Human kidney organoids produce functional renin
Authors: Anusha S. Shankar, Zhaoyu Du, Hector Tejeda Mora, Thierry P.P. van den Bosch, Sander S. Korevaar, Ingrid M. Van den Berg-Garrelds, Eric Bindels, Carmen Lopez-Iglesias, Marian C. Clahsen-van Groningen, Joost Gribnau, Carla C. Baan, A.H. Jan Danser, Ewout J. Hoorn, Martin J. Hoogduijn
Journal: Kidney International (2020)

Renin production by the kidney is of vital importance for salt, volume, and blood pressure homeostasis. The lack of human models hampers investigation into the regulation of renin and its relevance for kidney physiology. To develop such a model, we used human induced pluripotent stem cell–derived kidney organoids to study the role of renin and the renin-angiotensin system in the kidney. Extensive characterization of the kidney organoids revealed kidney-specific cell populations consisting of podocytes, proximal and distal tubular cells, stromal cells and endothelial cells. We examined the presence of various components of the renin-angiotensin system such as angiotensin II receptors, angiotensinogen, and angiotensin-converting enzymes 1 and 2. We identified by single-cell sequencing, immunohistochemistry, and functional assays that cyclic AMP stimulation induces a subset of pericytes to increase the synthesis and secretion of enzymatically active renin. Renin production by the organoids was responsive to regulation by parathyroid hormone. Subcutaneously implanted kidney organoids in immunodeficient IL2Ry-/-Rag2-/- mice were successfully vascularized, maintained tubular and glomerular structures, and retained capacity to produce renin two months after implantation. Thus, our results demonstrate that kidney organoids express renin and provide insights into the endocrine potential of human kidney organoids, which is important for regenerative medicine in the context of the endocrine system. Renin production by the kidney is of vital importance for salt, volume, and blood pressure homeostasis. The lack of human models hampers investigation into the regulation of renin and its relevance for kidney physiology. To develop such a model, we used human induced pluripotent stem cell–derived kidney organoids to study the role of renin and the renin-angiotensin system in the kidney. Extensive characterization of the kidney organoids revealed kidney-specific cell populations consisting of podocytes, proximal and distal tubular cells, stromal cells and endothelial cells. We examined the presence of various components of the renin-angiotensin system such as angiotensin II receptors, angiotensinogen, and angiotensin-converting enzymes 1 and 2. We identified by single-cell sequencing, immunohistochemistry, and functional assays that cyclic AMP stimulation induces a subset of pericytes to increase the synthesis and secretion of enzymatically active renin. Renin production by the organoids was responsive to regulation by parathyroid hormone. Subcutaneously implanted kidney organoids in immunodeficient IL2Ry-/-Rag2-/- mice were successfully vascularized, maintained tubular and glomerular structures, and retained capacity to produce renin two months after implantation. Thus, our results demonstrate that kidney organoids express renin and provide insights into the endocrine potential of human kidney organoids, which is important for regenerative medicine in the context of the endocrine system. Translational StatementThe advancement of knowledge and technology in the organoid field has allowed the generation of structurally complex and functional kidney organoids. We demonstrate that renin, a key factor in blood pressure regulation produced by the kidney, is produced and secreted by kidney organoids. This work demonstrates that kidney organoids can be used to test the effect of novel drugs on the renin-angiotensin system and contributes to the field of regenerative medicine to replace the hormonal system of injured kidneys. The advancement of knowledge and technology in the organoid field has allowed the generation of structurally complex and functional kidney organoids. We demonstrate that renin, a key factor in blood pressure regulation produced by the kidney, is produced and secreted by kidney organoids. This work demonstrates that kidney organoids can be used to test the effect of novel drugs on the renin-angiotensin system and contributes to the field of regenerative medicine to replace the hormonal system of injured kidneys. Recently several innovative protocols have been published that convincingly show the induction of induced pluripotent stem cells (iPSCs) into kidney organoids.1Homan K.A. Gupta N. Kroll K.T. et al.Flow-enhanced vascularization and maturation of kidney organoids in vitro.Nat Methods. 2019; 16: 255-262Crossref PubMed Scopus (356) Google Scholar, 2Takasato M. Little M.H. Making a kidney organoid using the directed differentiation of human pluripotent stem cells.Methods Mol Biol. 2017; 1597: 195-206Crossref PubMed Scopus (29) Google Scholar, 3Tanigawa S. Islam M. Sharmin S. et al.Organoids from nephrotic disease-derived iPSCs identify impaired NEPHRIN localization and slit diaphragm formation in kidney podocytes.Stem Cell Reports. 2018; 11: 727-740Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar Kidney organoids recapitulate the composition and organization of the kidney and display its functional properties. In the adult kidney, the cell types within the nephron are positioned in a defined arrangement that permits for volume, electrolyte, and acid-base regulation in the body through glomerular filtration and tubular transport. The kidney is also indispensable for the production or activation of various homeostatic hormones.4Kurtz A. Endocrine functions of the renal interstitium.Pflugers Arch. 2017; 469: 869-876Crossref PubMed Scopus (20) Google Scholar Owing to the nonredundant role of the kidney in these endocrine processes, it is of great interest to investigate whether in vitro–generated structures have the potential to repair the endocrine systems of the kidney. In this study we hypothesized that human kidney organoids are able to produce functional renin, the first and rate-limiting enzyme of the renin-angiotensin system (RAS). Renin produced by the adult kidney dictates salt reabsorption, blood pressure, and volume homeostasis.5Shaw I. Rider S. Mullins J. et al.Pericytes in the renal vasculature: roles in health and disease.Nat Rev Nephrol. 2018; 14: 521-534Crossref PubMed Scopus (63) Google Scholar During kidney development renin-producing cells can be observed in a diffuse distribution in the undifferentiated metanephric mesenchyme, descending from a common stromal progenitor population.6Sequeira-Lopez M.L. Lin E.E. Li M. et al.The earliest metanephric arteriolar progenitors and their role in kidney vascular development.Am J Physiol Regul Integr Comp Physiol. 2015; 308: R138-R149Crossref PubMed Scopus (76) Google Scholar Once vascularization of the kidney is initiated, renin-producing cells align along the newly formed large vessels and eventually become confined to the juxtaglomerular apparatus at the terminal part of the afferent arterioles. The exact mechanisms regulating renin expression during development are poorly understood. This is partly due to the lack of suitable human in vitro models to study the RAS, as juxtaglomerular cells are rare and rapidly lose their ability to produce renin in culture.7Stefanska A. Kenyon C. Christian H.C. et al.Human kidney pericytes produce renin.Kidney Int. 2016; 90: 1251-1261Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 8Castrop H. Hocherl K. Kurtz A. Schweda F. et al.Physiology of kidney renin.Physiol Rev. 2010; 90: 607-673Crossref PubMed Scopus (196) Google Scholar, 9Brunskill E.W. Sequeira-Lopez M.L. Pentz E.S. et al.Genes that confer the identity of the renin cell.J Am Soc Nephrol. 2011; 22: 2213-2225Crossref PubMed Scopus (88) Google Scholar Kidney organoids may represent a model to study the RAS in a human system. As current kidney organoids resemble the early developmental stages of the kidney, they could be used for examining the unique patterning of renin-producing cells and their contribution to the growth and differentiation of the kidney. Eventually kidney organoids with a functional endocrine system may be developed that have the capacity to normalize hormonal imbalances in patients with kidney failure. Our hypothesis asserts the existence of a functional RAS in human iPSC-derived kidney organoids. Hence, we investigated the regulation of renin production in kidney organoids and characterized the renin-producing cells using imaging techniques and single-cell RNA sequencing (scRNAseq) analyses. Finally, we showed that renin-producing cells in kidney organoids maintain their hormonal production after implantation. Kidney organoids were differentiated from human iPSCs through a protocol involving 7 days of monolayer culture followed by 18 days of 3-dimensional culture and the timely addition of CHIR99021 and fibroblast growth factor 9 (Figure 1a). At day 25, complex structures were visible by bright-field microscopy (Figure 1b and c). Intermediate mesoderm markers such as OSR1 and PAX2 showed a 90- and 3000-fold mRNA upregulation compared with iPSCs (Figure 1d). Wilms tumor 1 (WT1) and homeobox protein Hox-D11 (HOXD11), transcription factors crucial for the nephron lineage, increased 9000- and 20,000-fold, whereas glial-derived neurotrophic factor (GDNF), a marker for the ureteric epithelium, increased 18-fold after differentiation. The expression of pluripotent markers nanog homeobox (NANOG), C-MYC, octamer-binding transcription factor 4 (OCT-4), and SOX2 was downregulated, whereas expression of REX1 and Krüppel-like factor 4 (KLF4) remained unchanged (Figure 1d). At day 25, immunohistochemistry revealed that WT1+, Villin-1+, E-cadherin (ECAD+), and CD31+/CD34+ cells were present, suggesting the emergence of glomeruli, proximal tubuli, distal tubuli, ureteric epithelium and endothelial cells, respectively (Figure 1e and f). COL1A1 and platelet-derived growth factor receptor A (PDGFRα) marked the basement membranes and renal interstitium (Figure 1f). Electron microcopy showed the presence of proximal tubular structures containing a single layer of polarized epithelial cells connected by tight junctions on day 25 (Figure 1g). The tubuli contained an open lumen with brush border microvilli along with mitochondria, lysosomes, and small apical endocytic vacuoles. Sodium transporters sodium-proton-exchanger subtype 3 (NHE3) and sodium-potassium-chloride transporter 2 (NKCC2) were observed at the apical side of tubular structures, suggesting a loop of Henle population (Figure 1h). We observed upregulation of mRNA of transporters for small organic anions, OAT1 and OAT3, megalin, and cubilin (Figure 1i). The functionality of organic anion transporter 1 (OAT1) and OAT3 was demonstrated by the active transport of the organic anion fluorescein at 37 °C but not at 4 °C into the lumen of tubuli (Figure 1j). To profile the various cell populations in the kidney organoids, we performed scRNAseq on a day-25 kidney organoid (Figure 1k). Clustering confirmed the presence of podocytes (22.1% of the cells), proximal (22.3%) and distal tubular cells/loop of Henle (2.3%), endothelial cells (0.5%), pericytes (8.6%), and stromal cells (3.4%). Populations of neural cells (20.5%) and cell-cycling cells (20%) were also found. Expression levels of marker genes within each cluster and the proportion of cells expressing a gene were visualized in a dot plot (Figure 1l). These data demonstrate that iPSC-derived organoids contain the major cell types of the kidney. We then continued studying the presence of the RAS in the kidney organoids.Figure 1Human iPSC-derived kidney organoids contain essential renal structures and display tubular transport. (a) A schematic overview of the kidney organoid culture. (b) Representative bright-field images of different time points during kidney organoid culture. Bars = 100 μm. (c) Image of a whole organoid at day 25. Bar = 500 μm. (d) Gene expression analysis of intermediate mesoderm, kidney differentiation markers, and pluripotency markers. Plots depict gene expression changes relative to undifferentiated iPSC. Data are represented as mean ± SEM (n = 4 from 1 experiment). (e) Immunohistochemical analysis for markers of glomerular cells (WT1), proximal tubular cells (Villin-1), distal tubular cells (ECAD) and endothelial cells (CD31+). Bars = 100 μm. (f) Immunohistochemical analysis for markers of endothelial cells (CD34), basement membrane (COL1A1), and renal interstitium (PDGFRα). Bars = 100 μm. (g) Electron microscopic analysis for the presence of tubular structures. Tj, tight junctions; mv, microvilli; mi, mitochondria; lys, lysosomes; sav, small apical endocytic vacuoles. (h) Immunohistochemistry for markers of the proximal tubuli (NHE3) and the loop of Henle (NKCC2) in the kidney organoid (as indicated by black arrows). Bars = 100 μm. (i) Gene expression analysis of various kidney transporters (OAT1, OAT3, megalin, and cubilin) over the course of the organoid culture. Plots depict gene expression changes relative to undifferentiated iPSC. Data are represented as mean ± SEM (n = 4 from 1 experiment). (j) Fluorescein uptake assay showing uptake in lumen of tubuli (fluorescein, green; 4′,6-diamidino-2-phenylindole [DAPI], blue). Bars = 50 μm. The left panel shows fluorescein uptake at 37 °C for 1 hour (yellow arrow). The right panels shows the negative control at 4 °C. (k) Uniform manifold approximation and projection plot displaying 9 distinct clusters of cell populations found in the kidney organoid. Populations have distinct colors to indicate identity. Prox. Tub., proximal tubule; Distal Tub. & LOH, distal tubule and loop of Henle. (l) Dot plot visualization of representative marker genes per cluster of cells. ECAD, E-cadherin; iPSC, induced pluripotent stem cells; PDGFRα, platelet-derived growth factor receptor A; SEM, standard error of the mean. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the kidney organoids, renin mRNA increased 146-fold during differentiation of iPSC to intermediate mesoderm at day 7 (Figure 2a). Hereafter, the expression of renin in the organoids at day 25 decreased 45-fold. Expression of AGTR1 and AGTR2 mRNA was 80-fold and 26,000-fold increased, respectively, compared with undifferentiated iPSCs (Figure 2b). We also detected a 166-fold increase in angiotensinogen (AGT) mRNA in the kidney organoids (Figure 2c). ACE mRNA was detected throughout organoid differentiation (Figure 2c). Expression of renin was most pronounced in a small population of pericytes (Figure 2d). In this cluster, pericyte markers such as NG2, MEIS1, MEIS2, PDGFRα, PDGFRβ, COL1A1, and COL3A1 were highly expressed. Unexpectedly, this compartment along with the stromal cluster exhibited the highest expression of the genes AGTR1 and AGTR2, whereas the tubular cells, podocytes, and endothelial cells did not express these receptors. AGT mRNA was expressed in proximal and distal tubular cells; however, AGT protein was absent in the supernatant of the kidney organoids (data not shown, n = 8). ACE was expressed by a low number of cells in the podocyte, distal tubular, and endothelial cell clusters. scRNAseq analysis revealed components of the alternative RAS, including protease prolyl carboxypeptidase (PRCP) and prolyl endopeptidase (PREP) (Figure 2d). Glomerular and proximal tubular localization of alternative RAS components membrane metallo-endopeptidase (MME) and angiotensin-converting enzyme 2 (ACE2) was confirmed with immunohistochemistry in day 25 organoids (Figure 2e), as shown recently by Monteil et al.10Monteil V. Kwon H. Prado P. et al.Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2.Cell. 2020; 181: 905-913.e907Abstract Full Text Full Text PDF PubMed Scopus (1369) Google Scholar To examine the influence of the RAS components on the development of kidney organoids, we added 500 nM angiotensin II to kidney organoid cultures from day 12 onward in the presence or absence of AGTR1 antagonist losartan. We also added the direct renin inhibitor aliskiren throughout the culture period. Neither losartan nor aliskiren exerted any effect (Supplementary Figure S1). The expression of RAS components was not affected in angiotensin II–treated organoids, apart from a small increase in expression of AGTR1 (Supplementary Figure S1A). Angiotensin II drives fibrotic processes in the kidney,11Mezzano S.A. Ruiz-Ortega M. Egido J. Angiotensin II and renal fibrosis.Hypertension. 2001; 38: 635-638Crossref PubMed Scopus (427) Google Scholar but we found no apparent effect of angiotensin II on fibrotic pathways in the kidney organoids (Supplementary Figure S1B). Gene and protein expression of WT1, Villin-1, and ECAD remained stable in all conditions (Supplementary Figure S1C and D) and profibrotic COL1A1 did not change when the patient was treated with angiotensin II (Supplementary Figure S1D). There was a significant 305-fold increase in renin mRNA in forskolin-treated organoids (Figure 3a). In undifferentiated iPSCs, forskolin did not upregulate renin mRNA (Figure 3b). scRNAseq on a forskolin-stimulated organoid revealed that a significant subset of pericytes in the forskolin-treated organoid expressed renin (Figure 3c and d). To obtain a closer perspective on the identity of these pericytes, we examined our previous transcriptome data in which reninomas and murine developing kidney were analyzed for their renin-related gene signature12Martini A.G. Xa L.K. Lacombe M.J. et al.Transcriptome analysis of human reninomas as an approach to understanding juxtaglomerular cell biology.Hypertension. 2017; 69: 1145-1155Crossref PubMed Scopus (7) Google Scholar and identified core genes that constituted the gene profile of renin-expressing cells. Some of these genes were highly expressed in pericytes of the kidney organoid, including VIM, PDGFRβ, and CXCL12 (Figure 3e). Interestingly, in our scRNAseq data analysis we noticed that a relatively low level of renin expression also emerged in the proximal tubular and—to a lesser extent—in the distal tubular compartment. The increase in renin mRNA was visible at the protein level, as forskolin stimulation caused a drastic increase in renin positivity (Figure 3f). We observed a diffuse distribution of renin-positive cells that expressed renin in a granular pattern (Figure 3f). Immunogold labeling of renin visualized cells mainly at the periphery of the organoids treated with forskolin, which may be classified as pericytes based on previous literature (Figure 3g and h).7Stefanska A. Kenyon C. Christian H.C. et al.Human kidney pericytes produce renin.Kidney Int. 2016; 90: 1251-1261Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar,13Sequeira Lopez M.L. Pentz E.S. Nomasa T. et al.Renin cells are precursors for multiple cell types that switch to the renin phenotype when homeostasis is threatened.Dev Cell. 2004; 6: 719-728Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar Multivesicular bodies from the late endocytic/lysosomal pathway could be observed that may represent secretory lysosomes that release their content upon trigger14Peters P.J. Borst J. Oorschot V. et al.Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes.J Exp Med. 1991; 173: 1099-1109Crossref PubMed Scopus (524) Google Scholar (Figure 3g). Double immunofluorescence of the pericyte marker PDGFRα and renin revealed colocalization of both markers (Figure 3i). Consecutive stainings for renin and the glomerular marker WT1+ showed close proximity of the 2 proteins (Figure 3j). In a week-13 human fetal kidney, renin-producing cells surround the glomeruli in a similar fashion (Figure 3k). Next we demonstrated renin enzymatic activity in medium of organoids by the generation of angiotensin I, both before and after prorenin conversion to renin (Figure 3l). Aliskiren blocked more than 99% of the angiotensin I–generating activity (data not shown), supporting that renin was the angiotensin I–generating enzyme. The increase in angiotensin I–generating activity after prorenin activation (5.1 ng angiotensin I/ml/h) corresponded to 20% to 30% of the angiotensin I–generating activity before prorenin stimulation (18.8 ng angiotensin I/ml/h) (Figure 3l). Juxtaglomerular cells are known to release mainly renin upon acute stimulation of cyclic adenosine monophosphate, and in further studies we therefore focused on renin only. Stimulation of the kidney organoids with forskolin resulted in a 20-fold increase in intracellular renin activity compared with untreated cells (Figure 3m). The same rise in renin activity was observed in the conditioned culture medium (Figure 3n).Figure 3Forskolin stimulates renin production in kidney organoids. (a) Comparison of renin gene expression levels in unstimulated and forskolin-stimulated kidney organoids. Data are represented as mean ± SEM (n = 8 from 4 independent experiments). Statistical analysis was performed using the Mann-Whitney test (∗∗P < 0.01). (b) Gene expression analysis for renin in iPSC after forskolin stimulation. Data are represented as mean ± SEM (n = 4 from 1 experiment). Statistical analysis was performed using the Mann-Whitney test (ns, nonsignificant). (c) Representation of uniform manifold approximation and projection with superimposed renin expression in forskolin-treated organoid. (d) Dot plot visualization of renin expression in the different clusters of forskolin-treated organoid. (e) Dot plot visualization of renin-related gene expression levels based on published literature in forskolin-treated organoid. (f) Immunohistochemical analysis for renin protein expression in control and forskolin-treated organoid. Bars = 100 μM. (g). Immunogold labeling of renin shows that the cytoplasm of potential pericytes contains granules with renin immunogold particles. The inset depicts multivesicular bodies from the late endocytic/lysosomal pathway. (h) Immunogold labeling in the cytosol (upper image) and in multivesicular bodies from the late endocytic/lysosomal pathway (lower image, yellow arrows). (i) Double immunofluorescence staining for PDGFRα+ (red) and renin positivity (green) showing colocalization (yellow arrows). Bar = 50 μM. (j) Immunohistochemical analysis for a marker of glomerular cells (WT1) and renin in consecutive slides. Bars = 100 μM. (k) Immunohistochemical analysis for renin in a 13-week-old fetal kidney. Bars = 100 μM. (l) Activity assay for prorenin and renin in the medium of 5 unstimulated organoids represented as ng Ang I (angiotensin I) /ml/h. Each bar represents one organoid (n = 5 from 2 independent experiments). (m,n) Activity assay for renin depicted as ng Ang I/μg protein/h in the cell lysates (n = 5 from 3 independent experiments) and ng Ang I/ml/h in the supernatant after a 24-hour stimulation with forskolin (n = 7 from 4 independent experiments). Data are represented as mean ± SEM. Statistical analysis was performed using the Mann-Whitney test (∗∗P < 0.01). Distal Tub. & LOH, distal tubule and loop of Henle; iPSC, induced pluripotent stem cells; PDGFRα, platelet-derived growth factor receptor A; Prox. Tub., proximal tubule; SEM, standard error of the mean. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Next we sought to detect whether the renin-producing cells were responsive to hormonal stimuli. Initially we tested common regulators of renin to mimic signals from the macula densa to the juxtaglomerular apparatus. Organoids were stimulated with prostaglandins and cultured in low-salt conditions. No increase in renin was observed in these conditions (data not shown), most likely owing to the relative immaturity of the kidney organoids. Previous studies showed a relation between parathyroid hormone (PTH) stimulation and renin activity.15Saussine C. Judes C. Massfelder T. et al.Stimulatory action of parathyroid hormone on renin secretion in vitro: a study using isolated rat kidney, isolated rabbit glomeruli and superfused dispersed rat juxtaglomerular cells.Clin Sci (Lond). 1993; 84: 11-19Crossref PubMed Scopus (37) Google Scholar,16Smith J.M. Mouw D.R. Vander A.J. Effect of parathyroid hormone on plasma renin activity and sodium excretion.Am J Physiol. 1979; 236: F311-F319PubMed Google Scholar We found that type 1 PTH receptor (PTH1R) mRNA rises more than 100-fold during kidney organoid development (Figure 4a). scRNAseq revealed that in a subset of stromal cells in the forskolin-treated organoid there was co-expression of renin and PTH1R (Figure 4b). A 20-fold increase in renin mRNA in kidney organoids treated with 500 nM PTH indicated a functional PTH1R (Figure 4c). Next we hypothesized that PTH could facilitate renin production and secretion by activation of its receptor. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was added to avoid degradation of cyclic adenosine monophosphate. Immunoreactivity of renin after stimulation with PTH or IBMX alone was present in a few stromal cells dispersed throughout the organoids, whereas the combination of the 2 factors culminated in a higher frequency of stromal cells positive for renin (Figure 4d). Forskolin-treated organoids were used as a positive control. Renin synthesis was evident by the 7-fold increase in intracellular renin activity upon PTH stimulation, whereas stimulation with IBMX alone or in combination with PTH resulted in a 20- and 25-fold increase, respectively (Figure 4e). We sought to determine whether this increase would result in secretion of renin. A trend could be observed in renin secretion by the addition of PTH (4-fold, not significant) or IBMX (15-fold) (Figure 4e). The combination of the 2 factors caused a significant 34-fold rise of renin secretion into the supernatant (Figure 4e). To improve maturation status, organoids were subcutaneously implanted in the flanks of immune-deficient mice and analyzed after 2 months. The organoids were invaded by surrounding vessels (Figure 5a). Vascular structures contained erythrocytes, confirming vascularization (Figure 5b), and were of human origin as witnessed by the presence of human mitochondria (Figure 5c). Tubular and glomerular structures were maintained as immunohistochemistry showed putative WT1+ glomeruli, Villin-1+ proximal tubuli, and ECAD+ distal tubuli (Figure 5d). Forskolin stimulation of organoid explants resulted in a 3-fold increase in the excretion of renin compared with untreated explanted organoids (Figure 5e). Similar treatment of murine tissue served as a baseline control for the increase in renin activity. Immunostaining of renin in the explanted organoid revealed renin-positive cells near the juxtaglomerular localization (Figure 5f). We successfully generated kidney organoids that recapitulate essential renal structures and explored the presence and function of the RAS, a fundamental kidney endocrine system. To our knowledge, this is the first study to show in a comprehensive and detailed manner that iPSC-derived human kidney organoids contain a unique population of renin-producing pericytes and that renin production is an inducible process. These results offer exciting opportunities for future in vitro and in vivo studies focused on the distribution and function of renin-producing cells in the human kidney, which has not been possible to date. Kidney organoids do not contain vascularized glomeruli and the intricate signaling pathways between the macula densa and juxtaglomerular apparatus, as seen in the adult kidney. In addition, in the absence of glomerular filtration and tubular flow, NKCC2 may not sense sodium chloride, the starting point for renin release. In the current model it is therefore challenging to examine physiologic interactions with other important mediators of renin production. Previous studies have shown a direct stimulatory effect of PTH on renin secretion, but the exact mechanism underlying this relationship has not been elucidated yet.17Saussine C. Massfelder T. Parnin F. et al.Renin stimulating properties of parathyroid hormone-related peptide in the isolated perfused rat kidney.Kidney Int. 1993; 44: 764-773Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 18Grant F.D. Mandel S.J. Brown E.M. et al.Interrelationships between the renin-angiotensin-aldosterone and calcium homeostatic systems.J Clin Endocrinol Metab. 1992; 75: 988-992Crossref PubMed Scopus (0) Google Scholar, 19Helwig J.J. Musso M.J. Judes C. Nickols G.A. Parathyroid hormone and calcium: interactions in the control of renin secretion in the isolated, nonfiltering rat kidney.Endocrinology. 1991; 129: 1233-1242Crossref PubMed Scopus (43) Google Scholar, 20Broulik P.D. Horky K. Pacovsky V. Effect of parathyroid hormone on plasma renin activity in humans.Horm Metab Res. 1986; 18: 490-492Crossref PubMed Scopus (16) Google Scholar, 21Vaidya A. Brown J.M. Williams J.S. The renin-angiotensin-aldosterone system and calcium-regulatory hormones.J Hum Hypertens. 2015; 29: 515-521Crossref PubMed Scopus (60) Google Scholar We show that PTH has a direct and strong effect on renin production. To drive maturation, we implanted the organoids subcutaneously in mice, which led to vascularized renal structures. In this setting, a functional subset of renin-producing cells was maintained. The low levels of renin in implanted organoids may be due to fewer renin-positive cells after implantation, which is comparable to the adult kidney in which renin-producing cells constitute 0.01% of the cells.22Gomez R.A. Sequeira-Lopez M.L. Novel func