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We tested a hypothesis that superoxide, by inducing Sp3, increases renal renin activity, renal angiotensin II type 1 receptor (AT1R) function, and blood pressure (BP) in rats. Group 1 rats were treated with vehicle, saline. Group 2 rats were treated with superoxide dismutase (SOD) inhibitor diethylthiocarbamate (DETC). Group 3 rats were treated with DETC and an SOD mimetic, tempol. Group 4 rats were treated with tempol only. All four groups of rats were treated for 2 wk then anesthetized, and BP was recorded. Thereafter, diuresis and natriuresis in response to AT1R blocker candesartan were determined. When compared with vehicle rats, BP increased in DETC rats. The increased BP in DETC rats decreased with tempol. Diuresis and natriuresis in response to candesartan increased in controls, and this further increased in DETC rats and decreased with tempol. A second set of four groups of rats underwent the same treatment as above and were anesthetized, and their kidneys were obtained for biochemical studies. The levels of superoxide but not hydrogen peroxide increased, whereas SOD activities decreased further in the renal cortical tissues of DETC rats than vehicle rats. These effects were attenuated with tempol in DETC rats. Moreover, tissue renin activity and abundance of membranous AT1R proteins increased more in DETC rats than vehicle rats, and decreased with tempol in DETC rats. Furthermore, the levels of lysine-acetylated, but not serine-phosphorylated, Sp3 increased more in the nuclei of DETC rats than vehicle rats. The increased levels of Sp3 lysine acetylation decreased in DETC rats with tempol. Taken together, our results suggest that superoxide activates renal Sp3 via lysine acetylation increasing renin activity, AT1R function, and BP in rats. [ABSTRACT FROM AUTHOR]

The role and mechanism of renal protein disulfide isomerase (PDI) in blood pressure regulation has not been tested before. Here, we test this possibility in Sprague-Dawley rats. Rats were treated with PDI inhibitor bacitracin (100 mg·kg-1 ip·day-1 for 14 days), and then blood pressure and renal angiotensin II type 1 (AT1) receptor function were determined in anesthetized rats. Renal AT1 receptor function was determined as the ability of candesartan (an AT1 receptor blocker) to increase diuresis and natriuresis. A second set of vehicle- and bacitracin-treated rats was used to determine biochemical parameters. Systolic blood pressure as well as diastolic blood pressure increased in bacitracin-treated compared with vehicle-treated rats. Compared with vehicle, bacitracin-treated rats showed increased diuresis and natriuresis in response to candesartan (10μg iv bolus dose) suggesting higher AT1 receptor function in these rats. These were associated with higher renin activities in the plasma and renal tissues. Furthermore, urinary 8-isoprostane and kidney injury molecule-1 levels were higher and urinary antioxidant capacity was lower in bacitracin-treated rats. Renal protein carbonyl and nitrotyrosine levels also were higher in bacitracin- compared with vehicle-treated rats, suggesting oxidative stress burden in bacitracin treated rats. Moreover, PDI activity decreased and its protein levels increased in renal tissues of bacitracin-treated rats. Also, nuclear levels of Nrf2 transcription factor, which regulates redox homeostasis, were decreased in bacitracin-treated rats. Furthermore, tissue levels of Keap1, an Nrf2 inhibitory molecule, and tyrosine 216-phosphorylated GSK3β protein, an Nrf2 nuclear export protein, were increased in bacitracin-treated rats. These results suggest that renal PDI by regulating Keap1-Nrf2 pathway acts as an antioxidant, maintaining redox balance, renal AT1 receptor function, and blood pressure in rats. [ABSTRACT FROM AUTHOR]

A recent study demonstrated that the dopamine D1 receptor (D1R) is nonfunctional in human kidney cells, HK2 cells, in terms of their inability to couple to Gs protein in response to the D1R agonist fenoldopam. Since D1R also couples to Gq protein, we tested whether D1R is functional in HK2 cells in terms of their ability to couple to Gq and produce downstream signaling. For comparison, we also studied another receptor, angiotensin II type 1 receptor (AT1R) known to couple to Gq. Protein kinase C (PKC) and 86rubidium transport activities were determined as surrogate downstream signaling markers. Fenoldopam and angiotensin II increased PKC activity, which decreased in the presence of respective receptor antagonists (SCH23390 for D1R; candesartan for AT1R), PKC (chelerythrine chloride) and Gi protein (pertussis toxin) inhibitors and Gq/11α siRNA. Furthermore, fenoldopam and angiotensin II increased 35S-GTPαS binding, an index of receptor-G protein coupling, which decreased with pertussis toxin and in Gq/11α-depleted cells. Also, fenoldopam-mediated inhibition of 86rubidium transport (an index of Na-K-ATPase activity) was attenuated with SCH23390, chelerythrine chloride, pertussis toxin, and Gq/11α siRNA. Moreover, fenoldopam caused a decrease in cytosolic and increase in membranous abundance of Gq/11α. The immunoprecipitated levels of Gq/11α in the membranes were greater in fenoldopam-treated cells, and Giα coimmunoprecipitated with Gq/11α. Our results suggest that both D1R and AT1R are functional in HK2 cells, enabling Gq-mediated downstream signaling in a Gi dependent manner. [ABSTRACT FROM AUTHOR]

Kidneys play a vital role in long-term regulation of blood pressure. This is achieved by actions of many renal and nonrenal factors acting on the kidney that help maintain the body's water and electrolyte balance and thus control blood pressure. Several endogenously formed or circulating hormones/peptides, by acting within the kidney, regulate fluid and water homeostasis and blood pressure. Dopamine and angiotensin II are the two key renal factors that, via acting on their receptors and counterregulating each other's function, maintain water and sodium balance. In this review, we provide recent advances in the signaling cascades of these renal receptors, especially at the level of their cross talk, and discuss their roles in blood pressure regulation in the aging process. [ABSTRACT FROM AUTHOR]

Nuclear factor- κB (NF-κB) plays a role in inflammation. However, we recently reported an association between NF-κB and antioxidant enzymes in renal proximal tubules of exercise-trained rats, suggesting its role in antioxidant homeostasis (George L, Lokhandwala MF, Asghar M. Am J Physiol Renal Physiol 297: F1174-F1180, 2009). A direct role of NF-κB in antioxidant homeostasis in renal cells has not been elucidated and warrants investigation. Therefore, we examined whether NF-κB has a direct role in antioxidant homeostasis and redox balance in human kidney-2 cells overexpressing NF-κB-p65 and compared them with the cells overexpressing Nrf-2, a well-known transcription factor involved in antioxidant homeostasis. The ability of NF-κB-p65 to increase antioxidant enzymes, to reduce reactive oxygen species (ROS), and to rescue ROS-induced renal dopamine D1 receptor dysfunction, was studied. The transcription activity of NF-κB-p65 and Nrf-2, measured as luciferase reporter activity, increased in cells overexpressing these nuclear factors. The levels of mRNA and activity of glutathione peroxidase as well as the protein levels of superoxide dismutase-1 and glutamylcystein transferase were increased in cells overexpressing NF-κB-p65 and Nrf-2. Furthermore, the levels of ROS decreased and D1 receptor agonist SKF38393-mediated [35S]GTPγS binding (index of D1 receptor function) increased in the presence of hydrogen peroxide in cells overexpressing NF-κB-p65 and Nrf-2. These results suggest a direct role of NF-κB-p65 in antioxidant homeostasis, contributing to redox balance in renal cells. [ABSTRACT FROM AUTHOR]

Inflammation and oxidative stress are believed to contribute to hypertension in obesity/diabetes. Recently, we reported a role for the AT2 receptor in blood pressure control in obese Zucker rats. However, the role of AT2 receptors in inflammation and oxidative stress in obesity is not known. Therefore, in the present study, we tested the effects of the AT2 receptor agonist CGP-42112A on inflammation and oxidative stress in obese Zucker rats and compared them in their lean counterparts. Rats were systemically treated with either vehicle (control) or CGP-42112A (1 μg·kg-1·min-1; osmotic pump) for 2 wk. Markers of inflammation (CRP, MCP-1, TNF-α, and IL-6) and oxidative stress (HO-1, gp-91phox) as well as an antioxidant (SOD) were determined. Control obese rats had higher plasma levels of CRP, MCP-1, TNF-α, IL-6, and HO-1 compared with control lean rats. Conversely, plasma SOD activity was lower in control obese than in control lean rats. Furthermore, the protein levels of TNF-α and gp-91phox were higher in the kidney cortex of control obese rats. Interestingly, CGP-42112A treatment in obese rats reduced the plasma and kidney cortex inflammatory (TNF-α, IL-6) and oxidative stress (gp-91phox) markers and increased plasma SOD activity to the levels seen in lean control rats. However, CGP-42112A treatment in lean rats increased inflammatory (TNF-α, IL-6) and oxidative stress (gp-91phox) markers in the plasma and kidney cortex. Our present studies suggest anti-inflammatory and antioxidative functions of AT2 receptor in obese Zucker rats but proinflammatory and prooxidative functions in lean Zucker rats. [ABSTRACT FROM AUTHOR]

Aging is associated with an increase in oxidative stress and blood pressure (BP). Renal dopamine D1 (D1R) and angiotensin II AT1 (AT1R) receptors maintain sodium homeostasis and BP. We hypothesized that age-associated increase in oxidative stress causes altered D1R and AT1R functions and high BP in aging. To test this, adult (3 mo) and old (21 mo) Fischer 344 × Brown Norway F1 rats were supplemented without/with antioxidant tempol followed by determining oxidative stress markers (urinary antioxidant capacity, proximal tubular NADPH-gp91phox, and plasma 8-isoprostane), D1R and AT1R functions, and BP. The D1R and AT1R functions were determined by measuring diuretic and natriuretic responses to D1R agonist (SKF-38393; 1 μg·kg-1·min-1 iv) and AT1R antagonist (candesartan; 10 μg/kg iv), respectively. We found that the total urinary antioxidant capacity was lower in old rats, which increased with tempol treatment. In addition, tempol decreased the elevated NADPH-gp91phox and 8-isoprostane levels in old rats. Systolic, diastolic, and mean arterial BPs were higher in old rats and were reduced by tempol. Although SKF-38393 produced diuresis in both adult and old rats, urinary sodium excretion (UNaV) increased only in adult rats. While candesartan increased diuresis and UNaV in adult and old rats, the magnitude of response was greater in old rats. Tempol treatment in old rats reduced candesartan-induced increase in diuresis and UNaV. Our results demonstrate that diminished renal D1R and exaggerated AT1R functions are associated with high BP in old rats. Furthermore, oxidative stress may cause altered renal D1R and AT1R functions and high BP in old rats. [ABSTRACT FROM AUTHOR]

A positive association between renin-angiotensin system, especially AT1 receptor, and oxidative stress in the pathogenesis of hypertension and cardiovascular/renal diseases has been suggested. However, the role of oxidative stress, especially superoxide radicals in renal sodium handling in response to AT1 and AT2 receptors, is not known. Therefore, the present study was designed to investigate the role of NAD(P)H oxidase (NOX), a major superoxide radical producing enzyme, in AT1 and AT2 receptor function on natriuresis/diuresis in Sprague-Dawley rats. The rats under anesthesia were intravenously infused with NOX inhibitor apocynin (3.5 µg/kg-1min-1), the AT1 receptor antagonist candesartan (100 µg/kg; bolus), and the AT2 receptor agonist CGP-42112A (1 µg⋅kg-1) alone and in combinations. Candesartan alone significantly increased urinary flow (UF; µ/30 min) by 53 and urinary Na excretion (UNaV: µmol/min) by 0.4 over basal. Preinfusion of apocynin had no effect on the net increase in UF or UNaV in response to candesartan. On the other hand, apocynin preinfusion caused profound increases in CGP-42112A-induced UF by 72, UNaV by 1.14, and fractional excretion of Na by 7.8. Apocynin and CGP-42112A alone did not cause significant increase in UF or UNaV over the basal. CGP-42112A infusion in the presence of apocynin increased urinary nitrite/nitrates and cGMP over basal. The infusion of candesartan, apocynin, and CGP-42112A alone or in combinations had no effect on the blood pressure or the glomemlar filtration rate, suggesting tubular effects on natriuresis/ diuresis. The data suggest that NOX may have an antagonistic role in AT2 receptor-mediated natriuresis/diuresis possibly via neutralizing nitric oxide and thereby influence fluid-Na homeostasis. [ABSTRACT FROM AUTHOR]

We tested the effects of inflammation on renal dopamine D1 receptor signaling cascade, a key pathway that maintains sodium homeostasis and blood pressure during increased salt intake. Inflammation was produced by administering lipopolysaccharide (LPS; 4 mg/kg ip) to rats provided without (normal salt) and with 1% NaCl in drinking water for 2 wk (high salt). Control rats had saline injection and received tap water. We found that LPS increased the levels of inflammatory cytokines, interleukin-6, and tumor necrosis factor-α in the rats given either normalor high-salt intake. Also, these rats had higher levels of oxidative stress markers, malondialdehyde and nitrotyrosine, and lower levels of antioxidant enzyme superoxide dismutase in the renal proximal tubules (RPTs). The nuclear levels of transcription factors NP-κB increased and Nrf2 decreased in the RPTs in response to LPS in rats given normal and high salt. Furthermore, D1 receptor numbers, D1 receptor proteins, and D1 receptor agonist (SKF38393)-mediated 35S-GTPγS binding decreased in the RPTs in these rats. The basal activities of Na-K-ATPase in the RPTs were similar in control and LPS-treated rats given normal and high salt. SKF38393 caused inhibition of Na-K-ATPase activity in the primary cultures of RPTs treated with vehicle but not in the cultures treated with LPS. Furthermore, LPS caused an increase in blood pressure in the rats given high salt but not in the rats given normal salt. These results suggest that LPS differentially regulates NF-κB and Nrf2, produces inflammation, decreases antioxidant enzyme, increases oxidative stress, and causes D1 receptor dysfunction in the RPTs. The LPS-induced dysfunction of renal D1 receptors alters salt handling and causes hypertension in rats during salt overload. [ABSTRACT FROM AUTHOR]

We have previously reported that age-associated oxidative stress via protein kinase C (PKC) increases D1 receptor (D1R) phosphorylation and causes D1R-G protein uncoupling in renal proximal tubules (RPTs) of old Fischer 344 rats. This results in reduced ability of D1R agonist SKF-38393 to inhibit Na+-K+-ATPase in RPTs of old rats. Here, we studied the effect of treadmill exercise on markers of oxidative stress, PKC, DiR phosphorylation, D1R-G protein coupling, and Na+-K+-ATPase activity in RPTs of adult and old rats. We found increased levels of malondialdehyde, a marker of oxidative stress, in RPTs of old rats, which decreased during exercise. Nuclear levels of nuclear erythroid-related factor (Nrf)-2 and nuclear factor (NF)-κB in RPTs, transcription factors involved in antioxidant enzyme gene transcription, increased in exercised old rats. This was accompanied by an increase in the activity and expression of antioxidant enzymes, superoxide dismutase and heme oxygenase-1. Age-related decrease in the levels of D1R mRNAs and proteins was attenuated during exercise. Furthermore, exercise in old rats decreased PKC activity and D1R phosphorylation and increased SKF-38393-mediated [35S]guanosine 5′-O-(3-thiot-riphosphate) binding (an index of D1R-G protein coupling). SKF- 38393 also caused inhibition of Na+-K+-ATPase in these animals. Also, exercise caused a decrease in proteinuria and increase in phosphaturia in old rats. These results suggest beneficial effects of exercise in terms of increasing antioxidant defenses, decreasing oxidative stress, and improving kidney function in general and D1R function in particular in aging. Both Nrf-2 and NF-κB seem to play key role in this phenomenon. [ABSTRACT FROM AUTHOR]

Recently, we reported that oxidative stress decreases D1 receptor numbers and G protein activation in renal proximal tubules (RPT), resulting in diminished natriuretic response to dopamine in old rats. We tested the hypothesis that exercise in old rats will decrease oxidative stress and restore natriuretic response to D1 receptor agonist, SKF 38393. Old (23 mo) rats were subjected to rest (sedentary) or to treadmill exercise followed by measurement of oxidative stress [malondialdehyde (MDA)], inflammation [C-reactive protein (CRP)], anti-inflammation (IL-10), antioxidant enzyme [superoxide dismutase (SOD)], and natriuretic response to SKF 38393. We found that MDA levels decreased and total SOD activity and Cu/ZnSOD protein increased in RPT of exercised rats. Exercise increased the nuclear levels of Nrf2 transcription factor (which binds to anti-oxidant response elements) in RPT. The levels of CRP decreased and IL-10 increased in RPT of exercised rats. Furthermore, exercise increased the basal bindings of [³H]SCH 23390 and [35S]GTPγS (indexes of D1 receptor number and G protein activation, respectively) together with D1 receptor and Gαq proteins in RPT membranes. Moreover, SKF 38393 increased sodium excretion in exercised rats. Also, exercise decreased the levels of proteins in the urine of old rats. These results demonstrate that exercise decreases oxidative stress, inflammation, and proteinuria and increases anti-oxidant defense and D1 receptor function in old rats. Therefore, exercise may prove beneficial in preventing age-associated increases in oxidative stress, inflammation, and preserving kidney function, in general, and renal D1 receptor responsiveness, in particular. [ABSTRACT FROM AUTHOR]

Aging is associated with an increase in oxidative stress. Previously, we have reported that dopamine failed to inhibit proximal tubular Na-K-ATPase and to promote sodium excretion in old rats (Beheray S, Kansra V, Hussain T, and Lokhandwala MF. Kidney Int 58: 7 12-720, 2000). This was due to uncoupling of dopamine Dl receptors from G proteins resulting from hyperphosphorylation of Dl receptors. The present study was designed to test the role of oxidative stress in the age-related decline in renal dopamine Dl receptor function. We observed that old animals had increased malondialdehyde (MDA) levels, a biomarker of oxidative stress, and decreased Dl receptor number and protein in the proximal tubules (PT) compared with adult rats. In old rats, there was increased G protein-coupled receptor kinase-2 (GRK-2) abundance, increased basal serine phosphorylation of Dl receptors, and defective Dl receptor-G protein coupling in PT membranes. Interestingly, supplementation with an antioxidant, tempol (1 mmol/l in drinking water for 15 days), lowered MDA levels and normalized Dl receptor number and protein in old rats to the level seen in adult rats. Furthermore, tempol decreased GRK-2 abundance and Dl receptor serine phosphorylation and restored Dl receptor-G protein coupling in PT of old rats. The functional consequence of these changes was the restoration of the natriuretic response to Dl receptor activation in tempol-supplemented old rats. Therefore, in old rats, tempol reduces oxidative stress and prevents GRK-2 membranous abundance and hyperphosphorylation of Dl receptors, resulting in restoration of Dl receptor-G protein coupling and the natriuretic response to SKF- 38393. Thus tempol, by lowering oxidative stress, normalizes the age-related decline in dopamine receptor function. [ABSTRACT FROM AUTHOR]

Previously, we reported that natriuretic and diuretic response to dopamine is diminished in old Fischer 344 rats, which is due to higher basal protein kinase C (PKC) activity and hyperphosphorylation of Na-KATPase in the proximal tubules (PTs) of old rats. The present study was conducted to determine whether higher PKC activity could be due to altered expression of some of the PKC isoforms in the superficial cortex (rich in PTs) of old rats. Fluorimetric measurement showed almost twofold increase in the PKC activities in homogenates and membranes of old (24 mo) compared with adult (6 mo) rats. Interestingly, in the basal state PKC-βI was overexpressed in the membranes, whereas PKC-δ expression was increased in the cytosol of old compared with adult rats. Treatment of the cortical slices with either SKF-38393, a D1-like agonist, or PDBu, a direct activator of PKC, caused translocation of PKC-βI from cytosol to membranes in adult but not in old rats. Both of these drugs caused translocation of PKC-δ from membranes to cytosol in adult but not in old rats. These drugs had no effect on translocation of PKC-ζ in both adult and old rats. Both PKC-βI and -δ coimmunoprecipiated with α[sub 1]-subunit of NaK-ATPase in adult and old rats. These observations suggest that both SKF-38393 and PDBu differentially regulate PKC-βI and -δ in adult but not in old rats. Also, PKC-βI and -δ seem to interact with Na-K-ATPase in these animals. The overexpression of both PKC-βI and -δ in old rats could be responsible for a higher basal PKC activity, which causes the hyperphosphorylation of Na-K-ATPase and contributes to the diminished inhibition of Na-K-ATPase activity by dopamine in old rats. [ABSTRACT FROM AUTHOR]

Examines the basal serine phosphorylation of D[sub 1A] receptors in proximal tubules of rats. Reduction in agonist affinity of receptors; Discussion on the process of phosphorylation; Link between the alteration in the receptor desentization/resintization process to the development of disease.