Human kidney organoids produce functional renin

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