11wLS 0.819 ± 0.07, p = 0.024) was significantly decreased in older animals compared to young animals, both on a LS. mRNA expression of Cu/ZnSOD (4wLS 1.012 ± 0.02 vs. 4wHS 0.289 ± 0.17, p = 0.005) in young rats and expression of GPx4 (4wLS 1.299 ± 0.02 vs. 4wHS 0.457 ± 0.16, p = 0.031; 11wLS 0.819 ± 0.07 vs. 11wHS 0.644 ± 0.02, p = 0.041) in both age groups was significantly lower in HS groups. Expression of each SOD isoforms was increased in 11wHS rats compared to 4wHS rats (Cu/ZnSOD (4wHS 0.289 ± 0.017 vs. 11wHS 0.979 ± 0.05, p < 0.001), MnSOD (4wHS 0.022 ± 0.019 vs. 11wHS 0.792 ± 0.06, p < 0.001) and ecSOD (4wHS 0.080 ± 0.05 vs. 11wHS 0.510 ± 0.04, p < 0.001). Also the expression of MnSOD and ecSOD was significantly higher in 11wHS group compared to 4wLS group. Conclusions: The antioxidative genes expression increased with age. HS diet does not affect SOD isoform expression in older rats, but significantly decreases Cu/ZnSOD and GPx4 in young rats. Thus, harmful effects of HS intake may be more detrimental to vascular function in the younger age, due to presumably higher level of oxidative stress.