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 11 days. 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 10 days 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\u3c0.0001). The intervention with anakinra reduced BP in 1K/DOCA/salt-treated mice by approximately 20mmHg (n=16, P\u3c0.05), but had no effect in controls. In 1K/DOCA/salt-treated mice, anakinra modestly reduced ( approximately 30%) renal expression of some (CCL5, CCL2; n=7-8; P\u3c0.05) but not all (ICAM-1, IL-6) inflammatory markers, and had no effect on immune cell infiltration (n=7-8, P \u3e 0.05). Anakinra reduced renal collagen content (n=6, P\u3c0.01) but paradoxically appeared to exacerbate the renal and glomerular hypertrophy (n=8-9, P\u3c0.001) 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

Nitroxyl (HNO) Stimulates Soluble Guanylyl Cyclase to Suppress Cardiomyocyte Hypertrophy and Superoxide Generation

Background: New therapeutic targets for cardiac hypertrophy, an independent risk factor for heart failure and death, are essential. HNO is a novel redox sibling of NON attracting considerable attention for the treatment of cardiovascular disorders, eliciting cGMP-dependent vasodilatation yet cGMP-independent positive inotropy. The impact of HNO on cardiac hypertrophy (which is negatively regulated by cGMP) however has not been investigated. Methods: Neonatal rat cardiomyocytes were incubated with angiotensin II (Ang II) in the presence and absence of the HNO donor Angeli’s salt (sodium trioxodinitrate) or B-type natriuretic peptide, BNP (all 1 mmol/L). Hypertrophic responses and its triggers, as well as cGMP signaling, were determined. Results: We now demonstrate that Angeli’s salt inhibits Ang II-induced hypertrophic responses in cardiomyocytes, including increases in cardiomyocyte size, de novo protein synthesis and b-myosin heavy chain expression. Angeli’s salt also suppresses Ang II induction of key triggers of the cardiomyocyte hypertrophic response, including NADPH oxidase (on both Nox2 expression and superoxide generation), as well as p38 mitogen-activated protein kinase (p38MAPK). The antihypertrophic, superoxide-suppressing and cGMP-elevating effects of Angeli’s salt were mimicked by BNP. We also demonstrate that the effects of Angeli’s salt are specifically mediated by HNO (with no role for NON or nitrite), with subsequent activation of cardiomyocyte soluble guanylyl cyclase (sGC) and cGMP signaling (on both cGMP-dependen

The anti-fibrotic actions of relaxin are mediated through a NO-sGC-cGMP-dependent pathway in renal myofibroblasts<i> in vitro </i>and enhanced by the NO donor, diethylamine NONOate

INTRODUCTION: The anti-fibrotic hormone, relaxin, has been inferred to disrupt TGF-beta1/Smad2 phosphorylation (pSmad2) signal transduction and promote collagen-degrading gelatinase activity via a nitric oxide (NO)-dependent pathway. Here, we determined the extent to which NO, soluble guanylate cyclase (sGC) and cyclic guanosine monophosphate (cGMP) were directly involved in the anti-fibrotic actions of relaxin using a selective NO scavenger and sGC inhibitor, and comparing and combining relaxin’s effects with that of an NO donor. METHODS AND RESULTS: Primary renal cortical myofibroblasts isolated from injured rat kidneys were treated with human recombinant relaxin (RLX; 16.8nM), the NO donor, diethylamine NONOate (DEA/NO; 0.5-5uM) or the combined effects of RLX (16.8nM) and DEA/NO (5uM) over 72 hours. The effects of RLX (16.8nM) and DEA/NO (5uM) were also evaluated in the presence of the NO scavenger, hydroxocobalamin (HXC; 100uM) or sGC inhibitor, ODQ (5uM) over 72 hours. Furthermore, the effects of RLX (30nM), DEA/NO (5uM) and RLX (30nM)+DEA/NO (5uM) on cGMP levels were directly measured, in the presence or absence of ODQ (5uM). Changes in matrix metalloproteinase (MMP)-2, MMP-9 (cell media), pSmad2 and α-smooth muscle actin (α-SMA; a measure myofibroblast differentiation) (cell layer) were assessed by gelatin zymography and Western blotting, respectively. At the highest concentration tested, both RLX and DEA/NO promoted MMP-2 and MMP-9 levels by 25-33%, while inhibiting pSmad2 and α-SMA expression by up to 50% (all p<0.05 vs untreated and vehicle-treated cells). However, 5uM of DEA/NO was required to produce the effects seen with 16.8nM of RLX over 72 hours. The anti-fibrotic effects of RLX or DEA/NO alone were completely abrogated by HXC and ODQ (both p<0.01 vs RLX alone or DEA/NO alone), but were significantly enhanced when added in combination (all p<0.05 vs RLX alone). Additionally, the direct cGMP-promoting effects of RLX, DEA/NO and RLX+DEA/NO (which all increased cGMP levels by 12-16-fold over basal levels; all p<0.01 vs vehicle-treated cells) were significantly inhibited by pre-treatment of ODQ (all p<0.05 vs the respective treatments alone). CONCLUSIONS: These findings confirmed that RLX mediates its TGF-beta1-inhibitory and gelatinase-promoting effects via a NO-sGC-cGMP-dependent pathway, which was additively augmented by co-administration of DEA/NO

Hydrogen sulfide protects endothelial nitric oxide function under conditions of acute oxidative stress in vitro

The aim of this study was to examine the ability of H2S, released from NaHS to protect vascular endothelial function under conditions of acute oxidative stress by scavenging superoxide anions (O2−) and suppressing vascular superoxide anion production. O2− was generated in Krebs' solution by reacting hypoxanthine with xanthine oxidase (Hx-XO) or with the O2− generator pyrogallol to model acute oxidative stress in vitro. O2− generation was measured by lucigenin-enhanced chemiluminescence. Functional responses in mouse aortic rings were assessed using a small vessel myograph. NaHS scavenged O2− in a concentration-dependent manner. Isolated aortic rings exposed to either Hx-XO or pyrogallol displayed significantly attenuated maximum vasorelaxation responses to the endothelium-dependent vasodilator acetylcholine, and significantly reduced NO bioavailability, which was completely reversed if vessels were pre-incubated with NaHS (100 μM). NADPH-stimulated aortic O2− production was significantly attenuated by the NADPH oxidase inhibitor diphenyl iodonium. Prior treatment of vessels with NaHS (100 nM–100 μM; 30 min) inhibited NADPH-stimulated aortic O2− production in a concentration-dependent manner. This effect persisted when NaHS was washed out prior to measuring NADPH-stimulated O2− production. These data show for the first time that NaHS directly scavenges O2− and suppresses vascular NADPH oxidase-derived O2− production in vitro. Furthermore, these properties protect endothelial function and NO bioavailability in an in vitro model of acute oxidative stress. These results suggest that H2S can elicit vasoprotection by both scavenging O2− and by reducing vascular NADPH oxidase-derived O2− production