INTRODUCTION

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THE JUXTAGLOMERULAR (JG) CELL is one of the components of the JG apparatus. This cell is located in the wall of the afferent arteriole at the entrance to the glomerulus (15, 33). JG cells synthesize and release renin from storage granules (18, 21, 33). Renin is a hormone enzyme that initiates the enzymatic cascade that generates the angiotensin peptides that regulate blood pressure, renal hemodynamics, and electrolyte balance. In addition to renin, adult JG cells also contain myofilaments, peroxisomes, small electron dense vesicles, and few mitochondria (33). They are connected to arteriolar smooth muscle cells, endothelial cells, and other JG cells by gap and myoendothelial junctions. JG cells are round, plump, and epithelioid in nature (33). Although renin has been the characteristic marker of JG cells, other markers have been cloned such as Zis (Zinc finger Splicing factor), which is a developmentally regulated gene expressed in JG cells (19).

It has been postulated that JG cells derive from smooth muscle cells because in the adult mammal they contain myofilaments (33); however, no studies have been performed to determine the lineage of these cells.

In the fetal kidney of mammals, renin cells are widely distributed along the walls of large renal arteries and afferent arterioles (7, 13, 26, 28), in contrast to the typical adult JG localization (4, 34). An association between renin cells and the branching of renal arterioles has been described, suggesting that these cells play a role in the development of the kidney vasculature (27). We have observed that in the fetal rat at embyronic day 14 (E14) renin cells are also present in the kidney interstitium before vessel formation has occurred.

Although it has been suggested that glomerular capillaries develop from an intrinsic precursor (30), the origin of the renal arteriolar endothelium, the smooth muscle of the whole kidney vasculature, and the renin cells is unknown.

It is well known that embryonic kidneys (E12 mouse, E14 rat) in culture systems undergo nephrogenesis, developing tubules and glomeruli. Unfortunately, under the usual culture conditions, in this otherwise excellent model, there is no vessel formation and therefore renin cells do not assemble into arterioles, remaining dispersed in the interstitium. Although interstitial and glomerular capillaries may develop in vitro under certain specific conditions such as exposure to vascular endothelial growth factor (VEGF) (35) or to a low oxygen concentration (3% O₂) (36), renal arterioles do not form uniformly. Therefore, we chose a transplantation model of prevascular embryonic kidneys to define the embryonic origin of JG cells. Furthermore, we utilized single cell PCR and double immunostaining combined with lineage markers to define the lineage of the JG cell.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals

Timed-pregnant Sprague-Dawley rats were purchased from Hilltop Farms (Scottdale, PA). Embryos at 14 days of gestation were the source of fetal kidneys used to aspirate single cells and perform single cell RT-PCR to monitor the expression of cell identity markers. Time-dated pregnant mice and rats were mated overnight, and the females were checked for vaginal plugs the following morning. The day of detection of a vaginal plug was regarded as day 0 of gestation. All mice were fed regular mouse chow (Prolab 2000, PMI Feeds, St. Louis, MO) and tap water ad libitum and housed in a temperature-controlled (22 ± 2°C) environment with a 12-h light/dark cycle. All procedures were performed in accordance with the guidelines of the American Physiological Society and were approved by the University of Virginia Animal Care Committee.

Grafting of Embryonic Kidneys

Grafting into the anterior chamber of the eye. Allografts (n = 11) of fetal kidneys into the anterior eye chamber were performed as described previously (1). Briefly, adult C57 Bl6/6J hosts were anesthetized by intraperitoneal injection of a ketamine-xylazine combination (100 and 15 mg, respectively, per kg body wt), and then tropicamide was applied to the mouse eye to dilate the iris. The cornea was incised with a 27-gauge needle, and the incision was extended 2 mm with Vannas scissors. Freshly harvested embryonic (E12) prevascular kidneys were placed into the anterior eye chamber of a host mouse via the corneal incision and positioned over the iris. Antibiotic (neomycin and polymyxin B sulfates, and bacitracin zinc) ophthalmic ointment was applied to the eye, and grafts were allowed to develop in oculo for 8 days. After the animals were killed, grafts were removed, fixed, and embedded in paraffin as previously described (14, 37), and processed for immunohistochemistry as described in Immunohistochemistry.

Grafting under the kidney capsule. To define whether vascular progenitors originating within the metanephric mesenchyme differentiate in situ to JG cells, smooth muscle cells, and endothelial cells, we cross-transplanted kidneys between wild-type and transgenic mice expressing LacZ in endothelial cells (Flk1+/ LacZ mice) or all cells (Rosa 26 mice) and GFP in renin cells (see Table 1). Metanephric (E12) kidneys were grafted under the kidney capsule of adult mice (host: Rosa 26 or Flk1+/, donor: E12 kidneys from C57 Bl/6J and vice versa; and host: C57 Bl/6J, donor: E12 kidneys from Ren-GFP) (see Table 2). Donor and host mice were anesthetized by intraperitoneal injection of tribromoethanol (300 mg/kg) (11). Metanephric (E12) kidneys were dissected aseptically at 37°C in serum-free organ culture medium [DMEM/F-12 (GIBCO-BRL no. 430-2500EG) with 10 mM HEPES (Sigma H9136), 1.1 mg/ml NaHCO₃, 50 U/ml penicillin, 50 U/ml nystatin, insulin-transferrin-selenite (Sigma I1884; 5 µg/ml insulin and transferrin, 2.8 nM selenite), 25 ng/ml PGE₁, and 32 pg/ml triiodothyronine (T₃)]. In the host, an incision was made along the dorsal lumbar side above the kidney, the muscle layers overlaying the kidney were dissected, the left kidney was exteriorized, and a small incision was made in the renal capsule. A blunt 20-gauge needle was gently inserted into the incision to create a 1-cm subcapsular tunnel towards the upper pole of the kidney and another one towards the lower pole where the embryonic kidneys (E12) were placed with forceps. Usually, from two to three embryonic kidneys were transplanted in this fashion. The host kidney was replaced into the abdomen, and the muscle layers and the skin were sutured separately. The animals were allowed to recover from the anesthesia on a heating pad at 37°C. Subcapsular grafts were allowed to undergo nephrovascular development for 7-8 days.

5-Bromo-4-Chloro-3-Indolyl -D-Galactopyranoside Reaction

Kidneys from mice carrying transplants (left kidneys with grafts and right kidneys as controls) performed between Rosa 26 or Flk1+/ and C57 Bl/6J mice (Rosa 26  B6 and Flk1+/  B6), and kidneys from Flk1+/ and Tie2 mice were harvested from anesthetized mice as described above, decapsulated, sectioned in 2-mm slices, and fixed for 15 min in 3.7% formaldehyde. After being washed 3 times for 15 min each in detergent rinse (0.1 M phosphate buffer, pH 7.4, containing 2 mM MgCl₂, 0.01% sodium deoxycholate, and 0.02% tergitol NP-40), the tissue was placed in staining solution [detergent rinse, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mg/ml 5-bromo-4-chloro-3-indolyl -D-galactopyranoside (X-Gal; Fisher Biotech) in dimethylformamide] overnight in the dark at 37°C. The tissue was then washed three times for 15 min each in PBS, postfixed in 3.7% formaldehyde at 4°C overnight, dehydrated in graded alcohols to xylenes, and embedded in paraffin. On the X-Gal reaction thus performed, cells expressing -galactosidase turn blue. After the X-Gal reaction, the tissues were subjected to immunohistochemistry for renin and -smooth muscle actin (-SMA). Coincidences or discrepancies among blue cells and cells immunostained for those markers were evaluated.

Fluorescence Microscopy

Immunohistochemistry

Double immunostaining for both renin and -SMA on the same tissue section was performed on kidney sections from mice at different embryonic and postnatal (N) ages (E14-E16, E18, N1, N5, N10, N21, N45, and N70, n = 3 to 5 animals for each age). This procedure was performed as described above through the peroxidase immunohistochemistry reaction for renin. After the first reaction, the sections were microwaved (3 cycles, 1 min each) in antigen retrieval solution (0.01 M sodium citrate buffer, pH 6), and then a second immunodetection was performed by the method described above for -SMA using a peroxidase substrate, which generates a different color reaction product (VIC purple, Vector Lab). The tissue was not counterstained, and was directly dehydrated through graded alcohols to xylenes and mounted with Permount. Using this procedure, renin-containing cells are purple and smooth muscle cells will be brown or vice versa depending on which antibody was used first.

Single Cell RT-PCR of Cells Aspirated From Embryonic Kidneys

Nested PCR was performed on the RT reactions to test for the presence of the lineage marker mRNAs: -SMA (6, 24) and myosin heavy chain (MHC) (3) for smooth muscle cells, Ets1 (20) and vimentin (5, 16) for mesenchymal cells, and tenascin (2, 9) for interstitial cells. Each individual sample was subjected to two PCR reactions: one for renin and the second for a lineage marker. The basic PCR reaction (using either outer primers or nested primers) contained 1× PCR buffer, 0.1 mM dNTPs, and 1.5 units Taq DNA polymerase (Promega, Madison, Wisconsin) in a volume of 50 µl, and PCR amplification was carried out for 40 cycles. The volume of template was added, and the concentration of MgCl₂ and primers and the cycling parameters were adjusted for the marker in question. (See Table 3 for the primers, specific PCR conditions, and volume of template used.) Depending on the abundance of the mRNA to be detected, 2 to 20 µl of the RT reaction were used as a template in the first PCR reaction. Twenty microliters of the first PCR reaction were used as a template in the second PCR reaction with the nested primers.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Lineage of the JG Cell

Staining for lineage markers. Double immunostaining of mouse kidneys for both renin and -SMA at different embryonic and postnatal ages (E14-E18, N1, N5, N10, N21, N45, N70) showed that at early embryonic ages (E14, E15) renin-expressing cells are large, either round or oval shaped, and found among undifferentiated mesenchymal cells usually as single isolated cells or in small groups of two to three cells (Fig. 1). These cells are distributed in the mesenchyme throughout the entire kidney. They can be found close to forming vessels but definitely separated from smooth muscle cells (Fig. 1, inset). They are also seen inside the forming glomeruli (Fig. 1). At E16 we can identify two populations of renin-expressing cells: some are still isolated but others are found in groups close to the forming vessels and glomeruli (not shown). Some renin-expressing cells within the vessels contain -SMA (Fig. 2). Thus at this developmental stage three types of cells expressing renin and/or -SMA can be found: one type expressing only renin, another expressing only -SMA, and a third cell type expressing both markers. By 18 days of gestation, isolated cells expressing solely renin can no longer be found. As shown in Fig. 3, by E18, renin-expressing cells are mostly associated with the vasculature. However, they can still be found inside some glomeruli and in the interstitium. Renin cells at this embryonic age are found mainly in large arteries, whereas in the adult kidney they are found in their classic JG localization (Fig. 4). The above findings are supported by immunohistochemistry for renin or -SMA performed individually on consecutive sections at the same embryonic and postnatal ages as the double immunostaining referred to above. These experiments demonstrated that renin-expressing cells begin to express -SMA at E16, and expression of both proteins is maintained thereafter in the mature JG cells. As described below, the lack of coincidence between smooth muscle and renin expression in embryonic renal cells was confirmed by single cell RT-PCR experiments performed in rat embryonic kidneys at E14, a time where no arterioles are present in the rat kidney.

View larger version (112K): [in this window] [in a new window]   Fig. 1.   Embryonic day 15 (E15) mouse kidney double immunostained for renin and -smooth muscle actin (-SMA). Renin cells (brown, arrows) are distributed in the mesenchyme as single isolated cells. They are present inside the forming glomeruli and close to the vessels (identified by -SMA staining in purple). Inset: higher magnification of renin cell close to a forming vessel. g, Glomerulus. Bars: 100 µm; inset: 25 µm.

View larger version (128K): [in this window] [in a new window]   Fig. 2.   E16 mouse kidney double immunostained for renin and -SMA. At this stage, renin cells (brown) that have become part of a newly assembled vessel express either only renin (arrow) or both renin and -SMA (arrowheads). Bar: 25 µm.

View larger version (115K): [in this window] [in a new window]   Fig. 3.   E18 mouse kidney immunostained for renin and -SMA. Double immunostaining for renin (brown, arrow) and -SMA (purple). Renin cells are mainly associated with the vasculature (arrows). Inset left: higher magnification showing the colocalization of renin and -SMA. Inset right: consecutive section immunostained only for renin (brown). Renin is present in the vessel at the same location as -SMA. Bars: 50 µm; inset left: 25 µm; inset right: 50 µm.

View larger version (171K): [in this window] [in a new window]   Fig. 4.   Adult (P70) mouse kidney double immunostained for renin and -SMA. Renin cells (brown) are found in the typical juxtaglomerular location (arrows). Some of these cells also express -SMA (purple, arrowheads). Bar: 25 µm.

View larger version (151K): [in this window] [in a new window]   Fig. 5.   Endothelial cells and renin cells in 5-day-old Flk1+/ mouse kidney. The tissue was subjected first to the 5-bromo-4-chloro-3-indolyl -D-galactopyranoside (X-Gal) reaction and then immunostained for renin (brown). The immunolocalization of renin does not coincide with the blue endothelial cells. Bar: 25 µm.

View larger version (67K): [in this window] [in a new window]   Fig. 6.   Endothelial cells and smooth muscle cells in 5-day-old Flk1+/ (A) and adult Tie2 (B) mouse kidneys. The tissue was subjected first to the X-Gal reaction and then immunostained for -SMA. A: N5 Flk1+/ mouse kidney showing the immunolocalization of -SMA (purple) not coinciding with the blue endothelial cells. B: adult Tie2 mouse kidney immunostained for -SMA (brown) showing no coincidence with the blue endothelial cells. Bars: 25 µm.

View larger version (96K): [in this window] [in a new window]   Fig. 7.   Triple labeling of endothelial cells, renin cells, and smooth muscle cells in 5-day-old Flk1+/ mouse kidney. The tissue was subjected first to the X-Gal reaction and then double immunostained for renin (brown) and -SMA (purple). Blue endothelial cells do not coincide with either renin- or -SMA-containing cells. Some renin cells coexpress the -SMA protein (arrowheads). Peritubular cells at this developmental age still express -SMA protein. Bar: 50 µm.

View larger version (156K): [in this window] [in a new window]   Fig. 8.   Triple labeling of endothelial cells, renin cells, and smooth muscle cells in E12 Flk1+/ mouse kidney transplanted under the kidney capsule of an adult wild-type host for 7 days. The tissue was subjected first to the X-Gal reaction and then double immunostained for renin (brown) and -SMA (purple). Blue endothelial cells are derived from the grafted Flk1+/embryonic kidney, and they do not costain with either renin or -SMA. However, some renin cells also contain -SMA (arrowheads). Bar: 50 µm.

Single cell RT-PCR. To study the lineage of the JG cell we performed single cell RT-PCR of potential JG cell precursors from rat embryonic kidney cells at day 14 of gestation. Distribution and morphology of these cells resembled distribution of the renin cell. The potential JG cell precursors were selected by their large size and granular morphology as revealed by staining with the vital dye neutral red (Fig. 9). By immunostaining with renin antibody in the whole prevascular metanephric kidney, we previously found that some but not all of these large granulated cells contained renin. Therefore, these cells were chosen for microaspiration, and some of them did express renin (Table 4). As shown in Table 4, the experiments (1-5) were designed to test, in each single cell, the expression of renin and one of the following cell markers: -SMA, MHC, Ets1, vimentin, or tenascin. At this prevascular stage of kidney development, all the markers were already present in the metanephric kidney in a variety of cell types. In experiments 1 and 2, none of the cells that expressed smooth muscle markers (either -SMA or MHC) were positive for renin, and cells expressing renin tested negative for smooth muscle markers. Experiment 3 showed that 50% of renin-expressing cells coexpressed the Ets1 marker and 50% did not. The transcriptional factor Ets1, known to be present in most mesenchymal cells, was widely distributed among these embryonic cells, with more than one-half of all the studied cells (25/41) expressing Ets1. In experiment 4, 1 out of 8 cells expressing tenascin was also positive for renin, and in experiment 5, cells positive for vimentin did not express renin. Overall, the number of cells expressing renin for the combined five experiments were 18 out of 123 cells picked, which represents ~15%. These results confirmed the presence of progenitors of renin cells, as well as other cell types identified by different cell markers. In fact, they demonstrate the presence of vascular precursors for all cell types of the renal arteriole. Furthermore, renin cell progenitors at this prevascular stage of kidney development did not express smooth muscle markers. However, many of them did express the transcriptional factor Ets1, and there was one renin cell that also contained the interstitial marker tenascin. Among the markers studied, renin cells show a clear lineage relationship with mesenchymal cells in early development.

View larger version (154K): [in this window] [in a new window]   Fig. 9.   E14 rat kidney incubated with the vital dye neutral red. Large granulated cells that stained red with the dye were chosen for microaspiration to perform single cell RT-PCR.

Origin and Formation of Renal Arterioles

View larger version (83K): [in this window] [in a new window]   Fig. 10.   E12 mouse kidney transplanted into the anterior eye chamber of an adult mouse for 7 days. A: immunostaining for renin (brown) showing a normal distribution of renin cells in juxtaglomerular areas. B: immunostaining for -SMA (brown). The vessels developed in a similar pattern as the one found in the intact 19-day-old embryo. Bars: 25 µm (A); 100 µm (B).

Origin of Renin-Expressing, Smooth Muscle, and Endothelial Cells

View larger version (125K): [in this window] [in a new window]   Fig. 11.   Transplants of E12 kidneys under the kidney capsule of adult mice. Tissues were subjected to the X-Gal reaction and to immunohistochemistry for renin (B, C, and E) and -SMA (F). A: E12 wild-type embryonic kidney under the kidney capsule of a Rosa 26 host. After the X-Gal reaction, the wild-type embryonic kidney, in white, was not invaded by host vessels, whereas all host cells were completely blue. B and C: renin immunostaining of transplanted E12 wild type  Rosa 26 showing renin distribution along the arterioles (B) and in the juxtaglomerular area and inside the glomerulus (arrows, C). None of these cells are blue, indicating the intrinsic origin of the juxtaglomerular (JG) cells. D: E12 Rosa 26 embryonic kidney under the kidney capsule of a wild-type host. After the X-Gal reaction, the transplanted kidney turned completely blue. Renin immunostaining (dark brown, arrows, E) shows coincidence with blue, and -SMA immunostaining (dark brown, arrowheads, F) along the arterioles also coincides with the blue reaction product. The endothelial cells inside the immunostained vessels are also blue, confirming the intrinsic embryonic origin of all these cells. Magnification: (A and D) ×16. Bars: 100 µm (B); 25 µm (C and F); 12.5 µm (E).

Transplants of E12 Ren-GFP kidneys (both homozygous and heterozygous) grown under the kidney capsule of wild-type mice (Ren-GFP  B6) showed expression of GFP-labeled renin cells in the interstitium and along kidney microarterioles, confirming their intrinsic metanephric blastema embryonic origin (Fig. 12, A and B).

View larger version (64K): [in this window] [in a new window]   Fig. 12.   Transplant of E12 ren-green fluorescence protein (GFP) under the kidney capsule of a wild-type host. A: fluorescence microscopy (darkfield) showing green fluorescent cells labeling renin cells derived from the embryonic kidney. B: brightfield of A showing green cells are distributed along the arterioles and inside the glomerulus (arrows). g, Glomerulus; a, arterioles. Bars: 25 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study, we examined the embryonic origin and lineage of JG cells and their relationship with smooth muscle and endothelial cells. The results showed that all arteriolar cell precursors, including JG cells, are already present in the metanephric blastema at E11 and E12 before vessel development has occurred. Our transplantation experiments demonstrated that renin precursor cells are capable of assembling to the appropriate vessel type and segment (i.e., afferent arteriole). Finally, those transplantation experiments provided the first experimental evidence indicating that JG cells and renal vascular smooth muscle cells originate within the metanephric blastema rather than from an extrarenal source. As we determined that JG cell precursors are present in the embryonic rat kidney, at E14 before vascular structures are formed, we also confirmed that other cell markers such as -SMA, MHC, Ets1, vimentin, and tenascin are present at this prevascular stage of kidney development. These results demonstrate that before the vasculature has developed, the metanephric blastema possess renin cell progenitors as well as precursors for many other cell types. The present study agrees with those of others regarding the origin of endothelial cells (17, 22, 30). Using specific cell markers for endothelial cells, such as two of the receptors for VEGF (VEGF-R1 or Flt1 and VEGF-R2 or Flk1) involved in the commitment and differentiation of the endothelial cells (25) and Tie1 Rc (22), several investigators have identified the presence of endothelial cell precursors in the rodent kidney (17, 23, 30). We have previously shown that smooth muscle precursors are present in the primitive interstitium of fetal rat kidneys at 14 days of gestation (6) as well as Flk1- and Flt1-positive cells in the E12 mouse kidney (35). These results suggest that at the time that the ureteric bud begins its induction of the metanephric mesenchyme (E11 and E12) a variety of cell progenitors are already present, and contribute to both nephrogenesis and vasculogenesis. The molecular signals that define whether an undifferentiated mesenchymal cell follows one lineage pathway or another require further work.

In addition to demonstrating the intrinsic origin of JG, smooth muscle, and endothelial cells, our cross-transplantation experiments showed that JG cell progenitors were capable of assembling into preglomerular arterioles in a normal pattern. Interestingly, embryonic kidneys grown in vitro develop nephrons but they do not develop blood vessels, and renin cells remain dispersed in the interstitium. However, if these same embryonic kidneys are grown under the kidney capsule or in oculo, blood vessels (containing renin cells, smooth muscle, and endothelial cells) develop properly. Celio and collaborators (8) described that renin-containing cells were present in kidney transplants grown in the anterior eye chamber. However, in those studies rat E17-E19 kidneys were used, and arterial blood vessels containing renin-expressing cells were already developed at the time of transplant. Although no clear conclusions can be ascertained regarding the origin of those structures, it seems clear that the anterior eye chamber microenvironment provided the appropriate signals for the maintenance of the vascular structures and for renin cell localization. Our transplantation experiments using prevascular embryonic kidneys clearly suggest that signals from the environment provided the appropriate positional information for JG cell localization and arteriolar development. In addition, these experiments rendered further support to the hypothesis that JG cells, smooth muscle, and endothelial cells all originate from within the metanephric mesenchyme. The cross-transplantation studies of embryonic kidneys under the kidney capsule of adult mice (between Rosa 26 and C57 Bl/6J-strain mice) demonstrated that the JG cells, smooth muscle, and endothelial cells found within the grafted tissue developed in situ from the metanephric blastema and not from invading host cells, suggesting that JG cell precursors have the capability to, and in effect do, differentiate into JG cells. This finding can be related to those of Hyink et al. (17) who demonstrated that glomerular capillaries and mesangial cells originate in situ within the metanephric blastema. These data reveal that all vascular precursor cells are already present within the metanephric blastema. Further studies are needed to define the molecular mechanisms governing the lineage of kidney vascular cells.

Our previous work demonstrated that there is an association between renin-expressing cells and the branching of renal arterioles (27). In fact, inactivation of various components of the renin-angiotensin system using gene targeting results in aberrant renal arteriolar branching, suggesting that renin, acting through local generation of angiotensin, regulates renal vascular development. It remains to be determined whether JG cells, independent of renin, can contribute to vascular development. Furthermore, whether the assembling vessel contributes to differentiation of the JG cell and the signals involved in that process remains to be investigated.

Using single cell RT-PCR, we demonstrate that during early embryonic life, renin-expressing cells are not related to smooth muscle cells. In addition, immunostaining studies also showed that renin cells in early stages of kidney development (before E15) are unrelated to cells that express -SMA. Analysis at later ages (E16 to N70) revealed that some JG cells contained -SMA, indicating that acquisition of a smooth muscle phenotype is a later event in the differentiation of the JG cell. Although it has been suggested for many years that renin cells are derived from smooth muscle cells (33), this assertion has never been tested experimentally. The current experiments, however, suggest a different scenario in which at this stage there are at least two distinct populations of cells expressing either renin or smooth muscle markers but not both. Subsequently, the subpopulation of renin-expressing cells acquires the capacity to express smooth muscle markers. This finding suggests that renin cells are capable of giving rise to smooth muscle cells (arteriolar smooth muscle cells and very likely to other smooth muscle-like cells such as the interstitial pericyte and the glomerular mesangial cell), rather than originating from them. Interestingly, not all smooth muscle cells seem to originate from renin cells, suggesting that smooth muscle cells can also originate from another nonrenin precursor, including a distinct embryonic smooth muscle cell progenitor. Smooth muscle cells that have descended from renin precursors are very likely the ones that undergo metaplasia to renin cells when homeostasis is threatened with a need for more renin to preserve it (12). By contrast, during aging and long-term diabetes there are less JG cells, probably due to a retransformation of JG cells to smooth muscle cells (10). A brief conceptualization of the lineage of JG cells is shown in Fig. 13.

View larger version (32K): [in this window] [in a new window]   Fig. 13.   Conceptualization of the lineage of the JG cell. Metanephric mesenchymal cells (MCs) give origin to angioblasts, which in turn give origin to endothelial cells. MCs also give rise to smooth muscle cells and to renin precursor cells. During ontogeny the renin precursor has the capability to give rise to JG cells and to a subset of arteriolar smooth muscle cells. Smooth muscle cells that have descended from renin precursors are very likely the ones that undergo metaplasia to renin cells when the body needs more renin to preserve homeostasis. By contrast, during aging and long-term diabetes there are less JG cells, probably due to a transformation of JG cells to smooth muscle cells.

In summary, our data show that JG cells originate in situ within the metanephric kidney from mesenchymal cells unrelated to the endothelial or smooth muscle lineages. Interestingly, as they differentiate, they acquire smooth muscle markers that are maintained throughout adulthood. The mechanisms that direct JG cell development, and their acquisition of smooth muscle characteristics, remain to be determined.