The damaging effects of superoxides on renal physiology:
According to Muscoli, et. al, the list of pathophysiological conditions associated with the overproduction of superoxide expands every day. Loss of redox homestasis can contribute to pathological states including reduced vascular compliance and proteinuria. While the kidney utilizes regulation systems to keep ROS concentrations within a physiologically acceptable range, an imbalance of redox homeostasis can lead to a variety of renal diseases including hypertension and atherosclerosis.
In pathological conditions, an excess of ROS in tissue causes oxidative stress with various deleterious consequences such as fibrosis and inflammation.
Gill, et. al. used siRNA to downregulate a subunit of NADPH oxidase called P22 and observed a decrease in hypertension. Therefore, they showed that NADPH oxidase leads to the development of oxidative stress and hypertension.
A high salt diet can upregulate ANG II which leads to overexpression of ROS, resulting in hypertension.
Schluter et. al used lucigenin-enhanced chemiluminescence expression of NOX isoforms to show that increased NOX activity leads to hypertension in elderly patients. Increased activity of NADPH oxidase and increased levels of superoxides contribute to elevated blood pressure.
In elderly individuals, 60% of the superoxide formed in renal resistance arteries are derived from NOX2 and NOX4, and their activation can reduce endothelium-dependent vasodilation.
In a study of diabetic mice, NOX4 derived ROS contributed to fibrosis and diabetic nephropathy. In diabetic mice, NOX4 was upregulated in the renal cortex, MPT cells exhibited functionally active NOX-4 based NADPH, NOX4 was shown to be a major producer of ROS, and activation of fibrosis was modulated by NOX4 sensitive p38MAP kinase-dependent pathway. Overproduction of ROS can lead to endothelial dysfunction, vascular inflammation, and arterial remodeling.
Many cancer cells develop large amounts of ROS. Overexpression of NADPH oxidase increases superoxide generation which can lead to uncontrolled cell growth.
Recent therapeutic efforts have been focused towards targeting 02-. Evidence indicates that superoxide dismutase mimetics can be used therapeutically to treat disease. Superoxide dismutase catalyzes the conversion of superoxide to hydrogen peroxide and/or molecular oxygen. Thus superoxide dismutase mimetics can be used to minimize the damage from superoxides. Artificial enzymes have been used effectively to remove superoxides at a high rate without reacting with biologically relevant oxidizing species. These drugs can be used to treat hypertension and atherosclerosis.
Biologically, superoxide dismutase maintains cellular integrity by inactivating free radicals. Other systems that function as free radical scavengers include catalase, an enzyme that converts hydrogen peroxide to water and oxygen, and the glutathione redox cycle, which scavenges lipid peroxides and hydrogen peroxide.
superoxide production pathways
In the kidney, a slew of cells contribute to the production of reactive oxygen species (ROS). These cells include, fibroblasts, endothelial cells, vascular smooth muscle cells, mesangial cells, tubular cells and podocyte cells. The phagocytic NADPH oxidase and the NADPH subunits NOX-1 and NOX-4 are also expressed in the kidney, and are localized in renal vessels, glomeruli, podocytes, the thick ascending limb, macula densa, distal tubules, cortical interstitial fibroblasts, and collecting ducts. NADPH oxidase catalyzes the transfer of an electron from NADPH to 02 across a cell membrane. 02- will therefore be produced inside a phagosome or in the extracellular space. This function contrasts with other oxidases which produce ROS as a byproduct of other oxidative reactions, such as the electron transport chain.
Six homologues of the cytochrome subunit of NADPH oxidase are known. They are NOX1, NOX3, NOX4, NOX5, DUOX1, and DUOX2. These homologs are collectively known as the NOX family of NADPH oxidases. These enzymes transport electrons across the plasma membrane and generate ROS further downstream.
NOX is one of several isoforms of gp91phox, a subunit of NADPH oxidase. Each subunit (NOX1, NOX2, NOX3, and NOX4) depends on p22 phox, the catalytic subunit required for enzyme activity. Various isoforms of NADPH oxidase are expressed throughout the kidney. Of the NOX isoforms, NOX4 is the most abundant in the kidney. NOX produces reactive oxygen species that are involved in a variety of functions in the kidney, including gluconeogenesis, glucose transport, electrolyte transport, hemodynamic and tubuloglomerular feedback.
One of the known pathways of O2- in signaling is the activation of the RAS/RAC-RAF1-MAPK pathway. ANG II propagates 02- generation in endothelial cells via NADPH oxidase. Next, O2- activates the Raf-1-MAPK pathway to regulate gene expression. In the central nervous system, experiments in mice have shown that ANG II stimulates O2- production, resulting in the production of vasopressin and sympathetic outflow.
Huang et al. showed that ERK activation by PPAR-g (peroxisome proliferator-activated receptor gamma) agonists were inhibited by a SOD mimetic, but propagated by O2-.
Superoxides inhibit autoregulation:
Another harmful physiological effect of superoxides is that they hinder autoregulation during a high salt diet. According to Fellner, et. al, a high salt diet increases renal ROS generation partly due to NADPH oxidase. They observed that control afferent arterioles displayed normal autoregulatory behavior in response to acute increases in renal perfusion pressure, whereas arterioles from rats on a high salt diet had minimized responses. Furthermore, autoregulatory behavior in high salt rats was remediated by acute exposure to apocynin, an NADPH oxidase inhibitor. In HS rats, the kidney’s ability to maintain stable RBF and GFR in response to reduced renal perfusion pressure was lessened compared to rats fed a normal diet. Fellner et. al. concluded that the impaired ability of the kidney to autoregulate itself during a high salt diet is mediated by NADPH oxidase-derived ROS.
Sedeek, et. al., demonstrated that NADPH oxidases inhibit autoregulation in the afferent arteriole. For instance, NOX-derived ROS decreased juxtamedullary afferent arteriolar autoregulation in response to TGF-B.
Finally, Sharma et. al. discovered that TGF-beta attenuates autoregulation by increasing levels of reactive oxygen species. At high levels of TGF-B, autoregulation was completely blocked due to upregulation of reactive oxygen species. Basal autoregulation levels were returned to normal when an NADPH oxidase inhibitor was introduced.
Superoxides are required for autoregulation:
Superoxide function in the body is paradoxical because they are required for normal physiological function. Although superoxide and hydrogen peroxides are traditionally thought of as cytotoxic and mutagenic, recent evidence indicates that they play important biological roles. Despite their damaging effects, they regulate biologic processes such as cell defense, activation of G-protein coupled receptors, and hormone synthesis. They are also essential regulators of gene expression and transcription. In addition to their role in cell growth and apoptosis, oxidant species are heavily involved in intracellular and receptor signaling mechanisms that control vascular contractility. Superoxide production, metabolism and their role in cell signaling mechanisms are important aspects of vascular function. The absence of NOX isoforms leads to a wide range of pathological processes. For example, NOX deficiency can cause hypothyroidism, otoconogenesis, and immunosuppression.
The role of superoxides in the myogenic response:
Lai et al. discovered that superoxide regulates myogenic contraction in the afferent arterioles of mice. They tested the hypothesis that myogenic contraction is controlled in part by release of superoxides in response to afferent arteriole stretch. They used dihydroethidium fluorescence to show that the reduction in diameter of the afferent arteriole in response to increased perfusion pressure is caused by an increase in reactive oxygen species. Superoxide release in response to stretch was calcium and NO independent. They concluded that the vasoconstriction in response to the increased perfusion pressure was caused by the release of superoxides. According to Just et. al., ROS contributes to autoregulation by strengthening the myogenic response in the normal kidney. However, they noted that this response is mainly due to superoxide, whereas the contribution due to hydrogen peroxide is variable. nitrous oxide, a known vasodilator, hinders the vasoactive effects of superoxide.
Sung et. al. showed that elevated ROS enhances the myogenic response in isoflurane anesthetized SHR and SDR.
The role of superoxides in Tubuloglomerular feedback:
Not only does superoxide modulate the myogenic contraction, but it also plays a role in signaling during tubuloglomerular feedback. According to Just, et. al, reactive oxygen species are known to participate in acute agonist induced vasoconstriction in the kidney and to enhance tubuloglomerular feedback.
Similarly, Liu et al. showed that high levels of 02- production were associated with the maximum TGF response. Liu used the fluorescent dye dihydroethidium to detect 02- production at the macula densa. they discovered that increasing the concentration of NaCl leads to increased production of 02-. They concluded that increased concentrations of NaCl in the tubular lumen initiate 02- production during TGF. Furthermore, 02- generated at the macula densa is generated by NADPH oxidase and is induced by depolarization.
Acording to Liu, et al., superoxide amplifies TGF both in vivo and in vitro by scavenging nitric oxide in the macula densa. Their results indicate that 02-, instead of h202, enhances the TGF response directly by constricting the afferent arteriole, and also indirectly by scavenging NO in the macula densa.
O2- reacts at a diffusion-controlled rate with NO and inactivates it. Therefore, O2- indirectly causes vasoconstriction by eliminating the vasodilating effects of NO. Furthermore, the product of this reaction, ONOO-, can react with macromolecules downstream to regulate cellular function.
The interaction between 02- and NO in the macula densa will modify the TGF response. Superoxides enhance tubuloglomerular feedback directly by constricting the afferent arteriole and indirectly by scavenging nitrous oxides.
According to Sedeek et. al., silencing NOX2 (but not NOX4) in mouse macula densa decreased high salt induced superoxide production.
Sedeek et. al. used single cell RT-PCR to extrapolate that NOX2 and NOX4, but not NOX1, are expressed in the macula densa. These findings indicate that NOX isoforms play a role in TGF.
Zhang et. al. found that NOX2 and NOX4 isoforms are expressed at the macula densa. While NOX4 is responsible for basal level 02- production during homeostasis, NOX2 is responsible for producing 02- induced by increased NaCl concentrations.
Other autoregulatory effects of superoxides in the kidney:
The renin-angiotensin-aldosterone system utilizes ROS to produce vasoconstriction during volume contraction and hypotension. In vascular smooth muscle, NADPH oxidase is known to exist at basal levels in the absence of stimulation, but can be upregulated by ANG II, TNF, thrombin, and latosylceramide. Such stimulation would increase the concentration of superoxides in the vasculature.
According to Carlstrom et. al., Nox2-derived ROS regulates afferent arteriole tone and reduces vasodilatory effects.
According to Buetlet et al., O2- can alter cellular responses to vasoconstrictor hormones and thereby alter cellular function.
This increase in 02- production at the macula densa is also controlled by pH. Liu et. al. showed that elevated intracellular pH leads to 02- production by NADPH oxidase. They discovered that 02 – concentrations are maximized at pH near 8.0
According to Nistala et al., upregulation of the RAAS system (renin – angiotenin – aldosterone system) lead to increased production of ROS. ROS are also released in response to mechanical shear forces.
When renal afferent arterioles are exposed to ANG II, NADPH derived 02- levels increase in the renal cortex. At the same time, ACh-induced endothelium-dependent relaxation is impaired.