possibility that the kidney which is known to have a 'growth' inhibitor factor (Dicker,

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1 J. Physiol. (1980), 301, pp Printed in Great Britain ORIGIN OF THE HUMORAL FACTOR RESPONSIBLE FOR COMPENSATORY RENAL HYPERTROPHY BY S. E. DICKER AND CHRISTINE A. MORRIS From the Department of Chemistry, University College London, 20, Cordon Street, London WC1E 6BT (Received 17 July 1979) SUMMARY 1. Rats were unilaterally nephrectomized and the remaining kidney was removed 10 min after the operation. 2. Cortical slices from both kidneys were incubated in culture medium in the presence of freeze-dried plasma from a control animal, for 4 hr. When renal cortical slices from a normal animal were now added to and incubated in the medium in which cortical slices from the remaining kidney (i.e. the kidney removed 10 min after unilateral nephrectomy) had been incubated, they showed an increase of protein content and dry weight. This increase did not occur when slices from normal kidneys were incubated in culture medium in which the first kidney had been incubated. 3. The increase of protein content and dry weight of cortical slices from normal kidneys did not occur when incubated in culture medium in which cortical slices from the remaining kidney had been incubated when freeze-dried plasma from normal rats had been substituted by freeze-dried plasma from anephric rats. 4. Since normal plasma which does not normally promote protein accretion in normal cortical slices in vitro can do so after it has been incubated with cortical slices from a kidney removed 10 min after unilateral nephrectomy, it is suggested that normal plasma contains a precursor of renal origin which is 'activated' when in the presence of a remaining kidney removed shortly after unilateral nephrectomy. INTRODUCTION There is overwhelming evidence that compensatory renal hypertrophy is controlled by a humoral substance (for reference, see Dicker & Morris, 1980). So far, however, there is no indication as to its origin. In a previous paper, Dicker & Morris (1980) had shown that addition of freezedried plasma from a unilaterally nephrectomized rat to normal renal cortical slices incubated in a culture medium produced an increase of their protein content. Freeze-dried plasma either from normal or from bilaterally nephrectomized rats did not have that effect. This suggested that the existence of a renotrophic factor depended on the presence of renal tissue, a fact which had been reported previously by Preuss, Terryi & Keller (1970). If this is so, one would have to envisage the possibility that the kidney which is known to have a 'growth' inhibitor factor (Dicker, /80/ $ The Physiological Society I PHY AnT

2 2 S. E. DICKER AND C. A. MORRIS Morris & Shipolini, 1977) also possesses the property to increase its own size, directly or indirectly, when stimulated to do so. Since the existence of a 'growth' inhibitor factor, and the renotrophic effect of plasma from unilaterally nephrectomized rats had been demonstrated using tissue culture methods, available and currently used in this Department, it was thought of interest to see whether with similar methods it would be possible to investigate the possible origin of the humoral factor responsible for compensatory renal hypertrophy. METHODS Male Wistar rats of about 250 g body weight were used. Under anaesthesia, the left kidney (called hereafter AO) was removed and immediately put in culture medium 199 (Gibco-Biocult Ltd.) containing Earle's salts, L-glutamine and 25 mm-hepes buffer. Ten minutes later the remaining kidney (called hereafter BI0) was taken out and put into 199 culture medium.cortical slices from kidneys AO and BIo were cut with a Stadie-Riggs microtome and each slice from AO and B1o separately was put into a small vessel containing 5 ml of 199 culture medium to which had been added 30,ug mg-' wet tissue of freeze-dried plasma from either a normal or from a bilaterally nephrectomized rat. The plasma was obtained from blood withdrawn from the lower aorta as described by Dicker & Morris (1980). In the case of bilaterally nephrectomized animals, blood was withdrawn either 10 min or 2 hr after the operation. The wet weight of cortical slices AO and BLO was of the order of 35 mg. Each vessel containing one cortical slice in 5 ml culture medium was then put into a humidifier, gassed with a mixture of 02-CO2 and kept in a dark room at a temperature of 37 'C. After 4 hr incubation, the slices from kidneys AO and B1o were removed and the respective solutions of culture media called As0 and B810 were collected separately. Fresh cortical slices from normal rats (called hereafter C) were cut, as usual, accurately weighed and immersed into the medium in which slices from kidney AO or BIO had been incubated. After gassing with 02-CO2 mixture, the slices were incubated for 4 hr, at 37 'C, in the usual way (Dicker & Morris, 1980). At the end of the second incubation period, the slices were prepared for protein estimation or were transferred into a dessicator, over phosphorus pentoxide and dried at 104 'C for 48 hr. For protein estimation, cortical slices were homogenized in the presence of sodium deoxycholate and proteins were estimated according to Lowry's method, as described previously (Dicker & Morris, 1980). Results are given as means + s.e. of means. RESULTS The results of incubating normal renal cortical slices (C) in media Aso and BS10 (i.e. in media in which slices from kidneys Ao and B10 had been incubated in the presence of freeze-dried normal plasma) are shown in Table 1. It will be seen from this table that when cortical slices from a normal rat (C) were incubated for 4 hr in culture media (Aso and Bs10) slices in Bs1o showed an increase of dry weight and of protein content of the order of % when compared with slices incubated in Aso (Table 1A). Aso alone had no effect. When the same experiment was repeated but using freeze-dried plasma from anephric (instead of normal) rats, the results varied according to the time at which blood had been withdrawn after bilateral nephrectomy. Using freeze-dried plasma from blood withdrawn 10 min after bilateral nephrectomy, the increase of dry weight and protein content of slices (C) incubated in media Bs1 was much reduced; it was less than 3 % above that for control slices incubated in Aso medium (Table 1B). When, however, freeze-dried plasma from blood withdrawn 2 hr after double

3 ORIGIN OF RENOTROPHIC FACTOR 3 nephrectomy was used, no significant differences were found between the dry weight and protein content of slices incubated in media As. or Bs1. (Table 1C), thus suggesting that some activity present in plasma from anephric rats withdrawn 10 min after the operation had disappeared from plasma taken 2 hr later. TABLE 1. Effect of incubating normal cortical slices in media in which cortical slices from kidneys AO and B10 had been incubated in the presence of freeze-dried plasma from normal or bilaterally nephrectomized rats Average % difference between CA. CAs CB810 and. A_ 5, A \ ~~~~~~~~~CBF3Lo DW Protein n DW Protein n r- A mg g-1 mg g-1 mg g-1 mg g-1 DW Protein A Normal plasma *6 +1* B DN plasma (10 min) + 2l C DNplasma *5 1-6 (2 hr) + 2*0 + 2* (n.s.) (n.s.) D 199 medium (ns.) (n.s.) For explanation of kidneys AO and B10, CA5, and CB81,, see text (Methods). DN plasma (10 min), plasma from blood withdrawn 10 min after double nephrectomy; DN plasma (2 hr), plasma from blood withdrawn 2 hr after double nephrectomy; n, number of experiments; DW, dry weight; n.s., not significant. TABLE 2. Effect of incubating renal cortical and liver slices in culture medium B8,10 Renal cortical slices Liver slices DW Protein DW Protein mgg-' mgg1 n mgg 1 mgg-1 n I After incubation in A8o * solution II. Afterincubation in B * solution Average % increase between incubation in Aso and BEF0 (n.s.) (n.s.) For explanation of B8., and A8., see text (Methods). n, number of experiments; n.s., not significant. To see whether the 'activation' of normal plasma (Table 1A) was the result of a secretion from kidneys B10, similar experiments to those reported in Table I were performed in which cortical slices from kidneys AO and B10 were incubated in culture medium 199 in the absence of freeze-dried normal plasma. No significant increases in either dry weights or protein content of slices incubated in media Aso or BS~o were observed (Table 1 D). Since it would appear that during incubation of cortical slices from a kidney (B10) removed 10 min after unilateral nephrectomy with freeze-dried plasma from a 1-2

4 4 S. E. DICKER AND C. A. MORRIS normal rat there is the formation of a factor capable of producing an increase of protein of control cortical slices, it was of interest to see whether that factor would have a similar effect on slices from another tissue. To this end, experiments were modified so as to test the effect of solutions Bs,, and As. on liver slices. This was done by substituting liver slices for renal cortical slices during the second period of incubation, the rest of the procedure remaining the same as usual. The results, tabulated in Table 2, show clearly that whereas there was an increase of dry weight and protein content in renal cortical slices incubated in solution Bs.., the dry weight and protein content of liver slices did not show any increase. DISCUSSION The results of the present investigation show that when cortical slices from a kidney removed 10 min after unilateral nephrectomy (kidney B10) are incubated with freeze-dried normal plasma, the incubating medium becomes capable of promoting an increase of protein in fresh renal cortical slices from normal kidneys. In other words, normal plasma which normally does not promote protein increase of renal cortical slices in in vitro experiments, did so after incubation with cortical slices from a remaining kidney (B1o). It follows that a few minutes after unilateral nephrectomy, biochemical changes must have taken place in the remaining kidney which when in contact with normal plasma, transforms the latter from an inactive to an active form. This could be achieved if slices from a remaining kidney were producing, or secreting substance(s) capable of producing protein increase when in contact with cortical slices from normal kidneys. This, however, is unlikely since when slices from a remaining kidney (B1o) were incubated in a culture medium in the absence of normal plasma, the culture medium did not promote protein increase in slices from normal kidneys. It is clear therefore that not only was there no production of a renal active substance per se, but that whatever happened needed the presence of normal plasma. Furthermore, the 'activation' of plasma present in the culture medium had to be plasma from a normal animal (e.g. from an animal with its kidneys intact) since no 'activation' occurred with plasma from blood withdrawn 2 hr after double nephrectomy (e.g. from an animal without kidneys). The reason for using a kidney (B1o) which had been left in situ for 10 min after unilateral nephrectomy was based on the knowledge that biochemical changes have been shown to occur in the remaining kidney a few minutes after the removal of its congener. For instance the incorporation of [14C]choline into acid insoluble phospholipids (phosphatidylcholine, lysophosphophatidylcholine and sphingomyelin) is already increased 5 min after contralateral nephrectomy (Lowenstein & Toback, 1978) and the renal content of a nucleotide, cyclic-guanosine monophosphate, is markedly increased in less than 10 min after unilateral nephrectomy (Dicker & Greenbaum, 1977; Dicker, 1978). The increase of the latter which is known to act as a second messenger has been considered of potential importance for protein synthesis. It is therefore likely that it is during the first minutes after the removal of one kidney that the remaining kidney will show active changes in its metabolism, and this justified the timing chosen. What then is the interpretation of the present results? To be able to produce an

5 ORIGIN OF RENOTROPHIC FACTOR 5 increase of dry weight or protein content of incubated normal cortical slices, plasma had not only to be present but it had to be plasma from a normal animal with its kidneys intact. This strongly suggests that normal kidneys secrete into the blood a factor, which may be a precursor. When in contact with the remaining kidney, soon after unilateral nephrectomy, the plasma 'precursor' is activated and so becomes endowed with the ability of starting protein synthesis. This view is supported by the observation that plasma from blood withdrawn 10 min after double nephrectomy and incubated with cortical slices from remaining kidneys (B10) had still some activity, which had disappeared in plasma from blood taken 2 hr after double nephrectomy. This would suggest some kind of time lag for the complete disappearance of the plasma 'precursor', which is what one would expect. In conclusion, it would appear that besides a renal 'growth' inhibitor (Dicker et al. 1977), cortical cells produce a substance or factor which in normal conditions circulates in the blood in an inactive form; a few minutes after unilateral nephrectomy, the cortical cells of the remaining kidney can 'activate' the normally inactive substance or factor so as to make it capable of inducing an increase of protein. This factor or substance may conceivably be the first messenger in the chain of events leading to protein synthesis. We would like to thank the Medical Research Council for a grant to one of us (S.E.D.) and Professors M. McGlashan and C. Vernon for allowing us to do the work in the Department of Chemistry. REFERENCES DICKER, S. E. (1978). Changes in renal cyclic nucleotides as a trigger to the onset of compensatory renal hypertrophy. Yale J. Biol. Med. 51, DICKER, S. E. & GREENBAUM, A. L. (1977). Changes in renal cyclic nucleotide content as a possible trigger to the initiation of compensatory renal hypertrophy in rats. J. Phy8iol. 271, DICKER, S. E. & MORRIS, C. A. (1980). Presence of a renotrophic factor in plasma of unilaterally nephrectomized rats. J. Phy8iol. 299, DICKER, S. E., MORRIS, C. A. & SHIPOLINI, R. (1977). Regulation of compensatory kidney hypertrophy by its own products. J. Phyaiol. 269, LOWENSTEIN, L. M. & TOBACK, F. G. (1978). Metabolic response to renal compensatory growth. Yale J. Biol. Med. 51, PREUSs, H. G., TERRYI, E. F. & KELLER, A. I. (1970). Renotropic factor(s) in plasma from uninephrectomised rats. Nephron 7,