Human kidney pericytes produce renin
Authors: Ania Stefanska, Christopher Kenyon, Helen C. Christian, Charlotte Buckley, Isaac Shaw, John J. Mullins, Bruno Péault
Journal: Kidney International (2016)

Pericytes, perivascular cells embedded in the microvascular wall, are crucial for vascular homeostasis. These cells also play diverse roles in tissue development and regeneration as multi-lineage progenitors, immunomodulatory cells and as sources of trophic factors. Here, we establish that pericytes are renin producing cells in the human kidney. Renin was localized by immunohistochemistry in CD146 and NG2 expressing pericytes, surrounding juxtaglomerular and afferent arterioles. Similar to pericytes from other organs, CD146+CD34–CD45–CD56– renal fetal pericytes, sorted by flow cytometry, exhibited tri-lineage mesodermal differentiation potential in vitro. Additionally, renin expression was triggered in cultured kidney pericytes by cyclic AMP as confirmed by immuno-electron microscopy, and secretion of enzymatically functional renin, capable of generating angiotensin I. Pericytes derived from second trimester human placenta also expressed renin in an inducible fashion although the renin activity was much lower than in renal pericytes. Thus, our results confirm and extend the recently discovered developmental plasticity of microvascular pericytes, and may open new perspectives to the therapeutic regulation of the renin-angiotensin system. Pericytes, perivascular cells embedded in the microvascular wall, are crucial for vascular homeostasis. These cells also play diverse roles in tissue development and regeneration as multi-lineage progenitors, immunomodulatory cells and as sources of trophic factors. Here, we establish that pericytes are renin producing cells in the human kidney. Renin was localized by immunohistochemistry in CD146 and NG2 expressing pericytes, surrounding juxtaglomerular and afferent arterioles. Similar to pericytes from other organs, CD146+CD34–CD45–CD56– renal fetal pericytes, sorted by flow cytometry, exhibited tri-lineage mesodermal differentiation potential in vitro. Additionally, renin expression was triggered in cultured kidney pericytes by cyclic AMP as confirmed by immuno-electron microscopy, and secretion of enzymatically functional renin, capable of generating angiotensin I. Pericytes derived from second trimester human placenta also expressed renin in an inducible fashion although the renin activity was much lower than in renal pericytes. Thus, our results confirm and extend the recently discovered developmental plasticity of microvascular pericytes, and may open new perspectives to the therapeutic regulation of the renin-angiotensin system. Pericytes (also known as mural cells) wrap around microvessels and play a crucial role in vascular development, maintenance, and remodeling.1Bergers G. Song S. The role of pericytes in blood-vessel formation and maintenance.Neuro Oncol. 2005; 7: 452-464Crossref PubMed Scopus (1087) Google Scholar, 2Díaz-Flores L. Gutiérrez R. Varela H. et al.Microvascular pericytes: a review of their morphological and functional characteristics.Histol Histopathol. 1991; 6: 269-286PubMed Google Scholar, 3Hyde G.D. Canfield A.E. Pericytes: adaptable vascular progenitors.in: Abraham D. Handler C. Dashwood M. Coghlan G. Translational Vascular Medicine. Springer, New York2012: 3-15Crossref Scopus (1) Google Scholar Reciprocal communication between pericytes and endothelial cells exists because they directly interact through a shared basement membrane and long cytoplasmic processes that ensheath the vessel wall.4Armulik A. Abramsson A. Betsholtz C. Endothelial/pericyte interactions.Circ Res. 2005; 97: 512-523Crossref PubMed Scopus (1531) Google Scholar Specialized types of renal pericytes contribute mechanically to vascular tone and blood pressure regulation,5Pallone T.L. Silldorff E.P. Pericyte regulation of renal medullary blood flow.Exp Nephrol. 2001; 9: 165-170Crossref PubMed Scopus (108) Google Scholar, 6Hamilton N.B. Attwell D. Hall C.N. Pericyte-mediated regulation of capillary diameter: a component of neurovascular coupling in health and disease.Front Neuroenergetics. 2010; 2: 1-14Crossref PubMed Google Scholar and can modulate immune responses.7Schlöndorff D. Banas B. The mesangial cell revisited: no cell is an island.J Am Soc Nephrol. 2009; 20: 1179-1187Crossref PubMed Scopus (304) Google Scholar, 8Schrimpf C. Xin C. Campanholle G. et al.Pericyte TIMP3 and ADAMTS1 modulate vascular stability after kidney injury.J Am Soc Nephrol. 2012; 23: 868-883Crossref PubMed Scopus (150) Google Scholar Crisan et al.9Crisan M. Yap S. Casteilla L. et al.A perivascular origin for mesenchymal stem cells in multiple human organs.Cell Stem Cell. 2008; 3: 301-313Abstract Full Text Full Text PDF PubMed Scopus (3007) Google Scholar prospectively identified human vascular pericytes from multiple human organs based on the expression of CD146 (Mel-CAM), nerve/glial antigen 2 (NG2), and platelet-derived growth factor receptor-β (PDGFR-β), and demonstrated that pericytes are one of the origins of mesenchymal stem cells (MSCs). Pericytes show regenerative potential in injured skeletal muscle,9Crisan M. Yap S. Casteilla L. et al.A perivascular origin for mesenchymal stem cells in multiple human organs.Cell Stem Cell. 2008; 3: 301-313Abstract Full Text Full Text PDF PubMed Scopus (3007) Google Scholar, 10Dellavalle A. Maroli G. Covarello D. et al.Pericytes resident in postnatal skeletal muscle differentiate into muscle fibres and generate satellite cells.Nat Commun. 2011; 2: 411-499Crossref PubMed Scopus (326) Google Scholar ischemic heart,11Chen C.-W. Okada M. Proto J.D. et al.Human pericytes for ischemic heart repair.Stem Cells. 2013; 31: 305-316Crossref PubMed Scopus (190) Google Scholar, 12Chen W. Baily J. Corselli M. et al.Human myocardial pericytes: multipotent mesodermal precursors exhibiting cardiac specificity.Stem Cells. 2015; 33: 557-573Crossref PubMed Scopus (123) Google Scholar bone,13James A. Zara J. Corselli M. et al.An abundant perivascular source of stem cells for bone tissue engineering.Stem Cells Transl Med. 2012; 1 (673–668)Google Scholar, 14Chung C. James A. Asatrian G. et al.Human perivascular stem cell-based bone graft substitute induces rat spinal fusion.Stem Cells Transl Med. 2015; 4: 538Crossref PubMed Google Scholar adipose tissue,15Tang W. Zeve D. Suh J.M. et al.White fat progenitor cells reside in the adipose vasculature.Science. 2008; 322: 583-586Crossref PubMed Scopus (840) Google Scholar and dental pulp16Zhao H. Feng J. Seidel K. et al.Secretion of shh by a neurovascular bundle niche supports mesenchymal stem cell homeostasis in the adult mouse incisor.Cell Stem Cell. 2014; 14: 160-173Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar; therefore, they are regarded as a local reservoir of regenerative cells.17Corselli M. Chen C.-W. Crisan M. et al.Perivascular ancestors of adult multipotent stem cells.Arterioscler Thromb Vasc Biol. 2010; 30: 1104-1109Crossref PubMed Scopus (261) Google Scholar, 18Murray I.R. West C.C. Hardy W.R. et al.Natural history of mesenchymal stem cells, from vessel walls to culture vessels.Cell Mol Life Sci. 2013; 71: 1353-1374Crossref PubMed Scopus (210) Google Scholar In tissue fibrosis, pericytes have a pathological role in which they proliferate and differentiate into collagen-I–producing myofibroblasts.19Lin S.-L. Kisseleva T. Brenner D.A. et al.Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.Am J Pathol. 2008; 173: 1617-1627Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 20Humphreys B.D. Lin S.-L. Kobayashi A. et al.Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis.Am J Pathol. 2010; 176: 85-97Abstract Full Text Full Text PDF PubMed Scopus (1093) Google Scholar In aging, kidney pericyte loss leads to capillary dilation and vascular damage.21Stefanska A. Eng D. Kaverina N. et al.Interstitial pericytes decrease in aged mouse kidneys.Aging (Albany NY). 2015; 7: 370-382Crossref PubMed Scopus (31) Google Scholar Cells of renin lineage (CoRL), which originate from renin-positive precursors, include smooth muscle, mesangial, and some epithelial cells.22Sequeira López M.L.S. Pentz E.E. 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 Recently, a role for CoRL in disease has become evident. CoRL have been shown to regenerate parietal epithelial cells and/or podocytes in a podocyte depletion model,23Pippin J.W. Sparks M.A. Glenn S.T. et al.Cells of renin lineage are progenitors of podocytes and parietal epithelial cells in experimental glomerular disease.Am J Pathol. 2013; 183: 542-557Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar erythropoietin-producing cells in chronic hypoxia,24Kurt B. Paliege A. Willam C. et al.Deletion of von Hippel-Lindau protein converts renin-producing cells into erythropoietin-producing cells.J Am Soc Nephrol. 2013; 24: 433-444Crossref PubMed Scopus (39) Google Scholar and mesangial cells after injury.25Starke C. Betz H. Hickmann L. et al.Renin lineage cells repopulate the glomerular mesangium after Injury.J Am Soc Nephrol. 2014; 26: 48-54Crossref PubMed Scopus (60) Google Scholar CoRL and pericytes share gene expression and transcription factor regulatory circuits. Gene expression analysis of CoRL isolated from renin-reporter mice shows enrichment in the pericyte marker regulator of G-protein signaling 5.26Brunskill E.W. Sequeira-Lopez M.L.S. 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 Furthermore, the RBP-J transcriptional modulator is crucial for both renin expression and pericyte development.27Castellanos Rivera R.M. Monteagudo M.C. Pentz E.S. et al.Transcriptional regulator RBP-J regulates the number and plasticity of renin cells.Physiol Genomics. 2011; 43: 1021-1028Crossref PubMed Scopus (52) Google Scholar, 28Lin E.E. Sequeira-Lopez M.L.S. Gomez R.A. RBP-J in Foxd1+ renal stromal progenitors is crucial for the proper development and assembly of the kidney vasculature and glomerular mesangial cells.Am J Physiol Renal Physiol. 2013; 306: F249-F258Crossref PubMed Scopus (55) Google Scholar Deletion of RBP-J causes a reduction in renin mRNA in the kidney, with decreases in circulating renin levels and blood pressure, and an inability to recruit renin-expressing cells when homeostasis is threatened.27Castellanos Rivera R.M. Monteagudo M.C. Pentz E.S. et al.Transcriptional regulator RBP-J regulates the number and plasticity of renin cells.Physiol Genomics. 2011; 43: 1021-1028Crossref PubMed Scopus (52) Google Scholar Pericytes, which are Foxd1+ cell derivatives,29Hatini V. Huh S.O. Herzlinger D. et al.Essential role of stromal mesenchyme in kidney morphogenesis revealed by targeted disruption of Winged Helix transcription factor BF-2.Genes Dev. 1996; 10: 1467-1478Crossref PubMed Scopus (424) Google Scholar are severely affected by targeted disruption of RBP-J in the Foxd1 lineage.28Lin E.E. Sequeira-Lopez M.L.S. Gomez R.A. RBP-J in Foxd1+ renal stromal progenitors is crucial for the proper development and assembly of the kidney vasculature and glomerular mesangial cells.Am J Physiol Renal Physiol. 2013; 306: F249-F258Crossref PubMed Scopus (55) Google Scholar FOXD1RBPJ−/− mice die prematurely and show a decreased number of renal arteries and arterioles, and an absence of glomerular cells associated with extensive glomerulosclerosis postnatally. Recent studies of the lineage relationships revealed that all mural cells, including renin-expressing cells, are derived from Foxd1+ stromal cells.30Sequeira-Lopez M.L.S. 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 We hypothesized that the versatile nature of renin-expressing cells is related to their perivascular identity. Therefore, we investigated the developmental affiliation between pericytes and renin-expressing and/or -producing cells in the human kidney. For the first time, human kidney pericytes were typified, purified, cultured, and functionally characterized as MSCs. Principally, we showed that fetal renal pericytes natively express renin with functional enzyme activity. Pericyte identification on tissue sections requires (i) confirmation of vascular anatomy, (ii) localization of ≥2 pericyte markers, and (iii) counterstaining of an endothelial cell marker.31Armulik A. Genové G. Betsholtz C. Pericytes: developmental, physiological, and pathological perspectives, problems, and promises.Dev Cell. 2011; 21: 193-215Abstract Full Text Full Text PDF PubMed Scopus (1722) Google Scholar First, we identified pericytes in the human developing kidney by transmission electron microscopy (TEM). An ultrathin section of the fetal kidney shows “peg and socket” anchoring between pericytes and adjacent endothelial cells (Figure 1). Pericytes are partially embedded into the capillary basement membrane; therefore, they can be distinguished from fibroblasts that do not establish specific contacts with endothelial cells.32Kida Y. Duffield J.S. Kida Yujiro D.S.J. Pivotal role of pericytes in kidney fibrosis.Clin Exp Pharmacol Physiol. 2011; 38: 467-473Crossref PubMed Scopus (69) Google Scholar Secondly, we used CD146 and NG2 coexpression to label pericytes. In the human fetal kidney cortex, the pericyte markers CD146 and NG2 were localized to the mesangium, afferent arterioles, and interstitial capillaries (Figure 2a). In the medulla, pericytes were found in the vasa recta and peritubular capillaries (Figure 2b).Figure 2Identification of pericytes in human embryonic and fetal kidneys. (a) Fetal pericytes were labeled with antibodies against CD146 (green) and NG2 (red) in a 15-week-old kidney cortex. CD146+NG2+ pericytes were found in peritubular capillaries (arrow), mesangial cells (open arrowheads), and afferent arterioles (arrowheads). (b) In the kidney medulla, pericytes encircled peritubular capillaries (arrows) and the vasa recta (arrowheads). (c) Double staining for CD146 (red) and smooth muscle α-actin (αSMA) (green) shows that in the cortex, pericytes coexpress αSMA in the mesangium (open arrowheads) and afferent arterioles (arrowheads). In embryonic pericytes, sections of 7-week-old kidneys were stained for pericyte markers: CD146 (green), nerve/glial antigen 2 (NG2) (green), and αSMA (green), with the endothelial cell marker CD31 (red) (on consecutive sections). (d) αSMA immunoreactivity was sparse; only single vascular smooth muscle cells were found (inset, arrow). (e) NG2+ cells surrounded endothelial cells (inset, arrow). (f) CD146 staining was perivascular (inset, arrow) and in endothelial cells (inset, arrowhead). DAPI, 4′,6-diamidino-2-phenylindole.View Large Image Figure ViewerDownload (PPT) It has long been known that certain pericyte populations, such as those in the vasa recta, are associated with α-smooth muscle actin (αSMA) expression and contractility.5Pallone T.L. Silldorff E.P. Pericyte regulation of renal medullary blood flow.Exp Nephrol. 2001; 9: 165-170Crossref PubMed Scopus (108) Google Scholar, 33Joyce N.C. Haire M.F. Palade G.E. Contractile proteins in pericytes. II. Immunocytochemical evidence for the presence of two isomyosins in graded concentrations.J Cell Biol. 1985; 100: 1387-1395Crossref PubMed Scopus (153) Google Scholar Pericytes and vascular smooth muscle cells (αSMA+ cells) are related by their mesenchymal origin and are not readily distinguished from each other.4Armulik A. Abramsson A. Betsholtz C. Endothelial/pericyte interactions.Circ Res. 2005; 97: 512-523Crossref PubMed Scopus (1531) Google Scholar Transitional pericytes and/or vascular smooth muscle cells have been documented in arterioles and venules.2Díaz-Flores L. Gutiérrez R. Varela H. et al.Microvascular pericytes: a review of their morphological and functional characteristics.Histol Histopathol. 1991; 6: 269-286PubMed Google Scholar, 34Meyrick B. Reid L. Ultrastructural features of the distended pulmonary arteries of the normal rat.Anat Rec. 1979; 193: 71-97Crossref PubMed Scopus (57) Google Scholar To examine αSMA expression in relation to pericyte markers, cells were costained for αSMA and CD146. In the cortex, CD146+αSMA+ cells were detected in afferent arterioles and mesangium (Figure 2c), but they were not detected in the medulla (not shown). Next, we queried whether αSMA, NG2, and CD146 expression overlap at earlier stages of kidney development. Cells were costained with CD31 to visualize endothelium. Few αSMA expressing cells were observed in the 7-week-old kidney (Figure 2d). NG2 and CD146 expressions were more pronounced, both marking the developing vasculature (Figure 2e and f). Taken together, these results indicate that CD146 and NG2 expression precedes that of αSMA in human kidney development. First, TEM renin immunolabeling was used to determine renin expression in vivo. Immunogold labeling revealed numerous gold particles in the pericyte cytoplasm of fetal kidneys (Figure 3a and b). Second, confocal microscopy images for renin, CD146, and NG2 in the fetal kidney were obtained. CD146 staining was found in the mesangium and afferent arterioles, and renin was localized to the juxtaglomerular (JG) area and afferent arterioles, which presented a characteristic striped pattern of expression (Figure 4a). Coexpression of renin and CD146 was apparent on the abluminal side of the vessel (Figure 4a′), and none of the renin+ cells expressed the endothelial marker CD144 (Figure 4a′). NG2 staining was present in the mesangium and afferent arterioles; renin was colocalized in afferent arterioles (Figure 4b and b′). Third, renin immunoreactivity was investigated in adult kidney sections. Similar to the fetal kidney, renin in afferent arterioles coincided with the pericyte marker CD146, but not with endothelial CD34 (Figure 5a–d). In addition, a close look at the morphology of renin-expressing arterioles revealed that renin was restricted to the abluminal side of the vessel, but it was absent from the endothelial luminal surface (Figure 5e and f). In conclusion, we found that renin-expressing cells are pericytes in vivo.Figure 4Renin is colocalized with pericyte markers in the human fetal kidney. Triple staining of fetal kidney demonstrates that (a) renin immunoreactivity (red) coincides with pericyte marker CD146 (green) expression in afferent arterioles. CD146 staining is also present in the mesangium (open arrowhead). Characteristically, renin staining had a striped pattern. The inset (a′) shows an afferent arteriole at high magnification. Renin and CD146 stainings overlap on the abluminal side of the vessel (arrowheads), whereas immunoreactivity for CD144 (grey), an endothelial cell marker, is visible inside blood vessels (arrow). (b) Nerve/glial antigen 2 (NG2) (green) staining is found in the mesangium and afferent arteriole. Renin (red) is present in the JG area. Inset (b′) shows renin+ cells coexpressing NG2 (arrowheads) at higher magnification. JG, juxtaglomerular cell.View Large Image Figure ViewerDownload (PPT)Figure 5Renin-expressing cells in the human adult kidney are pericytes. Triple staining of human adult kidney sections shows that (a) renin-expressing cells (red) are colocalized with (b) CD146 pericyte marker (green) expression (arrowheads) in afferent arterioles, whereas (c) renin-expressing cells do not express the CD34 endothelial cell marker. (d) The merged image illustrates the overlap between renin and CD146 expressions, indicated by arrowheads. (e) Transverse section of the JG area in the adult kidney demonstrates renin immunoreactivity in an afferent arteriole (open arrowhead). Inset (e′) shows JG vascular structure at higher magnification. On the abluminal side of the vessel, pericytes (PC) (arrow) show characteristic ring-like distribution in the vascular wall, which coincides with renin expression. Endothelial cells (EC) are localized to the luminal side of the vessel (arrow). (f) No primary antibody control is included that showing negligible staining. Bars = 1 μm on (e), (e′), and (f). JG, juxtaglomerular cell.View Large Image Figure ViewerDownload (PPT) Primary cultures of fetal kidney cells were established to investigate the renin induction potential of kidney pericytes. A method for pericyte purification by flow cytometry from multiple human tissues has been described previously.35Crisan M. Deasy B. Gavina M. et al.Purification and long-term culture of multipotent progenitor cells affiliated with the walls of human blood vessels: myoendothelial cells and pericytes.Methods Cell Biol. 2008; 86: 295-309Crossref PubMed Scopus (104) Google Scholar, 36Corselli M. Crisan M. Murray I.R. et al.Identification of perivascular mesenchymal stromal / stem cells by flow cytometry.Cytometry. A. 2013; 83: 714-720Crossref PubMed Scopus (103) Google Scholar The gating strategy is shown in Figure 6a. CD146+CD34−CD56−CD45− pericytes were sorted at an average percentage from total live cells of 4.15 ± 2.61% (n = 22). Cytospun kidney pericytes were analyzed for renin immunoreactivity. Antirenin staining confirmed that freshly isolated pericytes included renin-expressing cells (Figure 6b), and that renin immunoreactivity was retained in culture; 0.003 ± 0.002% of cells were renin-positive after 48 hours (Figure 6c). Semiquantitative real-time polymerase chain reaction (RT-PCR) analysis showed that renal pericyte primary cultures express high, stable levels of CD146 and gradually increasing levels of NG2 and PDGFR-β across passages 1 to 6. Cultured pericytes were negative for CD34. Depletion of endothelial cells in sorted pericytes was based on (i) a CD34 negative selection on the fluorescence-activated cell sorter, (ii) additional negative selection of endothelial cells using CD144 in most of the sorts performed for this study (not shown), and (iii) verification by flow cytometry that renal pericyte cultures did not contain endothelial cells (CD31+) (not shown). CD56 was detected at passage 1 of sorted pericytes, but was not detected at later passages. We also confirmed that the FOXD1 stromal cell marker29Hatini V. Huh S.O. Herzlinger D. et al.Essential role of stromal mesenchyme in kidney morphogenesis revealed by targeted disruption of Winged Helix transcription factor BF-2.Genes Dev. 1996; 10: 1467-1478Crossref PubMed Scopus (424) Google Scholar was present in cultured pericytes as well as CRIM1, which plays a role in maintaining renal microvascular integrity37Wilkinson L. Gilbert T. Sipos A. et al.Loss of renal microvascular integrity in postnatal Crim1 hypomorphic transgenic mice.Kidney Int. 2009; 76: 1161-1171Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar (Figure 6d). During the initial phase of cell culture, pericytes displayed elongated, spindle-shaped morphology (Figure 6e, passage 0). However, after a few weeks in high glucose medium, cells displayed a more rounded shape (Figure 6e, passage 2). At passages 5 to 6, renal pericytes appeared homogenously elongated, with extended cell processes (Figure 6e, passage 6). Immunocytochemistry performed at passage 2 showed that cultured kidney pericytes contained low levels of αSMA with a high frequency of NG2+ cells and ubiquitous CD146 immunoreactivity (Figure 6f). MSCs are defined by their ability to differentiate into several mesodermal cell lineages. After 2 weeks, pericyte primary cultures with lineage-specific differentiating media exhibited features of adipogenic osteogenic or chondrogenic cell lineages (Figure 6g). To demonstrate that renal pericytes have the potential to produce and secrete renin, primary cell cultures were assayed for renin expression, immunoreactivity, and enzymatic activity after treatment with cyclic adenosine monophosphate (cAMP) inducers: forskolin and isobutyl-1-methylxanthine (IBMX).38Everett A.D. Carey R.M. Chevalier R.L. et al.Renin release and gene expression in intact rat kidney microvessels and single cells.J Clin Invest. 1990; 86: 169-175Crossref PubMed Scopus (57) Google Scholar, 39Fowler J.D. Johnson N.D. Haroldson T.A. et al.Regulated renin release from 3T3-L1 adipocytes.Am J Physiol Endocrinol Metab. 2009; 296: E1383-E1391Crossref PubMed Scopus (18) Google Scholar Control cells (vehicle: medium + vehicle; untreated cells: medium) demonstrated no renin immunoreactivity (Figure 7a), whereas 4.60 ± 2.50% of induced cells expressed renin (Figure 7b). Induced renal pericyte primary cultures showed substantial upregulation of renin expression after 24 hours (Figure 7c). Renin mRNA was virtually absent in untreated (0.01 ± 0.00) and vehicle-treated cells (0.02 ± 0.01) compared with cells treated with cAMP inducers (n = 5; 1.25 ± 0.33; P < 0.01). A renin activity assay confirmed that pericytes produce metabolically active renin (Figure 7d). In medium from control cells, renin activity was virtually undetectable (0.40 ± 0.40 and 0.02 ± 0.12 ng angiotensin I/ml/h in untreated and vehicle cells, respectively) compared with activity in medium from induced cells (n = 3; 5.62 ± 1.49 ng angiotensin I/ml/h; P < 0.05). In agreement with previous studies,26Brunskill E.W. Sequeira-Lopez M.L.S. 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, 40Kurtz A. Schweda F. Todorov V. et al.Physiology of kidney renin.Physiol Rev. 2010; 90: 607-673Crossref PubMed Scopus (196) Google Scholar it was noted that early passaged cells displayed greater potential for renin induction. Induction of renin expression and activity showed a dramatic decline beyond passage 2 (Supplementary Figure S1). TEM confirmed the presence of renin granules in induced kidney pericytes. Human pericyte ultrastructure was well preserved, and immunogold labeling for the pericyte marker NG2 was localized to the cytoplasm (Figure 7e). Immunogold labeling of renin was visible in pericytes treated with cAMP inducers (Figure 7g) but not in noninduced control cells (Figure 7f). The cytoplasm of induced pericytes appeared more vacuolated (Figure 7g) compared with controls (Figure 7e and f). To verify that renin-producing cells were pericytes, primary cultures of cells were stained for renin and CD146, NG2, and αSMA; 65.68 ± 7.40% of cells were renin+/αSMA+ (Figure 7h), and all renin+ cells were costained for CD146 (Figure 7i) and NG2 (Figure 7j). Therefore, renin-expressing cells in these experiments were defined as CD146+NG2+αSMA+/−. We concluded that NG2 is a better marker for the renin-producing cell population, because in vivo NG2 is mainly associated with arterioles (where renin is expressed) and capillaries.41Murfee W.L. Skalak T.C. Peirce S.M. Differential arterial/venous expression of NG2 proteoglycan in perivascular cells along microvessels: identifying a venule-specific phenotype.Microcirculation. 2005; 12: 151-160Crossref PubMed Scopus (110) Google Scholar Components of the renin angiotensin system (RAS) have been found in many human tissues and are commonly referred to as local RASs.42Paul M. Poyan Mehr A. Kreutz R. et al.Physiology of local renin-angiotensin systems.Physiol Rev. 2006; 86: 747-803Crossref PubMed Scopus (1350) Google Scholar We hypothesized that renin induction potential is an intrinsic feature of pericytes of renal and nonrenal origins. Renin gene expression was therefore compared in tissues and primary cultures of pericytes derived from second trimester tissues: fetal kidney, liver, adrenal glands, and placenta. High renin expression was found in fetal kidney and placenta tissue digests, lower levels of expression were present in cultured renal and placental pericytes, and the least amount was detected in fetal liver and adrenal gland digests (Supplementary Figure S2). Placental pericytes show renin immunoreactivity in vitro after incubation with cAMP inducers (Figure 8a and b). No renin positivity (0%) was observed in control cells, whereas 4.64 ± 2.02% of induced cells were positive. Primary placental pericytes had increased renin mRNA levels after 24-hour treatment with cAMP inducers (Figure 8c). Renin mRNA levels were low in untreated (0.38 ± 0.32), and vehicle-treated (0.28 ± 0.28) cells, but were significant after induction (2.11 ± 0.05; n = 2). Renin activity was measured in culture medium from primary cells after renin induction and was increased (0.74 ± 0.32; n = 3) compared with untreated (0.20 ± 0.13) and vehicle-treated cells (0.14 ± 0.1 ng angiotensin I/ml/h). However, renin gene expression did not correlate with renin activity (Figure 8d). This study provided definitive evidence that renin-producing cells are—at least some of them—pericytes. Previously, a lineage relationship between renin-expressing cells and pericytes was proposed based on microarray studies,26Brunskill E.W. Sequeira-Lopez M.L.S. 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 and recently, it was shown that renin-expressing cells and pericytes are derived from a common Foxd1+ progenitor.30Sequeira-Lopez M.L.S. 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 We used a human fetal kidney to demonstrate that renin-expressing cells are pericytes, as defined by anato