Knockdown of Add3 impairs the myogenic response of renal afferent arterioles and middle cerebral arteries

Abstract

We have reported that the myogenic response of the renal afferent arteriole (Af-art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow are impaired in Fawn-Hooded Hypertensive (FHH) rats. Transfer of a region of chromosome 1 containing γ-adducin (Add3) from the Brown Norway rat rescued the vascular dysfunction and the development of renal disease. To examine whether Add3 is a viable candidate gene altering renal and cerebral hemodynamics in FHH rats, we knocked down the expression of Add3 in rat Af-arts and MCAs cultured for 36-h using a 27-mer Dicer-substrate short interfering RNA (DsiRNA). Control Af-arts constricted by 10 ± 1% in response to an elevation in pressure from 60 to 120 mmHg but dilated by 4 ± 3% when treated with Add3 DsiRNA. Add3 DsiRNA had no effect on the vasoconstrictor response of the Af-art to norepinephrine (10⁻⁷ M). Add3 DsiRNA had a similar effect on the attenuation of the myogenic response in the MCA. Peak potassium currents were threefold higher in smooth muscle cells isolated from Af-arts or MCAs transfected with Add3 DsiRNA than in nontransfected cells isolated from the same vessels. This is the first study demonstrating that Add3 plays a role in the regulation of potassium channel function and vascular reactivity. It supports the hypothesis that sequence variants in Add3, which we previously identified in FHH rats, may play a causal role in the impaired myogenic response and autoregulation in the renal and cerebral circulation.

the fawn-hooded hypertensive (FHH) rat develops proteinuria, glomerulosclerosis, and mild systolic hypertension. (28, 37, 39) It has been reported that the myogenic response of the afferent arteriole (Af-art), autoregulation of renal blood flow (RBF), and glomerular capillary pressure (Pgc) are altered before the development of proteinuria and hypertension in FHH rats. (6, 37, 39). Recently, we found that the myogenic response of the middle cerebral artery (MCA) and autoregulation of cerebral blood flow (CBF) are also altered in these rats (16, 30). Substitution of a 2.4-Mbp region of Brown Norway (BN) rat chromosome (Chr) 1 containing 15 genes, including γ-adducin (Add3), onto the FHH genetic background restores the myogenic response and autoregulation of RBF and CBF (6, 16, 30). The impaired myogenic activity in FHH rats is associated with a fourfold increase in the iberiotoxin (IBTX)-sensitive potassium (BK) channel current in vascular smooth muscle cells (VSMCs) isolated from cerebral and renal arteries (31). Sequence analysis identified unique sequence variants in the Add3 gene in FHH relative to other strains (6, 14).

Adducin is a cytoskeletal protein consisting of α (Add1)-, β (Add2)-, or γ (Add3)- subunits. Add1 forms heterodimers with Add3 in most tissues, including the cerebral and renal vasculature (27). It promotes actin-spectrin interactions and regulates actin polymerization by end-capping (27). Adducin plays an important role in the organization of the cytoskeleton, signal transduction, membrane trafficking, cell-to-cell contact formation, and cell migration (27). Its function is calcium and calmodulin dependent (25) and is regulated by protein kinases A and C (26), tyrosine kinase (27), and Rho kinase (22). Sequence variants in either Add1 or Add3 could potentially alter the cytoskeleton, trafficking of ion channels, and vascular reactivity. Mutations in Add1 have been linked to the development of hypertension in Milan normotensive (MNS) rats and humans (2), but the role of Add3 in the regulation of vascular function, especially at the level of the Af-art or MCA, has not been directly studied.

The myogenic response of the Af-art or MCA normally protects the kidney or brain from barotrauma by preventing the transmission of elevated pressures to glomerular and cerebral capillaries (1, 7–9, 15, 17, 35). However, identification of genes responsible for regulating the myogenic response has been difficult due to the lack of techniques to specifically alter the expression of genes of interest in the renal and cerebral vasculature. Studies using in vivo gene therapy approaches, such as viral transduction or injection of siRNA into the kidney or brain, do not specifically target the renal or cerebral microcirculation. There have also been some attempts to generate VSMC-specific transgenic or knockout animals; however, the cerebral or renal vasculature has not been specifically targeted or studied (5).

The purpose of the present study was to determine whether Add3 is a viable candidate gene that plays a role in the impaired myogenic response and the development of renal and cerebral injury in FHH rats. We first developed a new approach to specifically knockdown the expression of Add3 (or any other gene) in microdissected renal and cerebral arterioles maintained in short-term organ culture using a 27-mer Dicer-substrate short interfering RNA (DsiRNA). We then validated the Add3 DsiRNA and examined its effect of the knockdown of the expression of Add3 on the myogenic response of the Af-art and MCA. In addition, positively transfected VSMCs were also isolated from these vessels and potassium currents were studied using patch-clamp techniques.

MATERIALS AND METHODS

General

Experiments were performed on 9- to 12-wk-old male Sprague-Dawley (SD) and FHH.1^BN rats bred in the colonies maintained at the University of Mississippi Medical Center (UMMC). The rats had free access to food and water throughout the study. The animal care facility at the UMMC is approved by the American Association for the Accreditation of Laboratory Animal Care. All protocols involving animals received prior approval by the Institutional Animal Care and Use Committees (IACUC) of the UMMC. Rat aorta smooth muscle (RASM) cells (R-ASM-580) were purchased from Lonza (Basel, Switzerland). A full-length rat Add3 cDNA expression plasmid (pCMV6-Entry.Add3) was purchased from OriGene (Rockville, MD).

Design of the Rat Add3 DsiRNA

Five Add3 DsiRNAs were designed using IDT SciTools software (Integrated DNA Technologies, Coralville, IA: http://www.idtdna.com/SciTools/SciTools.aspx) (29). Two isoforms of Add3 have been identified in rats and reported to NCBI nucleotide database (NM_001164103 and NM_031552). They differ by a lack of exon 14 in isoform 2 (NM_031552). The sites selected were not located on exon 14 so that they could target both isoforms. The selection was based on the combined elements of both “standard” 21-mer siRNA design rules as well as the new 27-mer design criteria as described in IDT Scitools. Briefly, a double-stranded (DS) RNA interference molecule associated with a selected single-stranded DNA target site in Add3 was extended to be a Dicer substrate by adding different base pairs to the 3′-end of the sense strand and the 5′-end of the antisense strand, creating an asymmetric blunt end and a 3′-overhang molecule. The final DsiRNA consists of a 25-mer sense strand and a 27-mer antisense strand that is processed by the Dicer enzyme into the desired 21-mer siRNA by incorporating the antisense strand into the mature RNA Induced Silencing Complex (RISC). A DS NC1 duplex (Integrated DNA Technologies) that does not target any part of the human, mouse, or rat transcriptomes was used as a negative control for concentration-dependent nonspecific RNA interference.

Evaluation of the Efficacy of Add3 DsiRNA in a RASM Cell Line

Transfection of Add3 DsiRNA and SiGLO red fluorescence indicator in a RASM cell line.

RASM cells were transfected with 25 or 45 nM Add3 DsiRNA or 20 nM Add3 DsiRNA plus 5 nM of a red fluorescent Dharmacon siGLO indicator (Thermo Scientific, Waltham, MA). The cells were seeded into six-well plates on the day before transfection at the density that achieved 80–90% confluency the next day in antibiotic-free Dulbecco's modified Eagle’s medium (DMEM; Thermo Scientific) supplemented with 10% fetal bovine serum (FBS; Thermo Scientific). Five microliters of Add3 DsiRNA with the indicator in 95 µl Opti-MEM I reduced serum medium (Thermo Scientific) and 5 µl DharmaFECT siRNA transfection reagent in the same medium were incubated separately for 5 min at room temperature. These solutions were then combined. The mixture was incubated for an additional 20 min at room temperature and the volume was brought up to 1 ml with the antibiotic-free complete medium. This transfection solution was used to replace the medium in the wells of a six-well plate. The cells were then incubated at 37°C in 5% CO₂ for 24 h for mRNA analysis or 48 h for protein analysis.

RT-qPCR.

RNA was extracted from the transfected RASM cells and cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) as described earlier (15, 16). Fast SYBR Green Real-Time PCR Master Mixes (Life Technologies, Grand Island, NY) were mixed with 4 ng of cDNA and 25 ng of forward (Add3FE12: 5′-CATCGTGCATCTCGTCCTTGC-3′) and reverse (Add3RE15: 5′-CATCGTGCATCTCGTCCTTGC-3′) primers. The PCR products were amplified using a real-time PCR system (Mx3000P; Stratagene, La Jolla, CA) in a 25-µl PCR reaction containing 20 mM Tris·HCl buffer (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 mM of each dNTP, and 0.5 U Taq DNA polymerase (QIAGEN). DNA contamination from PCR products synthesized in the presence of dUTP (23) was eliminated by a blend of dTTP/dUTP that is compatible with uracil N-glycosylase (UNG) contained in the Master Mixes. The data were analyzed with Mxpro qPCR software (Stratagene, La Jolla, CA) using the 2^−ΔΔCT method (24).

Western blot.

The cells were collected and homogenized in an ice-cold RIPA buffer (R0278; Sigma-Aldrich, St. Louis, MO) supplemented with protease and phosphatase inhibitors (cat. no. 88663; Thermo Scientific). The homogenates were centrifuged at 9, 000 g at 4°C for 15 min and the supernatant was collected. Aliquots of the protein (50 µg) were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes using Trans-Blot Turbo Transfer System (Bio-Rad) and the membranes were blocked at room temperature for 1 h in TBST blocking buffer containing 5% nonfat milk. The membranes were incubated overnight at 4°C with a 1:100 dilution of mouse anti-Add3 antibody (sc-365177; Santa Cruz Biotechnology, Santa Cruz, CA) or a 1:8,000 dilution of an anti-β-actin antibody (ab6276; Abcam, Cambridge, MA) followed by a 1:1,000 (Add3) or 1:20,000 (β-actin) dilution of a horseradish peroxidase coupled anti-mouse secondary antibody (sc-2005; Santa Cruz Biotechnology) for 1 h. The anti-Add3 antibody is specific for an epitope between amino acids 631–657 near the COOH terminus of human Add3 but cross reacts with the highly homologous region in rat and mouse Add3. The membranes were treated with SuperSignal West Dura Extended Duration Substrate (34076; Thermo Scientific) and the relative intensities of the bands at ~94 kDa for Add3 and 42 kD for β-actin were determined using a ChemiDoc Imager system (Bio-Rad).

Validation of Add3 DsiRNA in Isolated Vessels

These experiments were performed on the MCAs freshly dissected from 9-wk-old male control rats as previously described (15, 16). The MCAs were cut into small fragments using sterilized surgical instruments in ice-cold DMEM supplied with 10% FBS and antibiotics, placed into 6-well cell culture plates precoated with poly-l-lysine (Sigma), and incubated at 37°C in 5% CO₂ for 6 h. The medium was then removed, and the MCAs were carefully washed twice with antibiotic-free DMEM supplied with 10% FBS without disturbing their attachment at the bottom of the plates. The vessels were then transfected with various concentrations of Add3 DsiRNA plus 5 nM of a red fluorescent Dharmacon siGLO transfection indicator. The transfected MCAs were incubated at 37°C in 5% CO₂ for 24 h for RT-PCR analysis. A transfected MCA fragment was placed side-by-side with a nontransfected fragment and was imaged using a fluorescent microscope (Nikon TS-100; Nikon Instruments, Melville, NY).

The MCAs were washed with ice-cold PSS solution 24-h after transfection. The vessels were placed into TRIzol solution (Life Technologies, Grand Island, NY) and homogenized using a FastPrep-24 homogenizer (MP Biomedicals, Santa Ana, CA). RNA was extracted, cDNAs were synthesized, and PCR was performed using the Add3FE12 and Add3RE15 primers described above. The PCR products were separated on 2% agarose gel and analyzed using ChemiDoc MP Imaging System (Bio-Rad). GAPDH was amplified to serve as a loading control to normalize the expression of Add3 mRNA in various samples.

Effect of Add3 DsiRNA on the Myogenic Response of the Af-art

Nine-week-old SD rats were euthanized using 4% isoflurane. The kidneys were removed and sliced along the cortical-medullary axis. The tissue slice was placed in ice-cold minimum essential media (MEM; Sigma) containing 5% BSA. Single superficial Af-arts with attached glomeruli were microdissected under the stereomicroscope and transferred to six-well cell culture plates that were precoated with poly-l-lysine (Sigma). The Af-arts were incubated in DMEM supplemented with 10% FBS and antibiotics and incubated at 37°C for 6 h in an atmosphere containing air with 5% CO₂.

After a 6-h incubation, the medium was carefully removed and the microvessels were washed twice with antibiotic-free medium and cotransfected with Add3 DsiRNA a (20 nM) and siGLO red (5 nM) as described above. The transfected Af-arts were incubated at 37°C in 5% CO₂ for 24–36 h for RT-PCR analysis, measurement of the myogenic response, and isolation of VSMCs.

Twenty-four hours posttransfection, the Af-arts with attached glomeruli were placed side-by-side with cultured microvessels that were exposed to the transfection reagent on a glass slide. The samples were covered with anti-fade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories. Burlingame, CA), coverslipped, and then sealed around the perimeter with nail polish. Images were obtained using an inverted fluorescent microscope using light phase filters for DAPI (excitation 360 nm, emission 460 nm) and siGLO red (DY-547, 557 nm/570 nm).

The Af-arts were also collected 24–36 h after transfection and washed with an ice-cold physiological salt solution (PSS) containing the following (in mmol/l): 119 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.6 CaCl₂, 1.2 NaH₂PO₄, 18 NaHCO₃, 0.03 EDTA, 10 glucose, and 5 HEPES. The attached glomeruli were removed by microdissection using a stereomicroscope. The Af-arts were pooled and placed into TRIzol solution and the expression of Add3 mRNA was determined by RT-PCR as described above for the MCA.

Thirty-six hours after transfection, the Af-arts with attached glomeruli were transferred to a temperature-regulated chamber mounted on the stage of an inverted microscope using a micropipette. The Af-arts were incubated at 37°C in MEM (Sigma) containing 5% BSA and cannulated with an array of glass pipettes as described previously (12, 18, 20). Briefly, the proximal end of the Af-art was aspirated into the end of a holding pipette with an inner diameter of 20–25 µm. A perfusion pipette with a tip of an inner diameter of 8–10 µm was advanced into the lumen of the Af-art so that the shank of the perfusion pipette could seal the vessel against the inner wall of the holding pipette. The tip of an additional fine-tipped pipette was advanced into the vessel beyond the tip of the perfusion pipette to measure intraluminal pressure (20). After a 30-min equilibration period, the baseline of the inner diameter of the Af-art was measured at a perfusion pressure of 60 mmHg using a digital CCD camera (Andor, Concord, MA) and NIS-Elements software (Nikon Metrology). Perfusion pressure was then increased to 120 mmHg and the inner diameter of the Af-art was redetermined. The measurements were taken at the proximal or midportion of the Af-art where the maximal constriction occurred (33). Then, perfusion pressure was returned to 60 mmHg, and the vasoconstrictor responses to norepinephrine (NE; 10⁻⁷ M) was determined.

Effect of Add3 DsiRNA on the Myogenic Response of the MCA

Thirty-six hours after transfection, the MCAs were transferred to a temperature-regulated chamber, mounted on glass micropipettes in a myograph, and bathed in PSS solution maintained at 37°C. The bath was bubbled with 95% O₂-5% CO₂ to provide adequate oxygen and maintain a pH of 7.4_. The vessels were equilibrated for 60 min and then preconditioned by the change in the intraluminal pressure from 40 to 140 mmHg three times. The myogenic response was then determined by measuring the diameter of the MCAs in response to intraluminal pressures ranging from 40 to 140 mmHg in steps of 20 mmHg using a videomicrometer (VIA-100; Boeckeler Instruments), as we previously described (15, 16). Then, perfusion pressure was returned to 60 mmHg, and the vasoconstrictor responses to 5-HT (serotonin, 10⁻⁶ M) and 5-HT plus IBTX (10⁻⁷ M) were determined. Finally, the bath was replaced with calcium-free PSS solution and the passive pressure-diameter relationship was determined.

Patch-Clamp Studies

Isolation of VSMCs from Add3 DsiRNA-transfected microvessels.

These experiments were performed using pooled renal microvessels and MCAs that were transfected with Add3 DsiRNA. Renal interlobular arteries and Af-arts with the glomeruli removed were used to isolate a sufficient number of renal VSMCs for patch-clamp experiments since we have previously reported that both of these segments exhibit myogenic responses (6, 40). The VSMCs were isolated from the transfected vessels as previously described (6, 31). Briefly, 24–36 h posttransfected renal and cerebral microvessels were washed with ice-cold PSS. The vessels were pelleted at 1,000 RPM for 5 min and incubated with gentle rotation at 37°C for 12 min in PSS solution containing papain (22.5 units/ml; Sigma) and dithiothreitol (2 mg/ml). The vessels were pelleted and resuspended in fresh PSS solution containing collagenase (250 units/ml; Sigma), trypsin inhibitor (10,000 units/ml), and elastase (2.4 units/ml) and incubated with gentle rotation at 37°C for 12 min. Single cells were released by gentle pipetting of the digested tissue. The VSMCs released into the media were collected by centrifugation and the pellet was resuspended in fresh PSS solution and maintained at 4°C. Patch-clamp experiments were completed within 2–4 h after cell isolation.

Patch-clamp experiments.

A whole cell patch-clamp mode was used to record potassium channel currents at room temperature using a pipette solution containing the following (in mM): 130 potassium gluconate, 30 KCl, 10 NaCl, 1.8 CaCl₂, 1 MgCl₂, and 10 HEPES (pH 7.4), and a bath solution containing the following (in mM): 130 NaCl, 5 KCl, 2 CaCl2,1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4). An Axopatch 200B amplifier (Axon Instruments, Foster City, CA) was used to clamp pipette potential and record whole cell currents. Data acquisition and analysis were performed using Clampfit software (version 10.0, Axon Instruments). Positively transfected cells were identified by the appearance of red fluorescence and the cells that were not fluorescent were compared as nontransfected controls. Outward potassium currents before and after IBTX (10⁻⁷ M) treatment were elicited by a series of 20 mV voltage steps from −60 to +120 mV from a holding potential of −40 mV as we have previously described (6, 31). Peak current amplitudes were obtained by averaging 5–10 trials. Membrane capacitance was determined by integrating the average capacitance in response to a 5-mV pulse. Peak currents (in pA) were expressed as current density (pA/pF) to normalize for differences in the size of the VSMCs.

Statistics

Mean values ± SE are presented. The significance of differences in mean values between and within groups was analyzed using a two-way ANOVA for repeated measures and Holm-Sidak test for preplanned comparisons. A P value < 0.05 was considered statistically significant.

RESULTS

Design and Evaluation of Add3 DsiRNA Constructs in a RASM cell line

The duplex sequences of the five Add3 DsiRNAs (a, b, c, d, e) and NC1 negative control used in the present study are presented in Fig. 1A. They were designed to bind to different regions of the Add3 gene as indicated in Fig. 1B. These Add3 DsiRNAs were first used to transfect the RASM cell line at two concentrations with DharmaFECT siRNA transfection reagent 4 to compare the efficiency of the knockdown of the expression of Add3 mRNA. Add3 DsiRNA a produced the largest response and lowered Add3 mRNA expression by 80 ± 3 and 84 ± 3% at concentrations of 25 and 45 nM, respectively. In contrast, RASM cells treated with the NC1 negative control exhibited a much smaller nonspecific reduction in Add3 mRNA expression of 22 ± 7 and 35 ± 6% at concentrations of 25 and 45 nM, respectively (Fig. 1C).

Fig. 1.

Design and evaluation of rat Add3 Dicer-Substrate RNAi (DsiRNA) on rat aorta smooth muscle (RASM) cell line. A: duplex sequences of Add3 DsiRNA and negative control (NC1). The table presents the sequences of the 5 Add3 DsiRNAs (a, b, c, d, and e) and the negative NC1 duplex control compared in the present study. The final DsiRNA consists of a 25-mer sense strand and a 27-mer antisense strand. Numbers in the parentheses refer to the targeted nucleotide sites in the mRNA sequence of Add3 (NM_001164103). B: positions of Add3 DsiRNA. The targeting sites of the five DsiRNA (a, b, c, d, and e) on the Add3 gene (NM_001164103) are presented. Boxes indicate the exons. Filled boxes indicate coding regions. The DsiRNA a selected for use in the present studies targets a site located in exon 5 that is indicated in red. C: efficiency of the knockdown of the expression of Add3 mRNA on RASM cells. The efficiency of the knockdown of the expression of Add3 mRNA on RASM cells 24-h posttransfection with Add3 DsiRNA was detected using RT-qPCR. The relative expression of Add3 mRNA normalized with β-actin was 100% in untreated cells, 78 ± 7% in NC1 negative control cells, and 20 ± 3, 29 ± 5, 111 ± 13, 25 ± 1, and 48 ± 5%, in the cells transfected with 25 nM Add3 DsiRNA a, b, c, d, and e, respectively. The levels of Add3/Actin were 65 ± 6% in NC1 negative control cells and 16 ± 3, 19 ± 6, 63 ± 9, 19 ± 2, and 32 ± 4%, respectively, using 45 nM Add3 DsiRNA. Mean values ± SE are presented. Experiments were repeated 3 times in triplicate. *Significantly different from the corresponding value in NC1 (25 nM)-treated cells. †Significant difference from the corresponding value in NC1 (45 nM)-treated cells. D: comparison of the efficacy of different DharmaFECT transfection reagents. The efficacy of 4 different DharmaFECT transfection reagents was compared by testing their ability to reduce the expression of Add3 mRNA normalized with β-Actin in RASM cells transfected with 20 nM Add3 DsiRNA a and 5 nM of a red or green fluorescent Dharmacon siGLO transfection indicator. Reagent 2 was most effective and Add3 DsiRNA knocked down Add3 mRNA expression by 89 ± 1% in a RASM cell line in comparison with the other 3 reagents. The inserted images show that the red or green fluorescent indicators localize to the nucleus. Mean values ± SE are presented. Experiments were repeated 3 times in triplicate. *Significantly different from the corresponding value in untreated cells. E: Add3 DsiRNA knockdown the expression of Add3 at the protein level. Add3 DsiRNA a reduced the expression of the Add3 protein in RASM cells (lanes 3 and 6) in comparison to untreated cells (lanes 1 and 4). Protein extracted from RASM cells transfected with pCMV6-Entry. Add3 expressed a high level of Add3 protein (lanes 2 and 5) and was used as a positive control for the Western blot. Experiments were repeated 3 times in triplicate.

The DharmaFECT siRNA transfection reagent kit includes four unique cationic lipid formulations. We next compared the efficacy of the different transfection reagents by testing the relative knockdown of the expression of Add3 mRNA in RASM cells transfected with Add3 DsiRNA a as presented in Fig. 1D. A RISC-independent fluorescent Dharmacon siGLO transfection indicator (5 nM, red or green) that localizes in the nucleus (Fig. 1D, inest) was cotransfected with Add3 DsiRNA a at a concentration of 20 nM. Reagent 2 was more effective at reducing Add3 mRNA levels than the other reagents. Figure 1E presents data indicating that the Add3 DsiRNA a reduced the expression of the Add3 protein in RASM cells (Fig. 1E, lanes 3 and 6) in comparison to untreated cells (Fig. 1E, lanes 1 and 4). Protein extracted from RASM cells transfected with pCMV6-Entry. Add3 expressed a high level of Add3 protein (Fig. 1E, lanes 2 and 5) and was used as a positive control for the Western blot.

Fig. 3.

Representative images showing positively transfected MCAs and Af-arts. A: representative picture showing a MCA positively transfected with Add3 DsiRNA could be identified by a strong siGLO red fluorescent signal vs. weak autofluorescent signal in an untreated vessel. B: renal Af-arts with attached glomeruli transfected with Add3 DsiRNA and the siGLO red reporter can be identified by the presence of red fluorescence (red arrows). Yellow arrows indicate that the untreated Af-art exhibits no red fluorescence and is only visible in light phase or after staining with 4',6-diamidino-2-phenylindole (DAPI, blue).

Fig. 6.

Measurements of potassium currents in isolated VSMCs from transfected renal and cerebral microvessels. A: representative images of positively transfected red fluorescent cells in the wall of a cannulated and perfused Af-art. B: representative images of VSMCs isolated from transfected renal microvessels. Positively transfected cells (red arrows) and nontransfected control cells (yellow arrows and circled) isolated from the same vessel could be distinguished by the presence or absence of a siGLO red fluorescent signal. C: comparison of potassium channel current densities in transfected (Add3 DsiRNA) and nontransfected (Ctrl) VSMCs isolated from Add3 DsiRNA-treated renal microvessels. Data are presented as mean values ± SE. Numbers in parentheses indicate animals studied. *Significant difference in the corresponding values measured in nontransfected cells. D: comparison of potassium channel current densities in transfected (Add3 DsiRNA) and nontransfected (Ctrl) VSMCs isolated from Add3 DsiRNA-treated MCAs. Data are presented as mean values ± SE. Numbers in parentheses indicate animals studied. *Significant difference in the corresponding values measured in nontransfected cells.

Fig. 4.

Effect of Add3 DsiRNA on the myogenic response of the afferent arteriole (Af-art). A: representative images illustrating that the myogenic response remains intact in an Af-art transfected with NC1 and cultured for 36 h. The inner diameter of this Af-art decreased from 17.2 to 15.7 µm when perfusion pressure was increased from 60 to 120 mmHg. B: comparison of the changes in the inner diameter of the freshly isolated Af-art to those seen in vessels transfected with Add3 DsiRNA or a NC1 control in response to an elevation in transmural pressure from 60 to 120 mmHg. Mean values ± SE are presented. The dashed line represents the control diameter of MCA at 60 mmHg. Numbers in parentheses indicate animals studied. *†Significantly different from the corresponding value in Add3 DsiRNA-transfected vessels. C: comparison of the response to norepinephrine (NE; 10⁻⁷ M) in the same freshly isolated vessels to transfected with Add3 DsiRNA or NC1 that were studied in B. Transmural pressure was maintained at 60 mmHg. Mean values ± SE are presented. Numbers in the parentheses indicate the number of animals studied in each group. The dashed line represents the control diameter of MCA at 60 mmHg.

Fig. 2.

Add3 DsiRNA knockdown of the expression of Add3 mRNA in rat middle cerebral artery (MCA) and afferent arteriole (Af-art). A: representative RT-PCR gel illustrating dose-dependent knockdown of the expression of Add3 mRNA in rat MCAs. B: Add3 DsiRNA dose dependently reduced the expression of Add3 mRNA normalized with GAPDH in pooled MCA fragments cultured for 24 h. Samples were run in duplicate from pooled MCA fragments (n = 6 rats). Mean values ± SE are presented. *Significant difference from the corresponding value in NC1-treated vessels. C: the expression of Add3 mRNA normalized with GAPDH was reduced by 55 ± 14% in Af-arts transfected with Add3 DsiRNA (20 nM) and cultured up to 24 h. The attached glomeruli were removed from the vessels before extraction of the RNA from the vessel segments. Samples were run in triplicate from pooled Af-art (n = 6 rats). Mean values ± SE are presented. *Significant difference from the corresponding value in NC1-treated vessels.

Fig. 5.

Effect of Add3 DsiRNA on the myogenic response of the MCA. A: comparison of the pressure-diameter relationships in MCAs freshly isolated from Sprague-Dawley (SD) and fawn hooded hypertensive (FHH.1^BN) rats and from vessels isolated from FHH.1^BN rats that were transfected with NC1, a negative control for concentration-dependent nonspecific RNA interference that does not target any part of the human, mouse, or rat transcriptomes, that were cultured for 36 h. Mean values ± SE are presented. The dashed line represents the control diameter of MCA at 60 mmHg. Numbers in parentheses indicate animals studied. B: comparison of the pressure-diameter relationship of MCAs isolated from the pooled SD and FHH.1^BN rats that were untransfected (ctrl) and transfected with NC1 vs. Add3 DsiRNA in the presence and absence of calcium in the bath. Mean values ± SE are presented. The dashed line represents the control diameter of MCA at 60 mmHg. Numbers in parentheses indicate animals studied. *†Significantly different from the corresponding value in Add3 DsiRNA-transfected vessels. C. Comparison of the pressure-diameter relationship of MCAs isolated from the pooled SD and FHH.1^BN rats that were untransfected (ctrl), transfected with NC1 vs. Add3 DsiRNA in the presence of serotonin (5-HT) and iberiotoxin (IBTX) in the calcium solution. Mean values ± SE are presented. Numbers indicate animals studied. *Significantly different from the corresponding value in freshly isolated untreated control vessels. †Significantly different from the corresponding value in NC1 control DsiRNA-treated vessels. #Significant difference from the treatment with 5-HT only within the same group.

Add3 DsiRNA Knockdown the Expression of Add3 mRNA in the Af-art and MCA

The ability of Add3 DsiRNA a and DharmaFECT siRNA transfection reagent 2 to reduce the expression of Add3 in the isolated Af-art and MCA was evaluated. We first found that the fluorescence of the siGLO reporter reached a peak in the vessels 24 h after transfection. The maximum knockdown of the expression of Add3 mRNA was achieved 24–36 h after transfection (data not shown). Add3 DsiRNA at concentrations of 1, 5, 10, or 20 nM dose dependently reduced the expression of Add3 mRNA 24 h after transfection in MCA by 22 ± 13, 25 ± 6, 36 ± 6, and 57 ± 2%, respectively (Fig. 2, A and B). Similarly, Add3 DsiRNA (20 nM) knocked down the expression of Add3 mRNA by 55 ± 14% in microdissected Af-art cultured for 24 h as shown in Fig. 2C.

Identification of Positively Transfected MCAs and Af-arts

MCAs transfected with Add3 DsiRNA could be identified by the strong fluorescent signal of the siGLO red reporter, while the untreated MCAs exhibit only a weak autofluorescent signal (Fig. 3A). Similarly, a strong signal was detected in the wall of the Af-art transfected with Add3 DsiRNA (red arrows) relative to the untreated Af-art (yellow arrows), which could only be imaged in the light phase or after staining the vessel with 4',6-diamidino-2-phenylindole (DAPI) using a fluorescence microscope (Fig. 3B). A number of other cells surrounding the glomerulus were also transfected. From their localization, these are likely parietal epithelial cells in Bowman’s space.

Effect of Add3 DsiRNA on the myogenic response of the Af-art.

Representative images obtained from a control Af-art microdissected from control rats and transfected with NC1 and cultured for 36 h indicate that the myogenic response remains intact. The luminal diameter of this vessel decreased from 17.2 µm (Fig. 4A, left) to 15.7 µm (right panel) when perfusion pressure was increased from 60 to 120 mmHg (Fig. 4A). The inner diameter of the Af-art cultured for 36 h constricted by 11 ± 4 and 10 ± 1% in fresh Af-arts and in vessels transfected with NC1 (20 nM), respectively, but increased by 4 ± 3% in Add3 DsiRNA transfected vessels when the transmural pressure was increased from 60 to 120 mmHg (Fig. 4B). However, the vasoconstrictor response to NE (10⁻⁷ M) was not significantly different in fresh control, NC1, or Add3 DsiRNA-treated Af-arts (Fig. 4C).

Effect of Add3 DsiRNA on the myogenic response of the MCA.

The MCAs isolated from SD and FHH.1^BN rats that were transfected with the NC1 control (n = 8) and cultured for 36 h constricted to the same extent as MCAs freshly isolated from SD (n = 5) and FHH.1^BN (n = 15) rats in response to an elevation in perfusion pressure from 40 to 140 mmHg (Fig. 5A). In contrast, the diameter of vessels treated with Add3 DsiRNA did not constrict when the pressure was increased (Fig. 5B). We combined the vessels from outbred SD and inbred FHH.1^BN rats that both have a wild-type Add3 gene as controls. All of the transfected vessels exhibited passive pressure-diameter curves when the bath solution was replaced with a Ca²⁺ free PSS solution. The vasoconstrictor response to 5-HT (10⁻⁶ M) was significantly lower in Add3 DsiRNA-treated vessels (19 ± 3%) in comparison to freshly isolated (35 ± 3%) or NC1 (36 ± 2%)-treated vessels (Fig. 5C). However, the vasoconstriction was not significantly different in the fresh control, NC1, or Add3 DsiRNA-treated MCAs in the presence of IBTX (10⁻⁷ M; Fig. 5C).

Effect of Add3 DsiRNA on potassium currents in VSMCs isolated from transfected renal and cerebral microvessels.

Positively transfected VSMC cells could be clearly identified by the red fluorescent signal in the wall of an isolated perfused Af-art (Fig. 6A). Positively transfected (red arrows) and nontransfected control VSMCs (yellow arrows and circled) isolated from these vessels could be distinguished by the presence or absence of the red fluorescent signal (Fig. 6B). Potassium currents were approximately threefold higher in Add3 DsiRNA-transfected VSMCs isolated from renal microvessels as compared with the currents recorded from nontransfected cells (Fig. 6C). Similar results were obtained in VSMCs isolated from the MCAs transfected with Add3 DsiRNA (Fig. 6D). Inhibition of the BK channel with IBTX (10⁻⁷ M) decreased peak potassium current densities by ~3.5-fold and IBTX-sensitive peak potassium current densities were ~4-fold higher in Add3 DsiRNA-positive transfected VSMCs compared with nontransfected control cells (Fig. 7, A and B).

Figure 7.

Comparison of IBTX-sensitive potassium currents in isolated VSMCs from transfected cerebral microvessels. A: comparison of the effect of iberiotoxin (IBTX) on potassium channel current densities in transfected (Add3 DsiRNA) and nontransfected (Ctrl) VSMCs isolated from Add3 DsiRNA-treated MCAs. Data are presented as mean values ± SE. Numbers in parentheses indicate animals studied. *Significant difference in the corresponding values measured in nontransfected cells. †Significant difference in the corresponding values measured in the same group of cells after IBTX treatment. B: comparison of IBTX-sensitive potassium current densities in transfected (Add3 DsiRNA) and nontransfected (Ctrl) VSMCs isolated from Add3 DsiRNA-treated MCAs. Data are presented as mean values ± SE. Numbers in parentheses indicate animals studied. *Significant difference in the corresponding values measured in nontransfected cells.

DISCUSSION

We previously reported that the myogenic response of renal and cerebral arteries and autoregulation of RBF and CBF are impaired in FHH rats (6, 16, 39, 40). Transfer of a region of Chr 1 containing 15 genes including Add3 from BN rats rescues renal and cerebral vascular dysfunction and opposes the development of renal disease in this strain. We further identified a number of sequence variants in the coding region of the Add3 gene in FHH relative to other strains that may alter its activity (6, 14). Moreover, we have evidence that the expression of Add3 protein in the vasculature is reduced in FHH rats relative to inbred FHH.1^BN rats that contain a wild-type Add3 gene (14). In the present studies, we combined the vessels from outbred SD rats that also have a wild-type Add3 gene with inbred FHH.1^BN rats as controls, since there was no significant difference on their myogenic response and vasoconstrictor response (Fig. 5A). However, little is known about the role of Add3 in the control of renal and cerebral vascular function, especially at the levels of the Af-art and MCA. Thus the goal of the present study was to determine whether Add3 is a viable candidate gene for the alterations in cerebral and renal hemodynamics in FHH rats. To this end, we first developed a new general technique to specifically knock down the expression of Add3 (or any other gene) in renal and cerebral arterioles maintained in organ culture for 36-h using a 27-mer DsiRNA. We then examined the effect of the knockdown of the expression of Add3 on the myogenic response of the Af-art and MCA. In addition, positively transfected VSMCs were isolated from these vessels and potassium currents were studied using patch-clamp techniques.

Recent studies using microdissected skeletal muscle resistance (4) or cerebral arteries (42) demonstrated that these vessels can maintain their vascular responsiveness and pressure-induced myogenic response when maintained in organ culture for up to 48 h. Several investigators have also reported that the expression of various genes could be knocked down in vessels maintained in organ culture after transfection with siRNA using reverse permeabilization technique (10, 41, 42). Thus we first evaluated whether a similar approach could be used to study the effect of the knockdown of the expression of Add3 on the myogenic response of rat MCA since it is larger than the Af-art and easier to obtain sufficient mRNA to test the effectiveness. We initially used the reverse permeabilization transfection strategy and commercial available pooled 21-mer Add3 siRNA (42). However, the transfection efficiency was poor based on the uptake of a siGLO fluorescent reporter. Therefore, we tested other transfection methods. We found that DharmaFECT siRNA transfection reagent 2 was more effective than other reagents in knocking down the expression of Add3 in cultured RASM cells (Fig. 1D). Moreover, we found that the EC₅₀ of the commercial available pooled 21-mer Add3 siRNA that reduced the expression of Add3 in the MCA was 40 nM (data not shown). The introduction of such a high concentration of siRNA could have off-target effects and produce a generalized reduction in the expression of Add3 and other transcripts (3). Therefore, we designed a 27-mer Add3 DsiRNA using an approach developed by Dr. John J. Rossi and Integrated DNA Technologies (38). DsiRNAs can increase the potency of RNA interference up to 100-fold in comparison to conventional 21-mer siRNAs (21). Five Add3 DsiRNAs targeting different regions of the Add3 gene were designed and a NC1 duplex that does not target any part of the human, mouse, or rat transcriptomes was used as the negative control (Fig. 1, A and B). We validated the efficacy and efficiency of these DsiRNAs in the RASM cell line and found that DsiRNA a transfected with DharmaFect reagent 2 was the most effective combination for reducing the expression of Add3 at both the message and protein level (Fig. 1, C–E) in RASM cells. This combination specifically knocked down the expression of Add3 mRNA by ~60% in both MCA and Af-art cultured for 36 h (Fig. 2).

Fluorescein (FITC)-labeled siRNA has been previously employed as an indicator of positive transfection (42); however, elastin and collagen in vessels exhibit strong green autofluorescence and interfere with its detection. Therefore, we used a red fluorescent Dharmacon siGLO indicator that localizes in the nucleus of transfected cells. We found that the uptake of the fluorescent signal by labeled vessel peaked 24 h after transfection and the knockdown of the expression of Add3 was maximal 24–36 h after transfection. We could easily distinguish positively transfected renal Af-arts and MCAs from untreated vessels when imaged side-by-side using a fluorescent microscope. We could also identify transfected vs. nontransfected VSMCs isolated from the same vessels by the presence or absence of red fluorescence.

After determining the appropriate condition for knocking down the expression of Add3 in small vessels with 27-mer DsiRNA, we studied its effects on the myogenic response of the Af-art and MCA. First, we determined that the myogenic responses to elevations in transmural pressure of MCAs cultured for up to 36 h were similar to those seen in freshly microdissected vessels. Next, we verified that transfection of MCAs and Af-arts with 20 nM of NC1 did not alter the myogenic response relative to untreated vessels cultured for 36 h. Moreover, the myogenic response of vessels isolated from either outbred SD or inbred FHH.1^BN genetic control rats, which both have a wild-type Add3 gene, was not significantly different (Fig. 5A); thus the data from both of these strains were pooled and used as normal controls.

The microdissected Af-arts also retained the ability to constrict normally to elevations in transmural pressure and NE when cultured up to 36 h. However, the vasoconstriction to 5-HT was diminished in MCAs treated with Add3 DsiRNA and administration of additional IBTX restored the constriction in these vessels to the similar levels as seen in fresh vessels and in NC1 controls (Fig. 5C). This finding is consistent with our recent report (32) indicating that vasoconstriction to 5-HT is reduced in the MCA of FHH rats, which has a mutation in the Add3 gene, due to failure to inhibit BK channel activity in comparison with control animals. The magnitude of these responses was similar to results previously published by our laboratory and others (6, 20, 34). The Af-art and MCA constricted by 11 ± 4 or 10 ± 1% and 24 ± 4 or 17 ± 4%, respectively, in response to an elevation in pressure from 60 to 120 mmHg in fresh controls or when transfected with the NC1 negative control but dilated by 4 ± 3 and 3 ± 0%, respectively, when treated with Add3 DsiRNA a. The loss of the myogenic response in the Af-art and MCA of SD and FHH.1^BN rats after knockdown of the expression of Add3 is of the same magnitude as we have previously reported in FHH rats (6, 30). Peak potassium current was threefold higher in positively Add3 DsiRNA transfected VSMCs isolated from the Af-art and/or MCA than in nontransfected (nonfluorescent) cells obtained from these same vessels. The magnitude of the increase in K⁺ channel currents in the cerebral and renal VSMCs resembles that seen in VSMCs isolated from FHH rats relative to FHH.1^BN rats and other strains (6, 31). Knockdown of Add3 exhibits a significantly higher level of IBTX-sensitive peak potassium current densities since administration of IBTX decreased peak potassium current densities in Add3 DsiRNA positively transfected VSMCs compared with nontransfected control cells (Fig. 7, A and B). These results are consistent with our previous findings demonstrating that the elevation in potassium currents in VSMCs isolated from renal and cerebral arteries of FHH rats is largely due to an increase in the opening of BK channels as they were inhibited by IBTX but not by 4-aminopyridine (31).

The mechanism by which the adducin regulates K⁺ channel activity and vascular reactivity remains to be determined. Add3 is a cytoskeletal protein composed of heterodimers of Add1 and Add3 in the kidney and vasculature (27) that favors actin-spectrin binding and regulates actin polymerization (27). The activity of adducin is also regulated by calcium-calmodulin, PKA, PKC, tyrosine, and Rho kinases (2, 22, 25–27). Adducin plays an important role in the organization of the cytoskeleton, signal transduction, membrane trafficking, cell-to-cell contact formation, and cell migration (27). A G460T polymorphism in ADD1 that alters actin polymerization disrupts the cytoskeleton and enhances Na⁺-K⁺-ATPase activity and sodium transport in the kidney and has been linked to the development of hypertension, stroke, and cardiovascular disease in single gene association studies in humans (2, 13). An A386G polymorphism in ADD3 was also identified in humans, but it is not linked to hypertension. Rather, it exhibits an epistatic interaction with blood pressure and the distensibility of the brachial artery in patients carrying the ADD1 variant (36). Add3 has also been reported to influence the activity of the renal thiazide-sensitive NaCl cotransporter (NCC) in the distal nephron (11). The results of the present study indicate that knockdown of the expression of Add3 increases K⁺ channel activity and inhibits the myogenic response in rat renal and cerebral arteries. The findings in the present studies are consistent with our hypothesis and preliminary reports (14, 31) that the functional variants in the Add3 gene could regulate vascular function by altering the membrane trafficking and expression of BK channel subunits and possible other channels and proteins involved in myogenic activation of renal and cerebral arteries.

The myogenic response of the Af-art or MCA normally protects the kidney or brain from damage by preventing the transmission of elevated pressures to the renal and cerebral microcirculation (1, 7–9, 15, 17, 33). It protects the brain from increases in capillary pressure, blood-brain barrier leakage, cerebral edema, and neurological damage (9, 17, 33). An impaired myogenic response of the MCA is often found in aging, hypertensive, diabetic, and obese individuals that are highly correlated with stroke and a cognitive decline (9, 17, 19, 33). Loss of the myogenic response of the Af-art increases pulsatile pressure and contributes to damage to glomerular capillaries, endothelium, and podocytes (7, 8, 34). This contributes to loss of barrier function and promotes the development of proteinuria. Sustained elevations in the delivery of protein to the proximal tubule lead to tubular injury, activation of the release of inflammatory cytokines and profibrotic mediators, and the development of hypertension and chronic kidney disease (7, 8, 34).

Perspectives

In the present study, we modified and improved existing technologies to knock down the expression of Add3 in rat renal Af-arts and MCAs maintained in organ culture for up to 36 h using a 27-mer DsiRNA. We also examined the effect of Add3 DsiRNA on the myogenic response of these vessels. This approach can be applied to manipulate the expression of any gene of interest ex vivo, specifically at the level of arterioles and small arteries. Our results indicate that knockdown of the expression of Add3 impairs the myogenic response of both Af-art and MCA and is associated with an increase BK channel activity. This is the first study demonstrating that Add3 plays a role in the regulation of IBTX-sensitive potassium channel function and vascular reactivity. It supports the hypothesis that sequence variants in Add3, which we previously identified in FHH rats, may play a causal role in the impaired myogenic response and autoregulation in renal and cerebral circulation that underlie the development of hypertension, chronic renal, and cerebral vascular diseases in FHH rats.

GRANTS

This study was supported by National Institutes of Health Grants HL-36279 (to R. J. Roman), DK-104184 (Roman), 050049 (to F. Fan), P20-GM-104357 (cores B and C-Roman; Pilot-Fan) and American Heart Association Grant 16GRNT31200036 (to F. Fan). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

F.F. and R.J.R. conceived and designed research; F.F., M.R.P., Y.G., L.L., S.W., and P.N.M. performed experiments; F.F., M.R.P., and R.J.R. analyzed data; F.F., M.R.P., and R.J.R. interpreted results of experiments; F.F. prepared figures; F.F. drafted manuscript; F.F. and R.J.R. edited and revised manuscript; F.F., M.R.P., and R.J.R. approved final version of manuscript.