Reactivity of renal and mesenteric resistance vessels to angiotensin II is mediated by NOXA1/NOX1 and superoxide signaling.

Activation of NADPH oxidase (NOX) enzymes and the generation of reactive oxygen species and oxidative stress regulate vascular and renal function and contribute to the pathogenesis of hypertension. The present study examined the role of NOXA1/NOX1 function in vascular reactivity of renal and mesenteric resistance arteries/arterioles of wild-type and Noxa1-/- mice. A major finding was that renal blood flow is less sensitive to acute stimulation by angiotensin II (ANG II) in Noxa1-/- mice compared with wild-type mice, with a direct action on resistance arterioles independent of nitric oxide (NO) bioavailability. These functional results were reinforced by immunofluorescence evidence of NOXA1/NOX1 protein presence in renal arteries, afferent arterioles, and glomeruli as well as their upregulation by ANG II. In contrast, the renal vascular response to the thromboxane mimetic U46619 was effectively blunted by NO and was similar in both mouse genotypes and thus independent of NOXA1/NOX1 signaling. However, phenylephrine- and ANG II-induced contraction of isolated mesenteric arteries was less pronounced and buffering of vasoconstriction after acetylcholine and nitroprusside stimulation was reduced in Noxa1-/- mice, suggesting endothelial NO-dependent mechanisms. An involvement of NOXA1/NOX1/O2•- signaling in response to ANG II was demonstrated with the specific NOXA1/NOX1 assembly inhibitor C25 and the nonspecific NOX inhibitor diphenyleneiodonium chloride in cultured vascular smooth muscle cells and isolated mesenteric resistance arteries. Collectively, our data indicate that the NOX1/NOXA1/O2•- pathway contributes to acute vasoconstriction induced by ANG II in renal and mesenteric vascular beds and may contribute to ANG II-induced hypertension.NEW & NOTEWORTHY Renal reactivity to angiotensin II (ANG II) is mediated by superoxide signaling produced by NADPH oxidase (NOX)A1/NOX1. Acute vasoconstriction of renal arteries by ANG was blunted in Noxa1-/- compared with wild-type mice. NOXA1/NOX1/O2•- signaling was also observed in ANG II stimulation of vascular smooth muscle cells and isolated mesenteric resistance arteries, indicating that it contributes to ANG II-induced hypertension. A NOXA1/NOX1 assembly inhibitor (C25) has been characterized that inhibits superoxide production and ameliorates the effects of ANG II.

Renal autoregulation in health and disease.

Intrarenal autoregulatory mechanisms maintain renal blood flow (RBF) and glomerular filtration rate (GFR) independent of renal perfusion pressure (RPP) over a defined range (80-180 mmHg). Such autoregulation is mediated largely by the myogenic and the macula densa-tubuloglomerular feedback (MD-TGF) responses that regulate preglomerular vasomotor tone primarily of the afferent arteriole. Differences in response times allow separation of these mechanisms in the time and frequency domains. Mechanotransduction initiating the myogenic response requires a sensing mechanism activated by stretch of vascular smooth muscle cells (VSMCs) and coupled to intracellular signaling pathways eliciting plasma membrane depolarization and a rise in cytosolic free calcium concentration ([Ca(2+)]i). Proposed mechanosensors include epithelial sodium channels (ENaC), integrins, and/or transient receptor potential (TRP) channels. Increased [Ca(2+)]i occurs predominantly by Ca(2+) influx through L-type voltage-operated Ca(2+) channels (VOCC). Increased [Ca(2+)]i activates inositol trisphosphate receptors (IP3R) and ryanodine receptors (RyR) to mobilize Ca(2+) from sarcoplasmic reticular stores. Myogenic vasoconstriction is sustained by increased Ca(2+) sensitivity, mediated by protein kinase C and Rho/Rho-kinase that favors a positive balance between myosin light-chain kinase and phosphatase. Increased RPP activates MD-TGF by transducing a signal of epithelial MD salt reabsorption to adjust afferent arteriolar vasoconstriction. A combination of vascular and tubular mechanisms, novel to the kidney, provides for high autoregulatory efficiency that maintains RBF and GFR, stabilizes sodium excretion, and buffers transmission of RPP to sensitive glomerular capillaries, thereby protecting against hypertensive barotrauma. A unique aspect of the myogenic response in the renal vasculature is modulation of its strength and speed by the MD-TGF and by a connecting tubule glomerular feedback (CT-GF) mechanism. Reactive oxygen species and nitric oxide are modulators of myogenic and MD-TGF mechanisms. Attenuated renal autoregulation contributes to renal damage in many, but not all, models of renal, diabetic, and hypertensive diseases. This review provides a summary of our current knowledge regarding underlying mechanisms enabling renal autoregulation in health and disease and methods used for its study.

NOXA1-dependent NADPH oxidase 1 signaling mediates angiotensin II activation of the epithelial sodium channel.

The activity of the epithelial Na+ channel (ENaC) in principal cells of the distal nephron fine-tunes renal Na+ excretion. The renin-angiotensin-aldosterone system modulates ENaC activity to control blood pressure, in part, by influencing Na+ excretion. NADPH oxidase activator 1-dependent NADPH oxidase 1 (NOXA1/NOX1) signaling may play a key role in angiotensin II (ANG II)-dependent activation of ENaC. The present study aimed to explore the role of NOXA1/NOX1 signaling in ANG II-dependent activation of ENaC in renal principal cells. Patch-clamp electrophysiology and principal cell-specific Noxa1 knockout (PC-Noxa1 KO) mice were used to determine the role of NOXA1/NOX1 signaling in ANG II-dependent activation of ENaC. The activity of ENaC in the luminal plasma membrane of principal cells was quantified in freshly isolated split-opened tubules using voltage-clamp electrophysiology. ANG II significantly increased ENaC activity. This effect was robust and observed in response to both acute (40 min) and more chronic (48-72 h) ANG II treatment of isolated tubules and mice, respectively. Inhibition of ANG II type 1 receptors with losartan abolished ANG II-dependent stimulation of ENaC. Similarly, treatment with ML171, a specific inhibitor of NOX1, abolished stimulation of ENaC by ANG II. Treatment with ANG II failed to increase ENaC activity in principal cells in tubules isolated from the PC-Noxa1 KO mouse. Tubules from wild-type littermate controls, though, retained their ability to respond to ANG II with an increase in ENaC activity. These results indicate that NOXA1/NOX1 signaling mediates ANG II stimulation of ENaC in renal principal cells. As such, NOXA1/NOX1 signaling in the distal nephron plays a central role in Na+ homeostasis and control of blood pressure, particularly as it relates to regulation by the renin-ANG II axis.NEW & NOTEWORTHY Activity of the epithelial Na+ channel (ENaC) in the distal nephron fine-tunes renal Na+ excretion. Angiotensin II (ANG II) has been reported to enhance ENaC activity. Emerging evidence suggests that NADPH oxidase (NOX) signaling plays an important role in the stimulation of ENaC by ANG II in principal cells. The present findings indicate that NOX activator 1/NOX1 signaling mediates ANG II stimulation of ENaC in renal principal cells.

Evaluation of a SPLUNC1-derived peptide for the treatment of cystic fibrosis lung disease.

In cystic fibrosis (CF) lungs, epithelial Na+ channel (ENaC) hyperactivity causes a reduction in airway surface liquid volume, leading to decreased mucocilliary clearance, chronic bacterial infection, and lung damage. Inhibition of ENaC is an attractive therapeutic option. However, ENaC antagonists have failed clinically because of off-target effects in the kidney. The S18 peptide is a naturally occurring short palate lung and nasal epithelial clone 1 (SPLUNC1)-derived ENaC antagonist that restores airway surface liquid height for up to 24 h in CF human bronchial epithelial cultures. However, its efficacy and safety in vivo are unknown. To interrogate the potential clinical efficacy of S18, we assessed its safety and efficacy using human airway cultures and animal models. S18-mucus interactions were tested using superresolution microscopy, quartz crystal microbalance with dissipation, and confocal microscopy. Human and murine airway cultures were used to measure airway surface liquid height. Off-target effects were assessed in conscious mice and anesthetized rats. Morbidity and mortality were assessed in the ß-ENaC-transgenic (Tg) mouse model. Restoration of normal mucus clearance was measured in cystic fibrosis transmembrane conductance regulator inhibitor 172 [CFTR(inh)-172]-challenged sheep. We found that S18 does not interact with mucus and rapidly penetrated dehydrated CF mucus. Compared with amiloride, an early generation ENaC antagonist, S18 displayed a superior ability to slow airway surface liquid absorption, reverse CFTR(inh)-172-induced reduction of mucus transport, and reduce morbidity and mortality in the ß-ENaC-Tg mouse, all without inducing any detectable signs of renal toxicity. These data suggest that S18 is the first naturally occurring ENaC antagonist to show improved preclinical efficacy in animal models of CF with no signs of renal toxicity.

Modulation of the myogenic mechanism: concordant effects of NO synthesis inhibition and O2- dismutation on renal autoregulation in the time and frequency domains.

Renal blood flow autoregulation was investigated in anesthetized C57Bl6 mice using time- and frequency-domain analyses. Autoregulation was reestablished by 15 s in two stages after a 25-mmHg step increase in renal perfusion pressure (RPP). The renal vascular resistance (RVR) response did not include a contribution from the macula densa tubuloglomerular feedback mechanism. Inhibition of nitric oxide (NO) synthase [N(G)-nitro-l-arginine methyl ester (l-NAME)] reduced the time for complete autoregulation to 2 s and induced 0.25-Hz oscillations in RVR. Quenching of superoxide (SOD mimetic tempol) during l-NAME normalized the speed and strength of stage 1 of the RVR increase and abolished oscillations. The slope of stage 2 was unaffected by l-NAME or tempol. These effects of l-NAME and tempol were evaluated in the frequency domain during random fluctuations in RPP. NO synthase inhibition amplified the resonance peak in admittance gain at 0.25 Hz and markedly increased the gain slope at the upper myogenic frequency range (0.06-0.25 Hz, identified as stage 1), with reversal by tempol. The slope of admittance gain in the lower half of the myogenic frequency range (equated with stage 2) was not affected by l-NAME or tempol. Our data show that the myogenic mechanism alone can achieve complete renal blood flow autoregulation in the mouse kidney following a step increase in RPP. They suggest also that the principal inhibitory action of NO is quenching of superoxide, which otherwise potentiates dynamic components of the myogenic constriction in vivo. This primarily involves the first stage of a two-stage myogenic response.

Renal vasoconstriction by vasopressin V1a receptors is modulated by nitric oxide, prostanoids, and superoxide but not the ADP ribosyl cyclase CD38.

Renal blood flow (RBF) responses to arginine vasopressin (AVP) were tested in anesthetized wild-type (WT) and CD38(-/-) mice that lack the major calcium-mobilizing second messenger cyclic ADP ribose. AVP (3-25 ng) injected intravenously produced dose-dependent decreases in RBF, reaching a maximum of 25 ± 2% below basal RBF in WT and 27 ± 2% in CD38(-/-) mice with 25 ng of AVP. Renal vascular resistance (RVR) increased 75 ± 6% and 78 ± 6% in WT and CD38(-/-) mice. Inhibition of nitric oxide (NO) synthase with nitro-L-arginine methyl ester (L-NAME) increased the maximum RVR response to AVP to 308 ± 76% in WT and 388 ± 81% in CD38(-/-) (P < 0.001 for both). Cyclooxygenase inhibition with indomethacin increased the maximum RVR response to 125 ± 15% in WT and 120 ± 14% in CD38(-/-) mice (P < 0.001, <0.05). Superoxide suppression with tempol inhibited the maximum RVR response to AVP by 38% in both strains (P < 0.005) but was ineffective when administered after L-NAME. The rate of RBF recovery (relaxation) after AVP was slowed by L-NAME and indomethacin (P < 0.001, <0.005) but was unchanged by tempol. All vascular responses to AVP were abolished by an AVP V1a receptor antagonist. A V2 receptor agonist or antagonist had no effect on AVP-induced renal vasoconstriction. Taken together, the results indicate that renal vasoconstriction by AVP in the mouse is strongly buffered by vasodilatory actions of NO and prostanoids. The vasoconstriction depends on V1a receptor activation without involvement of CD38 or concomitant vasodilatation by V2 receptors. The role of superoxide is to enhance the contractile response to AVP, most likely by reducing the availability of NO rather than directly stimulating intracellular contraction signaling pathways.

Thromboxane-induced renal vasoconstriction is mediated by the ADP-ribosyl cyclase CD38 and superoxide anion.

The present renal hemodynamic study tested the hypothesis that CD38 and superoxide anion (O2(·-)) participate in the vasoconstriction produced by activation of thromboxane prostanoid (TP) receptors in the mouse kidney. CD38 is the major mammalian ADP-ribosyl cyclase contributing to vasomotor tone through the generation of cADP-ribose, a second messenger that activates ryanodine receptors to release Ca(2+) from the sarcoplasmic reticulum in vascular smooth muscle cells. We evaluated whether the stable thromboxane mimetic U-46619 causes less pronounced renal vasoconstriction in CD38-deficient mice and the involvement of O2(·-) in U-46619-induced renal vasoconstriction. Our results indicate that U-46619 activation of TP receptors causes renal vasoconstriction in part by activating cADP-ribose signaling in renal resistance arterioles. Based on maximal renal blood flow and renal vascular resistance responses to bolus injections of U-46619, CD38 contributes 30-40% of the TP receptor-induced vasoconstriction. We also found that the antioxidant SOD mimetic tempol attenuated the magnitude of vasoconstriction by U-46619 in both groups of mice, suggesting mediation by O2(·-). The degree of tempol blockage of U-46619-induced renal vasoconstriction was greater in wild-type mice, attenuating renal vasoconstriction by 40% compared with 30% in CD38-null mice. In other experiments, U-46619 rapidly stimulated O2(·-) production (dihydroethidium fluorescence) in isolated mouse afferent arterioles, an effect abolished by tempol. These observations provide the first in vivo demonstration of CD38 and O2(·-) involvement in the vasoconstrictor effects of TP receptor activation in the kidney and in vitro evidence for TP receptor stimulation of O2(·-) production by the afferent arteriole.

The proteoglycan syndecan 4 regulates transient receptor potential canonical 6 channels via RhoA/Rho-associated protein kinase signaling.

OBJECTIVE: Syndecan 4 (Sdc4) modulates signal transduction and regulates activity of protein channels. Sdc4 is essential for the regulation of cellular permeability. We hypothesized that Sdc4 may regulate transient receptor potential canonical 6 (TRPC6) channels, a determinant of glomerular permeability, in a RhoA/Rho-associated protein kinase-dependent manner. METHODS AND RESULTS: Sdc4 knockout (Sdc4(-/-)) mice showed increased glomerular filtration rate and ameliorated albuminuria under baseline conditions and after bovine serum albumin overload (each P<0.05). Using reverse transcription-polymerase chain reaction and immunoblotting, Sdc4(-/-) mice showed reduced TRPC6 mRNA by 79% and TRPC6 protein by 82% (each P<0.05). Sdc4(-/-) mice showed an increased RhoA activity by 87% and increased phosphorylation of ezrin in glomeruli by 48% (each P<0.05). Sdc4 knockdown in cultured podocytes reduced TRPC6 gene expression and reduced the association of TRPC6 with plasma membrane and TRPC6-mediated calcium influx and currents. Sdc4 knockdown inactivated negative regulatory protein Rho GTPase activating protein by 33%, accompanied by a 41% increase in RhoA activity and increased phosphorylation of ezrin (P<0.05). Conversely, overexpression of Sdc4 reduced RhoA activity and increased TRPC6 protein and TRPC6-mediated calcium influx and currents. CONCLUSIONS: Our results establish a previously unknown function of Sdc4 for regulation of TRPC6 channels and support the role of Sdc4 for the regulation of glomerular permeability.

Renal NOXA1/NOX1 Signaling Regulates Epithelial Sodium Channel and Sodium Retention in Angiotensin II-induced Hypertension.

Aims: NADPH oxidase (NOX)-derived reactive oxygen species (ROS) are implicated in the pathophysiology of hypertension in chronic kidney disease patients. Genetic deletion of NOX activator 1 (Noxa1) subunit of NOX1 decreases ROS under pathophysiological conditions. Here, we investigated the role of NOXA1-dependent NOX1 activity in the pathogenesis of angiotensin II (Ang II)-induced hypertension (AIH) and possible involvement of abnormal renal function. Results: NOXA1 is present in epithelial cells of Henle's thick ascending limb and distal nephron. Telemetry showed lower basal systolic blood pressure (BP) in Noxa1-/-versus wild-type mice. Ang II infusion for 1 and 14 days increased NOXA1/NOX1 expression and ROS in kidney of male but not female wild-type mice. Mean BP increased 30 mmHg in wild-type males, with smaller increases in Noxa1-deficient males and wild-type or Noxa1-/- females. In response to an acute salt load, Na+ excretion was similar in wild-type and Noxa1-/- mice before and 14 days after Ang II infusion. However, Na+ excretion was delayed after 1-2 days of Ang II in male wild-type versus Noxa1-/- mice. Ang II increased epithelial Na+ channel (ENaC) levels and activation in the collecting duct principal epithelial cells of wild-type but not Noxa1-/- mice. Aldosterone induced ROS levels and Noxa1 and Scnn1a expression and ENaC activity in a mouse renal epithelial cell line, responses abolished by Noxa1 small-interfering RNA. Innovation and Conclusion: Ang II activation of renal NOXA1/NOX1-dependent ROS enhances tubular ENaC expression and Na+ reabsorption, leading to increased BP. Attenuation of AIH in females is attributed to weaker NOXA1/NOX1-dependent ROS signaling and efficient natriuresis. Antioxid. Redox Signal. 36, 550-566.

Complex interactions of NO/cGMP/PKG systems on Ca2+ signaling in afferent arteriolar vascular smooth muscle.

Little is known about the effects of nitric oxide (NO) and the cyclic GMP (cGMP)/protein kinase G (PKG) system on Ca(2+) signaling in vascular smooth muscle cells (VSMC) of resistance vessels in general and afferent arterioles in particular. We tested the hypotheses that cGMP-, Ca(2+)-dependent big potassium channels (BK(Ca(2+))) buffer the Ca(2+) response to depolarization by high extracellular KCl and that NO inhibits adenosine diphosphoribose (ADPR) cyclase, thereby reducing the Ca(2+)-induced Ca(2+) release. We isolated rat afferent arterioles, utilizing the magnetized microsphere method, and measured cytosolic Ca(2+) concentration ([Ca(2+)](i)) with fura-2, a preparation in which endothelial cells do not participate in [Ca(2+)](i) responses. KCl (50 mM)-induced depolarization causes an immediate increase in [Ca(2+)](i) of 151 nM. The blockers N(omega)-nitro-L-arginine methyl ester (of nitric oxide synthase), 1,2,4-oxodiazolo-[4,3-a]quinoxalin-1-one (ODQ, of guanylyl cyclase), KT-5823 (of PKG activation), and iberiotoxin (IBX, of BK(Ca(2+)) activity) do not alter the [Ca(2+)](i) response to KCl, suggesting no discernible endogenous NO production under basal conditions. The NO donor sodium nitroprusside (SNP) reduces the [Ca(2+)](i) response to 77 nM; IBX restores the response to control values. These data show that activation of BK(Ca(2+)) in the presence of NO/cGMP provides a brake on KCl-induced [Ca(2+)](i) responses. Experiments with the inhibitor of cyclic ADPR 8-bromo-cyclic ADPR (8-Br-cADPR) and SNP + downstream inhibitors of PKG and BK(Ca(2+)) suggest that NO inhibits ADPR cyclase in intact arterioles. When we pretreat afferent arterioles with 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP; 10 muM), the response to KCl is 143 nM. However, in the presence of both IBX and 8-Br-cGMP, we observe a surprising doubling of the [Ca(2+)](i) response to KCl. In summary, we present evidence for effects of the NO/cGMP/PKG system to reduce [Ca(2+)](i), via activation of BK(Ca(2+)) and possibly by inhibition of ADPR cyclase, and to increase [Ca(2+)](i), by a mechanism(s) yet to be defined.

Connexin 40 mediates the tubuloglomerular feedback contribution to renal blood flow autoregulation.

Connexins are important in vascular development and function. Connexin 40 (Cx40), which plays a predominant role in the formation of gap junctions in the vasculature, participates in the autoregulation of renal blood flow (RBF), but the underlying mechanisms are unknown. Here, Cx40-deficient mice (Cx40-ko) had impaired steady-state autoregulation to a sudden step increase in renal perfusion pressure. Analysis of the mechanisms underlying this derangement suggested that a marked reduction in tubuloglomerular feedback (TGF) in Cx40-ko mice was responsible. In transgenic mice with Cx40 replaced by Cx45, steady-state autoregulation and TGF were weaker than those in wild-type mice but stronger than those in Cx40-ko mice. N omega-Nitro-L-arginine-methyl-ester (L-NAME) augmented the myogenic response similarly in all genotypes, leaving autoregulation impaired in transgenic animals. The responses of renovascular resistance and arterial pressure to norepinephrine and acetylcholine were similar in all groups before or after L-NAME inhibition. Systemic and renal vasoconstrictor responses to L-NAME were also similar in all genotypes. We conclude that Cx40 contributes to RBF autoregulation by transducing TGF-mediated signals to the afferent arteriole, a function that is independent of nitric oxide (NO). However, Cx40 is not required for the modulation of the renal myogenic response by NO, norepinephrine-induced renal vasoconstriction, and acetylcholine- or NO-induced vasodilation.

Mice lacking the ADP ribosyl cyclase CD38 exhibit attenuated renal vasoconstriction to angiotensin II, endothelin-1, and norepinephrine.

ADP ribosyl (ADPR) cyclases comprise a family of ectoenzymes recently shown to influence cytosolic Ca(2+) concentration in a variety of cell types. At least two ADPR cyclase family members have been identified in mammals: CD38 and CD157. We recently found reduced renal vascular reactivity to angiotensin II (ANG II), endothelin-1 (ET-1), and norepinephrine (NE) in the presence of the broad ADPR cyclase inhibitor nicotinamide. We hypothesized that CD38 mediates effects attributed to ADPR cyclase. We found expression of ADPR cyclases CD38 and CD157 mRNA in spleen, thymus, skin, and preglomerular arterioles of wild-type (WT) animals. Mice lacking CD38 showed decreased CD157 expression in most tissues tested. No difference in systolic or mean arterial pressure was observed between strains in either conscious or anesthetized states, whereas heart rate was reduced 10-20% in CD38-/- animals (P < 0.05). During anesthesia, CD38-/- mice had reduced basal renal blood flow (RBF) and urine excretion (P < 0.05). RBF responses to intravenous injection of ANG II, ET-1, and NE were attenuated approximately 50% in CD38-/- vs. WT mice (P < 0.01 for all). The systemic pressor response to ANG II was decreased in the absence of CD38 (P < 0.01), whereas that to NE was normal (P > 0.05); ET-1 was administered at a nonpressor dose. Nicotinamide effectively inhibited ANG II-induced renal vasoconstriction in WT mice (P < 0.001), but had no effect on renal responses to ANG II in CD38-/- mice (P > 0.5). Overall, our observations indicate the presence of two ADPR cyclase family members in renal preglomerular resistance arterioles and the importance of CD38 participation in acute vascular responses to all three vasoconstrictors in the renal microcirculation.

NAADP receptors mediate calcium signaling stimulated by endothelin-1 and norepinephrine in renal afferent arterioles.

The enzyme ADP-ribosyl (ADPR) cyclase plays a significant role in mediating increases in renal afferent arteriolar cytosolic calcium concentration ([Ca(2+)](i)) in vitro and renal vasoconstriction in vivo. ADPR cyclase produces cyclic ADP ribose, a second messenger that contributes importantly to ryanodine receptor-mediated Ca(2+) mobilization in renal vascular responses to several vasoconstrictors. Recent studies in nonrenal vascular smooth muscle cells (VSMC) have shown that nicotinic acid adenine dinucleotide phosphate (NAADP), another second messenger generated by ADPR cyclase, may contribute to Ca(2+) signaling. We tested the hypothesis that a Ca(2+) signaling pathway involving NAADP receptors participates in afferent arteriolar [Ca(2+)](i) responses to the G protein-coupled receptor agonists endothelin-1 (ET-1) and norepinephrine (NE). To test this, we isolated rat renal afferent arterioles and measured [Ca(2+)](I) using fura-2 fluorescence. We compared peak [Ca(2+)](i) increases stimulated by ET-1 and NE in the presence and absence of inhibitors of acidic organelle-dependent Ca(2+) signaling and NAADP receptors. Vacuolar H(+)-ATPase inhibitors bafilomycin A1 and concanamycin A, disruptors of pH and Ca(2+) stores of lysosomes and other acidic organelles, individually antagonized [Ca(2+)](i) responses to ET-1 and NE by 40-50% (P < 0.05). The recently discovered NAADP receptor inhibitor Ned-19 attenuated [Ca(2+)](i) responses to ET-1 or NE by 60-70% (P < 0.05). We conclude that NAADP receptors contribute to both ET-1- and NE-induced [Ca(2+)](i) responses in afferent arterioles, an effect likely dependent on acidic vesicle, possibly involving lysosome, signaling in VSMC in the renal microcirculation.

Regulation of the renal microcirculation by ryanodine receptors and calcium-induced calcium release.

PURPOSE OF REVIEW: Emerging evidence highlights the importance of physiological participation of ryanodine receptors (RyR) and Ca-induced-Ca-release (CICR) from the sarcoplasmic reticulum in Ca signaling and arteriolar contraction in the renal microcirculation. RECENT FINDINGS: Adenosine diphosphate -ribosyl (ADPR) cyclase and its endogenous metabolites cyclic adenosine diphosphate-ribose and nicotinic acid adenine dinucleotide phosphate mobilize intracellular Ca from sarcoplasmic reticulum stores in the renal vasculature via actions on RyR. The ADPR cyclase/cyclic adenosine diphosphate-ribose/RyR/CICR second messenger system mediates significant (>50%) changes in cytosolic Ca concentration ([Ca]i) and contractile function of preglomerular arteries/arterioles during angiotensin II and endothelin-1 stimulation of G-protein coupled receptors. These receptors rapidly activate ADPR cyclase via stimulation of superoxide (O2) production by nicotinamide adenine dinucleotide phosphate oxidases. Basal ADPR cyclase activity and RyR/CICR contribute to [Ca]i responses initiated by Ca entry and by inositol trisphosphate receptor-induced sarcoplasmic reticulum Ca release. Acute [Ca]i responses in isolated afferent arterioles and renal vasoconstriction in vivo are attenuated by more than 50% by pharmacological inhibition of ADPR cyclase or RyR. Similarly, renal vascular reactivity to angiotensin II, endothelin-1 and norepinephrine is attenuated by approximately 50% in mice lacking CD38, the main mammalian ADPR cyclase. CONCLUSION: RyR and CICR are important regulations of Ca signaling and contractile tone of renal resistance arterioles in healthy kidneys. The role of this novel-signaling pathway in pathophysiological mechanisms awaits investigation.

Activation of phospholipase C gamma 1 protects renal arteriolar VSMCs from H2O2-induced cell death.

BACKGROUND: We evaluated the effect of hydrogen peroxide (H2O2) on viability of vascular smooth muscle cells (VSMCs) of renal resistance arterioles and determined whether responses are modulated by activation of PLCgamma1. METHODS: Phospholipase C (PLC)-isozyme protein levels and activity were measured using Western blot analysis and enzymatic production of phosphoinositol 1,4,5-trisphosphate (IP3), respectively. Stimulation of PLCgamma1 was assessed by immunoblots of tyrosine phosphorylation. RESULTS: Cytotoxicity of H2O2 exposure was concentration-dependent (30% death with 250 microM; 87% with 500 microM at 8 h) and time-dependent (7% at 1 h; 30% at 8 h with 250 microM H2O2. Catalase abolished such relations. H2O2 increased PLCgamma1 expression more than that of PLCdelta1 and almost doubled total PLC enzymatic activity between 2 and 8 h, changes prevented by catalase. The PLC inhibitor U73112 (3 microM) enhanced the cytotoxic concentration and time effects of H2O2. In acute studies, H2O2 rapidly caused tyrosine phosphorylation of PLCgamma1. CONCLUSION: H2O2 increased PLCgamma1 expression and almost doubled total PLC activity, changes abolished by catalase. We conclude that H2O2 is cytotoxic to cultured VSMCs of renal preglomerular arterioles, a process that is attenuated by compensatory increases in PLCgamma1 protein level, tyrosine phosphorylation of PLCgamma1 and PLC enzymatic activity to generate IP3.

Phospholipase C expression and activity in smooth muscle cells of renal arterioles and aorta of young, spontaneously hypertensive rats during culture.

BACKGROUND: Phospholipase C (PLC)-beta(1) and -delta(1), but not -gamma(1), protein expressions in fresh renal arterioles and aorta are greater in 6-week-old, spontaneously hypertensive rats (SHRs) versus normotensive Wistar-Kyoto rats (WKYs). This PLC activity is also greater in both vessels of SHRs. In the present study, we tested whether cultured vascular smooth muscle cells (VSMCs) of preglomerular arterioles and aorta accurately reflect strain differences observed in fresh vessels, with VSMCs of SHRs predicted to have higher levels of PLC isozymes and enzyme activity. We assessed the stability of variables over passages 3 to 11. METHODS: The VSMCs were isolated and cultured using standard techniques. The PLC-isozyme protein levels and catalytic activity were determined by Western blot analysis and inositol 1,4,5-trisphosphate (IP(3)) production, respectively. RESULTS: Immunoblots showed expression of PLC-gamma(1) and -delta(1), but not PLC-beta(1), in VSMCs from both vessels. Arteriolar VSMCs of SHRs had three-to-fivefold higher levels of PLC-gamma(1) and -delta(1) during passages 3 to 8. Enzymatic activity in these VSMCs was higher in SHRs versus WKYs, especially during passages 6 to 11. In contrast, cultured aortic VSMCs of SHRs had two-to-threefold lower densities of PLC-gamma(1) and -delta(1) protein. CONCLUSIONS: Compared with fresh resistance arterioles and aorta, cultured VSMCs exhibit changes in PLC-isozyme protein levels and enzyme activity that vary with passage. The differences between cultured VSMCs of SHRs and WKYs do not accurately reflect those in fresh resistance and conduit vessels, either qualitatively or quantitatively. The results of VSMC culture studies should be interpreted with caution and should ideally be compared with more physiologically relevant fresh preparations.

Increased expression and activity of phospholipase C in renal arterioles of young spontaneously hypertensive rats.

BACKGROUND: The aims of this study were to document the presence of phospholipase C (PLC) isozymes beta(1), gamma(1), and delta(1) in freshly isolated renal glomeruli and resistance vessels, to compare their expression and activity to that in aorta, and to contrast values between 6-week-old Wistar-Kyoto (WKY) controls and 6-week-old spontaneously hypertensive rats (SHR) during the developmental phase of genetic hypertension. METHODS: Aorta, preglomerular arterioles, and glomeruli were isolated from 6-week-old rats using standard techniques. PLC isozyme protein level and activity were determined with Western blot analysis and by measuring inositol 1, 4, 5-trisphosphate (IP(3)) production, respectively. RESULTS: Immunoblots indicate that all three PLC isozymes examined are detectable in freshly isolated preglomerular arterioles, glomeruli, and aorta. Increased levels of PLC-beta(1), and -delta(1) were found in all tested vascular tissues of SHR v WKY. No strain difference was noted for PLC-gamma(1). The relative abundance for both groups was glomeruli > preglomerular arterioles = aorta. The strain difference in protein expression correlated with increased PLC activity in each vascular bed of SHR. CONCLUSIONS: Protein levels of PLC-beta(1) and -delta(1) and PLC activity are upregulated in the systemic and renal vasculature in 6-week-old SHR, suggesting a role in exaggerated vascular reactivity during the development of genetic hypertension. A more complete understanding of the physiologic roles of PLC isozymes and their contributions to specific aspects of cellular function should advance our understanding of vascular tone/reactivity and hypertrophy/remodeling in normal and hypertensive states.

Caffeine intake antagonizes salt sensitive hypertension through improvement of renal sodium handling.

High salt intake is a major risk factor for hypertension. Although acute caffeine intake produces moderate diuresis and natriuresis, caffeine increases the blood pressure (BP) through activating sympathetic activity. However, the long-term effects of caffeine on urinary sodium excretion and blood pressure are rarely investigated. Here, we investigated whether chronic caffeine administration antagonizes salt sensitive hypertension by promoting urinary sodium excretion. Dahl salt-sensitive (Dahl-S) rats were fed with high salt diet with or without 0.1% caffeine in drinking water for 15 days. The BP, heart rate and locomotor activity of rats was analyzed and urinary sodium excretion was determined. The renal epithelial Na(+) channel (ENaC) expression and function were measured by in vivo and in vitro experiments. Chronic consumption of caffeine attenuates hypertension induced by high salt without affecting sympathetic nerve activity in Dahl-S rats. The renal α-ENaC expression and ENaC activity of rats decreased after chronic caffeine administration. Caffeine increased phosphorylation of AMPK and decrease α-ENaC expression in cortical collecting duct cells. Inhibiting AMPK abolished the effect of caffeine on α-ENaC. Chronic caffeine intake prevented the development of salt-sensitive hypertension through promoting urinary sodium excretion, which was associated with activation of renal AMPK and inhibition of renal tubular ENaC.

Superoxide enhances Ca2+ entry through L-type channels in the renal afferent arteriole.

Reactive oxygen species regulate cardiovascular and renal function in health and disease. Superoxide participates in acute calcium signaling in afferent arterioles and renal vasoconstriction produced by angiotensin II, endothelin, thromboxane, and pressure-induced myogenic tone. Known mechanisms by which superoxide acts include quenching of nitric oxide and increased ADP ribosyl cyclase/ryanodine-mediated calcium mobilization. The effect(s) of superoxide on other calcium signaling pathways in the renal microcirculation is poorly understood. The present experiments examined the acute effect of superoxide generated by paraquat on calcium entry pathways in isolated rat afferent arterioles. The peak increase in cytosolic calcium concentration caused by KCl (40 mmol/L) was 99±14 nmol/L. The response to this membrane depolarization was mediated exclusively by L-type channels because it was abolished by nifedipine but was unaffected by the T-type channel blocker mibefradil. Paraquat increased superoxide production (dihydroethidium fluorescence), tripled the peak response to KCl to 314±68 nmol/L (P<0.001) and doubled the plateau response. These effects were abolished by tempol and nitroblue tetrazolium, but not by catalase, confirming actions of superoxide and not of hydrogen peroxide. Unaffected by paraquat and superoxide was calcium entry through store-operated calcium channels activated by thapsigargin-induced calcium depletion of sarcoplasmic reticular stores. Also unresponsive to paraquat was ryanodine receptor-mediated calcium-induced calcium release from the sarcoplasmic reticulum. Our results provide new evidence that superoxide enhances calcium entry through L-type channels activated by membrane depolarization in rat cortical afferent arterioles, without affecting calcium entry through store-operated entry or ryanodine receptor-mediated calcium mobilization.