Juxtaglomerular cells cultured on a reconstituted basement membrane BARBARA M. RAYSON Department of Physiology and Cardiovascular Center, Cornell University Medical College-New York Hospital, New York, New York 10021 Rayson, Barbara M. Juxtaglomerular cells cultured on a reconstituted basement membrane. Am. J. Physiol. 262 (Cell Physiol. 31): C563-C568, 1992.~Circulating renin levels are regulated by release from juxtaglomerular (JG) cells. Here, for the first time, we describe the primary culture of rat juxtaglomerular cells on a reconstituted basement membrane. In addition, primary cultures were transformed with a temperaturesensitive SV40 large T antigen gene to promote the development of a continuous JG cell line. Both primary cultures and transformed JG cells maintain a highly differentiated state and secrete active renin. These preparations now provide a system in which characterization of the cellular mechanisms of regulation of renin synthesis and release is possible. kidney;
renin; hypertension;
rats; extracellular
matrix
DISEASE and hypertension are major causes of death in Western societies, and the diseases appear to be of diverse etiology. Significant subpopulations of hypertensive patients have, however, been identified in which inappropriately high circulating renin levels maintain elevated levels of blood pressure (15, 16,
CARDIOVASCULAR
23)
Circulating renin levels are primarily regulated by release from juxtaglomerular (JG) cells, first identified in 1925 (21), in which renin is synthesized and stored (15, 16, 23). The physiological stimuli that regulate renin release from these cells have been well characterized (13, 15). Because these cells have not previously been cultured though, less is known about the intracellular mechanisms implicated in the regulation of rates of synthesis and release, because in vivo it is difficult both to experimentally manipulate JG cell second messenger systems and to quantify specific JG cell responses. JG cells constitute at most O.Ol-0.1% of the renal cortical cell population. We therefore maintained JG cells on a reconstituted basement membrane (Matrigel; Collaborative Research). This development was made possible by, first, the previous publication of a technique with which to generate suspensions of cells highly enriched in JG ceils (14) and, second, the identification of the importance of cell-substrate interactions and cell three-dimensional structures in the maintenance of tissue-specific gene expression in culture (5).’ Cells were grown on a reconstituted basement membrane preparation in an attempt to mimic their in vivo substrate, with the aim of also generating cell three-dimensional structures similar to those in vivo. ’ The substrate on which cells are grown appears to be an important determinant of cell and cytoskeletal/nuclear matrix structure. Cellsubstrate contact is maintained through specific cellular receptors which bind to defined substrate ligands. The density, the distribution, and the tensile properties of these interactions then, together with those of the intercellular associations, determine the three-dimensional structure of the cell and the cytoskeletal-nuclear matrices. 0363-6143/92
$2.00 Copyright
The technique was effective in sustaining the differentiated state of JG cells, over extensive periods of time, in culture. METHODS Generation of JG cell cultures. Highly enriched preparations of JG cells were generated by collagenase digestion of rat renal cortices and density gradient centrifugation, as previously described (14). Two to four rats (200-300 g) were used. Forty to fifty percent of the cells recovered between the densities of 1.051 and 1.064 g/ml were identifiable, on the basis of morphological criteria, as JG cells (Fig. 1): granules at three different stages in their development, a large number of mitochondria, large nuclei, and well-developed endoplasmic reticular and Golgi networks (2, 7,17). Protogranules manifest distinct crystalline structure as previously described (2). Subsequently, this enriched preparation of JG cells was plated in high density (5 ml, l-7 x lo6 cells/ml) in plastic culture dishes (Falcon, 60 x 15 mm), to which JG cells preferentially adhere, over 48-h intervals. Cells were recovered by incubation in the neutral protease preparation Dispase (Collaborative Research) over 2h incubations at 37°C. Subsequently, they were washed in fresh medium at least three times to remove all Dispase contamination. Generally, only lo-20% of the cells originally plated on plastic were recovered with Dispase treatment. The suspension generated was then characterized using immunohistochemistry. As indicated in the original description of the technique used (14), at least 80% of the cells recovered stained positively after treatment with a rat polyclonal antiserum generated against mouse renin. Subsequently, this suspension, highly enriched in JG cells, was plated on the commercially available basement membrane preparation Matrigel (2.5 ml,/60 X 15 mm dish, Collaborative Research) and maintained in RPM1 1640 (GIBCO) to which was added 25-mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (United States Biochemicals), 2 pg/ml corticosterone (Sigma), 50 U/ml penicillin (GIBCO), 70 pg/ml streptomycin (GIBCO), and 10 ml/l ITS+ Premix (Collaborative Research) (6.25 pg/ml insulin, 6.25 pg/ml transferrin, 6.25 pg/ml selenous acid, 1.25 mg/ml bovine serum albumin, and 5.35 mg/ml linoleic acid; final concentrations). Medium was replaced twice each week. Retrovirus infection of cells and isolationof JG cell line. Eighty percent suspensions of JG cells were infected with a retrovirus recombinant encoding a ts mutant of the SV40 large T antigen, as previously described (12). \k 2 (19) cells, which maintained the recombinant pZipSVtsa58 construct (l2), were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% (vol/vol) fetal calf serum plus penicillin (30 mg/l) and streptomycin (70 mg/l). Supernatants (48 h) from 90-100% confluent dishes of these \k 2 producer cells were then filtered through a 0.45-pm filter and used in infection experiments. Eighty percent JG cell suspensions used in infection studies were prepared as described above. Cells were then infected in suspension. They were incubated in a 2-ml viral supernatant,
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Fig. 1. Electron micrographs indicating the juxtaglomerular (JG) cell morphological characteristics used in their identification and in the development of the isolation procedure. This JG cell was prepared by centrifugation through a Percoll density gradient. Renin granules were distinguishable. Frame 1, X48,000; frame 2, a crystalline protogranule, x100,000.
which also contained 8 rg/ml polybrene (Aldrich Chemical), over a 3-h interval at 37”C, after which fresh medium was added such as to dilute the viral supernatant 15. Aliquots (2 ml) of this suspension were then plated on the Matrigel substrate as described above, to which was added a further 3 ml of fresh medium. After a 48-h interval, the medium was replaced with medium containing 250 pg/ml G418 (GIBCO), and the temperature at which cells were maintained was reduced to 33”C, to select for transformed cells. This medium was replaced once. Then, 1 wk later, the concentration of G418 used was increased to 1 mg/ml. Cells resistant to G418 continued to be maintained in medium containing 1 mg/ml G418, as described above, at 33”C, and have subsequently been passaged both by cloning techniques and by Dispase digestion, as described above. The process of cloning in these experiments involved the identification of either single or small groups of cells within the Matrigel substrate. One or a small number of cells was then collected in a volume of from 2 to 5 ~1, using an Eppendorf pipette and placed on a new Matrigel surface. Isolution of renin mRNA. Total RNA was isolated from freshly prepared JG cells, from primary cultures, and from transformed JG cells using the technique describedby Chomczynski and Sacchi (8). Slot-blot technology (Schleicher and Schuell) was then used to quantitate specific renin mRNA levels under the culture conditions described.Slot blots were hybridized (22) using a cDNA probe kindly provided by Dr. K. Lynch, Charlottesville, VA (6). After hybridization, blots were washedthree or four times in 2~ SSC/O.l% sodium dodecyl sulfate (SDS) at 68°Cfor 20-30 min, and once in 2~ SSC/O.l% SDS at 68°C for 30 min. Measurement of renin activity. Renin activity measurements were madeenzymatically by measuringrates of angiotensin I (ANG I) generated in the presence of angiotensinogen, as previously described(1). RESULTS
If highly enriched JG cell suspensions were maintained on plastic, cells died. After 7-10 days, only a very small
proportion remained. If, on the other hand, cells were recovered, subsequently plated on a reconstituted basement membrane preparation, and maintained in defined medium, very different growth characteristics were manifest (Fig. 2). After 2 wk, single cells had established cellcell attachments to form complex circular three-dimensional structures, which grew over 8-wk periods into networks of cells. The development of such structures was dependent on the JG cell content of the cell suspension plated. If 4050% JG cell suspensions were plated, no such structures were generated. To further characterize the state of differentiation of these cells over increasing times in culture, levels of specific renin mRNA were measured (Fig. 3). Levels clearly were sustained, an observation consistent with the maintenance of the rate of transcription of the renin gene, over 8-wk culture periods. It is also possible though that the degradation rate of specific renin mRNA is reduced, an alternative which needs to be checked. After 12 wk in primary culture, specific renin mRNA continued to be expressed. In addition, levels were manipulatable, using classic techniques with which rates of renin release are changed (9) by changing intracellular adenosine 3’,5’-cyclic monophosphate (CAMP) concentrations and free cytosolic levels of Ca2+. Cells were incubated for 2 h at 37”C, in control medium, medium containing lo-* M 8-(p-chlorophenylthio)cAMP [a permeable and nonmetabolizable form of CAMP (ll)], or medium containing low6 M ionomycin [a selective Ca2+ ionophore (18)]. High intracellular CAMP levels stimulated and high free cytosolic Ca2+ levels inhibited specific renin mRNA expression (Table 1). Thus, not only do the CAMP and Ca2+ second messenger systems regulate renin release, but we now demonstrate that they also regulate renin mRNA expression in directions that promote the respective release responses.
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Fig. 2. Growth of cells on a reconstituted basement membrane/8-wk (phase micrographs). Cells grown on plastic over 48 h (80% JG cell suspensions) remained a suspension of single cells together with some small clumps. A: x30. After 2-wk growth of these suspensions on Matrigel, multicellular structures began to develop. B: X24. C: X48. These structures continued to develop over 4-wk periods (D X24) and also after 8-wk periods (E: X24). Here a series of frames is included to demonstrate the complex three-dimensional structures generated.
JG cells in primary culture also secrete active renin into the incubation medium. An activity of 16.1 f 1.5 (SE) ng ANG I generated. h-l. ml-l (n = 2 preparations, 6 plates) was measured 4 days after plating. After 2 wk the levels decreased to 0.6 + 0.2 ng ANG 1. h-l. ml-l (n = 2 preparations, 5 plates) and were undetectable after 3 wk. If, however, cells were passaged, levels of renin secretion into the medium again approached levels ob-
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served soon after plating (Fig. 4A), 10.6 f 0.6 ng ANG I. h-‘*ml-i (n = 2 preparations, 6 plates). With continuous passaging of these primary cultures, however, peak levels decreased. We have not yet determined the effect of frequency of passaging on peak levels of secretion in these cultures. This successful establishment of primary cultures then led us to develop a continuous JG cell line by infection of JG cell suspensions with a retrovirus recombinant encoding a ts mutant of the SV40 large T antigen (12), which has previously been used successfully, in the immortalization of rodent embryonic fibroblasts (12) and renal epithelial (rabbit intercalated) cells (10). The temperature sensitivity of the expression of this gene provides the investigator with a tool with which one can artificially manipulate the growth rate and the state of differentiation of transformed cells. Incubation of transformed cells at the nonpermissive temperature (40°C) suppresses the expression of the large T antigen and promotes the expression of endogenous genes. JG cells transformed with this recombinant gene have now been maintained in culture, under the same conditions used for the maintenance of primary cultures, for more than 1 yr. They have been passaged by cloning 20 times and appear to sustain a highly differentiated state. High levels of specific renin mRNA are expressed. At 40°C expression of renin mRNA is enhanced; preliminary results indicate a 50% increase over a 24-h period. Expression after longer intervals has not yet been measured. In addition, the characteristics of the secretion of active renin are similar to those in primary cultures. Again, active renin levels secreted by transformed JG cells fall to undetectable levels -3 wk after plating. Levels are raised when cells are passaged. With continuous passaging though, in contrast to the primary cultures, peak levels of active renin secretion increase (Fig. 4B). Within 4 days of passaging, a level of 9.4 + 0.5 ng ANG I. h-l. ml-l (n = 4 passages, 13 plates) was measured. After between 1 and 2 wk postpassaging, the level dropped to 2.1 -C 0.6 ng ANG 1. h-l. ml-’ (n = 4 passages, 15 plates), and after 3 wk the level was further reduced to 0.8 + 0.4 ng ANG I. h-l *ml-’ (n = 3 passages, 9 plates). On passaging, the level was raised to one of 14.0 f 0.3 ng ANG I. h-l. ml-’ (n = 2 passages, 5 plates). The renin activity of the plasma rat pool, over the course of these renin activity measurements, remained at 13.5 + 0.1 ng ANG I. h-l. ml-‘. DISCUSSION
Our success in maintaining JG cells on a reconstituted basement membrane preparation confirms the importance of structural considerations in the maintenance of JG cells in culture. These JG cells have not established a monolayer, however, but rather a complex threedimensional cell network (Fig. 2). Cells appear to grow through the substrate and also in a number of layers on its surface. The topmost layers apparently grow without direct contact with the reconstituted basement membrane preparation. Thus these cultured cells may now be synthesizing endogenous basement membrane compo-
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40-50% supension
90-90% supension 28s -
18s -
matrigel - 4 weeks
Fig. 3. Slot-blot measurements of renin mRNA over increasing incubation times on Matrigel. Lane 1: slot blot indicating relative recovery of renin mRNA over increasing culture intervals; 10 ag of total RNA were applied to each slot. Lanes 2 and 3 indicate duplicate and triplicate samples, respectively. At no time over the course of these culture intervals were the cells confluent. A Northern blot, using the rat renin cDNA probe described above (6), and 20 pg total RNA prepared from kidney cortex were also indicated.
m&rig& _ 8 weeks
tnsf - 4 weeks
Table
1. Renin: 18s autoradiographic density ratios in 12-wk primary cultures Treatment
Density
Ratios
n
P Value
0.52kO.12 5 Control 2.67kO.36 3 0.05 ClPheS-CAMP (10e4 M) 0.16+0.06 3 0.05 Ionomycin/l.8 mM Ca2+ ( 1O-6 M) Kidneys of 4 rats were used in the initial preparation of cells cultured, n values reflect the number of slots used in the calculation of relative densities. ClPheS-CAMP, 8-(p-chlorophenylthio)adenosine 3’,5’-cyclic monophosphate. After 12-wk in culture, juxtaglomerular cell-specific renin mRNA levels could be manipulated by changing intracellular CAMP concentrations and free cytosolic Ca*+ levels. RNA was extracted (8) from cells which manifest elevated intracellular CAMP levels, or alternatively, elevated free cytosolic Ca2+ levels, and specific renin mRNA levels were estimated using slot-blot technology, a rat renin cDNA probe (6), and an 18s rRNA probe (20), which was used as a quantitative control. P values were determined by unpaired Student’s t test.
nents, as has been described in mammary epithelial cells (25), such as to promote cell-superstratum/substratum associations which in turn establish “in vivo-like” threedimensional structures in daughter cells. The observation that growth of JG cells on a reconstituted basement membrane preparation also promotes the maintenance of specific renin mRNA levels, over extensive culture periods, points to the importance of in vivolike three dimensional structure in the maintenance of JG cell-specific gene expression. Cellular three-dimensional structure in turn defines the three-dimensional structure of the nuclear matrix (3), which is thought to play a crucial role in the maintenance of cell-specific regulation of gene transcription. There is some suggestion of a requisite association between the nuclear matrix and all active chromatin regions (24, 26). In addition, the nuclear matrix has been implicated in a number of important nuclear processes including DNA replication
and hnRNA processing (4, 27, 28). The results derived from the transformed JG cells also confirm the importance of cell-substrate associations in the maintenance of JG cells in culture. The changes in rate of active renin secretion elicited by passaging (Fig. 4) remain to be defined mechanistically. However, it seems possible that cell-specific threedimensional structure may be even more important with respect to the processing of specific renin mRNA than it seems to be with respect to the maintenance of renin mRNA levels. If this were the case, then one might postulate that despite the maintenance of specific renin mRNA levels over long incubation periods on Matrigel, the substrate may not be optimal for the maintenance of renin mRNA processing and/or processing of the immature peptide generated. Thus, with passaging, structural constraints might be reduced such that mRNA processing and renin secretion rates are restored more closely to baseline levels. Subsequently, with replating of cells, the restoration of structural constraints would again reduce processing of the specific renin mRNA. Thus, if postpassaging reduction in active renin secretion is explained by the fact that the cells are grown on a suboptimal substrate, then alternative substrates will need to be investigated. Once the substrate matrix is optimized, however, the three systems of JG cells described, freshly prepared JG cells, primary cultures, and transformed JG cells, will provide us with the tools with which to investigate some of the basic cellular mechanisms that contribute to the regulation of the synthesis and release of renin. The concurrent use of the three systems will provide a number of in-built controls with which to establish artifacts attributable to either culture conditions and/or transformation. In both primary cultures and transformed cells, transcription of the renin gene is sustained (Fig. 3), and active renin is secreted into the incubation medium. In addition, second messenger systems that contribute to the regulation of renin
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Address for reprint requests: B. M. Rayson, Cornell University Medical College, 1300 York 10021. ’
A
Received
16 July
1991; accepted
in final
form
Dept. of Physiology, Ave., New York, NY 28 October
1991.
16
REFERENCES 4
4
4
weeks 16
eB
2
4
6
Fig. 4. Levels of renin activity detected in incubation medium after increasing times postpassaging. Arrows indicate points at which cells were passaged. Renin secretion by both primary cultures (A) and transformed cells (B) are indicated. Primary culture data represent that generated by 2 preparations, 6 plates. The 2 preparations are represented by the 2 symbols (*, 0). Transformed cell data represent that generated by 2 passages, 4 or 5 plates. The 2 passages are also represented by distinct symbols (a, 0).
release in vivo regulate specific renin mRNA levels in primary cultures, such as to promote the well-described effects on release. Thus JG cell responses to a spectrum of physiological and pathophysiological signals can now be addressed to promote some resolution of the cellular mechanisms implicated in the subpopulations of hypertensive patients which manifest renin dependency. I thank Dr. J. H. Laragh and Dr. J. Sealey for support and for renin activity measurements. I also thank Dr. W. Henrich, Southwestern Medical School, Dallas, TX for assistance with the JG cell isolation procedure, and Drs. P. Jat (Ludwig Institute Cancer Research, UK) and P. Sharp (Massachusetts Institute of Technology) who kindly provided the \k 2 recombinant virus producer cells used in the infection studies. Finally, I thank Dr. J. Gulati for assistance in the preparation of this manuscript. This work was supported by National Institutes of Health Grants DK33352 and HL18323.
1. Atlas, S. A., J. E. Sealey, T. E. Hesson, A. P. Caplan, J. Menard, P. Corvol, and J. H. Laragh. Biochemical similarity of partially purified inactive renins from human plasma and kidney. Hypertension Dallas 5, Suppl. II: 86-95, 1982. 2. Barajas, L. The development and ultrastructure of the juxtaglomerular cell granule. J. Ultrastruct. Res. 15: 400-413, 1965. 3. Berezney, R., and D. S. Coffey. Identification of a nuclear protein matrix. Biochem. Biophys. Res. Commun. 60: 1410-1417, 1974. 4. Berezney, R., and D. S. Coffey. Nuclear protein matrix: association with newly synthesized DNA. Science Wash. DC 189: 291-293,1975. 5. Bissell, M. J. The differentiated state of normal and malignant cells or how to define a “normal” cell in culture. Int. Reu. Cytol. 70: 27-100,198l. 6. Burnham, C. E., C. L. Hawelu-Johnson, B. M. Frank, and K. R. Lynch. Molecular cloning of rat renin cDNA and its gene. Proc. Natl. Acad. Sci. USA 84: 5605-5609, 1987. 7. Chandra, S., J. Castlema Hubbard, F. Skelton, L. Bernadis, and S. Kamura. Genesis of juxtaglomerular cell granules. Lab. Inuest. 14:1834-1847,1965. 8. Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987. 9. Churchill, P. C. Second messengers in renin secretion. Am. J. Physiol. 249 (Renal FZuid Electrolyte Physiol. 18): F175-F184,1985. 10. Edwards, J. C., M. Rater, and Q. Al-Awqati. Immortalization of rabbit cortical intercalated cells (Abstract). Proc. Am. Sot. Nephrol. 43A, 1989. 11. Hall, D. A., L. D. Barnes, and T. P. Dousa. Cyclic AMP in action of antidiuretic hormone: effects of exogenous cyclic AMP and its new analogue. Am. J. Physiol. 232 (Renal Fluid Electrolyte Physiol. 1): F368-F376, 1977. 12. Jat, P. S., and P. A. Sharp. Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the non-permissive temperature. Mol. Cell. Biol. 9: 1672~1681,1989. 13. Keeton, T. K., and W. B. Campbell. The pharmacologic alteration of renin release. Pharmacol. Reu. 31: 82-227, 1981. 14. Kurtz, A., R. Della Bruna, J. Pfeilshifter, R. Taugner, and C. Bauer. Atria1 natriuretic peptide inhibits renin release from juxtaglomerular cells by a cGMP-mediated process. Proc. NatZ. Acad. Sci. USA 83: 4769-4773, 1986. 15. Laragh, J. H., and J. E. Sealey. The renin-angiotensin-aldosterone hormonal system and regulation of sodium, potassium, and blood pressure. In: Handbook of Physiology. Renal Physiology. Bethesda, MD: Am. Physiol. Sot., 1973, sect. 8, chapt. 26, p. 831-908. 16. Laragh, J. H., J. E. Sealey, A. P. Niarchos, and T. G. Pickering. The vasoconstriction-volume spectrum in normotension and pathogenesis of hypertension. Federation Proc. 41: 24152423,1982. 17. Latta, H., and A. B. Maunsbach. The juxtaglomerular apparatus as studied electron microscopically. J. Ultrastruct. Res. 6: 5471962. 18. Liu, C. M., and T. E. Herman. Characterization of ionomycin as a calcium ionophore. J. Biol. Chem. 253: 5892-5894, 1978. 19. Mann, R., R. C. Mulligan, and D. Baltimore. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 33: 153-159, 1983. 20. Paynton, B. V., R. Rempel, and R. Bachvarova. Changes in state of adenylation and time course of degradation of maternal mRNAs during oocyte maturation and early embryonic development in the mouse. Deu. Biol. 129: 304-314,1988. 21. Ruyter, J. H. C. Uber einen merkwurdigen Abschnitt der Vasa afferentia in der Mauseniere. 2. Zellforsch. 2: 242-248, 1925. 22. Sambrook, J., E. F. Fritsch, and T. Maniatis. Molecular
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Cloniw. Cold Spring Harbor, NY: Cold Spring Harbor, 1989, p. 7. 53-7.57. 23. Sealey, J. E., J. D. Blumenfeld, G. M. S. C. Sommers, and J. H. Laragh. J. 1988. 24. Small, D., B. Nelkin, and B. Vogelstein.
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The association of transcribed genes with the nuclear matrix of Drosophila cells during heat shock. Nucleic Acids Res. 13: 2413-2431,1988. 25. Streuli, C. H., and M. J. Bissell. Expression of extracellular matrix components is regulated by substratum. J. Cell BioZ. 110:
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A., A. Moore, and J. Knowland. Attachment of transcriptionally active DNA sequences to the nucleoskeleton under isotonic conditions (Abstract). Nucleic Acids Res. 16: 7183,
26. Thorburn,
1988. 27. Zeitlin,
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and
A. Efstratiadis.
Pre-mRNA splicing and the nuclear matrix. 1Mol. Cell. Bill. 7: 1 ll120,1987. 28. Zeitlin,
S., R. C. Wilson, and A. Efstratiadis. Autonomous splicing and complementation of in vivo-assembled spliceosomes. J. CeZZBiol. 108: 765-777, 1989.
1405-1416,199O.
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renin; hypertension;
rats; extracellular
matrix
DISEASE and hypertension are major causes of death in Western societies, and the diseases appear to be of diverse etiology. Significant subpopulations of hypertensive patients have, however, been identified in which inappropriately high circulating renin levels maintain elevated levels of blood pressure (15, 16,
CARDIOVASCULAR
23)
Circulating renin levels are primarily regulated by release from juxtaglomerular (JG) cells, first identified in 1925 (21), in which renin is synthesized and stored (15, 16, 23). The physiological stimuli that regulate renin release from these cells have been well characterized (13, 15). Because these cells have not previously been cultured though, less is known about the intracellular mechanisms implicated in the regulation of rates of synthesis and release, because in vivo it is difficult both to experimentally manipulate JG cell second messenger systems and to quantify specific JG cell responses. JG cells constitute at most O.Ol-0.1% of the renal cortical cell population. We therefore maintained JG cells on a reconstituted basement membrane (Matrigel; Collaborative Research). This development was made possible by, first, the previous publication of a technique with which to generate suspensions of cells highly enriched in JG ceils (14) and, second, the identification of the importance of cell-substrate interactions and cell three-dimensional structures in the maintenance of tissue-specific gene expression in culture (5).’ Cells were grown on a reconstituted basement membrane preparation in an attempt to mimic their in vivo substrate, with the aim of also generating cell three-dimensional structures similar to those in vivo. ’ The substrate on which cells are grown appears to be an important determinant of cell and cytoskeletal/nuclear matrix structure. Cellsubstrate contact is maintained through specific cellular receptors which bind to defined substrate ligands. The density, the distribution, and the tensile properties of these interactions then, together with those of the intercellular associations, determine the three-dimensional structure of the cell and the cytoskeletal-nuclear matrices. 0363-6143/92
$2.00 Copyright
The technique was effective in sustaining the differentiated state of JG cells, over extensive periods of time, in culture. METHODS Generation of JG cell cultures. Highly enriched preparations of JG cells were generated by collagenase digestion of rat renal cortices and density gradient centrifugation, as previously described (14). Two to four rats (200-300 g) were used. Forty to fifty percent of the cells recovered between the densities of 1.051 and 1.064 g/ml were identifiable, on the basis of morphological criteria, as JG cells (Fig. 1): granules at three different stages in their development, a large number of mitochondria, large nuclei, and well-developed endoplasmic reticular and Golgi networks (2, 7,17). Protogranules manifest distinct crystalline structure as previously described (2). Subsequently, this enriched preparation of JG cells was plated in high density (5 ml, l-7 x lo6 cells/ml) in plastic culture dishes (Falcon, 60 x 15 mm), to which JG cells preferentially adhere, over 48-h intervals. Cells were recovered by incubation in the neutral protease preparation Dispase (Collaborative Research) over 2h incubations at 37°C. Subsequently, they were washed in fresh medium at least three times to remove all Dispase contamination. Generally, only lo-20% of the cells originally plated on plastic were recovered with Dispase treatment. The suspension generated was then characterized using immunohistochemistry. As indicated in the original description of the technique used (14), at least 80% of the cells recovered stained positively after treatment with a rat polyclonal antiserum generated against mouse renin. Subsequently, this suspension, highly enriched in JG cells, was plated on the commercially available basement membrane preparation Matrigel (2.5 ml,/60 X 15 mm dish, Collaborative Research) and maintained in RPM1 1640 (GIBCO) to which was added 25-mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (United States Biochemicals), 2 pg/ml corticosterone (Sigma), 50 U/ml penicillin (GIBCO), 70 pg/ml streptomycin (GIBCO), and 10 ml/l ITS+ Premix (Collaborative Research) (6.25 pg/ml insulin, 6.25 pg/ml transferrin, 6.25 pg/ml selenous acid, 1.25 mg/ml bovine serum albumin, and 5.35 mg/ml linoleic acid; final concentrations). Medium was replaced twice each week. Retrovirus infection of cells and isolationof JG cell line. Eighty percent suspensions of JG cells were infected with a retrovirus recombinant encoding a ts mutant of the SV40 large T antigen, as previously described (12). \k 2 (19) cells, which maintained the recombinant pZipSVtsa58 construct (l2), were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% (vol/vol) fetal calf serum plus penicillin (30 mg/l) and streptomycin (70 mg/l). Supernatants (48 h) from 90-100% confluent dishes of these \k 2 producer cells were then filtered through a 0.45-pm filter and used in infection experiments. Eighty percent JG cell suspensions used in infection studies were prepared as described above. Cells were then infected in suspension. They were incubated in a 2-ml viral supernatant,
0 1992 the American
Physiological
Society
C563
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Fig. 1. Electron micrographs indicating the juxtaglomerular (JG) cell morphological characteristics used in their identification and in the development of the isolation procedure. This JG cell was prepared by centrifugation through a Percoll density gradient. Renin granules were distinguishable. Frame 1, X48,000; frame 2, a crystalline protogranule, x100,000.
which also contained 8 rg/ml polybrene (Aldrich Chemical), over a 3-h interval at 37”C, after which fresh medium was added such as to dilute the viral supernatant 15. Aliquots (2 ml) of this suspension were then plated on the Matrigel substrate as described above, to which was added a further 3 ml of fresh medium. After a 48-h interval, the medium was replaced with medium containing 250 pg/ml G418 (GIBCO), and the temperature at which cells were maintained was reduced to 33”C, to select for transformed cells. This medium was replaced once. Then, 1 wk later, the concentration of G418 used was increased to 1 mg/ml. Cells resistant to G418 continued to be maintained in medium containing 1 mg/ml G418, as described above, at 33”C, and have subsequently been passaged both by cloning techniques and by Dispase digestion, as described above. The process of cloning in these experiments involved the identification of either single or small groups of cells within the Matrigel substrate. One or a small number of cells was then collected in a volume of from 2 to 5 ~1, using an Eppendorf pipette and placed on a new Matrigel surface. Isolution of renin mRNA. Total RNA was isolated from freshly prepared JG cells, from primary cultures, and from transformed JG cells using the technique describedby Chomczynski and Sacchi (8). Slot-blot technology (Schleicher and Schuell) was then used to quantitate specific renin mRNA levels under the culture conditions described.Slot blots were hybridized (22) using a cDNA probe kindly provided by Dr. K. Lynch, Charlottesville, VA (6). After hybridization, blots were washedthree or four times in 2~ SSC/O.l% sodium dodecyl sulfate (SDS) at 68°Cfor 20-30 min, and once in 2~ SSC/O.l% SDS at 68°C for 30 min. Measurement of renin activity. Renin activity measurements were madeenzymatically by measuringrates of angiotensin I (ANG I) generated in the presence of angiotensinogen, as previously described(1). RESULTS
If highly enriched JG cell suspensions were maintained on plastic, cells died. After 7-10 days, only a very small
proportion remained. If, on the other hand, cells were recovered, subsequently plated on a reconstituted basement membrane preparation, and maintained in defined medium, very different growth characteristics were manifest (Fig. 2). After 2 wk, single cells had established cellcell attachments to form complex circular three-dimensional structures, which grew over 8-wk periods into networks of cells. The development of such structures was dependent on the JG cell content of the cell suspension plated. If 4050% JG cell suspensions were plated, no such structures were generated. To further characterize the state of differentiation of these cells over increasing times in culture, levels of specific renin mRNA were measured (Fig. 3). Levels clearly were sustained, an observation consistent with the maintenance of the rate of transcription of the renin gene, over 8-wk culture periods. It is also possible though that the degradation rate of specific renin mRNA is reduced, an alternative which needs to be checked. After 12 wk in primary culture, specific renin mRNA continued to be expressed. In addition, levels were manipulatable, using classic techniques with which rates of renin release are changed (9) by changing intracellular adenosine 3’,5’-cyclic monophosphate (CAMP) concentrations and free cytosolic levels of Ca2+. Cells were incubated for 2 h at 37”C, in control medium, medium containing lo-* M 8-(p-chlorophenylthio)cAMP [a permeable and nonmetabolizable form of CAMP (ll)], or medium containing low6 M ionomycin [a selective Ca2+ ionophore (18)]. High intracellular CAMP levels stimulated and high free cytosolic Ca2+ levels inhibited specific renin mRNA expression (Table 1). Thus, not only do the CAMP and Ca2+ second messenger systems regulate renin release, but we now demonstrate that they also regulate renin mRNA expression in directions that promote the respective release responses.
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Fig. 2. Growth of cells on a reconstituted basement membrane/8-wk (phase micrographs). Cells grown on plastic over 48 h (80% JG cell suspensions) remained a suspension of single cells together with some small clumps. A: x30. After 2-wk growth of these suspensions on Matrigel, multicellular structures began to develop. B: X24. C: X48. These structures continued to develop over 4-wk periods (D X24) and also after 8-wk periods (E: X24). Here a series of frames is included to demonstrate the complex three-dimensional structures generated.
JG cells in primary culture also secrete active renin into the incubation medium. An activity of 16.1 f 1.5 (SE) ng ANG I generated. h-l. ml-l (n = 2 preparations, 6 plates) was measured 4 days after plating. After 2 wk the levels decreased to 0.6 + 0.2 ng ANG 1. h-l. ml-l (n = 2 preparations, 5 plates) and were undetectable after 3 wk. If, however, cells were passaged, levels of renin secretion into the medium again approached levels ob-
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served soon after plating (Fig. 4A), 10.6 f 0.6 ng ANG I. h-‘*ml-i (n = 2 preparations, 6 plates). With continuous passaging of these primary cultures, however, peak levels decreased. We have not yet determined the effect of frequency of passaging on peak levels of secretion in these cultures. This successful establishment of primary cultures then led us to develop a continuous JG cell line by infection of JG cell suspensions with a retrovirus recombinant encoding a ts mutant of the SV40 large T antigen (12), which has previously been used successfully, in the immortalization of rodent embryonic fibroblasts (12) and renal epithelial (rabbit intercalated) cells (10). The temperature sensitivity of the expression of this gene provides the investigator with a tool with which one can artificially manipulate the growth rate and the state of differentiation of transformed cells. Incubation of transformed cells at the nonpermissive temperature (40°C) suppresses the expression of the large T antigen and promotes the expression of endogenous genes. JG cells transformed with this recombinant gene have now been maintained in culture, under the same conditions used for the maintenance of primary cultures, for more than 1 yr. They have been passaged by cloning 20 times and appear to sustain a highly differentiated state. High levels of specific renin mRNA are expressed. At 40°C expression of renin mRNA is enhanced; preliminary results indicate a 50% increase over a 24-h period. Expression after longer intervals has not yet been measured. In addition, the characteristics of the secretion of active renin are similar to those in primary cultures. Again, active renin levels secreted by transformed JG cells fall to undetectable levels -3 wk after plating. Levels are raised when cells are passaged. With continuous passaging though, in contrast to the primary cultures, peak levels of active renin secretion increase (Fig. 4B). Within 4 days of passaging, a level of 9.4 + 0.5 ng ANG I. h-l. ml-l (n = 4 passages, 13 plates) was measured. After between 1 and 2 wk postpassaging, the level dropped to 2.1 -C 0.6 ng ANG 1. h-l. ml-’ (n = 4 passages, 15 plates), and after 3 wk the level was further reduced to 0.8 + 0.4 ng ANG I. h-l *ml-’ (n = 3 passages, 9 plates). On passaging, the level was raised to one of 14.0 f 0.3 ng ANG I. h-l. ml-’ (n = 2 passages, 5 plates). The renin activity of the plasma rat pool, over the course of these renin activity measurements, remained at 13.5 + 0.1 ng ANG I. h-l. ml-‘. DISCUSSION
Our success in maintaining JG cells on a reconstituted basement membrane preparation confirms the importance of structural considerations in the maintenance of JG cells in culture. These JG cells have not established a monolayer, however, but rather a complex threedimensional cell network (Fig. 2). Cells appear to grow through the substrate and also in a number of layers on its surface. The topmost layers apparently grow without direct contact with the reconstituted basement membrane preparation. Thus these cultured cells may now be synthesizing endogenous basement membrane compo-
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40-50% supension
90-90% supension 28s -
18s -
matrigel - 4 weeks
Fig. 3. Slot-blot measurements of renin mRNA over increasing incubation times on Matrigel. Lane 1: slot blot indicating relative recovery of renin mRNA over increasing culture intervals; 10 ag of total RNA were applied to each slot. Lanes 2 and 3 indicate duplicate and triplicate samples, respectively. At no time over the course of these culture intervals were the cells confluent. A Northern blot, using the rat renin cDNA probe described above (6), and 20 pg total RNA prepared from kidney cortex were also indicated.
m&rig& _ 8 weeks
tnsf - 4 weeks
Table
1. Renin: 18s autoradiographic density ratios in 12-wk primary cultures Treatment
Density
Ratios
n
P Value
0.52kO.12 5 Control 2.67kO.36 3 0.05 ClPheS-CAMP (10e4 M) 0.16+0.06 3 0.05 Ionomycin/l.8 mM Ca2+ ( 1O-6 M) Kidneys of 4 rats were used in the initial preparation of cells cultured, n values reflect the number of slots used in the calculation of relative densities. ClPheS-CAMP, 8-(p-chlorophenylthio)adenosine 3’,5’-cyclic monophosphate. After 12-wk in culture, juxtaglomerular cell-specific renin mRNA levels could be manipulated by changing intracellular CAMP concentrations and free cytosolic Ca*+ levels. RNA was extracted (8) from cells which manifest elevated intracellular CAMP levels, or alternatively, elevated free cytosolic Ca2+ levels, and specific renin mRNA levels were estimated using slot-blot technology, a rat renin cDNA probe (6), and an 18s rRNA probe (20), which was used as a quantitative control. P values were determined by unpaired Student’s t test.
nents, as has been described in mammary epithelial cells (25), such as to promote cell-superstratum/substratum associations which in turn establish “in vivo-like” threedimensional structures in daughter cells. The observation that growth of JG cells on a reconstituted basement membrane preparation also promotes the maintenance of specific renin mRNA levels, over extensive culture periods, points to the importance of in vivolike three dimensional structure in the maintenance of JG cell-specific gene expression. Cellular three-dimensional structure in turn defines the three-dimensional structure of the nuclear matrix (3), which is thought to play a crucial role in the maintenance of cell-specific regulation of gene transcription. There is some suggestion of a requisite association between the nuclear matrix and all active chromatin regions (24, 26). In addition, the nuclear matrix has been implicated in a number of important nuclear processes including DNA replication
and hnRNA processing (4, 27, 28). The results derived from the transformed JG cells also confirm the importance of cell-substrate associations in the maintenance of JG cells in culture. The changes in rate of active renin secretion elicited by passaging (Fig. 4) remain to be defined mechanistically. However, it seems possible that cell-specific threedimensional structure may be even more important with respect to the processing of specific renin mRNA than it seems to be with respect to the maintenance of renin mRNA levels. If this were the case, then one might postulate that despite the maintenance of specific renin mRNA levels over long incubation periods on Matrigel, the substrate may not be optimal for the maintenance of renin mRNA processing and/or processing of the immature peptide generated. Thus, with passaging, structural constraints might be reduced such that mRNA processing and renin secretion rates are restored more closely to baseline levels. Subsequently, with replating of cells, the restoration of structural constraints would again reduce processing of the specific renin mRNA. Thus, if postpassaging reduction in active renin secretion is explained by the fact that the cells are grown on a suboptimal substrate, then alternative substrates will need to be investigated. Once the substrate matrix is optimized, however, the three systems of JG cells described, freshly prepared JG cells, primary cultures, and transformed JG cells, will provide us with the tools with which to investigate some of the basic cellular mechanisms that contribute to the regulation of the synthesis and release of renin. The concurrent use of the three systems will provide a number of in-built controls with which to establish artifacts attributable to either culture conditions and/or transformation. In both primary cultures and transformed cells, transcription of the renin gene is sustained (Fig. 3), and active renin is secreted into the incubation medium. In addition, second messenger systems that contribute to the regulation of renin
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Address for reprint requests: B. M. Rayson, Cornell University Medical College, 1300 York 10021. ’
A
Received
16 July
1991; accepted
in final
form
Dept. of Physiology, Ave., New York, NY 28 October
1991.
16
REFERENCES 4
4
4
weeks 16
eB
2
4
6
Fig. 4. Levels of renin activity detected in incubation medium after increasing times postpassaging. Arrows indicate points at which cells were passaged. Renin secretion by both primary cultures (A) and transformed cells (B) are indicated. Primary culture data represent that generated by 2 preparations, 6 plates. The 2 preparations are represented by the 2 symbols (*, 0). Transformed cell data represent that generated by 2 passages, 4 or 5 plates. The 2 passages are also represented by distinct symbols (a, 0).
release in vivo regulate specific renin mRNA levels in primary cultures, such as to promote the well-described effects on release. Thus JG cell responses to a spectrum of physiological and pathophysiological signals can now be addressed to promote some resolution of the cellular mechanisms implicated in the subpopulations of hypertensive patients which manifest renin dependency. I thank Dr. J. H. Laragh and Dr. J. Sealey for support and for renin activity measurements. I also thank Dr. W. Henrich, Southwestern Medical School, Dallas, TX for assistance with the JG cell isolation procedure, and Drs. P. Jat (Ludwig Institute Cancer Research, UK) and P. Sharp (Massachusetts Institute of Technology) who kindly provided the \k 2 recombinant virus producer cells used in the infection studies. Finally, I thank Dr. J. Gulati for assistance in the preparation of this manuscript. This work was supported by National Institutes of Health Grants DK33352 and HL18323.
1. Atlas, S. A., J. E. Sealey, T. E. Hesson, A. P. Caplan, J. Menard, P. Corvol, and J. H. Laragh. Biochemical similarity of partially purified inactive renins from human plasma and kidney. Hypertension Dallas 5, Suppl. II: 86-95, 1982. 2. Barajas, L. The development and ultrastructure of the juxtaglomerular cell granule. J. Ultrastruct. Res. 15: 400-413, 1965. 3. Berezney, R., and D. S. Coffey. Identification of a nuclear protein matrix. Biochem. Biophys. Res. Commun. 60: 1410-1417, 1974. 4. Berezney, R., and D. S. Coffey. Nuclear protein matrix: association with newly synthesized DNA. Science Wash. DC 189: 291-293,1975. 5. Bissell, M. J. The differentiated state of normal and malignant cells or how to define a “normal” cell in culture. Int. Reu. Cytol. 70: 27-100,198l. 6. Burnham, C. E., C. L. Hawelu-Johnson, B. M. Frank, and K. R. Lynch. Molecular cloning of rat renin cDNA and its gene. Proc. Natl. Acad. Sci. USA 84: 5605-5609, 1987. 7. Chandra, S., J. Castlema Hubbard, F. Skelton, L. Bernadis, and S. Kamura. Genesis of juxtaglomerular cell granules. Lab. Inuest. 14:1834-1847,1965. 8. Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987. 9. Churchill, P. C. Second messengers in renin secretion. Am. J. Physiol. 249 (Renal FZuid Electrolyte Physiol. 18): F175-F184,1985. 10. Edwards, J. C., M. Rater, and Q. Al-Awqati. Immortalization of rabbit cortical intercalated cells (Abstract). Proc. Am. Sot. Nephrol. 43A, 1989. 11. Hall, D. A., L. D. Barnes, and T. P. Dousa. Cyclic AMP in action of antidiuretic hormone: effects of exogenous cyclic AMP and its new analogue. Am. J. Physiol. 232 (Renal Fluid Electrolyte Physiol. 1): F368-F376, 1977. 12. Jat, P. S., and P. A. Sharp. Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the non-permissive temperature. Mol. Cell. Biol. 9: 1672~1681,1989. 13. Keeton, T. K., and W. B. Campbell. The pharmacologic alteration of renin release. Pharmacol. Reu. 31: 82-227, 1981. 14. Kurtz, A., R. Della Bruna, J. Pfeilshifter, R. Taugner, and C. Bauer. Atria1 natriuretic peptide inhibits renin release from juxtaglomerular cells by a cGMP-mediated process. Proc. NatZ. Acad. Sci. USA 83: 4769-4773, 1986. 15. Laragh, J. H., and J. E. Sealey. The renin-angiotensin-aldosterone hormonal system and regulation of sodium, potassium, and blood pressure. In: Handbook of Physiology. Renal Physiology. Bethesda, MD: Am. Physiol. Sot., 1973, sect. 8, chapt. 26, p. 831-908. 16. Laragh, J. H., J. E. Sealey, A. P. Niarchos, and T. G. Pickering. The vasoconstriction-volume spectrum in normotension and pathogenesis of hypertension. Federation Proc. 41: 24152423,1982. 17. Latta, H., and A. B. Maunsbach. The juxtaglomerular apparatus as studied electron microscopically. J. Ultrastruct. Res. 6: 5471962. 18. Liu, C. M., and T. E. Herman. Characterization of ionomycin as a calcium ionophore. J. Biol. Chem. 253: 5892-5894, 1978. 19. Mann, R., R. C. Mulligan, and D. Baltimore. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 33: 153-159, 1983. 20. Paynton, B. V., R. Rempel, and R. Bachvarova. Changes in state of adenylation and time course of degradation of maternal mRNAs during oocyte maturation and early embryonic development in the mouse. Deu. Biol. 129: 304-314,1988. 21. Ruyter, J. H. C. Uber einen merkwurdigen Abschnitt der Vasa afferentia in der Mauseniere. 2. Zellforsch. 2: 242-248, 1925. 22. Sambrook, J., E. F. Fritsch, and T. Maniatis. Molecular
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