Enhanced expression and activity of Nox2 and Nox4 in the macula densa in ANG II-induced hypertensive mice

Telemetry transmitter implantation.

Briefly, C57BL/6 mice (18–20 g, Harlan) were anesthetized with inhaled isoflurane (Butler Chemicals). A small incision was made in the middle of the neck for the insertion of the telemetry transmitter (PA-C10, Data Sciences). The catheter of the transmitter was placed in the left carotid artery and advanced down to the aortic arch. The body of the transmitter was placed subcutaneously in the right ventral flank of the animal. Mice were allowed to recover for 10 days.

Minipump insertion.

Osmotic minipumps (Alzet) were primed and filled with either ANG II (600 ng·kg⁻¹·min⁻¹) or saline (control). On the day of surgery, the animals, which had 10 days to recover from the telemetry transmitter implantation, were again anesthetized with isoflurane. Under sterile conditions, a small incision was made in the midscapular area on the animal's back. Using a hemostat, the subcutaneous tissue was separated and spread so as to create a small pouch in which the minipump was inserted. After insertion, the wound was sutured, and animals were allowed to recover for a day. Measurements of mean arterial pressure (MAP) were started on the second day after minipump implantation at 10-min intervals.

Laser capture microdissection and real-time PCR.

Laser capture microdissection (LCM) and real-time PCR were used to isolate MD cells and to measure Nox2 and Nox4 mRNA levels with methods we have previously described (19, 37, 64). Briefly, kidneys from C57BL/6 mice were removed and snap frozen in Optimal Cutting Temperature medium. Frozen sections (8 μm thick) were obtained and then stained and dehydrated using an Arcturus Histogene frozen section staining kit (Life Technologies, Carlsbad, CA). MD cells were dissected with an Arcturus LCM System (Model Veritas). Real-time PCR was used to quantify mRNA levels of Nox2 and Nox4. Total RNA from MMDD1 cells was extracted with the RNeasy Micro kit (Qiagen) following the manufacturer's instructions. One microgram of total RNA was reverse transcribed at 25°C for 5 min, 42°C for 45 min, and 85°C for 5 min using the iScript cDNA Synthesis kit (Bio-Rad) following the manufacturer's instructions. Real-time PCR was performed in a C1000TM Thermal Cycler real-time PCR machine (Bio-Rad). β-Actin was used as a housekeeping gene.

Isolation and microperfusion of the JGA and measurement of O₂⁻ with dihydroethidium.

We used methods similar to those we have previously described to isolate and microperfuse the MD with the attached glomerulus (19, 36). Briefly, the kidneys were removed and sliced along the corticomedullary axis. Single superficial intact glomeruli were microdissected together with adherent tubular segments consisting of portions of the TAL, MD, and early distal tubule. Using a micropipette, samples were transferred to a temperature-regulated chamber mounted on an inverted microscope (TE2000-S, Nikon). The glomeruli were positioned so the MD could be visualized using a holding pipette. TALs were cannulated with an array of glass pipettes. MDs were perfused with physiological saline solution containing (in mmol/l) 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); NaCl was 10 or 80 mmol/l. The luminal perfusion rate was 40 nl/min and was controlled with a pump (11 plus, Harvard Apparatus, Holliston, MS). The bath consisted of modified essential medium containing 0.15% BSA and was exchanged continuously at a rate of 1 ml/min.

We used the O₂⁻-sensitive fluorescent dye dihydroethidium to detect O₂⁻ levels, as we have previously described (33, 34). Briefly, once the TAL was perfused, MD cells were loaded with 10 μM dihydroethidium in 0.1% DMSO plus 0.1% pluronic acid from the lumen for 30 min and then washed for 20 min. Loaded MD cells were exposed to 380- and 490-nm light to excite dihydroethidium and oxyethidium, respectively. Emitted fluorescence from dihydroethidium was recorded using a 420-nm dichroic mirror with a 460/50-nm band-pass filter; for oxyethidium, we used a 565-nm dichroic mirror with a 605/55-nm band-pass filter. Square-shaped regions of interest were set inside the cytoplasm of MD cells, and their mean intensity was recorded every 5 s for 2 min. We recorded and calculated the rate of the changes for oxyethidium/dihydroxyethidium as an indicator of O₂⁻ production. Since we have previously found that increased luminal NaCl induced NO release by the MD (32, 34), we added the NO synthase inhibitor N-nitro-l-arginine methyl ester (10 ⁻⁴ mol/l) to the bath and lumen while measuring O₂⁻ to eliminate its reaction with NO, as we have previously reported (33, 34).

Measurement of O₂⁻ with lucigenin-enhanced chemiluminescence in MMDD1 cells.

Our experiments used MMDD1 cells, a MD-like cell line (kindly provided by Dr. J. Schnermann, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD) (62), in a manner similar to our previously described studies (20, 64, 65). MMDD1 cells were stimulated by ANG II (10⁻⁶–10⁻¹⁰ mol/l) for 12 h. O₂⁻ production in MMDD1 cells was determined using a lucigenin-enhanced chemiluminescence assay, as previously described (20, 64). Briefly, MMDD1 cells (10-cm dish) were washed with PBS twice, trypsinized in the dish, and kept in 9 or 12 ml Krebs-HEPES buffer [which contained (in mmol/l) 115 NaCl, 20 HEPES, 1.17 K₂HPO₄, 1.17 MgSO₄, 4.3 KCl, 1.3 CaCl₂, 25 NaHCO₃, 11.7 glucose, and 0.1 NAD(P)H, with pH adjusted to 7.4]. Lucigenin (5 × 10⁻⁶ mol/l) was then added to the samples, which were incubated for 30 min at 37°C with oxygen bubbling. A 0.5-ml sample was transferred into 1.6-ml polypropylene 8 × 50-mm tubes, and, using a Sirius luminometer (Berthold Detection Systems, Pforzheim, Germany), O₂⁻ was then measured following the manufacturer's instructions. Luminescence was measured for 10 s with a delay of 5 s.

Preparations for small interfering RNA.

Nox2, Nox4, and p22^phox small interfering (si)RNAs were designed and synthesized by Santa Cruz Biotechnology (Santa Cruz, CA). siRNA transfection was performed following the manufacturer's instructions as previously described (64, 65). Briefly, the transfection of the siRNA was performed using TransMessenger Transfection Reagent from QIANGEN (Germantown, MD) according to the manufacturer's instructions. Scrambled siRNAs were synthesized and used as negative controls. Twenty-four hours before transfection, MMDD1 cells were transferred onto six-well plates and transfected with 2 μg of each siRNA duplex using TransMessenger Transfection Reagent for 3 h in medium devoid of serum and antibiotics. MMDD1 cells were then washed once with PBS and grown in complete medium. Gene silencing was monitored by measuring RNA after incubation for 24 h.

Statistical analysis.

Data were collected as repeated measures over time under different conditions. We tested only the effects of interest using ANOVA for repeated measures and a post hoc Fisher's least-significant-difference test or a Student's paired t-test when appropriate. Changes were considered to be significant if P < 0.05. Data are presented as means ± SE.

Subpressor ANG II infusion induced hypertension in mice.

To demonstrate the effect of a slow pressor dose of ANG II on blood pressure, we infused ANG II with a minipump at a dose of 600 ng·min⁻¹·kg⁻¹ for 2 wk and measured MAP with telemetry in C57BL/6 mice. We found that basal MAP was 101 ± 3.1 mmHg and that this started to gradually elevate from day 5 of minipump insertion in the ANG II-treated group. Blood pressure continued to rise and reached a peak of 123 ± 3.8 mmHg on days 10–14 of the infusion (n = 7, P < 0.01). MAP of control mice infused with saline did not vary significantly (n = 5; Fig. 1).

O₂⁻ generation by the MD is enhanced in ANG II-induced hypertensive mice.

To determine whether O₂⁻ generation by MD cells increases after ANG II infusion, we isolated and perfused the JGA and measured O₂⁻ generation using dihydroethidium in mice infused with ANG II or saline for 2 wk by minipumps. The MD was perfused with 80 mM NaCl solution in the presence of N-nitro-l-arginine methyl ester (10 ⁻⁴ mol/l) in the bath and lumen. O₂⁻ generation by the MD was 9.4 ± 0.9 U/min in control mice. It increased to 34.7 ± 1.8 U/min in ANG II-induced hypertensive mice (n = 6, P < 0.01; Fig. 2).

To determine the source of O₂⁻ generation, we used the Nox inhibitor apocynin and repeated the above protocol. In the presence of apocynin, O₂⁻ generation by the MD was 2.1 ± 0.3 U/min in control mice and increased to 12.4 ± 2.3 U/min in ANG II-induced hypertensive mice (n = 5, P < 0.01; Fig. 2). These data indicate that there is a significant increase in MD O₂⁻ generation in ANG II-hypertensive mice and that Nox is an important source of this O₂⁻.

ANG II increased O₂⁻ generation in cultured MMDD1 cells.

To further study the source of ANG II-induced O₂⁻ generation in the MD, we stimulated cultured MMDD1 cells with ANG II and measured O₂⁻ generation. First, we tested the dose-response curve. As shown in Fig. 3A, when MMDD1 cells were stimulated with different concentrations of ANG II for 12 h, ANG II at a concentration of 10⁻⁸ mol/l was the lowest concentration with a prominent effect on O₂⁻ generation. We then tested the time-response curve using 10⁻⁸ mol/l ANG II and found that 12 h of stimulation induced substantial O₂⁻ generation. Therefore, in the following experiments, we stimulated MMDD1 cells with 10⁻⁸ mol/l ANG II treatment for 12 h.

Under basal conditions, O₂⁻ generation was 921 ± 91 U·min⁻¹·10⁵ cells⁻¹. O₂⁻ generation increased to 3,687 ± 183 U·min⁻¹·10⁵ cells⁻¹. In the presence of the Nox inhibitor apocynin (10⁻⁵ mol/l), the xanthine oxidase inhibitor oxypurinol (10⁻⁴ mol/l), or cyclooxygenase-2 inhibitor NS-398 (10⁻⁶ mol/l), ANG II-stimulated O₂⁻ generation was 1,963 ± 122, 3,189 ± 83, and 3,246 ± 96 U·min⁻¹·10⁵ cells⁻¹, respectively (Fig. 4). These data again point to the fact that ANG II-stimulated O₂⁻ generation in MMDD1 cells arises primarily from Nox.

Nox2 and Nox4 mRNA levels in the MD are increased in ANG II-induced hypertension.

To determine the changes of Nox expression in the MD induced by ANG II, LCM techniques were used to isolate MD cells in mice infused with either subpressor ANG II or saline. Next, Nox2 and Nox4 levels were measured with real-time PCR. As shown in Fig. 5, Nox2 mRNA levels in the MD increased 3.7 ± 0.17-fold in ANG II-infused mice compared with control mice (n = 5, P < 0.01). Nox4 mRNA levels in the MD increased 2.6 ± 0.15-fold in the ANG II-infused mice compared with control mice (n = 5, P < 0.01; Fig. 5). These data indicate that both Nox2 and Nox4 isoforms of Nox in the MD significantly increased in subpressor ANG II-induced hypertension.

ANG II-generated O₂⁻ in the MD originates from both Nox2 and Nox4 isoforms of Nox in MMDD1 cells.

To determine which isoform(s) of Nox is the primary source of ANG II-stimulated O₂⁻ generation, we used Nox2 and Nox4 siRNAs to silence Nox2 and Nox4 genes and used p22^phox siRNA to knock down the function of both Nox2 and Nox4. ANG II stimulation increased O₂⁻ generation in MMDD1 cells treated with scrambled siRNA from 967 ± 42 to 3,278 ± 94 U·min⁻¹·10⁵ cells⁻¹. In MMDD1 cells treated with Nox2 or Nox4 siRNAs, ANG II-stimulated O₂⁻ generation was 1,632 ± 61 and 1,932 ± 57 U·min⁻¹·10⁵ cells⁻¹, respectively. In cells treated with p22^phox siRNA, ANG II-stimulated O₂⁻ generation was blocked (784 ± 83 U·min⁻¹·10⁵ cells⁻¹; Fig. 6A). To further confirm the contributions from Nox2 and Nox4, we treated MMDD1 cells with both Nox2 and Nox4 siRNAs. ANG II stimulation increased O₂⁻ generation in MMDD1 cells treated with scrambled siRNAs from 853 ± 51 to 2,841 ± 83 U·min⁻¹·10⁵ cells⁻¹. In MMDD1 cells treated with Nox2 and Nox4 siRNAs, ANG II-stimulated O₂⁻ generation was 718 ± 44 U·min⁻¹·10⁵ cells⁻¹ (Fig. 6B). These data indicate that ANG II enhances O₂⁻ generation from both Nox2 and Nox4 isoforms of Nox in MMDD1 cells.