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. Author manuscript; available in PMC: 2013 Jan 28.
Published in final edited form as: Histochem Cell Biol. 2010 Sep 19;134(4):355–369. doi: 10.1007/s00418-010-0741-7

Stem cell marker TRA-1-60 is expressed in foetal and adult kidney and upregulated in tubulo-interstitial disease

Irina Fesenko 1, Danielle Franklin 2,3, Paul Garnett 4, Paul Bass 5, Sara Campbell 6,7, Michelle Hardyman 8, David Wilson 9, Neil Hanley 10,11, Jane Collins 12,✉
PMCID: PMC3556812  EMSID: EMS34821  PMID: 20853169

Abstract

The kidney has an intrinsic ability to repair itself when injured. Epithelial cells of distal tubules may participate in regeneration. Stem cell marker, TRA-1-60 is linked to pluripotency in human embryonic stem cells and is lost upon differentiation. TRA-1-60 expression was mapped and quantified in serial sections of human foetal, adult and diseased kidneys. In 8- to 10-week human foetal kidney, the epitope was abundantly expressed on ureteric bud and structures derived therefrom including collecting duct epithelium. In adult kidney inner medulla/papilla, comparisons with reactivity to epithelial membrane antigen, aquaporin-2 and Tamm–Horsfall protein, confirmed extensive expression of TRA-1-60 in cells lining collecting ducts and thin limb of the loop of Henle, which may be significant since the papillae were proposed to harbour slow cycling cells involved in kidney homeostasis and repair. In the outer medulla and cortex there was rare, sporadic expression in tubular cells of the collecting ducts and nephron, with positive cells confined to the thin limb and thick ascending limb and distal convoluted tubules. Remarkably, in cortex displaying tubulo-interstitial injury, there was a dramatic increase in number of TRA-1-60 expressing individual cells and in small groups of cells in distal tubules. Dual staining showed that TRA-1-60 positive cells co-expressed Pax-2 and Ki-67, markers of tubular regeneration. Given the localization in foetal kidney and the distribution patterns in adults, it is tempting to speculate that TRA-1-60 may identify a population of cells contributing to repair of distal tubules in adult kidney.

Keywords: Kidney, Tubulo-interstitial, TRA-1-60, Stem cell, Foetal, Regeneration

Introduction

The renal tubular epithelium is exposed to injurious stimuli from various sources including nephrotoxins, ischemia–reperfusion, inflammation and immunological disorders often leading to acute kidney injury, which may progress to chronic kidney disease and fibrosis (Gobe and Johnson 2007). However, the kidney has the intrinsic ability to recover from tissue damage. Understanding the cellular and molecular basis of the mechanisms of nephron repair is critical for the design of new therapeutic approaches to treat kidney disease (Hopkins et al. 2009). The precise origin of the cells that replace injured tubular epithelia is not known (Humphreys and Bonventre 2007) but several lines of evidence point to intrarenal sources (Duffield et al.2005; Bussolati et al. 2005). In mouse studies, regeneration by proliferation of resident surviving kidney epithelial cells appeared to be the predominant mechanism of repair after transient ischemic tubular injury, although this did not completely exclude participation of the interstitial cell population (Humphreys et al. 2008). Renal epithelial responses to injury suggested the process of recovery is based on a sequence of events, including cell spreading, migration, cell dedifferentiation and proliferation, to restore cell number, followed by differentiation, which results in restoration of the functional integrity of the nephron (Witzgall et al. 1994; Bonventre 2003; Humphreys and Bonventre 2007). The dedifferentiated phenotype reflects changes in gene expression patterns that may recapitulate the pattern that occurs during kidney development (Bonventre 2003). Pax-2 expression, in combination with other transcription factors, is related to renal progenitor-type cell behaviour (Kim and Dressler 2005; Vigneau et al. 2007). Pax-2 plays a key role in nephrogenesis, being downregulated when nephrogenesis is complete (Dressler and Douglass 1992; Winyard et al. 1996; Eccles et al. 2002) and re-expressed in response to tubular damage to participate in tubular regeneration and cellular differentiation (Imgrund et al.1999; Maeshima et al. 2002). Currently it is unclear whether all renal epithelial cells have equal potential for regeneration, or a resident population of progenitor cells is mainly responsible (Humphreys and Bonventre 2007; Humphreys et al. 2008). In rat and mouse renal models, the papillae were proposed to harbour slow cycling cells that may contribute to kidney homeostasis and repair (Oliver et al. 2004, 2009). In other studies, cells were isolated from the interstitium and Bowman’s capsule of adult human kidney using expression of adult stem cell markers, CD133 and CD24. These cells possessed some characteristics of progenitor cells and were able to contribute to renal repair in animal models (Bussolati et al. 2005; Sagrinati et al.2006; Lazzeri et al. 2007).

The TRA-1-60 antigen is linked to dedifferentiation in embryonal carcinoma cells and currently one of a few antigens that are widely used in human stem cell research as positive indicators of a true pluripotent human stem cell (Schopperle and DeWolf 2007; Andrews et al. 1984; Badcock et al. 1999). In human kidney, localisation of TRA-1-60 was discussed, but the precise location was not defined (Andrews et al. 1984). The TRA-1-60 epitope was identified on an isoform of the proteoglycan, podocalyxin (Schopperle and DeWolf 2007). Evidence that podocalyxin was shown to be involved in renal epithelial cell polarisation and tubulogenesis (Meder et al. 2005; Nielsen et al. 2007; Cheng et al. 2005) prompts the suggestion that TRA-1-60 is expressed on renal tubular epithelial cells involved in tubular morphogenesis or repair. Therefore, the distribution of TRA-1-60 was investigated in human foetal and adult kidneys.

Materials and methods

Tissue collection and preparation

Cortical, medullary and papillary normal renal tissue was excised from adult nephrectomy specimens, diseased samples were obtained by needle aspiration biopsy (see Table 1) and foetal tissue was harvested from pregnancy terminations with full ethical approval and patient consent. Permission was granted by Southampton and South West Hampshire Research Ethics Committees. All samples were fixed in 10% formalin in phosphate buffer (pH 7.5), embedded in paraffin wax and sections cut at 4 μm and mounted on APES coated slides.

Table 1.

Biopsy samples used for staining and morphometry

Age
(years)		 Sex Diagnosis
35 M Acute tubular necrosis
75 F Glomerulosclerosis, type II diabetes
57 M Granulomatous interstitial nephritis
70 F Subacute-on-chronic tubulo-interstitial damage
40 M Severe parenchymal damage
64 F Crescentic glomerulonephritis
38 M Sclerosis of glomeruli, severe chronic
 tubulo-interstitial damage
80 M Sclerosis and ischemia of glomeruli,
 multifocal tubulo-interstitial damage
75 F Sclerosis of glomeruli, glomerular lesions,
 multifocal tubulo-interstitial damage
53 F Interstitial nephritis with an associated
 acute tubular insult
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Antibodies and labelling reagents

Primary mouse monoclonal anti-TRA-1-60 (Millipore, UK); anti-human epithelial membrane antigen (EMA, Dako Cytomation), clone E29, anti-human Ki-67 antigen (Dako Cytomation), clone MIB-1, rabbit anti-aquaporin 2 (Santa Cruz Biotech), rabbit anti-aquaporin-2 (Lifespan Biosciences) and sheep anti-Tamm–Horsfall glycoprotein (THP) (Millipore, UK) were used in single or dual immunostaining experiments of foetal and adult tissues with rabbit polyclonal anti-murine Pax2 antibody (Invitrogen) and anti-human Ki-67 antigen (Abcam). Control antibodies at similar concentrations to primary antibodies were used: rabbit IgG (Vector Laboratories) for Pax2, mouse IgM for TRA-1-60, mouse IgG1 for Ki 67 and mouse IgG2a for EMA, all from Sigma. Secondary biotinylated rabbit anti-mouse and swine anti-rabbit Fab fragments (Dako Cytomation) were used with goat anti-rabbit Alexa 488 and goat anti-mouse Alexa 548 (Invitrogen) for primary antibody detection. Biotinylated Lotus tetragonolobus (LTA) lectin (Sigma) was used with Hoechst 33258 (Sigma) as a nuclear counterstain.

Immunohistochemistry

Immunohistochemistry was performed using conventional techniques; briefly, sections were dewaxed in xylene and rehydrated through graded alcohols to 70%. Endogenous peroxidase activity was inhibited by incubation in hydrogen peroxide/methanol solution. Antigen retrieval was performed by boiling the sections in a microwave for 25 min in citric acid buffer (pH 6.0). Sections were sequentially incubated with avidin/biotin blocking kit (Vector Laboratories) and blocking medium (20% foetal calf serum, 1% bovine serum albumin in Dulbecco’s modified Eagle medium). Primary antibodies were applied in tris buffered saline (TBS) at 4°C overnight, biotinylated secondary antibodies were applied in TBS for 30 min at room temperature followed by 30 min incubation with streptavidin–biotin peroxidase complexes (Dako Cytomation) at room temperature. The signal was detected using 3,3′ diaminobenzidine (DAB) substrate (BioGenex). The sections were counterstained with Mayers haematoxylin, dehydrated through graded alcohols, cleared in Clearin (Surgipath, Catalog No. 03670) and mounted in DPX mountant (Electron Microscopy Sciences). Images were taken using Nikon light microscope.

Immunofluorescence

Sections were dewaxed and rehydrated through graded alcohols. Sections were microwaved for 25 min in citric acid buffer (pH 6.0) for antigen retrieval and blocked by incubation in 20% foetal calf serum, 1% BSA. Primary antibodies were applied in TBS at 4°C overnight, secondary antibodies were applied in TBS for 30 min at room temperature, and Hoechst solution in TBS was applied for 5 min before the last wash and sections were mounted in Mowiol (Harlow Chemical Company, UK). Images were taken using a Leica epifluorescence microscope.

Quantitation of TRA-1-60 staining

TRA-1-60 staining in the cortex of adult kidney tissue was quantified on immunohistochemically labelled wax sections. Using Zeiss KS 400 3.0 software, total percentage of TRA-1-60 labelled area was measured at 20× magnification per 10 fields on sections of adult kidneys and biopsies of substantial size and per entire area of two sections of smaller biopsies. Statistical analysis of the data was performed using SPSS statistical software. One-way ANOVA was followed by an unpaired Student’s t test.

Results

Expression of human embryonic stem cell marker TRA-1-60 in human foetal kidney

At Carnegie stage 21 and 8, 9.5 and 10 weeks of gestation, TRA-1-60 staining was localised on the apical surface of the epithelial cells lining the ureteric bud and nascent collecting ducts (Fig. 1a, c). The transcription factor Pax-2 regulates ureteric bud development (Torres et al. 1995) and Pax-2 staining was observed in the nuclei of epithelial cells lining the ureteric bud and bud-derived structures and in condensing metanephric mesenchyme (Fig. 1b, d). Dual immunofluorescent staining confirmed that TRA-1-60 positive structures co-expressed Pax-2 and that the Pax-2 positive condensing mesenchyme was negative for TRA-1-60 staining (Fig. 2a–c). Further, dual labelling showed that Ki-67, a marker of cell proliferation, was positive in Pax-2 expressing structures including the TRA-1-60 positive ampullae of ureteric buds (Fig. 2d–f).

Fig. 1.

Fig. 1

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TRA-1-60 and Pax-2 immunostaining of normal human kidney at 10 weeks of gestation. a and c show TRA-1-60 expression in the nephrogenic zone. a TRA-1-60 was intensely expressed on the apical surface of the ureteric bud and putative collecting ducts. c TRA-1-60 expression in the ureteric bud tip at higher magnification. b and d show Pax-2 expression in the nephrogenic zone. b Transcription factor Pax-2 was strongly expressed in the ureteric bud, collecting duct and also in condensing metanephric mesenchyme and derivatives including comma-shaped bodies. A lower level of expression was noticeable in maturing nephrons. d Pax-2 expression in the ureteric bud, condensing mesenchyme and S-shaped bodies at higher magnification. e Mouse IgM, isotype control for TRA-1-60 immunostaining. f Rabbit IgG negative control for Pax-2 staining. ub ureteric bud, cd collecting duct, mm metanephric mesenchyme, cb comma-shaped body, Sb S-shaped body, nephr nephron. Scale bars 60 μm

Fig. 2.

Fig. 2

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TRA-1-60, Pax-2 and Ki-67 expression in the epithelium of ureteric bud of normal human kidney at 10 weeks of gestation. a–c show dual immunofluorescent staining of the nephrogenic zone for TRA-1-60 and Pax-2. Expression of TRA-1-60 was restricted to the ureteric bud epithelium (a), where it was co-expressed with Pax-2 (b). c Shows corresponding counterstaining of the nuclei with Hoechst re-agent. d–f Show dual immunostaining of the nephrogenic zone for Ki-67 and Pax-2. Ki-67 (d arrows) was highly expressed in structures expressing Pax-2 (e), including the ureteric bud tips. f Shows corresponding counterstaining of the nuclei with Hoechst reagent. ub ureteric bud. Scale bar 30 μm

TRA-1-60 is expressed by small numbers of tubular cells in the cortex of human adult kidney

In adult human cortex, TRA-1-60 was detected on the apical surfaces of a small number of tubular epithelial cells that were sparsely scattered throughout the cortex (Figs. 3b, e, 7b), in contrast to the widespread expression seen in foetal kidneys. We first used markers of proximal and distal renal structures to roughly map TRA-1-60 expression in the cortex. LTA lectin (Silva et al. 1993) was used to identify proximal tubules and antibody to human epithelial membrane antigen (EMA) to label distal tubules and collecting ducts (Heyderman et al. 1979; Cordell et al. 1985). Serial sections stained with EMA (Figs. 3a, d, g, 7a), TRA-1-60 (Figs. 3b, e, h, 7b) and LTA (Figs. 3c, f, i, 7 c) demonstrated that the TRA-1-60 epitope was expressed exclusively in EMA labelled structures. Notably, TRA-1-60 positive staining was consistently seen in a region of the distal tubule adjacent to the vascular pole of Bowman’s capsule (Figs. 3a, b, 7a, b), where distal convoluted tubules are located. Similar to foetal kidney, staining was seen mainly at the apical surfaces of the cells, although in some tubules it was also in the cell cytoplasm. Mostly, there were only a few TRA-1-60 expressing cells incorporated into the tubular wall, although sometimes positive cells were covering the entire circumference of the tubule (Figs. 3b, e, 7b). However, TRA-1-60 staining was not observed expanding over large areas of the tubule surfaces where longitudinal sections of EMA labelled structures were seen (Fig. 3g, h, i).

Fig. 3.

Fig. 3

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TRA-1-60 epitope expression in the cortex of adult human kidney. Serial sections of adult kidneys were stained for EMA, to label distal tubules and collecting ducts (a, d, g), TRA-1-60 (b, e, h) and LTA lectin, a proximal tubule marker (c, f, i). b and e Show TRA-1-60 expression in tubular cells (arrows) in the areas of cortex with no signs of chronic tubulo-interstitial damage. On serial sections the TRA-1-60 positive tubules expressed EMA (a and d arrows) but not LTA lectin (c and f arrows). h Depicts a longitudinal section of a branched distal tubular structure (arrows) in the cortex, showing absence of TRA-1-60 expression over expansive areas of the tubular epithelial surface. On serial sections the tubule expressed EMA (g arrows) and did not express LTA lectin (i arrows). Scale bar 60 μm

Fig. 7.

Fig. 7

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Higher magnification of the images presented in Figs. 3 and 5 showing serial sections of renal cortex stained with TRA-1-60 (arrows in b, e, h and k) in normal kidney cortex (a–c), chronic tubulo-interstitial scarring (d–f), ATN (g–i) and GN (j–l). EMA staining (arrows in a, d, g and j) was observed in analogous TRA-1-60 positive tubules. LTA lectin staining, (arrows in c, f, i and l) indicated that tubules analogous to the TRA-1-60 positive tubules are LTA lectin negative. Scale bar 30 μm

TRA-1-60 is expressed in the thick ascending limb, thin limb and collecting ducts in the medulla and papilla of adult human kidney

In view of a recent report that TRA-1-60 reactivity was found in the epithelium of the entire collecting duct system in baboons (Gubhaju et al. 2008), human medullary and papillary regions were also stained for TRA-1-60 (see Table 2 for summary). In the inner medulla/papilla, the TRA-1-60 epitope was widely expressed in tubules, corresponding morphologically to the collecting ducts (ducts of Bellini). To confirm which structures were expressing TRA-1-60, serial sections of the medullary and papillary regions were stained for EMA (distal structures), THP (thick ascending limb, TAL), TRA-1-60 and aquaporin-2 (collecting duct) (Knepper et al. 1996; Deen and Knoers 1998). The staining revealed that the TRA-1-60 antigen was expressed in epithelium of the collecting ducts (Fig. 4c, arrows) identified by expression of EMA and aquaporin-2 (Fig. 4a, d, respectively, arrows). The localisation was apical or apical with cytoplasmic staining (Fig. 4c, arrows). The antigen was also found in the collecting ducts of the inner stripe of outer medulla (Fig. 4g, arrows) identified by positive staining for EMA and aquaporin-2 (Fig. 4e, h, respectively, arrows) and negative staining for THP (Fig. 4f, arrows). However, in the inner medulla/papilla the expression was more widespread than in the inner stripe of the outer medulla where it became restricted to smaller groups of cells or single cells (compare Fig. 4c and g). Throughout papilla, inner medulla and inner stripe of outer medulla TRA-1-60 expression was also found in some thinwalled tubules (Fig. 4c, arrowheads; o, arrow) expressing EMA, but not THP and aquaporin-2 (Fig. 4a, b, d, respectively, arrowheads; m, n, p, respectively, arrows), representing thin limb of the loop of Henle. Both in the inner (Fig. 4k, arrow) and outer (Fig. 4s, arrow) stripe of outer medulla TRA-1-60 staining were found in TAL. These tubules were labelled positively with EMA (Fig. 4i, q, respectively, arrows) and THP (Fig. 4j, r, respectively, arrows) and negatively with aquaporin-2 (Fig. 4l, t, respectively, arrows).

Table 2.

Summary of TRA-1-60 staining localisation in different anatomical zones of human kidney

Kidney anatomical zones Structures localised to the zone Structures expressing TRA-1-60
Inner medulla/Papilla Collecting duct, Henle thin limb Collecting duct, Henle thin limb
Outer medulla
 Inner stripe Collecting duct, Henle thin limb, thick ascending limb Collecting duct, Henle thin limb,
 thick ascending limb
 Outer stripe Collecting duct, thick ascending limb,
 proximal straight tubules		 Thick ascending limb
Cortex Collecting duct, thick ascending limb, distal convoluted tubule,
 proximal straight tubule, proximal convoluted tubule,
 renal corpuscle (including Bowman’s capsule and glomeruli)		 Thick ascending limb,
 distal convoluted tubule
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Fig. 4.

Fig. 4

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TRA-1-60 epitope localisation in specific areas of human medulla and papilla. Sections of human renal inner medulla/papilla and outer medulla were stained in series with antibodies against EMA (a, e, i, m, q), THP (b, f, j, n, r), TRA-1-60 (c, g, k, o, s) and aquaporin-2 (d, h, l, p, t). In the inner medulla/papilla (a–d) TRA-1-60 staining (c) corresponded to small thin-walled, EMA-positive tubules in the thin limb of the loop of Henle (arrowheads) and also to collecting ducts that were positive for aquaporin-2 (d) and EMA (a arrows). Here both types of tubules were negative for THP (b). In the inner stripe of the outer medulla (e–h; i–l and m–p), TRA-1-60 was localised to the collecting ducts (G arrows) corresponding positively with staining for EMA (e arrows) and aquaporin-2 (h arrows) and negatively with THP (f arrows), to the TAL (k arrow), corresponding positively with EMA (i arrow) and THP (j arrow) and negatively with aquaporin-2 staining (l arrow) and to the thin limb of the loop of Henle (o arrow) stained positively for EMA (m arrow) and negatively for THP (n arrow) and aquaporin-2 (p arrow). In the outer stripe of the outer medulla (q–t), TRA-1-60 was expressed in TAL (s arrow) corresponding positively with EMA (q arrow) and THP (r arrow) and negatively with aquaporin-2 (t arrow). Scale bar 60 μm (a and e), 30 μm (i, m and q)

TRA-1-60 is expressed in the thick ascending limb and distal tubule in kidney cortex

To identify more precisely the nature of TRA-1-60 expressing tubules in the cortex, serial sections were stained for EMA (Fig. 5a, e, i), THP (Fig. 5b, f, j), TRA-1-60 (Fig. 5c, g, k) and aquaporin-2 (Fig. 5d, h, l). Results of the staining are summarised in Table 2. TRA-1-60 staining (Fig. 5c, g, k, arrows) was found in tubules corresponding positively with EMA (Fig. 5a, e, i, arrows) and THP staining (Fig. 5b, f, j, arrows) and negatively with aquaporin-2 staining (Fig. 5d, h, l, arrows). Some TRA-1-60 positive tubules showed THP staining cytoplasmically and apically indicating TAL (Fig. 5f, g, j, k, arrows), whereas other tubules showed THP staining only at the apical surface, most likely representing distal convoluted tubules (Fig. 5b, c, arrows). We did not observe staining for TRA-1-60 on cortical collecting ducts identified by aquaporin-2 expression (Fig. 5, compare c and d, k and l, arrows indicate TRA-1-60 expressing tubules, arrowheads show collecting ducts).

Fig. 5.

Fig. 5

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Tubular location of TRA-1-60 epitope in the cortex of human kidney. Sections of human renal cortex were stained in series with antibodies against EMA (a, e, i), THP (b, f, j), TRA-1-60 (c, g, k) and aquaporin-2 (d, h, l). TRA-1-60 staining (c, g, k arrows) corresponded positively with EMA (a, e, i arrows) and THP staining (b, f, j arrows) and negatively with aquaporin-2 (d, h, l arrows). Some tubules showed THP staining cytoplasmically and apically indicative of TAL (f and j arrows). Some tubules showed THP staining only at the apical surface, most likely representing distal convoluted tubules (b arrows). Scale bar 30 μm

TRA-1-60 expression increases in cortical areas of chronically damaged and diseased kidneys

TRA-1-60 was expressed by only a small number of tubular cells in normal kidney; however, in areas of cortical tubulo-interstitial damage, there were more TRA-1-60 expressing cells in the tubules (Figs. 6b, 7e, arrows). Therefore, distribution of reactivity was assessed in acute tubulo-interstitial disease, interstitial inflammation or chronic parenchymal damage, including acute tubular necrosis (ATN) (Figs. 6e,7h) and glomerulonephritis (GN) (Figs. 6h, 7k). Again, serial wax sections were stained for EMA and LTA lectin. In all diseased samples examined we observed an increase in TRA-1-60 staining by tubular cells, when compared to normal kidneys (Fig. 6b, e, h, compared to Fig. 3b, e). The epitope was again expressed in EMA-positive tubules distal to the Loop of Henle (Figs. 6a–i, 5a, g–l). The increase in the percentage area of TRA-1-60 staining was statistically significant when diseased tissue (n = 10) (see Table 1) was compared with normal (n = 7, p ≤ 0.001) or chronically scarred normal (n = 5, p ≤ 0.05) and when comparing scarred tissue directly with normal tissue (p ≤ 0.05) (Fig. 8).

Fig. 6.

Fig. 6

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TRA-1-60 antigen expression in tubulo-interstitial damage. Serial sections of areas of renal cortex with tubulo-interstitial damage were stained for EMA (a, d, g), TRA-1-60 (b, e, h) and LTA (c, f, i). b Shows normal kidney with accumulation of TRA-1-60 expressing cells (arrows) in scarred areas marked by lymphocyte infiltration (arrowheads). The TRA-1-60 expressing tubules were also positive for EMA (a arrows) but not LTA lectin (c arrows) on serial sections. Increased TRA-1-60 expression by tubular cells in the cortex affected by ATN (e arrows) and GN (h arrows). On serial sections the TRA-1-60 expressing tubules were EMA positive (d and g, respectively, arrows) and LTA lectin negative (f and i, respectively, arrows). j–l Show negative controls: mouse IgG2 for EMA antibody (j), mouse IgM for TRA-1-60, with TBS only for LTA lectin. Scale bar 60 μm

Fig. 8.

Fig. 8

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Chart showing mean percentage area of TRA-1-60 staining in three renal tissue groups: normal cortex (n = 7), scarred areas of normal cortex (n = 5) and cortical biopsies of diseased kidneys (n = 10). Statistical analysis of the results of the measurements was performed using SPSS statistical software. One-way ANOVA was followed by an unpaired t test

TRA-1-60 expression is associated with increased cell proliferation in disease biopsies

To determine the regenerative status of diseased tubules containing TRA-1-60 positive cells, serial sections of biopsies were stained for TRA-1-60 and Ki-67. Both in ATN and GN, a significant increase in Ki-67 staining was observed in the tubules in comparison to normal adult kidney, where very little Ki-67 staining was found (data not shown). Dual labelling of GN biopsies for TRA-1-60 and Ki-67 showed that some TRA-1-60 positive cells were also positive for Ki-67 (Fig. 9a–c, d–f) thus indicating proliferation within TRA-1-60 positive cell population itself. Cell proliferation was not restricted only to TRA-1-60 expressing structures.

Fig. 9.

Fig. 9

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Co-expression of Ki-67 and Pax-2 in TRA-1-60 positive cells in diseased kidney. a–f Show dual immunofluorescent staining of renal cortex affected by GN for TRA-1-60 and Ki-67. Some of the tubular cells positive for TRA-1-60 (a, d arrows) co-expressed proliferation marker Ki-67 (b and e, respectively arrows). c and f show respective nuclei counterstained with Hoechst reagent (arrows point to the nuclei of cells co-expressing TRA-1-60 and Ki-67). g–l Show dual immunofluorescent staining for TRA-1-60 and Pax-2. TRA-1-60 (g arrow) and Pax-2 (h arrow) co-expression in tubular cells of renal cortex affected by ATN. I shows counterstaining of the nuclei with Hoechst reagent (arrow points to the nuclei of TRA-1-60 and Pax-2 co-expressing cell). TRA-1-60 (j arrows) and Pax-2 (k arrows) co-expression by tubular cells of renal cortex affected by GN. l Shows counterstaining of the nuclei with Hoechst reagent (arrows point to the nuclei of TRA-1-60 and Pax-2 co-expressing cells). Scale bar 30 μm

TRA-1-60 and Pax-2 expression in disease biopsies

Dual staining with TRA-1-60 and Pax-2 was done in order to further clarify the nature of TRA-1-60 expressing cells in ATN and GN biopsies. Pax-2 was abundantly expressed by tubular epithelial cells and the majority of TRA-1-60 positive cells co-expressed Pax-2 demonstrating that there was a significant overlap between Pax-2 and TRA-1-60 expressing populations of tubular cells (Fig. 9g–i, j–l). There were also cells that were positive only for Pax-2 (compare Fig. 9g, h).

Discussion

Development of the mammalian metanephric kidney begins when an outgrowth of the primary nephric duct, termed the ureteric bud, invades the surrounding metanephric mesenchyme and the ureteric bud starts to bifurcate, eventually forming the collecting duct system that will drain through the nephric duct into the bladder. At the same time, the metanephric mesenchyme condenses at the ureteric bud tips and generates the diverse epithelial cell types of the nephron and interstitial stromal cells between the developing tubules (Davies and Bard 1998; Dressler 2006). We looked at the expression of TRA-1-60 in human foetal kidneys in early gestation, between CS21 (about 7.5 weeks) and 10 weeks, soon after the beginning of nephrogenesis, which is the Wfth week of gestation (Haycock 1998). During this time, strong reproducible TRA-1-60 staining was observed on the apical surface of the epithelium of the ureteric bud and its derivatives. Cells in cycle were also identified in the ampullae of human ureteric bud with the antibody against Ki-67, a marker that confirmed that in foetal kidney TRA-1-60 staining was seen in growing, proliferating epithelium. In all these structures, TRA-1-60 was co-expressed with Pax-2, a key regulator of kidney development and ureteric bud in particular (Torres et al. 1995; Eccles et al. 2002; Ribes et al. 2003).

In adult kidney, widespread staining was seen in inner medulla/papilla in collecting ducts also known as the ducts of Bellini, and the thin limb of the loop of Henle. Conversely, in the adult cortex and outer medulla, TRA-1-60 staining was observed in very low numbers of cells confined to medullary collecting ducts and the nephron compartments: thin limb and TAL of Henle’s loop and the distal convoluted tubules, with no detectable staining in cortical collecting ducts.

Collecting ducts and nephron tubules develop from different rudiments and no cell mixing occurs at the nephron–collecting duct junction according to current data (Kobayashi et al. 2008). So it is most likely that TRA-1-60 expressing cells in collecting ducts and nephron tubules are unrelated. In the collecting ducts TRA-1-60 can be re-expressed later on or the expression may be retained after completion of the kidney development. In the baboon, the entire collecting duct system was shown to express TRA-1-60 at the end of the gestational term, when kidney development was complete (Gubhaju et al. 2008).

A dramatic increase in numbers of TRA-1-60 positive cells was observed in distal tubular structures in adult kidney showing inflammation or scarring and in biopsies affected by ATN and GN, diseases characterised by significant tubular or tubulo-interstitial damage. Increased TRA-1-60 expression in the biopsies did not extend to entire distal structures, but rather to specific tubular cells, suggesting that they might be involved in tubular repair. There was no TRA-1-60 staining in the interstitium, whereas staining of inflamed areas with CD68 (labelling macrophages) was positive for interstitial cells, suggesting that the TRA-1-60 positive cells were not macrophage in origin (Fig. S1, compare A and C to B and D, respectively). The TRA-1-60 epitope may be expressed in cells in response to injury, as distal parts of the nephron are known to respond to injury by alterations in gene expression (Lieberthal and Nigam 1998). TRA-1-60 expressing cells could have increased by proliferation, since it is known that distal tubular epithelial cells have regenerative capacity after injury (Gobe and Johnson 2007) and cells in diseased biopsies co-expressed TRA-1-60 and Ki-67, a marker of cells in cycle (Gerdes et al. 1983).

The TRA-1-60 epitope could be a marker of a resident stem or progenitor cell population/populations. A population of slow cycling cells was detected in the collecting ducts and interstitium of renal papilla (Oliver et al. 2004, 2009). A high proportion of TRA-1-60 positive cells in the biopsies were co-expressing the transcription factor Pax-2, which is linked to differentiation of renal tubules and collecting ducts (Kim and Dressler 2005; Vigneau et al. 2007), downregulated after nephrogenesis (Dressler and Douglass 1992; Winyard et al. 1996; Stayner et al. 2006) but re-expressed in response to injury during tubular regeneration (Imgrund et al. 1999; Maeshima et al. 2002). Pax-2 expression in the kidney correlates with cell proliferation in development and pathology (Winyard et al. 1996; Eccles et al.2002), in agreement with our data for Pax-2 and Ki-67 in foetal and adult kidney tissue (data not shown). Alternatively, TRA-1-60 may identify facultative progenitor cells appearing as differentiated cells that are activated in response to injury, as reported in lung, liver and brain (Cantz et al. 2008; Stripp and Reynolds 2008; Alvarez-Buylla et al. 2002). It is notable that TRA-1-60 positive cells mostly had morphologically well-preserved polarity and nuclei suggesting they had characteristics of viable differentiated cells.

The TRA-1-60 antibody was originally shown to recognise an epitope on an undefined sialylated keratan sulphate proteoglycan (Badcock et al. 1999). Sialylated proteoglycans are involved in negative regulation of cell–cell adhesion, attachment and increased cell motility (Funderburgh 2000; Takeda et al. 2000). In embryonal carcinoma cells, the epitope was found on the CD34 related 200,000-Mrisoform of podocalyxin, which is lost upon differentiation with reduction to a lower Mr form (Badcock et al. 1999; Andrews et al. 1984; Schopperle and DeWolf 2007). Podocalyxin functions as an ‘anti-adhesin’ (Takeda et al. 2000) and is implicated in renal tubulogenesis (Kerjaschki et al.1984; Cheng et al. 2005) and cell polarisation and in particular in apical domain formation (Meder et al. 2005; Nielsen et al. 2007). Together these data suggest that TRA-1-60 may react with a form of podocalyxin that is widely expressed during foetal kidney development, and may be involved in cell migration, spreading and epithelial morphogenesis during tubular repair in the adult (Bonventre 2003; Humphreys and Bonventre 2007). In this context it has been suggested that cells in the hypoxic renal papilla migrate towards other parts of the kidney and may participate in repair after renal injury (Oliver et al. 2004, 2009). The canine orthologue of podocalyxin, Gp135, is rapidly removed from the apical membrane and degraded to reappear by new synthesis at the new location when cells are induced to inverse their polarity during tubulogenesis (Ojakian et al. 1997; Wang et al. 1990; Pollack et al. 1998). This kind of dynamic behaviour of podocalyxin could be reflected in the intracellular localisation of the TRA-1-60 epitope in addition to apical expression.

Collectively these data indicate that the human ES cell marker TRA-1-60 is apically expressed on cells of the morphogenetically active epithelium of ureteric bud and nascent collecting ducts of human foetal kidney. In adult kidneys, a substantial TRA-1-60 expressing population was detected in the tubular structures of inner medulla/papillae and small populations of TRA-1-60 positive cells were identified in the collecting ducts and distal nephron structures of the outer medulla and in the distal nephron compartments of renal cortex. The cortical population increased in numbers with inflammation and scarring and expressed markers of tubular regeneration. This observation may be highly significant since a distinct stem cell population has not yet been identified in the human distal tubule and the papillae were recently proposed to be reservoir of slow cycling cells that may contribute to kidney homeostasis and regeneration (Oliver et al. 2004). This epitope may identify cells that contribute to mechanisms of distal tubular repair, but further study is needed to clarify the exact nature and function of TRA-1-60 expressing cells in adult and diseased human kidney.

Acknowledgments

J.E.C. and S.K.C. would like to thank the The University of Southampton and Wessex Renal Research and Transplant Unit, Queen Alexandra Hospital, Cosham, Portsmouth, UK, for project funding. N.H. would like to acknowledge the support of the Medical Research Council and the Wellcome Trust. We would like to thank Ron Lee and Susan Wilson from the University of Southampton Medical School Histochemistry Research Unit for technical support and Anton Page from the Southampton Medical School Bioimaging Unit for help with figures.

Footnotes

Electronic supplementary material The online version of this article (doi:10.1007/s00418-010-0741-7) contains supplementary material, which is available to authorized users.

Contributor Information

Irina Fesenko, Infection, Inflammation and Immunity Division, School of Medicine, University of Southampton, Southampton, UK.

Danielle Franklin, Infection, Inflammation and Immunity Division, School of Medicine, University of Southampton, Southampton, UK; Wessex Renal and Transplant Unit, Queen Alexandra Hospital, Portsmouth, UK.

Paul Garnett, Infection, Inflammation and Immunity Division, School of Medicine, University of Southampton, Southampton, UK.

Paul Bass, Infection, Inflammation and Immunity Division, School of Medicine, University of Southampton, Southampton, UK.

Sara Campbell, Infection, Inflammation and Immunity Division, School of Medicine, University of Southampton, Southampton, UK; Wessex Renal and Transplant Unit, Queen Alexandra Hospital, Portsmouth, UK.

Michelle Hardyman, Infection, Inflammation and Immunity Division, School of Medicine, University of Southampton, Southampton, UK.

David Wilson, Human Genetics Division, School of Medicine, University of Southampton, Southampton, UK.

Neil Hanley, Human Genetics Division, School of Medicine, University of Southampton, Southampton, UK; Endocrinology and Diabetes, Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK.

Jane Collins, Infection, Inflammation and Immunity Division, School of Medicine, University of Southampton, Southampton, UK, jec3@southampton.ac.uk.

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