telocytes and myoepithelial cells

Accepted Manuscript Title: Molecular phenotypes of the human kidney: Myoid stromal cells/telocytes and myoepithelial cells Authors: M.C. Rusu, L. Mogoant˘a, F. Pop, M.A. Dobra PII: DOI: Reference:

S0940-9602(18)30046-3 https://doi.org/10.1016/j.aanat.2017.12.015 AANAT 51249

To appear in: Received date: Revised date: Accepted date:

5-11-2017 9-12-2017 10-12-2017

Please cite this article as: Rusu, M.C., Mogoant˘a, L., Pop, F., Dobra, M.A., Molecular phenotypes of the human kidney: Myoid stromal cells/telocytes and myoepithelial cells.Annals of Anatomy https://doi.org/10.1016/j.aanat.2017.12.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Molecular phenotypes of the human kidney: myoid stromal cells/telocytes and myoepithelial cells Running title: Renal telocytes

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Rusu MC1*, Mogoantă L2, Pop F3, Dobra MA4

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MD, Ph.D., Dr.Hab.,Dr.Biol.,Prof., (a) Division of Anatomy, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania; (b) MEDCENTER – Center of Excellence in Laboratory Medicine and Pathology 2

MD, Ph.D., Prof., Department of Histology, University of Medicine and Pharmacy Craiova, Romania 3

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MD, Ph.D., Lect. (a) Division of Pathologic Anatomy, Faculty of Medicine, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania; (b) MEDCENTER – Center of Excellence in Laboratory Medicine and Pathology

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MD, Ph.D. stud., Division of Anatomy, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania

*Corresponding Author

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Work Address: “Carol Davila” University of Medicine and Pharmacy, 8 Eroilor Sanitari Blvd., RO-76241, Bucharest, Romania

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PHONE: +40722363705

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E-mail address: [email protected] (M.C.Rusu)

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Abstract

The connective stromal and epithelial compartments of the kidney have regenerative

potential and phenotypic flexibility. A few studies have shown that cells appertaining to both compartments can exhibit myoid phenotypes. The purpose of our study was to investigate the myoid pattern of kidney and its association with the kidney niches containing stromal cells/telocytes (SC/TCs). We performed an immunohistochemical study using a panel of endothelial, myoid, mesenchymal and stem/progenitor markers, namely CD31, CD34, CD105 1

(endoglin), CD117/c-kit, nestin, desmin, α-smooth muscle actin (α-SMA) and the heavy chain of smooth muscle myosin (SMM). We used histologically normal kidney samples, obtained after nephrectomy, from nine adult patients. The capsular SC/TCs had a strong CD34 and partial nestin and CD105 immunopositivity. Subcapsular and interstitial SC/TCs expressed ckit, nestin, CD105, but also α-SMA and SMM, therefore having a myoid phenotype. The endothelial SC/TCs phenotype was CD31+/ CD34+/ CD105+/ nestin±/ SMM±/ α-SMA±. All

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three myoid markers were expressed in periendothelial SC/TCs. We also found a scarce expression of nestin in parietal epithelial cells of Bowman’s capsule, and in podocytes. In

epithelial cells, we found a positive expression for CD31, CD117/c-kit, desmin, CD34, SMM,

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and CD105. In epithelial tubular cells, we found a predominant basal expression of the myoid markers (SMM and desmin). In conclusion, myoepithelial tubular cells, myoid endothelial cells

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and myoid SC/TCs are normal constituents of the kidney.

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Keywords: renal tubules; nephron; stem niche; mesangial cells; immunohistochemistry;

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stromal cells

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Introduction

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Telocytes (TCs) were indicated as a distinctive cell type by Popescu and FaussonePellegrini (2010) after being previously described as “Interstitial Cajal-Like Cells” (ICLCs). TCs

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were defined as interstitial (stromal) cells projecting long, thin and slender processes named telopodes; these prolongations are rather moniliform, with dilations (podoms) united by thin segments (podomers) (Grigoriu et al., 2016; Petre et al., 2016; Rusu et al., 2016; Rusu et al., 2014b; Rusu et al., 2017a; Rusu et al., 2014c; Rusu et al., 2012b; Rusu et al., 2012c; Vannucchi

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et al., 2016).

Various authors have described distinct TC subtypes, some of them located in the same

organ (Grigoriu et al., 2016; Rusu et al., 2017a; Vannucchi et al., 2016). There are only a few studies explicitly investigating TCs within the urinary system (Dobra et al., 2017; Gevaert et al., 2012; Metzger et al., 2004; Neuhaus et al., 2018; Qi et al., 2012; Rusu et al., 2014b; Rusu

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et al., 2017b; Traini et al., 2018; Wolnicki et al., 2016; Zheng et al., 2012). In the kidney, Zheng et al. (2012) found TCs exclusively in the subcapsular space (Zheng et al., 2012). However, Qi et al. (2012) reported TCs in the interstitium of the human kidney cortex. Renal TCs were found to express CD34, CD117/c-kit, and vimentin, with variable intensity (Qi et al., 2012). Li et al. suggested that renal TCs, which in their study were identified only morphologically, could protect tubular epithelial cells from ischemia-reperfusion injuries through an inflammation-

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independent mechanism involving growth factors (Li et al., 2014). As there are only a few reports on renal TCs, which are not enough to adequately

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characterize the topography and immunophenotypes of renal TCs, we aimed to use a larger panel of markers to locate the renal stromal cells/TCs (SC/TCs) and to evaluate their

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phenotype.

Material and Method

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Sample tissues of histopathologically normal kidneys were obtained from human adult patients (three men and six women, aged from 52 to 67 years) after nephrectomy (for kidney

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tumors). Informed consent for their use for research purposes was obtained before surgery. The study was conducted according to the principles of the Helsinki Declaration and relevant

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national regulations (Law 46/20003 regarding the rights of patients, and the Deontology Code of the Romanian College Board). The kidney samples were oriented, paraffin-embedded, fixed for 24 hours in buffered

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formalin (8%) and processed with an automatic histoprocessor (Diapath, Martinengo, BG, Italy), with paraffin embedding. Sections were cut manually at three μm and mounted on SuperFrost® electrostatic slides for immunohistochemistry (Thermo Scientific, Menzel-Gläser, Braunschweig, Germany). Histological evaluations used three μm thick sections stained with hematoxylin and eosin. Internal negative controls resulted when the primary antibodies were

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not applied to slides. The slides were screened for renal pathologies, and only nonpathological slides were used for the study. We used primary antibodies for the smooth muscle myosin heavy chain (SMM, clone S131, Novocastra-Leica, Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, UK, 1:100), CD34 (clone QBEnd/10, Biocare Medical, Concord, CA, USA, 1:50), endoglin/CD105 (polyclonal, Thermo Scientific, Pierce Biotechnology, Rockford, USA, 1:50), CD117/c-kit (clone

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Y145, Biocare Medical, Concord, CA, USA, 1:100), CD31 (PECAM-1, clone BC2, Biocare Medical, Concord, CA, USA, 1:200), nestin (clone 10c2, Santa Cruz Biotechnology, Santa

Cruz, CA, USA, 1:500), desmin (clone OV-TL 12/30, Biocare Medical, Concord, CA, USA,

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1:100) and alpha-smooth muscle actin (α-SMA, clone D33, Biocare Medical, Concord, CA, USA, 1:50).

Tissues were deparaffinized and rehydrated; then endogenous peroxidase was blocked

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using Peroxidase 1 (Biocare Medical, Concord, CA, USA). For the heat-induced epitope

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retrieval was used the Decloaking Chamber (Biocare Medical, Concord, CA, USA) and a

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retrieval solution at a pH of six (Biocare Medical, Concord, CA, USA). Background Blocker

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(Biocare Medical, Concord, CA, USA) was used to reduce non-specific background staining. The primary antibody was then applied. Different HRP-based detection systems were used:

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for CD34 and CD105, the two-steps detection used the 4plus detection system (Biocare Medical, Concord, CA, USA); for α-SMA, desmin and SMM was used MACH 4 (Biocare Medical, Concord, CA, USA); for all other primary antibodies we used the MACH 2 rabbit HRP polymer

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detection (Biocare Medical, Concord, CA, USA). An HRP-compatible chromogen (DAB) was applied. Sections were counterstained with hematoxylin and rinsed with deionized water. For

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washing steps was used TBS solution, pH 7.6. The microscopic slides were analyzed, and micrographs were acquired and scaled using

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a Zeiss working station that was described elsewhere (Rusu et al., 2012a).

Results Molecular anatomy of the kidney cortex stroma

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HE slides were used for analyzing the overall morphology of the kidney and its associated pathologies. The normal kidney cortex consisted of a fibrous, collagen capsule underneath which was a subcapsular connective layer and, below, the renal parenchyma. The renal parenchyma consisted of tubular epithelia covered by a basal lamina, renal corpuscles, and microvessels, all being embedded within an interstitial framework containing networks of delicate spindle-shaped stromal cells with telopodial prolongations.

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The SC/TCs from the kidney capsule and subcapsular stroma had slightly different

molecular phenotypes. The renal capsule appeared as a vascularized structure, in which

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SC/TCs were collagen-embedded, while in the subcapsular stroma SC/TCs formed a cell-rich, mesh-like structure.

The capsular SC/TCs did not express CD31 (fig.1), CD117/c-kit (fig.1), desmin (fig.2) or

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SMM (fig.6), and scarcely expressed nestin (fig.4) and CD105 (fig.7). These capsular SC/TCs

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were positive for CD34 (fig.3) and α-SMA (fig.5).

The subcapsular SC/TCs failed to express CD31 and CD117/c-kit (fig.1) or desmin (fig.2)

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but were positive for the other markers: CD34 (fig.3), nestin (fig.4), α-SMA (fig.5), SMM (fig.6)

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and CD105 (fig.7).

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The interstitial kidney SC/TCs were negative for CD31 (fig.1), CD117/c-kit (fig.1) and desmin (fig.2). Expression of SMM was scarcely positive (fig.6). These cells expressed CD34

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(fig.3), nestin (fig.4), α-SMA (fig.5), and CD105 (fig.7). Endothelial cells of renal cortex expressed CD31 (fig.1), CD34 (fig.3), and CD105 (fig.7).

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They also had scarce nestin (fig.4) and SMM immunopositivity (fig.6). In these cells, we could not find a positive expression for CD117/c-kit (fig.1) and α-SMA (fig.5). Molecular anatomy of the kidney medulla stroma

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Within the renal medulla, SC/TCs did not express CD31 (fig.8), CD34 (fig.9), nestin

(fig.10), CD117/c-kit (fig.10). However, the myoid markers, namely α-SMA and SMM, were focally expressed in stromal networks (fig.12). Endothelial cells expressed CD31 (fig.8), CD34 (fig.9), nestin (fig.10) but also α-SMA and SMM (fig.11). Isolated stromal cells, morphologically distinct from SC/TCs, expressed CD34 (fig.9). False telopodes belonging to endothelial tubes (fig.9) were accurately differentiated from true TCs. All the myoid markers we have used, 5

namely α-SMA, SMM, and desmin, were expressed in the periendothelial mural cells of microvessels. Tubular epithelial phenotypes

Scarce expression of nestin was found in parietal epithelial cells of the Bowman’s capsule and podocytes (fig.4). In these cells, all other markers were negative, except for

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desmin which was infrequently positive (fig.2). In all tested samples, we found either a complete or scarce expression of the following markers within the tubular epithelial cells: CD31 (fig.1), CD117/c-kit (fig.1, fig.10), desmin

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(fig.2), CD34 (fig.3), SMM (fig.6, fig.11) and CD105 (fig.7). All the others were negative in the tubular epithelia. Also, in the tubular epithelial cells, the expression of myoid markers (SMM

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and desmin), was preferentially located at the basal poles.

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Discussion

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The stromal cells/fibroblasts/fibrocytes/telocytes Telocytes, described by Popescu and Faussone-Pellegrini (2010) as a distinctive cell

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type, were previously characterized by various authors as ICLCs. One of the ICLC studies (Pieri et al., 2008) positively identified a CD34+ ICLC, using immune electron microscopy, and was

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used as an argument for the distinctiveness of these cells, and a reason for their rebranding as TC (Popescu and Faussone-Pellegrini, 2010). To our knowledge, that was the only IEM proof

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of a marker expression in cells with telopodes, even though TCs were often studied using transmission electron microscopy (TEM) (Cantarero Carmona et al., 2011; Cantarero et al., 2016; Manetti et al., 2013; Nicolescu and Popescu, 2012; Rusu et al., 2014c; Rusu et al., 2012c).

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Based on that study, CD34+ spindle-shaped stromal cells with telopodes could be considered TCs, even if telopodes can be hardly distinguished using light microscopy. As there is no obvious correlation between TEM appearance of TCs and a specific panel of immune markers, a positive diagnosis of TC using either single- or double-labeling of stromal cells with a TC-like morphology should be regarded with caution.

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The connective tissue of virtually all anatomic locations harbors CD34+ cells, which received different names depending on the research group, perhaps because the authors “were unaware of the fact that the cell population they investigated and described were identical” (Barth and Westhoff, 2007). The CD34+ stromal cells/telocytes (SC/TCs) were suggested to play essential functions in the maintenance and modulation of tissue homeostasis and morphogenesis/renewal/repair

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(Diaz-Flores et al., 2016). These CD34+ SC/TCs behave as quiescent progenitors within stromal

stem niches and have the potential to acquire an α-SMA+ phenotype (Diaz-Flores et al., 2016;

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Diaz-Flores et al., 2015; Díaz-Flores et al., 2016). The in vivo activation of CD34+ SC/TCs leads to a separation from the neighboring structures (mainly the vascular walls), and the development of organelles needed for synthesis, cell proliferation (transit-amplifying cells)

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and differentiation (Díaz-Flores et al., 2016).

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The findings of Pease (1968)

Pease (1968) found, in transmission electron microscopy, renal interstitial myoid cells

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that lacked organelles (except for ribosomes) and projected “long pseudopods” (Pease, 1968).

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These cells could be viewed as poorly differentiated cells (namely stem/progenitor cells). The prolongations of these myoid interstitial cells were poorly described in that study (Pease,

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1968), and therefore a retrospective analysis based on the criteria for telopodes was impossible. However, our results in light microscopy did identify myoid interstitial cells lining

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the tubules and renal corpuscles, which could be considered, by also taking into account the study mentioned above, as myoid SC/TCs.

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The SC/TCs within renal interstitium Johnson et al. found, in an IEM study, that α-SMA+ stromal cells of the kidney are

actively proliferating mesangial cells (Johnson et al., 1991). Other authors (Alpers et al., 1994;

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Nagle et al., 1975) have also identified, in various kidney disorders, an extensive expression of myoid filaments, such as α-SMA, in the renal interstitium, suggesting “that the whole kidney may function as a contractile organ” (Alpers et al., 1994), potentially influencing renal blood flow and pressure (Alpers et al., 1994). Some authors suggested that the interstitial upregulation of α-SMA could be correlated with the presence of interstitial fibrosis (Boukhalfa et al., 1996) and altered renal function (Saratlija Novakovic et al., 2012). 7

By corroborating these studies with our own, we could hypothesize that periendothelial SC/TCs could be pericytes, pericytes-derived myofibroblasts, or mesangial-like progenitor cells populating the interstitium. These distinctions should be further clarified in transmission electron microscopy. Some authors suggested that a α-SMA+ stromal cells phenotype could appear through a epithelial-mesenchymal transformation (EMT) from epithelial cells (Loeffler and Wolf, 2015; Masszi et al., 2003; Ng et al., 1998), a hypothesis that

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is supported by our study; however, other studies suggested these cells to have a stromal, and not an epithelial origin (Humphreys et al., 2010), which could be reinforced by the negative epithelial expression of α-SMA in our samples. Sun et al suggested the origin of these cells to

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be either from the bone marrow or the endothelial-mesenchymal transformation (Sun et al., 2016). As there is conflicting data, the actual origin of myoid SC/TCs, or myofibroblasts, is still

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controversial.

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The myoepithelial cells of the human kidney

Our results suggested a variable expression of myoid markers in renal epithelia. α-SMA

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was not expressed in epithelial cells, while the expression of SMM was inconstant in cortical tubules and collector ducts and was common in the loops of Henle. Desmin was often

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expressed in cortical tubules and loops of Henle but was scarce in the collector ducts. When positive, the myoid markers were preferentially located at the basal poles of the epithelial

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cells. Within the renal corpuscles, desmin was occasionally expressed in the parietal cells of the Bowman’s capsule, while the expression of SMM was occasionally positive in glomerular endothelium. These findings support the “contractile kidney” hypothesis (Alpers et al., 1994).

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Pease (1968) showed, using TEM, the myoid features of normal renal epithelia, which

were more preeminent in the parietal cells of Bowman’s capsule and the base of the proximal tubular cells, and scarce in distal tubular cells (Pease, 1968), and suggested that they should be regarded as being myoepithelial in nature (Pease, 1968). Ross and Reith confirmed the findings of Pease (1968) and found additional structures, the branching microvillous processes of the basal region of the tubular epithelial cells, postulating that these cell processes are 8

associated with fluid transport, and the myoid bands in the basal poles of the epithelial cells could act as devices controlling the movement of fluids (1970). Tanaka et al. gathered additional evidence and discussed that the thin and thick filaments they found in epithelial cells of Bowman’s capsule and proximal tubules consist of actin and myosin, respectively (1977). Trenchev et al., working on rat kidney, found by IEM that actin localized within the foot processes of podocytes, the basal processes of tubular epithelial cells and smooth muscle

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cells, and myosin was found only in vascular smooth muscle cells (1976). More recently, Saleem et al. showed that mature podocytes in vitro have a functional contractile smooth

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muscle cell phenotype with low levels of α-SMA (Saleem et al., 2008).

The myoid endothelial cells of the kidney

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Our study showed scarce positive expression of SMM in glomerular and interstitial endothelia and a lack of α-SMA endothelial expression. Several studies, quoted in Giacomelli

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et al. (1970), indicate the presence of functional contractile filaments in endothelial cells,

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(Giacomelli et al., 1970; Ragan et al., 1988; Rohlich and Olah, 1967; Yohro and Burnstock,

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1973). We believe that further demonstration of contractile endothelial cells of kidney should be done using TEM, in accordance with Hammersen who commented in 1976 that “a

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convincing demonstration of myosin in endothelial cells is still lacking” (Hammersen, 1976). Our study has shown that myosin is expressed in kidney endothelial cells, but the results

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should be reinforced by TEM.

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The renal capsular stem niche and telocytes Zheng et al. performed a TEM study, and primary cultures of TCs that were sorted only

on morphological criteria (the presence of telopodes), and found TCs in the subcapsular space

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of the kidney (2012). The authors did not investigate the renal capsule being a potential niche of TCs. Interestingly, in the above quoted TEM study, were also presented telopodesprojecting macrophages (Zheng et al., 2012), this being an additional argument for lack of specificity of morphological criteria to positively identify TCs. Some authors showed that the renal capsule acts as a stem niche which harbors mesenchymal stem cells potentially involved in renal repair (Park et al., 2010). As we showed that SC/TCs are present in these niches, they could be stem/progenitor cells, as was suggested by various studies in other organs (Grigoriu 9

et al., 2016; Petre et al., 2016; Rusu et al., 2017a). In neonatal kidneys, the renal stem/progenitor niche is in contact with a tunnel system widely spread between atypical smooth muscle cells at the inner side of the capsule (Minuth and Denk, 2014). We found SC/ TCs to have a different immunophenotype in the renal capsule compared to the subcapsular niche. The capsular SC/TCs variably expressed nestin and endoglin, were CD34-positive, and negative for all other markers. The subcapsular TCs

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expressed CD34, nestin, endoglin but also two of the myoid markers we used, namely α-SMA and SMM, being negative for other markers. This result suggests that subcapsular myoid

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SC/TCs are probably the atypical smooth muscle cells previously described on the inner side

of the renal capsule (Minuth and Denk, 2014). The positive expressions of nestin, CD34, and endoglin (CD105) reveals a stem/progenitor potential of the capsular and the subcapsular

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SC/TCs.

Interstitial myoid SC/TCs had an immunophenotype similar to the one found in the

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subcapsular space, suggesting a continuity of these niches. Our result supports the study of

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Alpers et al., who identified, in non-diseased kidneys, a population of myoid interstitial cells

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that can proliferate and migrate in sites of tubulointerstitial injury (1994). Qi et al. reported a positive expression of CD34, CD117/c-kit and vimentin in renal interstitial TCs (2012). We did

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not find c-kit+ interstitial SC/TCs; also, the presence of the mesenchymal and endothelial marker vimentin in the study mentioned above could suggest either the presence of TCs or

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endothelial cells (Qi et al., 2012). CD117/c-kit is a progenitor/stem cell marker (Didilescu et al., 2013; Rusu et al., 2014a); therefore, when a TC-like cell is positive with this marker, with

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or without a concomitant CD34 immunopositivity (Li et al., 2014), we should take into account a potential role of that cell in regeneration and repair. Noteworthy, as Anglani et al. commented, “in the kidney, as in other mesenchymal

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tissues, the need for a real stem cell compartment might be less important than the phenotypic flexibility of tubular cells” (Anglani et al., 2004). Our findings showed a myoid (and potentially contractile) potential, in both renal epithelial and interstitial cells, which should be seriously taken into account when the normal function of the kidney is studied.

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Acknowledgements

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All the authors have equally contributed to this paper.

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Figure Legends

Figure 1. Renal cortical expression of CD31 (A) and CD 117/c-kit (B). Capsular SC/TCs (red arrows) and cortical subcapsular telocytes (red arrowhead) are equally CD31-negative and ckit-negative. Endothelia (*) express CD31. Scarce tubular epithelial cells express CD31 (A,

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white arrows) as well as c-kit (B, white arrowheads).

Figure 2. Immunoexpression of desmin in human adult renal tissue. Usually, desmin was not

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expressed in renal corpuscles (A, arrow) but rarely parietal cells of Bowman’s capsule were

desmin-positive (B, arrow). Epithelial cells in proximal and distal tubules (A and B, arrowheads), as well as in the loops of Henle (C, arrowheads) displayed the basal positive expression of desmin. The basal positive expression of desmin was scarce in collector ducts

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(D, arrows). Renal telocytes were desmin-negative, including the capsular (A, *) and

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Figure 3. Immunoexpression of CD34 in human adult renal cortical tissue. The marker is expressed in interstitial and glomerular endothelial cells (white bullets), in capsular (white

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arrowheads).

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(*) and vascular smooth muscle cells (inset). Tubular epithelial cells also express CD34 (red

Figure 4. Nestin expression in human adult renal cortical tissue. A and B: Positive expression of nestin in capsular (arrowheads) and subcapsular telocytes (arrows). C and D: Perivascular networks of interstitial cells (arrows) neighbor vascular smooth muscle cells (arrowheads) and

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continue to surround tubules and renal corpuscles; an isolated expression of nestin in a parietal epithelial cell of Bowman’s capsule (double-headed arrow). E: Peritubular multipolar stromal cells project telopodes (arrows). F: Nestin is expressed in interstitial networks around the renal corpuscles (arrows), as well as in podocytes (arrowheads).

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Figure 5. Renal cortical expression of α-smooth muscle actin (α-SMA) is positive in capsular (double-headed arrow), subcapsular (white arrow) and interstitial periendothelial (arrowheads) cells projecting telopodes, as well as in renal glomeruli (*). Endothelial cells (black arrows) did not express α-SMA.

Figure 6. Renal cortical expression of the heavy chain of smooth muscle myosin is positive in

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subcapsular (A, arrow) but not in capsular (A, arrowhead) telocytes. The marker is also

expressed in perivascular and peritubular interstitia (B, C, arrows), vascular smooth muscle

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cells (C, arrowhead), tubular epithelial cells (D, arrowheads), pericapsular interstitia (E, arrow),

interstitial endothelia (E, double-headed arrow), and in glomerular endothelia (E, F,

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arrowheads).

Figure 7. Renal cortical expression of endoglin (CD105) is positive in subcapsular SC/TCs (A,

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in interstitial (A, double-headed arrow) and glomerular (C, arrows) endothelia.

Figure 8. Within the kidney medulla, endothelial cells (arrows) and scarce epithelial cells

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(double-headed arrow) express CD31.

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Figure 9. Positive expression of CD34 within the renal medulla is found in endothelial cells (arrowheads) and in isolated stromal cells with discrete filopodia (arrows). Stromal telocytes are CD34-negative (triple-headed arrows). False CD34-positive telocytes (double-headed

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arrow) are in fact tangentially cut endothelial tubes.

Figure 10. Expression of nestin (A) and CD117/c-kit (B) in the renal medulla. Endothelial cells (A, arrow) and vascular smooth muscle cells (A, arrowheads) express nestin. Epithelial cells express CD117/c-kit (B, arrows), only, or predominantly, in the basal poles.

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Figure 11. Expression of the heavy chain of smooth muscle myosin in renal medulla, which is positive in microvascular mural cells (arrowheads) and in epithelial cells, mostly at the basal poles (arrows).

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Figure 12. Myoid markers (left panel: α-smooth muscle actin, right panel: the heavy chain of

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