There is growing evidence that oxidative stress contributes to hypertension. Oxidative stress can precede the development of hypertension. In almost all models of hypertension, there is oxidative stress that, if corrected, lowers BP, whereas creation of oxidative stress in normal animals can cause hypertension. There is overexpression of the p22phox and Nox-1 components of NADPH oxidase and reduced expression of extracellular superoxide dismutase (EC-SOD) in the kidneys of ANG II-infused rodents, whereas there is overexpression of p47phox and gp91phox and reduced expression of intracellular SOD with salt loading. Several mechanisms have been identified that can make oxidative stress self-sustaining. Reactive oxygen species (ROS) can enhance afferent arteriolar tone and reactivity both indirectly via potentiation of tubuloglomerular feedback and directly by microvascular mechanisms that diminish endothelium-derived relaxation factor/nitric oxide responses, generate a cyclooxygenase-2-dependent endothelial-derived contracting factor that activates thromboxane-prostanoid receptors, and enhance vascular smooth muscle cells reactivity. ROS can diminish the efficiency with which the kidney uses O2 for Na+ transport and thereby diminish the Po2 within the kidney cortex. This may place a break on further ROS generation yet could further enhance vasculopathy and hypertension. There is a tight relationship between oxidative stress in the kidney and the development and maintenance of hypertension.
- reactive oxygen species
- superoxide anion
- nitric oxide synthase
- thromboxane-prostanoid receptors
- salt sensitivity
oxidative stress implies an increased production, or a decreased scavenging or metabolism, of reactive oxygen species (ROS). Pioneering work by Harrison, Griendling, and colleagues (53, 162) established that ANG II-induced hypertension in the rat is accompanied by oxidative stress in blood vessels. They showed further that administration of forms of superoxide dismutase (SOD) that interact with endothelial cells reduce blood pressure (BP) in this model but not in rats with norepinephrine-induced hypertension, which do not develop oxidative stress (102). They implicated increased activity of reduced NADP (NADPH) oxidase in the generation of oxidative stress in the blood vessel wall. Subsequent studies by Schiffrin and Touyz (76, 155, 161, 235), Harrison (193), Webb (11), Manning (122), Schmid-Schobein (201) and colleagues showed further that oxidative stress is engendered during mineralocorticoid (MC)-salt hypertension and salt-loaded Dahl salt-sensitive or spontaneously hypertensive stroke-prone rats (SHRsp), in which the renin-angiotensin-aldosterone system (RAAS) is suppressed, thereby widening the role of oxidative stress in hypertension. Indeed, an increase in salt intake without accompanying hypertension also can enhance renal NADPH oxidase, impair SOD, and cause oxidative stress (91).
Many authors have shown that antioxidants can diminish not only the increase in BP but also the inflammation, fibrosis, sclerosis, and dysfunction of the heart, kidneys, and other organs of certain hypertensive models (10, 50, 58, 59, 67, 68, 90, 92, 93, 100, 126, 158, 177, 195, 215–217, 258–261, 268). However, in one study of oxidative stress, caused by prolonged infusion of endothelin-1 (ET-1) into rats, the hypertension was resistant to an effective antioxidant regimen with Tempol (43). This conflicts with a prior study in which Tempol administered to ET-1 infused rats abolished the increase in blood pressure and renal vascular resistance, while it corrected the abnormal lipid peroxidation (186). The cellular and organ-sparing effects of antioxidants in hypertensive models appear to be at least partially independent of any BP lowering (25, 92, 256, 265). Studies of human subjects with essential or renovascular hypertension report evidence of oxidative stress (96) that underlies the endothelial dysfunction of forearm blood vessels observed in vivo (54, 154) or of isolated vessels studied ex vivo (66, 240). Inhibition of nitric oxide synthase (NOS) causes hypertension and renal and systemic vasoconstriction in normal human subjects, which demonstrates the importance of ongoing nitric oxide (NO) generation for the maintenance of normal BP and blood flow (21, 72, 224). The evidence of the primary role of the kidneys in setting the long-term level of BP (32, 64) has focused attention on renal mechanisms of hypertension mediated through oxidative stress and NO deficiency. This is the subject of the present review, which places special emphasis on studies from the author's laboratory that formed the basis for the Starling lecture.
Among ROS, attention has centered on the free radical, highly reactive superoxide anion (O2−·) and the more stable hydrogen peroxide (H2O2). Reaction of H2O2 with metals, notably Fe2+, leads directly to the formation of the highly reactive hydroxyl radical (·OH−). ·OH− is also generated by a reaction between O2−· and NO (74). The reactions of O2−· or ·OH− with NO not only lead to NO bioinactivation (63, 94), and thereby to endothelial dysfunction (173), but to the generation of highly oxidative and nitrosating species, including peroxynitrite (ONOO−) (221, 234). Hypochlorous acid (HOCl−) is generated by myeloperoxidase in activated phagocytes and can circulate to cause widespread endothelial dysfunction (42). These species represent only some of the proximate ROS. Their further reaction with cell products can lead to long-lasting mediators. These include oxidized low-density lipoprotein (LDL) formed from the interaction of H2O2 or ·OH− with LDL, or isoprostanes formed by the interaction of O2−· with arachidonic acid, or advanced glycation end products formed by the interaction of ROS with carbohydrate moieties, or carbonyl species formed by the interaction of ROS with proteins, or oxidized DNA and its products, or nitrosated tyrosine epitopes on proteins. Because presently less is known of the importance of these “second-order” oxidation products in the hypertensive kidney, precedence is given to O2−· and H2O2.
This review aims to summarize evidence linking ROS to hypertension, with an emphasis on renal and vascular mechanisms.
CAN ROS CAUSE HYPERTENSION?
Four lines of evidence indicate that ROS can cause hypertension. First, interventions designed to cause oxidative stress lead to the gradual development of hypertension. Vaziri and colleagues have documented two examples. Lead promotes ·OH−and O2−· generation and lipid peroxidation in cultured aortic and vascular smooth muscle cells (39, 138). Rats given lead in their drinking water develop oxidative stress and nitrotyrosine deposition in their blood vessels and organs (230) and hypertension (62), despite upregulation of endothelial nitric oxide synthase (eNOS) (228, 229). The effect appears to be a direct action of lead to induce ROS generation since there are no major changes in the expression of NADPH oxidase, glutathione peroxidase, SOD, or catalase (231). The hypertension depends on ROS, as it can be prevented by coadministration of a free radical scavenger, vitamin E (230) or of an OH− scavenger, dimethylthiourea (40). In a second model, rats given buthionine sulfoximine for 2 wk in the drinking water to deplete glutathione likewise develop oxidative stress, nitrotyrosine deposition, and hypertension (233, 282). Again, the hypertension apparently depends on ROS, as it is prevented by coadministering vitamins E and C (233).
Second are observations that oxidative stress can precede hypertension. Studies in 4-wk-old SHR show enhanced renal expression of the p47phox component of NADPH oxidase and increased plasma levels of lipid peroxidation products, whereas BP does not rise until after this age (25).
Third are studies of gene-deleted mice. Deletion of extracellular superoxide dismutase (EC-SOD) leads to oxidative stress and increased basal blood pressure whether measured while conscious with blood pressure telemetry or under anesthesia (31, 251). Gene transfer of EC-SOD into SHR reduces their BP (31). Furthermore, deletion of the gene for fibulin-5, which is essential for binding of EC-SOD to vascular tissue (137), also raises the BP of mice (135, 273). Because EC-SOD metabolizes O2−· to H2O2, these findings relate hypertension specifically to an increase in O2−·, or a decrease in H2O2, in the extracellular environment. EC-SOD activity depends on a supply of Ca2+ by the Ca2+-binding protein Atox-1. Remarkably, fibroblasts from Atox-1 -/- mice not only have defective function of EC-SOD but also have a greatly reduced EC-SOD expression, whereas both Atox-1 and EC-SOD are upregulated in blood vessels of mice with atherosclerosis (84). Clearly, Atox-1 and Fibulin-5 are important regulators of vascular EC-SOD, but their effects in the kidney have not been studied.
Fourth are many reports that the correction of the oxidative stress that accompanies a variety of hypertensive models also corrects hypertension (10, 50, 58, 59, 67, 68, 90, 92, 93, 126, 158, 177, 195, 215–217, 258–261, 268).
RENAL GENERATION OF ROS DURING HYPERTENSION
Evidence of increased ROS in the hypertensive kidney comes from three primary sources. First are observations of increased steady-state renal excretion of lipid peroxidation markers in hypertensive animals. This provides quantitative evidence of increased ROS, but increased renal excretion is not in itself evidence of renal ROS production. 8-Isoprostane PGF2α and malondialdehyde (MDA) excretion are increased by prolonged infusion of ANG II into rats (24, 248) or mice (89) and in ANG II-dependent forms of hypertension, including the spontaneously hypertensive rat (SHR) (256) and the two-kidney one-clip (2K,1C) Goldblatt model of renovascular hypertension (249). Equally impressive increases are found in volume-dependent models, including Dahl salt-sensitive (122, 124, 125, 210) and DOCA-salt (11) rats. The SHR and ANG II-infused rabbit have increased plasma levels of lipid peroxidation products (LPOs) (25, 238).
Second are observations of increased renal tissue generation of ROS or ROS-dependent products. There is increased renal nitrotyrosine immunoreactivity in kidneys of SHR (253), 2K,1C rats (13), those with aortic banding (9), lead-induced oxidative stress and hypertension (230), and chronic renal insufficiency (232). Interestingly, nitrotyrosine immunoreactivity in the 2K,1C model is located preferentially in the postclip kidney around cells of the macula densa, whereas in the contralateral kidney, it is located preferentially within the walls of the afferent arteriole (13). These are sites of elevated ANG II since the postclip kidney generates excessive renin within the juxtaglomerular apparatus (JGA), whereas the contralateral kidney is perfused with blood with high renin-angiotensin content. Because nitrotyrosine implies ROS interaction with NO, these findings indicate that ANG II-induced O2−· formation is increased in the JGA of the postclip kidney and the afferent arteriole of the contralateral kidney. ROS in the JGA could be quenched by reaction with NO generated by neuronal nitric oxide synthase (nNOS) expressed in macula densa cells (266), and ROS in the afferent arteriole could be quenched by NO generated by eNOS expressed in the afferent arteriolar endothelial cells (213).
Third are more direct measurements of ROS. Zou and Cowley (284) have shown O2−· production in the medulla of rats. Tojo and colleagues (145) demonstrated increased renal production of H2O2 by an ANG type 1 receptor (AT1-R)-dependent mechanism in rats with insulinopenic diabetes mellitus.
RENAL EXPRESSION OF NOSs, OXIDASES, AND ANTIOXIDANTS IN HYPERTENSION
Endothelial or type III NOS is expressed abundantly in renal vascular endothelium (213), in the thick ascending limb (TAL) of Henle's loop (151) and in the collecting ducts (196). Neuronal NOS (nNOS) type I is expressed abundantly in the macula densa cells (133, 266), Bowman's capsule and the inner medullary collecting ducts (211, 214). The mRNA and protein for both nNOS and eNOS are increased in the JGA of the SHR (252) (Fig. 1). This is accompanied by a two-fold increase in renal cortical nitrotyrosine deposition, indicating enhanced interaction of NO with ROS (253). Dietary salt restriction in normal rats increases the expression of nNOS in the kidney cortex and macula densa (14, 167, 184, 191, 212, 213) but not in the collecting ducts (191). This suggests differential regulation of NOS by salt in the renal cortex and medulla. Indeed, dietary salt restriction diminishes expression of all NOS isoforms in the renal inner medulla (123). Dietary salt restriction enhances the expression of eNOS in the kidney cortex and vascular endothelium (212). The effects of salt on renal cortical nNOS expression are independent of ANG II, since they are unaffected by AT1-receptor blockade during salt restriction or by ANG II infusion during salt loading, whereas similar maneuvers ascribe the effects of salt on the expression of eNOS to ANG II acting on AT1 receptors (212).
NADPH oxidase in activated neutrophils is composed of a membrane-associated heterodimer of the flavoprotein gp91phox with p22phox that is activated by binding of a cytoplasmic component, p47phox, which is itself activated by p67phox and rac-1. The role of p40phox is uncertain (5). NADPH oxidase activity can be reconstituted in vitro using the purified cytosolic factors p47phox, p67phox, and Rac with phospholipid-reconstituted gp91phox and p22phox (49). However, activity can be achieved in the absence of p47phox if there is a high concentration of p67phox and Rac (57). Moreover, homologous components can substitute of gp91phox (nox-1 or -4) (2), and homologs to p47phox and p67phox (p41nox and p51nox) can support O2−· production by NADPH oxidase in some cells (55). The components of neutrophil NADPH oxidase are all present in endothelial cells. However, in these cells, the majority of gp91phox, p22phox, p67phox, and p40phox and of NADPH oxidase activity is located on the intracellular cytoskeleton in a perinuclear region, whereas p47phox is expressed predominantly in cell membranes (108). The functions of these principal NADPH oxidase components have been evaluated thoroughly in the activated phagocyte, where gp91phox, p22phox, and p47phox, p67phox and rac-1 are required to support maximal rates of O2−· generation. Mutations in these subunits in patients with chronic granulomatous disease lead to defective phagocyte killing of bacteria and recurrent infection. However, the requirement of these components for O2−· generation at other sites is incompletely understood and is a subject of intense investigation.
Chabrashvili et al. (25) detected the mRNA and protein for gp91phox, p22phox, p47phox, and p67phox in the rat renal cortex. Immunohistochemical studies located prominent sites of expression of these proteins in the arterioles [vascular smooth muscle cells (VSMCs) and endothelium], the glomerulus, and the distal nephron. Remarkably, all four proteins are expressed strongly at the luminal border of the macula densa cells. Podocyte cells in the glomerulus are prominent sites for p47phox expression. The SHR kidney has increased expression of these components, notably of p47phox protein and mRNA. The increased p47phox is detected in the kidney, coincident with increased plasma LPO in 4-wk-old SHR before the establishment of hypertension.
O2−· and/or H2O2 also can be generated in the kidney and blood vessels by xanthine oxidoreductase (X-OR) (69, 200). When assessed by lucigenin-detected O2−· in response to specific substrates and enzyme antagonists, X-OR activity is negligible in homogenates from the rabbit renal cortex, but in the medulla, is equivalent to NADPH oxidase (238). However, X-OR appears to be regulated differently from NADPH oxidase. Thus NADPH oxidase activity in the kidney cortex (238, 248), but not in the outer medulla, of the rabbit or rat is upregulated by ANG II (238), but X-OR activity is unchanged at either site (238) (Fig. 2).
However, when assessed from fluorogenic oxidation of ethidium, NADH oxidase is of predominant importance in both the cortex and outer medulla of the rat kidney, although there is almost no oxidase activity in the papilla (284). This difference between the studies may relate to the availability of cofactors that determine the interconversion of NADH and NADPH oxidase activity. O2−·· also can be generated by lipoxygenase (78–80, 98, 187, 286), epoxygenase (46), and NOS (98, 286). Uncoupling of eNOS has been detected in blood vessels of hypertensive rats, where oxidation of tetrahydrobiopterin (BH4) to dihydrobiopterin (BH2) by ROS produced by NADPH oxidase (98) or by ·ONOO− (286) directs NOS to synthase O2−·· rather than NO. If a similar reaction occurs in the kidney, this would provide a ready mechanism, whereby generation of O2−·, for example by activation of NADPH oxidase, could further enhance ROS by oxidizing BH4 and uncoupling NOS (98).
There are several examples of discordant signaling by ROS. Although O2−· or ONOO− can oxidize BH4, which can lead to further O2−· generation from NOS, H2O2 can increase the synthesis of BH4 in cultured endothelial cells by transcriptional upregulation of guanosine triphosphate-cytohydrolase I, which is the limiting step in BH4 synthesis (187). O2−· biodegrades NO, whereas H2O2 enhances eNOS expression and NOS activity in cultured endothelial cells sufficiently to increase NO release (20). This can explain how the addition of ANG II to cultured endothelial cells paradoxically increases their NO activity, despite oxidative stress. This increase in NO is prevented by catalase, which metabolizes H2O2 and is absent from cells of p47phox knockout mice (20). Thus the interaction between ROS and NO in vascular tissue is complex and involves both direct effects and compensatory mechanisms. Despite these reservations, direct measurement of O2−· and NO in blood vessels using electron spin paramagnetic resonance demonstrates that 7 days of ANG II infusion into rats doubles the vascular activity of O2−·· but halves the vascular activity of NO (127). Apparently, bioactivation of NO by O2−· is of predominant importance in determining NO activity in blood vessels during prolonged exposure to ANG II in vivo (Fig. 3).
SOD is expressed as three isoforms: mitochondrial (Mn), intracellular (IC), and extracellular (EC) (50). All are expressed in the normal kidney (24, 248). SOD catalyzes the conversion of O2−· to H2O2, thereby changing a highly reactive radical that inactivates NO (87) into a more stable compound whose functions include the release of the endothelium-derived hyperpolarizing factor (EDHF) (71, 112, 175), upregulation of eNOS (20), and hypertrophic and remodeling responses in blood vessels (50). These two ROS have discordant effects in the renal medulla on the generation of hypertension, as BP is increased by medullary H2O2, rather than O2−·, both in acute and in more prolonged studies (30, 120, 121). However, the dominant effect of EC- and IC-SOD activity normally is to metabolize O2−· and thereby prevent hypertension. Thus blockade of glutathione production, which should increase intracellular H2O2, causes progressive hypertension (231, 233). EC-SOD knockout mice have a higher BP during the early phase of high-dose ANG II infusion that can be normalized by acute intravenous injection of EC-SOD (87). SOD mimetic nitroxides, such as Tempol, whether given acutely or during prolonged administration, lower the BP in multiple models of hypertension (176, 178). Therefore, interest is focused on overexpression of NADPH oxidase components, or underexpression of SOD, as potential prohypertensive mechanisms in models of ANG II, MC, or salt-induced hypertension. Indeed vascular EC-SOD is upregulated by NO and by exercise, which could be a component of the antihypertensive effects of these maneuvers (51).
Blood vessels from rats or rabbits infused with ANG II generate increased O2−· and have increased expression of p22phox, p67 phox, and a Nox homolog (24, 53, 56, 99, 127, 218, 237, 238) (Fig. 4). During ANG II infusion, one homolog of gp91 phox, Nox-1, is upregulated in VSMCs (73, 99, 127, 199) and the kidney (24). Another homolog, Nox-4, is upregulated in blood vessels of ANG II-infused rats (127) or in VSMCs from renin-transgenic rats (267) but is downregulated by ANG II in cultured VSMCs from normal rats (99). Studies of normal rats infused with ANG II alone, or with an AT1- or AT2-receptor antagonist, indicate that renal cortical mRNA expression for Nox-4 is downregulated strongly by ANG II. This is ascribed to an inhibitory action of ANG II on Nox-4 expression mediated via AT1-R (24). gp91phox is upregulated by ANG II in blood vessels and endothelial cells (86, 127) and in VSMCs of human resistance arteries (218), but little gp91phox is expressed in the media of human coronary blood vessels (194). Coronary arteries from patients with atherosclerosis have prominent expression of gp91phox in macrophages but prominent expression of Nox-1 in VSMCs (194).
Infusion of ANG II at a slow pressor rate increases NADPH oxidase activity in the renal cortex of the rat (24, 248, 281) and rabbit (238) without significant upregulation of NADH oxidase or X-OR (Fig. 2). There is increased expression of p22phox and Nox-1 and downregulation of Nox-4 (24) (Fig. 4). Renal afferent arterioles of rabbits infused with ANG II have enhanced expression of the mRNA for p22phox (237, 238). These effects of ANG II in the kidney are mediated by AT1-R, as they are prevented by candesartan (24). The upregulation of p22phox and Nox-1 is counteracted by stimulation of AT2-R, which also downregulates the expression of p67phox(24). ANG II infusion in the rat decreases the renal cortical mRNA for EC-SOD yet increases Mn-SOD (24). The decrease in EC-SOD is due to activation of AT1-R (Fig. 4). NADPH oxidase likely is of predominant importance for O2−· generation in the renal cortex since more O2−· is generated with NADPH than with NADH or xanthine as substrates (238, 284). In contrast, NADPH and XO contribute equally to O2−·-generating capacity in the outer medulla of the rabbits (238), but neither increases significantly at this site during prolonged ANG II infusion (238).
There is a marked increase in NO generation in the medulla of the rat during ANG II infusion. Quenching of O2−· by NO in the medulla may account for the absence of an increase in O2−· concentration during ANG II (37, 153, 166, 281, 285). This demonstrates important regional differences in the ANG II-dependent regulation of ROS and NOS, and hence the bioactivity of NO, in the kidney. Nevertheless, despite the absence of a notable increase in oxidative stress in the outer medulla or in isolated, perfused vasa recta of ANG II-infused rats, in situ studies demonstrate that ANG II infusion increases O2−· generation in medullary TAL segments. The O2−· generated at this site interacts with NO in vasa recta to mediate tubulo-vascular crosstalk (284). Also, unlike the renal cortex, dismutation of O2−· in the medulla by Tempol does not always dissipate the effects of local ROS. Apparently, Tempol enhances the medullary levels of H2O2 sufficiently to mediate vasoconstriction and enhance tubular Na+ reabsorption. When given into the medulla, Tempol must be coadministered with catalase to cause vasodilatation or to reduce BP in some studies (30, 120), although in others Tempol alone is effective (284). This is in contrast to the vasodilatation and antihypertensive effects of systemic Tempol, or Tempol applied directly to isolated blood vessels, where catalase is not required to elicit these effects.
A high salt intake increases the NADPH oxidase activity of the rat kidney cortex and increases the excretion of 8-isoprostane PGF2α and MDA, despite profound reductions in the circulating RAAS. Opposite changes occur during a low-salt intake (91). High salt increases the renal cortical expression of mRNA for gp91phox and p47phox and decreases IC- and mitochondriol-SOD. Thus both salt loading and prolonged ANG II can generate oxidative stress and engage the renal machinery for O2−· generation via NADPH oxidase while impairing O2−· metabolism via SOD, but by discrete changes in the components of these systems (Fig. 4). This may explain how infusion of ANG II during a high-salt intake may cause severe oxidative stress, hypertension, and renal damage (22). Remarkably, salt loading and ANG II regulate the same endpoint in these complex systems, but by engaging different components.
Early insulinopenic diabetes mellitus in the rat, which, like salt loading, is a model of low renin and volume excess, enhances the renal expression of gp91phox and p47phox (4). In this model, preventing membrane assembly of p47phox with apocynin prevents an increase in renal and glomerular H2O2 generation and excretion of lipid peroxidation products (4).
DOPAMINERGIC ACTIONS ON ROS
At high concentrations, dopamine and D1-receptor-like agonists act as prooxidants that increase O2−· and H2O2 (12, 26, 60, 117, 241, 257). ROS are produced both by dopamine transporter uptake-dependent and dopamine receptor-independent mechanisms (23, 115). Excessive stimulation of D2 receptors increases ROS production (269). D3 receptors stimulate phospholipase D activity which can activate NADPH oxidase (44).
At physiological concentrations, dopamine acts as an antioxidant. Activation of D4 receptors by apomorphine protects cells against the lethal effects of oxidative stress via generation of cycle guanosine 5′-monophosphate (cGMP)-operated Ca2+ channels (81). Dopamine, acting at the D1 and D5 receptor, decreases oxidative stress in brain cortical cells (139) and renal tubular and vascular smooth muscle cells (274, 277) by inhibition of phospholipase D activity (277) and stimulation of SOD and catalase activities (134). The D5 receptor, which is one of the two members of the mammalian D1-like receptor family, has antioxidant properties in renal proximal tubules and brain mediated by inhibition of phospholipase D2 and NADPH oxidase activity (274). D5 receptor−/− mice are hypertensive and have increased oxidative stress in the kidney and brain (75, 274). Apocynin, a drug that inhibits the assembly of NADPH oxidase subunits, normalizes blood pressure and oxidative stress in the kidney and brain of D5 receptor−/− mice (275). An additional antioxidant mechanism of the D5 receptor may derive from its ability to decrease the expression of the AT1 receptor in renal proximal tubules (279). Thus at physiological concentrations, dopamine protects against hypertension, in part, by activation of D5 receptors that prevent oxidative stress in the kidney.
SELF-SUSTAINING NATURE OF OXIDATIVE STRESS
Several mechanisms have been implicated in sustaining oxidative stress. Stretching blood vessels increases O2−· generation, thereby providing a direct link between hypertension and ROS production in conduit vessels (222). ANG II infusion increases renal cortical expression of the mRNA and protein for p22phox, yet prevention of oxidative stress with Tempol also prevents upregulation of p22phox and NADPH oxidase activity in the kidney (248). This indicates that oxidative stress may enhance expression of p22phox and NADPH oxidase activity itself. Indeed, generation of ROS or addition of H2O2 to endothelial cells in culture increases p38 mitogen-activated protein kinase and phosphatidylinosital-3-kinase and enhances expression of p22phox mRNA and protein (41). ANG II infusion downregulates EC-SOD in the kidney, which should enhance levels of O2−· (24). Oxidative stress can oxidize BH4 sufficiently to uncouple eNOS and thereby direct NOS to generate O2−· rather than NO in blood vessel walls (103, 286). ONOO− generated during oxidative stress can nitrosate and inactivate IC-SOD (1). Indeed, vascular SOD activity in stroke-prone SHR is increased, and NADPH oxidase activity is decreased, after prolonged treatment with vitamins C and E (27). H2O2 stabilizes TP receptors in the membrane, prevents recycling, and, thereby, increases the number of apparent binding sites for TP receptor agonists (223). ANG II infusion reduces prostaglandins and thromboxane A2 (TxA2) (116). Studies in the thromboxane-prostanoid-receptor (TP-R) knockout mouse show that this receptor mediates oxidative stress during a slow pressor infusion of ANG II (88). Thus ANG II induced H2O2 generation can perpetuate vasoconstriction and oxidative stress mediated via the TP. Antioxidants reduce the influx of inflammatory cells into the kidneys of SHR, thereby reducing a major source for further ROS generation (169). These are some of the pathways whereby oxidative stress could become self-sustaining (Fig. 5).
ROS AND RENAL FUNCTION
The role of ROS in the regulation of renal function has been studied from the consequences of enhancing ROS metabolism or inactivation. Intravenous injections (178) or prolonged administration (176) of the permanent SOD mimetic nitroxide, Tempol into SHR reduce BP and renal vascular resistance (RVR). Normotensive Wistar Kyoto (WKY) rats have a blunted acute response and no fall in BP with prolonged Tempol administration (176, 250). Blockade of NOS prevents most of the fall of BP during chronic Tempol infusion in the SHR (178), yet less than half of the response of a bolus dose of Tempol in control WKY (270), SHR (156), or in DOCA-salt rats (271, 271, 272). Thus, unlike chronic treatment, only a part of the acute response to Tempol can be attributed to enhanced bioactivity of NO by a reduction in tissue O2−· concentration. Indeed, the acute fall in MAP is accompanied by bradycardia (178, 271, 272), rather than the tachycardia that is provoked by an infusion of an NO donor compound such as sodium nitroprusside (SNP) (7). These NO-independent effects of acute Tempol have been attributed to reductions in O2−· in peripheral sympathetic nerves that inhibit their activity (270, 272). Direct application to peripheral nerves of the SOD inhibitor diethyldithiocarbamate increases their activity, whereas direct application of Tempol has the opposite effect (188).
The reductions in RVR of SHR infused with Tempol are in proportion to the falls in MAP, thereby maintaining renal blood flow. Similarly, long-term studies in which Tempol is given to SHR (176) or to 2K,1C renovascular hypertensive rats (249), or to ANG II-infused mice (89) or rats (248) show that Tempol is highly effective in moderating the increase in BP, yet reduces RVR only in proportion to the reduction in MAP. These studies in rats did not detect significant changes in the glomerular filtration rate (GFR) with Tempol, although Tempol does increase the GFR of ANG II-infused mice (89). Majid, Nishiyama, and colleagues (118, 119) studied anesthetized ANG II-infused dogs. They confirmed that Tempol reduces RVR acutely and leads to a diuresis and natriuresis. They showed further that the renal vasoconstriction and especially the natriuresis and diuresis with Tempol persist after NOS blockade. They concluded that the tubular effects of systemic Tempol are independent of NO.
Kawada et al. (89) reported that coadministration of Tempol to mice infused with a low dose of ANG II over 2 wk prevented the increase in renal excretion of 8-isoprostane PGF2α, the slow development of hypertension, and the increase in RVR. The reduction in RVR was attributed to preferential inhibition of preglomerular vasoconstriction, since Tempol increased GFR in this mouse model yet maintained the ANG II-induced increase in filtration fraction. The preferential vasoconstriction of afferent arterioles by O2−· in the mouse could represent a direct effect on the renal afferent arteriole or an indirect effect mediated through enhancement of the tubuloglomerular feedback (TGF) response (255, 260, 263).
H2O2 has complex effects on the tone of blood vessels. Preliminary studies in mice have disclosed a biphasic effect of H2O2 on mesenteric and cremasteric vessels in vivo (29). Superfusion of mesenteric blood vessels with H2O2 yields vasodilation in TP-R−/− mice. This may reflect release of an EDHF, as shown in human submucosal intestinal vessels challenged with Ach (71). The vasoconstriction seen in wild-type mice precedes the vasodilation and may reflect stabilization of TP-Rs in the membrane by H2O2, and, hence, facilitation of contractions mediated via the TP-R (223). H2O2 reduces the diameter of isolated, perfused renal afferent arterioles from the rabbit (179). These studies have shown that ROS can have both vasoconstrictor or vasodilator actions depending on conditions. SOD has an important regulatory role in converting O2−·, which is predominantly a vasoconstrictor, to H2O2, which has bifunctional vasodilator/vasoconstrictor properties.
Johnson and colleagues have shown that a 2-wk high dose ANG II infusion in the rat is followed in the succeeding weeks by salt sensitivity (113). Thus the BP of these rats is normal but rises progressively during a high-salt intake. The ANG II infusion leads to a persistent inflammatory cell infiltrate in the renal cortical interstitium that is linked both to the development of salt sensitivity and to oxidative stress in this model. Moreover, salt sensitivity, afferent arteriolar vasoconstriction and remodeling, and oxidative stress are prevented in these rats by mycophenolate mofetil that prevents the inflammatory infiltrate (47, 168). Thus ANG II may induce oxidative stress in the kidney either by upregulation of NADPH oxidase components resident in tubular, glomerular, and vascular cells or by causing a persistent influx of phagocytes that can generate substantial ROS. The interstitial influx of oxidase-bearing cells is particularly important in the oxidative stress that accompanies severe hypertension and renal damage induced by high levels of ANG II (114), whereas ROS generated by resident renal cells is likely predominant in less severe forms of hypertension.
There is a delayed increase in BP during ANG II infusion into p47phox knockout mice (97), whereas there is an exaggerated early increase in BP during ANG II infusion into EC-SOD knockout mice (87). This demonstrates the importance of NADPH oxidase-derived O2−· in the development of hypertension during ANG II infusion. TP-R knockout mice have a blunted rise in BP and no increase in RVR with ANG II (48, 88). Moreover, these mice fail to develop oxidative stress with ANG II infusion (88). Apparently, ANG II activates TP receptors to engage oxidative stress, which can account for about one half of the increase in BP and all of the increase in RVR. The increase in ROS may entail TP-R-dependent proinflammatory actions that enhance cellular immune responses (203). Additionally, TP-Rs can activate protein kinase C (PKC) in VSMCs and kidney glomeruli (33, 65) that upregulates NADPH oxidase and enhances ROS formation in resident renal and vascular cells (143, 244).
AFFERENT ARTERIOLE IN HYPERTENSION
Because structural and functional adaptation of the renal afferent arteriole can contribute to the development of hypertension and may be a focal point for prohypertensive actions of ROS in the kidneys, this review will include an overview of mechanisms of oxidative stress in blood vessels with specific reference to the afferent arteriole in hypertension.
Autopsy studies of patients with hypertension by Moritz and Oldt (130) reveal that the renal afferent arteriole is a preferential site for structural adaptation. The afferent arteriole shows narrowing of the lumen, hypertrophy of the wall, and hyalinization or myointimal proliferation in 98% of those known to have had hypertension during life, compared with only 12% of normotensive subjects. Blood vessels to other organs show a lesser frequency of structured remodeling in hypertension. Tracy and colleagues (219, 220) have perfusion-fixed kidneys and measured the mean afferent arteriolar diameter. They have documented a continuous relationship between reduced luminal diameter of cortical afferent arterioles and the premortem level of BP. Mulvany and colleagues (141) have shown that the lumen of the afferent arteriole is narrowed in hypertensive SHR and hypertensive primates (192). Among F1 hybrid crosses between SHR and WKY, the development of hypertension in adult life is predicted by a reduced afferent arteriolar diameter in the prehypertensive state (141).
The glomerular capillary hydraulic pressure (PGC) and blood flow to outer cortical glomeruli of SHR are well maintained despite the presence of hypertension (82). This implies an increased preglomerular vascular resistance. Studies have confirmed excellent autoregulation of outer cortical blood flow in the SHR during short-term step changes in perfusion pressure (82). However, there is less complete autoregulation of blood flow to juxtamedullary nephrons, which have an elevated PGC and an accelerated rate of glomerulosclerosis (82) and associated tubular damage (142). The extent to which this preglomerular vasoconstriction of SHR outer cortical glomeruli is an autoregulation to sustained hypertension or a primary defect that engenders hypertension is not clearly resolved. Guyton's model of circulatory homeostasis predicts that an increase in afferent arteriolar resistance or TGF responsiveness will displace the BP: body salt relationship to the right, leading to sustained, salt-resistant hypertension (64). This is consistent with the predominantly salt-resistant hypertension of the SHR (61), which has a narrowed afferent arteriole (141) and an enhanced TGF response (252). An enhanced distal NaCl reabsorption is required to add a component of salt sensitivity (32, 64). Moreover, because dietary salt loading can itself increase ROS generation in the kidney and blood vessels (91, 122) and ROS are linked to hypertension, it remains quite possible that oxidative stress could contribute to renal vascular mechanisms of both salt-resistant and salt-sensitive hypertension. Therefore, an enhanced reactivity or remodeling of afferent arterioles under the influence of ROS could underlie hypertension during renal oxidative stress. The importance of renal afferent arteriolar remodeling has been emphasized by Johnson and colleagues (85).
The ANG II slow pressor response is a gradual rise in BP over days or weeks during an infusion of ANG II at an initially subpressor rate. This response is seen in mice (89), rats (16, 105, 106, 116, 146, 147, 248), rabbits (38, 237, 238), and humans (3). It is relatively specific for ANG II, as infusions of norepinephrine lead to tachyphylaxis (3, 102), although a similar slow pressor response develops in rats infused with a TP-R mimetic, U-46,619 (245). The hypertension is preceded by a selective increase in RVR (77, 89). The responses in the rabbit, rat, and mouse are accompanied by increased ROS and increased excretion, or increased renal or vascular production, of lipid peroxidation products (24, 52, 53, 68, 88, 89, 97, 102, 146, 147, 162, 237, 238, 243, 248, 256, 261, 278) and increased nitrotyrosine deposition implying ONOO− generation (93). The hypertension and renal vasoconstriction depend on O2−· because they are prevented by coinfusion of a membrane-permeable form of SOD (102) or by the nitroxide SOD mimetic, Tempol (89, 146, 147, 248).
Further studies implicated vasoconstrictor PGs, including TxA2, whose excretion is increased during an ANG II slow pressor response (116). Coffman and colleagues (48) and Kawada and colleagues (88) have reported that the ANG II pressor response is blunted in mice with targeted disruption of the TP-R (182). Indeed TP-R−/− mice have a paradoxical reduction in RVR with ANG II and fail to develop oxidative stress. Thus TP-Rs mediate oxidative stress, hypertension, and renal vasoconstriction of afferent arterioles during an ANG II slow pressor response.
ROS AND TGF
TGF entails a predominant increase in preglomerular (afferent arteriolar) resistance, leading to a fall in PGC and single nephron glomerular filtration rate (SNGFR) during delivery and reabsorption of solute at the macula densa segment of the nephron (15). TGF contributes to renal autoregulation (160), and thereby guards glomerular capillaries against barotrauma during hypertension. Resetting of TGF permits the timely and efficient excretion of NaCl in response to changes in BP or salt intake and thereby guards against salt sensitivity and hypertension (18, 128, 144, 180). Wilcox et al. (266) and Mundel et al. (133) reported heavy expression of nNOS in the macula densa cells of the rat. Microperfusion of the NOS inhibitor NG-monomethyl-l-arginine (l-NMA) or of an NO scavenging molecule (pyocyanin) into the tubular lumen of the macula densa or into the blood supplying the interstitium of the JGA via the efferent arteriole enhances the TGF response (266). Microperfusion of l-NMA reduces PGC during a TGF response but is ineffective if the TGF response is quiescent during luminal furosemide or in the absence of luminal salt delivery (266). These studies establish that NO is generated in the macula densa during luminal NaCl reabsorption and blunts the TGF response. Use of the relatively nNOS-specific inhibitor 7 nitroindazole (7-NI) confirms that it is this isoform that participates in TGF responses in the rat (252, 253, 255). Studies of nNOS blockade show that nNOS is responsible for the resetting of TGF and glomerular hemodynamics after sustained changes in salt intake or proximal tubule reabsorption (18, 204, 205, 208). Moreover, blockade of nNOS in the normal rat reduces the GFR and leads to hypertension (144).
Studies of pharmacologic blockade of nNOS in the macula densa of the rat or of the nNOS knockout mouse (225) or of the isolated, doubly-perfused JGA preparation from the rabbit (151) have shown that nNOS blunts the reduction in PGC or SNGFR during macula densa reabsorption of solute. There is normally a lower SNGFR when assessed from tubular fluid samples drawn from the early distal tubule (DT), compared with the proximal tubule (PT), since PT sampling disrupts flow to the macula densa and thereby interrupts the contribution of macula densa activation and TGF to the reduction of SNGFR. Accordingly, the finding by Vallon, Schnermann and colleagues that this difference in SNGFR between PT and DT is enhanced in nNOS−/− mice implies that nNOS in the macula densa tonically enhances SNGFR via the macula densa (225). However, it is currently unclear why this group detected no differences in TGF responses to tubular fluid perfusion of the macula densa segment of nNOS−/− mice (225).
The finding that macula densa cells express both nNOS (266) and a complete complement of NADPH oxidase components (25) provoked the hypothesis that TGF responses are regulated by an interaction between O2−· and NO (253, 260). Despite a twofold upregulation of the protein and mRNA for nNOS in the renal cortex and immunocytochemical confirmation that this upregulation involves the macula densa cells (252) (Fig. 1), the SHR has diminished or absent responses of PGC to microperfusion of 7-NI into the macula densa, implying no role for nNOS-derived NO in blunting of TGF responses (253). This cannot be ascribed to a failure to deliver adequate substrate (l-arginine) or a critical cofactor (BH4), as luminal microperfusion of these substances fails to restore a normal response to nNOS inhibition in SHR nephrons. The NO donor compound SNP was perfused into the lumen of the MD to investigate the bioactivity of luminally delivered NO in the JGA (253, 256). SNP caused graded increase in PGC, consistent with blunting of TGF. The response to SNP was more sensitive, but not more responsive, in SHR nephrons. The response to SNP in SHR nephrons was enhanced and became similar to the WKY, during contemporary microperfusion of Tempol into the efferent arteriole supplying the test nephron.
The implication from these studies is that endogenous NO produced in the macula densa of the SHR is bioinactivated by O2−· in the JGA before exercising its influence to dampen TGF. This was examined directly from the increase in TGF responses during blockade of macula densa nNOS by luminal microperfusion of 7-NI (256) (Fig. 6). Microperfusion of 7-NI enhances TGF responses of WKY, but not of SHR, nephrons. Contemporary microperfusion of Tempol into the efferent arteriole does not modify the enhancement of TGF responses to luminal 7-NI in the WKY, yet results in a six-fold enhancement of the response to luminal 7-NI in the SHR. Parallel studies were undertaken in WKY and SHR that were administered the AT1-R antagonist, candesartan, or triple antihypertensive therapy with hydralazine, hydrochlorizide, and reserpine (HHR), which does not reduce renin or block AT1 receptors (256). Both treatments yielded equivalent reductions in BP in SHR to the levels of normal WKY. Neither treatment altered the TGF responses of WKY. In contrast, prolonged candesartan administration to SHR normalized the enhancement of TGF by luminal 7-NI, and completely prevented the augmented responses to luminal 7-NI in the SHR nephrons during efferent arteriolar microperfusion of Tempol. Whereas HHR had no significant effects, there was a tendency for a reduced TGF, which could relate to antioxidant effects of hydralazine. Apparently, TGF responses are enhanced in SHR nephrons because of a failure to blunt TGF by nNOS-derived NO. This can be ascribed to a local interaction of NO with O2−· within the JGA that leads to bioinactivation of NO. The enhanced generation of O2−· in the JGA of the SHR can be attributed to AT1-receptors. It occurs independent of changes in systemic BP, or local expression of nNOS in the macula densa (255, 256, 258, 260, 262, 265).
The interaction between O2−· and NO has been studied by Garvin and colleagues (164, 165) in the isolated, doubly perfused JGA and TAL segments (148–152, 159, 197, 226) of rabbit nephrons. NO inhibits the absorption of NaCl in the TAL by the generation of cGMP that inactivates the luminal Na+-K+-2Cl− transporter (148) and the Na+/H+ exchanger (152) . Higher concentrations of NO also inhibit the basolateral Na+-K+-ATPase (226). eNOS is the principal source of NO in the TAL (159), whereas nNOS is the principal source in the macula densa (258) and collecting ducts (197). O2−· enhances NaCl reabsorption in the TAL, in part, by inactivation of the effects of locally generated NO to prevent lumenal Na+ entry (149, 150) or to block Na+-K+-ATPase (226). Thus one mechanism whereby O2−· may increase afferent arteriolar tone is bioinactivation of NO within the JGA. This should augment TGF responses by enhancing the TGF signal generated within the macula densa cells during luminal solute delivery and absorption (164).
TGF responses depend on ANG II (181). Thus TGF is enhanced by infusion of ANG II (181), is blunted during administration of an angiotensin converting enzyme inhibitor or an angiotensin receptor blocker (ARB) to rats (198, 254) and is absent in the AT1A receptor knockout mouse (183). ANG II activates AT1 receptors that are expressed on macula densa cells (70), where it increases intracellular calcium concentration (ICCa2+) (83, 111) that can activate NOS and generate NO (206, 207). Likewise, ANG II activates AT1 receptors expressed on the renal afferent arteriole (238), where it also increases ICCa2+ (239, 283), releases NO (157, 206, 207), and generates O2−· (238). AT1- and TP-R, PKC-dependent activation of NADPH oxidase (33, 65, 127, 143, 244) could provide a mechanism for generation of ROS in macula densa cells and renal afferent arterioles during infusion of ANG II that offsets the effects of NO (237, 260).
REDOX REGULATION OF THE RENAL CORTICAL AFFERENT ARTERIOLE AND MEDULLARY VASA RECTA
Studies of isolated, pressurized, and perfused renal afferent arterioles from mice (157) and rabbits (237, 238) provide direct insight into the role of ROS in enhanced microvascular reactivity during prolonged ANG II infusion. These vessels from normal animals have a robust relaxation response to Ach that entails an endothelium-dependent relaxation factor (EDRF) and an EDHF. The EDRF depends on NOS, while the EDHF response is mediated by an epoxyeicosatrienoic acid (EET) derivative that activates a potassium conductance (236).
ROS are implicated in the short-term (minutes) and longer-term (days) responses of renal afferent arterioles to several vasoconstrictors. Incubation of rabbit afferent arterioles for 20 min with the TP-R mimetic U-46,619 induces dose-dependent contractions that are powerfully modulated by local generation of NO and ROS. Thus contractions are enhanced by blockade of NOS but dampened by metabolism of O2−· by Tempol (179).
Afferent arterioles dissected from the kidneys of rabbits infused with ANG II at a slow pressor rate exhibit an increased expression of mRNA for p22phox and COX-2, but a maintained expression of COX-1 and TP-R (264), and a reduced expression of AT1-R (237, 238). No transcripts for AT2-R are detected. Therefore, functional studies investigated the roles of O2−· derived from NADPH and PGs derived from COX-2. Contractions to ANG II are increased two-fold in arterioles from rabbits infused with ANG II despite downregulation of the AT1-R. This is accompanied by enhanced responsiveness to ET-1 and U-46,619 but persistent responses to norepinephrine (NE) and high [K+]. This maintained response to two vasoconstrictors indicates that the enhanced ANG II response in this model of early adaptation to infused ANG II is probably not secondary to structural remodeling. Removal of the endothelium of arterioles from normal rabbits enhances contractions to ANG II, thereby demonstrating that ANG II releases an EDRF from normal arterioles. In contrast, removal of the endothelium from arterioles of ANG II-infused rabbits blunts contractions to ANG II, thereby demonstrating that ANG II releases an endothelial-derived contracting factor (EDCF) from arterioles of ANG II-hypertensive rabbits (237).
The nature of the EDCF was studied from response to Ach in arterioles from ANG II-infused rabbits without preconstriction (Fig. 7). Vessels were incubated with l-nitro-arginine to block EDRF/NO and 14–15 epoxyeicosa-5 (Z)- enoic acid (14–15 EEZE) to block EET-induced EDHF (236). Vessels from sham-infused rabbits do not contract to Ach, whereas those from ANG II-infused rabbits show a graded contraction (Fig. 7). This contraction is abolished by endothelium removal and therefore is an EDCF response. The EDCF is moderated, but not abolished, by bath addition of Tempol (SOD mimetic) to metabolize O2−· or by SC-506 (COX-1 antagonist) or by OKY-046 (thromboxane A2 synthase antagonist) (TxA2-S). However, the contractions are almost abolished by bath addition of parecoxib to block COX-2 or by ifetroban to block TP-Rs (237).
These studies have disclosed two related processes that underlie enhanced contractility of afferent arterioles during an ANG II slow pressor response (Fig. 8). The first is an increased responsiveness of the VSMCs to a number of agonists including ANG II, ET-1, and TP-R mimetics that is dependent upon oxidative stress. The second is the absence of an EDRF response, due to bioinactivation of endothelial NO by O2−·, and the generation of an EDCF response that depends on COX-2 generation of a TP-R ligand. The nature of this ligand is presently unclear but TxA2 is not primary, as the EDCF response is only blunted by a TxA2-S antagonist. Isoprostanes are formed nonenzymatically by interaction of O2−· with arachidonate (131) and can activate TP receptors on renal arterioles (202). However, isoprostanes presumably are not the primary TP ligand either since the EDCF response is dependent on cyclooxygenase and is not abolished by metabolism of O2−· with Tempol. Likely candidates include the stable prostaglandin endoperoxide, PGH2 (110) or a COX metabolite of HETE (242), perhaps oxidatively metabolized to a polar form (170).
In contrast to the enhanced reactivity of the renal cortical afferent arteriole, there is diminished reactivity to ANG II of isolated vasa recta of ANG II-infused rats (153, 166, 281, 285). The addition of ANG II to the bath of isolated vasa recta generates a large excess of NO as shown by the NO-sensitive dye, 3-amino-4-aminomethyl-2′-7′-difluorofluoresin diacetate 2 DA (281). Studies with dihydroethidium loading to detect O2−· provide no evidence of oxidative stress in the vasa recta of ANG II-infused rats (281). Unlike the renal outer cortex (281), there is no increase in NADPH activity in the renal outer medulla of ANG II-infused rats (281) and no increase in NADPH-, NADH- or xanthine-oxidase in the outer medulla of rabbits undergoing an ANG II slow pressor response (238).
A limitation of some studies of the renal medulla is that they are not always performed under the unusual conditions of low Po2 encountered in the medulla in vivo. Moreover, it is possible that effects of ANG II to enhance O2−· generation via the activation of AT1-Rs are concealed by coincident activation of AT2-Rs. Indeed, an increase in ROS occurs in isolated vasa recta when ANG II is added to the bath in the presence of the AT2-R antagonist PD-123,319 (280).
Studies of intact rats have shown that inhibition of SOD in the renal medulla by diethylthiocarbamate causes oxidative stress, reduces medullary blood flow, and raises BP (121, 284). Likewise, local medullary infusion of H2O2 (120) raises the BP. Studies of rat renal outer medullary strips with fluorescent probes for O2−· and NO show clearly that the addition of ANG II increases O2−· generation in the medullary TAL segment, but not in pericytes of vasa recta (129). However, an increase in O2−· in vasa recta can derive from the adjacent TAL segments. These studies have shown how NADPH-oxidase-derived O2−· in the TAL, if not quenched by NO, can lead to oxidative stress in the adjacent vasa recta. Collectively, the evidence shows a role for O2−· in the renal cortex and medulla of ANG II-infused rats or rabbits to eclipse the effects of NOS-derived NO on renal afferent arterioles or to influence the tone of vasa recta in the renal medulla.
DEFECTS IN RENAL OXYGENATION DURING HYPERTENSION AND OXIDATIVE STRESS
ROS ultimately derive from molecular oxygen either as a byproduct of mitochondrial respiration where O2−· generation increases with Po2, or as the product of specific oxidases notably NADPH, NADH oxidase or xanthine oxidase, or by interaction with various cellular constituents such as Fe2+, whereby ·OH− is generated from H2O2 in the Fenton reaction. Therefore, the availability of O2 may regulate ROS generation, according to the functional Km of the specific system involved. Recent studies have examined the Po2 in the kidneys in vivo and its effects on ROS generation to address the hypothesis that Po2 could limit oxidative stress in the kidney.
Renal oxygen usage (QO2) normally increases linearly with tubular sodium transport (TNa) above a basal level (35, 209). The slope of this line (TNa:QO2 ) defines the efficiency with which the kidney uses O2 for chemical work, above a basal level. This efficiency depends markedly on NO and ROS (104).
Hintze and colleagues reported that NOS blockade in the dog increases the QO2, while reducing the GFR, and hence the TNa. Consequently, the TNa:QO2 declines sharply (104). Similar results are apparent in other tissues, such as the heart, where NOS blockade also enhances O2 usage at a given level of work (109). Although the molecular mechanisms responsible are unclear, NO can compete with O2 for mitochondrial respiration (17, 163, 174). Therefore, a reduction in NO may enhance mitochondrial O2 usage above the level required to satisfy the energy needs of the cell. Because the cell has a strictly limited capacity to store unused sources of chemical energy, this will lead to an inefficient utilization of O2 for chemical work.
The TNa:QO2 of the SHR kidney is reduced by 50%. As in normal kidneys after the NOS blockade (104), this is due to an enhanced O2 usage despite a reduced TNa (246). This inefficient O2 utilization in the SHR kidney can be corrected fully by 2 wk of administration of an ARB, whereas equiantihypertensive therapy with hydralazine, hydrochlorothiazide and reserpine is not effective (247). These results assign the inefficient O2 utilization in the SHR kidney to prolonged AT1 receptor activation and disassociate it from the accompanying hypertension. The early phase of 2K,1C Goldblatt hypertension in the rat is also characterized by ANG II-dependent hypertension and oxidative stress. The postclipped kidney also has a reduced TNa:QO2. However, in the 2K,1C model, the defect in O2 usage is not reversed fully by prolonged administration of an ARB but is normalized by correcting oxidative stress with Tempol (249).
The effects of ANG II infusion for 12 days at a slow pressor rate when given alone, or with the SOD mimetic Tempol, have been studied in the rat (248). ANG II increases BP, RVR, p22phox expression, and NADPH oxidase activity of the renal cortex, and decreases the TNa:QO2 and Po2 in the kidney (Fig. 9). Tempol alone has no effect, but when given with ANG II, Tempol prevents all of these changes. Remarkably, p22phox expression is reduced below baseline in ANG II-infused rats given Tempol (Fig. 9C). These results demonstrate that not only hypertension and renal vasoconstriction but also NADPH oxidase activation and defective oxygenation in the kidney during ANG II can be ascribed to the effects of O2−·. There is evidence of bioinactivation of NO by O2−· in the kidney of the SHR (253), the postclip kidney of the 2K,1C model (6, 13, 189, 190), and the kidney of the ANG II-infused rat (36). Therefore, enhanced mitochondrial O2 usage during functional NO deficiency as a consequence of oxidative stress could account for the inefficient utilization of O2 by the kidneys of these hypertensive rat models.
Recent studies of isolated mitochondria demonstrate that ROS can interact directly with mitochondrial energy generation (163). ROS, whether produced as a byproduct of mitochondrial respiration or within the cell cytoplasm, can diminish the activity of the mitochondrial uncoupling protein 2 (136). The ensuing proton leak across the mitochondrial membrane reduces the proton motive force that couples O2 usage to ATP synthesis. This provides an additional and more direct mechanism whereby O2−· may diminish the efficiency of oxidative metabolism and ATP generation. Indeed, prolonged blockade of ANG II generation or AT1 receptors markedly improves O2 usage and diminishes ROS formation in mitochondria isolated from the kidneys of aged rats (34).
Direct recording of the Po2 in the renal proximal and distal tubules and interstitium suggests that O2 is in diffusional equilibrium within the outer kidney cortex. The highest values of Po2 in the kidney of ∼40–45 mmHg are in the outer cortex, yet the Po2 at these sites is below the Po2 of 50–65 mmHg measured in the renal vein (246). This implies a preglomerular diffusional shunt for O2, confirming conclusions from earlier studies (107, 185). This O2 shunt pathway may be between the arcuate arteries and veins, which run a prolonged course in a countercurrent alignment to one another at the junction of the kidney cortex and the outer medulla (95). A shunt that supplies arteriolar O2 to the surrounding tissue, as well as the adjacent arcuate vein, could explain the increase in Po2 at the cortico-medullar border, and the relative sparing of this region around the arcuate veins, in a model of ischemic renal injury (171).
The kidneys of rats exposed to hypoxia show a marked accumulation of hypoxia inducible factor-1α and -2α (HIF-1α and -2α). However, these two isoforms are expressed in different cell populations (172). HIF-1α is induced relatively selectively in tubules, especially the collecting ducts. HIF-2α is not expressed in tubular cells but is detected in some glomerular endothelial cells and more widely in peritubular capillary endothelial cells and fibroblasts (172). The Po2 in the outer cortex is reduced in the kidneys of the SHR (246), the ANG II-infused rat (248), and the postclipped kidneys of the 2K,1C rat (249). The low Po2 and defective TNa:QO2 are normalized by administration of the ARB candesartan in the SHR and by Tempol in the 2K,1C kidney. Thus the efficiency with which the kidney uses O2 for TNa is likely a major determinant of its Po2 and thereby of the expression of HIF-1α and -2α. This efficiency is strongly dependent on ROS.
The effects of ROS to reduce renal parenchymal Po2 could have far-reaching consequences, one of which may be to provide a brake to ongoing oxidative stress (Fig. 10). Thus lipid peroxidation products (132) or O2−· generation from NADPH oxidase (28) increase with O2, yielding an apparent Km for O2 that spans values measured directly in the renal cortex and medulla. Consequently, ROS may inhibit their own production by limiting O2 availability in the kidney, and especially the medulla, of hypertensive animals. However, hypoxia-induced upregulation of HIFs could engage many genes that are implicated in inflammation and fibrosis. This could be a trade-off for the effects of hypoxia to further limit ROS generation (Fig. 10). In this way, hypoxia in the hypertensive kidney could be a switch for a number of potentially adverse mediators. Indeed, hypobaric hypoxia over 24 days to reduce the Po2 by almost 50% not only raises the BP, but leads to renal interstitial inflammation and preglomerular arteriopathy (8).
The expression of HIF-1α in renal medullary interstitial cells in culture is inhibited during coincubation with xanthine and xanthine oxidase to induce O2−·, whereas HIF-1α expression is upregulated by the SOD mimetic Tempol or polyethylene glycol complexed SOD or by blockade of NADPH oxidase with diphenyleneiodonium or apocynin (276). Because scavenging of ·OH− with tetramethylthiourea does not change HIF-1α, the authors concluded that O2−·, independent of H2O2 on ·OH−, destabilizes HIF-1α in RMCD cells. Thus there may be two conflicting consequences of an increase in O2−· in the kidney on HIF activity: a direct effect of O2−· to destabilize HIF-1α protein expression and indirect effects mediated by changes in NO activity or mitochondrial function that enhance O2 usage and reduce Po2, thereby increasing HIF-1α generation. Clearly, further studies are required to determine how these discordant regulatory events play out under conditions of hypertension and renal damage and how important are HIFs for mediating effects of ROS on structural and functional changes in the hypertensive kidney. This may be important because kidneys of rat models of diabetes (94) or chronic renal insufficiency (227) have oxidative stress. Norman and colleagues (45, 140) have proposed that hypoxia in the damaged kidney may activate cytokines that perpetuate renal damage.
Growing evidence from animal studies suggests that oxidative stress in the kidney could be a key factor in the development and persistence of hypertension. There is a spectrum of BP responses to antioxidants such as Tempol that range from complete prevention of the development of hypertension in the rat or mouse infused with ANG II at a slow pressor response to partial reversal of established hypertension in the SHR, to more modest antihypertensive effects in stroke-prone SHR. Further work is warranted to translate these findings into clinical investigation and therapeutics. This is of growing importance because all of the established or nontraditional cardiovascular risk factors have been associated in clinical or experimental studies with evidence of increased ROS, which may be linked not only to vasoconstriction, salt retention, and hypertension, but also to many other adverse long-term consequences (Fig. 11; Ref. 19).
This work has been supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK36079 and DK49870) and the National Heart, Lung, and Blood Institute (HL 68086) and by funds from George E. Schriener, chair of Nephrology. The manuscript was expertly prepared by Margaret Brierton.
Studies in the author's laboratory were codirected by William J. Welch. The author thanks him, and other colleagues, especially Tina Chabrashvili, Dan Wang, Yifan Chen, Nori Kawada, Prit Gill, Pedro José, Tom Pallone, Kathryn Sandberg, Akahiro Tojo, and the many fellows, postdoctoral scientists, and research staff for their ideas and work that have contributed to these studies.
↵1 Distinguished Lectureship Awards are named after outstanding contributors to the disciplinary areas of physiology represented by 12 APS Sections. The recipient is chosen by a Section as a representative of the best within the discipline. Lecturers present and are active participants at the Experimental Biology meeting. Each year, four of the 12 lecturers give plenary lectures that incorporate the main meeting topic. In the years that sections do not have plenary lectures, the lecturer presents one hour of a featured topic programmed by the section.
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