Compensatory renal hypertrophy. I. Evidence for a factor of renal origin inhibiting DNA synthesis'

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by MICHIGAN STATE UNIV on 01/25/17 For personal use only. Compensatory renal hyp...
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Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by MICHIGAN STATE UNIV on 01/25/17 For personal use only.

Compensatory renal hypertrophy. I. Evidence for a factor of renal origin inhibiting DNA synthesis' J. MARTEL-BELLETIER AND M. BERGERON Dhpnrtenrent de physiologic., Universitk de hfontrbal, Mantrckcll ('Quh.) Carzada HdC dT8

Received October 29, 1976 MARTEL-PELI-ETIER J., and BERGERON. M. 1977. Compensatory renal hypertrophy. I. Evidence for a factor of renal origin inhibiting DNA synthesis. Can. J. Physiol. Pharmacol. 55, 839-847. This study describes a method for the measurement and partial purification of a factor seemingly involved in the regulation of the renal nrass. After homogenization at 4 ° C rabbit kidneys were centrifuged for 100 min at 805 000 g. The resulting supernatant (S-105) was lyophilized and tested on kidney slices obtained from rats mononephrectomized 48 h previously. We have developed a method based on the inhibition of D N A synthesis to measure the activity of the S-105. Slices of renal cortex, undergoing con~pensatoryhypertrophy, were incubated in vitro in Hanks' medium at 37"C, p H 7.4, in an OTCO2 atmosphere in the presence of 0.144 p g (20 pCi ( 1 Ci .=. 37 GBq)) [%]thymidine. An inhibition of D N A uptake of ["Hlthymidine was noted in the presence of S-105. When other media (Hanks', sucrose, water) were used to extract S-105, the same type of inhibition was noted even though the sucrose buffer seemed ideal for the preservation of the inhibitory factor. The inhibitory effect was still observed after dialysis of S-105 against n~embranespermitting exclusion of molecules with molecular weight smaller than about 4000 (such as electrolytes and tissue thymidine). This inhibition seems to he specific, since other tissues such as liver in regeneration and rat intestine were not influenced by the dialyzed renal S-105. The dialyzable fraction did contain some inhibitors, but they were not specific for the kidney since they also acted on the liver and the jejunum. Our results suggest the existence, in the normal nephron. of a specific inhibitor of thymidine incorporation into D N A of kidneys undergoing a compensatory hypertrophy. This renal factor has a molecular weight of over 5000. MARTEL-BELLETIER. J. et BERGERON, M. 1977. Compensatory renal hypertrophy. I. Evidence for a factor of renal origin inhibiting D N A synthesis. Can. 9. Physiol. Pharmacol. 55,839-847. Notre Ctude dCcrit une m6thode de mesure et la purification partielle d'un facteur qui semble implique dans la rtgulation de la masse rknale. Aprks homogtnCisation 2t 4"C, des reins de lapin sont centrifuges pendant 100 min B 105 000 g. Le surnageant final (S-105) est lyophilise et test6 sur des tranches de rein de rat, ayant subi une mononCphrectomie 48 h auparavant. L'incubation in vitro de tranches de cortex renal en hypertrophie compensatrice est faite dans un milieu de Hanks A 37°C. ii p H 7.4, et dans une atmosph6re d'OlrCO, en prtsence de 0.144 pg (20 p C i ) de ["HIthymidine. On note une inhibition de l'incorporation de la ["Hlthyn~idinedans le D N A en presence du S-105. Divers milieux (Hanks, Tris-saccharose, eau) ont 6td employis pour l'extraction du S-105 et on note le m i m e type d'inhibition avec ces trois milieux bien que le Trissaccharose semble ideal pour la preservation du facteur inhibiteur. Aprks dialyse, contre une membrane laissant diffuser des molCcules de taille infirieure h environ 4000, dont la thymidine tissulaire, on observe encore l'effet inhibiteur du S-105. Cette inhibition semble spCcifique puisque d'autres tissus comme le foie en rtgtntration et l'intestin de rat ne sont pas influences par l'extrait rCnal dialyse. La fraction dialysable contient plusieurs inhibiteurs nonspecifiques au rein puisque l'on note dans celle-ci un effet sur le foie et l'intestin. Nos resultats suggkrent qu'il existe, dans le nCphron normal, ann inhibiteur sptcifique de la synthtse du D N A du rein en hypertrophie compensatrice; son poids moltculaire serait suptrieur ii 5000. ABBREVIATIONS: Tris, tris(hydroxymethy1)aminomethane; TCA, trichIoroacetic acid. 'This work was presented in part at the Canadian Society for Clinical Investigation, January 1976.

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CAN. J.

PHYSIOE. PHARMACOL. VOE. 55.

Following a nephrectomy, a compensatory hypertrophy of the remaining kidney ensues in most mammals. The initial mechanism underlying this phenomenon is still unknown. Many authors have suggested the existence of a renal or sehc stimulating factor (Ogawa and Nowinski 1958; Lowenstein and Stern 7963; Lowenstein and Lozner 1966; Vichi and Earle 1970; Lyons et nl. 1974; Preuss et ul. 1978; Preuss and Goldin 1975, 1976). However, no definite evidence was presented for such a factor. The cell proliferation following a reduction of the renal mass could also be caused by the suppression of some inhibitory factor(s) (SaetHen 1956; Simnett and Chopra 1969; Chopra and Simnett 1969, 1970, 1971 ; Dicker 1972, 1974; Dicker and Shirley 1971, 1972; Dicker and Morris 1974). In fact, the growth arrest seen in kidneys of normal adults could be due to a negative feedback mechanism. Such a model of growth and growth control was illustrated in mathematical terms by Weiss and Kavanau ( 1957 ) . These inhibitory factors, described as chalones (Bullough 1965 ) , are thought to possess the following characteristics: they act in vivo and in vitro; they act on the tissue by which they are produced; they are tissue but not species specific; they are not cytotoxic. Our results suggest the existence of a factor of renal origin that could regulate the renal mass by inhibiting DNA synthesis in the kidney. Materials and Methods Materials Experiments were performed on Sprague-Dawley female rats, less than 50 days old, and weighing 9Q k 10 g, and on New Zealand Albino female rabbits weighing 2-3 kg. All animals were fed a standard diet. [Me-"HIThymidine with a specific activity of 6.7 Ci/mM was purchased from New England Nuclear Corporation. Hanks' medium (BSS 10 X , Grand Island Biological Company, without sodium bicarbonate and without phenol red) was diluted to a normal concentration with double distilled water, and sterilized again before use. The Tris-sucrose medium contained sucrose (0.25 M ) and Tris (0.025 M ) ; HC1 (0.02 M ) was added until the pH reached 7.4. A freshly prepared solution of the diphenylamine reagent ( Dische 1955) was used each time.

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Reagent-grade chemicals were used throughout the experiments. Albumin bovine, fraction V, and unlabelled thymine, thymidine, and its phosphorylated derivatives (mono- di-, tri-) were purchased from Sigma, Sephadex G-25 (20-80 p m ) from Pharmacia, and Dowex A G SOW-X2 (280-400 mesh) from BioRad. Methods Rabbit kidneys were used to determine the inhibitory factor(s) and the activity measured on rat kidney slices; thus, species specificity and tissular specificity could be studied. Paeprrratiort of 105 000 g Rabbit Kidney Supeanatant Rabbits were killed by stunning. The kidneys were immediately removed, kept on ice for less than 10 min, then rinsed and homogenized in three volumes of cold distilled water. In different series of experiments, Trissucrose (300 mosmol) and Hanks' medium (388 mosmol) were used at p H 7.4, instead of water, to homogenize the rabbit kidneys. Successive centrifugations ( 4 ° C ) were done at 35 008 g for 38 min, then at 105 000 g for 100 min. The lipid layer was discarded and the supernatant (S-105) used for the biological assay, its protein concentration being determined by the method of Cowry et a!. (1951) using bovine albumin as standard. Gross Purification of the Rabbit Kidney Extract Dialysis of the S-185 was carried out at 4°C with a cellulose membrane (wall thickness of 8.025 m m ) having an average pore radius of 24 A. A larger volume of water was used as the exchange solvent and was changed five times during a 24-h period. Both the dialyzed and the nondialyzed fractions were studied separately. The nondialyzed fraction was immediately lyophilized and kept at -20°C as a powder; storage never exceeded 1 week. For the subsequent determination, the powder was diluted in water and centrifuged at 10 000 g for 28 min at 4°C; the clear supernatant was used (see below). The dialyzable fraction, comprised in our dialysis system of molecules that could pass through 24-W pores (molecular weight less than 4000), could not be used as such because of the excessively large volume. However, to verify the presence of a specific inhibitor in this fraction, a different procedure was used: the initial S-105 was passed through a Sephadex 6 - 2 5 column which fractionates peptides and globular proteins in the molecular weight range from 1000 to 5000. Pharmzeia columns (1 x 60 cm or 5 x 100 cm) containing fine Sephadex (3-25 equilibrated with a buffer of acetate ammonium (0.1 M ) were used at 4°C. The elution was carried out with the same solution. The chromatogram was determined by the optical density of each fraction at 280 rim. The void volume was measured from a water solution of Blue Dextran 2000 (3 mg/ml) and the total volume ( V , ) estimated according to the size of the gel. Each peak obtained was studied as described in the results section. Assay of the Various Extracts The crude S-1QS extract and the various fractions

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MARTEL-PELLETIER A N D BERGERON

obtained after dialysis or chromatography in SepHadex G-25 were assayed in vitro by measuring the incorporation of ["Hlthymidine into D N A of renal slices. Only renal slices from uninephrectomized rats were used and they were obtained in the following conditions. A right nephrectomy was performed in female rats; 48 h after the operation the remaining kidney was removed and placed in a cold isotonic saline solution ( 4 ° C ) . Slices (0.3-0.4 mm thick) were cut with a Stadie-Riggs microtome and placed in a sterilized Hanks' solution ( p H 7.4). Each slice was halved; one half was placed in the control flask and ifs corresponding half in the experimental one. Incubation was done at 37°C in a Dubnoff metabolic shaking incubator at 56 strokes/min in O r C O ? (95:5, v/v), in the presence of ["Hlthymidine. The experimental flasks contained a rabbit kidney extract which wzs added 5 or 20 min before the addition of the ['Hlthymidine; an equal volume of solvent was then added to the control flasks. the volume of this addition representing less than 20% of the total volume. Incubation was ended by placing the flasks in ice for 10 min. Men.srrr-c ofD N A Spccific Aclivity The incorporation of ["Mgthymidine into D N A was observed by measuring the specific radioactivity of DNA according to a method adapted from Verly er al. ( 197 1) . The slices were washed with unlabelled thymidine (100 pg/ml 10% T C A ) , centrifuged at 5000 g for 10 niin at 4°C. and the supernatant discarded. The pellet was energetically mixed (Vortex) in 10% TCA. centrifuged in the same manner as above, and the supernatant again discarded. Ethanol ( 9 5 % ) was added, mixed, and centrifuged at 5000 g for 10 min at room temperature and the supernatant discarded; this procedure was repeated twice. The pellet was again resuspended in 3 ml of 5 % T C A and heated (90°C for 15 min) with occasional stirring. The test tubes were then centrifuged for 10 min at 8008 g. The radioactivity of the supernatant was determined by liquid scintillation (Packard Tri-Carb) after addition of Aquasol (New EngIand Nuclear). The concentration of DNA was determined in the same supernatant. The specific radioactivity (SA) of the D N A for each solution was expressed in dpm per 10000,,,, and the inhibited fraction ( I F ) calculated using the equation: IF =

SA (experimental) - SA (control) SA (experimental)

Elimination o f Arrefactitol Inhibition Various artefacts could decrease the uptake of ['HIthymidine into D N A and therefore mimic the effect of an inhibitor, e.g., the impurity of commercial thymidine, the presence of tissular thymidine, the presence of enzymes acting on labelled thymidine, etc. The purity of commercial [3H]thymidine was verified by cationic resin exchange chromatography. This was carried out on a column ( 1 x 26 c m ) filled with Dowex A G 50W-Xz. The resin was equilibrated with a solution of ammonium formiate (0.1 M ) at pH 3.2 with formic acid. The elution was effected with a buffer of ammonium formiate and the optical density meas-

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ured at 260 nm.Although a major peak was obtained, a second minor peak appeared and was eliminated. Tissular thymidine already present in the kidney at the time of the preparation of the homogenates could also act as a false inhibitor by introducing a dilution effect on the labelled thymidine. In fact, because of its niolecular weight, most if not all tissular thymidine will dialyze through the 24-W pores of the sac and therefore cannot act as a false inhibitor in the nondialyzed fraction. For the dialyzable fraction studied in the Sephadex G-25 chromatogram, the peak of tissular thymidine was identified with the aid of purified labelled thymidine. An enzymatic effect of our extract could also mimic an inhibitor by transforming [%]thymidine into various compounds: thymine, and phosphorylated derivatives of thymidine such as the mono-, di-, or triphosphate forms. The S-185 extract and the various dialyzed fractions were incubated as above with ["I

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