10-6 M) or freeze-dried normal rat plasma (35 jug mg-' wet wt. tissue) did not show

Transcription

1 J. Physiol. (1981), 315, pp With 1 text-figure Printed in Great Britain ROLE OF A RENAL ARGINYLESTEROPEPTIDASE IN THE PRODUCTION OF A RENOTROPHIC FACTOR IN UNILATERALLY NEPHRECTOMIZED RATS BY THE LATE S. E. DICKER, CHRISTINE A. MORRIS AND F. L. PEARCE From the Department of Chemistry, University College London, 20 Gordon Street, London WClH OAJ (Received 21 July 1980) SUMMARY 1. Renal cortical slices incubated for 4 hr in culture medium in the presence of either dibutyrylguanosine-3': 5'-cyclic monophosphate (dibutyryl cyclic GMP, 10-6 M) or freeze-dried normal rat plasma (35 jug mg-' wet wt. tissue) did not show any change in dry weight or protein content. However, addition of freeze-dried normal plasma together with dibutyryl cyclic GMP led to an increase in both parameters. 2. Unilateral nephrectomy produced a marked increase in the level of arginylesteropeptidase in the renal cortex of the remaining kidney. A similar increase was observed in renal cortical slices incubated with dibutyryl cyclic GMP in vitro. 3. The renal cortical esteropeptidase was inhibited by phenylmethylsulphonyl fluoride (PMSF, 2 mm). The inhibitor did not, however, prevent the normal increase of dry weight and protein content of renal cortical slices incubated with plasma from unilaterally nephrectomized rats. Similarly, control normal plasma incubated with cortical slices from a kidney removed 10 min after unilateral nephrectomy became endowed with renotrophic activity (Dicker & Morris, 1980b) but this activation was abolished by PMSF. 4. Control plasma treated with a purified arginylesteropeptidase and incubated for 4 hr with renal cortical slices produced a hypertrophy of the slices similar to that evoked by plasma from a unilaterally nephrectomized rat. 5. Since following unilateral nephrectomy there is a rapid increase in the level of cyclic GMP in the renal cortical tissue of the remaining kidney (Dicker & Greenbaum, 1977) it is suggested that this increase leads to the induction of a specific arginylesteropeptidase. The possibility that the enzyme then cleaves a renotrophic precursor normally present in the plasma, so converting it to an active form, is discussed. INTRODUCTION Previous investigations in our and other laboratories have shown that compensatory renal hypertrophy is produced by a humoral factor which is present in the blood plasma following unilateral nephrectomy (Van Vroonhoven, Soler-Montesinos & Malt, 1972; Shames, Murphy & Berkowitz, 1976; Dicker, 1978). The study of this factor was facilitated by the development of a simple in vitro assay system in which /81/ $07.50 ( 1981 The Physiological Society

2 414 S. E. DICKER, C. A. MORRIS AND F. L. PEARCE the plasma from unilaterally nephrectomized rats, but not that from control animals, produced an increase in both the dry weight and protein content of excised cortical slices (Dicker & Morris, 1980a). This result suggested that normal plasma contained a renotrophic precursor which could be converted to an active form by contact with the remaining kidney in a unilaterally nephrectomized animal. As is well known. many polypeptide hormones are generated from high molecular weight precursors by the proteolytic action of specific arginylesteropeptidases (Steiner, 1976: Server & Shooter, 1976; Berger & Shooter, 1977; Schachter, 1969). This phenomenon may provide a general post-translational regulatory mechanism in hormone biosynthesis (Taylor, Mitchell & Cohen, 1974). We have therefore examined the role of such enzymes in the generation of the humoral factor responsible for renal hy7pertrophy. Since the initial event in this process may be a transient increase in the intracellular level of guanosine-3': 5'-cyclic monophosphate (cyclic GMP, Schlondorff & Weber, 1976; Dicker & Greenbaum, 1977), the effect of this nucleotide on the putative enzymes was also investigated. METHODS Male Wistar rats of body weight ca. 250 g were used. Left unilateral nephrectomy was performed as described previously (Dicker & Morris, 1980a) and the kidney placed immediately in tissue culture medium 199 (Gibco-Biocult) containing Earles' salts, i,-glutamine and 25 mm-hepes buffer. Ten min later the remaining kidney was removed and put separately in the same solution. Cortical slices (average wet wvt. 35 mg) were cut using a Stadie-Riggs microtome. Each slice was immersed separately in culture medium (4 ml.) and incubated for 4 hr. unless otherwise specified. under the conditions previously described (Iicker & Morris, 1980a). Freeze-dried plasma (35 /ug mg-1 wet wt. tissue) from normal or unilaterally nephrectomized rats or N2,(2'_ dibutyrylguanosine-3':5'-cyclic monophosphate (dibutyryl cyclic GIP, 10-6 Ai; Sigma) were alde(1 to the culture media as indicated. A stock solution (0-2 M) of the esteropeptidase inhibitor phenylmethylsulphonyl fluoride (PMVSF; Sigma) was prepared in dimethyl sulphoxide (DIMISO) and aliquots included, as noted, in buffers and media to a final concentration of 2 mm. At the final dilution used, DNISO itself was shown to have no effect on any of the parameters measured. Levels of cyclic GMP in the kidney slices were estimated after 20 min of culture. as previously reported by Dicker & Greenbaum (1977). The protein content and dry weight of the tissue slices were determined, in the usual way, after 4 hr of incubation (Dicker & Morris, 1980a. b). Arginylesteropeptidase activity was measured in freshly excised or cultured (4 hr) cortical slices after homogenization of the tissues in 4 times their volume of Tris-(hydroxymethyl)- aminomethane/hci buffer (0-05 M, ph 7-4). After centrifugation (30,000 gy 60 min. 4 C(). the supernatant fluid was collected and esterase activity was assayed against N-a-benzoyl-i,-arginine ethyl ester (BAEE, Calbiochem) as substrate. essentially according to the method described by Greene, Shooter & Varon (1969). One unit of enzyme was defined in terms of the amount which hydrolysed I n-mole of substrate per min: the specific activity of the extract was expressed as the number of units ge1 wet wt. tissue. To determine whether any generated esteropeptidase remained confined within the renal cells. or partially escaped into the surrounding milieu, appropriate culture media were harvested at the end of the incubation period and tested for enzymic activity. The effect of PMSF on the activation of control plasma, produced by inculbation (4 hr) with slices from a kidney removed 10 min after unilateral nephrectomy (Dicker & Morris b). was also examined. The incubation was carried out in the presence of the inhibitor and the media recovered. re-oxygenated and used for the culture of fresh slices from a normal kidney. At the end of the secon(l incubation period (4 hr), the dry weight and protein content of the tissue were measured. Finally, the effect of specific proteases on the activity of normal rat plasma was investigated. Pure arginylesteropeptidase (the y-subunit from nerve growth factor) was isolated from the submaxillary salivary glands of mice as previously described (Carstairs, Edwards, 1Pearce, \Vernon

3 ARGINYLESTERASE AND RENAL HYPERTROPHY 415 & Walter, 1977). In a given experiment, freeze-dried normal plasma (2 mg) was incubated (37 TC, 30 min) in culture medium 199 (8 ml.) containing the enzyme (06 mg). The solution was then divided into two samples, to each of which were added two or four renal cortical slices from a normal rat. At the end of a further period of incubation (4 hr) the dry weight and protein content of the slices were determined. As control experiments, cortical slices from an unoperated rat were incubated with untreated, freeze-dried normal plasma or plasma from an animal killed 10 min or 24 hr after unilateral nephrectomy. To examine the specificity of the effect, trypsin (Sigma) was used in place of the salivary peptidase in some experiments. All values are given as means+s.e. of means for the number (n) of kidney slices examined. In every case, these data are derived from at least four separate experimental animals. Values of P < 0 05 were considered to be statistically significant. RESULTS Effect of cyclic GMP and plasma on the dry weight and protein content of renal cortical slices Incubation of renal cortical slices with dibutyryl cyclic GMP (10-6 M) for 20 min led to a significant increase in the tissue content of the nucleotide, the levels in the control and treated slices being (n = 16) and (n = 16) p-mole g-1 wet wt. respectively (P < 005). The dry weights (201 ± P8 mg g-1, n = 12) and protein content ( mg g-1, n = 12) of the experimental slices estimated after 4 hr of incubation were, however, not significantly different from those of the controls ( P2, n = 12 and , n = 12, respectively). Addition of freeze-dried plasma (35,g mg-1 wet wt. tissue) from normal animals to cortical slices incubated for 4 hr in culture medium also produced no significant increase in the dry weight or protein content of the slices, confirming previous findings (Dicker & Morris, 1980a). However, addition of freeze-dried normal plasma together with dibutyryl cyclic GMP (10-6 M) led to a marked increase in these parameters, the dry weight rising from mg g-1 (n = 82) to P2 mg g-1 (n = 22, P < 0 05) and the protein content from P8 mg g-1 (n = 18) to mg g-1 (n = 18, P < 0 05). These increases are similar to those observed when normal renal cortical slices are incubated with freeze-dried plasma from rats killed 10 min after unilateral nephrectomy (Dicker & Morris, 1980a, b). Arginylesteropeptidase activity in renal cortical slices (a) in vivo. The amounts of arginylesteropeptidase activity in the renal cortex of normal adult rats were too small to be estimated ( < 5 units g-1 wet wt., Fig. 1). In contrast, kidneys removed 10 min after unilateral nephrectomy exhibited a strikingly enhanced activity ( units g-1 wet wt., n = 14) which produced a typical linear hydrolysis of the substrate (Fig. 1). This activity was abolished by addition of PMSF (2 mm) to the supernatant fluid. (b) in vitro. No measurable esterase activity ( < 5 units g-1 wet wt.) was found in normal renal cortical slices cultured in the presence or absence of normal freeze-dried plasma or of freeze-dried plasma from rats killed 10 min after unilateral nephrectomy. However, normal slices incubated in the presence of dibutyryl cyclic GMP showed an increased level of the esteropeptidase activity (50' units g-1 wet wt., n = 12) similar to that observed in cortical slices from a remaining kidney removed after unilateral nephrectomy (see above). This increase was abolished by addition of PMSF

4 416 S. E. DICKER, C. A. MORRIS AND F. L. PEARCE to the incubation fluid. These preparations, as did cortical slices from unilaterally nephrectomized animals, released some of the enzyme into the culture medium. This activity could again be blocked by PMSF. PMSF, however, had no effect on the renotrophic activity of freeze-dried plasma from rats which had been unilaterally nephrectomized 10 min or 24 hr previously (Dicker & Morris, 1980a, b). Normal cortical slices incubated for 4 hr with such plasma underwent a similar increase in 0* * Time (min) Fig. 1. Determination of arginylesteropeptidase activity by the hydrolysis of BAEE. Trypsin (0 2,ug, positive control) or a centrifuged homogenate of the renal cortex from a kidney (equivalent to 300 mg wet wt. tissue) removed 10 min after unilateral nephrectomy (A) or from an unoperated animal ( O) were separately added to 1 'tmole BAEE dissolved in 3 ml. buffer and contained in a cuvette of 1 cm path length. The rate of hydrolysis was measured by recording the optical density (O.D.) at 253 nm as a function of time. The absorbance change corresponding to complete hydrolysis of the substrate was taken from the end-point of the standard curve constructed using the enzyme trypsin (Greene et al. 1969). Specific activities were then expressed as n-mole of substrate hydrolysed per min per g wet wt. tissue. Graphs show the result of a typical experiment. dry weight and/or protein content in the presence or absence of the inhibitor. For instance, using plasma from rats operated for 24 hr, the dry weights in the presence of PMSF were mg g-1 (n = 18) and in the absence of PMSF were mg g-1 (n =72). The protein contents in the presence of PMSF were mg g-1 (n = 12) and in the absence of PMSF were mg g-1 (n = 48). These values were significantly (P < 005) greater than the corresponding control values for dry weight ( mgg-1, n = 72) and protein content (I mg g-1, n = 48). In sharp contrast, PMSF totally prevented the activation of control plasma after incubation with cortical slices from a unilaterally nephrectomized animal. Such plasma produced values of dry weight (199±+ 12 mg g-1, n = 12) and protein content ( mg g-1, n = 12) which were not significantly different from the respective controls (see above).

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6 418 S. E. DICKER, C. A. MORRIS AND F. L. PEARCE Activation of normal plasma with a purified arginylesteropeptidase Cortical slices, incubated in the presence of freeze-dried normal plasma which had been pre-treated with purified arginylesteropeptidase from mouse salivary gland, showed an increase in dry weight and protein content similar to that induced by plasma recovered from a rat 24 hr after unilateral nephrectomy (Table 1). The activation process was specific: it was not produced by incubation of the plasma with trypsin under identical conditions. DISCUSSION Previous investigations (Schlondorff & Weber, 1976; Dicker & Greenbaum, 1977; Dicker, 1978) have shown that a few minutes after unilateral nephrectomy in the rat, there is a sharp increase in the content of cyclic GMP in the remaining kidney. This increase was interpreted as a trigger to the onset of compensatory renal hypertrophy, but no further explanation as to the role of the nucleotide in the process was given. In the present investigation, it was found that incubation of normal renal cortical slices with dibutyryl cyclic GMP in vitro led to an increase in the cellular content of the free nucleotide but produced itself no increase in dry weight or protein content of renal slices. When, however, the slices were incubated with dibutyryl cyclic GMP together with normal plasma (which alone had no effect on the tissue), there was a marked increase in both the dry weight and protein content of the sections. This response was similar in magnitude to that observed when normal cortical slices were incubated with plasma from a unilaterally nephrectomized rat (Dicker & Morris, 1980a). These results strongly suggested that an increased intracellular level of cyclic GMP, either produced in vivo following unilateral nephrectomy or in vitro by incubation with the dibutyryl derivative of the nucleotide, led to biochemical changes in the cortical tissue, rendering it capable of activating normal plasma and so endowing the latter with renotrophic activity. Since many humoral factors are generated from biosynthetic precursors by the action of specific arginylesteropeptidases (see Introduction), the possible role of these enzymes in the mechanism of compensatory renal hypertrophy was examined. Unilateral nephrectomy was found to produce a rapid increase of esteropeptidase activity in the remaining kidney and subsequent appearance of renotrophic activity in the plasma (Dicker & Morris, 1980a). A similar induction of the enzyme was observed in vitro by incubating normal cortical slices with dibutyryl cyclic GMP. The same effect of the nucleotide has recently been reported in isolated rat peritoneal mast cells (Rothschild, 1980). The activity of the renal esteropeptidase was blocked by the esterase inhibitor PMSF. This inhibitor had no effect on the renotrophic activity of plasma from unilaterally nephrectomized rats but prevented the activation of control normal plasma after incubation with cortical slices from such animals. Thus, it would appear that the action of the enzyme is confined to the activation process, this step being complete in plasma taken after unilateral nephrectomy. Further, normal plasma could be endowed with renotrophic activity by incubation with a purified arginylesteropeptidase (the y-subunit from the nerve growth factor present in mouse salivary gland). This enzyme is believed to be involved in the conversion of the biosynthetic precursor for nerve growth factor into its active form (Berger & Shooter, 1977) and

7 ARGIN YLESTERASE AND RENAL H YPERTROPH Y 419 it is of considerable interest that its activity extends to the present situation. Thus, from the results of the current and previous investigations, it would appear that immediately after unilateral nephrectomy, there is a rapid increase in the level of cyclic GMP in the cortical tissue ofthe remaining kidney (Dicker & Greenbaum, 1977). This in turn induces the production of a specific arginylesteropeptidase which cleaves a renotrophic precursor normally present in the plasma, so converting it into an active form. The latter then produces the observed hypertrophic response. The events which lead to the initial increase in cyclic GMP and the biochemical characterization of the renal esterase (and in particular its relationship to the y-subunit from mouse nerve growth factor) are of obvious interest and await further study. One of us (S. E. D.) would like to thank the Medical Research Council without whose financial help this investigation would not have been possible. REFERENCES BERGER, E. A. & SHOOTER, E. M. (1977). Evidence for pro-f-nerve growth factor, a biosynthetic precursor to fl-nerve growth factor. Proc. natn. Acad. Sci. U.S.A. 74, CARSTAIRS, J. R., EDWARDS, D. C., PEARCE, F. L., VERNON, C. A. & WALTER, S. J. (1977). Immunogenic contaminants in mouse nerve growth factor. Eur. J. Biochem. 77, 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. Physiol. 271, DICKER, S. E. & MORRIS, C. A. (1980a). Presence of a renotrophic factor in plasma of unilaterally nephrectomized rats. J. Physiol. 299, DICKER, S. E. & MORRIS, C. A. (1980b). Origin of the humoral factor responsible for compensatory renal hypertrophy. J. Physiol. 301, 1-5. GREENE, L. A., SHOOTER, E. M. & VARON, S. (1969). Subunit interaction and enzymatic activity of mouse 7S nerve growth factor. Biochemistry 8, ROTHSCHILD, A. M. (1980). Hydrolysis of arginine and tyrosine esters in mast cells exposed to epinephrine. Biochem. Pharmac. 29, SCHACHTER, M. (1969). Kallikreins and kinins. Physiol. Rev. 49, SCHLONDORFF, D. & WEBER, H. (1976). Cyclic nucleotide metabolism in compensatory renal hypertrophy and neonatal kidney growth. Proc. natn. Acad. Sci. U.S.A. 73, SERVER, A. C. & SHOOTER, E. M. (1976). Comparison of the arginine esteropeptidases associated with the nerve and epidermal growth factors. J. biol. Chem. 251, SHAMES, D., MURPHY, J. J. & BERKOWITZ, H. (1976). Evidence of a humoral factor in unilaterally nephrectomised dogs stimulating renal growth in isolated canine kidneys. Surgery, St. Louis 79, STEINER, D. F. (1976). Peptide hormone precursors: biosynthesis, processing and significance. Peptide Hormones, ed. PARSONS, J. A., pp London and Basingstoke: Macmillan Press. TAYLOR, J. M., MITCHELL, W. M. & COHEN, S. (1974). Characterization of the binding protein for epidermal growth factor. J. biol. Chem. 249, VAN VROONHOVEN, T. J., SOLER-MONTESINOS, L. & MALT, R. A. (1972). Humoral regulation ofrenal mass. Surgery, St. Louis 72,