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Recombination Signal Binding for Ig-kJ Region Regulates Juxtaglomerular Cell Phenotype by Activating the Myo-Endocrine Program and Suppressing Ectopic Expression

† † ‡ Ruth M. Castellanos-Rivera,* Ellen S. Pentz,* Eugene Lin,* Kenneth W. Gross, † Silvia Medrano,* Jing Yu,§ Maria Luisa S. Sequeira-Lopez,* and R. Ariel Gomez*

*Department of Pediatrics, School of Medicine, †Department of Biology, Graduate School of Arts and Sciences, and §Department of Cell Biology, University of Virginia, Charlottesville, Virginia; and ‡Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York

ABSTRACT Recombination signal binding protein for Ig-kJ region (RBP-J), the major downstream effector of Notch signaling, is necessary to maintain the number of renin-positive juxtaglomerular cells and the plasticity of arteriolar smooth muscle cells to re-express renin when homeostasis is threatened. We hypothesized that RBP-J controls a repertoire of that defines the phenotype of the renin cell. Mice bearing a bacterial artificial reporter with a mutated RBP-J binding site in the renin promoter had markedly reduced reporter expression at the basal state and in response to a homeostatic challenge. Mice with conditional deletion of RBP-J in renin cells had decreased expression of endocrine (renin and Akr1b7)and smooth muscle (Acta2, Myh11, Cnn1,andSmtn) genes and regulators of smooth muscle expression (miR- 145, SRF, Nfatc4, and Crip1). To determine whether RBP-J deletion decreased the endowment of renin cells, we traced the fate of these cells in RBP-J conditional deletion mice. Notably, the lineage staining patterns in mutant and control kidneys were identical, although mutant kidneys had fewer or no renin- expressing cells in the juxtaglomerular apparatus. Microarray analysis of mutant arterioles revealed upreg- ulation of genes usually expressed in hematopoietic cells. Thus, these results suggest that RBP-J maintains the identity of the renin cell by not only activating genes characteristic of the myo-endocrine phenotype but also, preventing ectopic and adoption of an aberrant phenotype, which could have severe consequences for the control of homeostasis.

J Am Soc Nephrol 26: 67–80, 2015. doi: 10.1681/ASN.2013101045

Juxtaglomerular (JG) cells are crucial for the JG cells express a unique set of genes character- maintenance of BP and fluid/electrolyte homeosta- istic of both endocrine and SM cells.7 This dual sis. These cells act as sensors that constantly monitor endocrine–contractile phenotype is crucial for the the physiologic status of the animal and convey rapid regulation of BP and renal hemodynamics. information to adjacent cells within the JG appa- However, the factors controlling this dual phenotype ratus and along the renal arterioles. Under normal circumstances, renin secretion by JG cells suffices to maintain homeostasis. However, when there is a Received October 4, 2013. Accepted April 4, 2014. more profound physiologic challenge, to restore Published online ahead of print. Publication date available at homeostasis, preexistent smooth muscle (SM) cells www.jasn.org. along the arterioles, mesangial cells, and interstitial Correspondence: Dr. R. Ariel Gomez, University of Virginia, School pericytes1 gradually dedifferentiate into renin- of Medicine, 409 Lane Road, MR4 Building 2001, Charlottesville, producing cells in a pattern resembling the one VA 22908. Email: [email protected] – found in fetal life.2 6 Copyright © 2015 by the American Society of Nephrology

J Am Soc Nephrol 26: 67–80, 2015 ISSN : 1046-6673/2601-67 67 BASIC RESEARCH www.jasn.org and the mechanisms that regulate the ability of SM cells along Mut-BAC than treated WT-BAC mice (Figure 1F). As expec- the afferent arterioles to reacquire the renin phenotype have ted, renin staining and JGA index for renin were similar in not been fully defined. both groups, indicating that introduction of the BAC trans- JG cells and adjacent cells along the arterioles express all the gene did not disrupt the expression of the endogenous renin components of the Notch signaling pathway,7 an ancestral cell– gene (Figure 1, E and F). Together, these data indicate that cell communication system involved in cell fate decisions. We RBP-J regulates the renin promoter directly (Figure 7A) and hypothesized that the Notch pathway through its major tran- is involved in the ability of SM cells along the arteriole to scriptional effector, recombination signal binding protein for reacquire the renin phenotype. Ig-kJ region (RBP-J), maintains the identity of the JG cell and the ability of cells along the arterioles to regain the renin phe- RBP-J Deletion Does Not Affect the Endowment of notype when homeostasis is threatened. In fact, mice with Cells from the Renin Lineage conditional deletion of RBP-J (conditional knockout [cKO]) To determine whether the marked diminution in the number in cells from the renin lineage have very few JG cells, and SM of JG cells resulted from a decreased population or a change in cells along the afferent arterioles cannot reacquire the renin the distribution of cells from the renin lineage, we performed phenotype on a physiologic challenge.8 However, it was lineage studies in cKO and control mice harboring the R26R10 unclear whether in vivo Notch/RBP-J regulated the renin reporter. After cre-mediated recombination in the control and promoter directly and/or the expression of genes known to cKO mice, cells of the renin lineage express b-galactosidase be characteristic of or responsible for the dual endocrine– (b-gal), effectively labeling renin cells and their descendants. contractile phenotype of the renin cell. Therefore, we We found that, as expected, these RBP-J cKO;R26R mice had designed a series of experiments to test the hypothesis reduced renin expression (Supplemental Table 1) as previ- that RBP-J regulates a gene network that controls the dual ously described in cKO mice lacking the reporter.8 Interest- endocrine–contractile identity of the JG cell and the ability ingly, the distribution of b-gal–positive cells was identical in of cells upstream from the glomerulus to reacquire the renin the control and cKO kidneys (Figure 2), and the b-gal JGA phenotype. indices were the same (Supplemental Figure 1). In contrast to controls, costaining for renin verified that cKO mice had few or no renin-expressing cells in the JGAs, but they were still b-gal– RESULTS positive (Figure 2F, Supplemental Table 1). Furthermore, assays for apoptosis (Supplemental Table 2) and cell proliferation (not RBP-J Activates the Renin Promoter shown) showed no difference between control and cKO mice. To determine whether RBP-J directly affects renin expression, These data indicate that the decrease in the number of renin- we used a bacterial artificial chromosome (BAC) system to expressing cells was not caused by an increase in the percentage generate control wild-type BAC (WT-BAC) transgenic mice, in of dead cells or a decrease in the number and/or location of the which the first exon of the Ren1c gene was replaced with an renin precursors and subsequent progeny of renin-derived enhanced green fluorescent protein (GFP), and mutant BAC cells. Therefore, former renin-expressing cells and their de- (Mut-BAC) mice, in which the four nucleotides in the RBP-J scendants are still present in the appropriate locations in cKO consensus sequence critical for its binding9 were substituted in mice, although they are no longer capable of expressing renin, the BAC construct (Figure 1A). suggesting the possibility that they have adopted a different We studied two independent transgenic lines each contain- phenotype. ing two copies of the WT-BAC or Mut-BAC transgene. In the basal state, Mut-BAC mice had 87% lower GFP mRNA RBP-J Deletion Affects the Myo-Endocrine Phenotype expression than WT-BAC mice (Figure 1B). To investigate of Cells of the Renin Lineage whether Mut-BAC mice can increase the expression of GFP Given that cells of the renin lineage were still present in the under a physiologic threat, we treated Mut-BAC and WT-BAC appropriate locations in RBP-J cKO mice, suggesting that they mice with captopril and sodium depletion for 5 days, a ma- may have switched their phenotype, led us to investigate nipulation known to induce arteriolar SM cells upstream from whether repression of renin was accompanied by alterations the JG area (JGA) to reacquire the renin phenotype. Treated in the expression of other genes characteristic of renin cells.7 Mut-BAC mice had diminished capacity to increase GFP ex- Aldo-keto reductase 1b7 (Akr1b7), an independent endocrine pression (only 30% of that in treated WT-BAC mice) (Figure marker of JG cells, is coexpressed at almost the same level as 1C). In correspondence with the smaller increase in GFP ex- renin in JG cells throughout development and in response to pression in the treated Mut-BAC mice, immunohistochemis- physiologic manipulations.7 Akr1b7 immunostaining in cKO try for GFP showed that, in contrast to treated WT-BAC mice, mice was markedly diminished with respect to controls (Fig- treated Mut-BAC mice did not have GFP-positive cells up- ure 3A), and quantitation showed a significantly lower Akr1b7 stream of the arterioles distant from the glomeruli (Figure JGA index (Figure 3B). The decrease in the number of JGAs 1D). Quantification of the immunostaining results showed expressing Akr1b7 in the cKO mice was accompanied by a re- that the JGA index for GFP was significantly lower in treated duction in Akr1b7 mRNA expression to the same level as renin

68 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 67–80, 2015 www.jasn.org BASIC RESEARCH

Figure 1. RBP-J regulates the renin promoter in vivo. Mutation of the RBP-J site in the renin promoter diminishes GFP expression, a surrogate of renin expression. (A) Diagram of the BAC reporter construct used to generate BAC transgenic mice. The dual kanamycin/ streptomycin selection strategy was used to replace the first exon of renin with enhanced GFP, insert the renin gene 39 untranslated region (UTR), and modify the RBP-J binding site (nucleotides are in red). (B) Quantitative RT-PCR for GFP mRNA expression in kidneys from mice harboring the mutated RBP-J reporter (Mut-BAC) is significantly lower than in the control (WT-BAC) mice at basal state. (C–F) Mice were treated with low sodium diet+captopril for 5 days to induce re-expression of renin along arterioles. (C) Mut-BAC mice do not increase GFP mRNA to the same level as WT-BAC mice. (D and E) Immunohistochemistry for GFP and renin expression in response to homeostatic challenge. (D) In WT-BAC mice, GFP (brown) is in JG cells, small arterioles, and along the arterioles, recapitulating the renin pattern. Mut-BAC mice have no GFP along the arterioles and few GFP-positive JGAs (arrows). (E) Renin staining in WT-BAC and Mut-BAC mice shows that, as expected, the endogenous, nonmutated renin gene responds by increasing the number of renin-positive cells along the arterioles properly and equally in both groups. *Glomeruli with GFP-positive or renin-positive JGA. (F) The JGA index for renin in both transgenic mice shows that harboring the BAC transgene does not affect expression of the endogenous renin gene. Values are means6SEMs. **P,0.01; ***P,0.001. mRNA (Figure 3C). The effects of RBP-J on the endocrine chain11 (SM-MHC), a-SM actin (a-SMA), and calponin1 phenotype of the JG cell are illustrated in Figure 7A. (Cnn1) showed decreased expression in cKO mice: cells were The expression of SM genes is an important defining thinner with lesser amounts of staining (Figure 3E), particu- characteristic of the JG cells, which must have contractile larly in the interlobular arteries and upstream of the afferent function to properly regulate renal hemodynamics.7 We eval- arterioles. Interestingly, the larger intrarenal arteries had fewer uated the effect of RBP-J deletion on expression of specificSM SM-MHC–positive cells, whereas staining for a-SMA and genes in the JG and the upstream segments of the afferent Cnn1 was similar to controls (Figure 3E). Although immu- arterioles and along larger intrarenal arteries (diagram in Fig- nostaining for a-SMA and Cnn1 in large arteries appeared to ure 3D). Immunohistochemistry for SM myosin heavy not be different from controls, their expression was

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whether the expression of SM genes was specifically affected in the cells of the JGA. Using double immunostaining for renin and a-SMA (Figure 4), we found that a sig- nificant proportion of JGAs in the cKO mice was not stained with either antibody. We quantified the costaining for renin and a-SMA as follows. Five JG cells/JGA were scored and divided into four categories based on the expression of one or both markers or no expression: (1) renin only, (2) a-SMA only, (3) overlapping staining (renin and a-SMA), and (4) unstained cells (Figure 4, B–D). As expected, the cKO mice had a significant decrease in the number of renin-positive cells (Figure 4C). Interest- ingly, cKO mice had a 2.9-fold increase in the total number of unstained (with either antibody) cells compared with controls (Figure 4C). We then took a closer look at the unstained category in which five cells/ JGA were evaluated and found that cKO animals had a significant number of JGAs with two to five cells that have lost the ex- pression of renin and SM (Figure 4D). Furthermore, double staining for Cnn1 and renin showed a similar result: a Figure 2. RBP-J deletion does not affect the endowment of cells from the renin lineage. 2.4-fold increase in the number of un- RBP-J R26R Kidneys from control and cKO; adult mice were subjected to the X-gal re- stained JGA cells in cKO mice (Supplemental action to detect b-gal, which marks cells that expressed renin earlier in development +/+ Figure 2). Altogether, these data indicated (blue). (A and B) In RBP-J control kidneys, blue staining is seen in the JGA (JG), along the afferent arteriole (aa), and in a large vessel (V). (C and D) RBP-J cKO kidneys have the that deletion of RBP-J in JG cells affects not same blue staining pattern as controls. (E and F) Double staining for b-gal (blue) and only expression of renin and Akr1b7 but renin (brown). Whereas b-gal labels the developmental history of renin expression (thus, also, the expression of genes characteris- blue staining is observed along arterioles and glomeruli), the actual expression of renin tic of the SM phenotype, suggesting that (brown) in adult animals is confined to JG cells. In cKO mice, the distribution of blue cells RBP-J also maintains the contractile ma- in the JGA and arterioles is not different from controls; however, cKO mice have fewer chinery of the JG cells. This point is illus- renin+ JGAs (arrows) than in control mice. *Glomeruli with renin+ JGAs. trated in Figure 7A. diminished in arterioles (Figure 3E). Thus, deletion of RBP-J RBP-J Regulates the SM Program of the JG Cell has a differential effect on the expression of SM proteins de- We previously reported that microRNAs (miRs), a group of pending on the blood vessel size, with greater effect on the small noncoding RNAs,11 are necessary to maintain the phe- expression of SM-MHC in large arteries than a-SMA and notype of renin-expressing cells and the integrity of the kidney Cnn1. vasculature.12 Given that miR-145–5p is prominently ex- The decreased expression of SM markers in the upstream pressed in the kidney arterioles and may be important to sus- portion of arterioles was accompanied by lower mRNA levels. tain the SM phenotype of JG cells, we evaluated whether Measurement of a-SMA, SM-MHC,andsmoothelin (a marker expression of miR-145–5p was altered by deletion of RBP-J. for terminally differentiated SM cells) using quantitative RT- In situ hybridization for miR-145–5p in RBP-J cKO mice PCR and semiquantitative PCR in total kidney and/or isolated revealed a significant decrease in the number of miR-145– arterioles showed that, in cKO mice, total kidney a-SMA 5p-positive cells in the JGA and renal arterioles (Figure 5A) mRNA was 44% lower than in controls (Figure 3F), and in and a significantly lower miR-145–5p JGA index (Figure 5B). isolated arterioles, expression of a-SMA, SM-MHC,and Toexamine whether deletion of RBP-J affects the expression smoothelin was 2.4-, 1.5-, and 2.3-fold lower, respectively, of additional key SM regulators, we studied the expression of compared with controls. (Figure 3, G–I). SRF, Crip1, and NF of activated T cells 4 (Nfatc4). SRF binds The observation that the expression of the SM genes was the CArG box in the promoters of SM genes to activate tran- reduced led us to further investigate in topological detail scription. SRF forms a complex with myocardin,13,14 which is

70 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 67–80, 2015 www.jasn.org BASIC RESEARCH

Figure 3. Deletion of RBP-J affects expression of genes marking the dual endocrine and SM phenotype of renin cells. (A–C) Expression +/+ of Akr1b7, an endocrine phenotype marker. (A) Immunostaining for Akr1b7 shows that RBP-J mice express Akr1b7 in the JGAs (arrows in left panel). RBP-J cKO mice have few or no Akr1b7-positive JGAs (arrow in right panel). (B) In RBP-J cKO mice, the JGA indices for both Akr1b7 and renin are significantly reduced. (C) Akr1b7 and renin mRNA levels in RBP-J cKO mice decreased signif- icantly to the same extent compared with controls. (D–I) Expression of SM phenotype markers assessed by (D and E) immunostaining and (F–I) mRNA expression. (D) The sketch illustrates the location of JG cells (yellow) in the adult animal and the location of VSMCs upstream in the afferent arterioles, where expression of SM genes is reduced in RBP-J cKO mice. The diagram also indicates that the SM cells (SMCs) have the plasticity to re-express the renin phenotype when confronted with a homeostatic threat. (E) Consecutive sections stained with renin and SM-MHC show that RBP-J cKO mice had few renin-positive cells, and the expression of SM-MHC was markedly reduced, particularly in the interlobular arteries and upstream portion of the afferent arterioles. Consecutive sections stained for a-SMA and Cnn1 show that a-SMA staining upstream in the afferent arterioles is thinner with decreased intensity and extent of expression. Cnn1 staining shows an overall decrease in the number of Cnn1-positive cells. In large arteries (right panels), cKO mice had fewer SM-MHC–positive cells around the artery, but localization of a-SMA and Cnn1 was not different from controls. Notice that

J Am Soc Nephrol 26: 67–80, 2015 RBP-J and Renin Cell Fate 71 BASIC RESEARCH www.jasn.org positively regulated by miR-145–5p and also modulates miR- significantly decreased in arterioles from cKO mice (Supple- 145–5p in a positive feedback loop (Figure 7A).15,16 SRF mental Figure 4). Results from the microarray analysis were mRNA expression in cKO mice was significantly lower than striking: arterioles from cKO mice had increased representa- in controls (Figure 5C). To further investigate whether expres- tion of genes that belong to the immune response system (Fig- sion of SRF is altered by deletion of miR-145–5p,weper- ure 6A). A closer look at the top significantly upregulated formed quantitative RT-PCR for SRF in miR-145–5p KO genes in the cKO arterioles shows that many of these genes mice. Results show that kidney cortex from miR-145–5p KO are normally expressed in hematopoietic cells (B cells: Ccl9, Il6, mice had significantly reduced SRF expression (Figure 5D), Fcamr, Bcl2a1c, Igk-C, Rgs1, H2Aa,andCd72; T cells: Timd2, suggesting that both RBP-J and miR-145–5p are necessary to Ccl9, Il6,andCcl5) (Figure 6B, Supplemental Table 3). We maintain SRF expression in the kidney vasculature. In addi- performed RT-PCR analysis and confirmed the increased tion to the SRF–myocardin complex, SRF associates with the expression of Timd2, Ccl2, Ccl5, Fcamr, and Abp1 in kidneys SM marker Crip1, a double zinc finger-like molecule that me- from cKO mice (Figure 6C). These genes and numerous other diates protein–protein interactions to promote the contractile upregulated genes are involved in B cell differentiation, direct- phenotype15,17 (Figure 7A). Crip1 distribution in cKO mice ing migration of monocytes, macrophages, or T cells, and im- was diminished in both JGA and arteriolar SM cells, indicating mune and inflammatory responses. Thus, these data suggest that RBP-J controls the expression of another key regulator of that cells of the renin lineage lacking RBP-J now are expressing the SM phenotype (Supplemental Figure 3). genes normally confined to cells of the hematopoietic/immune Nfactc4, another gene known to be important in regulating response system. the expression of SM genes, is a that mod- ulates SM gene transcription in a Ca2+- dependent manner18 Discussion (Figure 7A). We found that cKO mice had a significant de- This series of studies shows that RBP-J, the main effector of the crease in the number of JGAs positive for Nfatc4 compared Notch pathway, (1) controls the genetic program that confers with controls (Figure 5F). Interestingly, in Nfatc4-negative the dual endocrine–contractile phenotype of the JG cell, (2) JGAs (controls as well as cKO), their corresponding arterioles sustains the memory of SM cells upstream in the arterioles to had Nfatc4-positive cells farther upstream from the glomeruli reacquire the renin phenotype, and (3) suppresses the extem- (Figure 5E). Although expression of Nfatc4 was heteroge- poraneous ectopic expression of genes from other lineages. neous, the distance from the junction where the arteriole en- These findings are illustrated in Figure 7. ters the glomerulus to the beginning of the Nfatc4-positive The expression of two crucial genes characteristic of the JG cells along the afferent arteriole (diagram in Figure 5E) was cell endocrine phenotype, renin and Akr1b7, is profoundly 1.6-fold greater in cKO mice (Figure 5G). Thus, control and diminished by RBP-J deletion in both the basal unstressed cKO mice exhibit heterogeneous Nfatc4 distribution within state and response to the homeostatic challenge of sodium the afferent arteriole, but deletion of RBP-J results in a de- depletion and captopril treatment. Using BAC-GFP reporter crease in the number of Nfatc4-positive JGAs and fewer mice, we show that RBP-J directly regulates the renin pro- Nfatc4-positive cells along the renal arterioles. moter in vivo. This effect is manifested in the unstressed animal as well as in response to the physiologic challenge men- Deletion of RBP-J Alters the Fate of Cells from the tioned above, indicating that RBP-J controls the phenotype of Renin Lineage the JG cell as well as the response of the SM cells upstream In an attempt to define the identity of the JG cells that have lost from the glomerulus. These results agree with in vitro studies their endocrine–contractile phenotype, we screened for the showing that RBP-J activates the renin promoter.19 Using expression of markers characteristic of stem cells (Oct-4 and chromatin immunoprecipitation, we showed that RBP-J is ex- Nanog) and mesenchymal (vimentin), endothelial (PECAM), pressed in JG cells and binds the renin promoter,7 and now, it and epithelial (cytokeratin) cells but found no changes in provides a functional correlate in vivo. In other systems, RBP-J staining in the JGA of cKO mice (data not shown). Given those is a repressor or activator depending on the tissue context.20 negative results and with the goal of defining the repertoire of Our results show, for the first time, that, in vivo, RBP-J acts as a genes enriched in renin lineage cells lacking RBP-J,weper- direct transcriptional activator of the renin gene. formed mRNA microarray analysis in purified arterioles from In addition to renin, RBP-J regulates the expression of control and cKO mice. We first confirmed by semiquantitative Akr1b7, a newly discovered marker for renin cells.7 Akr1b7, PCR that, as expected, renin and RBP-J expressions were which also possesses RBP-J binding sites in its promoter, is

SM-MHC in mutant mice had a scant and dot-like distribution of SM-MHC–positive cells around the artery. (F) a-SMA mRNA expression was reduced to 56% of control in kidneys from cKO mice. (G–I) Semiquantitative PCR in isolated kidney arterioles showed that a-SMA, SM-MHC,andsmoothelin expression decreased 2.4-, 1.5-, and 2.3-fold, respectively, in the RBP-J cKO mice. Values are means6SEMs. Scale bars, 50 mm. *P,0.05; **P,0.01; ***P,0.001.

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expressed in kidney cells programmed to the renin phenotype, even when the renin gene has been deleted.7 Akr1b7 belongs to the Akr superfamily of enzymes that catalyzes the reduction to alcohol of harmful aldehydes and ketones generated by hor- mone synthesizing cells.21 This detoxifying function of Akr1b7 seems crucial to protect renin cells from those harmful com- pounds and promote cell survival. In the cKO mice, Akr1b7 mRNA and protein expression are reduced in parallel with renin. This finding is in agreement with our previous obser- vation that Akr1b7 is expressed together with renin in JG cells. When there is a challenge to BP/fluid electrolyte homeostasis, there is a recruitment of cells coexpressing both proteins (Akr1b7+/renin+ cells) along the kidney vasculature, indicat- ing that SM cells along the arterioles, upstream from the glo- merulus, are again transformed to the endocrine phenotype, a situation resembling the fetal pattern of expression for both genes. Interestingly, we have found that, when the renin gene is deleted, there is still an unsuccessful attempt to increase the number of renin cells. The expression of Akr1b7 in those cells is simultaneously upregulated along the arterioles, indicating that Akr1b7 is part of a genetic program that coactivates both genes as well as the machinery that controls renin granule formation when the endocrine phenotype is turned on.7,8 Un- til recently, the molecules that governed the expression of the endocrine phenotype of the JG cell were unknown. Our find- ings indicate that RBP-J is at least one of such molecules and that RBP-J is fundamental to sustain the endocrine status of the renin cell (Figure 7A). The actions of RBP-J are not limited to the endocrine functions of the renin cell. In addition to renin, JG cells express numerous SM genes7 crucial to maintain the contractile phe- notype required to regulate glomerular blood flow and re- sponse to physiologic cues. Here, we show that RBP-J deletion affects the expression of SM genes and their master regulators. Expression of SM genes was markedly reduced in afferent ar- terioles and interlobular arteries. Interestingly, SM-MHC ex- pression was severely decreased in large arteries, indicating a heterogeneous response of the SM genes along the kidney mi- crovasculature. Of note, SM-MHC has five predicted RBP-J binding sites in its promoter, whereas both a-SMA and Cnn1 have only one site (Figure 7A), suggesting that the number of RBP-J binding sites may influence the level of expression of individual SM genes. Additional studies are needed to answer this question. In agreement with the present results, other studies show that Notch signaling by RBP-J directly and/or

Figure 4. Deletion of RBP-J increases the number of JG cells that had an increased number of unstained cells. (D) The number of express neither renin nor SM markers. (A) Double staining for JGAs with one to five cells unstained with either antibody. RBP-J renin (brown) and a-SMA (purple) shows that cKO mice have few cKO mice had more JGAs with two to five unstained cells, whereas or no renin-positive JG cells (arrows) and diminished or absent the number of JGAs with one unstained cell is similar in RBP-J cKO a-SMA expression in JGAs. *Glomeruli. (B) Five cells in each JGA and control animals. In total, 219 JGA cells were counted in the were counted and categorized as (1)renin+ only, (2) a-SMA+ only, controls (n=2), and 216 JGA cells were counted in the mutants (3)bothrenin+ and a-SMA+,and(4) not stained. (C) A summary of (n=2). Each tick indicates a JGA. Cells in yellow are cells stained the number of cells in each category shows that RBP-J cKO mice with either antibody.

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indirectly affects adult vascular SM cell (VSMC) differentiation in a cell type- specific manner and that RBP-J has a role in activating the SM-MHC and a-SMA genes in VSMCs.22,23 In silico analyses showed that multiple SM genes expressed in the JG cells have RBP-J binding sites (Figure 7A). Although RBP-J can directly control SM gene transcription,22,23 it is likely that RBP-J also regulates a network that reinforces the acquisition and mainte- nance of the JG cell SM program. We, there- fore, examined whether lack of RBP-J affected the expression of key upstream reg- ulators of the SM program. One such regu- lator is miR-145–5p, which is important for muscle development in other systems24 and specifically expressed in JG cells and SM cells along the afferent arterioles.12 We found reduced miR-145–5p expression in cKO renal arterioles and JG cells, suggesting that RBP-J regulates miR-145–5p directly through its promoter. In fact, the human miR-145–5p promoter has seven predicted RBP-J binding sites,25 and studies in human aortic VSMCs showed that activation of the canonical Notch signaling pathway is re- quired to maintain miR-145–5p transcrip- tion.25 These findings, together with our observations, suggest that miR-145–5p functions to maintain the SM phenotype of JG cells and arteriolar VSMCs. miR- 145–5p may promote the contractile phe- notype by upregulating myocardin,25 which forms a complex with the transcription fac- tor SRF to activate SM genes13 (Figure 7A). Increasing miR-145–5p expression in fibro- blasts in vitro reprograms them into VSMCs, in part by the positive effect of miR-145–5p on myocardin in a feedback loop in which miR-145–5p upregulates myo- RBP-J miR-145–5p cardin, allowing the formation of the Figure 5. Deletion of reduces expression of and its downstream – SM gene targets. (A) In situ hybridization for miR-145–5p. In control mice, miR-145–5p SRF myocardin complex, and subsequent – is expressed in SM cells along the arterioles and in major arteries (arrows), whereas the SRF expression reinforces miR-145 5p to 15 RBP-J cKO mice exhibit a significant decrease in the overall expression of miR-145– maintain myocardin expression. In fact, 5p. (B) In addition, the number of JGAs positive for miR-145–5p was decreased. (C) lack of RBP-J resulted in a decrease in SRF SRF mRNA expression is reduced significantly in both RBP-J cKO and miR-145–5p KO mRNA, suggesting the possibility that mice. (D–G) Nfatc4 expression in RBP-J cKO mice. (E) Staining for Nfatc4 showed that RBP-J deletion decreased miR-145–5p some JGAs (arrows) were Nfatc4-positive and that some were negative; however, the upstream portion of the arterioles was positive. The diagram illustrates the distribution of Nfatc4-positive cells. (F) JGAs with a visible arteriole were examined in control and cKO mice, and it was seen that the percent of Nfatc4-positive JGAs is reduced in cKO mice, the majority of the Nfatc4-positive RBP-J cKO mice. (G) To define whether RBP-J deletion altered the distribution (distance) cells were farther upstream of the JGA com- of Nfatc4-positive cells from the glomerulus, we measured the distance from the JGA to pared with controls. Values are means6SEMs. the first Nfatc4-positive cells along the arteriole upstream from the glomerulus. In RBP-J **P,0.01; ***P,0.001.

74 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 67–80, 2015 www.jasn.org BASIC RESEARCH

Figure 6. Genes associated with the hematopoietic immune response are upregulated in arterioles from RBP-J cKO mice. (A) analysis of the mRNA microarray data in isolated arterioles from RBP-J cKO mice shows increased representation of biologic processes related to the immune response. (B) Immune response-related genes upregulated in RBP-J cKO arterioles. (C) Validation of upregulated genes by PCR of RNA from control and RBP-J cKO kidneys. GAPDH was used for normalization. levels, and therefore, miR-145–5p could not reinforce expres- miR-145–5p KO mice suggest that RBP-J and miR-145–5p sion of SRF. In support of this conclusion, we found that miR- jointly regulate SRF expression and therefore, SM expression 145–5p KO mice had a marked decrease in SRF expression, in JG cells. thus supporting the hypothesis that miR-145–5p serves as a Our laboratory has shown that Nfatc4 is expressed in JG positive regulator of SRF and the SM phenotype in JG cells cells as well as the kidney arterioles, suggesting that Nfatc4 (Figure 7A). Furthermore, our data showing that SRF mRNA contributes to maintenance of the contractile phenotype.7 levels are decreased significantly in both RBP-J cKO and Nfat proteins are transcription factors important for the

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development of the cardiovascular sys- tem26 and the migration and proliferation of SMCs.27 Nfatc4 also positively regulates a-SMA expression through its promoter.28 We found that the number of Nfatc4- positive cells in cKO mice is diminished sig- nificantly in the JGA and along the afferent arterioles. Our observation that Nfatc4 de- creased in cKO mice provides an additional link between miR-145–5p and Nfatc4 activ- ity, because Nfatc4 is a target of miR-145– 5p (Figure 7A).29 It is likely that decreased Nfatc4 in the RBP-J cKO is caused by di- minished miR-145–5p expression, al- though additional experiments will be required to confirm whether miR-145–5p directly regulates Nfatc4 in JG cells. Remarkably, the decreased number of renin-expressing cells found in RBP-J cKO mice was not accompanied by apoptosis, necrosis, and/or changes in proliferation. More important, lineage tracing clearly showed that, in cKO mice, cells of the renin lineage were present in all of their normal locations. Altogether, those findings indi- cate that cells from the renin lineage lack- ing RBP-J did not die but switched their phenotype to another cell type. Because Notch/RBP-J has been proposed to control binary cell fate decisions,30,31 we expected Figure 7. RBP-J acts as a master regulator that maintains the identity of the JG cell. (A) that suppression of renin would simply RBP-J regulates a network of genes that confers the endocrine–contractile phenotype cause a switch to the SM phenotype. How- of the JG cell. Red bars located in the promoters of target genes represent RBP-J ever, this result was not the case. In fact, binding sites. Endocrine genes, such as renin and Akr1b7, and generation of renin expression of SM genes (and the expression granules are indicated on the left side of the diagram. Genes necessary to maintain of their main regulators) was also markedly the contractile phenotype are indicated on the right side. RBP-J regulates the SM suppressed. How can these findings be bet- genes directly through their promoter regions and indirectly by upregulating the ter understood? Although the phylogenet- expression of miR-145–5p and SRF. miR-145–5p positively regulates SRF, myocardin, ically conserved role of Notch is to link the Nfatc4 15 SRF and , and they act together to activate the expression of SM genes. binds fate choice of one cell to that of neighbor- SRF miR-145–5p to CArG sites located in the SM genes. Given that is an target gene ing cells, this outcome is contextual, de- and has two RBP-J sites in its promoter, it is likely that both miR-145–5p and RBP-J pending on the proliferative state, the regulate the transcriptional activity of SRF.Inaddition,miR-145–5p also promotes the expression of SM genes by repressing , a transcription factor that forms a complex degree of differentiation of interacting with SRF to prevent transcriptional activity of SM genes.46 Crip1, another RBP-J cells, and the physiologic and maturational predicted target gene, is an SM marker known to form a complex with GATA 6 and status of the surrounding tissue where SRF to promote the contractile phenotype. We hypothesize that the canonical Notch those cells exist. Recent information from signaling pathway is involved in maintaining the myo-endocrine phenotype of the JG several laboratories clearly illustrates this cell. The ligand–Notch interaction (yet to be identified) results in the release point and helps in understanding our find- of the Notch intracellular domain (pink boxes), allowing its translocation to the nu- ings: we have recently shown that deletion cleus, where it binds RBP-J to activate transcription. CaM, calmodulin; Cn, calcineurin; of RBP-J in renal stromal Foxd1+ cells, the P, phosphate; Smtn, smoothelin. (B) RBP-J maintains the identity of the JG cells by not earliest progenitors for all the mural cells of only activating genes characteristic of their myo-endocrine phenotype but also, pre- the kidney vasculature,32 leads to decreased venting the undesirable ectopic expression of genes from other lineages. endowment of renin cells, mesangial cells, and SM cells, resulting in aberrant mor- phogenesis of arterioles and the formation of glomerular aneurysms.33 In this study,

76 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 67–80, 2015 www.jasn.org BASIC RESEARCH however, deletion of RBP-J in cells already committed to a differentiated (being, in fact, a pool of cells that can be called renin cell fate (and therefore, located downstream in the into action when more renin is needed to control homeosta- Foxd1 lineage hierarchy) does not result in overt morphogenetic/ sis).6 This hypothesis remains to be tested. developmental defects, but it does lead to a remarkable switch in Ourfindings also point to a repressor role of Notch/RBP-J. A the fate/identity of the adult JG cells. Thus, mutation of RBP-J in close look at the microanatomy of the afferent arterioles where the earliest progenitor leads to a decrease in the pool of descen- they abut the glomeruli showed that cKO mice have a dants, whereas deletion in the more differentiated renin precur- significant number of cells in the JG segment that expresses sors results in a significant change in the choice of cell identity. In neither SM nor renin. Those dually negative cells did not line with our own observations and contrary to the situation in express endothelial, epithelial, or stem cell markers. However, the embryo, Notch signaling in the adult gut influences cell fate arteriolar cells express transcripts characteristic of hemato- without altering morphogenesis.34 In those studies, ectopic poietic cells, indicating that, in renin cells, RBP-J normally Notch signaling in the adult mouse intestinal progenitors di- suppresses the ectopic expression of genes characteristic of rected cells away from a secretory fate, whereas in the embryo, other lineages. In retrospect, perhaps these findings should not Notch overexpression resulted in defects in gut morphogenesis. be as surprising: canonical Notch/RBP-J signaling is essential Altogether, the aforementioned experiments indicate that the for the development and function of lymphocytes,41 and in actions of Notch/RBP-J are influenced by the degree of and po- fact, JG cells share several transcription factors crucial for the tential for differentiation of the target cells. commitment and differentiation of hematopoietic cells, As mentioned above, our findings support the notion that including E2A, EBF1, Ikaros, and RBP-J.7 Altogether, this in- RBP-J sustains the memory of SM cells upstream in the formation suggests a remarkable conservation of the tran- arterioles to reacquire the renin phenotype. Under normal scriptional machinery and a possible lineage relationship circumstances, renin secretion from JG cells is sufficient to between these cell types. How RBP-J suppresses the expression respond to the minute-to-minute physiologicdemands of daily of hematopoietic genes in renin cells remains to be deter- life. However, when there is a more profound challenge to mined. However, it should be noted that a number of the homeostasis (such as sodium depletion and diminished upregulated genes in our cKO mice (e.g., Abp1, Ccl2, Ccl5, angiotensin actions as in these experiments), preexisting SM Ccl9, Fcamr,andTimd2) have in silico predicted RBP-J bind- cells along the renal arterioles are gradually called on to ing sites in their promoters and are likely to be regulated di- produce renin and restore homeostasis. This process, histor- rectly by RBP-J. Overall, our findings uncovered an unforeseen ically mislabeled as recruitment, is not because of proliferation and powerful function of RBP-J in renin cells: the control of and/or migration of new cells, but it is achieved instead by the their identity not only by stimulating the expression of myo- reacquisition of the capability of SM cells to synthesize renin as epithelioid genes as discussed above, but also by its function they did in fetal life.4,35,36 We show here that SM cells harbor- as a repressor. This view is portrayed schematically in Figure 7B. ing the deletion are incapable of such transformation, suggest- Finally, given that Notch signaling is a cell-to-cell commu- ing the distinct possibility that RBP-J controls the molecular nication system, a crucial question that remains to be explored machinery responsible for the switch in cell type. The intricate is the identity of the ligand(s) and receptor(s) involved in mechanism has yet to be delineated, but our previous obser- initiating and sustaining the response in both the basal state and vations in vitro and in vivo indicate that histone acetyl trans- response to major acute and chronic physiologic challenges, ferases such as CBP/p300, which partner with RBP-J, may be such as dehydration, sodium depletion, or inhibition of involved.37 Because Notch receptors are activated in SM cells angiotensin actions. It is very likely that the same ligand(s) and RBP-J moves to the nucleus, it interacts with several part- and receptor(s) involved in the maintenance of the JG cell are ners, including CBP/p300. We have proposed that histone H4 involved in a graded manner to acute and chronic challenges to acetylation with the involvement of the Creb/CBP/p300 path- homeostasis. This hypothesis, however, remains to be tested. way leads to the opening of chromatin, allowing transcription Our own assessment so far indicates that Jagged1 and Jagged 2 of the renin gene to proceed.37–39 Furthermore, our observa- are both expressed in renin cells, and both may contribute to tions using histone deacetylase inhibitors in JG cells in culture the response. We are currently exploring this possibility and and deletion of CBP/p300 in vivo support this possibility.37–39 delineating the nature of the receptor(s) involved. Additional work will be needed, however, to delineate the In summary, in the normal animal, RBP-J maintains the chain of molecular events responsible for the SM cells’ plas- identity of the JG cells by not only activating genes character- ticity. Lastly, the findings raise an intriguing hypothesis. It has isticof their myo-endocrine phenotype but also,preventing the been suggested that modulation of the Notch pathway in cells undesirable ectopic expression of genes from other lineages that are not terminally differentiated provides them with the and the adoption of an aberrant phenotype with potentially plasticity to change fate.40 Because SM cells along the arterio- devastating consequences for the control of homeostasis. les are capable of transiently alternating between the SM and Furthermore, RBP-J controls the memory of cells to reacquire the renin phenotype, their chromatin must be constantly the renin phenotype when homeostasis is challenged. poised to allow such changes to occur, suggesting the possi- Clearly, regulation of renin cell identity by the Notch bility that SM cells of the renal arterioles are not terminally pathway is a complex process, whereby RBP-J, a master

J Am Soc Nephrol 26: 67–80, 2015 RBP-J and Renin Cell Fate 77 BASIC RESEARCH www.jasn.org transcriptional regulator, governs a broad network of genes, Kidney Microvessel Isolation which acting in concert, culminates with the precise physio- Preglomerular microvessels were isolated using iron oxide perfusion logically able phenotype of the renin cell. and magnet separation of the vessels as previously described.38 The isolated microvessels were passed through 21- and 23-gauge needles to remove the glomeruli from the vessels, and the arterioles were CONCISE METHODS collected on a 100-mm filter.

Mice Generation of the BAC Construct with Mutated RBP-J fl fl The RBP-J / mice42 were the gift of Tasuku Honjo, and miR-145–5-p Binding Site KO mice43 were obtained from Eric N. Olson. The R26R reporter TheBACcloneRP2388K7containingthereningenewaspurchasedfrom mouse, which expresses b-gal in a cre-inducible manner,10 was used CHORI (Oakland, CA). We used the dual kanamycin and streptomycin 44 fi to trace the cells of the renin lineage in the RBP-J cKO mice. To selection method to replace the rst exon of the renin gene with dcre/+ fl/fl +/2 fl/fl fl generate the Ren1 ;RBP-J ;R26R study animals, RBP-J ; enhanced green uorescent protein (EGFP) (Clontech Laboratories, +/+ dcre/cre fl/fl 9 R26R mice were crossed with Ren1 ;RBP-J mice. The control Mountain View, CA) and the renin 3 untranslated region for the WT 2 mice, Ren1dcre/+;RBP-J+/+;R26R+/ ,areWTforRBP-J and heterozygous reporter (WT-BAC) and then introduced the mutation in the RBP-J for both Ren1dcre and R26R. The BAC transgenic mice were generated binding site in the renin promoter for the Mut-BAC. Supplemental using the services of the University of Virginia Gene Targeting and Material has details of the BAC constructs and primers used. Transgenic Facility. Founder mice carrying the BAC transgene were identified using primer sets targeting the area of GFP insertion, yield- DNA and RNA Extraction ing an 860-bp product (59-GAAGGAGAGCAAAAGGTAAGAG-39 To determine the transgene copy number in the transgenic mice, we and 59-GCCGATGGGGGTGTTCTG-39). The presence of the intended extracted DNA from mouse tails using a proteinase K digestion plus mutation in the RBP-J binding site in the Mut-BAC mice was confirmed phenol/chloroform extraction protocol (The Jackson Laboratory, Bar by sequencing (Supplemental Material, Supplemental Figure 5). The Harbor, ME). The phenol/chloroform extraction step was repeated control WT-BAC and Mut-BAC transgenic founder mice were bred two times to obtain optimal 260/230 ratios. to C57BL/6 mice, and GFP expression was quantified in 3.5-month- RNA was extracted from kidneys and isolated arterioles using Trizol, 8 fi old animals from the F1 generation. and cDNA was prepared as previously described. For GFP quanti ca- m All procedures followed the National Institutes of Health Guide for tion, cDNA was prepared from 1 g total kidney RNA using the High the Care and Use of Laboratory Animals and were approved by the Capacity cDNA Reverse Transcription Kit (part no. 4368814; Applied ’ Animal Care and Use Committee of the University of Virginia. Biosystems, Carlsbad, CA) according to the manufacturer s instructions. Animals were anesthetized with tribromoethanol, and the kidneys were removed, weighed, and preserved for different purposes. For RT-PCR and Quantitative RT-PCR Analyses RNA extraction, kidneys were stored in RNA later (Ambion, Austin, Semiquantitative RT-PCR was assessed using the integrated OD value fi TX). For immunohistochemistry in paraffin sections, they were fixed (de ned as a pixel value) by drawing equal sized boxes (for each sample) in Bouin solution (overnight) or 10% formalin for 30 minutes, and for around bands, with background values (water) taken for each sample. frozen sections, they were fixed in 4% paraformaldehyde in PBS for 1 Quantitative real-time PCR using SYBR Green (Invitrogen) to measure GAPDH and renin levels was performed as previously described.8 EGFP hour, cryoprotected in 30% sucrose in PBS with 2 mM MgCl2 at 4°C overnight, and then embedded in OCT compound embedding me- and GAPDH in the transgenic mice were measured by quantitative RT- dium (Miles, Elkhart, IL). PCR using the TaqMan Universal Master MixII and TaqMan expression assay primers for glyceraldehyde 3-phosphate dehydrogenase Immunohistochemistry and In Situ Hybridization (GAPDH) (4331182) and EGFP (4400291; Applied Biosystems). Immunohistochemistry was performed as previously described6 using The copy number of the BAC transgene in the transgenic mice was antibodies against a-SMA (isotype IgG2a, clone 1A4, dilution 1:10,000; determined using the services of the University of Virginia Bio- Sigma-Aldrich,St.Louis,MO),Cnn1(1806–1, dilution 1:500; Epitomics molecular Research Facility using TaqMan copy number assays Inc., Burlingame, CA), SM-MHC11 (5121–1, dilution 1:1000; Epitomics (Applied Biosystems) for EGFP (4400291) and Tfrc (4458366; trans- Inc.), Nfatc4 (ab62613, dilution 1:500; Abcam, Inc., Cambridge, MA), ferrin receptor) as the single-copy genomic standard. rabbit anti-mouse renin antibody37 (dilution 1:500), and Akr1b7 an- tibody (dilution 1:200, with citrate antigen retrieval; Santa Cruz Bio- RBP-J Binding Site Prediction technology, Santa Cruz, CA). For GFP detection, we used an anti-GFP To identify potential RBP-J binding sites in the promoter region of antibody (A6455, dilution 1:1000; Invitrogen, Grand Island, NY) and genes, we performed in silico analysis using the Transcription Element the TSA Biotin System protocol (NEL700A001KT; PerkinElmer, Search System program (http://www.cbil.upenn.edu/cgi-bin/tess/ Waltham, MA) to amplify the GFP signal. tess?RQ=WELCOME) or MatInspector.45 b-Gal expression was evaluated in cryosections (7 mm) as pre- viously described.6 Microarray Analyses In situ hybridization to detect miR-145–5p expression in kidney To identify genes altered by deletion of RBP-J, we performed mRNA sections was performed as previously described.12 microarray analysis in isolated arterioles from control and RBP-J cKO

78 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 67–80, 2015 www.jasn.org BASIC RESEARCH mice. Total RNA was extracted from microvessels isolated from adult 9. Tun T, Hamaguchi Y, Matsunami N, Furukawa T, Honjo T, Kawaichi M: mice (as described above), and hybridization to the Mouse Gene 1.0 Recognition sequence of a highly conserved DNA binding protein Nucleic Acids Res – ST array (Affymetrix, Santa Clara, CA) was performed by the Bio- RBP-J kappa. 22: 965 971, 1994 10. Soriano P: Generalized lacZ expression with the ROSA26 Cre reporter molecular Research Facility at the University of Virginia. Two groups strain. Nat Genet 21: 70–71, 1999 were used (control versus mutant). Each group was a pool of RNA 11. Bartel DP: MicroRNAs: Genomics, biogenesis, mechanism, and func- samples (controls: n=3; mutants: n=4). Microarray data were ana- tion. Cell 116: 281–297, 2004 lyzed through the use of Ingenuity Pathway Analysis (Ingenuity Sys- 12. Sequeira-Lopez ML, Weatherford ET, Borges GR, Monteagudo MC, tems) with a significance cutoff of P,0.05. Pentz ES, Harfe BD, Carretero O, Sigmund CD, Gomez RA: The microRNA-processing enzyme dicer maintains juxtaglomerular cells. JAmSocNephrol21: 460–467, 2010 Statistical Analyses 13. Sun Q, Taurin S, Sethakorn N, Long X, Imamura M, Wang DZ, Zimmer Data are expressed as means6SEMs. Significance was determined by WE, Dulin NO, Miano JM: Myocardin-dependent activation of the t test. P,0.05 was considered significant. CArG box-rich smooth muscle gamma-actin gene: Preferential utiliza- tion of a single CArG element through functional association with the NKX3.1 homeodomain protein. JBiolChem284: 32582–32590, 2009 14. Wang Z, Wang DZ, Pipes GC, Olson EN: Myocardin is a master regu- ACKNOWLEDGMENTS lator of smooth muscle gene expression. Proc Natl Acad Sci U S A 100: 7129–7134, 2003 15. Cordes KR, Sheehy NT, White MP, Berry EC, Morton SU, Muth AN, Lee The authors thank Takele Yazew and Kimberly Hilsen-Durette for TH, Miano JM, Ivey KN, Srivastava D: miR-145 and miR-143 regulate technical assistance with the mouse work. smooth muscle cell fate and plasticity. Nature 460: 705–710, 2009 This work was supported by National Institutes of Health Grants 16. Wang Z, Wang DZ, Hockemeyer D, McAnally J, Nordheim A, Olson EN: DK075481 (to M.L.S.S.-L.), R37-HL066242 (Diversity Supplement Myocardin and ternary complex factors compete for SRF to control Nature – GraduateResearchAssistant;toR.A.G),andR01-HL096735(toR.A.G.). smooth muscle gene expression. 428: 185 189, 2004 17. Henderson JR, Macalma T, Brown D, Richardson JA, Olson EN, Part of this work was presented at the High Blood Pressure Research Beckerle MC: The LIM protein, CRP1, is a smooth muscle marker. 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80 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 67–80, 2015