The protective effect of Bergamot Polyphenolic Fraction on reno-cardiac damage induced by DOCA-salt and unilateral renal artery ligation in rats.

To date, the complex pathological interactions between renal and cardiovascular systems represent a real global epidemic in both developed and developing countries. In this context, renovascular hypertension (RVH) remains among the most prevalent, but also potentially reversible, risk factor for numerous reno-cardiac diseases in humans and pets. Here, we investigated the anti-inflammatory and reno-cardiac protective effects of a polyphenol-rich fraction of bergamot (BPF) in an experimental model of hypertension induced by unilateral renal artery ligation. Adult male Wistar rats underwent unilateral renal artery ligation and treatment with deoxycorticosterone acetate (DOCA) (20 mg/kg, s.c.), twice a week for a period of 4 weeks, and 1% sodium chloride (NaCl) water (n = 10). A subgroup of hypertensive rats received BPF (100 mg/kg/day for 28 consecutive days, n = 10) by gavage. Another group of animals was treated with a sub-cutaneous injection of vehicle (that served as control, n = 8). Unilateral renal artery ligation followed by treatment with DOCA and 1% NaCl water resulted in a significant increase in mean arterial blood pressure (MAP; p< 0.05. vs CTRL) which strongly increased the resistive index (RI; p<0.05 vs CTRL) of contralateral renal artery flow and kidney volume after 4 weeks (p<0.001 vs CTRL). Renal dysfunction also led to a dysfunction of cardiac tissue strain associated with overt dyssynchrony in cardiac wall motion when compared to CTRL group, as shown by the increased time-to-peak (T2P; p<0.05) and the decreased whole peak capacity (Pk; p<0.01) in displacement and strain rate (p<0.05, respectively) in longitudinal motion. Consequently, the hearts of RAL DOCA-Salt rats showed a larger time delay between the fastest and the lowest region (Maximum Opposite Wall Delay-MOWD) when compared to CTRL group (p<0.05 in displacement and p <0.01 in strain rate). Furthermore, a significant increase in the levels of the circulating pro-inflammatory cytokines and chemokines (p< 0.05 for IL-12(40), p< 0.01 for GM-CSF, KC, IL-13, and TNF- α) and in the NGAL expression of the ligated kidney (p< 0.001) was observed compared to CTRL group. Interestingly, this pathological condition is prevented by BPF treatment. In particular, BPF treatment prevents the increase of blood pressure in RAL DOCA-Salt rats (p< 0.05) and exerts a protective effect on the volume of the contralateral kidney (p <0.01). Moreover, BPF ameliorates cardiac tissue strain dysfunction by increasing Pk in displacement (p <0.01) and reducing the T2P in strain rate motion (p<0.05). These latter effects significantly improve MOWD (p <0.05) preventing the overt dyssynchrony in cardiac wall motion. Finally, the reno-cardiac protective effect of BPF was associated with a significant reduction in serum level of some pro-inflammatory cytokines and chemokines (p<0.05 for KC and IL-12(40), p<0.01 for GM-CSF, IL-13, and TNF- α) restoring physiological levels of renal neutrophil gelatinase-associated lipocalin (NGAL, p<0.05) protein of the tethered kidney. In conclusion, the present results show, for the first time, that BPF promotes an efficient renovascular protection preventing the progression of inflammation and reno-cardiac damage. Overall, these data point to a potential clinical and veterinary role of dietary supplementation with the polyphenol-rich fraction of citrus bergamot in counteracting hypertension-induced reno-cardiac syndrome.

Elucidation of Anti-Hypertensive Mechanism by a Novel Lactobacillus rhamnosus AC1 Fermented Soymilk in the Deoxycorticosterone Acetate-Salt Hypertensive Rats.

Dietary intake of fermented soymilk is associated with hypotensive effects, but the mechanisms involved have not been fully elucidated. We investigated the anti-hypertensive effects of soymilk fermented by L. rhamnosus AC1 on DOCA-salt hypertension from the point of view of oxidative stress, inflammatory response and alteration of the gut microbiome. The antioxidant assays in vitro indicated the ethanol extract (EE) of L. rhamnosus AC1 fermented soymilk showed better antioxidative effects than the water extract (WE). Those extracts displayed a hypotensive effect using a tail-cuff approach to measuring blood pressure and improved nitric oxide (NO), angiotensin II (Ang II), tumor necrosis factor-α (TNF-α) and interleukin factor-6 (IL-6) on DOCA-salt hypertensive rats. Furthermore, cardiac and renal fibrosis were attenuated by those extracts. The gut microbiota analysis revealed that they significantly reduced the abundance of phylum Proteobacteria, its family Enterobacteriaceae and genus Escherichia-Shigella. Moreover, metabolomic profiling revealed several potential gut microbiota-related metabolites which appeared to involve in the development and recovery of hypertension. In conclusion, fermented soymilk is a promising nutritional intervention strategy to improve hypertension via reducing inflammation and reverting dysbiotic microbiota.

Secreted protein acidic and rich in cysteine (SPARC) and a disintegrin and metalloproteinase with thrombospondin type 1 motif (ADAMTS1) increments by the renin-angiotensin system induce renal fibrosis in deoxycorticosterone acetate-salt hypertensive rats.

Secreted protein acidic and rich in cysteine (SPARC), an extracellular matrix (ECM) protein, was recently shown to induce collagen deposition through the production of a disintegrin and metalloproteinase with thrombospondin type 1 motif (ADAMTS1) in the aging heart. ADAMTS1 regulates ECM turnover by degrading ECM components, and its excessive activation contributes to various pathological states, including fibrosis. The present study investigated the pathophysiological regulation and role of SPARC and ADAMTS1 in renal fibrosis using uninephrectomized rats treated with deoxycorticosterone acetate (DOCA, 40 mg/kg/week, subcutaneously) and salt (1% in drinking water). The administration of DOCA and salt gradually and significantly elevated systolic blood pressure during the 3-week treatment period, induced proteinuria, decreased creatinine clearance, and increased NADPH oxidase-derived superoxide production, malondialdehyde concentrations, and monocyte chemoattractant protein-1 and osteopontin expression in the kidneys. Glomerulosclerosis, fibrillar collagen deposition, and transforming growth factor-ß expression increased in a time-dependent manner, and SPARC and ADAMTS1 expression showed a similar pattern to these changes. The angiotensin II type-1 receptor blocker losartan suppressed the overexpression of SPARC and ADAMTS1, and an in vitro exposure to angiotensin II induced the production of both SPARC and ADAMTS1 in renal fibroblast NRK-49F cells. Knockdown of the SPARC gene with small interfering RNA reduced all forms (the 110-kDa latent and 87- and 65-kDa bioactive forms) of ADAMTS1 expression as well as collagen production. These results suggest that SPARC is induced by the renin-angiotensin system and may be a fibrogenic factor, at least in part, by producing ADAMTS1 in hypertensive renal disease.

Molecular Targeting of VEGF with a Suramin Fragment-DOCA Conjugate by Mimicking the Action of Low Molecular Weight Heparins.

Molecular targeting of growth factors has shown great therapeutic potential in pharmaceutical research due to their roles in pathological conditions. In the present study, we developed a novel suramin fragment and deoxycholic acid conjugate (SFD) that exhibited the potential to bind to the heparin-binding site (HBD) of vascular endothelial growth factor (VEGF) and to inhibit its pathogenic action for the first time. Notably, SFD was optimally designed for binding to the HBD of VEGF using the naphthalenetrisulfonate group, allowing to observe its excellent binding efficacy in a surface plasmon resonance (SPR) study, showing remarkable binding affinity (KD = 3.8 nM) as a small molecule inhibitor. In the tubular formation assay, it was observed that SFD could bind to HBD and exhibit antiangiogenic efficacy by inhibiting VEGF, such as heparins. The cellular treatment of SFD resulted in VEGF-inhibitory effects in human umbilical vein endothelial cells (HUVECs). Therefore, we propose that SFD can be employed as a novel drug candidate to inhibit the pathophysiological action of VEGF in diseases. Consequently, SFD, which has a molecular structure optimized for binding to HBD, is put forward as a new chemical VEGF inhibitor.

Metformin improves diastolic function in an HFpEF-like mouse model by increasing titin compliance.

Heart failure with preserved ejection fraction (HFpEF) is a complex syndrome characterized by a preserved ejection fraction but increased diastolic stiffness and abnormalities of filling. Although the prevalence of HFpEF is high and continues to rise, no effective therapies exist; however, the diabetic drug metformin has been associated with improved diastolic function in diabetic patients. Here we determine the therapeutic potential of metformin for improving diastolic function in a mouse model with HFpEF-like symptoms. We combine transverse aortic constriction (TAC) surgery with deoxycorticosterone acetate (DOCA) supplementation to obtain a mouse model with increased diastolic stiffness and exercise intolerance. Echocardiography and pressure-volume analysis reveal that providing metformin to TAC/DOCA mice improves diastolic function in the left ventricular (LV) chamber. Muscle mechanics show that metformin lowers passive stiffness of the LV wall muscle. Concomitant with this improvement in diastolic function, metformin-treated TAC/DOCA mice also demonstrate preserved exercise capacity. No metformin effects are seen in sham operated mice. Extraction experiments on skinned ventricular muscle strips show that the metformin-induced reduction of passive stiffness in TAC/DOCA mice is due to an increase in titin compliance. Using phospho-site-specific antibodies, we assay the phosphorylation of titin's PEVK and N2B spring elements. Metformin-treated mice have unaltered PEVK phosphorylation but increased phosphorylation of PKA sites in the N2B element, a change which has previously been shown to lower titin's stiffness. Consistent with this result, experiments with a mouse model deficient in the N2B element reveal that the beneficial effect of metformin on LV chamber and muscle stiffness requires the presence of the N2B element. We conclude that metformin offers therapeutic benefit during HFpEF by lowering titin-based passive stiffness.

The effects of LXR agonist GW3965 on vascular reactivity and inflammation in hypertensive rat aorta.

AIMS: Liver X receptors (LXRs) play an important role in the regulation of cholesterol, fatty acid and glucose metabolisms together with inflammatory processes. In the present study, the effects of LXR agonist GW3965 on vascular reactivity and expression of functional proteins in DOCA-Salt induced hypertension were examined. MAIN METHODS: Hypertension was induced through unilateral nephrectomy and deoxycorticosterone-acetate (DOCA) injection (20 mg/kg, twice a week) for 6 weeks in male Wistar albino rats (8 weeks old). An LXR agonist GW3965 (10 mg/kg/day, i.p.) was administered to animals for last seven days. KEY FINDINGS: GW3965 treatment reduced systolic blood pressures in hypertensive rats. Acetylcholine-induced endothelium-dependent and sodium nitroprusside-induced endothelium-independent vasorelaxations were decreased in hypertensive rats but not affected by GW3965. GW3965 treatment enhanced plasma nitrite levels in normotensive rats. KCl and phenylephrine (Phe)-induced vasocontractions were reduced in hypertensive groups and increased with GW3965 treatment. Decreased sarco/endoplasmic reticulum Ca2+-ATPase2 (SERCA2) expression in the hypertensive aorta was not changed by GW3965 treatment. Expression of inositoltrisphosphate receptor1 (IP3R1) was increased by GW3965 in normotensive animals. The nuclear factor kappaB (NF-κB) and tumor necrosis factor alpha (TNF-α) expressions were increased in hypertensive rats and reduced by GW3965 treatment. SIGNIFICANCE: The results of study indicate that the LXR agonist, GW3965, exhibited a beneficial effect on increased blood pressure and improved hypertension-induced impairment in contractile activity of vessel and inflammatory markers in vascular tissue. Therefore, these effects of LXR agonists on vessel should be taken into account in experimental or therapeutic approaches to hypertension.

Anakinra reduces blood pressure and renal fibrosis in one kidney/DOCA/salt-induced hypertension.

OBJECTIVE: To determine whether a clinically-utilised IL-1 receptor antagonist, anakinra, reduces renal inflammation, structural damage and blood pressure (BP) in mice with established hypertension. METHODS: Hypertension was induced in male mice by uninephrectomy, deoxycorticosterone acetate (2.4mg/d,s.c.) and replacement of drinking water with saline (1K/DOCA/salt). Control mice received uninephrectomy, a placebo pellet and normal drinking water. 10days post-surgery, mice commenced treatment with anakinra (75mg/kg/d, i.p.) or vehicle (0.9% saline, i.p.) for 11days. Systolic BP was measured by tail cuff while qPCR, immunohistochemistry and flow cytometry were used to measure inflammatory markers, collagen and immune cell infiltration in the kidneys. RESULTS: By 10days post-surgery, 1K/DOCA/salt-treated mice displayed elevated systolic BP (148.3±2.4mmHg) compared to control mice (121.7±2.7mmHg; n=18, P<0.0001). The intervention with anakinra reduced BP in 1K/DOCA/salt-treated mice by ∼20mmHg (n=16, P<0.05), but had no effect in controls. In 1K/DOCA/salt-treated mice, anakinra modestly reduced (∼30%) renal expression of some (CCL5, CCL2; n=7-8; P<0.05) but not all (ICAM-1, IL-6) inflammatory markers, and had no effect on immune cell infiltration (n=7-8, P>0.05). Anakinra reduced renal collagen content (n=6, P<0.01) but paradoxically appeared to exacerbate the renal and glomerular hypertrophy (n=8-9, P<0.001) that accompanied 1K/DOCA/salt-induced hypertension. CONCLUSION: Despite its anti-hypertensive and renal anti-fibrotic actions, anakinra had minimal effects on inflammation and leukocyte infiltration in mice with 1K/DOCA/salt-induced hypertension. Future studies will assess whether the anti-hypertensive actions of anakinra are mediated by protective actions in other BP-regulating or salt-handling organs such as the arteries, skin and brain.

Protective effects of MCR-1329, a dual α1 and angII receptor antagonist, in mineralocorticoid-induced hypertension.

BACKGROUND: With the prototypical structures of losartan and prazosin as the axis of our research, MCR-1329 emerged as a potential designed multiple ligand from a series of compounds designed to possess dual antagonistic activity on the α1 and AT1 receptor. After confirming the activity of MCR-1329 in in vitro and acute in vivo models, the present study was undertaken to determine the efficacy of MCR-1329 in a mammalian test system. METHODS: A rat model of deoxycorticosterone acetate (DOCA)-salt induced renal hypertension following unilateral nephrectomy was utilized to determine the effect of MCR-1329. For mechanistic evaluations, MCR-1329 was evaluated on rat aortic strips in vitro and on rat aortic smooth muscle cells to determine the role of MCR-1329 on phosphoinositide 3 kinase (PI3K) signaling. RESULTS: Results of the study showed that MCR-1329 prevents development of arterial hypertension. It was also observed that MCR-1329 upheld the intimal structures of major arteries like the thoracic aorta. Acetylcholine (Ach)-mediated relaxation remained intact in arteries from MCR-1329 treated animals. It was observed that MCR-1329 partially prevents Thr-308 phosphorylation of Akt following ligand-mediated receptor stimulation in vascular smooth muscle cells. Addition of LY294002 to the reaction medium caused a near-complete inhibition of Akt-phosphorylation. This suggested that MCR-1329 elicits its antihypertensive role by blocking activation of receptor-mediated PI3K-Akt downstream signaling as well as through preservation of arterial integrity. CONCLUSIONS: MCR-1329 has the potential to be evaluated further for clinical development as a potential antihypertensive agent with multiple mechanisms of action.

PGC-1α overexpression suppresses blood pressure elevation in DOCA-salt hypertensive mice.

Increasing evidences have accumulated that endothelial dysfunction is involved in the pathogenesis of hypertension. Peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) has been identified as an essential factor that protects against endothelial dysfunction in vascular pathologies. However, the functional role of PGC-1α in hypertension is not well understood. Using an adenovirus infection model, we tested the hypothesis that PGC-1α overexpression retards the progression of hypertension in deoxycorticosterone acetate (DOCA)-salt mice model through preservation of the function of endothelium. We first demonstrated that PGC-1α expression not only in conductance and resistance arteries but also in endothelial cells was decreased after DOCA-salt treatment. In PGC-1α adenovirus-infected mice, the elevation of blood pressure in DOCA-salt mice was attenuated, as determined using tail-cuff measurement. Furthermore, PGC-1α overexpression inhibited the decrease in nitric oxide (NO) generation and the increase in superoxide anion (O2 (-)) production in DOCA-salt-treated mice, in parallel with improved endothelium-dependent relaxation. Rather than affecting endothelial NO synthase (eNOS) total expression and phosphorylation, PGC-1α significantly inhibited eNOS uncoupling, as evidenced by increased eNOS homodimerization, BH4 levels, GTP-cyclohydrolase 1 (GTPCH1) and dihydrofolate reductase (DHFR) expression and heat-shock protein (Hsp)90-eNOS interaction. Our findings demonstrate that PGC-1α overexpression preserves eNOS coupling, enhances NO generation, improves endothelium-dependent relaxation and thus lowers blood pressure, suggesting that up-regulation of PGC-1α may be a novel strategy to prevent and treat hypertension.

Brain endoplasmic reticulum stress mechanistically distinguishes the saline-intake and hypertensive response to deoxycorticosterone acetate-salt.

Endoplasmic reticulum stress has become an important mechanism in hypertension. We examined the role of endoplasmic reticulum stress in mediating the increased saline-intake and hypertensive effects in response to deoxycorticosterone acetate (DOCA)-salt. Intracerebroventricular delivery of the endoplasmic reticulum stress-reducing chemical chaperone tauroursodeoxycholic acid did not affect the magnitude of hypertension, but markedly decreased saline-intake in response to DOCA-salt. Increased saline-intake returned after tauroursodeoxycholic acid was terminated. Decreased saline-intake was also observed after intracerebroventricular infusion of 4-phenylbutyrate, another chemical chaperone. Immunoreactivity to CCAAT homologous binding protein, a marker of irremediable endoplasmic reticulum stress, was increased in the subfornical organ and supraoptic nucleus of DOCA-salt mice, but the signal was absent in control and CCAAT homologous binding protein-deficient mice. Electron microscopy revealed abnormalities in endoplasmic reticulum structure (decrease in membrane length, swollen membranes, and decreased ribosome numbers) in the subfornical organ consistent with endoplasmic reticulum stress. Subfornical organ-targeted adenoviral delivery of GRP78, a resident endoplasmic reticulum chaperone, decreased DOCA-salt-induced saline-intake. The increase in saline-intake in response to DOCA-salt was blunted in CCAAT homologous binding protein-deficient mice, but these mice exhibited a normal hypertensive response. We conclude that (1) brain endoplasmic reticulum stress mediates the saline-intake, but not blood pressure response to DOCA-salt, (2) DOCA-salt causes endoplasmic reticulum stress in the subfornical organ, which when attenuated by GRP78 blunts saline-intake, and (3) CCAAT homologous binding protein may play a functional role in DOCA-salt-induced saline-intake. The results suggest a mechanistic distinction between the importance of endoplasmic reticulum stress in mediating effects of DOCA-salt on saline-intake and blood pressure.

Neuron-specific (pro)renin receptor knockout prevents the development of salt-sensitive hypertension.

The (pro)renin receptor (PRR), which binds both renin and prorenin, is a newly discovered component of the renin-angiotensin system that is highly expressed in the central nervous system. The significance of brain PRRs in mediating local angiotensin II formation and regulating blood pressure remains unclear. The current study was performed to test the hypothesis that PRR-mediated, nonproteolytic activation of prorenin is the main source of angiotensin II in the brain. Thus, PRR knockout in the brain is expected to prevent angiotensin II formation and development of deoxycorticosterone acetate-salt-induced hypertension. A neuron-specific PRR (ATP6AP2) knockout mouse model was generated using the Cre-LoxP system. Physiological parameters were recorded by telemetry. PRR expression, detected by immunostaining and reverse transcription-polymerase chain reaction, was significantly decreased in the brains of knockout mice compared with wild-type mice. Intracerebroventricular infusion of mouse prorenin increased blood pressure and angiotensin II formation in wild-type mice. This hypertensive response was abolished in PRR-knockout mice in association with a reduction in angiotensin II levels. Deoxycorticosterone acetate-salt increased PRR expression and angiotensin II formation in the brains of wild-type mice, an effect that was attenuated in PRR-knockout mice. PRR knockout in neurons prevented the development of deoxycorticosterone acetate-salt-induced hypertension as well as activation of cardiac and vasomotor sympathetic tone. In conclusion, nonproteolytic activation of prorenin through binding to the PRR mediates angiotensin II formation in the brain. Neuron-specific PRR knockout prevents the development of deoxycorticosterone acetate-salt-induced hypertension, possibly through diminished angiotensin II formation.