Human kidney pericytes produce renin

Identification of pericytes in the human fetal kidney

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.31 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.32 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).

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.5, 33 Pericytes and vascular smooth muscle cells (αSMA⁺ cells) are related by their mesenchymal origin and are not readily distinguished from each other.4 Transitional pericytes and/or vascular smooth muscle cells have been documented in arterioles and venules.2, 34 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.

Renin-expressing cells in the human fetal and adult kidney are pericytes

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.

Purification of pericytes from human fetal kidney

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.35, 36 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).

Phenotyping human renal pericytes

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 marker29 was present in cultured pericytes as well as CRIM1, which plays a role in maintaining renal microvascular integrity37 (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).

Cultured human fetal renal pericytes produce renin

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).38, 39 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,26, 40 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).

Renin-expressing cells are CD146⁺NG2⁺αSMA^+/− cells

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.41

Nonrenal pericytes also express and produce renin

Components of the renin angiotensin system (RAS) have been found in many human tissues and are commonly referred to as local RASs.42 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).

Human tissue collection

Human kidneys (6–18 weeks of development) and second trimester placenta were obtained after elective abortions, with informed consent from the donor and in compliance with the rules established by the South East Scotland Research Ethics Committee. Developmental age was estimated from the crown to rump distance measurements and medical history. Eight weeks of development is the limit between embryonic and fetal life in humans.

Normal human adult kidney biopsies were obtained from nephrectomy.

Tissue processing for cell sorting

Tissues were minced and incubated in Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Paisley, UK)/20% fetal bovine serum (FBS; Invitrogen)/1% penicillin, streptomycin (PS; Invitrogen) with 0.5 mg/ml of each collagenase (IA, II, and IV; Sigma-Aldrich, Dorset, UK) at 37 °C in a shaking water bath. After a 70-minute incubation, dispersed cells were passed through 100- and 70-μm filters (Fisher Scientific, Loughborough, UK) and centrifuged (10 minutes at room temperature). Cells were resuspended in red blood cell lysis buffer (9:1 ratio of stock 1 [8.3-g ammonium chloride/L water]; stock 2 [20.59-g Tris base/LH20]) at 37 °C for 5 minutes, then washed, resuspended in phosphate-buffered saline (PBS; Invitrogen)/2% FBS/1% PS, and filtered through a 40-μm cell strainer.

Cell suspensions (approximately 10⁸/ml) were incubated on ice for 20 minutes in the dark with fluorescence-conjugated antibodies (diluted 1:100): CD146-Alexa Fluor 647 (Abd Serotec, Kidlington, UK), and CD34-PE, CD45-APC-Cy7, and CD56-PE-Cy7 (BD Biosciences, Oxford, UK). The initial step for each flow analysis was to create a standard side scatter/forward scatter dot plot to eliminate cell aggregates, debris, and dead cells. Dead cells were excluded from the analysis using 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Kidney pericytes were sorted as a CD146⁺CD34⁻CD45⁻CD56⁻ population. Placental pericytes were obtained using the same protocol. Cell sorting was performed using a FACSAria flow cytometer (BD Biosciences).

Primary cell culture of kidney pericytes

Isolated pericytes were seeded in endothelial cell growth medium-2 (EGM-2, Cambrex, Bio Science, Wokingham, UK) on 0.2% gelatin-coated culture dishes (Sigma-Aldrich) at a cell density of 2 × 10⁴ cells/cm² and cultured at 37 °C, with 5% carbon dioxide. After reaching confluence, cells were passaged and maintained in high glucose DMEM/20% FBS/1% PS.

Multilineage differentiation assays to promote osteogenic, adipogenic, and chondrogenic phenotypes were performed as described previously.9, 12

Real-time polymerase chain reaction and quantitative real-time polymerase chain reaction

RNA was extracted using the phenol-chloroform method,63 and cDNA was generated with a SuperScript III Reverse Transcriptase system kit (Invitrogen). RT-PCR was performed with Taq DNA polymerase (Bioline) and carried out using primers listed in Supplementary Table S1.

Quantitative PCR was performed with Universal Probe Library System Technology (Roche Applied Science, Welwyn Garden City, UK). The following primers were used: glyceraldehyde-3-phosphate dehydrogenase: forward 5′-AGCCACATCGCTCAGACAC-3′; glyceraldehyde-3-phosphate dehydrogenase reverse 5′-GCCCAATACGACCAAATCC-3′; renin forward 5′-TACCTTTGGTCTCCCGACAG-3′; and renin reverse 5′- TTGAGGGCATTCTCTTGAGG-3′ (Eurofins MWG Operon, Ebersberg, Germany).

Immunohistochemistry and immunocytochemistry

Human developing kidneys were snap-frozen in optimal cutting temperature freezing medium. Tissue sections were fixed in acetone/methanol (1:1) for 10 minutes, then incubated at 4 °C overnight with the following antihuman primary antibodies: anti-CD146 (BD Pharmingen 1:100); anti-CD146-FITC (AbD Serotec 1:100); anti-NG2 (BD Pharmingen 1:300); anti-αSMA-FITC (Sigma 1:200); anti-renin (R&D Systems 1:100; R&D Systems, Abingdon, UK); anti-CD144 (Thermo Scientific 1:200); and anti-CD31 (Abcam 1:300). This was followed by 1-hour incubation with secondary antibodies conjugated with Alexa Fluor 488, 555, or 647 (Invitrogen 1:1000). To preserve the sections and visualize nuclei, DAPI-mounting medium (DAPI-Fluoromount-G; SouthernBiotech, Cambridge, UK) was used. The same fixation and staining protocol was used for immunocytochemistry and cytospins.

For immunocytochemistry, pericytes were seeded onto a 24-well plate (10,000 cells/well) with coverslips (diameter 13 mm; Van Waters & Rogers) placed at the well bottoms, and maintained in the culture until confluent.

For cytospin preparations, 2 × 10⁵ pericytes sorted from 17-week-old fetal kidneys were centrifuged and immediately suspended in PBS/2% FBS (5 × 10⁴ cells/100 μl). Subsequently, cells were loaded into cytospin cuvettes and centrifuged for 3 minutes at 300g.

Sections of paraffin-embedded adult human kidneys were dewaxed in xylene and rehydrated through a graded series of alcohol to water. For fluorescent imaging, overnight incubations at 4 °C were performed sequentially with antirenin (1:100), anti-CD146 (Abcam 1:50), and anti-CD34 antibody (BD Pharmingen 1:100). For better visualization of tissue morphology, antirenin (60 minutes at room temperature, 1:100) staining was performed with 3,3′-diaminobenzadine (Dako, Cambridge, UK).

Negative controls were performed for each immunohistochemistry experiment, consisting of isotype-matched negative control or omitting the primary antibody.

Renin induction

Confluent pericyte cultures were serum-starved (DMEM/1% PS) for 24 hours. Renin induction was stimulated with cAMP inducers (10-μm forskolin; Cell Signalling, Leiden, Netherlands, and 100 μm IBMX; Sigma-Aldrich) in DMEM/1% PS. Controls consisted of vehicle (medium/ethanol/0.01% dimethyl sulfoxide) and serum-free medium with no additives (DMEM/1% PS). After 24 hours, culture media were collected for renin activity assay. After a PBS wash, cells were harvested for RNA extraction.

Renin activity assay

Renin activity in media from primary pericytes cultures was determined by radioimmunoassay of angiotensin I generated by incubation with angiotensinogen as previously described. Media aliquots derived from renin-induced pericyte primary cultures and media containing 0 to 400 pmol angiotensin I standards (Sigma-Aldrich) were incubated at 37 °C and at 0 °C for 1 hour with an assay buffer (50-mM Tris/hydrogen chloride, 0.2% neomycin sulphate, and 0.1% human serum albumin, pH 7.4) that contained porcine renin substrate and angiotensin I antibody. Renin activity was calculated by subtracting the amount of angiotensin I in samples incubated at 0 °C from values of samples incubated at 37 °C.

Electron microscopy

Control and induced-treated pericytes cultured on polyester filters were fixed with 3% paraformaldehyde/0.05% glutaraldehyde in 0.1-M phosphate buffer (pH 7.2) for 10 minutes at 37 °C. Filters were stained with uranyl acetate (2% weight/volume in distilled water), dehydrated through increasing concentrations of methanol (70%–100%) at −20 C, and embedded in LR Gold resin (London Resin Company, Reading UK). Ultrathin sections (50–80 nm) were prepared with a Reichart-Jung ultracut microtome. For immunogold labeling of renin and the pericyte marker NG2, sections were incubated for 2 hours at 37 °C with primary antihuman renin (1:1000) or NG2 antibodies (1:500), then for 1 hour with 15-nm Protein A gold-conjugate (1:60; British Biocell, Cardiff, Wales, UK). No labeling was observed in negative control sections in which the primary antibody was replaced with PBS/0.1% egg albumin. Sections were counterstained with lead citrate and uranyl acetate, and examined on a JOEL 1010 TEM (JOEL USA Inc., Peabody, Massachusetts, USA).

Statistics

Data are expressed as mean ± SE. One-way analysis of variance was used to determine statistical significance.