Sex differences in hippocampal ß-adrenergic receptor subtypes drive retrieval, retention, and learning of cocaine-associated memories.

Background: Drug seeking behavior occurs in response to environmental contexts and drug-associated cues. The presence of these pervasive stimuli impedes abstinence success. ß-adrenergic receptors (ß-ARs) have a long-standing historical implication in driving processes associated with contextual memories, including drug-associated memories in substance use disorders. However, sex differences in the role of ß-adrenergic receptors in drug memories remain unknown. Hypothesis: Prior reports indicate a selective role for ß2-ARs in retrieval and retention of contextual drug memories in males, and substantial sex differences exist in the expression of ß-ARs of male and female rats. Therefore, we hypothesized that there are sex differences in selective recruitment of ß-ARs during different stages of memory encoding and retrieval. Methods: The role of ß-ARs in driving retrieval and learning of contextual cocaine memories was investigated using cocaine conditioned place preference (CPP) in adult male and female Sprague-Dawley rats. Rats were infused directly to the dorsal hippocampus with Propranolol (ß1 and ß2) or ICI-118,551 (ß1) and/or Betaxolol (ß2), immediately prior to testing (retrieval), or paired to each cocaine (10 mg/kp, IP) conditioning session (learning). Results: In males, administration of either ß1, ß2, or combined ß1 and ß2-ARs before the initial CPP testing reduced the expression of a CPP compared to vehicle administration. In females, ß2-ARs transiently decreased CPP memories, whereas ß1 had long lasting but not immediate effects to decrease CPP memories. Additionally, ß1 and combined ß1 and ß2-ARs had immediate and persistent effects to decrease CPP memory expression. DG Fos + neurons predicted cocaine CPP expression in males, whereas CA1 and CA3 Fos + neurons predicted cocaine CPP expression in females. Conclusion: There are significant sex differences in the role of dorsal hippocampus ß-ARs in the encoding and expression of cocaine conditioned place preference. Furthermore, sub regions of the dorsal hippocampus appear to activate differently between male and female rats during CPP. Therefore DG, CA3, and CA1 may have separate region- and sex-specific impacts on driving drug- associated, or context-associated cues.

Early superoxide scavenging accelerates renal microvascular rarefaction and damage in the stenotic kidney.

Renal artery stenosis (RAS), the main cause of chronic renovascular disease (RVD), is associated with significant oxidative stress. Chronic RVD induces renal injury partly by promoting renal microvascular (MV) damage and blunting MV repair in the stenotic kidney. We tested the hypothesis that superoxide anion plays a pivotal role in MV dysfunction, reduction of MV density, and progression of renal injury in the stenotic kidney. RAS was induced in 14 domestic pigs and observed for 6 wk. Seven RAS pigs were chronically treated with the superoxide dismutase mimetic tempol (RAS+T) to reduce oxidative stress. Single-kidney hemodynamics and function were quantified in vivo using multidetector computer tomography (CT) and renal MV density was quantified ex vivo using micro-CT. Expression of angiogenic, inflammatory, and apoptotic factors was measured in renal tissue, and renal apoptosis and fibrosis were quantified in tissue sections. The degree of RAS and blood pressure were similarly increased in RAS and RAS+T. Renal blood flow (RBF) and glomerular filtration rate (GFR) were reduced in the stenotic kidney (280.1 ± 36.8 and 34.2 ± 3.1 ml/min, P < 0.05 vs. control). RAS+T kidneys showed preserved GFR (58.5 ± 6.3 ml/min, P = not significant vs. control) but a similar decreases in RBF (293.6 ± 85.2 ml/min) and further decreases in MV density compared with RAS. These changes were accompanied by blunted angiogenic signaling and increased apoptosis and fibrosis in the stenotic kidney of RAS+T compared with RAS. The current study shows that tempol administration provided limited protection to the stenotic kidney. Despite preserved GFR, renal perfusion was not improved by tempol, and MV density was further reduced compared with untreated RAS, associated with increased renal apoptosis and fibrosis. These results suggest that a tight balance of the renal redox status is necessary for a normal MV repair response to injury, at least at the early stage of RVD, and raise caution regarding antioxidant strategies in RAS.

Reversal of renal dysfunction by targeted administration of VEGF into the stenotic kidney: a novel potential therapeutic approach.

Renal microvascular (MV) damage and loss contribute to the progression of renal injury in renovascular disease (RVD). Whether a targeted intervention in renal microcirculation could reverse renal damage is unknown. We hypothesized that intrarenal vascular endothelial growth factor (VEGF) therapy will reverse renal dysfunction and decrease renal injury in experimental RVD. Unilateral renal artery stenosis (RAS) was induced in 14 pigs, as a surrogate of chronic RVD. Six weeks later, renal blood flow (RBF) and glomerular filtration rate (GFR) were quantified in vivo in the stenotic kidney using multidetector computed tomography (CT). Then, intrarenal rhVEGF-165 or vehicle was randomly administered into the stenotic kidneys (n = 7/group), they were observed for 4 additional wk, in vivo studies were repeated, and then renal MV density was quantified by 3D micro-CT, and expression of angiogenic factors and fibrosis was determined. RBF and GFR, MV density, and renal expression of VEGF and downstream mediators such as p-ERK 1/2, Akt, and eNOS were significantly reduced after 6 and at 10 wk of untreated RAS compared with normal controls. Remarkably, administration of VEGF at 6 wk normalized RBF (from 393.6 ± 50.3 to 607.0 ± 45.33 ml/min, P < 0.05 vs. RAS) and GFR (from 43.4 ± 3.4 to 66.6 ± 10.3 ml/min, P < 0.05 vs. RAS) at 10 wk, accompanied by increased angiogenic signaling, augmented renal MV density, and attenuated renal scarring. This study shows promising therapeutic effects of a targeted renal intervention, using an established clinically relevant large-animal model of chronic RAS. It also implies that disruption of renal MV integrity and function plays a pivotal role in the progression of renal injury in the stenotic kidney. Furthermore, it shows a high level of plasticity of renal microvessels to a single-dose VEGF-targeted intervention after established renal injury, supporting promising renoprotective effects of a novel potential therapeutic intervention to treat chronic RVD.

Endothelin-A receptor blockade slows the progression of renal injury in experimental renovascular disease.

Endothelin (ET)-1, a potent renal vasoconstrictor with mitogenic properties, is upregulated by ischemia and has been shown to induce renal injury via the ET-A receptor. The potential role of ET-A blockade in chronic renovascular disease (RVD) has not, to our knowledge, been previously reported. We hypothesized that chronic ET-A receptor blockade would preserve renal hemodynamics and slow the progression of injury of the stenotic kidney in experimental RVD. Renal artery stenosis, a major cause of chronic RVD, was induced in 14 pigs and observed for 6 wk. In half of the pigs, chronic ET-A blockade was initiated (RVD+ET-A, 0.75 mg·kg(-1)·day(-1)) at the onset of RVD. Single-kidney renal blood flow, glomerular filtration rate, and perfusion were quantified in vivo after 6 wk using multidetector computer tomography. Renal microvascular density was quantified ex vivo using three-dimensional microcomputer tomography, and growth factors, inflammation, apoptosis, and fibrosis were determined in renal tissue. The degree of stenosis and increase in blood pressure were similar in RVD and RVD+ET-A pigs. Renal hemodynamics, function, and microvascular density were decreased in the stenotic kidney but preserved by ET-A blockade, accompanied by increased renal expression of vascular endothelial growth factor, hepatocyte growth factor, and downstream mediators such as phosphorilated-Akt, angiopoietins, and endothelial nitric oxide synthase. ET-A blockade also reduced renal apoptosis, inflammation, and glomerulosclerosis. This study shows that ET-A blockade slows the progression of renal injury in experimental RVD and preserves renal hemodynamics, function, and microvascular density in the stenotic kidney. These results support a role for ET-1/ET-A as a potential therapeutic target in chronic RVD.

Renal microvascular disease determines the responses to revascularization in experimental renovascular disease.

BACKGROUND: Percutaneous transluminal renal angioplasty (PTRA) is the most frequent therapeutic approach to resolving renal artery stenosis (RAS). However, renal function recovers in only 30% of the cases. The causes of these poor outcomes are still unknown. We hypothesized that preserving the renal microcirculation distal to RAS will improve the responses to PTRA. METHODS AND RESULTS: RAS was induced in 28 pigs. In 14, vascular endothelial growth factor (VEGF)-165 0.05 microg/kg was infused intrarenally (RAS+VEGF). Single-kidney function was assessed in all pigs in vivo using ultrafast CT after 6 weeks. Observation of half of the RAS and RAS+VEGF pigs was completed. The other half underwent PTRA and repeated VEGF, and CT studies were repeated 4 weeks later. Pigs were then euthanized, the stenotic kidney removed, renal microvascular (MV) architecture reconstructed ex vivo using 3D micro-CT, and renal fibrosis quantified. The degree of RAS and hypertension were similar in RAS and RAS+VEGF. Renal function and MV density were decreased in RAS but improved in RAS+VEGF. PTRA largely resolved RAS, but the improvements of hypertension and renal function were greater in RAS+VEGF+PTRA than in RAS+PTRA, accompanied by a 34% increase in MV density and decreased fibrosis. CONCLUSIONS: Preservation of the MV architecture and function in the stenotic kidney improved the responses to PTRA, indicating that renal MV integrity plays a role in determining the responses to PTRA. This study indicates that damage and early loss of renal MV is an important determinant of the progression of renal injury in RAS and instigates often irreversible damage.

Role of renal microcirculation in experimental renovascular disease.

BACKGROUND: Renal artery stenosis (RAS) causes renal injury partly via microvascular (MV) endothelial dysfunction and damage. Vascular endothelial growth factor (VEGF) is crucial for preservation of microvasculature and promotes vascular proliferation and endothelial repair. We have previously shown that MV rarefaction is associated with decreased VEGF in the kidney exposed to chronic RAS, accompanied by deteriorated renal function and fibrosis. We hypothesized that preserving the renal microcirculation in the stenotic kidney will halt the progression of renal damage. METHODS: Unilateral RAS was induced in 16 pigs. In eight, VEGF (0.05 micrograms/kg) was infused intra-renally at the onset of RAS. After 6 weeks, single-kidney haemodynamics and function were assessed using in vivo multi-detector computed tomography (CT). Renal microvessels, angiogenic pathways and morphology were investigated ex vivo using micro-CT, real-time PCR and histology. RESULTS: Blood pressure and degree of RAS was similar in RAS and RAS + VEGF pigs. Single-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were reduced in RAS compared to Normal (221.1 +/- 46.5 and 29.9 +/- 3.8 vs. 522.5 +/- 60.9 and 49.3 +/- 3.4 mL/min, respectively, P < 0.05), accompanied by decreased cortical MV density and increased renal fibrosis. Pre-emptive administration of VEGF preserved MV architecture, attenuated fibrosis and normalized RBF and GFR (510.8 +/- 50.9 and 39.9.1 +/- 4.1 mL/min, P = not significant vs. Normal). CONCLUSIONS: This study underscores the importance of the renal microcirculation in renovascular disease. Intra-renal administration of VEGF preserved renal MV architecture and function of the stenotic kidney, which in turn preserved renal haemodynamics and function and decreased renal fibrosis. These observations suggest that preventing renal MV loss may be a potential target for therapeutic approaches for patients with chronic renovascular disease.

Inhibition of biliverdin reductase increases ANG II-dependent superoxide levels in cultured renal tubular epithelial cells.

Induction of heme oxygenase-1 (HO-1) in the renal medulla increases carbon monoxide and bilirubin production and decreases ANG II-mediated superoxide production. The goal of this study was to determine the importance of increases in bilirubin to the antioxidant effects of HO-1 induction in cultured mouse thick ascending loop of Henle (TALH) and inner medullary collecting duct (IMCD3) cells. Bilirubin levels were decreased by using small interfering RNAs (siRNAs) targeted to biliverdin reductase (BVR), which is the cellular enzyme responsible for the conversion of biliverdin to bilirubin. Treatment of cultured TALH or IMCD-3 cells with BVR siRNA (50 or 100 nM) resulted in an 80% decrease in the level of BVR protein and decreased cellular bilirubin levels from 46 +/- 5 to 23 +/- 4 nM (n = 4). We then determined the effects of inhibition of BVR on ANG II-mediated superoxide production. Superoxide production induced by ANG II (10(-9) M) significantly increased in both TALH and IMCD-3 cells. Treatment of TALH cells with BVR siRNA resulted in a significant increase in ouabain-sensitive rubidium uptake from 95 +/- 6 to 122 +/- 5% control (n = 4, P < 0.05). Lastly, inhibition of BVR with siRNA did not prevent the decrease in superoxide levels observed in cells pretreated with the HO-1 inducer, hemin. We conclude that decreased levels of cellular bilirubin increase ANG II-mediated superoxide production and sodium transport; however, increases in bilirubin are not necessary for HO-1 induction to attenuate ANG II-mediated superoxide production.

Heme oxygenase attenuates angiotensin II-mediated superoxide production in cultured mouse thick ascending loop of Henle cells.

Heme oxygenase (HO)-1 induction can attenuate the development of angiotensin II (ANG II)-dependent hypertension. However, the mechanism by which HO-1 lowers blood pressure is not clear. The goal of this study was to test the hypothesis that induction of HO-1 can reduce the ANG II-mediated increase in superoxide production in cultured thick ascending loop of Henle (TALH) cells. Studies were performed on an immortalized cell line of mouse TALH (mTALH) cells. HO-1 was induced in cultured mTALH cells by treatment with cobalt protoporphyrin (CoPP, 10 microM) or hemin (50 microM) or by transfection with a plasmid containing the human HO-1 isoform. Treatment of mTALH cells with 10(-9) M ANG II increased dihydroethidium (DHE) fluorescence (an index of superoxide levels) from 35.5+/-5 to 136+/-18 relative fluorescence units (RFU)/microm2. Induction of HO-1 via CoPP, hemin, or overexpression of the human HO-1 isoform significantly reduced ANG II-induced DHE fluorescence to 64+/-5, 64+/-8, and 41+/-4 RFU/microm2, respectively. To determine which metabolite of HO-1 is responsible for reducing ANG II-mediated increases in superoxide production in mTALH cells, cells were preincubated with bilirubin or carbon monoxide (CO)-releasing molecule (CORM)-A1 (each at 100 microM) before exposure to ANG II. DHE fluorescence averaged 80+/-7 RFU/microm2 after incubation with ANG II and was significantly decreased to 55+/-7 and 53+/-4 RFU/microm2 after pretreatment with bilirubin and CORM-A1. These results demonstrate that induction of HO-1 in mTALH cells reduces the levels of ANG II-mediated superoxide production through the production of both bilirubin and CO.

Kidney-specific induction of heme oxygenase-1 prevents angiotensin II hypertension.

The main goal of this study was to determine whether kidney-specific induction of heme oxygenase-1 (HO-1) can prevent the development of angiotensin (Ang) II-dependent hypertension. To test this hypothesis, intrarenal medullary interstitial catheters were implanted into the left kidney of uninephrectomized mice. Infusion of cobalt protoporphyrin (CoPP; 250 microg/mL; at 50 microL/h for 48 hours) resulted in significant induction of HO-1 in the renal medulla when examined 2 weeks after the infusion with no induction observed in other organs, such as the heart or liver. Next, we examined the effect of renal-specific induction of HO-1 on the development of Ang II-dependent hypertension. CoPP or vehicle (0.1 mol/L NaOH [pH 8.3]) was infused as indicated above 2 days before implantation of an osmotic minipump, which delivered Ang II or saline vehicle at a rate of 1 microg/kg per minute. Mean arterial pressure was measured in conscious, unrestrained mice for 3 consecutive days starting on day 7 after implantation of the minipumps. Mean arterial pressure averaged 114+/-5, 122+/-4, 162+/-2, and 125+/-6 mm Hg in vehicle-, intrarenal medullary interstitial CoPP-, Ang II-, and Ang II + intrarenal medullary interstitial CoPP-treated mice, respectively (n=6 or 7). These results demonstrate that kidney-specific induction of HO-1 prevents the development of Ang II-dependent hypertension and that induction of HO-1 in the kidney may be the mechanism by which systemic delivery of CoPP lowers blood pressure in Ang II-dependent hypertension.

Heme oxygenase-1 induction does not improve vascular relaxation in angiotensin II hypertensive mice.

BACKGROUND: Induction of heme oxygenase-1 (HO-1) attenuates the development of angiotensin II (Ang II)-dependent hypertension in mice. However, the mechanism by which HO-1 lowers blood pressure in this model is not clear. This study was designed to determine whether induction of HO-1 results in an improvement in vascular relaxation in Ang II hypertensive mice. METHODS: Mice were treated with either of the vehicles (control), the HO-1 inducer cobalt protoporphyrin (CoPP;50 mg/kg), Ang II(1 microg/kg/min, 14 days), or Ang II + CoPP. CoPP was administered as a single bolus dose 2 days prior to subcutaneous implantation of the osmotic minipump containing Ang II. Vascular relaxation was examined in isolated carotid arteries precontracted with the thromboxane mimetic U46619 (0.4 microg/ml). RESULTS: Endothelial dependent relaxation to acetylcholine (ACh; 1 micromol/l) was significantly impaired in Ang II-treated mice compared to control mice (56 +/- 3% vs. 40 +/- 4%, P < 0.05, n > or = 6). Similarly, endothelial independent relaxation to sodium nitroprusside (SNP; 1 micromol/l) was significantly impaired in Ang II mice (56 +/- 6% vs. 28 +/- 6%, P < 0.05, n > or = 6). Relaxation in response to the carbon monoxide donor, CORM-A1 (100 micromol/l), was attenuated after Ang II treatment (75 +/- 7% vs. 59 +/- 7%,P < 0.05, n > or = 6). CoPP treatment induced HO-1 but not HO-2 protein in the aorta, as measured by western blot analysis. CoPP treatment had no effect on vascular responses in control mice and did not improve ACh (26 +/- 5%, n = 15), SNP (23 +/- 4%, n = 15), or CORM-A1 (46 +/- 7%, n = 10) dependent relaxation in Ang II treated mice. CONCLUSIONS: These results suggest that induction of HO-1 lowers Ang II-dependent hypertension through a mechanism independent of improved vascular relaxation.

HO-1 induction lowers blood pressure and superoxide production in the renal medulla of angiotensin II hypertensive mice.

Heme oxygenase-1 (HO-1) induction can attenuate the development of angiotensin II (ANG II)-dependent hypertension. However, the mechanism by which HO-1 lowers blood pressure in this model is not clear. The goal of this study was to test the hypothesis that induction of HO-1 in the kidney can attenuate the increase in reactive oxygen species (ROS) generation in the kidney that occurs during ANG II-dependent hypertension. Mice were divided into four groups, control (Con), cobalt protoporphyrin (CoPP), ANG II, and ANG II + CoPP. CoPP treatment (50 mg/kg) was administered in a single subcutaneous injection 2 days prior to implantation of an osmotic minipump that infused ANG II at a rate of 1 microg x kg(-1) x min(-1). At the end of this period, mean arterial blood pressure (MAP) averaged 93 +/- 5, 90 +/- 5, 146 +/- 8, and 105 +/- 6 mmHg in Con, CoPP-, ANG II-, and ANG II + CoPP-treated mice. To determine whether HO-1 induction resulted in a decrease in ANG II-stimulated ROS generation in the renal medulla, superoxide production was measured. Medullary superoxide production was increased by ANG II infusion and normalized in mice pretreated with CoPP. The reduction in ANG II-mediated superoxide production in the medulla with CoPP was associated with a decrease in extracellular superoxide dismutase protein but an increase in catalase protein and activity. These results suggest that reduction in superoxide and possibly hydrogen peroxide production in the renal medulla may be a potential mechanism by which induction of HO-1 with CoPP lowers blood pressure in ANG-II dependent hypertension.