Online Methods

MS#1400/R1

ONLINE METHODS

All animal care and use were according to Danish law as well as the guidelines of the National Institutes of Health. Male Sprague-Dawley rats 190-250 g (Animal Facility, University of Southern Denmark) were maintained on standard chow (Altromin C 1000) and had free access to tap water.

Cells. Preglomerular vascular smooth muscle cells (VSMC) were prepared as described by Dubey et al. (1). Renal microvessels were isolated from rat kidneys after iron oxide particle infusion and separated by a magnetic field. After enzyme digestion (1 mg/mL collagenase type II, 0.3 mg/mL trypsin inhibitor and 0.3 mg/mL dithiothreitol), and magnetic separation, the preparation was passed through syringes (18 and 20 gauge), washed and suspended in 5 mL RPMI 1640 (10 % fetal calf serum, penicillin 100 U/mL and streptomycin 100 mg/mL). When cells were confluent, RNA was isolated (RN´easy kit, Qiagen, Denmark). The vascular smooth muscle cells were characterized by immunohistochemical staining for smooth muscle a-actin (clone 1A4) (Sigma, St. Louis, MO) (Figure 1). Mesangial cells were obtained by outgrowth from isolated glomeruli as previously described (2). Only cells from first passage were used for RNA extraction. The aortic cell line A7r5 cells were from American Tissue Type Culture Co. and cultured according to instructions. VSMC, mesangial cells and A7r5 cells were incubated for 24 hours in the absence of FCS with 0,1% BSA prior to harvest of RNA.

Microdissection of rat preglomerular vessels and vasa recta. Renal vessels for RNA extraction were obtained by dissection of rat kidney tissue from 8 rats according to the protocol of Yang et al. (3). Two consecutive divisions of preglomerular vessels were isolated. Vasa recta bundles were isolated from outer medulla. Of the preglomerular samples, 40-50 ”branching points” were pooled, whereas the vasa recta were measured with a micrometer scale built into the ocular. Total RNA was isolated according to a microadapted protocol of Chomczynski and Sacchi (4). Final RNA pellets were dissolved in diethylpyrocarbonate-treated water.

RT-PCR and cloning. RT-PCR analysis was performed to facilitate cloning the a1A primers were synthesized with BamHI and EcoRI restriction sites were added to all oligomers (DNA Technology, Rodovre, Denmark).

a1A: Forward: 5´att aca tcc tga acc´3, reverse 5´ctt caa ctt agg cag c´3, covering bases 3564-3929, 383 bp (GenBank Accession no. M64373).

b-actin: copied from Yu et al. (5).

The cDNA used corresponded to 5-10 branching points (microdissected vessels), 100 ng total RNA (smooth muscle cells and whole kidney) or 1 mm (vasa recta). The RT-PCR products were inserted in vector pSP73 (Promega, Madison, WI) for heat-shock uptake by competent E. Coli (DH5a, GIBCO) (10). Plasmid DNA was extracted using the QIAGEN Plasmid Maxi Kit. Inserts were sequenced using T7 and SP6 promoter primers on an ABI PRISM 350 sequencer (Perkin Elmer, Wellesley, MA).

Southern blotting. PCR products were separated by agarose gel electrophoresis, and blotted to Zeta Probe GT membranes (Bio-Rad, Hercules, CA) using standard capillary blotting procedures (transfer buffer: 0.4 mol/L NaOH). Hybridization was allowed overnight to specific probe, in vitro labeled with a-32P-dCTP, all according to Sambrook et al.(6). Autoradiography was performed for 2-4 hours on Kodak Biomax MS film.

Western blotting. Tissues were dissected, snap frozen and homogenized in 0.3 mol/L sucrose, 25 mmol/L Imidazol, CompleteÔ, pH 7.2, and centrifuged at 4000 X g for 15 minutes. Protein concentrations were determined using the Bio-Rad protein assay, with BSA as standard. Cultured cells were rinsed twice in TBS (Tris-HCl 20 mmol/L, NaCl 137 mmol/L, pH 7.6), suspended in 100 mL lysis buffer (0.1 % triton X, 1 tablet Complete Mini (Roche Molecular Biochemicals, Nutley, NJ)/10 mL) and quick-frozen. In both preparations, 1 vol. 2x loading buffer was added (100 mmol/L Tris×Cl (pH 6.8), 5 % SDS, 0.2 % bromophenolblue, 20 % glycerol, 200 mmol/L DTT), samples boiled for 5 minutes at 95 °C, centrifuged for 15 minutes at 10,000 X g and the supernatant was transfered to a fresh tube. Proteins were separated by SDS-PAGE, Laemmli system, 6 % gel using Bio-Rad Protean III, and transferred to Immobilin P (PVDF) membrane by semi-dry blotting. Transfer buffer: 1xTGS (Bio-Rad) + 20 % ethanol (v/v). Membranes were blocked with 5 % dry milk in TTBS (TBS + 0.05% Tween-20). The primary antibody was anti-a1A subunit (Alomone Labs, Israel) diluted 1:200 in TTBS with 2 % dry milk. Secondary antibody was goat-anti-rabbit IgG, Horse Radish Peroxidase (HRP)-labeled (NENÔ), diluted 1:5000 in TTBS with 2 % dry milk. Proteins were detected using Renaissance Chemiluminescent Reagent Plus (NENä), according to the manufacturer’s instructions.

Immunostaining. Renal vascular trees were HCl maceration-microdissected from rats (7). They were fixed in 3.7 % paraformaldehyde and permeabilised with methanol + 0.006 % H2O2. The primary antibodies were rabbit anti-rat a1A antibody diluted 1: 50 in TTBS and rabbit anti-mouse renin antibody diluted 1: 333 in TTBS (final concentration 1 mg/mL). The secondary antibody was goat-anti-rabbit IgG, HRP-labeled, diluted 1:1000 in TTBS. Staining was with diaminobenzidine (DAB+ substrate-chromogen system, DAKO). Negative controls were no primary antibody and pre-incubation of primary antibody with the antigen peptide. Cultures of A7r5 cells were fixed, permeabilised and immunolabeled with anti-rat a1A antibody in a similar way, and then stained with hematoxylin.

Patch-Clamp Experiments: One glass coverslip with A7r5 cells was superfused with an isotonic physiological salt solution (in mmol/L: 140 NaCl, 2.8 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES-CsOH, 11 glucose, 10 sucrose, pH 7.26 (22.5°C)), transferred to the recording chamber, and supplemented with buffer to a volume of » 250 µl. Experiments were performed at room temperature in the tight-seal whole-cell configuration of the patch-clamp technique (8) with heat-polished, Sylgard (silicone elastomer)-coated patch pipettes with resistances of 3-5.5 MW. The pipette was filled with a solution with the following composition (in mmol/L: 120 CsCl, 3 MgCl2, 5 HEPES-CsOH, 5 MgATP, 10 EGTA, pH 6.00 (24.1°C)). Series resistances were in the range of 6-20 MW and seal resistances were in the range of 2-15 GW. High-resolution membrane currents were recorded with an EPC-9 patch-clamp amplifier (HEKA) controlled by the PULSE v8.11 software on a Power Macintosh G3 computer. High-resolution currents were low-pass filtered at 2.9 kHz and acquired at a sampling rate of 20 kHz. The reference electrode was an Ag/AgCl pellet connected to the bath solution through a 150 mmol/L NaCl/agar bridge. Immediately after the whole-cell configuration was obtained, the cells were superfused with a solution that facilitated calcium currents (in mmol/L: 148 TEA-acetate, 2.8 KCl, 1 MgCl2, 10.8 BaCl2, 10 HEPES-CsOH; pH 7.23 (21.4°C)) for a 1-2 minutes. Then the cells were superfused with the same solution supplemented with w-Agatoxin IVA (10-8 mol/L). External solution changes were made with a ValveBank™ 8II (AutoMate Scientific, Inc.) perfusion system.

Intracellular calcium measurements in afferent arterioles: Cytosolic-free calcium ([Ca2+]i) was monitored in afferent arterioles by digital fluorescence-imaging microscopy using the fluorescent calcium probe Fura2-AM. Ten microdissected afferent arterioles from 7 rabbits (2-2.5 kg, Kolding Technical School, Kolding, Denmark) were placed in a perfusion chamber (Luigs & Neuman, Ratingen, Germany) mounted on an inverted microscope (Zeiss Axiovert 35, Oberkochen, Germany) and secured by two holding pipettes. The arterioles were incubated in PSS containing 5mM fura-2/AM for 45 min at room temperature. The excitation light at 350 and 380 nm was provided by a monochromator (J&M, Aalen, Germany) and the emitted light (510 nm) was collected through a x40 oil immersion objective by a CCD camera and light intensifier (MTI-Dage, Michigan City IN). The camera output was fed into a digital image processor where video frames were digitized and integrated at real time using Metamorph/Metafluor software (Universal Imaging, West Chester, PA). The image acquisition was performed at one frame/s. Determination of ratio and [Ca2+] calculation were performed pixel by pixel on pairs of corresponding 350 and 380 images according to Grynkiewicz et al. (9). Rmax and Rmin were determined using the potassium salt of fura-2 (5 mmol/L) in solutions with 2 mM calcium or 10 mM EGTA, respectively. Thus, Rmax represented the ratio of 350 nm/ 380 nm signals under saturating conditions of calcium, whereas Rmin represented the same ratio in the absence of calcium. Values used for calibration included Rmax = 0.96, Rmin = 0.14, Kd = 224 nM, and f380min/f380max (ratio of fluorescence at high calcium to that at low calcium) = 2.1. Temporal plots were calculated from ratio images. In 10 experiments changes in intracellular calcium concentration were measured after addition of 100 mM K+. Phentolamine (10-5 mol/L) was present in all experiments to exclude activation of vascular a1-receptors by depolarization and subsequent release of norepinephrine from reminant nerve endnings. After a 5 min wash in PSS the vessel was exposed to w-Agatoxin-IVA (10-8 mol/L) for 1 min and then the response to K+ was measured again, now in the presence of w-Agatoxin-IVA. Physiological salt solution (PSS) had the following composition (mmol/L): NaCl 115, NaHCO3 25, K2HPO4 2.5, CaCl2 1.3, MgSO4 1.2, glucose 5.5. High potassium solution contained (mmol/L): NaHCO3 25, NaCl 20, KCl 95, MgSO4 1.2, K2HPO4 2.5, CaCl2 1.3, and glucose 5.5 with the addition of 0.1 % BSA at pH 7.4.

Isolation of rabbit afferent arterioles and microperfusion protocols. Afferent arterioles were microdissected from 10 rabbits and perfused essentially as described (10). A test stimulus of 100 mmol/L K+ was given initially to assure viability of the vessel (with phentolamine 10-5 mol/L present). Repetitive depolarizations of each of the perfused afferent arterioles were done in the presence of increasing concentrations of w-Agatoxin-IVA (10-20 mol/L-10-10 mol/L). After preincubation with toxin for 1 min, K+ (100 mmol/L) was added for 1 minute. This was repeated at each concentration. Reversibility of the responses was always tested by addition of K+ at the end of the experiment. Experiments were recorded on videotape at a magnification of 400 times. The video sequences of interest were digitized with a Matrox frame grabber and intraluminal vessel diameters were assessed using imaging software (Metamorph, Universal Imaging, West Chester, PA 19380).

REFERENCES

1. Dubey RK, Roy A, Overbeck HW. Culture of renal arteriolar smooth muscle cells. Mitogenic response to Angiotensin II. Circ Res. 1992;71:1143-1152

2. Kurtz A, Jelkmann W, Bauer C. Mesangial cells from rat glomeruli produce an erythropoiesis stimulating factor in cell culture: FEBS Lett 1982;137:129-132.

3. Yang T, Singh I, Pham H, Sun D, Smart A, Schnermann JB, Briggs JP. Regulation of cyclooxygenase expression in the kidney by dietary salt intake. Am J Physiol. 1998;274: F481-F489

4. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium-thiocyanate-phenol-chlororform extraction. Anal Biochem. 1987;162:156-159

5. Yu AS, Hebert SC, Brenner BM, Lytton J. Molecular characterization and nephron distribution of a familiy of transcripts encoding the pore-forming subunit of Ca2+ channels in the kidney. Proc Natl Acad Sci U S A. 1992;89:10494-10498.

6. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory Manual(2nd edition). Cold Spring Harbor Laboratory Press, 1989 Cold Spring Harbor, NY.

7. Casellas D, Dupont M, Bouriquet N, Moore LC, Artuso A, Mimran A. Anatomic pairing of afferent arterioles and renin cell distribution in rat kidneys. Am J Physiol. 1994;267:F931-936

8. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recordings from cells and cell-free membrane patches. Pflugers Arch. 1981; 391: 85-100.

9. Grynkiewicz, G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260: 3440-3450

10. Hansen PB, Jensen BL, Skøtt O. Chloride regulates afferent arteriolar contraction in response to depolarization. Hypertension. 1998;32:1066-1070

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