Calcineurin inhibitor effects on kidney electrolyte handling and blood pressure: tacrolimus versus voclosporin.

BACKGROUND AND HYPOTHESIS: Calcineurin inhibitors affect kidney electrolyte handling and blood pressure through an effect on the distal tubule. The second generation calcineurin inhibitor voclosporin causes hypomagnesemia and hypercalciuria less often than tacrolimus. This suggests different effects on the distal tubule, but this has not yet been investigated experimentally. METHODS: Rats were treated with voclosporin, tacrolimus or vehicle for 28 days. Dosing was based on a pilot experiment to achieve clinically therapeutic concentrations. Drug effects were assessed by electrolyte handling at day 18 and 28, thiazide testing at day 20, telemetric blood pressure recordings, and analysis of mRNA and protein levels of distal tubular transporters at day 28. RESULTS: Compared to vehicle, tacrolimus but not voclosporin significantly increased the fractional excretions of calcium (>4-fold), magnesium and chloride (both 1.5-fold) and caused hypomagnesemia. Tacrolimus but not voclosporin significantly reduced distal tubular transporters at mRNA and/or protein level, including the sodium-chloride cotransporter, transient receptor melastatin 6, transient receptor potential vanilloid 5, cyclin M2, sodium-calcium exchanger and calbindin-D28K. Tacrolimus but not voclosporin reduced the mRNA level and urinary excretion of epidermal growth factor. The saluretic response to hydrochlorothiazide at day 20 was similar in the voclosporin and vehicle groups, whereas it was lower in the tacrolimus group. The phosphorylated form of the sodium-chloride cotransporter was significantly higher at day 28 in rats treated with voclosporin than in those treated with tacrolimus. Tacrolimus transiently increased blood pressure, whereas voclosporin caused a gradual but persistent increase in blood pressure which was further characterized by high renin, normal aldosterone, and low endothelin-1. CONCLUSIONS: In contrast to tacrolimus, voclosporin does not cause hypercalciuria and hypomagnesemia, but similarly causes hypertension. Our data reveal differences between the distal tubular effects of tacrolimus and voclosporin and provide a pathophysiological basis for the clinically observed differences between the two calcineurin inhibitors.

Cyclooxygenase-2 inhibition prevents renal toxicity but not hypertension during sunitinib treatment.

BACKGROUND: Anticancer angiogenesis inhibitors cause hypertension and renal injury. Previously we observed in rats that high-dose aspirin (capable of blocking cyclooxygenase (COX)-1 and-2) was superior to low-dose aspirin (blocking COX-1 only) to prevent these side-effects during treatment with the angiogenesis inhibitor sunitinib, suggesting a role for COX-2. High-dose aspirin additionally prevented the rise in COX-derived prostacyclin (PGI2). Therefore, we studied the preventive effects of selective COX-2 inhibition and the hypothesized contributing role of PGI2 during angiogenesis inhibition. METHODS: Male WKY rats received vehicle, sunitinib ((SU), 14 mg/kg/day) alone or combined with COX-2 inhibition (celecoxib, 10 mg/kg/day) or a PGI2 analogue (iloprost, 100 µg/kg/day) for 8 days (n = 8-9 per group). Mean arterial pressure (MAP) was measured via radiotelemetry, biochemical measurements were performed via ELISA and vascular function was assessed via wire myography. RESULTS: SU increased MAP (17±1mmHg versus 3±1mmHg after vehicle on day 4, P < 0.002), which could not be significantly blunted by celecoxib (+12±3mmHg on day 4, P = 0.247), but was temporarily attenuated by iloprost (treatment days 1 + 2 only). Urinary PGI2 (996 ± 112 versus 51 ± 11ng/24h after vehicle, P < 0.001), but not circulating PGI2 increased during SU, which remained unaffected by celecoxib and iloprost. Celecoxib reduced sunitinib-induced albuminuria (0.36 ± 0.05 versus 0.58 ± 0.05mg/24h after SU, P = 0.005). Wire myography demonstrated increased vasoconstriction to endothelin-1 after SU (Emax P = 0.005 versus vehicle), which remained unaffected by celecoxib or iloprost. CONCLUSION: Selective COX-2 inhibition ameliorates albuminuria during angiogenesis inhibition with sunitinib, which most likely acts independently of PGI2. To combat angiogenesis inhibitor-induced hypertension, dual rather than selective COX-1/2 blockade seems preferential.

Combining renin-angiotensin system blockade and sodium-glucose cotransporter-2 inhibition in experimental diabetes results in synergistic beneficial effects.

BACKGROUND: Sodium-glucose cotransporter-2 (SGLT2) inhibition exerts cardioprotective and renoprotective effects, often on top of renin-angiotensin system (RAS) blockade. We investigated this in diabetic hypertensive (mREN2)27 rats. METHODS: Rats were made diabetic with streptozotocin and treated with vehicle, the angiotensin receptor blocker valsartan, the SGLT2 inhibitor empagliflozin, or their combination. Blood pressure (BP) was measured by telemetry. RESULTS: Diabetes resulted in albuminuria, accompanied by glomerulosclerosis, without a change in glomerular filtration rate. Empagliflozin did not lower BP, while valsartan did, and when combined the BP drop was largest. Only dual blockade reduced cardiac hypertrophy and prevented left ventricular dilatation. Valsartan, but not empagliflozin, increased renin, and the largest renin rise occurred during dual blockade, resulting in plasma angiotensin II [but not angiotensin-(1-7)] upregulation. In contrast, in the kidney, valsartan lowered angiotensin II and angiotensin-(1-7), and empagliflozin did not alter this. Although both valsartan and empagliflozin alone tended to diminish albuminuria, the reduction was significant only when both drugs were combined. This was accompanied by reduced glomerulosclerosis, no change in glomerular filtration rate, and a favorable expression pattern of fibrosis and inflammatory markers (including SGLT2) in the kidney. CONCLUSION: RAS blockade and SGLT2 inhibition display synergistic beneficial effects on BP, kidney injury and cardiac hypertrophy in a rat with hypertension and diabetes. The synergy does not involve upregulation of angiotensin-(1-7), but may relate to direct RAS-independent effects of empagliflozin in the heart and kidney.

Blood pressure-independent renoprotective effects of small interference RNA targeting liver angiotensinogen in experimental diabetes.

BACKGROUND AND PURPOSE: Small interfering RNA (siRNA) targeting liver angiotensinogen lowers blood pressure, but its effects in hypertensive diabetes are unknown. EXPERIMENTAL APPROACH: To address this, TGR (mRen2)27 rats (angiotensin II-dependent hypertension model) were made diabetic with streptozotocin over 18 weeks and treated with either vehicle, angiotensinogen siRNA, the AT1 antagonist valsartan, the ACE inhibitor captopril, valsartan + siRNA or valsartan + captopril for the final 3 weeks. Mean arterial pressure (MAP) was measured via radiotelemetry. KEY RESULTS: MAP before treatment was 153 ± 2 mmHg. Diabetes resulted in albuminuria, accompanied by glomerulosclerosis and podocyte effacement, without a change in glomerular filtration rate. All treatments lowered MAP and cardiac hypertrophy, and the largest drop in MAP was observed with siRNA + valsartan. Treatment with siRNA lowered circulating angiotensinogen by >99%, and the lowest circulating angiotensin II and aldosterone levels occurred in the dual treatment groups. Angiotensinogen siRNA did not affect renal angiotensinogen mRNA expression, confirming its liver-specificity. Furthermore, only siRNA with or without valsartan lowered renal angiotensin I. All treatments lowered renal angiotensin II and the reduction was largest (>95%) in the siRNA + valsartan group. All treatments identically lowered albuminuria, whereas only siRNA with or without valsartan restored podocyte foot processes and reduced glomerulosclerosis. CONCLUSION AND IMPLICATIONS: Angiotensinogen siRNA exerts renoprotection in diabetic TGR (mRen2)27 rats and this relies, at least in part, on the suppression of renal angiotensin II formation from liver-derived angiotensinogen. Clinical trials should now address whether this is also beneficial in human diabetic kidney disease.

Conventional Vasopressor and Vasopressor-Sparing Strategies to Counteract the Blood Pressure-Lowering Effect of Small Interfering RNA Targeting Angiotensinogen.

Background A single dose of small interfering RNA (siRNA) targeting liver angiotensinogen eliminates hepatic angiotensinogen and lowers blood pressure. Angiotensinogen elimination raises concerns for clinical application because an angiotensin rise is needed to maintain perfusion pressure during hypovolemia. Here, we investigated whether conventional vasopressors can raise arterial pressure after angiotensinogen depletion. Methods and Results Spontaneously hypertensive rats on a low-salt diet were treated with siRNA (10 mg/kg fortnightly) for 4 weeks, supplemented during the final 2 weeks with fludrocortisone (6 mg/kg per day), the α-adrenergic agonist midodrine (4 mg/kg per day), or a high-salt diet (all groups n=6-7). Pressor responsiveness to angiotensin II and norepinephrine was assessed before and after siRNA administration. Blood pressure was measured via radiotelemetry. Depletion of liver angiotensinogen by siRNA lowered plasma angiotensinogen concentrations by 99.2±0.1% and mean arterial pressure by 19 mm Hg. siRNA-mediated blood pressure lowering was rapidly reversed by intravenous angiotensin II or norepinephrine, or gradually reversed by fludrocortisone or high salt intake. Midodrine had no effect. Unexpectedly, fludrocortisone partially restored plasma angiotensinogen concentrations in siRNA-treated rats, and nearly abolished plasma renin concentrations. To investigate whether this angiotensinogen originated from nonhepatic sources, fludrocortisone was administered to mice lacking hepatic angiotensinogen. Fludrocortisone did not increase angiotensinogen in these mice, implying that the rise in angiotensinogen in the siRNA-treated rats must have depended on the liver, most likely reflecting diminished cleavage by renin. Conclusions Intact pressor responsiveness to conventional vasopressors provides pharmacological means to regulate the blood pressure-lowering effect of angiotensinogen siRNA and may support future therapeutic implementation of siRNA.

Differential effects of cyclo-oxygenase 1 and 2 inhibition on angiogenesis inhibitor-induced hypertension and kidney damage.

Vascular endothelial growth factor antagonism with angiogenesis inhibitors in cancer patients induces a 'preeclampsia-like' syndrome including hypertension, proteinuria and elevated endothelin (ET)-1. Cyclo-oxygenase (COX) inhibition with aspirin is known to prevent the onset of preeclampsia in high-risk patients. In the present study, we hypothesised that treatment with aspirin would prevent the development of angiogenesis inhibitor-induced hypertension and kidney damage. Our aims were to compare the effects of low-dose (COX-1 inhibition) and high-dose (dual COX-1 and COX-2 inhibition) aspirin on blood pressure, vascular function, oxidative stress, ET-1 and prostanoid levels and kidney damage during angiogenesis-inhibitor therapy in rodents. To this end, Wistar Kyoto rats were treated with vehicle, angiogenesis inhibitor (sunitinib) alone or in combination with low- or high-dose aspirin for 8 days (n=5-7/group). Our results demonstrated that prostacyclin (PGI2) and ET-1 were increased during angiogenesis-inhibitor therapy, while thromboxane (TXA2) was unchanged. Both low- and high-dose aspirin blunted angiogenesis inhibitor-induced hypertension and vascular superoxide production to a similar extent, whereas only high-dose aspirin prevented albuminuria. While circulating TXA2 and prostaglandin F2α levels were reduced by both low- and high-dose aspirin, circulating and urinary levels PGI2 were only reduced by high-dose aspirin. Lastly, treatment with aspirin did not significantly affect ET-1 or vascular function. Collectively our findings suggest that prostanoids contribute to the development of angiogenesis inhibitor-induced hypertension and renal damage and that targeting the prostanoid pathway could be an effective strategy to mitigate the unwanted cardiovascular and renal toxicities associated with angiogenesis inhibitors.

Vascular Ageing Features Caused by Selective DNA Damage in Smooth Muscle Cell.

Persistently unrepaired DNA damage has been identified as a causative factor for vascular ageing. We have previously shown that a defect in the function or expression of the DNA repair endonuclease ERCC1 (excision repair cross complement 1) in mice leads to accelerated, nonatherosclerotic ageing of the vascular system from as early as 8 weeks after birth. Removal of ERCC1 from endothelial alone partly explains this ageing, as shown in endothelial-specific Ercc1 knockout mice. In this study, we determined vascular ageing due to DNA damage in vascular smooth muscle cells, as achieved by smooth muscle-selective genetic removal of ERCC1 DNA repair in mice (SMC-KO: SM22αCre+ Ercc1fl/-). Vascular ageing features in SMC-KO and their wild-type littermates (WT: SM22αCre+ Ercc1fl/+) were examined at the age of 14 weeks and 25 weeks. Both SMC-KO and WT mice were normotensive. Compared to WT, SMC-KO showed a reduced heart rate, fractional shortening, and cardiac output. SMC-KO showed progressive features of nonatherosclerotic vascular ageing as they aged from 14 to 25 weeks. Decreased subcutaneous microvascular dilatation and increased carotid artery stiffness were observed. Vasodilator responses measured in aortic rings in organ baths showed decreased endothelium-dependent and endothelium-independent responses, mostly due to decreased NO-cGMP signaling. NADPH oxidase 2 and phosphodiesterase 1 inhibition improved dilations. SMC-KO mice showed elevated levels of various cytokines that indicate a balance shift in pro- and anti-inflammatory pathways. In conclusion, SMC-KO mice showed a progressive vascular ageing phenotype in resistant and conduit arteries that is associated with cardiac remodeling and contractile dysfunction. The changes induced by DNA damage might be limited to VSMC but eventually affect EC-mediated responses. The fact that NADPH oxidase 2 as wells as phosphodiesterase 1 inhibition restores vasodilation suggests that both decreased NO bioavailability and cGMP degradation play a role in local vascular smooth muscle cell ageing induced by DNA damage.

Renoprotective Effects of Small Interfering RNA Targeting Liver Angiotensinogen in Experimental Chronic Kidney Disease.

[Figure: see text].

Dietary salt modifies the blood pressure response to renin-angiotensin inhibition in experimental chronic kidney disease.

Chronic kidney disease contributes to hypertension, but the mechanisms are incompletely understood. To address this, we applied the 5/6th nephrectomy rat model to characterize hypertension and the response to dietary salt and renin-angiotensin inhibition. 5/6th nephrectomy caused low-renin, salt-sensitive hypertension with hyperkalemia and unsuppressed aldosterone. Compared with sham rats, 5/6th nephrectomized rats had lower Na+/H+ exchanger isoform 3, Na+-K+-2Cl- cotransporter, Na+-Cl- cotransporter, α-epithelial Na+ channel (ENaC), and Kir4.1 levels but higher serum and glucocorticoid-regulated kinase 1, prostasin, γ-ENaC, and Kir5.1 levels. These differences correlated with plasma renin, aldosterone, and/or K+. On a normal-salt diet, adrenalectomy (0 ± 9 mmHg) and spironolactone (-11 ± 10 mmHg) prevented a progressive rise in blood pressure (10 ± 8 mmHg), and this was enhanced in combination with losartan (-41 ± 12 and -43 ± 9 mmHg). A high-salt diet caused skin Na+ and water accumulation and aggravated hypertension that could only be attenuated by spironolactone (-16 ± 7 mmHg) and in which the additive effect of losartan was lost. Spironolactone also increased natriuresis, reduced skin water accumulation, and restored vasorelaxation. In summary, in the 5/6th nephrectomy rat chronic kidney disease model, salt-sensitive hypertension develops with a selective increase in γ-ENaC and despite appropriate transporter adaptations to low renin and hyperkalemia. With a normal-salt diet, hypertension in 5/6th nephrectomy depends on angiotensin II and aldosterone, whereas a high-salt diet causes more severe hypertension mediated through the mineralocorticoid receptor.NEW & NOTEWORTHY Chronic kidney disease (CKD) causes salt-sensitive hypertension, but the interactions between dietary salt and the renin-angiotensin system are incompletely understood. In rats with CKD on a normal-salt diet targeting aldosterone, the mineralocorticoid receptor (MR) and especially angiotensin II reduced blood pressure. On a high-salt diet, however, only MR blockade attenuated hypertension. These results reiterate the importance of dietary salt restriction to maintain renin-angiotensin system inhibitor efficacy and specify the MR as a target in CKD.

No evidence for brain renin-angiotensin system activation during DOCA-salt hypertension.

Brain renin-angiotensin system (RAS) activation is thought to mediate deoxycorticosterone acetate (DOCA)-salt hypertension, an animal model for human primary hyperaldosteronism. Here, we determined whether brainstem angiotensin II is generated from locally synthesized angiotensinogen and mediates DOCA-salt hypertension. To this end, chronic DOCA-salt-hypertensive rats were treated with liver-directed siRNA targeted to angiotensinogen, the angiotensin II type 1 receptor antagonist valsartan, or the mineralocorticoid receptor antagonist spironolactone (n = 6-8/group). We quantified circulating angiotensinogen and renin by enzyme-kinetic assay, tissue angiotensinogen by Western blotting, and angiotensin metabolites by LC-MS/MS. In rats without DOCA-salt, circulating angiotensin II was detected in all rats, whereas brainstem angiotensin II was detected in 5 out of 7 rats. DOCA-salt increased mean arterial pressure by 19 ± 1 mmHg and suppressed circulating renin and angiotensin II by >90%, while brainstem angiotensin II became undetectable in 5 out of 7 rats (<6 fmol/g). Gene silencing of liver angiotensinogen using siRNA lowered circulating angiotensinogen by 97 ± 0.3%, and made brainstem angiotensin II undetectable in all rats (P<0.05 vs. non-DOCA-salt), although brainstem angiotensinogen remained intact. As expected for this model, neither siRNA nor valsartan attenuated the hypertensive response to DOCA-salt, whereas spironolactone normalized blood pressure and restored brain angiotensin II together with circulating renin and angiotensin II. In conclusion, despite local synthesis of angiotensinogen in the brain, brain angiotensin II depended on circulating angiotensinogen. That DOCA-salt suppressed circulating and brain angiotensin II in parallel, while spironolactone simultaneously increased brain angiotensin II and lowered blood pressure, indicates that DOCA-salt hypertension is not mediated by brain RAS activation.

Local endothelial DNA repair deficiency causes aging-resembling endothelial-specific dysfunction.

We previously identified genomic instability as a causative factor for vascular aging. In the present study, we determined which vascular aging outcomes are due to local endothelial DNA damage, which was accomplished by genetic removal of ERCC1 (excision repair cross-complementation group 1) DNA repair in mice (EC-knockout (EC-KO) mice). EC-KO showed a progressive decrease in microvascular dilation of the skin, increased microvascular leakage in the kidney, decreased lung perfusion, and increased aortic stiffness compared with wild-type (WT). EC-KO showed expression of DNA damage and potential senescence marker p21 exclusively in the endothelium, as demonstrated in aorta. Also the kidney showed p21-positive cells. Vasodilator responses measured in organ baths were decreased in aorta, iliac and coronary artery EC-KO compared with WT, of which coronary artery was the earliest to be affected. Nitric oxide-mediated endothelium-dependent vasodilation was abolished in aorta and coronary artery, whereas endothelium-derived hyperpolarization and responses to exogenous nitric oxide (NO) were intact. EC-KO showed increased superoxide production compared with WT, as measured in lung tissue, rich in endothelial cells (ECs). Arterial systolic blood pressure (BP) was increased at 3 months, but normal at 5 months, at which age cardiac output (CO) was decreased. Since no further signs of cardiac dysfunction were detected, this decrease might be an adaptation to prevent an increase in BP. In summary, a selective DNA repair defect in the endothelium produces features of age-related endothelial dysfunction, largely attributed to loss of endothelium-derived NO. Increased superoxide generation might contribute to the observed changes affecting end organ perfusion, as demonstrated in kidney and lung.

Angiotensin-neprilysin inhibition confers renoprotection in rats with diabetes and hypertension by limiting podocyte injury.

OBJECTIVES: Combined angiotensin receptor--neprilysin inhibition (ARNI) reduces glomerulosclerosis better than single angiotensin receptor blockade (ARB) in diabetic, hypertensive rats. The renoprotective mechanism remains unknown, but may depend on superior blood pressure control, improved renal hemodynamics, suppressed renal inflammation or prevention of podocyte loss. METHODS: To address this, TGR(mREN2)27 rats (a model of angiotensin II-dependent hypertension) were made diabetic for 12 weeks and treated with vehicle (n = 10), valsartan (ARB; n = 7) or sacubitril/valsartan (ARNI; n = 8) for the final 3 weeks. Arterial pressure was measured via radiotelemetry. RESULTS: Sacubitril/valsartan lowered mean arterial pressure by -50 ±â€Š4 mmHg and valsartan by -43 ±â€Š4 mmHg (P = 0.3). Both treatments lowered albuminuria, but only sacubitril/valsartan maintained high urinary atrial natriuretic peptide, improved glycemic control and protected podocyte integrity, reflected by increased nephrin expression and suppression of transient receptor potential canonical 6 and regulator of calcineurin 1. This resulted in markedly reduced glomerulosclerosis (P < 0.05 vs. control and valsartan). Despite higher effective renal plasma flow and glomerular filtration rates, sacubitril/valsartan did neither improve filtration fraction nor renal immune cell infiltration. CONCLUSION: Sacubitril/valsartan offers drug-class-specific renoprotection in a preclinical model of diabetes and hypertension. Renoprotection is unrelated to antihypertensive efficacy, renal hemodynamics or inflammation, but may be related to protective effects of natriuretic peptides on podocyte integrity.

Selective ETA vs. dual ETA/B receptor blockade for the prevention of sunitinib-induced hypertension and albuminuria in WKY rats.

AIMS: Although effective in preventing tumour growth, angiogenesis inhibitors cause off-target effects including cardiovascular toxicity and renal injury, most likely via endothelin (ET)-1 up-regulation. ET-1 via stimulation of the ETA receptor has pro-hypertensive actions whereas stimulation of the ETB receptor can elicit both pro- or anti-hypertensive effects. In this study, our aim was to determine the efficacy of selective ETA vs. dual ETA/B receptor blockade for the prevention of angiogenesis inhibitor-induced hypertension and albuminuria. METHODS AND RESULTS: Male Wistar Kyoto (WKY) rats were treated with vehicle, sunitinib (angiogenesis inhibitor; 14 mg/kg/day) alone or in combination with macitentan (ETA/B receptor antagonist; 30 mg/kg/day) or sitaxentan (selective ETA receptor antagonist; 30 or 100 mg/kg/day) for 8 days. Compared with vehicle, sunitinib treatment caused a rapid and sustained increase in mean arterial pressure of ∼25 mmHg. Co-treatment with macitentan or sitaxentan abolished the pressor response to sunitinib. Sunitinib did not induce endothelial dysfunction. However, it was associated with increased aortic, mesenteric, and renal oxidative stress, an effect that was absent in mesenteric arteries of the macitentan and sitaxentan co-treated groups. Albuminuria was greater in the sunitinib- than vehicle-treated group. Co-treatment with sitaxentan, but not macitentan, prevented this increase in albuminuria. Sunitinib treatment increased circulating and urinary prostacyclin levels and had no effect on thromboxane levels. These increases in prostacyclin were blunted by co-treatment with sitaxentan. CONCLUSIONS: Our results demonstrate that both selective ETA and dual ETA/B receptor antagonism prevents sunitinib-induced hypertension, whereas sunitinib-induced albuminuria was only prevented by selective ETA receptor antagonism. In addition, our results uncover a role for prostacyclin in the development of these effects. In conclusion, selective ETA receptor antagonism is sufficient for the prevention of sunitinib-induced hypertension and renal injury.

Strong and Sustained Antihypertensive Effect of Small Interfering RNA Targeting Liver Angiotensinogen.

Small interfering RNAs (siRNAs) targeting hepatic angiotensinogen ( Agt) may provide long-lasting antihypertensive effects, but the optimal approach remains unclear. Here, we assessed the efficacy of a novel AGT siRNA in spontaneously hypertensive rats. Rats were treated with vehicle, siRNA (10 mg/kg fortnightly; subcutaneous), valsartan (31 mg/kg per day; oral), captopril (100 mg/kg per day; oral), valsartan+siRNA, or captopril+valsartan for 4 weeks (all groups, n=8). Mean arterial pressure (recorded via radiotelemetry) was lowered the most by valsartan+siRNA (-68±4 mm Hg), followed by captopril+valsartan (-54±4 mm Hg), captopril (-23±2 mm Hg), siRNA (-14±2 mm Hg), and valsartan (-10±2 mm Hg). siRNA and captopril monotherapies improved cardiac hypertrophy equally, but less than the dual therapies, which also lowered NT-proBNP (N-terminal pro-B-type natriuretic peptide). Glomerular filtration rate, urinary NGAL (neutrophil gelatinase-associated lipocalin), and albuminuria were unaffected by treatment. siRNA lowered circulating AGT by 97.9±1.0%, and by 99.8±0.1% in combination with valsartan. Although siRNA greatly reduced renal Ang (angiotensin) I, only valsartan+siRNA suppressed circulating and renal Ang II. This coincided with decreased renal sodium hydrogen exchanger type 3 and phosphorylated sodium chloride cotransporter abundances. Renin and plasma K+ increased with every treatment, but especially during valsartan+siRNA; no effects on aldosterone were observed. Collectively, these data indicate that Ang II elimination requires >99% suppression of circulating AGT. Maximal blockade of the renin-angiotensin system, achieved by valsartan+siRNA, yielded the greatest reduction in blood pressure and cardiac hypertrophy, whereas AGT lowering alone was as effective as conventional renin-angiotensin system inhibitors. Given its stable and sustained efficacy, lasting weeks, RNA interference may offer a unique approach to improving therapy adherence and treating hypertension.

Blood pressure-independent renoprotection in diabetic rats treated with AT1 receptor-neprilysin inhibition compared with AT1 receptor blockade alone.

ARNI [dual AT1 (angiotensin II type 1) receptor-neprilysin inhibition] exerts beneficial effects on blood pressure and kidney function in heart failure, compared with ARB (AT1 receptor blockade) alone. We hypothesized that ARNI improves cardiac and kidney parameters in diabetic TGR(mREN2)27 rats, an angiotensin II-dependent hypertension model. Rats were made diabetic with streptozotocin for 5 or 12 weeks. In the final 3 weeks, rats were treated with vehicle, irbesartan (ARB) or irbesartan+thiorphan (ARNI). Blood pressure, measured by telemetry in the 5-week group, was lowered identically by ARB and ARNI. The heart weight/tibia length ratio in 12-week diabetic animals was lower after ARNI compared with after ARB. Proteinuria and albuminuria were observed from 8 weeks of diabetes onwards. ARNI reduced proteinuria more strongly than ARB, and a similar trend was seen for albuminuria. Kidneys of ARNI-treated animals showed less severe segmental glomerulosclerosis than those of ARB-treated animals. After 12 weeks, no differences between ARNI- and ARB-treated animals were found regarding diuresis, natriuresis, plasma endothelin-1, vascular reactivity (acetylcholine response) or kidney sodium transporters. Only ARNI-treated rats displayed endothelin type B receptor-mediated vasodilation. In conclusion, ARNI reduces proteinuria, glomerulosclerosis and heart weight in diabetic TGR(mREN2)27 rats more strongly than does ARB, and this occurs independently of blood pressure.

AT1-receptor blockade, but not renin inhibition, reduces aneurysm growth and cardiac failure in fibulin-4 mice.

AIMS: Increasing evidence supports a role for the angiotensin II-AT1-receptor axis in aneurysm development. Here, we studied whether counteracting this axis via stimulation of AT2 receptors is beneficial. Such stimulation occurs naturally during AT1-receptor blockade with losartan, but not during renin inhibition with aliskiren. METHODS AND RESULTS: Aneurysmal homozygous fibulin-4 mice, displaying a four-fold reduced fibulin-4 expression, were treated with placebo, losartan, aliskiren, or the ß-blocker propranolol from day 35 to 100. Their phenotype includes cystic media degeneration, aortic regurgitation, left ventricular dilation, reduced ejection fraction, and fractional shortening. Although losartan and aliskiren reduced hemodynamic stress and increased renin similarly, only losartan increased survival. Propranolol had no effect. No drug rescued elastic fiber fragmentation in established aneurysms, although losartan did reduce aneurysm size. Losartan also increased ejection fraction, decreased LV diameter, and reduced cardiac pSmad2 signaling. None of these effects were seen with aliskiren or propranolol. Longitudinal micro-CT measurements, a novel method in which each mouse serves as its own control, revealed that losartan reduced LV growth more than aneurysm growth, presumably because the heart profits both from the local (cardiac) effects of losartan and its effects on aortic root remodeling. CONCLUSION: Losartan, but not aliskiren or propranolol, improved survival in fibulin-4 mice. This most likely relates to its capacity to improve structure and function of both aorta and heart. The absence of this effect during aliskiren treatment, despite a similar degree of blood pressure reduction and renin-angiotensin system blockade, suggests that it might be because of AT2-receptor stimulation.

Angiotensin II type 2 receptor- and acetylcholine-mediated relaxation: essential contribution of female sex hormones and chromosomes.

Angiotensin-induced vasodilation, involving type 2 receptor (AT2R)-induced generation of nitric oxide (NO; by endothelial NO synthase) and endothelium-derived hyperpolarizing factors, may be limited to women. To distinguish the contribution of female sex hormones and chromosomes to AT2R function and endothelium-derived hyperpolarizing factor-mediated vasodilation, we made use of the four-core genotype model, where the testis-determining Sry gene has been deleted (Y(-)) from the Y chromosome, allowing XY(-) mice to develop a female gonadal phenotype. Simultaneously, by incorporating the Sry gene onto an autosome, XY(-)Sry and XXSry transgenic mice develop into gonadal male mice. Four-core genotype mice underwent a sham or gonadectomy (GDX) operation, and after 8 weeks, iliac arteries were collected to assess vascular function. XY(-)Sry male mice responded more strongly to angiotensin than XX female mice, and the AT2R antagonist PD123319 revealed that this was because of a dilator AT2R-mediated effect occurring exclusively in XX female mice. The latter could not be demonstrated in XXSry male and XY(-) female mice nor in XX female mice after GDX, suggesting that it depends on both sex hormones and chromosomes. Indeed, treating C57bl/6 GDX male mice with estrogen could not restore angiotensin-mediated, AT2R-dependent relaxation. To block acetylcholine-induced relaxation of iliac arteries obtained from four-core genotype XX mice, both endothelial NO synthase and endothelium-derived hyperpolarizing factor inhibition were required, whereas in four-core genotype XY animals, endothelial NO synthase inhibition alone was sufficient. These findings were independent of gonadal sex and unaltered after GDX. In conclusion, AT2R-induced relaxation requires both estrogen and the XX chromosome sex complement, whereas only the latter is required for endothelium-derived hyperpolarizing factors.

Optimum AT1 receptor-neprilysin inhibition has superior cardioprotective effects compared with AT1 receptor blockade alone in hypertensive rats.

Neprilysin inhibitors prevent the breakdown of bradykinin and natriuretic peptides, promoting vasodilation and natriuresis. However, they also increase angiotensin II and endothelin-1. Here we studied the effects of a low and a high dose of the neprilysin inhibitor thiorphan on top of AT1 receptor blockade with irbesartan versus vehicle in TGR(mREN2)27 rats with high renin hypertension. Mean arterial blood pressure was unaffected by vehicle or thiorphan alone. Irbesartan lowered blood pressure, but after 7 days pressure started to increase again. Low- but not high-dose thiorphan prevented this rise. Only during exposure to low-dose thiorphan plus irbesartan did heart weight/body weight ratio, cardiac atrial natriuretic peptide expression, and myocyte size decrease significantly. Circulating endothelin-1 was not affected by low-dose thiorphan with or without irbesartan, but increased after treatment with high-dose thiorphan plus irbesartan. This endothelin-1 rise was accompanied by an increase in renal sodium-hydrogen exchanger 3 protein abundance, and an upregulation of constrictor vascular endothelin type B receptors. Consequently, the endothelin type B receptor antagonist BQ788 no longer enhanced endothelin-1-induced vasoconstriction (indicative of endothelin type B receptor-mediated vasodilation), but prevented it. Thus, optimal neprilysin inhibitor dosing reveals additional cardioprotective effects on top of AT1 receptor blockade in renin-dependent hypertension.

Effect of a stable Angiotensin-(1-7) analogue on progenitor cell recruitment and cardiovascular function post myocardial infarction.

BACKGROUND: Angiotensin-(1-7) improves cardiac function and remodeling after myocardial infarction (MI). This may involve recruitment of hematopoietic progenitor cells that support angiogenesis. However, angiotensin-(1-7) is rapidly metabolized in plasma and tissue. The authors investigated in mice the effect of a metabolically stable angiotensin-(1-7) analogue, cyclic angiotensin-(1-7), on progenitor cell recruitment and on the heart post MI, when given in the angiogenesis phase of remodeling. METHODS AND RESULTS: Angiogenic progenitor cell recruitment was measured by using flow cytometry 24 and 72 hours after a daily bolus injection of cyclic angiotensin-(1-7) in healthy C57BL/6 mice. Further, mice underwent MI or sham surgery and subsequently received saline or 2 different doses of cyclic angiotensin-(1-7) for 3 or 9 weeks. Cyclic angiotensin-(1-7) increased circulating hematopoietic progenitor cells at 24 hours but not 72 hours. Post MI, cyclic angiotensin-(1-7) diminished cardiomyocyte hypertrophy and reduced myogenic tone, without altering cardiovascular function or cardiac histology at 9 weeks. Importantly, cyclic angiotensin-(1-7)-treated mice had reduced cardiac capillary density at 3 weeks after MI but not after 9 weeks. Finally, cyclic angiotensin-(1-7) decreased tube formation by cultured human umbilical vein endothelial cells. CONCLUSIONS: Our results suggest that cyclic angiotensin-(1-7), when given early after MI, recruits progenitor cells but does not lead to improved angiogenesis, most likely because it simultaneously exerts antiangiogenic effect in adult endothelial cells. Apparently, optimal treatment with cyclic angiotensin-(1-7) depends on the time point of onset of application after MI.

Combined renin inhibition/(pro)renin receptor blockade in diabetic retinopathy--a study in transgenic (mREN2)27 rats.

Dysfunction of renin-angiotensin system (RAS) contributes to the pathogenesis of diabetic retinopathy (DR). Prorenin, the precursor of renin is highly elevated in ocular fluid of diabetic patients with proliferative retinopathy. Prorenin may exert local effects in the eye by binding to the so-called (pro)renin receptor ((P)RR). Here we investigated the combined effects of the renin inhibitor aliskiren and the putative (P)RR blocker handle-region peptide (HRP) on diabetic retinopathy in streptozotocin (STZ)-induced diabetic transgenic (mRen2)27 rats (a model with high plasma prorenin levels) as well as prorenin stimulated cytokine expression in cultured Müller cells. Adult (mRen2)27 rats were randomly divided into the following groups: (1) non-diabetic; (2) diabetic treated with vehicle; (3) diabetic treated with aliskiren (10 mg/kg per day); and (4) diabetic treated with aliskiren+HRP (1 mg/kg per day). Age-matched non-diabetic wildtype Sprague-Dawley rats were used as control. Drugs were administered by osmotic minipumps for three weeks. Transgenic (mRen2)27 rat retinas showed increased apoptotic cell death of both inner retinal neurons and photoreceptors, increased loss of capillaries, as well as increased expression of inflammatory cytokines. These pathological changes were further exacerbated by diabetes. Aliskiren treatment of diabetic (mRen2)27 rats prevented retinal gliosis, and reduced retinal apoptotic cell death, acellular capillaries and the expression of inflammatory cytokines. HRP on top of aliskiren did not provide additional protection. In cultured Müller cells, prorenin significantly increased the expression levels of IL-1α and TNF-α, and this was completely blocked by aliskiren or HRP, their combination, (P)RR siRNA and the AT1R blocker losartan, suggesting that these effects entirely depended on Ang II generation by (P)RR-bound prorenin. In conclusion, the lack of effect of HRP on top of aliskiren, and the Ang II-dependency of the ocular effects of prorenin in vitro, argue against the combined application of (P)RR blockade and renin inhibition in diabetic retinopathy.