In response to a homeostatic threat circulating renin increases by increasing the number of cells expressing renin by dedifferentiation and re-expression of renin in arteriolar smooth muscle cells (aSMCs) that descended from cells that expressed renin in early life. However, the mechanisms that govern the maintenance and reacquisition of the renin phenotype are not well understood. The cAMP pathway is important for renin synthesis and release: the transcriptional effects are mediated by binding of cAMP responsive element binding protein with its co-activators, CBP and p300, to the cAMP response element in the renin promoter. We have shown previously that mice with conditional deletion of CBP and p300 (cKO) in renin cells had severely reduced renin expression in adult life. In this study we investigated when the loss of renin-expressing cells in the cKO occurred and found that the loss of renin expression becomes evident after differentiation of the kidney is completed during postnatal life. To determine whether CBP/p300 is necessary for re-expression of renin we subjected cKO mice to low sodium diet + captopril to induce retransformation of aSMCs to the renin phenotype. The cKO mice did not increase circulating renin, their renin mRNA and protein expression were greatly diminished compared with controls, and only a few aSMCs re-expressed renin. These studies underline the crucial importance of the CREB/CBP/p300 complex for the ability of renin cells to retain their cellular memory and regain renin expression, a fundamental survival mechanism, in response to a threat to homeostasis.
- renin re-expression
- phenotypic switch
- adenosine 3′,5′-cyclic monophosphate
renin, the key enzyme and hormone of the renin-angiotensin system, is crucial for the regulation and maintenance of blood pressure and fluid-electrolyte homeostasis (10; 14). Renin is synthesized and released by juxtaglomerular (JG) cells in the adult kidney. In embryonic development, the undifferentiated metanephric blastema contains renin cells before the kidney vasculature has developed (25). Later in fetal life renin-expressing cells are broadly distributed throughout the intrarenal arterial tree, inside the glomeruli and in the interstitium. As maturation continues, renin expression becomes restricted to the a few myoepithelioid, granulated JG cells in the wall of the afferent arteriole at the entrance to the glomerulus (9). The other non-JG cells along the afferent arteriole and within the glomerulus that expressed renin during early development differentiate into smooth muscle and mesangial cells in the adult (24). When fluid/electrolyte homeostasis is threatened in adult life (due to hypotension, dehydration, hemorrhage, sodium depletion) circulating renin increases. The increase in circulating renin is accomplished mainly by an increase in the number of cells expressing renin (7; 8; 15). The cells along the kidney vasculature, in the glomeruli and in the interstitium, that expressed renin in early life de-differentiate and synthesize renin again (24). In fact, not all cells can re-express renin, and the increase in the number of renin-expressing cells seems to be limited to those that belong to the renin cell lineage and retain the memory to reacquire the renin cell identity. Therefore, in response to homeostatic challenges, the capacity to retain this cellular memory and regain renin expression is a fundamental survival mechanism.
The mechanisms that govern the maintenance and reacquisition of the renin phenotype are not well understood. It has been shown that the cAMP pathway is crucial in the regulation of renin synthesis and release (4–6; 16; 18; 19; 22; 23). Most of the transcriptional effects of cAMP are mediated by the binding of the phosphorylated form of cAMP responsive element binding protein (CREB) associated with its co-activators CBP and p300 (1; 17) to the cAMP response element present in the upstream region of a variety of genes, including the renin gene (19). CBP and p300 have histone acetyltransferase activity, which may facilitate access of transcription factors to gene promoters.
We tested the in vivo role of the histone acetyl transferases CBP and p300 in the expression of the renin gene and the maintenance of renin cell identity by deleting these genes from cells of the renin lineage using mice with floxed alleles of CBP and p300 crossed to our Ren1d-cre mouse (11). The resulting adult CBP/p300 conditional knockout (cKO) mice had a severe reduction in renin expression and kidney morphological abnormalities (11). These results support a role for CBP and p300 in JG cell identity and maintenance of normal kidney morphology in the adult. It is not known, however, whether deletion of CBP and p300 results in the loss of renin expression early in development or if the severity of the phenotype increases with age. In the present study we examine renin expression and kidney morphology at earlier stages of development.
The ability of cells to re-acquire the renin phenotype when fluid/electrolyte balance is challenged is crucial for the maintenance of homeostasis. We demonstrated previously in vitro that the re-acquisition of the renin phenotype is mediated by cAMP and acetylation of histones at the cAMP response element of the renin gene in smooth muscle cells of the renin lineage (22), implicating a role for CBP and p300 in this process. We tested the in vivo role of CBP and p300 in the re-acquisition of the renin phenotype in our cKO mice maintained on low sodium diet + captopril in the drinking water, a treatment known to induce re-transformation of smooth muscle cells to the renin phenotype. The cKO mice had a severely blunted response: renin expression in JG cells and, more importantly, in smooth muscle cells along the afferent arterioles was severely diminished.
These studies underline the crucial importance of the CREB/CBP/p300 complex for the ability of renin cells to retain their cellular memory and regain renin expression, a fundamental survival mechanism, in response to a threat to homeostasis.
MATERIAL AND METHODS
Housing and experimental use of the mice conformed to the Guiding Principles in the Care and Use of Animals approved by the Council of the American Physiological Society and with federal laws and regulations. All protocols have been reviewed and approved by the Animal Care and Use Committee of the University of Virginia. The University of Virginia is an Association for Assessment and Accreditation of Laboratory Animal Care-accredited institution.
Generation of experimental animals.
To delete the co-activators CBP and p300 simultaneously in renin cells, we used three strains of animals: our Ren-cre mice (24), which express cre in renin cells, and mice with floxed alleles of the co-activators CBP (CBPflox) (12) and p300 (p300flox) (13). These animals were interbred as described previously to produce the cKO (CBPfl/fl;p300fl/fl; Ren1dcre/+) and the control (CBP+/+;p300+/+;Ren1dcre/+) animals (11). Genotyping was performed by standard PCR on DNA extracted from tail biopsies as described previously (11).
Stimulation of renin expression.
To elicit re-expression of renin 2- to 4-month-old mice were administered a low-sodium diet (0.05%; Harlan, Madison, WI) plus captopril (0.5 g/l; Sigma, St. Louis, MO) in the drinking water for 8 days. Blood pressures were recorded from the control and cKO study mice before and after the treatment. The study animals were weighed and then anesthetized with tribromoethanol (300 mg/kg) (24). Blood was drawn via cardiac puncture and placed into heparinized plasma separator tubes for plasma renin assays and EDTA plasma-separator tubes for basic metabolic panel analysis (Microtainer; Becton Dickinson, Franklin Lakes, NJ). The kidneys were removed, weighed, and preserved for RNA extraction in RNAlater Solution (Ambion, Austin, TX) or fixed for immunohistochemistry.
Blood pressure measurements.
Blood pressure was measured in conscious mice using a CODA noninvasive blood pressure system (Kent Scientific Corporation, Torrington, CT). Systolic blood pressure values are reported.
Plasma renin concentration and blood chemistry measurements.
These assays were performed as described previously (20; 21).
RNA extraction, reverse transcription, and quantitative real-time PCR analysis.
These procedures were performed as described previously (11). Relative expression of renin mRNA was determined using the standard ΔΔCt method.
Kidneys were fixed in Bouin's solution and embedded in paraffin, and sections prepared for immunohistochemistry. Immunohistochemistry for renin and α-smooth muscle actin (α-SMA) was performed as described previously (25) using a rabbit anti-mouse renin antibody (1:500 dilution) (11) and a monoclonal anti-α-SMA specific antibody (isotype IgG2a, dilution 1:10,000; clone 1A4; Sigma) and the appropriate Vectastain ABC kit (Vector Laboratories, Burlingame, CA).
The number of renin positive JG areas (JGAs) was counted in each kidney section and is expressed as a percentage of the total number of glomeruli per section, a value designated as the JG index. In addition, the appearance and number of individual renin-positive cells within the JGAs was noted. In the renin re-expression studies the number of afferent arterioles having extension of renin expression beyond the JGA (15) was also counted in each kidney section and normalized to the total number of glomeruli per section.
Values are expressed as means ± SE. Significance was determined by t-test or Mann-Whitney Rank Sum test as appropriate using Sigma Stat version 3.0.1.
Ontogeny of renin expression and morphological abnormalities in cKO mice.
We reported previously that 2.5-month-old cKO mice had greatly reduced renin mRNA expression, very few renin positive JGAs, and small, morphologically abnormal kidneys when CBP and p300 were absent from renin cells (11). In this study, further characterization of these mice revealed that the plasma renin concentration (PRC) was significantly reduced to 52% of control levels in the cKO mice reflecting the previously observed reductions in renin mRNA and protein expression (Table 1). In addition, the systolic blood pressure in the cKO mice was 26 mmHg lower than that in the control mice (Table 1). The kidney morphological abnormalities in the cKO mice were also reflected functionally: both blood urea nitrogen and serum creatinine were significantly elevated (Table 1).
To determine whether the reduction in renin expression and the kidney abnormalities observed in 2.5-month-old cKO mice were already evident in the neonatal period or progressed with age, we examined mice at 5 days (N5) and 30 days (N30) of postnatal life. At N5 renin mRNA and protein expression in the cKO mice were similar to that of the control mice and the kidneys exhibited no morphological abnormalities (Fig. 1, A–C). By N30, significant changes had developed. Renin expression was diminished, although, unlike in the older mice (11), the kidney size was not yet affected [the weight of both kidneys/body weight (×100) controls: 1.09 ± 0.04, n = 3; cKO: 1.03 ± 0.06, n = 5, P = 0.493]. PRC in N30 cKO mice was significantly lower compared with control mice [control: 0.74 ± 0.10 μg ANG I·ml−1·h−1 (n = 2) vs. cKO: 0.19 ± 0.08 μg ANG I·ml−1·h−1 (n = 3); P = 0.02]. Renin mRNA levels were an average of 30% of the control level (Fig. 2A) and this was reflected in the reduced renin protein staining with the JG index in the cKO being 43% of that in the control mice (Fig. 2, B and D). There was some variation in the severity of the phenotype in the cKO mice with some animals having a higher JG index, thus more renin, than others, but all of them lower that the controls (Fig. 2C). Staining for α-SMA showed that the N30 cKO kidneys had some areas of α-SMA expression in the interstitium indicating fibrotic changes (Fig. 2E). In addition there were cystic and dilated tubules. On the whole, the N30 cKO kidneys had somewhat higher renin mRNA and protein levels and less severe morphological abnormalities than those seen in the 2- to 4-month-old cKO kidneys.
cKO mice have a blunted response to homeostatic challenge.
To test the role of CBP/p300 in the ability of arteriolar smooth muscle cells to retransform to the renin phenotype in response to homeostatic challenge, we treated control and cKO mice with low-sodium diet plus captopril to elicit renin re-expression. The treatment resulted in a significant reduction in systolic blood pressure in both groups [control: before treatment 136.2 ± 11.2 mmHg, after treatment 81.6 ± 7.3 mmHg (n = 5), P = 0.023; cKO: before treatment 114.6 ± 3.4 mmHg, after treatment 80.5 ± 5.7 mmHg (n = 5; P = 0.006)], indicating a marked hypotensive response to the treatment. Circulating renin levels (PRC) in the treated control mice increased 36-fold compared with untreated control animals (Fig. 3E). In contrast, the cKO mice were unable to increase circulating renin. The already low PRC increased less than threefold, which was not significantly different from the levels found in untreated cKO mice (Fig. 3E).
After treatment, the kidney renin mRNA levels in the control mice increased an average of 17-fold relative to untreated control mice (Fig. 3A). In the treated cKO mice the renin mRNA levels also increased relative to untreated cKO mice, but not to the same extent as in the control mice (Fig. 3A). Staining for renin in kidney sections showed that the JG index increased significantly in the treated animals and each individual animal had a similar JG index (Fig. 3, B and C). The renin-positive cells were plump with renin and renin expression extended to smooth muscle cells of the renin lineage along the afferent arterioles (Fig. 4, A and C). The cKO mice, however, evidenced diminished recruitment of renin-positive cells. The treated cKO mice also had a somewhat higher number of renin-positive JGAs than untreated cKO animals (Fig. 3, B and C); however, the difference between the treated and untreated cKO animals, due to variability of the response, did not reach significance. The average JG index in treated cKO mice was only 44% of the treated control mice. Renin expression, as evaluated by the JG index, varied somewhat among the individual treated cKO animals (Fig. 3C) but was always lower than the treated controls. In addition, the JG cells in the treated cKO mice generally did not acquire the plump appearance of the control JG cells (Fig. 4B). Furthermore, the average number of positive cells per JGA (1.8–2.5 cells/JGA) in the cKO was still fewer than the usual 3 to 4 cells/JGA found in untreated control animals and was little changed from the untreated cKO (1.5 cells/JGA). In addition, the treated cKO mice evidenced much less renin expression in smooth muscle cells (SMCs) along the afferent arterioles: there were 77% fewer afferent arterioles with extension of renin expression compared with the response in treated control mice (Fig. 3D). Although there was a range of renin expression in the renin-positive afferent arterioles in the cKO mice, most exhibited thinner renin-positive cells and the cells were confined closer to the glomerulus in contrast with the control afferent arterioles, which often had plump renin cells along a significant portion of their length (Fig. 4D). Thus it appears that some JG-localized renin cells in the cKO animals were able to somewhat increase their renin expression. However, the cells of the renin lineage along the renal arterioles, which are the source of recruited renin-expressing cells in control mice, responded poorly or not at all. The cKO mice have a severely blunted recruitment response indicating the impossibility for these mice to transform any other cell in the kidney to a renin cell.
The present study shows that the histone acetyl transferases CBP and p300 are essential for maintenance of renin cells in adult mice and, more importantly, for the ability of adult renin cells to respond to a homeostatic challenge.
Our previous study of 2- to 4-month-old adult cKO mice showed that at this age the reduction in renin and the morphological alterations were extensive and severe. However, it was not clear whether deletion of CBP and p300 affected the establishment of the normal endowment of renin cells. The finding that cKO animals at 5 days of age have normal renin mRNA expression, protein localization, and kidney morphology suggests that this initial endowment of renin cells is not affected. This is perhaps not surprising since the expression of cre in renin cells, which is required for deletion of CBP and p300, is dependent on renin promoter activation. It might be expected, then, that the initial population of renin cells would be established normally as the first expression of renin is in cells having CBP and p300 intact. However, if the loss of CBP/p300 immediately halted the development of that initial cell, there would be an effect on the endowment of renin cells. The results in newborn animals show that the initial endowment of renin cells in the cKO is normal and, furthermore, that there is no significant depletion of these cells during embryonic development and early postnatal life. It is possible that the cre-induced depletion of CBP/p300 levels has only a modest effect on cells that transiently express renin during development and that diminution of renin expression in JG cells is effected when the adult state is achieved. The results in 1-month-old mice support this interpretation. By 1 month of age significant changes have occurred. Renin mRNA and protein expression are substantially reduced and kidney morphological abnormalities are evident, although not as extensive as in the older cKO mice. Thus, as demand for renin expression by the JG cells continues with maturation, the capacity of the cells to synthesize it diminishes, suggesting that the role of CBP/p300 is likely more important for the maintenance of the renin phenotype in these cells.
Given that CBP/p300 are such powerful regulators of multiple cellular processes, it is important to determine whether the decrease in renin cell number was due to cell death. This possibility seems unlikely: the number of apoptotic cells in N5 cKO kidneys was not different from controls and in the N30 cKO kidneys apoptotic cells were few in number and only found in the tubules, which have evident abnormalities at this age, but not in the JGAs or vessels (not shown). It is also possible that lack of CBP/p300 causes more renin cell descendants to enter the pathway to SMC, mesangial, and interstitial cells, of which renin cells are progenitors, thus diminishing the JG cell population. Lineage tracing studies would be required to determine whether this occurs. However, if more cells had entered the SMC pathway, there would be a larger population of renin-lineage cells to potentially mobilize for renin re-expression in response to a homeostatic challenge. If lack of CBP/p300 reduces their capability to re-express renin, we would predict a phenotype similar to the one we observed: inability of the SMCs to revert to the renin phenotype in response to a homeostatic challenge.
In the adult cKO mice all measures of renin expression were significantly reduced. PRC was 52% of the control and renin mRNA and protein (as reflected by the JG index) were even further diminished. The cKO adult JG index reported here (11%), which is higher than in our previous report (2%) (11), includes more animals and shows the phenotypic heterogeneity. In addition the cKO adults had a lower BP than controls, indicating that their reduced renin expression is a primary effect of the lack of CBP/p300 and not due to blood pressure/hemodynamic alterations, which could suppress renin expression. The very low JG index and number of cells staining for renin in the cKO suggests that most of the renin made by these cells is secreted to the circulation in an attempt to maintain normal blood pressure.
The loss of renin expression with age and the phenotypic variability among cKO animals may reflect heterogeneity in the efficiency of complete deletion of all CBP and p300 alleles in individual renin cells. We showed in our previous report (11) that only in animals having both alleles of CBP and p300 floxed (making it possible to delete both of these genes in the renin lineage) is there an effect on renin expression and kidney morphology. Thus in the cKO mice there may be individual renin cells that retain some expression of CBP and/or p300. However, as the JG renin cells are called upon over time to express renin (and thus also express cre), deletion of CBP and p300 will eventually be complete in all of the renin cells and they can no longer make renin. We have seen a similar pattern of progression in our studies with conditional deletion of Dicer in renin cells using the same Ren1d-cre mouse to delete Dicer (unpublished).
As mentioned above, cells from the renin lineage differentiate into arteriolar smooth muscle cells, mesangial cells, interstitial cells, and a subset of tubular cells (24). The observed progression with age in the severity of the morphological defects in the vascular, interstitial, and tubular compartments may be due to the chronic lack of CBP and p300 in these renin cell descendants. In silico analysis of genes expressed in renin cells that we identified by mRNA microarray analysis (2) showed that a number of them have potential CREB binding sites in their promoters and thus can be regulated by CREB/CBP/p300. It is likely that in the renin cell descendants there is alteration of the expression of these genes, and this contributes to the progression of the morphological abnormalities.
The ontogeny studies demonstrate the importance of CBP/p300 for the maintenance of renin cells in the basal state. When fluid/electrolyte homeostasis is challenged in a normal animal, the re-establishment of homeostasis is mediated by an elevation in renin expression, mainly by increasing the number of cells expressing renin (15). The increase in the number of cells occurs by 1) increase in the number of JGAs expressing renin, 2) increase in the number of renin positive cells per JGA, and 3) re-transformation of (non-JGA) vascular SMCs upstream from the glomeruli along the afferent arterioles into renin-expressing cells (7; 24; 26; 27). We, therefore, tested whether CBP/p300 are important for the ability of cells of the renin lineage to respond in this manner to homeostatic challenge.
Sodium depletion + captopril treatment, which presents a very strong challenge to blood pressure and fluid-electrolyte homeostasis, caused the expected reduction in BP in control and cKO animals to the same level. It should be stated that as the animals reach a low BP, other compensatory mechanisms, such as enhanced sympathetic activity, come into play to maintain blood pressure at a level compatible with tissue perfusion when the action(s) of the renin angiotensin system are blocked. The observation that the blood pressure in the cKO did not drop further than the control is likely due to the fact that cKO mice have had all of their adult life to adapt to a renin depleted state before the challenge and have likely already activated other compensatory systems. This possibility remains to be tested.
In control animals PRC and kidney renin are highly elevated in an attempt to re-establish homeostasis. The cKO mice, however, exhibited a severely blunted response. Although the kidney renin mRNA and protein expression of the mice did increase over the basal state, indicating an attempt to increase renin, the response was inadequate, and they were unable to increase circulating renin. The renin-expressing JG cells in the cKO did not acquire the usual plump appearance of normal JG cells responding to a homeostatic challenge. Instead they retained the thin appearance seen in the untreated cKO mice, and so, despite having a small increase in renin positive JGAs, the cumulative effect was a minimal increase in renin. We have recently seen a similar response when RBP-J, the final effector of the Notch pathway, which has a binding site in the renin promoter, is deleted in renin cells (3).
In addition to the poor response of the JG cells, in the cKO mice very few of the cells of the renin lineage in the afferent arterioles were capable of re-expressing renin, and the afferent arterioles that did exhibit re-expression generally had fewer and thinner renin cells. The observation that the arteriolar SMCs in the cKO cannot respond by appropriately re-expressing renin supports the importance of the role of CBP and p300 in this process. The continued presence of CBP/p300 is required for aSMCs of the renin lineage to retain the plasticity to de-differentiate into renin cells when the need arises. In the absence of these proteins these cells may become terminally differentiated, precluding a return to the endocrine, renin-expressing phenotype. Alternatively, the chronic lack of CBP/p300 may alter the fate of renin cells so that they differentiate into cells not normally part of the renin lineage and thus cannot respond. Analysis of the renin cell lineage in the cKO mice would be required to address this question. Taken together these observations underline the importance of CBP and p300 in the ability of renin cells to re-express renin when the need arises and reveal the crucial importance of the CREB/CBP/p300 complex for renin cells to retain the ability to respond to a threat to homeostasis.
In summary, these studies confirm the important role of the histone acetyltransferases, CBP and p300, in renin cells. CBP and p300 are crucial for renin cells to maintain their ability to express renin during adult life and to retain the plasticity to return to the endocrine renin phenotype and re-express renin in response to a homeostatic challenge.
This work was supported by National Institutes of Health Grants R37-HL-066242 and R01-HL-096735 to R. A. Gomez and K08-DK-75481 to M. L. S. Sequeira Lopez.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: E.S.P., M.L.S.S.-L., and R.A.G. conception and design of research; E.S.P., M.C., O.A.C., A.E.T., and M.L.S.S.-L. performed experiments; E.S.P., M.C., A.E.T., M.L.S.S.-L., and R.A.G. analyzed data; E.S.P., A.E.T., M.L.S.S.-L., and R.A.G. interpreted results of experiments; E.S.P. prepared figures; E.S.P. drafted manuscript; E.S.P., M.L.S.S.-L., and R.A.G. edited and revised manuscript; E.S.P. and R.A.G. approved final version of manuscript.
We thank Kim Hilsen-Durette for technical assistance and for breeding the mice, Dr. Paul Brindle and St. Jude's Children's Research Hospital for providing the floxp300 mice, Dr. Jan van Duersen (Mayo Clinic) for providing the floxCBP mice, and Dr. Silvia Medrano for helpful comments on the manuscript.
- Copyright © 2012 the American Physiological Society