Background People with the human immunodeficiency virus (PWH) have microvascular disease. Because perivascular adipose tissue (PVAT) regulates microvascular function and adipose tissue is inflamed in PWH, we tested the hypothesis that PWH have inflamed PVAT that impairs the function of their small vessels. Methods Subcutaneous small arteries were dissected with or without PVAT from a gluteal skin biopsy from 11 women with treated HIV (WWH) aged < 50 years and 10 matched women without HIV, and studied on isometric myographs. Nitric oxide (NO) and reactive oxygen species (ROS) were measured by fluorescence microscopy. Adipokines and markers of inflammation and ROS were assayed in PVAT. Results PVAT surrounding the small arteries in control women significantly (P < .05) enhanced acetylcholine-induced endothelium-dependent relaxation and NO, and reduced contractions to thromboxane and endothelin-1. However, these effects of PVAT were reduced significantly (P < .05) in WWH whose PVAT released less adiponectin but more markers of ROS and inflammation. Moderation of contractions by PVAT were correlated positively with adipose adiponectin. Conclusions PVAT from WWH has oxidative stress, inflammation, and reduced release of adiponectin, which may contribute to enhanced contractions and therefore could promote small-artery dysfunction.

Nuclear factor erythroid factor E2-related factor 2 (Nrf2) transcribes antioxidant genes that reduce the blood pressure (BP), yet its activation with tert-butylhydroquinone (tBHQ) in mice infused with angiotensin II (Ang II) increased mean arterial pressure (MAP) over the first 4 days of the infusion. Since tBHQ enhanced cyclooxygenase (COX) 2 expression in vascular smooth muscle cells (VSMCs), we tested the hypothesis that tBHQ administration during an ongoing Ang II infusion causes an early increase in microvascular COX-dependent reactive oxygen species (ROS) and contractility. Mesenteric microarteriolar contractility was assessed on a myograph, and ROS by RatioMaster™. Three days of oral tBHQ administration during the infusion of Ang II increased the mesenteric microarteriolar mRNA for p47phox, the endothelin type A receptor and thromboxane A2 synthase, and increased the excretion of 8-isoprostane F2α and the microarteriolar ROS and contractions to a thromboxane A2 (TxA2) agonist (U-46,619) and endothelin 1 (ET1). These were all prevented in Nrf2 knockout mice. Moreover, the increases in ROS and contractility were prevented in COX1 knockout mice with blockade of COX2 and by blockade of thromboxane prostanoid receptors (TPRs). In conclusion, the activation of Nrf2 over 3 days of Ang II infusion enhances microarteriolar ROS and contractility, which are dependent on COX1, COX2 and TPRs. Therefore, the blockade of these pathways may diminish the early adverse cardiovascular disease events that have been recorded during the initiation of Nrf2 therapy.

Angiotensin II can cause oxidative stress and increased blood pressure that result in long term cardiovascular pathologies. Here we evaluated the contribution of cellular senescence to the effect of chronic exposure to low dose angiotensin II in a model that mimics long term tissue damage. We utilized the INK-ATTAC (p16 Ink4a –Apoptosis Through Targeted Activation of Caspase 8) transgenic mouse model that allows for conditional elimination of p16 Ink4a -dependent senescent cells by administration of AP20187. Angiotensin II treatment for 3 weeks induced ATTAC transgene expression in kidneys but not in lung, spleen and brain tissues. In the kidneys increased expression of ATM, p15 and p21 matched with angiotensin II induction of senescence-associated secretory phenotype genes MMP3, FGF2, IGFBP2, and tPA. Senescent cells in the kidneys were identified as endothelial cells by detection of GFP expressed from the ATTAC transgene and increased expression of angiopoietin 2 and von Willebrand Factor, indicative of endothelial cell damage. Furthermore, angiotensin II induced expression of the inflammation-related glycoprotein versican and immune cell recruitment to the kidneys. AP20187-mediated elimination of p16-dependent senescent cells prevented physiologic, cellular and molecular responses to angiotensin II and provides mechanistic evidence of cellular senescence as a driver of angiotensin II effects.

Introduction: Furosemide reduces the glomerular filtration rate (GFR) and increases the renal vascular resistance (RVR) despite inhibiting tubuloglomerular feedback but increases proximal tubule pressure, renin release, and renal nerve activity. Objective: This study tested the hypothesis that the fall in GFR with furosemide is due to volume depletion or activation of angiotensin type 1 (AT1) receptors or renal nerves. Methods: Furosemide was infused for 60 min at 1.0 mg·kg−1·h−1 in groups of 5–8 anesthetized rats. Additional groups received intravenous volume replacement to prevent fluid and Na+ losses or volume replacement plus losartan or plus sham denervation or plus renal denervation or renal nerve deafferentation. Results: At 60 min of infusion, furosemide alone reduced the GFR (–37 ± 4%; p < 0.01). This fall was not prevented by volume replacement or pretreatment with losartan, although losartan moderated the increase in RVR with furosemide (+44 ± 3 vs. +82 ± 7%; p < 0.01). Whereas the GFR fell after furosemide in rats after sham procedure (–31 ± 2%), it was not changed significantly after prior renal deafferentation. Proximal tubule pressure increased significantly but returned towards baseline over 60 min of furosemide, while urine output remained elevated, and GFR and renal blood flow depressed. Conclusions: The fall in GFR over 60 min of furosemide infusion is independent of volume depletion or activation of AT1 receptors but is largely dependent on renal afferent nerves.

Hypothesis: A lack of contraction of cerebral microarterioles to Ang II (“resilience”) depends on cyclooxygenase (COX) and lipocalin type prostaglandin D sythase L-PGDS producing PGD2 that activates prostaglandin D type 1 receptors (DP1Rs) and nitric oxide synthase (NOS). Materials & Methods: Contractions were assessed in isolated, perfused vessels and NO by fluorescence microscopy. Results: The mRNAs of penetrating intraparenchymal cerebral microarterioles versus renal afferent arterioles were >3000-fold greater for L-PGDS and DP1R and 5-fold for NOS III and COX 2. Larger cerebral arteries contracted with Ang II. However, cerebral microarterioles were entirely unresponsive but contracted with endothelin 1 and perfusion pressure. Ang II contractions were evoked in cerebral microarterioles from COX1 –/– mice or after blockade of COX2, L-PGDS or NOS and in deendothelialized vessels but effects of deendothelialization were lost during COX blockade. NO generation with Ang II depended on COX and also was increased by DP1R activation. Conclusion: The resilience of cerebral arterioles to Ang II contractions is specific for intraparenchymal microarterioles and depends on endothelial COX1 and two products that are metabolized by L-PGDS to generate PGD2 that signals via DP1Rs and NO.

BackgroundThe cerebral circulation requires mechanisms to protect it against vasoconstriction since prolonged reductions in cerebral blood flow predispose to stroke or cognitive decline. However, although cerebral microarterioles (CAs) are normally resilient to angiotensin II (Ang II) contractions, prolonged Ang II causes oxidative stress and enhanced contractility in many vascular beds, but direct effects on CAs are not known. In contrast, the renal circulation requires robust responses to adjust renal function to the body’s needs. We reported that contractions of renal afferent arterioles (Affs) are enhanced by superoxide from p47phox/NOX2 or from reduced superoxide dismutase (SOD) expression but diminished by H2O2 from POLDIP2/NOX4. We tested the hypothesis that hypertension itself enhances vasoconstriction of CAs via p47phox/NOX2 or POLDIP2/NOX4 and contrasted Ang II contractions in CAs with Affs.MethodsMice fed high salt were infused with Ang II (1000 ng ·Kg−1 · min−1 sc) or implanted with DOCA (vs sham procedures) for 4 weeks. The MAP of these mice was increased >25 mmHg. Intraparenchymal CAs were isolated from within the frontal cortex and Affs from the outer renal cortex. Vessels were perfused at 40 mmHg and tested with graded Ang II (10−12 to 10−6 M) vs vehicle.ResultsCAs from hypertensive mice had > 50% enhanced expression of p47phox and > 50% reduced expression of SOD1 and 3 but unchanged POLDIP2 and NOX4. CAs from sham mice failed to contract even with the highest concentration of Ang II (10−6 mol · l−1; change in diameter 0 ± 0%) whereas Affs contracted strongly. Ang II contractions were evoked by Ang II in CAs from hypertensive mice infused with Ang II (−7 ± 2%; P < 0.01) or implanted with DOCA (−18 ± 3%; P < 0.01) whereas contractions in Affs were unchanged. However, preventing hypertension with hydralazine, hydrochlorothiazide and reserpine reduced Ang II contractions in Ang II infused mice (−2 ± 0 vs −7 ± 1; P < 0.01) or DOCA mice (−4 ± 1 vs −18 ± 3; P < 0.01). Enhanced Ang II contractions of CAs were prevented in Ang II infused p47phox −/− mice (−1 ± 1 vs −7 ± 3%; P < 0.05) but were maintained in Ang II infused POLDIP2 +/− mice (−7 ± 1 vs −7 ± 3%; NS).ConclusionThe protection of CAs from Ang II contractions breaks down during hypertension per se because of enhanced expression of p47phox/NOX2 and reduced expression of SODs rather than POLDIP2/NOX4 highlighting targets for cerebral protection from hypertensive damage or dementia.

Nitric oxide prevents hypertension yet enhances proximal tubule Na+ reabsorption. Nitric oxide synthase is inhibited by asymmetric dimethylarginine (ADMA) that is metabolized by dimethylarginine dimethylaminohydrolase (DDAH) whose type 1 isoform is expressed abundantly in the proximal tubule (PT). We hypothesize that ADMA metabolized by DDAH-1 inhibits fluid reabsorbtion (Jv) by the proximal tubule. S2 segments of the PT were microperfused between blocks in vivo to assess Jv in anesthetized rats. Compared with vehicle, microperfusion of ADMA or Nω-nitro-l-arginine methyl ester (l-NAME) in the proximal tubule reduced Jv dose dependently. At 10-4 mol/l both reduced Jv by ~40% (vehicle: 3.2 ± 0.7 vs. ADMA: 2.1 ± 0.5, P < 0.01 vs. l-NAME: 1.9 ± 0.4 nl·min-1·mm-1, P < 0.01; n = 10). Selective inhibition of DDAH-1 in rats with intravenous L-257 (60 mg/kg) given 2 h before and L-257 (10-5 mol/l) perfused in the proximal tubule for 5 min reduced Jv by 32 ± 4% (vehicle: 3.2 ± 0.5 vs. L-257: 2.2 ± 0.5 nl·min-1·mm-1; P < 0.01) and increased plasma ADMA by ≈50% (vehicle: 0.46 ± 0.03 vs. L-257: 0.67 ± 0.03 µmol/l, P < 0.0001) without changing plasma symmetric dimethylarginine. Compared with nontargeted control small-interference RNA, knock down of DDAH-1 in mice by 60% with targeted small-interference RNAs (siRNA) reduced Jv by 29 ± 5% (nontargeted siRNA: 2.8 ± 0.20 vs. DDAH-1 knockdown: 1.9 ± 0.31 nl·min-1·mm-1, P < 0.05). In conclusion, fluid reabsorption in the proximal tubule is reduced by tubular ADMA or by blocking its metabolism by DDAH-1. L-257 is a novel regulator of proximal tubule fluid reabsorption.

High salt, Ang II (angiotensin II), and reactive oxygen species enhance progression of chronic kidney disease. We tested the hypothesis that a high salt intake generates specific reactive oxygen species to enhance Ang II contractions of afferent arterioles from mice with reduced renal mass (RRM). C57BL/6 mice were subjected to surgical RRM or sham operations and received 6% or 0.4% NaCl salt diet for 3 months. Ang II contractions were measured in perfused afferent arterioles and superoxide (O2-) and hydrogen peroxide (H2O2) by fluorescence microscopy. RRM enhanced the afferent arteriolar gene expression for p47phox and neutrophil oxidase (NOX) 2 and high salt intake in RRM mice enhanced gene expression for angiotensin type 1 receptors, POLDIP2 and NOX4 and reduced catalase. High salt in mice with RRM enhanced arteriolar O2- and H2O2 generation and maximal contractions to Ang II (10-6 mol/L) that were dependent on O2- because they were prevented by gene deletion of p47phox and on H2O2 because they were prevented by transgenic smooth muscle cell expression of catalase (tgCAT-SMC) and POLDIP2 gene deletion. Three months of tempol normalized arteriolar reactive oxygen species and Ang II contractions. However, arteriolar contractions to lower concentrations of Ang II (10-8 to 10-11 mol/L) were paradoxically inhibited by H2O2 and POLDIP2. In conclusion, both O2- from p47phox/NOX2 and H2O2 from NOX4/POLDIP2 enhance maximal arteriolar Ang II contractions from RRM mice during high salt, but H2O2 and NOX4/POLDIP2 reduce the sensitivity to lower concentrations of Ang II by >100-fold. Tempol prevents all of these changes in function.

Fibroblast growth factors (FGFs) participate in organ development and tissue maintenance, as well as the control of vascular function. The paracrine-acting FGFs are stored in the extracellular matrix, and their release is controlled by a secreted FGF-binding protein (FGF-BP, FGFBP1, and BP1) that modulates FGF receptor signaling. A genetic polymorphism in the human FGFBP1 gene was associated with higher gene expression and an increased risk of familial hypertension. Here, we report on the effects of inducible BP1 expression in a transgenic mouse model. Induction of BP1 expression in adult animals leads to a sustained rise in mean arterial pressure by >30 mm Hg. The hypertensive effect of BP1 expression is prevented by candesartan, an angiotensin II (AngII) receptor antagonist, or by tempol, an inhibitor of reactive oxygen species. In vivo, BP1 expression sensitizes peripheral resistance vessels to AngII constriction by 20-fold but does not alter adrenergic vasoconstriction. FGF receptor kinase inhibition reverses the sensitization to AngII. Also, constriction of isolated renal afferent arterioles by AngII is enhanced after BP1 expression and blocked by FGF receptor kinase inhibition. Furthermore, AngII-mediated constriction of renal afferent arterioles is abolished in FGF2 −/− mice but can be restored by add-back of FGF2 plus BP1 proteins. In contrast to AngII, adrenergic constriction is not affected in the FGF2 −/− model. Proteomics and gene expression analysis of kidney tissues after BP1 induction show that MAPK (mitogen-activated protein kinase) signaling via MKK4 (MAPK kinase 4), p38, and JNK (c-Jun N-terminal kinase) integrates the crosstalk of the FGF receptor and AngII pathways and thus impact vascular tone and blood pressure.

The brain depends on a continuous supply of blood for its oxygenation, whereas the kidney is over-perfused for its metabolic needs in order to provide sufficient plasma to form a glomerular filtrate. Thus, the brain requires resilience to vasoconstriction to prevent ischemia and vascular cognitive impairment, but the mechanisms are unclear. Methods and Results: Single penetrating cerebral microarterioles (CMAs, 12-18μM) were dissected from the frontal cortex and single renal afferent arterioles (RAAs, 8-12μM) from the kidney cortex to investigate the hypothesis that CMAs deploy unique mechanisms to provide resilience to Ang II vasoconstriction. Individual arteriolar genes were assessed by RNAseq or RT-PCR of endothelial cells (ECs). The mRNA for lipocalin type PGD 2 synthase (LPGDS) and the PGD 1 receptor (PD1R) were > 3,000-fold higher in CMAs than RAAs whereas RAAs expressed 3-fold more mRNA for thromboxane A 2 synthase. Both microarterioles had similar expression of AT1Rs. Endothelial cells cultured from these vessels had similar patterns of gene expression. Single isolated perfused RAAs contracted strongly with Ang II (at 10 -6 mol·l -1 ; -47 ± 2%; P<0.005) whereas CMAs were totally resistant to Ang II (0.1 ± 0.1%; NS), yet both contracted similarly to endothelin I or perfusion pressure (n = 6 per group). However, CMAs from COX 1 -/- (vs +/+) mice did contract with Ang II (-15 ± 2 vs 0.1 ± 0.1%; P<0.01) and contracted with Ang II after incubation with parecoxib (vs vehicle) to block COX2 (-7 ± 3 vs 0.1 ± 0.1%; P<0.01) or after dual blockade of COX1 + 2 (-20 ± 2%; P<0.01) or after incubation with AT-56 (vs vehicle) to block LPGDS (-20 ± 3 vs 0.1 ± 0.1%; P<0.01). During LPGDS blockade, incubation of CMAs with BW245c (stable PD1R agonist) reduced Ang II contraction > 65% (-8 ± 2%; P<0.01). In contrast, COX blockade reduced Ang II contractions of RAAs, indicating opposing effects of PGs on cerebral and renal vessels. Measurements of cerebral and renal blood flow and MAP in anesthetized mice confirmed selective renal vasoconstriction with Ang II, yet selective cerebral vasodilation with BW245c. Perspective: Resilience against Ang II vasoconstriction in cerebral arterioles depends on the generation of PGD 2 and could be a therapeutic target for vascular dementia and stroke.

High salt intake, angiotensin II (Ang II), and reactive oxygen species (ROS) enhance progression of chronic kidney disease (CKD). We reported that myogenic contractions of renal afferent arterioles (RAAs) were enhanced by superoxide (O 2 ·- ) generated from p47 phox /NOX2 but inhibited by H 2 O 2 generated from POLDIP2/NOX4. We tested the hypothesis that feeding a high salt diet to mice with the reduced renal mass (RRM) model of CKD generates specific ROS in their RAAs that enhances Ang II contractions. Methods and Results: C57BL/6 mice received surgical RRM or sham operations and 6% or 0.4% NaCl salt for 3 months. Ang II contractions were measured in RAAs perfused at 45 mmHg and superoxide (O 2 ·- ) and H 2 O 2 by fluorescence microscopy. RRM enhanced the gene expression in RAAs for p47 phox and NOX2 and high salt intake in mice with RRM enhanced the gene expression for AT1Rs, POLDIP2 and NOX4 and reduced the gene expression for catalase. Mice with RRM fed a normal salt diet had contractions to 10 -6 mol·l -1 Ang II similar to sham (-56 ± 5 vs -52 ± 5 %; NS). However, RRM mice fed a high salt diet had an enhanced O 2 ·- and H 2 O 2 generation (P<0.005) with Ang II in RAAs and enhanced Ang II maximal contractions (-72 ± 2 vs -45 ± 2%; P<0.005) that were dependent on O 2 ·- from NOX2 since they were prevented in p47 phox -/- mice and on H 2 O 2 from NOX4 since they were prevented in mice with transgenic smooth muscle cell expression of catalase (tg CAT-SMC ), and in POLDIP2 +/- mice. However, RAA contractions to lower concentrations of Ang II (10 -8 to 10 -11 mol·l -1 ) were paradoxically enhanced in tg CAT-SMC vs Wt mice (-17 ± 2 vs -1 ± 1%; P<0.01) and in POLDIP2 +/- vs +/+ mice (-22 ± 3 vs -5 ± 3; P<0.01). Tempol normalized the ROS and Ang II contractions in RAAs from mice with RRM. In conclusion, both O 2 ·- from p47 phox /NOX2 and H 2 O 2 from NOX4/POLDIP2 enhance maximal Ang II contractions of RAAs from mice with RRM fed a high salt diet but H 2 O 2 from NOX4/POLDIP2 reduces the sensitivity to lower concentrations of Ang II by >100-fold and tempol prevents all of these changes Thus, although a high salt intake reduces circulating Ang II, blockade of angiotensin receptors or ROS may prove beneficial for patients with CKD unable to restrict salt.

Endothelial cells (ECs) lose their endothelial specification and gain mesenchymal cell features during endothelial-mesenchymal transition (EndMT). Post-developmental EndMT disrupts EC homeostasis, leading to vascular dysfunction. We found that afferent arterioles from deoxycorticosterone acetate (DOCA)-salt treated mice had > 5 fold upregulation of mRNAs for preproendothelin-1, p47phox ,NOX2 and TGF-β accompanied by microvascular dysfunction. Since these may cause EndMT, we investigated the mechanism in human glomerular endothelial cells (HGECs) treated for 7-21 days with high salt and DOCA. Endothelin-1 (ET-1) in the medium was increased 2.8±0.2 fold by day 7 while the cells gained multiple mesenchymal markers with increased mRNA for alpha-smooth muscle actin (1.78±0.19 and 2.96±0.32 fold, P<0.05 and 0.01, n=3) and transgelin ( 1.96±0.14 and 2.91±0.28 fold, p<0.05 and 0.01, n=3) on day 7 and 21, respectively, and markedly downregulated mRNA for endothelial markers with decreased vascular endothelial cadherin ( 1.99± 0.27 and 2.12±0.24 fold, P<0.05 and 0.005, n=3) and platelet endothelial cell adhesion molecule 1 (1.78±0.26 and 1.94±0.23 fold, P<0.05 and 0.005, n=3). There were parallel changes in protein expression. Dihydroethidium and MitSox fluorescence probes were used to determine intracellular and mitochondria ROS. The fluorescent intensities were increased by 1.89±0.27 and 1.62±0.22 fold (P<0.01, N=6) respectively in the cells treated for 7 days with DOCA-salt accompanied by increased expression of TGF-β and phosphorylated-extracellular signal-regulated kinases (P-ERK 1/2). In conclusion, human glomerular endothelial cells treated with high salt and DOCA for 1-3 weeks have increased cellular and mitochondrial ROS, ET-1, TGF-β and P-ERK that could account for adverse changes of endothelial-mesenchymal transition and associated microvascular dysfunction.

Nuclear factor erythyroid factor 2 (Nrf2) transcribes genes in cultured endothelial cells that reduce reactive oxygen species (ROS) and generate nitric oxide (NO) or metabolize asymmetric dimethylarginine (ADMA), which inhibits NO synthase (NOS). Therefore, we undertook a functional study to test the hypothesis that activation of Nrf2 by tert-butylhydroquinone (tBHQ) preserves microvascular endothelial function during oxidative stress. Wild-type CB57BL/6 (wt), Nrf2 wt (+/+), or knockout (-/-) mice received vehicle (Veh) or tBHQ (0.1%; activator of Nrf2) during 14-day infusions of ANG II (to induce oxidative stress) or sham. MAP was recorded by telemetry. Mesenteric resistance arterioles were studied on isometric myographs and vascular NO and ROS by fluorescence microscopy. ANG II increased the mean arterial pressure (112 ± 5 vs. 145 ± 5 mmHg; P < 0.01) and excretion of 8-isoprostane F2α (2.8 ± 0.3 vs. 3.8 ± 0.3 ng/mg creatinine; P < 0.05) at 12-14 days. However, 12 days of ANG II reduced endothelium-derived relaxation (27 ± 5 vs. 17 ± 3%; P < 0.01) and NO (0.38 ± 0.07 vs. 0.18 ± 0.03 units; P < 0.01) but increased microvascular remodeling, endothelium-derived contractions (7.5 ± 0.5 vs. 13.0 ± 1.7%; P < 0.01), superoxide (0.09 ± 0.03 vs. 0.29 ± 0.08 units; P < 0.05), and contractions to U-46,619 (87 ± 6 vs. 118 ± 3%; P < 0.05), and endothelin-1(89 ± 4 vs. 123 ± 12%; P < 0.05). tBHQ prevented all of these effects of ANG II at 12-14 days in Nrf2+/+ mice but not in Nrf2-/- mice. In conclusion, tBHQ activates Nrf2 to prevent microvascular endothelial dysfunction, remodeling, and contractility, and moderate ADMA and hypertension at 12-14 days of ANG II infusion, thereby preserving endothelial function and preventing hypertension.

Myogenic contractions protect kidneys from barotrauma but are impaired in chronic kidney disease (CKD). Since myogenic contractions are enhanced by superoxide but impaired by hydrogen peroxide, we tested the hypothesis that they are counterregulated by superoxide and H2O2 from NOX2/p47phox and/or NOX4/POLDIP2 in CKD. Myogenic contraction in isolated perfused afferent arterioles from mice with surgical 5/6 nephrectomy or sham operations fed a 6% sodium chloride diet was measured directly while superoxide and H2O2 were measured by fluorescence microscopy. Compared to sham-operated animals, an increase in perfusion pressure of arterioles from CKD mice doubled superoxide (21 versus 11%), increased H2O2 seven-fold (29 versus 4%), and reduced myogenic contractions profoundly (-1 versus -14%). Myogenic contractions were impaired further by PEG-superoxide dismutase or in arterioles from p47phox-/- (versus wild type) mice but became supra-normal by PEG-catalase or in mice with transgenic expression of catalase in vascular smooth muscle cells (-11 versus -1%). Single arterioles from mice with CKD expressed over 40% more mRNA and protein for NOX4 and POLDIP2. Myogenic responses in arterioles from POLDIP2 +/- (versus wild type) mice with CKD had over an 85% reduction in H2O2, but preserved superoxide and a normal myogenic response. Tempol administration to CKD mice for 3 months decreased afferent arteriolar superoxide and H2O2 and maintained myogenic contractions. Thus, afferent arteriolar superoxide generated by NOX2/p47phox opposes H2O2 generated by NOX4/POLDIP2 whose upregulation in afferent arterioles from mice with CKD accounts for impaired myogenic contractions.

The Na+-K+-2Cl(-) cotransporter (NKCC2) on the loop of Henle is the site of action of furosemide. Because outer medullary potassium channel (ROMK) inhibitors prevent reabsorption by NKCC2, we tested the hypothesis that ROMK inhibition with a novel selective ROMK inhibitor (compound C) blocks tubuloglomerular feedback (TGF) and reduces vascular resistance. Loop perfusion of either ROMK inhibitor or furosemide caused dose-dependent blunting of TGF, but the response to furosemide was 10-fold more sensitive (IC50 = 10(-6) M for furosemide and IC50 = 10(-5) M for compound C). During systemic infusion, both diuretics inhibited TGF, but ROMK inhibitor was 10-fold more sensitive (compound C: 63% inhibition; furosemide: 32% inhibition). Despite blockade of TGF, 1 h of constant systemic infusion of both diuretics reduced the glomerular filtration rate (GFR) and renal blood flow (RBF) by 40-60% and increased renal vascular resistance (RVR) by 100-200%. Neither diuretic altered blood pressure or hematocrit. Proximal tubule hydrostatic pressures (PPT) increased transiently with both diuretics (compound C: 56% increase; furosemide: 70% increase) but returned to baseline. ROMK inhibitor caused more natriuresis (3,400 vs. 1,600% increase) and calciuresis (1,200 vs. 800% increase) but less kaliuresis (33 vs. 167% increase) than furosemide. In conclusion, blockade of ROMK or Na+-K+-2Cl(-) transport inhibits TGF yet increases renal vascular resistance. The renal vasoconstriction was independent of volume depletion, blood pressure, TGF, or P-PT.

Progression of chronic kidney disease (CKD) is related to reactive oxygen species (ROS), impaired arteriolar myogenic contraction and angiotensin II (Ang II). Hence, we tested the hypothesis that ROS modulate the Ang II contractions of afferent arterioles from mice with reduced renal mass (RRM). C57BL/6 mice were subjected to 5/6 surgical nephrectomy or sham operations and fed a 6% salt diet for 3 months. Alternate groups received 2 mmol ·l −1 tempol in drink water. Ang II contractions were measured directly in afferent arterioles and O 2 − and H 2 O 2 by fluorescence microscopy during applying 10 −6 mol·L −1 of Ang II. Compared to sham, mice with RRM had increased O 2 − and (45.2 ± 4.9% versus 15.0 ± 3.2%; p<0.01), increased H 2 O 2 (35.5 ± 5.0% versus 5.6 ± 0.4%, p<0.01) and enhanced contractions (−72.1 ± 2.5% versus −45.9 ± 3.6%; p<0.05) with 10 −6 M of Ang II. Reduction of arteriolar O 2 − (3.7 ± 2.4 versus 45.2 ± 4.9%, p<0.005) in p47 phox −/− mice with RRM blunted the contractions (−35.3 ± 6.0 versus −72.1 ± 2.5%, p<0.005) independent of Ang II concentrations. In contrast, arteriolar contractions in mice with RRM were blunted at high dose (10 −6 mol·L −1 ) but enhanced at low dose (10 −9 mol·L −1 ) of Ang II by metabolism of H 2 O 2 with bath addition of PEG‐catalase (high dose, −55.1 ± 7.1% versus −72.1 ± 2.5%, p<0.005; low dose, −31.1 ± 7.8% versus 10.1 ± 6.8%, p<0.05) or by transgenic overexpression of catalase in vascular smooth muscle cells (high dose, −52.8 ± 1.8% versus −72.1 ± 2.5%, p<0.005; low dose, −19.2 ± 4.6% versus 10.1 ± 6.8%, p<0.05). Therefore, arteriolar H 2 O 2 had Ang II concentration‐dependent effects that contractions are enhanced at high dose but blunted at low dose. Ang II contractions in POLDIP‐2 +/− mice with RRM that did not show an increase in arteriolar H 2 O 2 were similar to POLDIP‐2 +/− sham mice. Three months administration of tempol normalized ROS generation and contractions. The afferent arterioles in mice with RRM expressed much more mRNAs for p47 Phox (155 ± 14% versus 100 ± 3, p<0.05), NOX2 (151 ± 16% versus 100 ± 5, p<0.05), Poldip2 (150 ± 17% versus 100 ± 3%, p<0.05), NOX4 (138 ± 12% versus 100 ± 4%, p<0.05) and AT 1 R (192 ± 6% versus 100 ± 15%, p<0.005) but no change in AT 2 R mRNA. We concluded that afferent arterioles from mice with RRM had a ROS and/or AT 1 R associated and Ang II concentration‐dependent enhancement of arteriolar contraction, which were normalized by tempol.

Background: DOCA/uninephrectomy/high salt (DOCA) is a model of hypertensive nephropathy. Afferent arteriolar myogenic responses prevent hypertensive renal barotrauma but myogenic tone is blocked by vascular generation of H 2 O 2 . Since thromboxane-prostanoid receptors (TP-Rs) generate H 2 O 2 , we tested the hypothesis that they mediate hypertensive nephropathy. Methods: DOCA and Sham TP-R +/+ and -/- mice (n=6/group) were studied at 2 weeks and myogenic responses recorded from the diameter of perfused single afferent arterioles studied in a bath preparation during increased perfusion pressure (40 to 80 mmHg). Results: DOCA treatment in TP-R +/+ mice increased (p<0.001) 24-hour excretion of H 2 O 2 (45 ± 3 vs 220 + 15 nmol) , TxB 2 (4 ± 2 vs 29 ± 4 pmol) and albumin (20 ± 5 vs 270 ± 20 mg) and increased MAP by 35 ± 5 mmHg. However, all effects of DOCA were prevented in TP-R -/- mice. Sham treatment had no effect in TPR +/+ or -/- mice. Myogenic responses were severely impaired in DOCA vs sham WT mice (Δ diameter: -4 ± 1 vs -8 ± 1%; p< 0.005). Myogenic responses also were reduced by incubation of arterioles with 10 -10 mol·l -1 of the TP-R mimetic, U-46,619 vs vehicle added to the bath for 10 minutes (Δ diameter: -7 ± 1 vs -10 ± 1%; p<0.01) and in WT mice infused for 3 days with U-46,619 (500 ng·kg -1 ·d -1 x 3) vs vehicle (Δ diameter: -3 ± 1 vs -10 ± 1%; p<0.005). Conclusion: Hypertensive nephropathy is dependent on TP-Rs that mediate the increase in H 2 O 2 and blood pressure and likely the impaired myogenic responses that expose the kidney to barotrauma

Myogenic contraction is the principal component of renal autoregulation that protects the kidney from hypertensive barotrauma. Contractions are initiated by a rise in perfusion pressure that signals a reduction in membrane potential (Em) of vascular smooth muscle cells to activate voltage-operated Ca(2+) channels. Since ROS have variable effects on myogenic tone, we investigated the hypothesis that superoxide (O2 (·-)) and H2O2 differentially impact myogenic contractions. The myogenic contractions of mouse isolated and perfused single afferent arterioles were assessed from changes in luminal diameter with increasing perfusion pressure (40-80 mmHg). O2 (·-), H2O2, and Em were assessed by fluorescence microscopy during incubation with paraquat to increase O2 (·-) or with H2O2 Paraquat enhanced O2 (·-) generation and myogenic contractions (-42 ± 4% vs. -19 ± 4%, P < 0.005) that were blocked by SOD but not by catalase and signaled via PKC. In contrast, H2O2 inhibited the effects of paraquat and reduced myogenic contractions (-10 ± 1% vs. -19 ± 2%, P < 0.005) and signaled via PKG. O2 (·-) activated Ca(2+)-activated Cl(-) channels that reduced Em, whereas H2O2 activated Ca(2+)-activated and voltage-gated K(+) channels that increased Em Blockade of voltage-operated Ca(2+) channels prevented the enhanced myogenic contractions with paraquat without preventing the reduction in Em Myogenic contractions were independent of the endothelium and largely independent of nitric oxide. We conclude that O2 (·-) and H2O2 activate different signaling pathways in vascular smooth muscle cells linked to discreet membrane channels with opposite effects on Em and voltage-operated Ca(2+) channels and therefore have opposite effects on myogenic contractions.