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Effects of the rat renotropic system on 14C-uridine incorporation into RNA and RNA precursors
Life Sciences, Vol . 25, pp . 497-506 Printed in the U .S .A .
Pergamon Press
EFFECTS OF THE RAT RENOTROPIC SYSTEM ON 14 C-URIDINE INCORPORATION INTO RNA AND RNA PRECURSORS Harry G . Preuas and Herzl Goldin Renal Division, Departments of Medicine and Pathology Georgetown University Schôol of Medicine, Washington, D .C . 20007 (Received in final form Juge 25, 1979)
We used the incorporation of l4 C-urldine into RNA of incubating kidney fragments from normal control rats to eval w to RNA metabolism . Sera from unilaterally nephrectomized rats (uni) obtained 20 hrs post-oparetively stimulate iliC-urldine incorporation into RNA slgnifiuntly mere than sera from Differently, sera from uni and sham-operated rats (sham) . sham rats have little influence on specific activities of endogenous urldine nucleotide pool in renal fragments . Renal extracts ware obtained by homogenizing kidneys in saline . Extracts from kidneys of uni and sham rata 20 hrs post operation depress incorporation markedly, and each depresses to a similar extent, but kidney extracts dilute the specific activities of urldine pools . Correcting for the latter dilution deoqnstrates that kidney extracts atone have little effect on l 4 C-urldine Incorporation into RNA . We then followed the results when these sera and extracts Compared to fragments incubating in sham were combined . sera and sham extracts, substitution of uni extracts or both uni extracts and uni sera enhances 14C-uridtne into renal RNA, whether or not results are corrected for changes in the specific activities of the uridtne pools . We conclude that after uninephrectomy there is a concurrent elevation in circulating renotropin and a tissue activating factor In the remaining kidney . The tissue factor wn only form an excitor to 14C-uridtne incorporation into RNA when serum is present . The rat renotropic system that anhancea ncorporation of 3 H-thymidtne into DNA also can stimulate 1~-urldine incorporation Into renal RNA . Following loss of renal mass (unilateral nephrectomy), the remaining viable kidney tissue of rats grown by hypertrophy (cell enlargement) and hyperplasta (cell division) (1) . Changes in renal RNA and DNA synthesis It is generally accepted that changes in these occur within hours (2,3) . mechanisms may set compensatory renal growth into motion . Tha exact uuse : that incite and regulate these synthetic mechanisms ors unknown although many investigators favor the concept that humoral stimulators play sans role (4-12) . Recently, we demonstrated the presence of different factors In the circulating blood and renal tissue of unilaterally nephrectomized rats that enhance 3H-thymidtne incorporation into renal DNA In vitro within a 90 minute incubation (13) . The present investigationsxt~ the original studies in order to determine if these factors also affect iliC-uridtne incorporation Into renal RNA . 0024-3205/79/060497-09$02 .00/0 Copyright (c) 1979 Pergasxm Press Ltd
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MATERIALS AHD METHODS We used 200-250 g male Sprague-Dawley rats that were housed in a constant temperature room with a light-dark phase of 14 and 10 hrs . Rats received food and water _ad llbltum . Unilateral nephrectomy was tattled out by a left flank incision Sham operation consisted of exposure of the left kidney . In the sham procedures, care was taken not to handle excessively the left kidneys . Operations were performed between 2-3 p .m ., and materials for assay were obtained the next morning between 9-10 a .m . Rats were lightly anesthetized with ether . Following a midline abdominal incision, blood was drawn from the lower aorta into glass syringes and allowed to clot in plastic centrifuge tubes . Blood was immediately centrifuged in the cold . We added sera for assay to each flask in a concentration of 12$ V/V . To obtain renal extracts, whole kidneys were removed from sham-operated rats or those with a single remaining kidney approximately 20 hrs after unilateral nephrectamy (uninephrectomy), and homogenized in the same cold medium used for incubation (1 g kidney/5 ml medium) . We homogenized the tissue with a motor driven teflon pestle . After centrifugation at 500 g, supernates were collected and kept cold until initiation of study . Supernates from the remaining growing kidneys of uninephrectamized rats and kidneys of shamoperated rats were added (12$ V/V) along with 14C-uridine to incubating fragments . Controls (addition of 12$ V/V medium only) were assayed simultaneously . The in vitro assay to estimate 14 C-uridine incorporation into RNA has been described in detail elsewhere (14) . In brief, we obtained kidney frog- . ments by forcing cortices diced into small pieces with scissors, through a nylon sieve (2 x 1 .5 mn) . This nylon sieve was obtained from the framework of a twin coil diâlyzer (Travenol Laboratories, Morton Grove, IL) . The bottom of the sieve was scraped with a spatula to recover as much tissue as possible, and the material was placed in cold oxygenated medium . We allowed the tissue to settle twice by mixing with cold oxygenated medium . All fragments for assay were obtained from control rats . The basic medium used in washing and incubating fragments was modified Krebs-Ringer solution composed of the following anions and cations : Na+ 126 mM, K+ 5 mM, Mg++ 1 .2 mM, Ca++ 1 .0 mM,~50~, ~ 1 .2 mM, Ci- 133 mM, phosphate buffer (pH 7 .4) 10 mM, and gassed with 100$ 0 . To each flask containing 3 ml of medium and 30-60 mg wet wt of tissue2 ftagments, approximately 1 .5 uCi of uridine -2 -14C (specific activity 529 mCi/mmol) was added at the beginning of incubation . In this system, less than 12$ of the total counts disappears from the medium over the course of incubation . At the end of a 90 minute incubation at 25o C, cortical fragments were trapped with suction on circular filter paper (Whatman 40, 2 .4-cm radius) . We extracted RNA in the fragments from the filter paper by the method of Fleck and Munto (15), as modified by Halliburton and Thomson (16) . We estimated the concentrations of RNA by measuring the difference in absorbante of the RNA extracts at wave lengths of 260 mu and 310 mu on a Bnckmen DU spectrophotometer and by comparing readings to standard curves of a hydrolysate of pure reagent grade ribose nucleic acid (Sigma Chemical Company, St . Louis, MO) . To estimate specific activity ôf the uridine mono-, di-, and triphosphate pools at the end of 90 minute incubations, acid soluble fragments (4$ PCA) were boiled for 40 minutes to convert all the above to uridine monophosphate (17) . Çharcoal was added to the cooled extracts, and the nucleotides were subsequently eluted with a mixture to ethanol and ammonium hydroxide (18) .
ool.
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Iacorporation
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49 9
After evaporation, the solids were dissolved in 0 .01 N NH OH . The UMP was separated on PEI cellulose F with 0 .1 N formic acid as th~ carrier . The spots corresponding to uridine monophosphate were scraped from the plates into centrifuge tubes and the nucleotides eluted with 0 .01 N HC1 . After elution for 1 hr, the eluates ware read with a Beckman DU spectrophotometer at 262 mu and 310 mu to estimate the uridine monophosphate concentrations . In addition, radioactivity of the elutes was determined by ß counting . In a single experiment, the conversion step was eliminated and a uridine triphosphate pool was isolated . We performed ß counting on a Packard Trt-Carb 2425 liquid scintillation counter (Packard Instrument Co ., Inc ., Downers Grove, IL) with samples corrected back to 100$ absolute efficiency using a standard quench curve which relates automatic external standard counts to absolûte efficiency . Dioxane was used as a solvent, 2,5-diphenyloxazole was the primary scintillstor, and 1,4-bis (2-4 methyl-5-phenyloxazolyl) benzene, the secondary scintillator . In a few studies where the uridine pools were Isolated, the specific activities of RNA were corrected for the specific activities of the pools (17) . ThrougFiout this manuscript, we often refer to the sham operated rats themselves and extracts and sera from the sham-operated rats as "sham rats, sham sera, and sham extracts" . In a like manner, their uninsphrectamlzed counterparts are a lied "uni rats, uni sera, and uni extracts" . Each value listed in the tables represents the average value of specific activities measured in 3-4 flasks . Statistics shown here are by Student's "t" test using group or paired analysts . A11 statistics were checked by analysis of variance which essentially gave, the same results . Statistt u 1 significance is set at p < 0 .05 . RESULTS In the first 13 studies (Table I), the specific activities of RNA in renal fragments from normal untouched rats incubating in medium alone (control), sera, èxtracts, and sera plus extracts from sham or unl rats, were measured . All samples were obtained 20 hrs post operation . Products from sham (6 experiments) or uni rats (7 experiments) were assayed separately in different experiments and obviously on different fragments . The effects of sera, extracts, and the combinations were compared to control within each separate experiment . Attempts were made to randomize studies using sham and unl products . While sham sera enhanced 14C-uridine incorporation into RNA, uni sera caused a significantly greater stimulation . Extracts from sham operated and cant rats depressed specific activity to a similar extent . When the extracts and sera were combined, those from sham-0peratad rats still depressed, while those from uninephroctomized rats were mots likely to stimulate . Three of the seven experiments with the uni combinations showed definite stimulation whereas none of the experiments using the sham combinations showed stimulation . The differences between the ratios of the two sets were statistically significant (p < 0 .01) . Although the experiments were performed randomly, the fragments from the normal rats used for assay of the sham sera and extracts had higher control specific activities than those fragments used in the unI studies, necessitating comparisons to be made by percent change in stimulation . However, in those experiments where the baselines were comparable, the same trends generally persisted - uni serum has greater stimulatory effects than sham serum, both sham and uni extracts depress, and unl combinations either stimulate or do little canpared to control . Sham combinations generally depress compared to control .
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TABLE I EFFECTS OF SERA AND TISSUE EXTRACTS FROM UNINEPHRECTOMIZED AND -It I NTÔ~RÉIEAA~Ô~TR ~ÔRT~Ôf'IiÂ1r1EEi~t~ô?'Pt71t . ~F1C~RÉIiTS / Expts
Control Spec . Act . DPM/ uq RNA
Sera
Sera + Rana} Extract
Rsnal Extract
Spec . Act . $ CMnye DPM/ uq RNA from Control
Spec . Act . $Change Spec, Act . $Change DPH~+q RNA from DPM/uq RNA f ram Control Control
from sham ôpereted rats 20 hours post operation (6 expt) 6
37 .5 + 8 .7
48 .5+10 .5$
31 .7
21 .3 + 3 .1*
21 .9 + 2 .g*
-66
-72
from rats unTnephrectomized 20 hours earlier (7 expt) 48 .1+10,6$ 88 .4 24 7 27 .0 + 6,2
16 .5 + 2 .7*
-56 .6
.7+
30 .3 + 5 .3 °
SEM shown, °p > ,OS < .1, *p < 0 .01, $p < 0,01 compared to control (patted analysis) ; +p < 0,01 from value above by group analysis
In the next 10 studies (Table 11), the influences of various comblnattons of sera and extracts from both unl and sham rats were compared pn the same normal control fragments in each experiment . The effects upon 14 C urtdtne incorporation Into RNA of the following combinations - uni sera plus sham extracts, sham sera plus unl extracts, and uni sera plus unt extracts were compared to the effects of sham sera plus sham extracts .
TABLE II RAT
EFFECTS OF COMBINATION OF SERA AND RENAL EXTRACTS FROM UNI E HRECTOMI E OR SHAM-0 RA ED 0 H S RL ER C-URIDINE I NC ON I NT H A L FIEAGF1EliT~
Sham Sera Sham Extract
Uni Sera Sham Extract
Uni Sara Unt Extract
Spec . Act . DPM/uq RNA
Spec . Act . $Change DPM/yq RNA from Control
Spec . Act . DPM/u4 RNA
12 .6 + l .0
18 .0 + 2 .8k
18 .8 + 2 .9k
43 .5
$Change from Control 47 .6
. .Uni Sera Uni Extract Spac . Act . $Change DPM/uq RNA from Control 17 .0 + 2 .3*
35 .9
SEM shown, *p < .OS compared to fragments incubating in sham sera and sham extracts (pelted analysts) (10 experiments) .
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Unlike the design of the first experiments, the effects of cant and sham sera and extracts were compared directly on the same fragments . Compared to fragments Incubated in sham sera and extracts, the substitution of either cant sera or cant extracts for sham, or a combined substitution of both cant sera and cant extracts res lied in a statistically significant augmentation to the incorporation of l~C-uridine into RNA . Unlike the other studies where 3H-thymidina was used as isotope (13), a combination of cant sera and extracts compared to sham sera and extracts showed no greater stimulation to Isotope incorporation then when uni . sera or cant extracts alone were substituted Into the system . However, it is important to note that the addition of renal extracts from remaining kidneys of cant rats when combined with sham sera ca~ared to the addition of extracts and sera from sham operated rats enhances specific activity . Another series of studies evaluated the Influences of our various procedures on the specific activities of the uridine pools . i n an experiment that compared sham and cant sera, we saw little affect by either on the specific activities of uridine precursor pools . The specific activities of those fragments incubating in sham sera were only slightly higher than control (+10$), whereas fragments incubating in cant sera had little change in their pool specific activities compared to control (-9$) . Corrected for pool size, sham serum slightly depressed l 4 C-uridine incorporation Into RNA (-4$) while cant serum stimulated (+37$), This was near the uncorrected values - no stimulation in flasks containing control sera and +26$ in those flasks with cant sera .' Another study followed the effects of a renal extract from a remaining klds~y 20 hrs post untnephrectomy . Uni extract caused a -61$ depression of C-uridine incorporation into RNA but also diluted the specific activity of the uridine pool, -55$ . jhe overall result corrected for pool dilution, showed a decrease in I C-uridine incorporation into RNA of only -2$ . The effects of sham sera plus sham extracts and cant sera plus cant extracts were compared to the control situat ton In Table III . The comparat b effects of sham sera and sham extracts on 14 C-uridtne incorporation into renal RNA resembled the previous studies (see Table I) with the exception of experiments lA and 6A where there was a +44$ and +17$ stimulation by the sham combination over control . The other 4 experiments showed depression to Incorporation . i n this series of 6 studies, cant sera plus cant extracts significantly stimulate isotope incorporation (p < 0 .05) . Additionally, the specific activities of the combined pools of uridine monophosphate, uridine diphosphate and uridine triphosphate were estimated . In the first 5 experiments, the specific activities of the uridine phosphate pools were lower in the fragments incubating to the sera and fragments from the sham and cant rats canpared to control (p < 0 .05) . The combinations from the cant and sham rats caused comparable depressions to the specific activities of the uridine pools In 4 of the experiments . i n the exceptional experiment (3A), the cant combination cab~pared to the sham combination depressed the specific In 6A, the UTP pool was activities of the uridine pools relatively more . Corrected results tsol~ted rather than the entire uridine phosphate pool . for 4 C-uridine incorporation Into RNA were similar to those seen in the previous experiments . Corrections for pools sizes show the cant combination to be stimulatory to 1~C-uridine tncorporstion Pnto renal RIU (p< 0 .05 .
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TABLE III THE EFFECTS OF THE RENOTROPIC SYSTEM ON RAT RENAL . FRAGMENT V1TT' 0 U N OOL 1~TAli0LI3li
Expt,/ lA 2A 3A 4A
6A
Condition Control SS uu Control SS UU Control SS UU Control SS UU Control SS UU Control SS uU
Spec . Act . of UMP in Actd Sol . Fraction+
Spec . Acta of RNA*-
66 .9 48 .9 50 .1 37 .7 22,4 25 .3 58 .0 54 .2 337 52 .1 42 .3 45 .7 156 .1 98 .1 104 .5 189 .7 ° 178 .0 96 .0
Spec . Act . of RNA (corn)* 10,0 19,7 (+97) 26,5 (+165)
10 .0 14,4 (+44) 19 .8 (+g8) 40,7 50 .2 49-.2 91,6 49,9 38 .9 69 .5 21,1 13 .5 31,4
(+33) (- 2) (+82) (-28) (+39) (-5b) (+49)
61,8 (+17) 63 .0 (+19)
.
60,6 50,2 52 .7 157,b 49 .9 47 .9 79 .3 21,1 21 .5 46,7
(+99) (+5) (+214) (-4) (+59) (+2) (+121)
65,9 (+25) 124,5 (+135)
+ Spec . Act . of the pool of urldine mono-, di-, and trtphosphate (DPM/mmole) o5pec . Act . of the pool of urldine trtphosphate (DpM/mmole) . $Dlsintegratlons per minute per ug RNA . *Disintegrattons per minute per ug RNA after correction for the specific Correction based upon activity of the urtdtne phosphate or UTP pool, control . SS ~ sham sere + sham extract . UU ~ unt sera + unt extract . C ~ control . Each result is average of 4 flasks . ( ) ~ $ change from control,
DISCUSSION After untlaterai nephrectomy, RNA and DNA synthesis increase within hours to the remaining kidneys of healthy rats (2, 3) . An attractive hypothetsis is that humoral factors initiate and/or regulate the early changes In RNA and DNA synthesis (4-13) . Many studies have added information to the pros and cons favoring existence of hiunoral factors stimulating compensatory renal DNA synthesis (4-13), Some utilized 3H-thymidinn incorporation into renal DNA of living antenais to assess growth (5,7) . A few followed the In 1958, Ogawa influences of humoral substances on DNA synthesis i- vitro . and Nowtnski (4) found that sera from unilaterally nep~ectomized rats In 1970, we stimulate mitoses in tissue cultures of renal medullary cells . showed that plasma from unilaterally nephrectomized rats compared to plasma from sham-operated rats stimulate 3H-thymtdtne monophosphate incorporation Into the DNA of incubating rat kidney slices (8), An additional flndtng was that "untnephrectomtzed plasma" did not enhance DNA synthesis of liver, spleen, and lung slices .
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Malt placed the synthesis of RNA Into a prominent and important spot to the schema of compensatory renal growth (19, 20) . However, invastigattans on in vitro renotroptc Influenua on RNA metabolism have bean fewer than oMs on ~A métsboltsm . Two s~udies reported that sarr from unilaterally nephrectamized animals enhance i C-uridTne incorporation Into RNA of incubating kidney slices (8) and Into tissue cultures of renal cortex (11) . Because the effects of the whole renotroplc system were investigated only for DNA metabolism and because RNA metabolism plays a major role In compensatory renal hypertrophy (19, 20), our pr~v Tous studies wars extended to Include RNA . Wa followed the incorporation of 14 C-urtdine into RNA, Our first experiments explored the s stems derived from sham-operated and uninephrectamtzed rats separately . 1 ~C-uridine Incorporation into RNA of fragments utilized for the control baseline was htghar to those experiments amploylng sham products, although uré was taken to randomize studies . The difference between control baseline in the two groups probably relate to many factors, including the following . Tissue : from different rats on different days may differ in isotope incorporation, in addition, technical reproducibility of exact dally conditions may vary . This in vitro system is anaiogous to ono whara PAN transport is studied, Tn that dâTty var atlons have led to the use of Internal controls for comparisons (Z1, 22) . Using internal controls, can) sera compared to sham sera cause greater stimulation relative to baseline . Some avldencs exists from other studies that this indirect canparison is valid . Previws studies comparing Inftuenus of cant and sham sera on isotope incorporation into RNA of kidney sites from the wale rat corroborate that unl sera has r lativaly more stimulation (8)~. Extracts from cant and sham kidneys depress 1 ~C-urTdins incorporation to the same degree and the addition of sham sera to fragments incubating in sham extracts doss not overcome this depression . In contrast, unl sera and extracts increase incorporation, a marked turnabout from what uni extracts do alone . The results from these investigations mimic ones pravtously (13) . However, this sortes of studies does not prove that the unl combination has any more stimulatory properties than unT sera alone, Our second studies ware deslgnéd to resolve this, Experiments dapictsd in Table 11 compared tM Influences of sham and unl combinations head to head, I .e ., on the same fragments . In these studies, the magnitude of Isotopic Incorporation by fragments in each separate experiment era closer . Throe points can 6e made . First, when added to fragments incubating In sham extract: uni sera compared directly to sham sera stimulate i~C-uridins incorporation into RNA . This Is not unexpected when one considers the studies whsra sera alone were investigated . Second, cant extracts compared to sham extracts Incubating in the same sera stimulate l~C-uridlne incorporation into RNA of renal fragments, This finding is IMportant because tt shows that unl extracts are relatively stimulatory when serum, even normal control serua, is present . Third, stimulation by unl sera and cant extracts is no greater than stimulation by unl sera with sham extracts or unl extracts with sham sera, Un11ka studios on DNA metabolism 1~ vitro (13), the separate stimulatory properties of unl sera and extracts era not additive in vitro . Using labelled nucleotides to estimate RNA and DNA metabolism has problems connected with It - principally that isotope dilution rather than actwl synthesis may be responsible for changes in nucleotide specific activities . In our syaem, dilution by extracallular factors could not cause our stimulatory results for 2 reasons . First ; If sera or plasma stimulate by non specific dilution of lsotopa alone, this would cause higher specific activltlss of RNA and DNA in many tissues other than kidney, and this does not occur (8, 12) . Sera from untnephrectomtzed rats stimulate only kidney tissue (8,12,23) , whereas sera from partially hepatectomized rats are relatively specific for
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hepatic tissue (24) . Interestingly, sera from uninephrectomizad rats can stimulate hepatic 3H-thymidine incorporation into DNA when extracts from the growing kidney aro added to the medium (23) . Second, dialysis of sera, whiçh should remove all dilutlonal precursors, does not alter the stimulation of 3H-thymldine incorporation Into DNA 6y un1 sera (13) . While the above rules out extracellular dilutlonal factors, there is a slight possibility that endogenous nucleotide precursor pools might change In the same tissue under the varying conditions of study . For example, if unt sera and unl sera plus uni extracts decrease endogenous uridine pools, the added 14C-u~idina would produce a precursur pool with a higher specific acttvtty . This in turn, could theoretically create a higher RNA specific activity despite no alteration to the rate of RNA labelling . When we measured the specific activities of uridine pools (UMP, UDP, UTP) in fragments incubating to unt or shpn.sera, there was little change from control . Differently, and not wholly unexpected, addition of renal extracts dilutes the final specific activftlss of uridine precursor pools . When the specific activities of RNA are corrected for dilution, uni extracts Rather, have no apparent effect on 1 ~C-uridine incorporation into RNA alone . the lower Incorporation of .laC-uridine into RNA are probably secondary to isotope dilutto~ by nucleotides present in the extracts . Lastly, ws found in a more extensive study that both sera and extracts lower specific activities of uridine precursor pools, no doubt secondary to importantly, pool dilutions by the the effects of the extracts (vide supra) . uni co~apa~ed to sham combinations are either of different or ev6n greater . Therefore, we can state with certainty, that l~C-uridine Incorporation into RNA is rslatlvely greater with uni sera and extracts than with sham sera and extracts . More precisely, correcting for the specific activities of uridine pools shows that uni combinations stimulate compared to control, whereas sham combinations have lass effect . Two previous studies'hava indicated that the uridine triphosphâte pool may be increased early on In growth (3, 25), perhaps explaining the greater dilution of specific acttvtty by the unl cambinatlon in experiment 6A, Table iii . Cortes et al (25) could not reproduce earlier results showing enhanced 14C-uridine incorporation Into RNA in vitro by sera from untnephrectomized serum In their assay, a level that rats (8,11) . However, they used SO~ÎI fs far above maximum for stimulation (11,23) . Additionally, they incubated at 37oC instead of 25oC (8) . We have never shown much stimulation at the higher temperature . These two variations between assays may explain the differences, at least in part . Also, difference in oxidative substrates added to the medium directly or in the saran could cause differences . In summary, our findings suggest that unj sera alone and extracts from uni kidneys fn the presence of sera enhance 1 C-uridine incorporation into RNA . Unlike st .imulatlon to Isotope Incorporation into renal DNA, combined uni sera and extracts stimulate no more than uni sera alone or unf extracts plus control sera under the conditions of our assay . How these factors relate or interact in vlvo cannot be discerned from these studies . However, both factors appearÎn greater cancentrattons following renal mass loss . ACKNOWLEDGEMENTS The authors wish to thank Ms . Elizabeth Phillips for statistical help and Susan Dreux and Martiyn Davts for their excellent secretarial help .
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H .A, JOHNSON and J,M,, VERA ROMAN. Am . J . Pathol . 4~:1-13 (1966) . F, G, TOBACK and L. M. LOWENSTEIN, Growth :35-44 (~J4), F,G, TOBACK and L .M, LOWENSTEIN, Growth :17- ;4 (1974), K. OGAWA and W.W, NOWINSKI, Proc . Soc. Exp. Bio1 . Mad, ~350-354 (1958) . L.M. LOWENSTEIN and A. STERN. Science (Washington, D .C .) 142:1479-1480 (1963) . H.P .R BURY, W.A .J . CRANE, and L.P . DUTTA. Brit . J . Urol . :201-210 (1965) . M,R. SILK, G,E . HOMSY, and T . MERZ . J. Urol, 98 :36- ;9 (1967) . H,G. PREUSS, E .F, TERRYI, and A,1 . KELLER, Nap~FOn x:459-470 (1970), N,L,R, BÜCHER, and R.A, MALT, r~t ~ Liver a-d Kidney , Little, Brown and Co Inc ., Boston pp, , T,J, VAN VROONHOVEN, L. SOLER-MONTESINOS, and R, A. WILT . Surgery (st, Louis) 2 " 30o-305 (1972) . H.J, LYONS, ~: EVAN, L,C, MCLAREN, and S, SOLOMON, Nephron x:198-211 (1974), H.G, PREUSS, and H . GOLDIN, Msd, Clin . No . Mien . ~771-780 (1975) . H.G, PREUSS, and H . GOLDIN, J . Clln, Invest . ,5~ 9 - O1 (1976) . H. GOLDIN, M. ZMUDKA, F. TIO, A. VASQUEZ, AND H .7:, PREUSS . Proc . Soc, Exp, Blol, Med . 144:692-696 (1973) . A. FLECK and H,M,~OEIR0 . Blochim, Blophys, Acta, x:571-583 (1962) . I,W, HALLIBURTON and R,Y, THOMSON, Cancer Ras, 2 2-1887 (1965), R. BASERGA, R,O, PETERSEN, and R,D . ESTENSEN, B ochim, Biophy :. Acte, 1:259-270 (1960 , . , GREENMAN, R .C . HUANG, M, SMITH, and L,M. FURROW . Anal, Blocham, :348-359 (1969) . ,A . MALT and D .A . LEMAITRE, Amer, J . Physlol . 214 :1041-1047 (1968), R,A, MALT, nsato Re:fal ertr h , Eds.~~l . Nowinskl and R,J, 6oss, a am c ress -~ew or , ew ork, pp . 131-156 (1969) . T, KOISHI, Jap, J., of Pharm, 8 :101-12; (1959) . E.P . ORRINGER, F. R. WEISS, and A. G, PREUSS, Clin, Scl . 40 :159-169 (1971) . H,G, PREUSS, H. GOLDIH, and M, SHIVERS . Yale J, Biol, and Msd, 51 :403412, (1978) . H.G . PREUSS, R, PARRIS, and H. GOLDIN, Expsrientia ßs630-631 (1977), P. CORTES, N .W, LEVIN, and P,R, MARTIN, Biochim, J . x:457-470 (1976),
Pergamon Press
EFFECTS OF THE RAT RENOTROPIC SYSTEM ON 14 C-URIDINE INCORPORATION INTO RNA AND RNA PRECURSORS Harry G . Preuas and Herzl Goldin Renal Division, Departments of Medicine and Pathology Georgetown University Schôol of Medicine, Washington, D .C . 20007 (Received in final form Juge 25, 1979)
We used the incorporation of l4 C-urldine into RNA of incubating kidney fragments from normal control rats to eval w to RNA metabolism . Sera from unilaterally nephrectomized rats (uni) obtained 20 hrs post-oparetively stimulate iliC-urldine incorporation into RNA slgnifiuntly mere than sera from Differently, sera from uni and sham-operated rats (sham) . sham rats have little influence on specific activities of endogenous urldine nucleotide pool in renal fragments . Renal extracts ware obtained by homogenizing kidneys in saline . Extracts from kidneys of uni and sham rata 20 hrs post operation depress incorporation markedly, and each depresses to a similar extent, but kidney extracts dilute the specific activities of urldine pools . Correcting for the latter dilution deoqnstrates that kidney extracts atone have little effect on l 4 C-urldine Incorporation into RNA . We then followed the results when these sera and extracts Compared to fragments incubating in sham were combined . sera and sham extracts, substitution of uni extracts or both uni extracts and uni sera enhances 14C-uridtne into renal RNA, whether or not results are corrected for changes in the specific activities of the uridtne pools . We conclude that after uninephrectomy there is a concurrent elevation in circulating renotropin and a tissue activating factor In the remaining kidney . The tissue factor wn only form an excitor to 14C-uridtne incorporation into RNA when serum is present . The rat renotropic system that anhancea ncorporation of 3 H-thymidtne into DNA also can stimulate 1~-urldine incorporation Into renal RNA . Following loss of renal mass (unilateral nephrectomy), the remaining viable kidney tissue of rats grown by hypertrophy (cell enlargement) and hyperplasta (cell division) (1) . Changes in renal RNA and DNA synthesis It is generally accepted that changes in these occur within hours (2,3) . mechanisms may set compensatory renal growth into motion . Tha exact uuse : that incite and regulate these synthetic mechanisms ors unknown although many investigators favor the concept that humoral stimulators play sans role (4-12) . Recently, we demonstrated the presence of different factors In the circulating blood and renal tissue of unilaterally nephrectomized rats that enhance 3H-thymidtne incorporation into renal DNA In vitro within a 90 minute incubation (13) . The present investigationsxt~ the original studies in order to determine if these factors also affect iliC-uridtne incorporation Into renal RNA . 0024-3205/79/060497-09$02 .00/0 Copyright (c) 1979 Pergasxm Press Ltd
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14 C-Uridine Incorporation Into RNA
Vol . 25, No . 6, 1979
MATERIALS AHD METHODS We used 200-250 g male Sprague-Dawley rats that were housed in a constant temperature room with a light-dark phase of 14 and 10 hrs . Rats received food and water _ad llbltum . Unilateral nephrectomy was tattled out by a left flank incision Sham operation consisted of exposure of the left kidney . In the sham procedures, care was taken not to handle excessively the left kidneys . Operations were performed between 2-3 p .m ., and materials for assay were obtained the next morning between 9-10 a .m . Rats were lightly anesthetized with ether . Following a midline abdominal incision, blood was drawn from the lower aorta into glass syringes and allowed to clot in plastic centrifuge tubes . Blood was immediately centrifuged in the cold . We added sera for assay to each flask in a concentration of 12$ V/V . To obtain renal extracts, whole kidneys were removed from sham-operated rats or those with a single remaining kidney approximately 20 hrs after unilateral nephrectamy (uninephrectomy), and homogenized in the same cold medium used for incubation (1 g kidney/5 ml medium) . We homogenized the tissue with a motor driven teflon pestle . After centrifugation at 500 g, supernates were collected and kept cold until initiation of study . Supernates from the remaining growing kidneys of uninephrectamized rats and kidneys of shamoperated rats were added (12$ V/V) along with 14C-uridine to incubating fragments . Controls (addition of 12$ V/V medium only) were assayed simultaneously . The in vitro assay to estimate 14 C-uridine incorporation into RNA has been described in detail elsewhere (14) . In brief, we obtained kidney frog- . ments by forcing cortices diced into small pieces with scissors, through a nylon sieve (2 x 1 .5 mn) . This nylon sieve was obtained from the framework of a twin coil diâlyzer (Travenol Laboratories, Morton Grove, IL) . The bottom of the sieve was scraped with a spatula to recover as much tissue as possible, and the material was placed in cold oxygenated medium . We allowed the tissue to settle twice by mixing with cold oxygenated medium . All fragments for assay were obtained from control rats . The basic medium used in washing and incubating fragments was modified Krebs-Ringer solution composed of the following anions and cations : Na+ 126 mM, K+ 5 mM, Mg++ 1 .2 mM, Ca++ 1 .0 mM,~50~, ~ 1 .2 mM, Ci- 133 mM, phosphate buffer (pH 7 .4) 10 mM, and gassed with 100$ 0 . To each flask containing 3 ml of medium and 30-60 mg wet wt of tissue2 ftagments, approximately 1 .5 uCi of uridine -2 -14C (specific activity 529 mCi/mmol) was added at the beginning of incubation . In this system, less than 12$ of the total counts disappears from the medium over the course of incubation . At the end of a 90 minute incubation at 25o C, cortical fragments were trapped with suction on circular filter paper (Whatman 40, 2 .4-cm radius) . We extracted RNA in the fragments from the filter paper by the method of Fleck and Munto (15), as modified by Halliburton and Thomson (16) . We estimated the concentrations of RNA by measuring the difference in absorbante of the RNA extracts at wave lengths of 260 mu and 310 mu on a Bnckmen DU spectrophotometer and by comparing readings to standard curves of a hydrolysate of pure reagent grade ribose nucleic acid (Sigma Chemical Company, St . Louis, MO) . To estimate specific activity ôf the uridine mono-, di-, and triphosphate pools at the end of 90 minute incubations, acid soluble fragments (4$ PCA) were boiled for 40 minutes to convert all the above to uridine monophosphate (17) . Çharcoal was added to the cooled extracts, and the nucleotides were subsequently eluted with a mixture to ethanol and ammonium hydroxide (18) .
ool.
25, No . 6, 1979
14 C- Uridias
Iacorporation
into 131zA
49 9
After evaporation, the solids were dissolved in 0 .01 N NH OH . The UMP was separated on PEI cellulose F with 0 .1 N formic acid as th~ carrier . The spots corresponding to uridine monophosphate were scraped from the plates into centrifuge tubes and the nucleotides eluted with 0 .01 N HC1 . After elution for 1 hr, the eluates ware read with a Beckman DU spectrophotometer at 262 mu and 310 mu to estimate the uridine monophosphate concentrations . In addition, radioactivity of the elutes was determined by ß counting . In a single experiment, the conversion step was eliminated and a uridine triphosphate pool was isolated . We performed ß counting on a Packard Trt-Carb 2425 liquid scintillation counter (Packard Instrument Co ., Inc ., Downers Grove, IL) with samples corrected back to 100$ absolute efficiency using a standard quench curve which relates automatic external standard counts to absolûte efficiency . Dioxane was used as a solvent, 2,5-diphenyloxazole was the primary scintillstor, and 1,4-bis (2-4 methyl-5-phenyloxazolyl) benzene, the secondary scintillator . In a few studies where the uridine pools were Isolated, the specific activities of RNA were corrected for the specific activities of the pools (17) . ThrougFiout this manuscript, we often refer to the sham operated rats themselves and extracts and sera from the sham-operated rats as "sham rats, sham sera, and sham extracts" . In a like manner, their uninsphrectamlzed counterparts are a lied "uni rats, uni sera, and uni extracts" . Each value listed in the tables represents the average value of specific activities measured in 3-4 flasks . Statistics shown here are by Student's "t" test using group or paired analysts . A11 statistics were checked by analysis of variance which essentially gave, the same results . Statistt u 1 significance is set at p < 0 .05 . RESULTS In the first 13 studies (Table I), the specific activities of RNA in renal fragments from normal untouched rats incubating in medium alone (control), sera, èxtracts, and sera plus extracts from sham or unl rats, were measured . All samples were obtained 20 hrs post operation . Products from sham (6 experiments) or uni rats (7 experiments) were assayed separately in different experiments and obviously on different fragments . The effects of sera, extracts, and the combinations were compared to control within each separate experiment . Attempts were made to randomize studies using sham and unl products . While sham sera enhanced 14C-uridine incorporation into RNA, uni sera caused a significantly greater stimulation . Extracts from sham operated and cant rats depressed specific activity to a similar extent . When the extracts and sera were combined, those from sham-0peratad rats still depressed, while those from uninephroctomized rats were mots likely to stimulate . Three of the seven experiments with the uni combinations showed definite stimulation whereas none of the experiments using the sham combinations showed stimulation . The differences between the ratios of the two sets were statistically significant (p < 0 .01) . Although the experiments were performed randomly, the fragments from the normal rats used for assay of the sham sera and extracts had higher control specific activities than those fragments used in the unI studies, necessitating comparisons to be made by percent change in stimulation . However, in those experiments where the baselines were comparable, the same trends generally persisted - uni serum has greater stimulatory effects than sham serum, both sham and uni extracts depress, and unl combinations either stimulate or do little canpared to control . Sham combinations generally depress compared to control .
1 ``C-Uridine Incorporation into RNA
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Vol . 25, No . 6, 1979
TABLE I EFFECTS OF SERA AND TISSUE EXTRACTS FROM UNINEPHRECTOMIZED AND -It I NTÔ~RÉIEAA~Ô~TR ~ÔRT~Ôf'IiÂ1r1EEi~t~ô?'Pt71t . ~F1C~RÉIiTS / Expts
Control Spec . Act . DPM/ uq RNA
Sera
Sera + Rana} Extract
Rsnal Extract
Spec . Act . $ CMnye DPM/ uq RNA from Control
Spec . Act . $Change Spec, Act . $Change DPH~+q RNA from DPM/uq RNA f ram Control Control
from sham ôpereted rats 20 hours post operation (6 expt) 6
37 .5 + 8 .7
48 .5+10 .5$
31 .7
21 .3 + 3 .1*
21 .9 + 2 .g*
-66
-72
from rats unTnephrectomized 20 hours earlier (7 expt) 48 .1+10,6$ 88 .4 24 7 27 .0 + 6,2
16 .5 + 2 .7*
-56 .6
.7+
30 .3 + 5 .3 °
SEM shown, °p > ,OS < .1, *p < 0 .01, $p < 0,01 compared to control (patted analysis) ; +p < 0,01 from value above by group analysis
In the next 10 studies (Table 11), the influences of various comblnattons of sera and extracts from both unl and sham rats were compared pn the same normal control fragments in each experiment . The effects upon 14 C urtdtne incorporation Into RNA of the following combinations - uni sera plus sham extracts, sham sera plus unl extracts, and uni sera plus unt extracts were compared to the effects of sham sera plus sham extracts .
TABLE II RAT
EFFECTS OF COMBINATION OF SERA AND RENAL EXTRACTS FROM UNI E HRECTOMI E OR SHAM-0 RA ED 0 H S RL ER C-URIDINE I NC ON I NT H A L FIEAGF1EliT~
Sham Sera Sham Extract
Uni Sera Sham Extract
Uni Sara Unt Extract
Spec . Act . DPM/uq RNA
Spec . Act . $Change DPM/yq RNA from Control
Spec . Act . DPM/u4 RNA
12 .6 + l .0
18 .0 + 2 .8k
18 .8 + 2 .9k
43 .5
$Change from Control 47 .6
. .Uni Sera Uni Extract Spac . Act . $Change DPM/uq RNA from Control 17 .0 + 2 .3*
35 .9
SEM shown, *p < .OS compared to fragments incubating in sham sera and sham extracts (pelted analysts) (10 experiments) .
Vol . 25, No . 6, 1979
14 C-Uridine Incorporation into 1iNA
50 1
Unlike the design of the first experiments, the effects of cant and sham sera and extracts were compared directly on the same fragments . Compared to fragments Incubated in sham sera and extracts, the substitution of either cant sera or cant extracts for sham, or a combined substitution of both cant sera and cant extracts res lied in a statistically significant augmentation to the incorporation of l~C-uridine into RNA . Unlike the other studies where 3H-thymidina was used as isotope (13), a combination of cant sera and extracts compared to sham sera and extracts showed no greater stimulation to Isotope incorporation then when uni . sera or cant extracts alone were substituted Into the system . However, it is important to note that the addition of renal extracts from remaining kidneys of cant rats when combined with sham sera ca~ared to the addition of extracts and sera from sham operated rats enhances specific activity . Another series of studies evaluated the Influences of our various procedures on the specific activities of the uridine pools . i n an experiment that compared sham and cant sera, we saw little affect by either on the specific activities of uridine precursor pools . The specific activities of those fragments incubating in sham sera were only slightly higher than control (+10$), whereas fragments incubating in cant sera had little change in their pool specific activities compared to control (-9$) . Corrected for pool size, sham serum slightly depressed l 4 C-uridine incorporation Into RNA (-4$) while cant serum stimulated (+37$), This was near the uncorrected values - no stimulation in flasks containing control sera and +26$ in those flasks with cant sera .' Another study followed the effects of a renal extract from a remaining klds~y 20 hrs post untnephrectomy . Uni extract caused a -61$ depression of C-uridine incorporation into RNA but also diluted the specific activity of the uridine pool, -55$ . jhe overall result corrected for pool dilution, showed a decrease in I C-uridine incorporation into RNA of only -2$ . The effects of sham sera plus sham extracts and cant sera plus cant extracts were compared to the control situat ton In Table III . The comparat b effects of sham sera and sham extracts on 14 C-uridtne incorporation into renal RNA resembled the previous studies (see Table I) with the exception of experiments lA and 6A where there was a +44$ and +17$ stimulation by the sham combination over control . The other 4 experiments showed depression to Incorporation . i n this series of 6 studies, cant sera plus cant extracts significantly stimulate isotope incorporation (p < 0 .05) . Additionally, the specific activities of the combined pools of uridine monophosphate, uridine diphosphate and uridine triphosphate were estimated . In the first 5 experiments, the specific activities of the uridine phosphate pools were lower in the fragments incubating to the sera and fragments from the sham and cant rats canpared to control (p < 0 .05) . The combinations from the cant and sham rats caused comparable depressions to the specific activities of the uridine pools In 4 of the experiments . i n the exceptional experiment (3A), the cant combination cab~pared to the sham combination depressed the specific In 6A, the UTP pool was activities of the uridine pools relatively more . Corrected results tsol~ted rather than the entire uridine phosphate pool . for 4 C-uridine incorporation Into RNA were similar to those seen in the previous experiments . Corrections for pools sizes show the cant combination to be stimulatory to 1~C-uridine tncorporstion Pnto renal RIU (p< 0 .05 .
14 ~_Uridine Incorporation into RNA
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Vol . 25, No . 6, 1979
TABLE III THE EFFECTS OF THE RENOTROPIC SYSTEM ON RAT RENAL . FRAGMENT V1TT' 0 U N OOL 1~TAli0LI3li
Expt,/ lA 2A 3A 4A
6A
Condition Control SS uu Control SS UU Control SS UU Control SS UU Control SS UU Control SS uU
Spec . Act . of UMP in Actd Sol . Fraction+
Spec . Acta of RNA*-
66 .9 48 .9 50 .1 37 .7 22,4 25 .3 58 .0 54 .2 337 52 .1 42 .3 45 .7 156 .1 98 .1 104 .5 189 .7 ° 178 .0 96 .0
Spec . Act . of RNA (corn)* 10,0 19,7 (+97) 26,5 (+165)
10 .0 14,4 (+44) 19 .8 (+g8) 40,7 50 .2 49-.2 91,6 49,9 38 .9 69 .5 21,1 13 .5 31,4
(+33) (- 2) (+82) (-28) (+39) (-5b) (+49)
61,8 (+17) 63 .0 (+19)
.
60,6 50,2 52 .7 157,b 49 .9 47 .9 79 .3 21,1 21 .5 46,7
(+99) (+5) (+214) (-4) (+59) (+2) (+121)
65,9 (+25) 124,5 (+135)
+ Spec . Act . of the pool of urldine mono-, di-, and trtphosphate (DPM/mmole) o5pec . Act . of the pool of urldine trtphosphate (DpM/mmole) . $Dlsintegratlons per minute per ug RNA . *Disintegrattons per minute per ug RNA after correction for the specific Correction based upon activity of the urtdtne phosphate or UTP pool, control . SS ~ sham sere + sham extract . UU ~ unt sera + unt extract . C ~ control . Each result is average of 4 flasks . ( ) ~ $ change from control,
DISCUSSION After untlaterai nephrectomy, RNA and DNA synthesis increase within hours to the remaining kidneys of healthy rats (2, 3) . An attractive hypothetsis is that humoral factors initiate and/or regulate the early changes In RNA and DNA synthesis (4-13) . Many studies have added information to the pros and cons favoring existence of hiunoral factors stimulating compensatory renal DNA synthesis (4-13), Some utilized 3H-thymidinn incorporation into renal DNA of living antenais to assess growth (5,7) . A few followed the In 1958, Ogawa influences of humoral substances on DNA synthesis i- vitro . and Nowtnski (4) found that sera from unilaterally nep~ectomized rats In 1970, we stimulate mitoses in tissue cultures of renal medullary cells . showed that plasma from unilaterally nephrectomized rats compared to plasma from sham-operated rats stimulate 3H-thymtdtne monophosphate incorporation Into the DNA of incubating rat kidney slices (8), An additional flndtng was that "untnephrectomtzed plasma" did not enhance DNA synthesis of liver, spleen, and lung slices .
Vol . 25, No . 6, 1979
14 C-Uridine
Incorporation
into l3ä11
50 3
Malt placed the synthesis of RNA Into a prominent and important spot to the schema of compensatory renal growth (19, 20) . However, invastigattans on in vitro renotroptc Influenua on RNA metabolism have bean fewer than oMs on ~A métsboltsm . Two s~udies reported that sarr from unilaterally nephrectamized animals enhance i C-uridTne incorporation Into RNA of incubating kidney slices (8) and Into tissue cultures of renal cortex (11) . Because the effects of the whole renotroplc system were investigated only for DNA metabolism and because RNA metabolism plays a major role In compensatory renal hypertrophy (19, 20), our pr~v Tous studies wars extended to Include RNA . Wa followed the incorporation of 14 C-urtdine into RNA, Our first experiments explored the s stems derived from sham-operated and uninephrectamtzed rats separately . 1 ~C-uridine Incorporation into RNA of fragments utilized for the control baseline was htghar to those experiments amploylng sham products, although uré was taken to randomize studies . The difference between control baseline in the two groups probably relate to many factors, including the following . Tissue : from different rats on different days may differ in isotope incorporation, in addition, technical reproducibility of exact dally conditions may vary . This in vitro system is anaiogous to ono whara PAN transport is studied, Tn that dâTty var atlons have led to the use of Internal controls for comparisons (Z1, 22) . Using internal controls, can) sera compared to sham sera cause greater stimulation relative to baseline . Some avldencs exists from other studies that this indirect canparison is valid . Previws studies comparing Inftuenus of cant and sham sera on isotope incorporation into RNA of kidney sites from the wale rat corroborate that unl sera has r lativaly more stimulation (8)~. Extracts from cant and sham kidneys depress 1 ~C-urTdins incorporation to the same degree and the addition of sham sera to fragments incubating in sham extracts doss not overcome this depression . In contrast, unl sera and extracts increase incorporation, a marked turnabout from what uni extracts do alone . The results from these investigations mimic ones pravtously (13) . However, this sortes of studies does not prove that the unl combination has any more stimulatory properties than unT sera alone, Our second studies ware deslgnéd to resolve this, Experiments dapictsd in Table 11 compared tM Influences of sham and unl combinations head to head, I .e ., on the same fragments . In these studies, the magnitude of Isotopic Incorporation by fragments in each separate experiment era closer . Throe points can 6e made . First, when added to fragments incubating In sham extract: uni sera compared directly to sham sera stimulate i~C-uridins incorporation into RNA . This Is not unexpected when one considers the studies whsra sera alone were investigated . Second, cant extracts compared to sham extracts Incubating in the same sera stimulate l~C-uridlne incorporation into RNA of renal fragments, This finding is IMportant because tt shows that unl extracts are relatively stimulatory when serum, even normal control serua, is present . Third, stimulation by unl sera and cant extracts is no greater than stimulation by unl sera with sham extracts or unl extracts with sham sera, Un11ka studios on DNA metabolism 1~ vitro (13), the separate stimulatory properties of unl sera and extracts era not additive in vitro . Using labelled nucleotides to estimate RNA and DNA metabolism has problems connected with It - principally that isotope dilution rather than actwl synthesis may be responsible for changes in nucleotide specific activities . In our syaem, dilution by extracallular factors could not cause our stimulatory results for 2 reasons . First ; If sera or plasma stimulate by non specific dilution of lsotopa alone, this would cause higher specific activltlss of RNA and DNA in many tissues other than kidney, and this does not occur (8, 12) . Sera from untnephrectomtzed rats stimulate only kidney tissue (8,12,23) , whereas sera from partially hepatectomized rats are relatively specific for
504
14 C-Uridiva Incorporation Into RNA
Vol . 25, No . 6, 1979
hepatic tissue (24) . Interestingly, sera from uninephrectomizad rats can stimulate hepatic 3H-thymidine incorporation into DNA when extracts from the growing kidney aro added to the medium (23) . Second, dialysis of sera, whiçh should remove all dilutlonal precursors, does not alter the stimulation of 3H-thymldine incorporation Into DNA 6y un1 sera (13) . While the above rules out extracellular dilutlonal factors, there is a slight possibility that endogenous nucleotide precursor pools might change In the same tissue under the varying conditions of study . For example, if unt sera and unl sera plus uni extracts decrease endogenous uridine pools, the added 14C-u~idina would produce a precursur pool with a higher specific acttvtty . This in turn, could theoretically create a higher RNA specific activity despite no alteration to the rate of RNA labelling . When we measured the specific activities of uridine pools (UMP, UDP, UTP) in fragments incubating to unt or shpn.sera, there was little change from control . Differently, and not wholly unexpected, addition of renal extracts dilutes the final specific activftlss of uridine precursor pools . When the specific activities of RNA are corrected for dilution, uni extracts Rather, have no apparent effect on 1 ~C-uridine incorporation into RNA alone . the lower Incorporation of .laC-uridine into RNA are probably secondary to isotope dilutto~ by nucleotides present in the extracts . Lastly, ws found in a more extensive study that both sera and extracts lower specific activities of uridine precursor pools, no doubt secondary to importantly, pool dilutions by the the effects of the extracts (vide supra) . uni co~apa~ed to sham combinations are either of different or ev6n greater . Therefore, we can state with certainty, that l~C-uridine Incorporation into RNA is rslatlvely greater with uni sera and extracts than with sham sera and extracts . More precisely, correcting for the specific activities of uridine pools shows that uni combinations stimulate compared to control, whereas sham combinations have lass effect . Two previous studies'hava indicated that the uridine triphosphâte pool may be increased early on In growth (3, 25), perhaps explaining the greater dilution of specific acttvtty by the unl cambinatlon in experiment 6A, Table iii . Cortes et al (25) could not reproduce earlier results showing enhanced 14C-uridine incorporation Into RNA in vitro by sera from untnephrectomized serum In their assay, a level that rats (8,11) . However, they used SO~ÎI fs far above maximum for stimulation (11,23) . Additionally, they incubated at 37oC instead of 25oC (8) . We have never shown much stimulation at the higher temperature . These two variations between assays may explain the differences, at least in part . Also, difference in oxidative substrates added to the medium directly or in the saran could cause differences . In summary, our findings suggest that unj sera alone and extracts from uni kidneys fn the presence of sera enhance 1 C-uridine incorporation into RNA . Unlike st .imulatlon to Isotope Incorporation into renal DNA, combined uni sera and extracts stimulate no more than uni sera alone or unf extracts plus control sera under the conditions of our assay . How these factors relate or interact in vlvo cannot be discerned from these studies . However, both factors appearÎn greater cancentrattons following renal mass loss . ACKNOWLEDGEMENTS The authors wish to thank Ms . Elizabeth Phillips for statistical help and Susan Dreux and Martiyn Davts for their excellent secretarial help .
Vol. 25, No . 6, 1979
14 C-IIridine Incorporation into RNA
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