Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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ABSTRACT |
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We recently reported that
NADH oxidase is one of the major enzymes responsible for
superoxide (O
oxidative stress; reactive oxygen species; renal tubule; hypoxia; redox signaling
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INTRODUCTION |
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REACTIVE OXYGEN SPECIES (ROS) are conventionally considered as cytotoxic byproducts of cellular metabolism. Recently, evidence has been rapidly accumulating that ROS play an important role in the intracellular signaling, whereby ROS may participate in the regulation of cell function or activity under physiological conditions. In this regard, ROS have been reported to be involved in the growth, division, transformation, apoptosis, and senescence of a variety of mammalian cells, in addition to their physiological role in the respiratory burst of phagocytic cells. Many studies have indicated that ROS participate in the regulation of ion transport systems, protein phosphorylation, phospholipase activity, and gene expression (12, 13, 23).
More recently, ROS signaling has been indicated to contribute to the control of vascular tone or vasomotor response. It has been demonstrated that ROS serve as a vascular O2-sensing factor to control the vascular reactivity in response to tissue metabolic activity (39). Moreover, ROS were found to play a role in the response of vessels to pressure, flow, and receptor agonists (39). With respect to their pathological actions, ROS have been reported to participate in the development of atherosclerosis, hypertension, and progressive renal disease (10, 17, 20, 40, 43).
Although several pathways may account for the production of
O
Despite intensive studies of NADH oxidase-mediated production and
action of O
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MATERIALS AND METHODS |
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Microdissection of nephron segments in the rat kidney. Microdissection was performed as we described previously (45). Briefly, male Sprague-Dawley rats weighing between 250 and 300 g were anesthetized with pentobarbital sodium (80 mg/kg body wt ip), and the aorta below the left renal artery was isolated and cannulated. After the aorta was ligated at a site between the origin of left and right renal arteries, the left kidney was flushed with 20 ml ice-cold dissection solution containing (in mM) 135 NaCl, 3 KCl, 1.5 CaCl2, 1 MgSO4, 2 KH2PO4, 5.5 glucose, 5 L-alanine, and 5 HEPES (pH 7.4). Then, the kidney was perfused with 10 ml digestion solution, which was prepared by adding 1 mg/ml collagenase (243 U/mg, Worthington) and 1 mg/ml bovine albumin to the dissection solution. After perfusion, the kidney was removed and cut into 1- to 2-mm-thick sections containing the entire corticomedullary axis. The sections were incubated at 37°C for 30 min in the same digestion solution with gentle shaking. During incubation, the samples were bubbled with 95% O2-5% CO2. The sections were then rinsed twice with collagenase-free dissection solution and transferred into petri dishes filled with ice-cold dissection solution containing 0.1 mg/ml trypsin inhibitor and 20 µg/ml aprotinin. A petri dish was mounted on the microscope stage and maintained at 4°C during dissection.
Microdissection was performed under a Leica MZ8 stereomicroscope with darkfield illumination. The nephron segments including glomeruli, proximal convoluted tubule (PCT), proximal straight tubule (PST), cortical thick ascending limb of Henle's loop (cTALH), medullary thick ascending limb of Henle's loop (mTALH), thin limb of Henle's loop, cortical collecting duct (CCD), and medullary collecting duct (MCD) were dissected. The time period for dissection was limited to 1 h.O
Analysis of different pathways for O
Regulation of O
Expression of mRNA for NADH oxidase subunits in cTALHs. RT-PCR was performed to determine mRNA expression of NADH oxidase in cTALHs. The mRNAs of 5 NADH oxidase subunits, including p22phox, p40phox, p47phox, p67phox and Gp91phox (where phox indicates phagocyte oxidase), were PCR amplified and detected in this tubular segment. RNA extraction and RT-PCR were performed as we described previously (45). In brief, total RNA from microdissected segments (20 mm) was extracted using 450 µl TRIzol reagent (GIBCO BRL, Life Technologies). The resultant RNA was resuspended in 8 µl of RNase-free water. A first-strand cDNA synthesis kit (Pharmacia Biotech) was used to synthesize cDNA by RT from mRNA. According to the instruction of the manufacturer, 8 µl of total RNA, 0.2 µg random hexadeoxynucleotides, and 100 U Maloney murine leukemia virus RT were used. The reaction mixture was incubated at 37°C for 60 min and then heated to 65°C for 10 min to inactivate the activity of RT and to denature cDNA hybrids.
PCR reactions were performed in a total volume of 50 µl using a PCR Supermix kit (GIBCO BRL) containing 22 mM Tris · HCl (pH 8.4), 55 mM KCl, 1.65 mM MgCl2, 200 µM dNTPs, 5 µl RT reaction mixture, 22 U recombinant Taq DNA polymerase, and 400 pmol of the specific primer pairs for NADH oxidase subunits or GAPDH as we described previously (45, 47). The reactions were cycled 30 times from 94°C for 1 min to 52°C (for subunits p22phox, p47phox, and Gp91phox) or 55°C (for subunits p40phox and p67phox) for 1 min, and then 72°C for 1.5 min. Samples were incubated at 72°C for an additional 5 min after the last cycle was completed. NADH oxidase subunits p22phox, p40phox, p47phox, p67phox, p91phox, and GAPDH primers spanned fragments of 134, 611, 394, 599, 459, and 555 bp from their respective cDNAs. Negative control PCRs with a substitution of dissection solution or total RNA of TALHs without RT reaction were performed in parallel. The structure of the primers was as follows: p22: sense 5'-CGG GGA AAG AGG AAA AAG and antisense 5'-GGC ACG GAC AGC AGT AAG; p40: sense 5'-CCG CCG CTA TCG CCA GTT CTA and antisense 5'-CCT CCT CCA CCG CAA TGT CCT; p47: sense 5'-GCC TGA TGA CCT GAA ACT and antisense 5'-GGC TTC ACC CTC AGA CAG; p67: sense 5'-CCC AAA ACC CCA GAA ATC and antisense 5'-CCC ACC GTA TGC TCA CAC; Gp91: sense 5'-ATG AGG TGG TGA TGT TAG TGG and antisense 5'-AGT TGG AGA TGC TTT GTT TAC; and GAPDH: sense 5'-GTG AAG GTC GGT GTG AAC GGA TTT and antisense 5'-CAC AGT CTT CTG AGT GGC AGT GAT. All primers were synthesized by Operon Technologies (Alameda, CA). The base sequences of these primer pairs were based on previous work showing a high efficiency for PCR products in different tissues (4, 15, 21, 24, 39). PCR products were separated by a 1.5% agarose gel electrophoresis (200 V for 1 h) in 1× TBE buffer, stained with Eth bromide (0.5 µg/ml), and visualized under ultraviolet light; then, a photograph was made. The identity of the PCR products of NADH oxidase subunits was determined by TA cloning and sequencing as we described previously (45). Briefly, RT-PCR products of NADH oxidase subunits from TALHs were ligated into pCR II TOPO vector (Invitrogen, San Diego, CA), and the subsequent plasmid DNA was purified using an ion-exchange column (Qiagen, Chatsworth, CA). The plasmid DNA was digested with EcorI to confirm the positive clones, and the inserts in these positive clones were sequenced by Genemed Synthesis (San Francisco, CA).Immunohistochemistry for NADH oxidase within the kidney. Immunohistochemistry was performed as described previously (25). In brief, the rat kidneys were removed and fixed in 10% neutral formalin and embedded in paraffin. Then, kidney sections were prepared, incubated with xylene, and hydrated through several washes in ethanol and distilled water to remove the paraffin. Endogenous peroxidase activity was blocked by incubation of the sections with 3% H2O2 in methyl alcohol for 30 min and several rinses with distilled water. DAKO target antigen retrieval solutions were used to retrieve antigen by incubation of the sections in a water bath at 95-100°C for 30 min, and endogenous biotin was blocked by a DAKO biotin-blocking system. After a nonspecific staining block, a primary monoclonal antibody against Gp91 subunit of NADH oxidase (Transduction Laboratory, Lexington, KY) was used for immunostaining with a dilution of 1:100 in DAKO antibody diluents with background-reducing components. The kidney sections were incubated with the primary antibody overnight at room temperature in a humidified chamber, and a kidney section was incubated with vehicle (DAKO antibody diluent) as a negative control. Secondary antibody biotinylated cocktails were incubated with the sections for 60 min and then with streptavidin peroxidase conjugates (60 min; DAKO, Carpinteria, CA). The immunoreactive bands were detected with a diaminobenzidine-chromogen solution. Then, the sections were rinsed, nuclear stained, dehydrated, cleared, and mounted with permount media.
Statistical analysis. Data are presented as means ± SE. The significance of differences within and between multiple groups was evaluated using one- or two-way ANOVA followed by a post hoc test (Duncan's multiple range test; SigmaStat, San Rafael, CA). A P value <0.05 was considered statistically significant.
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RESULTS |
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O
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Different pathways responsible for O
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Inhibition of NADH oxidase by DPI and blockade of
O
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Identification of mRNA of NADH oxidase subunits in cTALHs.
Figure 4 illustrates a representative
photograph of Eth bromide-stained RT-PCR products of five subunits of
NADH oxidase and GAPDH in cTALHs. Four of these subunits were detected
in total RNA extracted from cTALHs (n = 5). The sizes
of the RT-PCR products of p40phox, p47phox,
p22phox, and Gp91phox were 611, 394, 134 and 459 bp, respectively, which were identical to the predicted sizes based on
their cDNA sequences deposited in GenBank. Direct addition of total RNA
from cTALHs into PCR reaction without reverse transcription or
dissection solution used for PCR did not produce any signal
[lane RT () and lane DS], suggesting that PCR
products are derived from cDNA synthesized by RT. Subunit
p67phox with a predicted size of 599 bp was not detected.
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Immunohistochemistry of NADH oxidase subunit Gp91phox within the
rat kidney.
Identification of NADH oxidase mRNA described above provided strong
evidence showing the mRNA expression of this enzyme in the TALHs.
However, the presence of mRNA does not necessarily indicate the
expression of enzyme proteins. Therefore, we performed immunohistochemical examination to identify the protein expression of
this enzyme in the TALHs. Using a monoclonal antibody against a major
membrane subunit of NADH oxidase, Gp91phox, a high level of
expression of this NADH oxidase subunit (brown staining) was found in
the renal outer medulla with the high abundance on the cell
membrane of the TALHs (n = 4). Lesser brown staining
was found in the renal cortex, and no signal could be detected in the
renal papilla (inner medulla; Fig. 5).
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Effects of hypoxia on O
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Effects of ANG II on O
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DISCUSSION |
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The present study detected O
Using this assay of intracellular O
In previous studies, NADH oxidase has been shown to be the major
oxidase in vascular tissue and in cardiac cells compared with the
production of ROS from xanthine oxidase, arachidonic acid, and
mitochodrial oxidases (5, 16, 23, 27, 29, 31). Although
early work demonstrated xanthine oxidase as a prime source of
endothelium-dependent O
In the present study, increased intracellular O
To further confirm that an increase in intracellular
O
It has been demonstrated that the activity of O1 · l · s
1 to 2 × 109 mol
1 · l
· s
1 (39). Previous studies have
shown that only picomolar O
NADH or NADPH oxidase in mammalian professional phagocytes is well
characterized among the O
To further determine the effects of tissue or cell oxygenation on the
production of O
The present study also determined whether the production of
O
In summary, the present study demonstrated that NADH oxidase was the
major enzyme responsible for O
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ACKNOWLEDGEMENTS |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-54927 and American Heart Association Grant 96007310.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A.-P. Zou, Dept. of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: azou{at}post.its.mcw.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 29, 2001;10.1152/ajprenal.00218.2001
Received 9 July 2001; accepted in final form 7 January 2002.
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