Salt-sensitive Hypertension Research Articles

S-38-1: EPIGENETIC MECHANISM OF SALT-SENSITIVE HYPERTENSION

Nutritional and environmental factors contribute to the development of salt sensitive hypertension. DNA methylation, the most extensively studied epigenetic marker, may have more stable, longer lasting effects than transient changes in gene expression. Protein malnutrition during pregnancy causes prenatal programmed salt sensitive hypertension via aberrant DNA methylation of the angiotensin receptor, which is induced by exposure to excessive glucocorticoid in the fetal hypothalamus. In adults, salt sensitive hypertension induced by obesity is attributable to increased renal activity of Rac1 and mineralocorticoid receptor (MR) pathway, and adverse prenatal exposure induces transgenerational hypertension through activation of Rac1 and MR pathway by an epigenetic regulated mechanism. Finally, DNA methylation of Klotho in aged kidneys leads to a reduction in the circulating levels of soluble Klotho, and in turn, resulting in the activation of noncanonical Wnt5a and RhoA signaling in the vasculature and the development of aging associated salt sensitive hypertension. These findings indicate that epigenetic modulation has an important role in the development of salt sensitive hypertension throughout life.

S-18-3: SGLT2 INHIBITOR CANAGLIFLOZIN ANTAGONIZES SALT-SENSITIVE HYPERTENSION THROUGH MODIFYING TRPC3 MEDIATED VASCULAR CALCIUM HANDLING

Objectiv: Salt-sensitive hypertension is highly prevalent and associated with cardiorenal damage. Large clinical trials have demonstrated that Sodium-glucose cotransporter 2 (SGLT2) inhibitors exert hypotensive effect and cardiorenal protective benefits in hypertensive patients with and without diabetes. However, the underlying mechanism remains elusive. Methods: Dahl salt-sensitive (SS) rats and salt-insensitive controls were fed with 8% high salt diet to establish salt-sensitive hypertensive model, some of them were treated with canagliflozin. The blood pressure, urinary sodium excretion, vascular function and remodeling were detected. Rat aortic proteomics and TRPC3 knockout mice were used to explain the mechanism of the effect of canagliflozin on salt-sensitive hypertension. Results: Canagliflozin treatment significantly reduced high salt induced hypertension and this effect was not totally dependent on urinary sodium excretion in SS hypertensive rats. Assay of vascular function and proteomics showed that canagliflozin significantly inhibited vascular cytoplasmic calcium increase and vasoconstriction in response to high salt diet. High salt intake increased vascular expression of TRPC3 in salt-sensitive rats, which could be alleviated by canagliflozin treatment. Overexpression of TRPC3 mimicked salt-induced vascular cytosolic calcium increase in vitro and knockout of TRPC3 erased the anti-hypertensive effect of canagliflozin. Mechanistically, high-salt-induced activation of sodium/calcium exchanger 1 (NCX1) reverse mode increased cytoplasmic calcium level and vasoconstriction, which required TRPC3, and this process could be blocked by canagliflozin. Conclusions: We define a previously unrecognized role of TRPC3/NCX1 mediated vascular calcium dysfunction in the development of high salt induced hypertension, which can be improved by canagliflozin treatment. This pathway is potentially a novel therapeutic target to antagonize salt sensitive hypertension.

Salt Restriction and Angiotensin-Converting Enzyme Inhibitor Improve the Responsiveness of the Small Artery in Salt-Sensitive Hypertension.

For salt-sensitive hypertension (SSH), salt restriction and angiotensin-converting enzyme (ACE) inhibitors are essential treatments, but their effect on the function of resistance arteries is unclear. Here, we present an intravital study to detect the effect of salt restriction and ACE inhibitors on the function of the mesenteric small artery (MSA) in SSH. Dahl salt-sensitive rats were randomized into the following groups: ACE inhibitor gavage, salt restriction, ACE inhibitor combined with salt restriction, and high-salt diet. After a 12-week intervention, the mesenteric vessels maintained their perfusion in vivo, and the changes in the diameter and blood perfusion of the MSAs to norepinephrine (NE) and acetylcholine (ACh) were detected. Switching from a high-salt diet to a low-salt diet (i.e., salt restriction) attenuated the vasoconstriction of the MSAs to NE and promoted the vasodilatation to ACh, while ACE inhibitor improved the vasodilatation more obviously. Pathologically, changes in local ACE, AT1R, and eNOS expression were involved in these processes induced by a high-salt diet. Our study suggests that salt restriction and ACE inhibitor treatment improve high salt intake-induced MSA dysfunction in SSH, and salt restriction is a feasible and effective treatment. Our findings may provide a scientific basis for the treatment of hypertension.

S-27-6: ADAPTIVE IMMUNITY REGULATES RENAL SALT-RETENTION AND BLOOD PRESSURE

Hypertension is a major public health problem, which contributes to a plethora of other cardiovascular diseases. A long-time postulate is that a defect in the kidney plays a key role in the development of hypertension. Years of intense research have confirmed that distal nephron plays important role in excessive salt retention, which contributes to the pathogenesis of hypertension. Yet the precise identity of this kidney defect and the downstream signaling mechanisms that impair sodium handling remain unclear. In the past decade, T cells, in particular CD8+ T cells (CD8Ts), have garnered attention as an important contributor to hypertension. T cells infiltrate into the kidneys of hypertensive animals, possibly driven by elevated perfusion pressure. Subsequently elevation of blood pressure and salt content stimulate T cell-activation and cytokine production. We recently reported a new mechanism by which renal infiltrated CD8Ts contribute to salt retention and hypertension: CD8Ts upregulate the sodium-chloride cotransporter (NCC) in the renal distal tubules, resulting in excessive salt retention via direct interactions between CD8Ts and renal distal convoluted tubule cells (DCTs) (Image in enclosed figure). This finding raises the exciting prospect that kidney-accumulation of CD8Ts and their interactions with DCTs represent one kidney defect in the pathogenesis of salt-sensitive hypertension. Although blocking NCC using thiazide temporarily lowered blood pressure in hypertensive animals, it failed to diminish T cell-homing to the kidneys, and hypertension returned after withdrawal of thiazide. Thus, in order to design effective therapeutics for salt-sensitive hypertension which interrupt the initial stimuli that trigger renal sodium retention, it is critical for us to define mechanisms of T cell-homing to the kidney and identify the key molecules mediating the CD8T-DCT interaction. Here, we further identified critical molecular players and demonstrated their roles in augmenting the CD8T-DCT interaction leading to salt-sensitive hypertension. We found that activated CD8Ts exhibit enhanced interaction with DCTs via IFNγ-induced upregulation of MHC-I and PDL1 in DCTs, thereby stimulating higher expression of NCC in DCTs to cause excessive salt retention and progressive elevation of blood pressure. Eliminating IFNγ or renal tubule-specific knockdown of PDL1 prevented T cell homing into the kidney, thereby attenuating hypertension in two different mouse models. These findings provide a new mechanism for T cell involvement in the pathogenesis of hypertension and reveal novel therapeutic targets.

JS-WHL-ISH-RTSL-2: WHY SHOULD WE WORRY ABOUT SODIUM INTAKE, IF WE INCLUDE A DIURETIC IN THE HYPERTENSION TREATMENT REGIMEN ?

The causal role of sodium intake on blood pressure level has been long recognized. While the pathophysiological link between hypertension and cardiovascular, cerebrovascular, and renal disease events are clear, there is evidence that sodium has an independent direct effect on target organs through multiple mechanisms including vascular damage, oxidative stress, hormonal alterations, the immune system and the gut microbiome. Thus, reduction in salt and sodium intake is an important global strategy to reduce disease burden. Certainly sodium is an essential element for homeostasis, but the levels of consumption far exceed the amount needed for physiological function. Likewise, considerable evidence indicates reduction in sodium consumption is associated with significant reduction in blood pressure in all individuals at the population level. Individual response to sodium intake is variable, but should be considered a risk factor when consumption is excessive. Interventions such as the DASH-sodium (Dietary Approaches to Stop Hypertension) study has clearly demonstrated benefit from sodium reduction. In nearly all populations acculturated to an industrialized lifestyle, blood pressure increases with age resulting with an increasing hypertension prevalence. Thus, salt intake becomes increasingly relevant at all ages.. Nonetheless the levels of salt consumption that are recommended remain debated. Contributing to the varying study results are the individual differences in salt sensitivity. Therefore, for salt-sensitive risk groups in the population, stricter limits of salt consumption should be implemented, with a specific target to the young before target organ damage. Maximum prevention benefit for reduced sodium consumption should target children. The magnitude of blood pressure lowering achieved with sodium reduction has a dose-response relationship with greater impact in older, non-white, and hypertensive populations. Many factors contribute to blood pressure and risk reduction with salt consumption leading to unclear and less precise results and conclusions. Individuals with salt-sensitive hypertension and/or low-renin hypertension typically have factors conducive to treatment with a diuretic. As salt-sensitive hypertension is more common in black patients, older adults, patients with more severe hypertension, and patients with co-morbidities, diuretics have been shown to be particularly effective with salt reduction adding additional benefit.

S-18-1: THE USUAL HEMODYNAMIC MECHANISM CAUSING SALT-INDUCED HYPERTENSION: ABNORMALLY INCREASED CARDIAC OUTPUT OR VASCULAR RESISTANCE?

Two competing theories have been proposed for the hemodynamic mechanism that usually mediates salt sensitivity and the initiation of salt-induced hypertension. The historical natriuretic dysfunction theory holds that impaired renal excretion of sodium initiates hypertension by causing abnormally large increases in cardiac output secondary to abnormally large increases in salt and water retention. In contrast, the contemporary vasodysfunction theory holds that abnormal vascular resistance responses to salt, not abnormal increases in cardiac output, mediate salt sensitivity and the initiation of salt-induced hypertension. Primary aldosteronism is one of the most common causes of salt sensitivity and salt-dependent hypertension. This presentation will discuss new studies showing that hyperaldosteronism promotes salt sensitivity and initiation of salt-dependent hypertension by amplifying salt-induced increases in systemic vascular resistance, not cardiac output. The findings are consistent with the results of other studies showing that an abnormal vascular resistance response to salt (vasodysfunction) is also usually responsible for the initiation of salt-induced hypertension in humans and animals without hyperaldosteronism. The vasodysfunction framework for the pathogenesis of salt sensitivity and salt-induced hypertension has implications for: 1) understanding how various genetic and non-genetic factors influence BP responses to high salt diets, and 2) development of new methods for preventing and treating salt-dependent hypertension.

PS-C09-11: THE EFFICACY OF SACUBITRIL/VALSARTAN FOR NONDIPPER AND EXTREME DIPPER HYPERTENSION USING 24 AMBULATORY BP MEASUREMENT

Sacubitril/Valsartan increases natriuresis and diuresis and makes superior blood pressure control compared with usual RAS inhibitors. In Japanese clinical cicumstances,ideal blood pressure control to attain BP < 130/80mmHg is difficult for next reasons. 1, Japanese predisposition towards high sodium intake. 2, Salt sensitivity due to Gene Polymorphism. 3, Increasing morbidity of obesity and Metabolic syndrome. 4, Clinical inertia to attain ideal blood pressure control especially in high risk hypertension patients ex. CKD and DM. These high risk patients often suggest nocturnal hypertension by 24 hour ambulatory blood pressure monitoring,24Hr ABPM. Nocturnal hypertension consists of non-dipper pattern, riser pattern, and often associated with cardiovascular event. On the contrary, extreme dipper pattern is sometimes found by24 Hr ABPM and it suggests high systolic BP during daytime in spite of night systolic BP decline. iIt sometimes results in stroke. Therefore antihypertensive therapies to these high risk patients by monitoring only clinic BP are surely insufficient to prevent CVD. Furthermore checking Home BP immediately after wake up is off course necessary, but still inadequate. Without monitoring by 24hr ABPM during nighttime,it is impossible to distinguish between non-dipper pattern, riser pattern and morning surge pattern. Quoting from literature, superiority of Ambulatory over Clinic Blood pressure measurement is proved in predicting mortality,The Dublin Outcome Study. It is essential to check 24hr ABPM to high risk patients. These high risk CKD or DM patients are accompanied by the diminished renal capacity to excrete sodium during daytime and compensate sodium excretion by elevating night blood pressure. This mechanism could be a component of the non dipper or riser pattern. Sacubitril/Valsartan `s effect of natriuresis and diuresis effect could be effective to excrete sodium excretion at daytime and Np makes vasodilatory effect and prohibits of aldosterone production.,and valsartan is anAT1 receptor blockade and prohibits of angiotensin2 effect. Sacubitril/Valsartan is very effective for characteristic “salt sensitive hypertension” Japanese patients. In our clinic, we prescribed 250 patients,120 patients are RAS inhibitor naive, and the rest of130 patients are switched from RAS inhibitor. We present 2 clinical cases prescribing Sacubitril/Valsartan 400 mg every morning and monitored 24hrABPM before and after prescription. 1st case is 48yrs old male, HT, DM and CKD showing non dipper-pattern. 2nd case is 86 yrs female, HT and old cerebral lacuna and left common iliac aneurysma showing extreme dipper pattern. 2 weeks after administration of Sacubitril/Valsartan, 24hr ABPM were checked and non dipper pattern disappeared in Ist patients and extreme dipper pattern disappeared in 2nd case,. Normal circadian rhythms are attained in both patients.

PS-B02-12: EFFECTS OF ULTRAVIOLET LIGHT ON BLOOD PRESSURE AND LEFT VENTRICULAR FUNCTION

Ultraviolet light (UV), induces the activation of constitutive nitric oxide synthases, which produces nitric oxide (NO) of vascular relaxation factor. We hypothesize that UV-induced production of NO could have beneficial effect on hypertension. Twelve 6-week-old salt-sensitive hypertensive rats (Dahl-Iwai salt-sensitive DIS/Eis, Japan Society for the Study of Hypertension, Shizuoka, Japan) were used in this study. These rats were fed a high-salt diet containing 8%NaCl until 10 weeks of age and were then randomly divided into three groups i.e. baseline group (group non-UV), 0 h post UV (group UV 0 h), and 6 h post UV (group UV 6 h). The rats were irradiated with UVA and UVB lamps at an intensity of 100 mJ/cm2. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured by using tail-cuff method (BP-98A; Softron Co., Ltd., Tokyo, Japan). We evaluated left ventricular (LV) systolic and diastolic function using echocardiography with a 12 MHz transducer connected to a Xario ultrasound system (Toshiba Medical Systems Co., Tochigi, Japan). Interventricular septal thickness, LV posterior wall thickness, and LV end-diastolic and end-systolic diameters (LVDd and LVDs) were obtained from a short-axis view. Percent LV fractional shortening (%LVFS) was calculated as an index of LV systolic function. Peak flow velocities at the mitral level during rapid filling (E) and deceleration time (Dct), and peak early diastolic mitral annular velocity which indicate LV diastolic function, were measured using pulsed and tissue Doppler echocardiography. Histological skin tissue sections were fixed with 4% paraformaldehyde phosphate buffer solution for 24 h at room temperature, embedded in paraffin, sectioned transversely (thickness, 3 μm), and stained with hematoxylin-eosin to evaluate the impairments of skin. The SBP and DBP were lower in group UV 6 h than group non-UV and group UV 0 h. In contrast, there were no significant differences in heart rate and body weight among the three groups. The peak early diastolic mitral annular velocity was greater in group UV 6 h than group non-UV and group UV 0 h. LVDd, LVDs, and %LVFS were not significantly different among the three groups. We did not find the impairments in skin after UV irradiation. UVA and UVB may play an important role in regulating not only blood pressure but also LV function.

PS-B05-4: COMPARATIVE EFFECTS OF RENAL DENERVATION IN DIFFERENT STAGES OF HYPERTENSION ON CARDIAC, RENAL, AND ADIPOSE PATHOLOGY IN DAHL SALT-SENSITIVE RATS

Objective: Renal denervation (RDN) has emerged as a novel therapy for resistant hypertension. In Dahl salt-sensitive (DahlS) rats, RDN does not prevent the development of hypertension but attenuates the progression of hypertension. We have now investigated the comparative effects of RDN in early versus advanced stages of hypertension on cardiac, renal, and adipose tissue pathology in DahlS rats. Design and method: Male DahlS rats fed an 8% NaCl diet after 6 weeks of age were subjected to RDN or sham surgery at 7 (early stage) or 9 (advanced stage) weeks. In the RDN groups, all visible nerves along the vessels were cut and the vessels were painted with a solution of 10% phenol in absolute ethanol. In contrast, in the sham-operated groups, the kidneys were exposed but the vessels were not stripped or coated with phenol. At 12 weeks, left ventricular (LV), renal, and adipose tissue were excised for analysis. Results: Successful RDN was confirmed by determination of renal norepinephrine content in each rat subjected to RDN less than 10% of the mean values of those in the sham-operated groups. Although both stages of RDN modestly attenuated blood pressure elevation, they improved LV diastolic function and fibrosis and attenuated macrophage infiltration and expression of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) genes in the heart, to a similar extent. In addition, both stages of RDN similarly reduced macrophage infiltration in glomeruli, the glomerulosclerosis index, the tubulointerstitial injury score as well as expression of MCP-1 and TNF-α genes in the kidney. The early stage group showed reductions in both retroperitoneal fat mass and adipocyte cross-sectional area, whereas the advanced stage group did an increase in fat mass without any change in adipocyte size. Conclusions: Both early and advanced stages of RDN mildly attenuated hypertension as well as ameliorated cardiac and renal injury, to a similar extent, in salt-loaded DahlS rats. The anti-hypertensive effect of RDN is thus likely attributable primarily to improved renal pathology. These results also suggest a possible crosstalk among the kidney, cardiovascular system, and adipose tissue via neural mechanisms in salt-sensitive hypertension.

PS-BPB10-5: POSSIBLE INVOLVEMENT OF RENAL TUBULAR NFAT5 IN AGING-ASSOCIATED RENAL DYSFUNCTION AND SALT-SENSITIVE HYPERTENSION

Objective: Japan is currently facing a super-aging society, where an incidence of lifestyle-related diseases has become a greater social burden. The number of patients with chronic kidney disease (CKD) and hypertension is being increased with age; however, it is not clearly defined why and how aging causes renal dysfunction and hypertension. Nuclear factor of activated T-cells 5 (NFAT5) is a transcription factor that is activated upon hypertonic conditions as observed in the renal medulla. We have shown that the renal tubular cell-specific NFAT5 conditional knockout (KO) mice exhibit salt-sensitive hypertension, while the mice exhibit impaired urine concentrating ability and are susceptible to renal fibrosis. These phenotypes resemble aging-associated renal dysfunction, i.e., urine concentrating disorder, salt-sensitive hypertension, and renal fibrosis. We therefore investigated the possible involvement of NFAT5 in aging-related changes of the kidney. Methods: Male wild-type (WT) and KO mice (12 weeks old) were subjected to unilateral ureteral obstruction (UUO) or sham operation. After 7 days, mice were sacrificed and kidneys were removed for analysis. Renal fibrosis was evaluated by AZAN staining. Senescence-associated beta-galactosidase (SA-β;-Gal) activity was examined by fluorescence study in the whole kidney slices. mRNA expression for senescence-associated secretory phenotype (SASP)-related factors (TGF-β;1, COL1A1, ICAM1, PAI-1, p21, p16, and p53) in the whole kidney tissue was examined by real-time PCR. Results: AZAN staining showed that UUO induced renal fibrosis both in WT and KO mice. The UUO-induced fibrotic area was significantly larger in KO mice than in WT mice in the medulla. In sham-operated mice, overall SA-β;-Gal activity was larger in KO mice, in which the activity was dominant in the corticomedullary region. In UUO groups, overall SA-β;-Gal activity was low compared to sham-operated groups. The mRNA expression of SASP-related factors was not different between WT and KO mice in sham-operated groups. UUO upregulated the mRNA expression of all SASP-related factors both in WT and KO mice. In UUO groups, the expression of TGF-β;1, COL1A1, ICAM1, and PAI-1 was significantly larger in KO mice than in WT mice. The expression of p21, p16, and p53 tended to be larger in KO mice than in WT mice. Conclusions: Renal tubular NFAT5 deficiency showed senescence-related phenotypes at baseline. Deficit of renal tubular NFAT5 may further induce cell senescence upon insult, which might accelerate aging-associated renal inflammation and dysfunction in CKD.

S-40-5: ROLE OF INTERFERON GAMMA IN T CELL MEDIATED SALT-RETENTION IN THE KIDNEY AND PROGRESSION OF HYPERTENSION

Objective: In accordance with previous studies implicating the immune system in the development of hypertension, we found that CD8+ T cells (CD8Ts) infiltrate the kidneys and stimulate distal convoluted tubular cells (DCTs), to upregulate sodium-chloride co-transporter (NCC) activity, leading to excessive salt retention. However, the precise molecules driving this direct interaction between CD8Ts and DCTs have not yet been identified. Here we hypothesize that gamma interferon (IFNg), released by activated CD8Ts, primes the tubular cell to promote interactions between activated CD8Ts and DCTs during the development of salt-sensitive hypertension. We predict blocking this molecular pathway will reduce CD8T-homing into the kidney, lowering blood pressure. Design & Method: In co-culture of mouse CD8Ts and DCTs, recombinant mIFNg, IFNg-neutralizing antibody, and specific siRNAs against the IFNg-receptor or PDL1 were used to determine the role of IFNg-PDL1 pathway in the CD8T-DCT interaction. To validate these studies, we employed IFNg knockout (KO) mice, mice with systemically blocked PDL1 using anti-PDL1 antibody or with renal tubule-specific knockdown of PDL1 (mediated via nanoparticles) in the DOCA-salt model and the CD8T adoptive transfer hypertension models. Blood pressure was monitored using radio-biotelemetry. Endpoint analysis involved flow cytometry, RT-qPCR, and western blot, and immunohistochemistry. Results: We found that CD8Ts isolated from DOCA-salt treated hypertensive mice produce more IFNg and exhibit augmented ability to interact with DCTs compared to those from sham normotensive mice. IFNg stimulation to mDCTs increased expression of CD8-specific antigen presenting molecule MHC-I and CD8T-co-signaling molecule PDL1, which enhanced the interaction between CD8Ts and DCTs, consequently upregulating NCC expression in DCTs, leading to enhanced salt retention. These effects on MHC-I, PDL1, or NCC expression were abolished by neutralizing IFNg in co-culture of DCTs and activated CD8Ts, and knockdown of PDL1 in DCTs blunted NCC expression and sodium retention. In-vivo results verified the in-vitro studies. We found that in both IFNg-KO mice and WT mice receiving PDL1-antagonizing antibodies, DOCA-salt treatment failed to elevate their blood pressure and reduced T cell infiltration and NCC expression within their kidneys compared to WT mice receiving the same treatment (anti-PDL1 results shown in Figure 1). Similar results were observed in DOCA-salt treated mice with renal tubule-specific knockdown of PDL1. Conclusion: In salt-sensitive hypertension, activated CD8Ts demonstrate enhanced ability to interact with DCTs leading to increased expression of NCC and sodium retention through a IFNg-PDL1 pathway mediated mechanism. Blocking the IFNg-PDL1 pathway prevents CD8T-DCT interaction both in vitro and in vivo, ameliorating hypertension.

PS-B08-13: PLASMIN INHIBITION ALLEVIATES HYPERTENSION AND PODOCYTE INJURY IN DAHL SALT-SENSITIVE RATS

Objective: The epithelial sodium channel (ENaC) in the kidney plays pivotal roles in blood pressure regulation, and the γ subunit is activated by extracellular serine proteases. In proteinuric renal diseases, plasmin filtered through injured glomeruli proteolytically activates γENaC. In addition, filtered plasmin directly causes podocyte injury. We previously reported that Dahl salt-sensitive (DS) rats fed a high-salt (HS) diet developed severe hypertension and proteinuria together with γENaC activation, and that a synthetic serine protease inhibitor, camostat mesilate, mitigated these changes. However, the role of plasmin in DS rats remains unclear. In this study, we evaluated the relationship between plasmin and hypertension as well as glomerular injury and the effects of plasmin inhibitors in DS rats. Design and method: Five-week-old male DS rats were divided into normal-salt (NS) diet, HS diet, and HS + plasmin inhibitors [tranexamic acid (TA, 2 mg/mL TA in drinking water) and synthetic plasmin inhibitor YO-2 (4 mg/kg/day, intraperitoneal injection)] groups. After systolic blood pressure measurement and 24-h urine collection over time for 5 weeks, the rats were sacrificed for biochemical examination. Results: The HS group displayed severe hypertension and proteinuria together with activation of plasmin in urine and γENaC in the kidney, which were not attenuated by TA. On the other hand, YO-2 mitigated both hypertension and proteinuria [SBP (mmHg): NS, 130.8 ± 7.5; HS, 209.0 ± 14.2; HS+YO-2, 183.8 ± 8.4; Urinary protein (mg/day): NS, 15.4 ± 3.2; HS 259.0 ± 129.3; HS+YO-2, 94.5 ± 31.5]. YO-2 inhibited the attachment of plasmin(ogen) to podocytes and alleviated podocyte injury by reducing glomerular apoptotic cells. Furthermore, YO-2 suppressed the upregulation of protease-activated receptor-1 and phosphorylated ERK1/2 as well as mRNA expression of inflammatory and pro-fibrotic cytokines in the kidney. Conclusion: These results indicate that plasmin plays important roles in the development of salt-sensitive hypertension and glomerular injuries in a rat model of hypertension, and suggest that plasmin inhibition could be a potential therapeutic strategy against salt-sensitive hypertension.

S-18-6: SALT AND AGE-RELATED HYPERTENSION

Hypertension is the greatest risk factor for cardiovascular disease and stroke, and its incidence increases with age. Clinical studies have shown that salt intake is involved in the development of age-related hypertension and that salt sensitivity of blood pressure (BP) increases with age, but the mechanism was unknown. On the other hand, it was reported that circulating Klotho level, an anti-aging factor protein, decreased with age, and there was an inverse relationship between circulating Klotho and mean BP changes after saline infusion. We hypothesized that the decrease in circulating Klotho levels might be involved in the increase in salt sensitivity of BP with aging. We found that circulating Klotho significantly decreased in aged mice and Klotho heterozygous knockout (KO) mice than in young wild-type mice. They showed salt-sensitive hypertension unlike young wild-type mice, suggesting that a decrease in circulating Klotho was involved in the pathogenesis of salt-sensitive hypertension in aged mice. In both aged mice and Klotho heterozygous KO mice, it was revealed that vascular Wnt5a-RhoA pathway was activated and vasoconstriction was enhanced during high salt intake, resulting in hypertension. We also demonstrated that Klotho supplementation and Wnt and Rho kinase inhibitors significantly suppressed the BP elevation during high salt intake in these mice. Furthermore, we found that Wnt5a was essential for the activation of RhoA by Angiotensin II, an important agent in the formation of hypertension. Since increased vascular resistance of the renal arteries and decreased renal blood flow are involved in the pathogenesis of salt-sensitive hypertension. We analyzed the effects of Angiotensin II and RhoA stimulators to the renal arteries of high-salt fed aged mice and Klotho heterozygous KO mice. Marked vasoconstriction of the renal arteries and decrease in renal blood flow with BP elevation were observed, while responses were less in young mice. Augmented responses observed in aged mice and Klotho heterozygous KO mice were normalized by Klotho supplementation. These findings suggest that in aged mice with low circulating Klotho, salt intake causes activation of the vascular Wnt5a-RhoA pathway, resulting in increased contraction of the renal arteries as well as peripheral blood vessels, which reduces renal blood flow and eventually caused hypertension. We suggest that reducing salt consumption is highly important and lifestyle habits preserving adequate levels of Klotho are useful in the prevention of age-related hypertension. Wnt and Rho inhibitors may be new treatment options. Therapeutic development focusing on the unique background of aging may be applied to other age-related diseases.

PS-BPB10-6: THE RELATION BETWEEN PERICYTE DETACHMENT AND INTERSTITIAL FIBROSIS IN DAHL SALT-SENSITIVE HYPERTENSIVE (DAHLS) RATS

Background: Congestive heart failure produces the state of fluid volume overload, central venous pressure (CVP) elevation, and consequently renal venous pressure (RVP) elevation. We have recently developed a novel rat unilateral renal venous congestion model, in which ligation of the inferior vena cava between both renal veins artificially induced renal congestion in the left kidney only. In this model, pericyte detachment and pericyte-myofibroblast transition (PMT) around the vasa recta were involved in interstitial injury and fibrosis. Dahl salt-sensitive hypertensive (DahlS) rats on high salt diets exhibit hypertension, congestive heart failure, fluid volume overload, CVP and RVP elevation, and renal damage, considering to be a non-artificial renal congestion model. The relation, however, between renal interstitial injury and fibrosis, elevated RVP, and pericyte detachment in DahlS rats has not been reported. In the present study, we assessed the relation of pericyte detachment around the vasa recta and PMT with interstitial fibrosis in DahlS rats. Methods: Eight-week-old DahlS rats (Dahl/Mcwi) were divided into three groups: normal diet, 4% NaCl diet, and 8% NaCl diet groups, and their kidneys were analyzed after 2 weeks. Total mRNA and protein expression levels were quantified by real-time PCR and western blot, respectively. Tubulointerstitial damage and fibrosis were identified by histological analysis. Furthermore, we evaluated pericyte detachment around the vasa recta by low-vacuum scanning electron microscopy (LV-SEM). Results: The expression of KIM1, a tubulointerstitial damage marker; CNN1 and αSMA, interstitial fibrosis markers; and TAGLN, a smooth muscle protein22 and PMT marker; was significantly increased in a salt-dependent manner in DahlS rats. In histological analysis, hyperplasia of the extracellular matrix and renal tubular injury were detected in 4% and 8% NaCl diet groups. The positive area of PMT markers in the interstitial space around the vasa recta was also increased in the NaCl diet groups. Pericyte detachment from the vasa recta was observed in the NaCl diet groups by LV-SEM. Conclusions: Pericyte detachment and PMT are involved in tubulointerstitial injury and fibrosis of DahlS rats. Renal venous congestion and subsequent physiological changes could have a role in renal damage and fibrosis. Arterial hypertension has been emphasized for a long time in renal injury, and venous hypertension must also be focused on with the concept of renal congestion. Further understanding of this concept may lead to new therapeutic strategies for renal injury.

PS-BPB03-1: ASSOCIATION OF ANTIHYPERTENSIVE EFFECTS OF ESAXERENONE WITH THE INTERNAL SODIUM BALANCE IN DAHL SALT-SENSITIVE HYPERTENSIVE RATS

Background: We have recently shown that the nonsteroidal mineralocorticoid receptor blocker, esaxerenone elicits effective blood pressure reduction in Dahl salt-sensitive hypertensive (DSS) rats. Here, we investigated the potential contribution of alterations in sodium homeostasis to esaxerenone-induced blood pressure reduction. Methods: In the different experimental setups, we evaluated blood pressure by the radio-telemetry system and sodium homeostasis by an approach of sodium in (food intake) and out (urinary excretion) in a low-salt diet (LSD, 0.3% NaCl), high-salt diet (HSD, 8% NaCl), HSD with 0.001 % esaxerenone (w/w), and HSD with 0.05% furosemide fed DSS rats. Results: HSD-fed DSS rats exhibited a non-dipper type hypertension, while esaxerenone treatment but not furosemide significantly reduced the BP with a dipper pattern. The cumulative urinary sodium excretion was significantly elevated from day 6 onward both in esaxerenone- and furosemide-treated rats compared to their HSD-fed counterparts in the active period where a significant increase in Na+/K+ ratio was evident for only the esaxerenone treatment group. Sodium content in the skin, skinned carcass as well as total body level tended to reduce in esaxerenone-treated rats compared to the HSD-fed matching parts, while these values were not changed in furosemide-treated rats. Consistently, sodium balance tended to be reduced in esaxerenone-treated rats during the active period. Histological evaluation revealed that esaxerenone but not furosemide treatment significantly attenuated glomerulosclerosis and tubulointerstitial fibrosis in HSD-fed DSS rats. Collectively, these findings suggest that esaxerenone-induced blood pressure reduction is associated with the body sodium homeostasis in salt-loaded DSS rats.

PS-B10-4: AFFERENT RENAL NERVOUS SIGNALS CONTRIBUTE TO THE DEVELOPMENT OF HFPEF WITH SYMPATHOEXCITATION IN DAHL SALT-SENSITIVE HYPERTENSIVE RATS

Objective: Sympathetic activation is involved in the pathophysiology of heart failure with preserved ejection fraction (HFpEF). Renal nerves contain sympathetic efferent and sensory afferent fibers. Excitation of the renal efferent nerves increases renal renin release and sodium/water retention. On the other hand, the renal afferent nerves are activated by the renal injury associated with hypertension, and acute stimulation of the afferent nerves increases sympathetic output from the brain. In this study, we investigated whether the signals from the renal afferent nerves contribute to the development of hypertensive HFpEF using a novel method of selective afferent renal nerve denervation (ARDN) in Dahl salt-sensitive rats, a widely used animal model of HFpEF. Design and method: Male Dahl salt-sensitive rats were fed 0.3% low-salt diet (LS) or 8% high-salt diet (HS) from 7 weeks of age. At 9 weeks, LS-fed rats received sham operation (LS-Sham), and HS-fed rats were randomly divided into ARDN (HS-ARDN) or sham operation (HS-Sham). At 19 weeks, blood pressure, echocardiographic parameters, organ weights, and plasma norepinephrine levels were measured, and the histological and biochemical analysis of the heart were performed. Results: Systolic blood pressure was significantly increased in HS-Sham compared to LS-Sham, and this increase was not affected in HS-ARDN. By echocardiography, left ventricular (LV) fractional shortening did not differ between the three groups; however, E/e’ ratio was significantly increased in HS-Sham compared to LS-Sham and was attenuated in HS-ARDN. LV weight / body weight ratio and lung weight / body weigh ratio were significantly increased in HS-Sham compared to LS-Sham, and were decreased in HS-ARDN. Plasma norepinephrine levels were significantly higher in HS-Sham than LS-Sham, and this increase tended to be inhibited in HS-ARDN. LV fibrosis evaluated by Masson trichrome staining was significantly increased in HS-Sham compared to LS-Sham and was attenuated in HS-ARDN. ANP mRNA expression in LV tissue was extremely increased in HS-Sham compared to LS-Sham and was significantly attenuated in HS-ARDN. Conclusion: The signals from afferent renal nerves contribute to the development of HFpEF with sympathoexcitation in Dahl salt-sensitive hypertensive rats.

S-51-5: DIFFERENT CONTRIBUTION OF AFFERENT RENAL NERVES TO DEVELOPMENT OF HYPERTENSION AND HYPERTENSIVE HEART FAILURE IN SEVERAL RAT MODELS OF HYPERTENSION

Objective: Renal nerves contain efferent and afferent fibers. Acute stimulation of the afferent nerves increases central sympathetic outflow. This study aimed to investigate the contribution of afferent renal nerves to the development of hypertension and hypertensive heart failure in several rat models of hypertension. Design and method: Selective afferent renal nerve denervation (ARDN) or sham operation was performed in the following models: (1) SHRSP at 9 weeks of age; (2) 4% high-salt diet (HS)-fed SHRSP at 8 weeks (HS from 8 weeks); (3) 8% HS-fed Dahl salt-sensitive rats at 9 weeks (HS from 6 weeks); and (4) 8% HS-fed Dahl salt-sensitive rats at 9 weeks (HS from 7 weeks). Dahl rats fed 0.3% low-salt diet (LS) were used as controls for (3) and (4). Blood pressure (BP) was measured by telemetry in (1) and (2), and by tail-cuff method in (3) and (4). The experimental period ended at the following age: (1) 11 weeks; (2) 11 weeks; (3) 16 weeks (“HFrEF’’ phase); and (4) 19 weeks (“HFpEF’’ phase). Results: (1) Mean BP was significantly elevated from 9 weeks to 11 weeks in sham-SHRSP (155.2 ± 2.0 vs 144.0 ± 1.9 mmHg, n = 10, p < 0.05). Mean BP in ARDN-SHRSP (157.8 ± 2.7 mmHg, n = 10) was not different compared to sham-SHRSP at 11 weeks. (2) Mean BP was significantly increased from 8 weeks to 11 weeks in sham-HS-SHRSP (191.5 ± 8.1 vs 126.5 ± 4.0 mmHg, n = 6, p < 0.05). Mean BP in ARDN-HS-SHRSP (186.0 ± 7.1 mmHg, n = 6) did not differ from sham-HS-SHRSP at 11 weeks. (3) Systolic BP was significantly increased (243.6 ± 7.0 vs 140.0 ± 2.7 mmHg, n = 9 each, p < 0.05) and echocardiographic left ventricular fractional shortening (LVFS) was significantly decreased (25.9 ± 1.7 vs 48.8 ± 0.4 %, p < 0.05) in sham-HS-Dahl rats compared to LS-Dahl rats at 16 weeks. Compared to sham-HS-Dahl rats, systolic BP was not different (223.4 ± 5.5 mmHg, n = 7); however, the decreased LVFS was significantly attenuated (42.2 ± 1.2 %, p < 0.05) in ARDN-HS-Dahl rats. (4) Systolic BP was significantly increased in sham-HS-Dahl rats compared to LS-Dahl rats at 19 weeks (207.8 ± 4.5 vs 135.7 ± 3.2 mmHg, n = 8 and 6, p < 0.05), and this increase was not affected in ARDN-HS-Dahl rats (220.7 ± 2.9 mmHg, n = 7). By echocardiography, LVFS did not differ between the three groups, whereas E/e’ ratio was significantly increased in sham-HS-Dahl rats compared to LS-Dahl rats and was attenuated in ARDN-HS-Dahl rats. Conclusion: The signals from afferent renal nerves contribute to the development of HFrEF and HFpEF without affecting BP in Dahl salt-sensitive hypertensive rats, but not of hypertension in SHRSP and HS-fed SHRSP.

Intrarenal Dopaminergic System Is Dysregulated in SS-Resp18mutant Rats.

The genetic and molecular basis of developing high blood pressure and renal disease are not well known. Resp18mutant Dahl salt-sensitive (SS-Resp18mutant) rats fed a 2% NaCl diet for six weeks have high blood pressure, increased renal fibrosis, and decreased mean survival time. Impairment of the dopaminergic system also leads to hypertension that involves renal and non-renal mechanisms. Deletion of any of the five dopamine receptors may lead to salt-sensitive hypertension. Therefore, we investigated the interaction between Resp18 and renal dopamine in SS-Resp18mutant and Dahl salt-sensitive (SS) rats. We found that SS-Resp18mutant rats had vascular dysfunction, as evidenced by a decrease in vasorelaxation in response to sodium nitroprusside. The pressure-natriuresis curve in SS-Resp18mutant rats was shifted down and to the right of SS rats. SS-Resp18mutant rats had decreased glomerular filtration rate and dopamine receptor subtypes, D1R and D5R. Renal dopamine levels were decreased, but urinary dopamine levels were increased, which may be the consequence of increased renal dopamine production, followed by secretion into the tubular lumen. The increased renal dopamine production in SS-Resp18mutant rats in vivo was substantiated by the increased dopamine production in renal proximal tubule cells treated with L-DOPA. Overall, our study provides evidence that targeted disruption of the Resp18 locus in the SS rat dysregulates the renal dopaminergic system.

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.

S-44-1: SALTING A BIRD'S TAIL

It is needless to stress high-salt intake threaten cardiovascular diseases because of inrement of hypertension and social activities to reduce salt intake is beneficial for healthy longetivity. The pathophysiology of salt-induced hypertension has been studied and innapropriate handling of salt in the kidney, vascular reaction to the salt and recently interstitial accumulation of sodium are proposed mechanism. In addition to raise blood pressure, too much salt may cause systemic effect such as regulation of immune responses. Not all patients are salt-sensitive in terms of blood pressure and we do not know if there are salt-sensitive and resistant in other physiological effect of salt or not. It is required to develop the diagnositic tools to find who is salt-sensitive and tools to keep patients under appropriate salt intake chronically. In this review, I am going to discuss salt-sensitivity, pathological effect of high salt intake and possible markers for salt-sensitive hypertension.