A novel acetyltransferase p300 inhibitor ameliorates hypertension-associated cardio-renal fibrosis

Hypertension-associated end-organ damage commonly leads to cardiac and renal fibrosis. As no effective anti-fibrotic therapy currently exists, the unchecked progression of fibrogenesis manifests as cardio-renal failure and early death. We have previously shown that FATp300—p300 with intrinsic factor acetyltransferase activity—is an essential epigenetic regulator of fibrogenesis, and is elevated in several fibrotic tissues. In this report, we investigate the therapeutic efficacy of a novel FATp300 inhibitor, L002, in a murine model of hypertensive cardio-renal fibrosis. Additionally, we examine the effects of L002 on cellular pro-fibrogenic processes and provide mechanistic insights into its antifibrogenic action. Utilizing cardiac fibroblasts, podocytes, and mesangial cells, we demonstrate that L002 blunts FATp300-mediated acetylation of specific histones. Further, incubating cells with L002 suppresses several pro-fibrogenic processes including cellular proliferation, migration, myofibroblast differentiation and collagen synthesis. Importantly, systemic administration of L002 in mice reduces hypertension-associated pathological hypertrophy, cardiac fibrosis and renal fibrosis. The anti-hypertrophic and anti-fibrotic effects of L002 were independent of blood pressure regulation. Our work solidifies the role of epigenetic regulator FATp300 in fibrogenesis and establishes it as a pharmacological target for reducing pathological matrix remodeling and associated pathologies. Additionally, we discover a new therapeutic role of L002, as it ameliorates hypertension-induced cardio-renal fibrosis and antagonizes pro-fibrogenic responses in fibroblasts, podocytes and mesangial cells.     

Vesicle-associated membrane protein-2 (VAMP2) mediates cAMP-stimulated renin release in mouse juxtaglomerular cells

Renin is essential for blood pressure control. Renin is stored in granules in juxtaglomerular (JG) cells, located in the pole of the renal afferent arterioles. The second messenger cAMP stimulates renin release. However, it is unclear whether fusion and exocytosis of renin-containing granules is involved. In addition, the role of the fusion proteins, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment proteins), in renin release from JG cells has not been studied. The vesicle SNARE proteins VAMP2 (vesicle associated membrane protein 2) and VAMP3 mediate cAMP-stimulated exocytosis in other endocrine cells. Thus, we hypothesized that VAMP2 and/or -3 mediate cAMP-stimulated renin release from JG cells. By fluorescence-activated cell sorting, we isolated JG cells expressing green fluorescent protein and compared the relative abundance of VAMP2/3 in JG cells versus total mouse kidney mRNA by quantitative PCR. We found that VAMP2 and VAMP3 mRNA are expressed and enriched in JG cells. Confocal imaging of primary cultures of JG cells showed that VAMP2 (but not VAMP3) co-localized with renin-containing granules. Cleavage of VAMP2 and VAMP3 with tetanus toxin blocked cAMP-stimulated renin release from JG cells by ~50% and impaired cAMP-stimulated exocytosis by ~50%, as monitored with FM1-43. Then we specifically knocked down VAMP2 or VAMP3 by adenoviral-mediated delivery of short hairpin silencing RNA. We found that silencing VAMP2 blocked cAMP-induced renin release by ~50%. In contrast, silencing VAMP3 had no effect on basal or cAMP-stimulated renin release. We conclude that VAMP2 and VAMP3 are expressed in JG cells, but only VAMP2 is targeted to renin-containing granules and mediates the stimulatory effect of cAMP on renin exocytosis.     

Vascular mechanisms involved in angiotensin II-induced venoconstriction in hypertensive rats

To investigate the venoconstrictor effect of angiotensin II (Ang II) in spontaneously hypertensive rats (SHR), we used preparations of mesenteric venular beds and the circular muscle of the portal veins. Vessels were tested with Ang II in the presence or absence of losartan, PD 123319, HOE 140, L-NAME, indomethacin, or celecoxib. In the mesenteric venular bed of SHR, the effect of Ang II (0.1 nmol) was nearly abolished by losartan and enhanced by HOE 140, indomethacin, and celecoxib, while PD123319 and L-NAME had no effect. In portal vein preparations, cumulative-concentration response curves (CCRC) to Ang II (0.1–100 nmol/L) exhibited a lower maximal response (E_max) in SHR compared to Wistar rats. AT₁ receptor expression was similar in the two strains, while AT₂ receptor levels were lower in SHR portal veins when compared to Wistar. In SHR portal veins, losartan shifted the CCRC to Ang II to the right, while indomethacin and HOE 140 increased the E_max to Ang II. PD 123319, celecoxib, and L-NAME had no effect. Taken together, our results suggest that Ang II-induced venoconstriction in SHR is mediated by activation of AT₁ receptors and this effect may be counterbalanced by kinin B₂ receptor and COX metabolites. Furthermore, our data indicate that there are different cellular and molecular mechanisms involved in the regulation of venous tonus of normotensive and hypertensive rats. These differences probably reflect distinct factors that influence arterial and venous bed in hypertension.     

Regulation of with‐no‐lysine kinase signaling by Kelch‐like proteins

In 2001, with‐no‐lysine (WNK) kinases were identified as the genes responsible for the human hereditary hypertensive disease pseudohypoaldosteronism type II (PHAII). It took a further 6 years to clarify that WNK kinases participate in a signaling cascade with oxidative stress‐responsive gene 1 (OSR1), Ste20‐related proline‐alanine‐rich kinase (SPAK), and thiazide‐sensitive NaCl cotransporter (NCC) in the kidney and the constitutive activation of this signaling cascade is the molecular basis of PHAII. Since this discovery, the WNK–OSR1/SPAK–NCC signaling cascade has been shown to be involved not only in PHAII but also in the regulation of blood pressure under normal and pathogenic conditions, such as hyperinsulinemia. However, the molecular mechanisms of WNK kinase regulation by dietary and hormonal factors and by PHAII‐causing mutations remain poorly understood. In 2012, two additional genes responsible for PHAII, Kelch‐like 3 (KLHL3) and Cullin3, were identified. At the time of their discovery, the molecular mechanisms underlying the interaction between these genes and their involvement in PHAII were unknown. Here we review the pathophysiological roles of the WNK signaling cascade clarified to date and introduce a new mechanism of WNK kinase regulation by KLHL3 and Cullin3, which provides insight on previously unknown mechanisms of WNK kinase regulation.     

NEDD4L基因变异体与内蒙古地区蒙古族人群中高血压 的相关分析

目的：探讨内蒙古地区蒙古族人群中NEDD4L基因多态性位点rs4149601(G/A)突变与高血压的相关性。方法：应用病例对 照方法研究包头地区蒙古族高血压病个体308 例及蒙古族血压正常个体454 例。检测所有个体舒张压,收缩压。使用TaqMan PCR技术进行rs4149601 多态基因分型。结果：rs4149601 多态基因型及等位基因分布在GG 基因型、GA 基因型、AA 等位基因的 频率在高血压组及对照组分别为54.7%,92.8%;11.4%及56.2%;71.4%,7.9%。rs4149601 多态基因型及等位基因分布与对照组差 异有显著性。应用多元logistic 回归分析对性别、年龄进行校正后发现rs4149601 多态基因与高血压病患病风险相关。结论：上皮 细胞钠通道亚单位基因多态性(rs4149601)同内蒙古地区蒙古族人群高血压病发病有关。     

超声心动图对高血压心室肥厚患者左心功能的评价及意义

目的:本研究利用超声心动图检测高血压心室肥厚患者左心房结构,探讨当左心结构发生变化时心脏功能所受到的影响,为高血压及其并发症的临床诊断提供检测及诊断参考。方法:选取2011年5月-2013年1月在我院接受检查的高血压心室肥厚患者76例作为观察组,另选取同期经体检的健康人群60例为健康对照组,利用超声心动图观察左心功能和结构,比较两组研究对象的左心房内径(LAD)、心肌质量(LVMM)、舒张末容积(LVEDV)、收缩末容积(LVESV)、左心室射血分数(LVEF)及二尖瓣口舒张末期流速比值(E/A)。结果:两组间心室收缩功能无显著性差异(P0.05);高血压组LAD高于对照组,LVEF及E/A低于对照组,差异具有统计学意义(P0.05);高血压Ⅰ期、Ⅱ期、Ⅲ期患者间比较,左房内径随血压的升高逐渐递增,而左心室射血分数和二尖瓣口舒张期流速比值则逐渐递减,差异具有统计学意义(P0.05)。结论:超声心动图可以直观的显示高血压心室肥厚患者左心功能及血流动力学的变化,对临床诊断具有积极的意义。     

缬沙坦联合吲达帕胺治疗高血压的效果观察

目的:探讨缬沙坦和吲达帕胺治疗高血压的临床效果,为临床治疗提供可借鉴的方法。方法:选取2012年4月-2014年1月在我院接受治疗的87例高血压患者的临床资料,根据治疗方式的不同将所选患者分为缬沙坦组、吲达帕胺组和联合用药组,每组27例。缬沙坦组患者采用口服缬沙坦单药治疗,吲达帕胺组患者采用口服吲达帕胺单药治疗,联合用药组采用缬沙坦+吲达帕胺缓释片治疗。观察三组患者治疗前后的血压变化、治疗总有效率及不良反应的发生情况。结果:治疗后,三组患者的血压均不同程度降低(P0.05);联合用药组患者血压下降幅度明显高于缬沙坦单药治疗组和吲达帕胺单药治疗组,差异具有统计学意义(P0.05)。联合用药组患者治疗的总有效率明显高于缬沙坦单药治疗组和吲达帕胺单药治疗组,差异具有统计学意义(P0.05)。三组患者不良反应的发生率无显著差异(P0.05)。结论:缬沙坦胶囊与吲哒帕胺缓释片联合应用治疗高血压具有显著的临床意义,能够有效的控制患者的血压,值得推广采用。     

老年高血压合并2 型糖尿病患者血清同型半胱氨酸、血尿酸水平 变化及临床意义

目的：探讨老年高血压合并2 型糖尿病患者血清同型半胱氨酸（Homocysteine, Hcy）、血尿酸（Serum uric acid, SUA）水平变 化及其临床意义。方法：2012 年9 月至2013 年9 月期间,我院诊治的40 例单纯高血压和40 例高血压合并2 型糖尿病患者,分别 作为对照组和研究组,检测两组血清Hcy、SUA水平。结果：两组患者收缩压、舒张压比较无统计学差异（P>0.05）。研究组空腹血 糖、餐后2h 血糖、血清Hcy、SUA 均显著高于对照组（P<0.05）。研究组中血管并发症患者血清Hcy、SUA为（25.0± 5.0）umol/L 和 （390.0± 65.0）mmol/L显著高于无血管并发症患者（17.0± 4.0）umol/L 和（330.0± 55.0）mmol/L,血管并发症患者FBG、餐后2h 血 糖与无血管并发症患者比较无统计学差异（P>0.05）。结论：高血压合并2 型糖尿病患者血清Hcy、SUA异常升高,且存在慢性血 管并发症患者两者水平更高,血清Hcy、SUA是老年高血压合并2 型糖尿病的危险因素。     

Hsp70 regulation on Nox4/p22phox and cytoskeletal integrity as an effect of losartan in vascular smooth muscle cells

A series of signaling cascades are activated after angiotensin II binds to angiotensin II type I receptor (AT₁R), a peptide that is an important mediator of oxidative stress. Hsp70 regulates a diverse set of signaling pathways through interactions with proteins. Here, we tested the hypothesis of angiotensin II AT₁R inhibition effect on Hsp70 interaction with Nox4/p22phox complex and Hsp70 leading to actin cytoskeleton modulation in spontaneously hypertensive rats (SHR) vascular smooth muscle cells (VSMCs). SHR and Wistar–Kyotto rats (VSMCs from 8 to 10 weeks) were stimulated with angiotensin II (100 nmol/L) for 15 min (AII), treated with losartan (100 nmol/L) for 90 min (L), and with losartan for 90 min plus angiotensin in the last 15 min (L + AII). Whereas SHR VSMCs exposure to angiotensin II overexpressed AT₁R and Nox4 nicotinamide–adenine dinucleotide phosphate (NADPH) oxidase and slightly downregulated caveolin-1 expression, losartan decreased AT₁R protein levels and increased caveolin-1 and Hsp70 expression in SHR VSMC membranes. Immunoprecipitation and immunofluorescence confocal microscopy proved interaction and colocalization of membrane translocated Hsp70 and Nox4/p22phox. Increased levels of Hsp70 contrast with the decreased immunoprecipitation of Nox4/p22phox and RhoA in membranes from SHR VSMCs (L) vs SHR VSMCs (AII). Hsp72 depletion resulted in higher Nox4 expression and increased NADPH oxidase activity in VSMCs (L + AII) from SHR when contrasted with nontransfected VSMCs (L + AII). After Hsp72 knockdown in SHR VSMCs, losartan could not impair angiotensin II-enhanced stress fiber formation and focal adhesion assembly. In conclusion, our data showing a negative regulation of Hsp70 on Nox4/p22phox demonstrates a possible mechanism in explaining the antioxidative function joined to cytoskeletal integrity modulation within the effects of losartan in VSMCs from SHR.