Published online before print February 9, 2009, doi: 10.1161/HYPERTENSIONAHA.108.124594
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
(Hypertension. 2009;53:556.)
© 2009 American Heart Association, Inc.
Original Articles |
Isoforms and Functions of NAD(P)H Oxidase at the Macula Densa
From the Department of Physiology and Biophysics (R.Z., R.J., L.A.J., R.L.) and Division of Nephrology, Department of Medicine (L.A.J., R.L.), University of Mississippi Medical Center, Jackson; and the Hypertension and Vascular Research Division (P.H., J.L.G.) and Biostatistics and Research Epidemiology (E.P.), Henry Ford Hospital, Detroit Mich. Current address (R.Z.): Shandong Medical College, Jinan, China.
Correspondence to Ruisheng Liu, Department of Physiology and Biophysics, University of Mississippi Medical Center, 2500 N State St, Jackson, MS 39216. E-mail rliu{at}physiology.umsmed.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Macula densa cells produce superoxide (O2–) during tubuloglomerular feedback primarily via NAD(P)H oxidase (NOX). The purpose of the present study was to determine NOXs expressed by the macula densa and the role of each one in NaCl-induced O2– production. To identify which isoforms are expressed, we applied single-cell RT-PCR to macula densa cells isolated by laser capture microdissection and to MMDD1 cells (a macula densa-like cell line). The captured cells expressed neuronal NOS (marker of macula densa), NOX2, and NOX4 but not NOX1. Expression of the NOXs and neuronal NOS was essentially identical in the MMDD1 cells. Thus, we used MMDD1 cells to investigate which isoform is responsible for NaCl-induced O2– production. We used small-interfering RNA to knock down NOX2 or NOX4 in MMDD1 cells and measured O2– exposed to low-salt solution (LS; 70 mmol/L of NaCl) or high-salt solution (HS; 140 mmol/L of NaCl). Exposing control cells (scrambled small-interfering RNA) to HS increased O2– concentrations from 0.75±0.28 to 1.48±0.46 U/min per 105 cells in LS and HS, respectively (P<0.001). Inhibiting NOX2 blocked the HS-induced increase in O2– (0.62±0.39 versus 0.76±0.31 U/min per 105 cells in LS and HS groups, respectively). Blocking NOX4 did not affect HS-induced O2– levels. O2– levels in the control cells during LS and HS were 0.80±0.30 and 1.56±0.49 U/min per 105 cells, respectively (P<0.001); whereas O2– levels in NOX4-small-interfering RNA–treated cells during LS and HS were 0.40±0.25 and 1.26±0.51 U/min per 105 cells, respectively (P<0.001). We conclude that, whereas macula densa cells express the NOX2 and NOX4 isoforms, NOX2 is primarily responsible for NaCl-induced O2– generation.
Key Words: NAD(P)H oxidase • superoxide • macula densa • tubuloglomerular feedback
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Macula densa cells are modified epithelial cells located at the end portion of the thick ascending limb, at the hilus of its own glomerulus, where they are in close contact with the glomerular arterioles. In response to changes in luminal NaCl delivery, they initiate signaling pathways that adjust glomerular filtration rate (a process called tubuloglomerular feedback [TGF]) and regulate renin release. These 2 processes are among the most important mechanisms that regulate the renal microcirculation and sodium excretion1–3; consequently, they are tightly regulated by various factors, such as angiotensin II, NO, and superoxide (O2–).
O2– is produced by various cytosolic enzymes and by mitochondria. The major source of O2– in the kidney is NAD(P)H oxidase,4,5 which is present in the vasculature, cortex, and medulla6–9 and has been implicated in the pathogenesis of several models of hypertension.10 Renal NAD(P)H oxidase activity is increased by angiotensin II and by salt loading.11 Macula densa cells have been found to have main units of NAD(P)H oxidase12 and produce O2– in response to increased NaCl delivery.6,13 Macula densa–derived O2– can modulate single nephron glomerular filtration rate during infusion of angiotensin II,14 and we reported recently that O2– in the macula densa augments the TGF response, primarily by scavenging NO.6 Accordingly, O2– is an important modulator of TGF, renal hemodynamics, and sodium excretion. However, which isoforms of NAD(P)H oxidase (NOXs) are produced at the macula densa cells and the function of each isoform are unknown. Performing such studies has been difficult, because macula densa cells represent a very small percentage of renal cells and are located in very small clusters. However, the recent development of laser capture microdissection (LCM),15–17 a novel technique that facilitates separating and harvesting of specific cells, now allows us to isolate and study specific renal cells, including macula densa cells.
In the present study, we isolated macula densa cells from frozen rat kidney using LCM. We identified the NOXs expressed by these captured macula densa cells and compared them with those expressed by a macula densa-like cell line (MMDD1). In addition, we investigated which isoform is responsible for NaCl-induced O2– generation in the macula densa. We found that captured macula densa and MMDD1 cells express NOX2 and NOX4 and that the NOX2 is the isoform primarily responsible for NaCl-induced O2– production by the macula densa.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
All of the experiments were approved by the Henry Ford Hospital Animal Care and Use Committee before performing any procedures on animals. Studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals and the Guidelines of the Animal Welfare Act. Experiments were undertaken on renal tissue obtained from male Sprague-Dawley rats and in MMDD1 cells, a renal epithelial cell line with properties of macula densa cells (kindly provided by Dr J. Schnermann, National Institutes of Health). All of the chemical compounds were purchased from Sigma, except DMSO and lucigenin, which were obtained from Invitrogen.
LCM of Macula Densa Cells
Sprague-Dawley rats weighing between 100 and 120 g were anesthetized with ketamine (50 mg/kg IP) and xylazine (50 mg/kg IP). The abdomen was opened and the kidneys removed. Sections of kidney were snap frozen in optimal cutting temperature medium using an isopentane bath, and 8-µm frozen sections were stained and dehydrated using a Histogene frozen-section staining kit (Arcturus) according to the manufacturer’s instructions. The sections were then viewed with the LCM microscope. The macula densa cells, which were identified by their anatomic location and morphology, were then "painted" (marked on computer screen) and dissected with the laser under the x20 objective, using a beam width of 7.5 µm and a beam intensity of 50 mW. We subsequently used RT-PCR to measure the NOX expression in the captured cells and neuronal NOS (nNOS), a marker for macula densa cells,18 to confirm that the captured cells were macula densa.
MMDD1 Cells
We used MMDD1 cells, a renal epithelial cell line with properties of macula densa cells, developed and kindly supplied by Dr J. Schnermann (National Institutes of Health).19 These cells were derived from SV40 transgenic mice and acquired using fluorescence-activated cell sorting of renal tubular cells labeled with segment-specific lectins. This cell line has been shown to express well-known macula densa markers, eg, cyclooxygenase 2 (COX-2), nNOS, ROMK, and NKCC2.19–21 In the present studies, MMDD1 cells at passages 15 to 20 were routinely trypsinized and suspended in DMEM nutrient mixture-Ham’s F-12, supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL). The cells were plated onto culture dishes and incubated at 37°C in a humidified atmosphere of 95% room air-5% CO2. The medium was changed every 2 days, and once the cells reached confluence (typically in 3 to 4 days), the cells were ready for small-interfering RNA (siRNA) and O2– experiments.
RT-PCR for Macula Densa Cells Isolated by LCM
We used the single-cell RT-PCR kit (Ambion), as described below. Despite the potential for measuring mRNA from single cells, our pilot experiments suggested that 
20 cells captured from the frozen slide were required to extract enough mRNA for RT-PCR. Total RNA was extracted using a PicoPure RNA isolation kit (Arcturus) according to the manufacturer’s instructions. Five microliters of RNA were reverse transcribed for 30 minutes at 45°C using 50 µmol/L of random primers (Invitrogen) and a MessageSensor reverse transcription (RT) kit (Ambion) and then heated for 10 minutes at 95°C and subsequently placed on ice. The resultant RT product was then amplified by PCR using the following protocol. Five µL of RT product and 0.5 µmol/L of gene-specific primers were added to 1 U of SuperTaq (Ambion) and heated to 95°C for 5 minutes. The samples were then cycled 40 times as follows: 15 seconds at 95°C, 30 seconds at 58°C, and 1 minute at 72°C. The final extension was for 10 minutes at 72°C.
RT-PCR for MMDD1 Cells
Total RNA was extracted using the RNeasy Micro kit (Qiagen) according to the manufacturer’s instructions. Briefly, 0.5 µg of total RNA were reverse transcribed for 1 hour at 37°C using 10 µmol/L of random primers (Invitrogen) and an Omniscript RT kit (Qiagen). The resultant RT product was then amplified by PCR by adding 5 µL of the RT reaction and 1 µmol/L of the gene-specific primers to the PCR Master Mix kit (Promega). The mixed samples were then heated to 94°C for 5 minutes and cycled at 94°C for 45 seconds, 58°C for 45 seconds, and 72°C for 1 minute for 35 cycles. Final extension was for 10 minutes at 72°C.
The amplified products of the single-cell RT-PCR and MMDD1-RT-PCR were run on 1.5% agarose gels containing ethidium bromide (0.5 µg/mL) and visualized under UV light. GAPDH, as a housekeeping gene, was set up as an internal loading control. All of the steps for RT-PCR are the same according to the manufacturer’s instruction. Samples that were not reverse transcribed were used as a negative control, and samples from kidney cortex were used as a positive control. A 100-bp DNA ladder marker was used to identify the molecular weight of the targeted DNA. Primer sequences, expected band, and GenBank No. are listed in the Table.
|
Preparations for siRNA
All of the siRNAs were designed and synthesized by Santa Cruz Biotechnology. siRNA transfection was performed using a siRNA Reagent System (Santa Cruz Biotechnology) according to the manufacturer’s instructions. Scrambled siRNAs were synthesized and used as negative controls. To achieve optimal transfection efficiency, various parameters, including the amounts of transfection reagent, RNA, and trans-mRNA complexes, the cell density, and the length of exposure of cells to trans-mRNA complexes, were optimized. At 24 hours before transfection, MMDD1 cells were transferred onto 6-well plates (5x105 cells per well) and transfected with 0.8 µg each of siRNA duplex using trans-mRNA transfection reagent for 4 hours in medium devoid of serum and antibiotics. This procedure does not affect cell viability. Macula densa cells were washed once with PBS and grown in complete medium. Gene silencing was monitored by measuring RNA after incubation for 24 to 72 hours.
Measurement of O2– With Lucigenin
We measured O2– production in the MMDD1 cell line using a lucigenin-enhanced chemiluminescence assay, as described previously.22–24 Briefly, confluent MMDD1 cells were rinsed twice in PBS solution. The cells were then trypsinized and suspended in 5 mL containing either high- or low-NaCl solutions. The high-NaCl solution contained (in mmol/L): 140 NaCl, 10 HEPES, 1.0 CaCO3, 0.5 K2HPO4, 4.0 KHCO3, 1.2 MgSO4, 5.5 glucose, 0.5 Na acetate, and 0.5 Na lactate (pH 7.4). The low-NaCl solution was the same as high-NaCl solution except that the NaCl was reduced to 70 mmol/L and mannitol was used to maintain the same osmolarity as the high-NaCl solution. Lucigenin (5 µmol/L) was added to each of the samples, which was then placed in 1.6-mL polypropylene 8x50-mm tubes (Evergreen Scientific). After allowing the samples to equilibrate with lucigenin at 37°C for 15 minutes, the tubes were placed in a luminometer (TD-20e; Turner Designs) with the light chamber maintained at 37°C. Luminescence measurements were integrated for 30-second periods and the cycle repeated 9 times, averaging 10 values. At the end of each experiment, the cell-permeant O2– scavenger Tiron (10 mmol/L) was added and 15 more cycles read; the final 8 values were averaged. O2– was expressed as units per minute per 105 cells. All of the O2– measurements were performed in the presence of 10–4 mmol/L N-nitro-L-arginine methyl ester, a, NOS inhibitor, to eliminate O2– quenching by NO.
Statistics
We used a 2-way ANOVAs to assess whether a NOX or the level of salt affected O2– production and whether the NOX affects salt-induced changes. The design had 2 main effects, NOX and salt concentration, and one 2-way interaction. If the interaction was significant, we checked for salt effects using paired t tests on each isoform separately and for NOX effects using Student t tests on each salt concentration separately.
The examination of 3 repeated measures was accomplished with ANOVA for repeated measures. The interest in this analysis was primarily directed at the 3 pairwise comparisons. These were done using paired t tests with a Hochberg’s adjustment for multiple testing. Data are expressed as the means plus or minus the SE, and an adjusted P value <0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
NOXs Expressed in LCM-Captured Macula Densa and MMDD1 Cells
We first demonstrated the feasibility of isolating and capturing macula densa cells using LCM. Figure 1 shows a representative example of a glomerulus with its macula densa, before and after the macula densa has been captured with LCM; the isolated macula densa cells are shown in Figure 1C. As seen in the figure, macula densa cells were readily identifiable by their anatomic location and morphology. These captured cells expressed nNOS (see below and Figure 2) but not endothelial NOS (expressed by the thick ascending limb and vasculatures), thus further confirming that the captured cells were macula densa cells and not contaminated by surrounding cells.
|
|
We next used RT-PCR techniques to identify which NOXs are expressed by the macula densa cells. Figure 2 shows representative blots for the NOX1, NOX2, and NOX4 isoforms, as well as for nNOS, in renal cortex, laser-captured macula densa cells, and MMDD1 cells. Figure 2A shows that the macula densa cells collected using LCM clearly expressed the NOX2 and NOX4, as well as nNOS (they did not express NOX1), indicating that NOX2 and NOX4 are the main isoforms present in macula densa cells.
Because the MMDD1 cell line may have some differences with macula densa cells isolated from the in vivo renal cortex, yet we would be using this cell line to study the function of each NOX in these cells, we tested whether the MMDD1 cells expressed the same NOXs as the laser-captured macula densa cells. The representative blot depicted in Figure 2B shows that the expression profile for the NOXs and nNOS in the MMDD1 cells was essentially identical to that of the laser-captured macula densa cells. Thus, these results demonstrate that the laser-captured macula densa cells and MMDD1 cells exhibit the same NAD(P)H oxidase expression profile, and together they provide strong evidence that macula densa cells express the NOX2 and NOX4 isoforms. Figure 2C shows that the renal cortex expressed all 3 of the NOXs and nNOS, thus verifying the efficacy of all of our primers to detect the NOXs and nNOS.
Comparative Function of Macula Densa–Derived NOX2 and NOX4
To study the functions of individual NOXs, we used siRNA to knock down the NOX2 and NOX4 mRNA in the MMDD1 cell line. We first determined the efficacy of the siRNA in reducing their target NOX mRNA. Figure 3A shows a representative blot for NOX2 mRNA in MMDD1 cells with the siRNA-NOX2 or the scramble siRNA control, whereas Figure 3B shows an analogous blot for NOX4 mRNA. The bottom graphs in this figure show the corresponding densitometric data. Incubating the MMDD1 cells for 48 hours with 0.8 µg of siRNA duplex transfection reagent was very effective at knocking down the mRNA of its intended target; the siRNA-NOX2 knocked down NOX2 mRNA by 91±0.5%, NOX2 mRNA knocked down NOX4-siRNA mRNA only by 6±5.1% (as compared with scramble NOX2; Figure 3), and the siRNA-NOX4 knocked down NOX4 mRNA by 86±2.1%. To test the specificity of the siRNAs that we used, we measured NOX2 mRNA in siRNA-NOX4–treated cells and measured NOX4 mRNA in siRNA-NOX2–treated cells. In siRNA-NOX4–treated cells, NOX2 mRNA was 94.5±5.2% compared with the cells treated with scrambled siRNA. In siRNA-NOX2–treated cells, NOX4 mRNA was 103.1±5.6% compared with the control (Figure 3; n=5). Accordingly, we used this dose and incubation time in the remaining experiments.
|
One of the primary stimuli for NOX-derived O2– production in macula densa cells is the increased luminal NaCl. To determine which NOX isoform is responsible for this increase in O2–, we tested whether knocking down either NOX2 or NOX4 mRNA in MMDD1 cells prevented high-NaCl–induced increases in O2–. The effect of knocking down NOX2 on O2– concentration levels in MMDD1 cells is shown in Figure 4. A high-NaCl solution caused O2– concentration to increase in control MMDD1 cells (treated with scrambled NOX2 siRNA); the O2– concentration was 0.88±0.11 and 1.74±0.17 U/min per 105 cells in the low- (70 mmol/L) and high- (140 mmol/L) NaCl solutions, respectively (P<0.001). Knocking down NOX2 did not alter basal O2– levels but blocked high-NaCl–induced increases in O2– (O2– concentrations were 0.73±0.20 and 0.90±0.15 U/min per 105 cells in the low- and high-NaCl solutions, respectively; n=7). On the other hand, knocking down NOX4 had no effect on high-NaCl–induced O2– production (Figure 5). The O2– concentration in the control cells (treated with scrambled NOX4 siRNA) was 0.94±0.12 and 1.82±0.17 U/min per 105 cells in the low- and high-NaCl groups, respectively (P<0.001). The high-NaCl solution caused a similar increase in O2– concentration in NOX4 siRNA-treated cells; O2– was 0.51±0.12 and 1.58±0.24 U/min per 105 cells in the low- and high-NaCl groups, respectively (P<0.001; n=9). These data indicate that NOX2 is the primary isoform responsible for NaCl-induced O2– generation in the macula densa, and NOX4 is an isoform responsible for basal O2– generation.
|
|
We reported previously that NaCl-induced O2– generation in isolated perfused MD cells is mainly attributable to NAD(P)H oxidase.13,25 To establish whether NAD(P)H oxidase was also the main source of O2– in MMDD1 cells, we determined the relative contribution of NAD(P)H oxidase, xanthine oxidase, and COX-2 to NaCl-induced O2– generation in MMDD1 using an antagonist of NAD(P)H oxidase, xanthine oxidase, and COX-2 (Figure 6). First, we tested the role NAD(P)H oxidase in NaCl-induced O2– generation in MMDD1. The O2– concentration in MMDD1 cells on low- and high-NaCl solution was 0.50±0.05 and 1.36±0.09 U/105 cells (n=7; P<0.01). Adding the NAD(P)H oxidase inhibitor apocynin (10–5 M) for 30 minutes to high-NaCl MMDD1 cells caused the O2– concentration to decrease to 0.68±0.05 U/105 cells (n=19; P<0.01). Thus, blocking NAD(P)H oxidase blunted NaCl-induced increases in O2– concentration, suggesting that NAD(P)H is an important source of O2– production in these cells. In contrast, blocking xanthine oxidase with oxypurinol did not significantly alter O2– concentrations. In these experiments, the O2– concentrations in the cells maintained in low- and high-NaCl solutions were 0.59±0.05 and 1.32±0.12 U/105 cells, respectively (P<0.001), and 1.14±0.12 U/105 cells in the cells treated with oxypurinol (10–4 mmol/L) for 30 minutes. Finally, blocking COX-2 with NS-398 also did not affect the NaCl-induced increase in MMDD1 O2– concentration. O2– concentrations were 0.52±0.03 and 1.36±0.09 U/min per 105 cells during low- and high-NaCl solutions, respectively (n=14; P<0.01) and 1.25±0.09 U/min per 105 cells in the cells treated with NS-398 (10–6 mmol/L) for 30 minutes. These data indicate that NaCl-induced increases in O2– in MMDD1 cells, like that in freshly isolated and perfused macula densa cells, are predominantly via NAD(P)H oxidase.13,25
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, we successfully applied LCM to isolate macula densa cells from a frozen rat kidney cortex. These cells expressed NOX2 and NOX4 but not NOX1. This expression profile was essentially identical to that of the MMDD1 cells, further verifying the similarity of these 2 cell types and their suitability for macula densa research. Finally, using the MMDD1 cells, we found that the NOX2 is the main source of NaCl-induced O2–, and NOX4 is responsible for basal O2– production.
The macula densa plays a major role in NaCl-dependent regulation of glomerular arteriolar tone and renin release, and, thus, has been the subject of much study.18,26,27 However, it is difficult to acquire macula densa cells in quantities sufficient enough to perform the necessary biochemical analysis required to study cellular and molecular mechanisms. The recent development of LCM provides us with a tool that can be used to isolate macula densa.15 LCM is a novel technique based on the adherence of visually selected cells to a thermoplastic membrane (overlying the dehydrated tissue section), which is focally melted by triggering a low-energy infrared laser pulse. The melted membrane forms a composite with the selected tissue area that is removed by simply lifting off the membrane. The size of the laser spot can be selected as 30.0, 15.0, or 7.5 µm, facilitating dissection of groups of cells or even single cells. Consequently, LCM can be applied to a wide range of cell and tissue preparations, including frozen-tissue sections.16,17 The first objective of the present study was to use LCM to harvest macula densa cells from frozen rat kidneys. We identified macula densa cells in the tissue sections and found that we were readily able to dissect and harvest them using LCM. We then confirmed that the captured cells were macula densa cells by verifying that they expressed nNOS,18 demonstrating that LCM can be used effectively to harvest macula densa cells. Thus, we next used LCM-harvested macula densa cells to investigate the source of NaCl-induced O2– generation.
Emerging evidence suggests that O2– produced by the macula densa plays an important role in regulating TGF. We reported recently that increasing tubular NaCl induced O2– production by the macula densa primary from NAD(P)H oxidase. O2– produced by the macula densa augmented TGF by scavenging NO.6 This contention is further supported by Chabrashvili et al,12 who found that the macula densa expresses the main components of NAD(P)H oxidase, some of which are overexpressed in the spontaneously hypertensive rat, an experimental model of hypertension that has enhanced oxidant stress and TGF. These studies indicate the existence and functional importance of NAD(P)H oxidase at the macula densa cells. However, they did not establish which NOXs are responsible for macula densa–derived O2–. Five isoforms of NOX proteins with distinct tissue distributions have been found: NOX1 is expressed mainly in the colonic epithelium and vascular smooth muscle cells,28–30 NOX2 (gp91phox) in phagocytes, NOX3 in the embryonic kidney,31 NOX4 in the renal cortex,32,33 and NOX5 in T and B lymphocytes of the spleen and lymph nodes, as well as sperm precursors in the testis.34 Recently, 2 new members of the gp91phox-homologue family have been reported,35 dual oxidase 1/thyroid oxidase 1, found primarily in the thyroid and lung, and dual oxidase 2/thyroid oxidase 2, primarily located in the thyroid and colon.36,37 The potential NAD(P)H oxidase isoforms expressed in the adult kidney are NOX1, NOX2, and NOX4, because NOX3, NOX5, and dual oxidase are not likely to be present.31,34–36 We tested for the presence of these 3 isoforms in the macula densa. Our RT-PCR studies in renal cortex confirmed the presence of the 3 isoforms. Of these, only NOX2 and NOX4 were expressed in the LCM-captured macula densa cells. Likewise, these same 2 isoforms were expressed in a similar manner in the MMDD1 cells, providing substantiate that MMDD1 cells are suitable to study O2– generation of the macula densa. The major reason we chose the MMDD1 cell line was because it is difficult to knock down NOX2 and NOX4 at the macula densa in vivo.
We next used the MMDD1 cells to investigate which NOX is responsible for NaCl-induced O2– production. We arbitrarily set low- and high-NaCl solution as 70 and 140 mmol/L of NaCl according to the literature,19 in which they found that low NaCl stimulates prostaglandin estrogen 2 release, COX-2 expression, and activation of mitogen-activated protein kinases. We blocked the expression of either NOX2 or NOX4 with a specific siRNA and measured O2– concentration during exposure to low- and high-NaCl solutions. Our data indicate that NOX2 is the primary source of O2– at the macula densa induced by NaCl. Although they do not rule out that NOX4 may contribute significantly during other conditions, it does not seem to play a role in NaCl-induced O2– production. These results are consistent with our previous study in which we found that NaCl-induced O2– production in the macula densa cells was blocked by apocynin, an NAD(P)H oxidase inhibitor that acts by blocking serine phosphorylation of the cytosolic p47phox subunit. Our present study has established that, whereas NOX4 contributes to basal O2– production in the macula densa, NOX2 is the primary source for O2– induced by NaCl.
The different roles for the NOXs are not unexpected, for the following reasons. First, NOX2 and NOX4 localize to different subcellular compartments.38,39 Because O2– cellular signaling likely depends on both the amount and site of O2– production,38,40,41 it seems likely that NOX2 and NOX4 may mediate distinct signaling pathways. In addition, they are regulated differently. Activation of NOX2 requires agonist stimulation to induce translocation of cytoplasmic proteins to the plasma membrane and small GTPase RAC activation, raising the possibility that angiotensin II–induced enhancement of macula densa O2– production (and TGF) may also be via NOX2. Conversely, NOX4 is not controlled by RAC or the known cytosolic NOX components and does not require or use the NOX1-associated proteins NOXO1 and NOXA1. NOX4 has been reported to colocalize with p22phox to internal membranes and to be constitutively active.41 Hence, the mechanisms by which macula densa NOX4 expression and function are regulated and the biological significance remain unknown. Cardounel et al42 showed that, when nNOS is studied under depleted arginine conditions, blockade with arginine derivatives dictates O2– production. In the present study, we cannot exclude the possibility of enhanced O2– production from nNOS in MMDD1 cells when we used NG-nitro-L-arginine methyl ester. However, we believe that the O2– from nNOS is not significant, because inhibition of NAD(P)H oxidase with apocynin blocks O2– production (see Figure 6).
In summary, we found that NOX2 and NOX4 are the isoforms of NAD(P)H oxidase expressed at the macula densa. NOX2 is the primary source of O2– induced by NaCl, whereas NOX4 is responsible for basal O2– production.
Perspectives
We used a novel approach that combined LCM of macula densa cells with functional studies in MMDD1 cells, a macula densa-like cell line, to facilitate the study of macula densa cellular function. We applied LCM to isolate and harvest macula densa cells from frozen kidney tissue and then identified that these captured macula densa cells express the NOX2 and NOX4 isoforms of NAD(P)H oxidase. The expression profile of these cells was essentially the same as that of the MMDD1 cells. The NOX2 is the main source for NaCl-induced O2–, whereas the NOX4 contribute to the basal O2– production. Thus, conditions in which the activity of either of these NOXs is altered may contribute to enhanced TGF and abnormal NaCl homeostasis.
|
Acknowledgments |
|---|
Sources of Funding
This work was supported by the National Institutes of Health grants RO1-HL086767 (to R.L.) and RO1-DK073401 (to L.A.J.).
Disclosures
None.
|
Footnotes |
|---|
This paper was sent to Richard J. Roman, associate editor, for review by expert referees, editorial decision, and final disposition.
Received October 3, 2008; first decision October 21, 2008; accepted January 6, 2009.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Wilcox CS, Welch WJ, Murad F, Gross SS, Taylor G, Levi R, Schmidt HHW. Nitric oxide synthase in macula densa regulates glomerular capillary pressure. Proc Natl Acad Sci U S A. 1992; 89: 11993–11997.
2. Schnermann J, Persson AE, Agerup B. Tubuloglomerular feedback. Nonlinear relation between glomerular hydrostatic pressure and loop of Henle perfusion rate. J Clin Invest. 1973; 52: 862–869.[Medline] [Order article via Infotrieve]
3. Persson AEG, Salomonsson M, Westerlund P, Greger R, Schlatter E, Gonzalez E. Macula densa cell function. Kidney Int. 1991; 39: S-39–S-44.[CrossRef]
4. Wilcox CS. Oxidative stress and nitric oxide deficiency in the kidney: a critical link to hypertension? Am J Physiol Regul Integr Comp Physiol. 2005; 289: R913–R935.
5. Gill PS, Wilcox CS. NADPH oxidases in the kidney. Antioxid Redox Signal. 2006; 8: 1597–1607.[CrossRef][Medline] [Order article via Infotrieve]
6. Liu R, Ren Y, Garvin JL, Carretero OA. Superoxide enhances tubuloglomerular feedback by constricting the afferent arteriole. Kidney Int. 2004; 66: 268–274.[CrossRef][Medline] [Order article via Infotrieve]
7. Mitchell KD, Navar LG. Enhanced tubuloglomerular feedback during peritubular infusions of angiotensins I and II. Am J Physiol. 1988; 255: F383–F390.[Medline] [Order article via Infotrieve]
8. Modlinger P, Chabrashvili T, Gill PS, Mendonca M, Harrison DG, Griendling KK, Li M, Raggio J, Wellstein A, Chen Y, Welch WJ, Wilcox CS. RNA silencing in vivo reveals role of p22phox in rat angiotensin slow pressor response. Hypertension. 2006; 47: 238–244.
9. Mundel P, Bachmann S, Bader M, Fischer A, Kummer W, Mayer B, Kriz W. Expression of nitric oxide synthase in kidney macula densa cells. Kidney Int. 1992; 42: 1017–1019.[Medline] [Order article via Infotrieve]
10. Harrison DG, Cai H, Landmesser U, Griendling KK. Interactions of angiotensin II with NAD(P)H oxidase, oxidant stress and cardiovascular disease. J Renin Angiotensin Aldosterone Syst. 2003; 4: 51–61.
11. Kitiyakara C, Chabrashvili T, Chen Y, Blau J, Karber A, Aslam S, Welch WJ, Wilcox CS. Salt intake, oxidative stress, and renal expression of NADPH oxidase and superoxide dismutase. J Am Soc Nephrol. 2003; 14: 2775–2782.
12. Chabrashvili T, Tojo A, Onozato ML, Kitiyakara C, Quinn MT, Fujita T, Welch WJ, Wilcox CS. Expression and cellular localization of classic NADPH oxidase subunits in the spontaneously hypertensive rat kidney. Hypertension. 2002; 39: 269–274.
13. Liu R, Garvin JL, Ren Y, Pagano PJ, Carretero OA. Depolarization of the macula densa induces superoxide production via NAD(P)H oxidase. Am J Physiol Renal Physiol. 2007; 292: 1867–1872.[CrossRef]
14. Nouri P, Gill P, Li M, Wilcox CS, Welch WJ. p22phox in the macula densa regulates single nephron GFR during angiotensin II infusion in rats. Am J Physiol Heart Circ Physiol. 2007; 292: H1685–H1689.
15. Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang Z, Goldstein SR, Weiss RA, Liotta LA. Laser capture microdissection. Science. 1996; 274: 998–1001.
16. Curran S, McKay JA, McLeod HL, Murray GI. Laser capture microscopy. Mol Pathol. 2000; 53: 64–68.
17. Fend F, Raffeld M. Laser capture microdissection in pathology. J Clin Pathol. 2000; 53: 666–672.
18. Tojo A, Gross SS, Zhang L, Tisher CC, Schmidt HHHW, Wilcox CS, Madsen KM. Immunocytochemical localization of distinct isoforms of nitric oxide synthase in the juxtaglomerular apparatus of the kidney. J Am Soc Nephrol. 1994; 4: 1438–1447.[Abstract]
19. Yang T, Park JM, Arend L, Huang Y, Topaloglu R, Pasumarthy A, Praetorius H, Spring K, Briggs JP, Schnermann J. Low chloride stimulation of prostaglandin E2 release and cyclooxygenase-2 expression in a mouse macula densa cell line. J Biol Chem. 2000; 275: 37922–37929.
20. Paliege A, Mizel D, Medina C, Pasumarthy A, Huang YG, Bachmann S, Briggs JP, Schnermann JB, Yang T. Inhibition of nNOS expression in the macula densa by COX-2-derived prostaglandin E(2). Am J Physiol Renal Physiol. 2004; 287: F152–F159.
21. He H, Podymow T, Zimpelmann J, Burns KD. NO inhibits Na+-K+-2Cl- cotransport via a cytochrome P-450-dependent pathway in renal epithelial cells (MMDD1). Am J Physiol Renal Physiol. 2003; 284: F1235–F1244.
22. Cifuentes ME, Rey FE, Carretero OA, Pagano PJ. Upregulation of p67phox and gp91phox in aortas from angiotensin II-infused mice. Am J Physiol Heart Circ Physiol. 2000; 279: H2234–H2240.
23. Lieberthal W, Triaca V, Koh JS, Pagano PJ, Levine JS. Role of superoxide in apoptosis induced by growth factor withdrawal. Am J Physiol. 1998; 275: F691–F702.[Medline] [Order article via Infotrieve]
24. Pagano PJ, Ito Y, Tornheim K, Gallop PM, Tauber AI, Cohen RA. An NADPH oxidase superoxide-generating system in the rabbit aorta. Am J Physiol. 1995; 268: H2274–H2280.[Medline] [Order article via Infotrieve]
25. Liu R, Carretero OA, Ren Y, Wang H, Garvin JL. Intracellular pH regulates superoxide production by the macula densa. Am J Physiol Renal Physiol. 2008; 295: F851–F856.
26. Navar LG. The regulation of glomerular filtration rate in mammalian kidneys. In: Andreoli TE, Hoffman JF, Fanestil DD, eds. Physiology of Membrane Disorders. New York, NY: Plenum Press; 1978: 593–627.
27. Schnermann J, Levine DZ. Paracrine factors in tubuloglomerular feedback: adenosine, ATP, and nitric oxide. Annu Rev Physiol. 2003; 65: 501–529.[CrossRef][Medline] [Order article via Infotrieve]
28. Suh Y-A, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D, Chung AB, Griendling KK, Lambeth JD. Cell transformation by the superoxide-generating oxidase Mox1 [letter]. Nature. 1999; 401: 79–82.[CrossRef][Medline] [Order article via Infotrieve]
29. Banfi B, Maturana A, Jaconi S, Arnaudeau S, Laforge T, Sinha B, Ligeti E, Demaurex N, Krause KH. A mammalian H+ channel generated through alternative splicing of the NADPH oxidase homolog NOH-1. Science. 2000; 287: 138–142.
30. Touyz RM, Chen X, Tabet F, Yao G, He G, Quinn MT, Pagano PJ, Schiffrin EL. Expression of a functionally active gp91phox-containing neutrophil-type NAD(P)H oxidase in smooth muscle cells from human resistance arteries. Regulation by angiotensin II. Circ Res. 2002; 90: 1205–1213.
31. Kikuchi H, Hikage M, Miyashita H, Fukumoto M. NADPH oxidase subunit, 6p91(phox) homologue, preferentially expressed in human colon epithelial cells. Gene. 2000; 254: 237–243.[CrossRef][Medline] [Order article via Infotrieve]
32. Geiszt M, Kopp JB, Várnai P, Leto TL. Identification of Renox, an NAD(P)H oxidase in kidney. Proc Natl Acad Sci U S A. 2000; 97: 8010–8014.
33. Shiose A, Kuroda J, Tsuruya K, Hirai M, Hirakata H, Naito S, Hattori M, Sakaki Y, Sumimoto H. A novel superoxide-producing NAD(P)H oxidase in kidney. J Biol Chem. 2001; 276: 1417–1423.
34. Banfi B, Molnar G, Maturana A, Steger K, Hegedus B, Demaurex N, Krause KH. A Ca(2+)-activated NADPH oxidase in testis, spleen, and lymph nodes. J Biol Chem. 2001; 276: 37594–37601.
35. De Deken X, Wang D, Many MC, Costagliola S, Libert F, Vassart G, Dumont JE, Miot F. Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family. J Biol Chem. 2000; 275: 23227–23233.
36. Dupuy C, Ohayon R, Valent A, Noel-Hudson MS, Deme D, Virion A. Purification of a novel flavoprotein involved in the thyroid NADPH oxidase. Cloning of the porcine and human cDNAs. J Biol Chem. 1999; 274: 37265–37269.
37. Edens WA, Sharling L, Cheng G, Shapira R, Kinkade JM, Lee T, Edens HA, Tang X, Sullards C, Flaherty DB, Benian GM, Lambeth JD. Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox. J Cell Biol. 2001; 154: 879–891.
38. Hilenski LL, Clempus RE, Quinn MT, Lambeth JD, Griendling KK. Distinct subcellular localizations of Nox1 and Nox4 in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2004; 24: 677–683.
39. van Buul JD, Fernandez-Borja M, Anthony EC, Hordijk PL. Expression and localization of NOX2 and NOX4 in primary human endothelial cells. Antioxid Redox Signal. 2005; 7: 308–317.[CrossRef][Medline] [Order article via Infotrieve]
40. Terada LS. Specificity in reactive oxidant signaling: think globally, act locally. J Cell Biol. 2006; 174: 615–623.
41. Martyn KD, Frederick LM, von LK, Dinauer MC, Knaus UG. Functional analysis of Nox4 reveals unique characteristics compared to other NADPH oxidases. Cell Signal. 2006; 18: 69–82.[CrossRef][Medline] [Order article via Infotrieve]
42. Cardounel AJ, Xia Y, Zweier JL. Endogenous methylarginines modulate superoxide as well as nitric oxide generation from neuronal nitric-oxide synthase: differences in the effects of monomethyl- and dimethylarginines in the presence and absence of tetrahydrobiopterin. J Biol Chem. 2005; 280: 7540–7549.
Related Article:
Hypertension 2009 53: 456-457.
This article has been cited by other articles:
 |
|
 |
|
  S. C. Young, M. V. Storm, J. S. Speed, S. Kelsen, C. V. Tiller, T. Vera, H. A. Drummond, and D. E. Stec Inhibition of biliverdin reductase increases ANG II-dependent superoxide levels in cultured renal tubular epithelial cells Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2009; 297(5): R1546 - R1553. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Carlstrom and A. E. G. Persson Important Role of NAD(P)H Oxidase 2 in the Regulation of the Tubuloglomerular Feedback Hypertension, March 1, 2009; 53(3): 456 - 457. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||