Methods

Animal study.

All experiments used male Sprague-Dawley rats with an initial age of 8 wk. The rats were weighed and deprived of water overnight. At 16 hr later, on the day of the experiment, the rats were lightly anesthetized with ether and a 1:1 (v/v) solution of glycerol and saline was injected (10 ml/kg) into the hind limb musculature, such that each limb received one-half of the required dose. Rats in the non-ARF group were injected with an equal volume of normal saline. After this, the animals had free access to food and water. Those to be administered S-0139 (30–3 mg/kg) were given it in three i.p. injections at 4-hr interval. The first dosing occurred 1 hr after the injection of glycerol. S-0139 dissolved in saline and control rats were administered saline (1 ml/kg i.p.). The doses of S-0139 chosen have preliminary tested and can be predicted to give a pharmacological effect because S-0139 at 30 mg/kg single i.v. injection caused a significant hypotensive effect to normotensive rats (Ninomiya et al., unpublished data). At 24 hr after glycerol injection, spontaneously voided 5-hr urine was collected using an individual metabolism cage, as previously described (Shimizuet al., 1988). At the end of the urine collection, each animal was again anesthetized with pentobarbital, laparotomy was performed and a blood sample was taken from the abdominal aorta.

In next protocol, the estimation of mortality was assessed over 7 days in the postnephritic period. For preparation of periodic RNA samples of the kidney, other animals were killed under pentobarbital anesthesia at 0, 1, 3, 6 10 and 24 hr after glycerol injection.

Further two additional experiments were performed to assess whether the drug treatment had any effect on a more moderate level of failure. First, water was allowed ad libitum, then the rats were subjected without hydropenia before the study. Second, 5 ml/kg of 50% glycerol/saline, which was a half dose as described above, was injected intramuscularly into only one hind limb following a 16-hr period of water deprivation.

Biochemical study.

The creatinine concentrations of urine and plasma were determined by the alkaline picrate method using commercial kits (Wako Pure Chemical Industries Ltd., Osaka Japan). The renal function was estimated from the endogenous creatinine clearance, which was calculated by employing a standard formula.

Immunoreactive ET-1 levels in plasma and urine were measured according to the procedure of Suzuki et al. (1990) with slight modification. Samples of 2 ml each of plasma and urine were mixed with 6 ml of 8% acetic acid and boiled for 15 min to abolish intrinsic proteolytic activity. The mixtures were centrifuged at 10,000 ×g for 15 min. The supernatants were extracted on Sep-pac C18 cartridges (Waters Chromatography Division, Millipore Corp., Milford, MA) preactivated with 6 ml of acetonitrile and 12 ml of water. The columns were washed with 6 ml water and then ET was eluted with 3 ml of 60% acetonitrile. The solvent was evaporated with a centrifugal evaporator and the dry residue was dissolved with 1 ml of radioimmunoassay buffer. ET-1 levels were determined using an ET-1 assay system (Amersham, Buckinghamshire, UK).

Total RNA extraction and quantitative RT-PCR analysis.

Total RNA was extracted from the renal cortex and medulla by the acid-guanidinium thiocyanate-phenol-chloroform method (Shomczynski and Sacchi, 1987). Tissue weighing 50 to 100 mg was dispersed at 4°C in 4 ml of 4 M guanidine thiocyanate, containing 0.5% sodium sarcosyl and 0.7% 2-mercaptoethanol, with a Polytron homogenizer. The homogenate was mixed with 0.4 ml of 2 M sodium acetate, pH 4.0, 4 ml of phenol and 0.8 ml of chloroform-isoamylalcohol (49:1, v/v) and kept on ice for 20 min. The sample was centrifuged at 10,000 × g for 20 min, and the aqueous phase was subjected to phenol-chloroform extraction. RNA in the aqueous phase was precipitated with isopropanol, collected by centrifugation at 15,000 × g for 20 min and washed with 75% ethanol. The RNA pellet was dissolved in 0.1% diethylpyrocarbonate-treated water and stored at −70°C until use. The concentration of RNA isolated was calculated on the basis of absorbance at 260 nm.

The samples (2 μg of total RNA/30 μl) were heated to 70°C for 10 min and cooled on ice for physical extension of the mRNA. Twenty microliters of the RT reaction mixture containing reaction buffer, 10 mM DTT, 1 mM dNTP, 50 ng of oligo-dT12–18 primer, 40 units of RNase inhibitor and 200 U of Super Script reverse transcriptase (GIBCO/BRL, Gaitherburg, MD) was added. The reaction mixture (50 μl) was incubated at 37°C for 60 min. At the end of the incubation, the reaction mixture was heated to 95°C for 5 min to inactivate the RTase activity and to denature RNA-cDNA hybrids. The samples were treated with 30 U of RNase H (Takara Shuzo Corp., Kyoto, Japan) at 37°C for 30 min.

Five microliters of the RT-reaction mixture was used for PCR amplification with ppET-1, ETAR, ETBR and β-actin specific oligonucleotide primers. PCR primers were selected from published cDNA sequences (Sakurai et al., 1991, Lin et al., 1991, Sakurai et al., 1990; Krapf and Solioz, 1991) and commercially synthesized (Takara Shuzo Corp.). Prepro ET-1 primer 1 (forward) was defined by bases 157–176, sequence 5′-TGCTCCTGCTCCTCCTTGAT-3′; primer 2 (reverse), bases 608–627, sequence 5′-CACCACGGGGCTCTGTAGTC-3′. The cDNA amplification product was predicted to be 471 bp in length. ETAR primer 1 (forward) was defined by bases 671–690, sequence 5′-TCGTCATGGTACCCTTCGAA-3′; primer 2 (reverse), bases 1282–1301, sequence 5′-CTCGGTGCTTCTGCACAGGG-3′. The cDNA amplification product was predicted to be 631 bp in length. ETBR primer 1 (forward) was defined by bases 801–820, sequence 5′-TTACAAGACAGCCAAAGACT-3′; primer 2 (reverse), bases 1346–1365, sequence 5′-CACGATGAGGACAATGAGAT-3′. The cDNA amplification product was predicted to be 565 bp in length. Beta-actin primer 1 (forward) was defined by bases 2170–2194, sequence 5′-CTGATCCACATCTGCTGGAAGGTGG-3′; primer 2 (reverse), bases 3055–3079, sequence 5′- ACCTTCAACACCCCAGCCATGTACG-3′. Beta-actin primers spanned two introns and resulted in a 703 bp product. Fifty picomoles each of primers 1 and 2 were used for each reaction for prepro ET-1, ETAR, ETBR and β-actin. Five units of Taq DNA polymerase (Takara Shuzo Corp.), reaction buffer and 2 mM dNTP were used for each PCR amplification. One microliter of [α-³²P]dCTP (10 μCi, 370 kBq/μl, Amersham) was added to the reaction mixture to label the PCR products. The reaction mixture (50 μl) was overlaid with 50 μl of mineral oil. The tubes were placed in a Program Temp Control System (ASTEC, PC-800, Fukuoka, Japan) programed as follows: 27 cycles (pp ET-1), 27 cycles (ETAR), 24 cycles (ETBR) and 18 cycles (β-actin) of the sequential steps of 94°C for 1 min (denaturation), 60°C for 1 min (annealing), 72°C for 1 min (extension).

Five microliters of the PCR products were size-fractionated with 3% agarose gel electrophoresis and the labeled DNA bands were blotted onto a nylon membrane with a Gel Drying Processor (Atto, AE-3700, Tokyo, Japan). Autoradiography with an imaging plate was performed at room temperature for 5 min. The radioactivity of the labeled cDNA bands on the imaging plate was measured using a Bioimage Analyzer (Fujix, BAS2000II, Fuji Film Inc. Tokyo, Japan). The radio activity of ppET-1, ETAR and ETBR was normalized to that of β-actin. The levels expressed as a percent of the values at the basal level.

As PCR amplification generally lacks quantitative reliance, we optimized the quantitative PCR in advance. The optimum number of amplification cycles was chosen within the exponential phase on the basis of pilot experiments. We decided to estimate the amount of amplified product for ppET-1, ERAR, ETBR and β-actin at 27, 27, 24 and 18 cycles, respectively. To establish the quantitative analysis of mRNA levels with the use of these settings, we confirmed the linearity between the quantity of starting material (total RNA) and that of the amplified product (cDNA). Quantitative analysis was first performed by serial dilution of total RNA isolated from normal kidney cortex as the starting material. A linear regression relationship was obtained for ppET-1, ETAR, ETBR and β-actin within 80, 160, 40 and 40 ng of total RNA, respectively. Our practical use of total RNA in RT-PCR amplification was 20 ng: the initial total RNA (2 μg) was finally diluted 100 times as described above.

In situ hybridization study.

Antisense and sense single-strand cRNAs were synthesized from cDNA fragments encoding ppET-1 constructed using RT-PCR as mentioned above and subcloned into pCR II vector (Invitrogen, San Diego, CA). The template was linearized with the restriction enzyme BamH1 (antisense probe) orXho1 (sense probe) and labeled RNA probes were synthesized with T7 (antisense probe) and Sp6 (sense probe) RNA polymerase in the presence of DIG-labeled UTP (DIG Labeling Kit, Boehringer Mannheim, Indianapolis, IN). The probes were precipitated and DIG incorporation was assessed by dot blotting as previously described (Panoskaltsis-Mortari and Par Bucy, 1995).

Under pentobarbital anesthesia, rats were flushed with PBS through the abdominal aorta followed by perfusion with 4% paraformaldehyde buffered with .1 M PBS (pH 7.4) at 10 hr after glycerol or saline injection after dehydration overnight. The kidneys were swiftly removed and cut into small pieces. The renal cortex tissue was immediately dissected and immersed into a fresh portion of the same fixative at 4°C overnight. All steps were carried out with care to avoid contamination with RNase. Diethylpyrocarbonate-treated water was used at 0.1% to prepare each buffer. Fixed samples were thoroughly rinsed with 0.1 M PBS (pH 7.4) and subsequently dehydrated by passage through an alcohol series diluted with diethylpyrocarbonate-treated water (30, 50, 70 and 90%, for 60 min in each), followed by another alcohol series (95%, 99.5%, and absolute, for 90 min in each) and alcohol-xylene mixed series (25, 50 and 75%, for 90 min in each) and 100% xylene (for 16 hr). The preparations were then passed into paraffin (Paraffin pistilles m.p. 52°C, Merck Co., Rahway, NJ) at 56°C. In situ hybridization was performed on paraffin-embedded sections as described by Angerer et al. (1987) with some modifications. Transversal tissue sections (4 μm thick) were cut from embedded blocks and mounted on slides freshly coated with 3-aminopropyltriethoxysilane. After dewaxing, sections were incubated with 0.2 M HCl for 15 min followed by 1% Triton X-100 detergent for 15 min, and permeabilized with 10 μg/ml proteinase K at 37°C for 20 min (Boehringer Mannheim). Next, sections were further fixed with 4% paraformaldehyde buffered with 0.1 M PBS (pH 7.4). They were then briefly washed with .1 M PBS, rinsed in 0.1 M triethanolamine (pH 8.0) and then acetylated with 0.1 M triethanolamine containing 0.25% acetic anhydride for 10 min.

Hybridization was conducted by the method of Lan et al. (1996). Briefly, sections were prehybridized, and then hybridized with 200 ng/ml DIG-labeled anti-sense or sense cRNA probe for 16 hr at 50°C in a hybridization buffer containing 50% deionized formamide, 1X Denhardt’s solution, 10% dextran sulfate, 600 mM NaCl, .25% SDS, 1 mM EDTA (pH 8.0), 0.2 mg/ml yeast tRNA and 10 mM Tris-HCl (pH 7.6). During hybridization, the slides were covered with parafilm and kept in a closed moist chamber with a 50% formamide atmosphere. After hybridization, the samples were rinsed four times with 5X SSC for 10 min at 50°C. Free labeled cRNA was digested by 10 μg/ml of RNase A in 10 mM Tris-HCl (pH 7.6) containing .5 M NaCl and 1 mM EDTA for 30 min at 37°C. The samples were stringently washed with the same buffer except for the RNase A, rinsed once with 2X SSC for 30 min at 50°C and thoroughly washed twice with 0.2X SSC for 20 min at 50°C. Then the samples were then immersed in 1.5% blocking reagent dissolved in DIG buffer 1 (100 mM Tris-HCl, pH 7.5, containing 150 mM NaCl) for 60 min at room temperature, preincubated in normal mouse serum at a dilution of 1:500 in DIG buffer 1 for 30 min, and subsequently incubated in anti-DIG sheep polyclonal antibodies at a dilution of 1:1000 in DIG buffer 1 for 30 min at room temperature, and rinsed with DIG buffer 1. These samples were then incubated in biotinylated anti-sheep mouse polyclonal antibodies at a dilution of 1:1000 in DIG buffer 1 for 30 min at room temperature and rinsed again with DIG buffer 1. The samples were next treated with avidin-biotinylated horseradish peroxidase complex solution (Vector Laboratories Inc., Burlingame, CA) at room temperature for 60 min. After immunological incubation, the samples were extensively washed with DIG buffer 1, then were processed using 0.1% 3,3′-diaminobenzidine hydrochloride substrate dissolved in 50 mM Tris-HCl (pH 7.4) containing 0.05% H₂O₂ for 5 min at room temperature in the dark and examined for expression of the specific gene of interest. After a brownish color developed, the reaction was stopped by rinsing in TE buffer (10 mM Tris-HCl, pH 7.6, containing 1 mM EDTA), and the target mRNA signals were visualized. The sections were counterstained with periodic acid-Schiff reagent, dehydrated and finally mounted in Entellan new (Merck Co.). The slides were examined under bright field microscopy with particular attention paid to evaluation of the glomerular cross views.

Statistical analysis.

All results are expressed as mean ± S.E. Statistical analysis was performed using Student’st test for unpaired samples. P < .05 was considered statistically significant. Statistical evaluation of the curves in survival studies was conducted using a log-rank test of Kaplan-Meier method.