Sites of renin production in fetal neonatal and postnatal syrian hamster kidneys.код для вставкиСкачать
THE ANATOMICAL RECORD 235:144-150 (1993) Sites of Renin Production in Fetal, Neonatal, and Postnatal Syrian Hamster Kidneys ALICE H. DODGE Department Basic Sciences-Anatomical Science, California College of Podiatric Medicine, S a n Francisco, California 941 15 ABSTRACT Renin is first observed in the 14-dayfetal kidney. There is a sharp increase in the number of renin positive cells in the 15-day fetal kidney. Renin is located in the smooth muscle cells of arterioles, interlobular arteries, and branches of the renal artery. In the neonatal kidney, the amount of renin appears to be equal to that observed in the 15-day fetal kidney and is still located in the same blood vessels. In the 24-hour postnatal kidney, there is a sharp decrease in the total amount of renin. Renin positive cells are now observed at the vascular pole. In the 48-hour postnatal kidney, there is a sharp increase in the total amount of renin. Most of the renin positive cells are located at the vascular poles; however, a few renin positive cells are seen in the interlobular arteries. 0 1993 Wiley-Liss, Inc. Key words: Hamster, Kidney, Immunohistochemistry,Renin, Fetal, Neonatal, Postnatal Renin is a small protolytic enzyme produced and stored as prorenin by vascular smooth muscle. Renin is produced primarily by the smooth muscle cells of the juxtaglomerular complex of the adult kidney. Active renin when released into the blood splits angiotensin from renin substrate. Angiotensin displays a vasoconstrictor action on small blood vessels. The renin-angiotensin-vasoconstriction system is activated in a matter of minutes (Guyton, 1991). Renin production has been reported to begin in fetuses in several animal species (guinea pigs-Raimbach and Thomas, 1990; rats-Gomez et al., 1989; sheep-Brace and Cheung, 1986; humans-Brar et al., 1985, 1987; Symonds et al., 1985; Gomez et al., 1991). In guinea pigs, the fetal plasma renin concentration is higher near term than the circulating renin level in the adult male guinea pig (Raimbach and Thomas, 1990). In rats, renin mRNA levels are higher in fetal and newborn kidneys than in adult kidneys. The fetal mRNA is found in the vascular poles, afferent arterioles, interlobular arteries, and arcuate arteries (Gomez et al., 1989). In pregnant women, circulating fetal renin levels have been found to be higher than maternal levels in some cases of pregnancy-induced hypertension (Brar et al., 1987). In other humans, fetal kidney renin activity is greater than adult kidney renin (Gomez et al., 1991). A recent study demonstrated that circulating renin levels were elevated when the host animal was treated with the synthetic estrogen diethylstilbestrol (DES). DES treatment of adult male hamsters resulted in the production of renin by nonjuxtaglomerular renal vascular smooth muscle cells. Renin positive cells were noted in renal artery branches, interlobular arteries, and veins (Dodge et al., 1990). 0 1993 WILEY-LISS, INC The purpose of the present study was to determine when fetal hamster kidneys produce renin and what cells produced it first. Another purpose was t o determine if renin production in fetal kidneys would parallel renin production in DES-treated adult kidneys. MATERIALS AND METHODS The fetal, neonatal, and postnatal Syrian hamsters used in this study were obtained from the Dodge hamster colony. The maintenance of this colony has been described previously (Dodge et al., 1988). The day and time of all hamster breedings were recorded. Fetal day one was calculated to have begun 24 hours after the hamsters were mated. Subsequent fetal days were calculated a t 24-hour intervals. The gestation period for the hamsters in the Dodge colony ranged from 16 to 17 days. Fetal kidneys were collected from fetuses at the beginning of 13, 14, and 15 days. Neonatal kidneys were collected from newborns, and postnatal kidneys were collected from 24-hour postnatal and 48-hour postnatal hamsters. A total of 27 kidneys were obtained and investigated. For each age group, the kidneys were subjected to light microscopic and electron microscopic procedures. There were six 13-day fetal kidneys, six 14-day fetal kidneys, five 15-day fetal kidneys, four neonatal kidneys, four 24-hour postnatal kidneys, and three 48-hour postnatal kidneys. Collection of Fetal Kidneys The hamster mothers were sacrificed by means of intrathoracic doses of sodium pentobarbital. The uteri Received January 24, 1992; accepted June 1, 1992 HAMSTER RENIN PRODUCTION Fig. 1 , A 7-km section of a 13-day fetal kidney reacted with a n antibody to rat kidney renin. No renin positive cells are observed, x 382. Fig, 2.A 7-km section of a 14-day fetal kidney reacted with an antibody to rat kidney renin. A few renin positive cells are observed (open arrowhead), x 489. 145 146 A.H. DODGE were removed and rinsed in phosphate-buffered saline (PBS). The fetuses were dissected free and placed in PBS. Each fetus was floated in PBS in the well of a maximov slide; the kidneys were dissected free while viewed with a dissecting microscope. Collection of Neonatal and Postnatal Kidneys Neonates, 24-hour postnatal, and 48-hour postnatal hamsters were sacrificed by means of a quickly severed spinal cord. The kidneys were dissected free and rinsed in PBS. Microscopic Procedures The dissected kidneys were fixed in either Bouin’s fixative, 4% paraformaldehyde/ Tris HCL pH 7.6 fixative, or formaldehyde-glutaraldehydecresol fixative (FGC). The kidneys were used for electron microscopic and immunohistochemical studies. Electron Microscopy All FGC fixed kidneys were postfixed in 1% OsO4, stained in block with 10% uranyl acetate in methyl alcohol, processed routinely, and embedded in Epon. Pale gold to silver sections were double stained with hot 2% uranyl acetate and sodium bismuth. The sections were viewed in a RCA EMU-3F microscope. lmmunohistochemistry Photomicroscopy Immunohistochemical-stained sections were photographed with Kodak ektaphan film #4162. A #80A Wratten gelatin filter was used; the filter enhanced the contrast between the DAB-stained renin deposits and the hematoxylin-stained nuclei. RESULTS In the developing hamster kidney, renin was observed in nonjuxtaglomerular vascular smooth muscle. In the 13-day fetal kidney, tubular structures and nondifferentiated masses of cells were observed, but no renin positive cells were found (Fig. 1). In the 14-day fetal kidney, a few renin positive cells were observed located in the walls of tubularlike structures (Fig. 2). In the 15-day fetal kidney, there was a sharp increase in renin positive cells. Renin was located in the smooth muscle cells of arterioles, interlobular arteries, and branches of the renal artery (Figs. 3-5). In the neonatal kidney, the total renin expression was similar to that observed in the 15-day fetal kidney. Renin positive cells were observed in the developing arteries (Fig. 6). In the 24-four postnatal kidney, the total renin expression was sharply reduced. Secretory granules were observed in smooth muscle cells at the vascular pole (Fig. 7). A small number of renin positive juxtaglomerular complexes were observed (Fig. 8). In the 48-hour postnatal kidney, there were more renin positive cells than had been observed in the 24-hour postnatal kidney. Renin positive juxtaglomerular complexes were observed and a few renin positive cells were observed in interlobular arteries (Fig. 9). The 4% paraformaldehyde fixed kidneys were processed as previously described (Dodge et al., 1988). Sections (7 pm) were reacted with antibody to r a t kidney renin supplied by Dr. T. Inagami (Department of BioDISCUSSION chemistry, Vanderbilt University School of Medicine, Renin, a proteolytic enzyme, is thought to be proNashville, TN). This antibody to rat kidney renin has been proven effective in demonstrating hamster kidney duced principally in juxtaglomerular cells in the adult animal. The results reported in this study support the renin (Dodge et al., 1988). The kidney sections were processed using Vector’s hypothesis that nonjuxtaglomerular vascular smooth biotinylated secondary antibody performed avidin DH- muscle of the hamster kidney can produce renin. In the biotinylated peroxidase procedure (Vector Elite ABC developing hamster kidney, vascular smooth muscle PK-6101 Rabbit IgG immunoperoxidase kit, Vector cells located in the developing arterioles, interlobular Laboratories, Burlingame, CA). The kidney sections arteries, and branches of the renal artery were renin were reacted with a 1:1,000 dilution of the antibody to positive. These vascular smooth muscle cells continued rat kidney renin in 1%NaCl PBS. Dilution of the pri- to produce renin in the neonatal kidney and then mary antibody in this NaCl concentration resulted in abruptly reduced renin production by 24 hours postnalittle to no nonspecific staining. The peroxidase-stained tal. Renin production increased in the 48-hour kidney components in the sections were demonstrated by and was produced primarily by the juxtaglomerular means of Vector’s peroxidase substrate DAB kit cells. Gomez et al. (1991) reported that under the approSK-4100. Renin positive cells stained a browdblack priate physiological stimulus adult renal vasculature color. The sections were counterstained with GillBaker-Mayer hematoxylin solution, routinely pro- could revert to a fetal pattern of renin distribution. The cessed, and mounted with coverslips. In order to make sure that each immunohistochemical staining reaction was positive, rat kidney sections were processed with the hamster sections. Renin positive juxtaglomerular Fig. 3. A 7-km section of a 15-day fetal kidney reacted with an sites in the rat kidneys indicated a positive staining antibody to rat kidney renin. Renin positive cells are observed in the reaction. Control kidney sections were reacted with wall of a branch of the renal artery (open arrowhead), x 443. Dako’s normal rabbit serum (NRS) #902 diluted 1: Fig. 4. A 7 pm section as a 15-day fetal kidney reacted with an 1,000 with 1% NaCl PBS. The control sections were antibody to rat kidney renin. Renin positive cells are observed in the processed similarly to the antibody to RKR-treated sec- wall of an interlobular artery (open arrowhead), x 550. tions. No renin positive cells were observed. Substrate Fig. 5. A 7-pm section of a 15-day fetal kidney reacted with an only controls and secondary antibody plus Vector’s antibody to rat kidney renin. There are numerous renin positive cells ABC plus substrate controls were negative. in the wall of several interlobular arteries (open arrowhead), x 458. Figs. 3-5. Fig. 6.A 7-(rm section of a neonatal kidney reacted with a n antibody to rat kidney renin. There are numerous renin positive cells in a branching interlobular artery (open arrowhead), x 367. Fig. 7. A 24-hour postnatal kidney section in which is seen an afferent arteriole. The vascular smooth muscle contains secretory granules (S.G.), x 15,000. Fig. 8. A 7-pm section of a 24-hour postnatal kidney reacted with a n antibody to rat kidney renin. A small amount of renin positive material is observed in the wall of the afferent arteriole at the vascular pole (open arrowhead), x 489. Fig. 9. A 7-pm section of a 48-hour postnatal kidney reacted with an antibody to rat kidney renin. Renin positive cells (open arrowheads) are observed in the walls of afferent arterioles and in the wall of the interlobular artery (*I, x 475. 150 A.H. DODGE DES-induced renin distribution pattern in the adult hamster kidney reported by Dodge et al. (1990) is similar to the developing renal renin pattern described in this report. For example, renin positive cells were demonstrated in branches of the renal artery i n the DEStreated adult kidneys and the 15-day fetal kidneys. It can be concluded that the adult renal renin pattern can revert to the developing pattern. The hamster DES animal model provides a system that can be used to study reversion to a fetal pattern. LITERATURE CITED Brace, R.A., and C.Y. Cheung 1986 Fetal cardiovascular and endocrine responses to prolonged fetal hemorrhage. Am. J. Physiol. 251 (2 PtL):R417-424. Brar, H.S., S.L. Kjos, W. Dougherty, Y.S. Do, H.B. Tam, and W.A. Hsuch 1987 Increased fetal olacental active renin Droduction in pregnancy-induced hypertension. Am. J . Obstit. Gynecol., 157(2):363-367. Brar, H.S., Y.S. Do, H.B. Tam, G.J. Valenzula, R.D. Murray, L.D. Longo, M.L. Yonekura, and W.A. Hsuch 1985 Uteroplacental unit as a source of elevated circulating prorenin levels in normal pregnancy. Am. J. Obstet. Gynecol., 155(6):1223-1226. Dodge, A.H., LA. Reid, and T. Inagami 1990 DES-induced renin production by hamster renal vascular smooth muscle. Thirtieth Annual Meeting of the American Society for Cell Biology, p. 345a. Dodge, A.H., M. Brownfield, I.A. Reid, and T. Inagami, 1988 Immunohistochemical renin study of DES-induced renal tumor in the Syrian hamster. Am. J . Anat., 182:347-352. Gomez, R.A., C. Pupilli, and A.D. Everett 1991 Molecular and cellular aspects of renin during kidney ontogeny. Pediat. Nephrology, 5(1):80-87. Gomez, R.A., K.R. Lynch, B.C. Sturgill, R.L. Chevalier, R.M. Carey, and M.J. Peach 1989 Distribution of renin mRNA and its protein in the developing kidney. Am. J. Physiol. 257:F850-858. Guyton, A.C. 1991 Blood pressure control-special role of the kidneys and body fluids. Science, 252:1813-1816. Raimbach, S.J., and A.L. Thomas, 1990 Renin and angiotensin converting enzyme concentrations in the fetal and neonatal guinea pig. J . Physiol., 423:441-451. Symonds, E.M., D.J. Craven, and C.H. Redeck 1985 Fetal plasma renin and renin substrate in midtrimester pregnancy. Br. J . Obstet. Gynaecol., 92(6):618-621.