Shengyu Mu

Epigenetic modulation of the renal β-adrenergic–WNK4 pathway in salt-sensitive hypertension

How high salt intake increases blood pressure is a key question in the study of hypertension. Salt intake induces increased renal sympathetic activity resulting in sodium retention. However, the mechanisms underlying the sympathetic control of renal sodium excretion remain unclear. In this study, we found that β2-adrenergic receptor (β2AR) stimulation led to decreased transcription of the gene encoding WNK4, a regulator of sodium reabsorption. β2AR stimulation resulted in cyclic AMP-dependent inhibition of histone deacetylase-8 (HDAC8) activity and increased histone acetylation, leading to binding of the glucocorticoid receptor to a negative glucocorticoid−responsive element in the promoter region. In rat models of salt-sensitive hypertension and sympathetic overactivity, salt loading suppressed renal WNK4 expression, activated the Na+-Cl− cotransporter and induced salt-dependent hypertension. These findings implicate the epigenetic modulation of WNK4 transcription in the development of salt-sensitive hypertension. The renal β2AR-WNK4 pathway may be a therapeutic target for salt-sensitive hypertension.

Rac1 GTPase in rodent kidneys is essential for salt-sensitive hypertension via a mineralocorticoid receptor–dependent pathway

Hypertension is a leading contributor to cardiovascular mortality worldwide. Despite this, its underlying mechanism(s) and the role of excess salt in cardiorenal dysfunction are unclear. Previously, we have identified cross-talk between mineralocorticoid receptor (MR), a nuclear transcription factor regulated by the steroid aldosterone, and the small GTPase Rac1, which is implicated in proteinuric kidney disease. We here show that high-salt loading activates Rac1 in the kidneys in rodent models of salt-sensitive hypertension, leading to blood pressure elevation and renal injury via an MR-dependent pathway. We found that a high-salt diet caused renal Rac1 upregulation in salt-sensitive Dahl (Dahl-S) rats and downregulation in salt-insensitive Dahl (Dahl-R) rats. Despite a reduction of serum aldosterone levels, salt-loaded Dahl-S rats showed increased MR signaling in the kidneys, and Rac1 inhibition prevented hypertension and renal damage with MR repression. We further demonstrated in aldosterone-infused rats as well as adrenalectomized Dahl-S rats with aldosterone supplementation that salt-induced Rac1 and aldosterone acted interdependently to cause MR overactivity and hypertension. Finally, we confirmed the key role of Rac1 in modulating salt susceptibility in mice lacking Rho GDP-dissociation inhibitor α. Therefore, our data identify Rac1 as a determinant of salt sensitivity and provide insights into the mechanism of salt-induced hypertension and kidney injury.

Fibroblast growth factor 23 accelerates phosphate-induced vascular calcification in the absence of Klotho deficiency

Fibroblast growth factor 23 (FGF23) is a phosphate-regulating hormone that acts primarily on the kidney and parathyroid. With declining kidney function there is an increase in circulating FGF23 levels, which is associated with vascular calcification and mortality in chronic kidney disease. Whether FGF23 exerts direct effects on vasculature is unclear. We evaluated the expression of Klotho and FGF receptors in rat aortic rings and rat aorta vascular smooth muscle cells maintained in culture by reverse transcription-PCR, western blotting, and immunostaining. Signaling pathways underlying FGF23 effects were assessed by western blotting, and effects of FGF23 on osteogenic markers and phosphate transporters were assessed by real-time reverse transcription-PCR. We detected Klotho and FGFR1 in total aorta but not in vascular smooth muscle cells. FGF23 augmented phosphate-induced vascular calcification in the aortic rings from uremic rats and dose dependently increased ERK1/2 phosphorylation in Klotho-overexpressing but not naive vascular smooth muscle cells. FGF23-induced ERK1/2 phosphorylation was inhibited by SU5402 (FGFR1 inhibitor) and U0126 (MEK inhibitor). FGF23 enhanced phosphate-induced calcification in Klotho-overexpressing vascular smooth muscle cells and increased osteoblastic marker expression, which was inhibited by U0126. In contrast, phosphate transporter expression was not affected by phosphate or FGF23. Thus, FGF23 enhances phosphate-induced vascular calcification by promoting osteoblastic differentiation involving the ERK1/2 pathway.

NADPH oxidase-mediated Rac1 GTP activity is necessary for nongenomic actions of the mineralocorticoid receptor in the CA1 region of the rat hippocampus

Mineralocorticoid receptors (MRs) in the central nervous system play important roles in spatial memory, fear memory, salt sensitivity, and hypertension. Corticosterone binds to MRs to induce presynaptic vesicle release and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor aggregation, which are necessary for induction of long-term potentiation under psychological stress. On the other hand, cognitive dysfunction is an important problem clinically in patients with hypertension, diabetes, and cerebral infarction, and all of these conditions are associated with an increase in reactive oxygen species (ROS) generation. Oxidative stress has been shown to modify the genomic actions of MRs in the peripheral organs; however, there have been no reports until now about the relation between the nongenomic actions of MRs and ROS in the central nervous system. In this study, we investigated the relationship between ROS and the nongenomic actions of MR. We examined the nongenomic actions of MR by measuring the slope of the field excitatory postsynaptic potentials and found that ROS induced an additive increase of these potentials, which was accompanied by Rac1 GTP activation and ERK1/2 phosphorylation. An NADPH oxidase inhibitor, apocynin, blocked the nongenomic actions of MRs. A Rac1 inhibitor, NSC23766, was also found to block synaptic enhancement and ERK1/2 phosphorylation induced by NADPH and corticosterone. We concluded that NADPH oxidase activity and Rac1 GTP activity are indispensable for the nongenomic actions of MRs and that Rac1 GTP activation induces ERK1/2 phosphorylation in the brain.

Oxidative stress augments pulmonary hypertension in chronically hypoxic mice overexpressing the oxidized LDL receptor

Chronic hypoxia is one of the main causes of pulmonary hypertension (PH) associated with ROS production. Lectin-like oxidized low-density lipoprotein receptor (LOX)-1 is known to be an endothelial receptor of oxidized low-density lipoprotein, which is assumed to play a role in the initiation of ROS generation. We investigated the role of LOX-1 and ROS generation in PH and vascular remodeling in LOX-1 transgenic (TG) mice. We maintained 8- to 10-wk-old male LOX-1 TG mice and wild-type (WT) mice in normoxia (room air) or hypoxia (10% O2 chambers) for 3 wk. Right ventricular (RV) systolic pressure (RVSP) was comparable between the two groups under normoxic conditions; however, chronic hypoxia significantly increased RVSP and RV hypertrophy in LOX-1 TG mice compared with WT mice. Medial wall thickness of the pulmonary arteries was significantly greater in LOX-1 TG mice than in WT mice. Furthermore, hypoxia enhanced ROS production and nitrotyrosine expression in LOX-1 TG mice, supporting the observed pathological changes. Administration of the NADPH oxidase inhibitor apocynin caused a significant reduction in PH and vascular remodeling in LOX-1 TG mice. Our results suggest that LOX-1-ROS generation induces the development and progression of PH.

High-salt in addition to high-fat diet may enhance inflammation and fibrosis in liver steatosis induced by oxidative stress and dyslipidemia in mice

BackgroundIt is widely known that salt is an accelerating factor for the progression of metabolic syndrome and causes cardiovascular diseases, most likely due to its pro-oxidant properties. We hypothesized that excessive salt intake also facilitates the development of nonalcoholic steatohepatitis (NASH), which is frequently associated with metabolic syndrome.MethodsWe examined the exacerbating effect of high-salt diet on high-fat diet-induced liver injury in a susceptible model to oxidative stress, apoE knockout and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) transgenic mice.ResultsHigh-salt diet led to NASH in high-fat diet-fed LOX-1 transgenic/apoE knockout mice without affecting high-fat diet-induced dyslipidemia or hepatic triglyceride accumulation. Additionally, a high-salt and high-fat diet stimulated oxidative stress production and inflammatory reaction to a greater extent than did a high-fat diet in the liver of LOX-1 transgenic/apoE knockout mice.ConclusionsWe demonstrated that high-salt diet exacerbated NASH in high-fat diet-fed LOX-1 transgenic /apoE knockout mice and that this effect was associated with the stimulation of oxidative and inflammatory processes; this is the first study to suggest the important role of excessive salt intake in the development of NASH.

The kidney and hypertension: pathogenesis of salt-sensitive hypertension.

Salt-sensitive hypertension is closely related with natriuretic capacity of the kidney. Besides several genome-wide research reported candidate gene or gene polymorphism responsible for salt-sensitive hypertension, recently, several new factors for acquired salt-sensitive hypertension are reported. Among them, we have identified that rac1, a small GTPase, activates mineralocorticoid receptor in aldosterone-independent fashion and induces salt-sensitive hypertension in several rodent model. On the other hand, sympathoactivation in the brain and/or kidney regulate sodium handlings in the kidney. Recently it is reported that oxidative stress in the brain or in the kidney may modulate sympathetic tone. Moreover, we reported that β2 adrenoceptor alters histone acetylation and further regulates sodium resorption at distal tubules via activating glucocorticoid receptor. These regulations are to be confirmed in humans and the future, and may open a new door for diagnosis and treatment of salt-sensitive hypertension or moreover preventing development of salt-sensitive hypertension.

Renal Denervation Improves Cardiac Diastolic Dysfunction by Restoring Serca2a Transcription in Uninephrectomized Rats

Background: The mortality and morbidity of heart failure with preserved ejection fraction has increased. Sarcoplasmic reticulum Ca2+-ATPase type 2a (SERCA2a) regulates cardiac functions, and a reduction in SERCA2a expression has been documented in left ventricular (LV) diastolic dysfunction. By contrast, SERCA2a overexpression improves LV diastolic dysfunction. Thus, transcriptional regulation of SERCA2a may be a new therapeutic target. The aim of this study was to determine whether renal denervation, a treatment for resistant hypertension, is a regulator of SERCA2a transcription in vivo. Methods: Uninephrectomy and 6-week salt loading in three-week-old male Sprague-Dawley rats were used to devise a cardiac diastolic dysfunction model, and mechanical renal denervation was performed. The expression of SERCA2a and related molecules was evaluated with quantitative polymerase chain reaction and western blot analyses. The maximal positive LV pressure development (+dP/dtmax) and time constant at the isovolumic relaxation phase (Tau) were determined with cardiac catheters. Results: Uninephrectomy combined with a high-salt diet significantly reduced the messenger RNA expression and protein abundance of SERCA2a, which were restored by renal denervation. In accordance with changes in SERCA2a transcription, uninephrectomy and the high-salt diet decreased LV diastolic function, which was evaluated by Tau and restored by renal denervation. LV systolic function, measured with +dp/dtmax, was preserved. Renal denervation did not lower blood pressure, urinary protein levels, cardiac hypertrophy, or fibrosis. Conclusions: We found that renal denervation is a regulator of SERCA2a transcription in vivo. Our data may provide new therapeutic insights into LV diastolic dysfunction and warrant further study.

588 STRICT FLUID MANAGEMENT PREVENTED LEFT VENTRICULAR HYPERTROPHY IN LONG-TERM PERITONEAL DIALYSIS PATIENTS

Backgrounds: It is still unclear whether strict salt restriction and blood pressure control could prevent left ventricular hypertrophy (LVH) in peritoneal dialysis (PD) patients with declining residual renal function (RRF). Methods: 45 patients who started PD in our hospital between Jan 2005 and July 2010 were investigated retrospectively. Clinical data of these patients including left ventricular mass index (LVMI) evaluated by echocardiographywere studied from the baseline (after 6 months of beginning PD)and thereafter every 6 months. Results: The patients included 32 males and 13 females (age 58.2 ± 11.6 years, observational period 33.7 ± 16.7 months). At baseline, systemic blood pressure (SBP) was 127.6 ± 18.5 mmHg, prevalence of LVH was 46.6% and LVMI was 115.1 ± 32.5 g/m2. We instructed salt restriction less than 6 g/day in daily clinical practice. At the end of follow-up, RRF declined significantly; weekly renal Kt/V from 1.16 ± 0.49 to 0.58 ± 0.54 (P < 0.0001). However, SBP (133.6 ± 20.3 mmHg, P = 0.14) and LVMI (119.7 ± 30.5 g/m2, P = 0.48) were almost unchanged. Stepwise multiple regression analysis showed that SBP (standard &bgr; = 0.607, P < 0.0001), CTR (standard &bgr; = 0.447, P =0.0002) and intact-PTH (standard &bgr; = 0.240, P = 0.02) were independently associated with LVMI (r2 = 0.616). Conclusions: Our report demonstrates for the first time that we could prevent the development of LVH in PD patients with declining RRF, as long as their fluid and blood pressure were kept under good control.

952 FIBROBLAST GROWTH FACTOR 23 ACCELERATES PHOSPHATE-INDUCED CALCIFICATION OF VASCULAR SMOOTH MUSCLE CELLS

Background: Fibroblast growth factor 23 (FGF23) is a phosphate (P) -regulating hormone. Clinical studies suggest that increased level of FGF23 is associated with vascular calcification among chronic kidney disease patients but its direct effect on vasculature is unknown. Methods: Vascular smooth muscle cells (VSMCs) were extracted from Sprague-Dawley rats. The protein expressions of Klotho, co-receptor essential for FGF23 signalling, in VSMCs and the aorta were analyzed by western blot and immunohistochemistry. ERK 1/2 phosphorylation in normal VSMCs or Klotho-overexpressed VSMCs (Klotho-VSMCs) by recombinant hFGF23 was analyzed by western blot. Calcification of Klotho-VSMCs was induced by high-P medium in the presence or absence of FGF23. The calcium depositions were visualized by calcification staining and quantified by methylxylenol blue method. The mRNA expressions of osteoblastic transcription factors and sodium dependent P transporters were evaluated by real-time RT-PCR. Results: Klotho protein was detected in the medial layer of the aorta but not in the VSMCs. ERK 1/2 phosphorylation by FGF23 was increased in Klotho-VSMCs in a dose dependent manner and inhibited by FGFR1 inhibitor or MEK inhibitor U0126. FGF23 accelerated P-induced calcification of Klotho-VSMCs in a dose dependent manner. P induced Msx2 and osterix expressions were enhanced by FGF23 and U0126 inhibited the additive effect of FGF23 on the expression of these osteoblastic markers. The expressions of Pit1 and Pit 2 were not affected by either increased P level or FGF23. Conclusions: FGF23 enhances P-induced calcification in Klotho-VSMCs by promoting osteoblastic differentiation and its effect might be mediated by the ERK 1/2 pathway.