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Premature birth imposes considerable stress on the kidney during a critical period of nephron formation and differentiation. Consequences of this stress include nephrocalcinosis, which has been reported to occur in approximately 15% of premature infants and may be associated with impaired renal function in later childhood (1–4). Decreased nephron mass in young adults who were born prematurely is associated with hypertension and impaired renal function (5). Studies of mammalian nephrogenesis have identified transcription factors, growth factors, and signaling proteins, which control the complex series of interactions resulting in kidney formation. Some factors that play a role in nephrogenesis can be re-expressed in adulthood during renal injury and may play a role in progressive renal damage (6). Although many of the basic mechanisms of nephrogenesis have been elucidated, there is very little information regarding immuno-localization of these factors in the developing human kidney.

CD44 is a cell surface glycoprotein receptor and adhesion molecule which is expressed in many human cell types and has been implicated in a variety of physiologic and pathologic processes including lymphocyte homing, wound healing, cell migration, and tumor growth and metastasis (7–10). CD44 in normal mature human kidney is limited to endothelial cells, interstitial cells, and circulating leukocytes. However, up-regulation of CD44 in tubular and glomerular epithelia because of renal injury has been observed in both human kidney disease and animal models of renal injury (11–16). CD44 is observed during renal embryogenesis at the ureteric bud, where reciprocal interaction of CD44 with hyaluronan (HA) plays a role in branching morphogenesis and early nephron differentiation (17–19). The CD44 ligand osteopontin (OPN) is an acidic glycoprotein, which has been characterized as a low-affinity, high-capacity calcium-binding protein (20). HA, also an important CD44 ligand, is a glycosaminoglycan abundant in renal medullary interstitium, is up-regulated in inflammatory renal disease and is a major crystal binding molecule at the surface of Madin-Darby canine kidney strain-1 cells (21,22). Based on the affinity of these ligands for calcium, and the propensity for calcium oxalate to adhere to injured renal epithelia, a role for CD44 in promoting renal accumulation of calcium oxalate has been proposed. In this regard, Verhulst et al. (23) has suggested that preconditioning of renal epithelia by OPN and HA precedes the development of nephrocalcinosis in premature infants. However, no data are available regarding immuno-localization of CD44 in developing tubular epithelia.

This study was designed to examine the immuno-localization of CD44 and OPN in the developing human kidney between 18 and 40 wk of gestation.

METHODS

This study was based on archival autopsy material and approved by the Institutional Review Board of Baystate Medical Center. Informed written consent was obtained at the time of autopsy with explicit understanding that tissue might be used for research without further need for consent. All cases were de-identified in compliance with HIPAA. The renal tissue studied was obtained after death because of intrauterine spontaneous fetal demise, or from infants who died because of the complications of prematurity. Fetal tissue was not studied in cases of elective termination of pregnancy.

A search of the pathology files from 1992 to 2006 was performed for autopsies between 18 and 40 wk of intrauterine development. Hematoxylin and eosin stained kidney tissue sections were reviewed to evaluate for adequacy of tissue preservation based on the criteria of Genest (24) and for presence of an intact renal capsule. Exclusion criteria included genetic abnormalities (cytogenetic testing or morphology), and inadequate preservation of renal histomorphology. Control tissue included normal adult renal tissue obtained from neoplastic nephrectomy specimens (n = 4), and a failed renal allograft with acute rejection.

Immunohistochemistry and morphology.

Immunohistochemical staining was performed on a DakoCytomation AutoStainer using the Signet Acuity Polymer Detection System HRP/DAB protocol in accordance with Signet Acuity data sheet (Dako, Carpenteria, CA). The primary antibodies used were: mouse anti-human MAb to PAN-CD44 (SFF-2, 1:10,000; Chemicon International, Temecula, CA); rabbit anti-human polyclonal antibody to OPN (aa75-90; 1:1500 dilutions; Chemicon International); mouse anti-human MAb to epithelial membrane antigen (anti-EMA) (E29, 1:3200; Dako). Tissue sections were cut at 4 μm thickness, deparaffinized in xylene, cleared and rehydrated in graded ethanols as per standard procedures. Antigen retrieval was performed in a microwave using a 1× working concentration of Biogenex Concentrated (10×) Antigen Retrieval Citra Solution Plus heated to and maintained at 98°C for 10 min. Slides were incubated with the primary antibody for 30 min, rinsed, and labeled polymer was applied for 25 min. Antigen-antibody reaction was visualized using diaminobenzidine chromogen (DAB) applied for 7 min, after which sections were counterstained in hematoxylin for 5 min and dehydrated in ethanol and in xylene before manual cover slipping. Positive and negative controls, omitting the primary antibody, were run in parallel.

In the subcapsular nephrogenic zone, CD44 and OPN immuno-localization were evaluated in areas beneath an intact capsule. For each renal tissue specimen, the percentage of CD44 and OPN positive vesicular bodies, C-shaped bodies, S-shaped bodies, and ureteric buds were calculated after the total number of these nephron structures was counted. The change in the percentage of positive structures as a function of gestational age (GA) was assessed using correlation, significance set at p < 0.05.

In the cortical regions of the kidney, tubules were classified as proximal or distal based on morphology. Proximal tubules were identified by cuboidal to low columnar cells with abundant eosinophilic granular cytoplasm, centrally located nuclei, and an apical brush border. Distal tubules were identified by cuboidal and pale cells with scant cytoplasm, apically located nuclei and absent brush border. We assessed the reliability of these morphologic characteristics in developing kidneys by immunohistochemical EMA stain, which is specific for distal renal tubules (25). In the medulla, loop of Henle tubules were identified by small diameter and a low cuboidal or thin epithelial lining and absence of luminal red blood cells. Collecting ducts were identified by larger diameter and a cuboidal to columnar epithelial lining with well-defined cell borders. Proximal tubules, distal tubules, Henle's loop tubules, and collecting ducts were evaluated for CD44 and OPN immuno-localization using a semi-quantitative approach. To avoid bias, we examined 10 high power fields of representative regions of the entire renal tissue section. In each of these high power fields we counted proximal tubules, distal tubules, Henle's loop tubules, and collecting ducts, and identified these structures as positive or negative for CD44 and OPN. Having determined the percentage of CD44 and OPN positive structures in these 10 high power fields, each renal tissue section was classified using a four-tiered scoring system as follows: none: no tubules or ducts positive for CD44 or OPN; 1+: <10% of tubules or ducts positive; 2+: between 10% and 50% of tubules or ducts positive; or 3+: >50% of tubules or ducts positive.

RESULTS

We identified 43 cases between 18 and 40 wk GA for which tissue blocks were available and meeting the selection criteria. The cases were distributed as follows: 39 cases between 18 and 34 wk GA with presence of nephrogenic zone, and four cases between 35 and 40 wk GA with absence of nephrogenic zone. The sex distribution was 13 female, 30 male. The cause of death in 38 cases was intrauterine fetal demise, whereas in five cases there was extra-uterine survival for up to 8 d.

Differentiation of nephrogenic and tubular structures in the developing kidney.

Figure 1A demonstrates the histomorphology of the nephrogenic zone at 20 wk of development with recognizable Wolffian duct and metanephric structures. Tubules that stained positive for EMA had morphologic characteristics of distal tubules, whereas EMA negative tubules had morphologic characteristics of proximal tubules (Fig. 2A–C). Therefore, in sections stained for CD44 and OPN, classification of tubules as proximal or distal was based on morphologic criteria.

Figure 1
figure 1

CD44 immuno-localization in developing human kidney. (A) Hematoxylin and eosin stained 20 wk gestational age (GA) kidney, showing different stages of renal development in the subcapsular nephrogenic zone. (B–E) Immunohistochemical stain showing CD44 in a membranous and cytoplasmic pattern in ureteric bud (UB) structures in 20 wk GA (B–C), 26 wk GA (D) and 30 wk GA (E), decreasing in intensity with advancing age. Metanephric structures including blastema, vesicle (V) and S-shaped structure (S), proximal tubule (PT), distal tubule (DT), and glomeruli (G) do not demonstrate CD44. Scattered CD44 positive endothelial cells and mononuclear cells are seen in B. (F) CD44 staining is further reduced at 34 wk GA. CD, collecting duct. Scale bars = 50 μm.

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Figure 2
figure 2

EMA and OPN immuno-localization in developing human kidney. (A) Immunohistochemical EMA apical and membranous staining of distal tubules (DT) and collecting ducts (CD) in 20-wk GA fetal kidney. (B) Higher power of A showing EMA positive distal tubule, some adjacent to glomerular hilum (insert). Proximal tubules (PT) and glomeruli (G) are EMA negative. (C) EMA staining in DT in 40 wk GA, some adjacent to glomerular hilum (insert). (D–F) OPN cytoplasmic and perinuclear staining (inserts) in proximal tubules in 20 wk GA (D), 30 wk GA (E), and 40 wk GA (F). Insert in F shows distal tubule at glomerular hilum with weak OPN cytoplasmic stain compared with PT (lower half). Scale bars = 50 μm.

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CD44 immuno-localization in developing human kidney.

CD44 staining was not seen in normal adult renal tissue, whereas there was diffuse and strong CD44 staining of tubular epithelial cells in the rejected renal allograft (not shown). In the nephrogenic zone, immuno-localization of CD44 was seen in Wolffian duct-derived ureteric buds in all 39 tissue specimens between 18 and 34 wk GA (Fig. 1B–E), whereas metanephric structures (vesicular bodies, C-shaped bodies, S-shaped bodies) were negative for CD44. The percentage of CD44 positive ureteric buds among all cases ranged from 5 to 94% and decreased with increasing GA (p = 0.02) (Fig. 3). Immuno-localization of CD44 in the medulla was minimal throughout development. CD44 immuno-localization is summarized in Table 1.

Figure 3
figure 3

Correlation of ureteric bud CD44 staining with postconceptual age. ○, infants who lived for up to 8 d. •, intrauterine demise. The percentage of ureteric buds staining positive for CD44 significantly decreased with increasing postconceptual age. p < 0.05.

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Table 1 CD44 staining in developing human kidney between 20 and 40 weeks postconceptual age
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OPN immuno-localization in developing human kidney.

All adult controls demonstrated OPN staining in 3+ distal tubules in an apical plasma membrane pattern, and OPN staining of 2+ proximal tubules in a perinuclear granular pattern (not shown).

In the nephrogenic zone, there was no OPN staining in vesicular bodies, C-shaped bodies or S-shaped bodies at any GA. Ureteric bud immuno-localization of OPN was 1+ in 12/43 (28%) tissue specimens in a membranous pattern.

Between 18 and 40 wk of development, all tissue specimens were positive for OPN staining in proximal tubules. In 35/43 (81%) of tissue specimens, there was perinuclear OPN staining in 3+ proximal tubules (Fig. 2 D–F), whereas 8/43 (19%) of specimens had 1+ or 2+ OPN staining. In contrast, 14/43 specimens demonstrated no OPN staining of distal tubules. Remaining cases showed 1+ and 2+ distal tubules with apical membrane OPN staining in 11/43 and 10/43 tissue specimens, respectively. There was OPN staining in 3+ distal tubules in only 8/43 (19%) of tissue specimens. In the medulla, OPN staining was predominantly noted in loops of Henle. OPN staining patterns are summarized in Table 2.

Table 2 Osteopontin staining in developing human kidney between 20 and 40 weeks postconceptual age
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We did not attempt to make statistical inferences about the effect of brief survival in the neonatal intensive care unit on the immuno-localization of theses factors, because of the small number of infants (n = 5) in this group.

DISCUSSION

We are the first to demonstrate the immuno-localization of CD44 in renal tissue from infants 18 to 40 wk GA, representing the last periods of the metanephric phase of human kidney development (5). We show that CD44 immuno-localization is limited to Wolffian duct-derived ureteric buds in the nephrogenic zone until 32 wk GA, after which ureteric buds become infrequent and disappear. CD44 was not observed in proximal tubules, rarely seen in distal tubules and never seen in early structures derived from metanephric blastema. These observations suggest that the role of CD44 in human renal development is limited to early mesenchymal-epithelial interaction, as has been reported in animal models (5,17–19). Our observation of CD44 immuno-localization in human fetal kidney ureteric bud suggests that the interaction of CD44 with HA or other potential ligands present in the surrounding mesenchyme may play a role in early human renal development. After 30 wk GA, CD44 staining was weak and noted in only a few ureteric buds, paralleling the exhaustion of metanephric mesenchyme and decrease in interstitial HA (17–19). These observations suggest that diminishing CD44 interactions with HA or other transcriptional target molecules such as Wnt in metanephric mesenchyme result in the termination of nephrogenesis (26).

A critical role for the CD44 ligand HA in renal development has been demonstrated using isolated ureteric buds and metanephric kidney cultures. Concentrations of HA below 1.5% at the ureteric bud tip promote branching and differentiation, whereas higher molecular weight and concentration inhibit branching. By this mechanism, local HA molecular weight and concentration may promote ureteric bud branching early in development, and bring about the cessation of branching at the appropriate time. Local differences in HA molecular weight and concentration may be regulated via developmental and topographic expression of enzymes responsible for HA synthesis and degradation (27). These observations suggest a critical role for HA during early renal development. Because CD44 is the principle HA cell surface receptor, our observations of CD44 at the ureteric bud tip suggest a role for CD44 in human renal development. However, a developmental decrease in the number of CD44-positive ureteric buds does not necessarily lend substantial support to a critical role for CD44 in early epithelial-mesenchymal interaction. Indeed, mice targeted for deletion of the CD44 gene develop grossly normal kidneys (28,29). The CD44 ligand HA is diffusely present in the developing human kidney. Verhulst et al. (23) have shown that HA is present in the interstitium of fetal kidneys throughout the period of nephrogenesis, decreasing along with interstitial volume as tubular growth occurs, and also observed HA at the luminal surface of developing tubules. Perhaps CD44-HA interaction regulates renal ureteric bud branching by promoting or inhibiting movement of other signaling molecules to the tips of the ureteric bud.

Although CD44 immuno-localization in developing tubules was rare, we observed diffuse OPN staining in developing tubules, especially proximal tubules. Animal studies have demonstrated the importance of OPN in tubule development. OPN is up-regulated in tubules of rat embryos with advancing development, and OPN inhibition by anti-OPN antibody disrupts tubule development (30–32). The ontogeny of OPN in human renal development has been reported in two studies. Hudkins et al. (33) examined fetuses between seven and 18 wk GA and observed that before 80 d of development OPN staining is weak and localized to the luminal surface of some tubules and some ureteric buds. After 80 d of development, increased OPN staining was noted throughout the tubular cytoplasm. Verhulst et al. (23) demonstrated OPN staining of cortical and medullary tubules in infants between 24 and 40 wk GA. These reports did not address differential staining of OPN in proximal and distal tubules. In cultures of adult human proximal and distal tubules, different staining patterns were noted by immunocytochemical and confocal microscopy (34). Perinuclear golgi apparatus OPN staining was noted in proximal tubules, whereas apical plasma membrane OPN staining was noted in distal tubules.

We were able to differentiate proximal and distal tubules as early as 18 wk GA and we observed most OPN staining in proximal rather than distal tubules during early human renal development. We observed OPN immuno-localization in EMA negative proximal tubules in a perinuclear granular pattern, whereas EMA positive distal tubules demonstrated apical plasma membrane staining. In mature normal kidney, OPN is present in distal tubules in an apical plasma membrane pattern suggesting export of OPN into the tubular lumen (35). In contrast, the normal adult kidney displays little or no proximal tubule staining of OPN. However, strong proximal tubule OPN staining in a cytoplasmic pattern is seen in diabetic kidney, acute allograft rejection and cyclosporine toxicity (36–40). These patterns of OPN staining suggest that OPN up-regulation in proximal tubules occurs in response to injury. In our studies, we noted OPN staining pattern in developing kidney similar to that seen in adult renal injury. It is tempting to speculate that proximal tubule OPN staining in injured adult kidney represents a re-appearance of the fetal pattern of OPN expression.

We have shown that premature infants have decreased urinary OPN concentration compared with adults (41). Our current study suggests minimal synthesis and cellular export of OPN into cortical distal tubular segments where there is a high concentration of stone-forming constituents. The lack of OPN staining at this distal nephron site may be in agreement with our data showing decreased urinary excretion of OPN in premature infants.

Damaged and regenerating tubular epithelial cells have a propensity to bind calcium oxalate crystals (42). Binding of CD44 to OPN or HA has been shown to promote calcium oxalate crystal adherence to Madin-Darby canine kidney cells (22). Asselman and coworkers have proposed a model of nephrocalcinosis in which CD44, up-regulated in injured epithelia, binds OPN or HA, which in turn bind calcium oxalate crystals in the tubular lumen (43,44). Verhulst et al. (23) emphasized the importance of OPN in the pathogenesis of nephrocalcinosis in premature infants, pointing out that presence of OPN precedes the development of nephrocalcinosis in these infants. In our study, we noted only rare (<10% of distal tubules) CD44 staining in 35% of tissue samples, whereas 65% of these tissue specimens demonstrated no tubular CD44 staining at all. Thus, our observations do not support a critical role for constitutive CD44 expression in the pathogenesis of nephrocalcinosis in premature infants. It is possible that premature infants who suffer repeated bouts of hypoxemia, hypotension, and infection have up-regulation of tubular CD44 expression. It is likely that the etiology of nephrocalcinosis in premature infants is multi-factorial, with developmental, pharmacologic, and nutritional factors contributing to calcium oxalate accumulation.

In summary, we demonstrate the immuno-localization of CD44 in developing kidney between 18 and 40 wk of human development. CD44 staining is seen in ureteric bud epithelium in the subcapsular nephrogenic zone, with diminishing staining as nephron formation progresses to completion. These observations lend support to the concept that CD44 plays a role in early epithelial-mesenchymal interactions. Lack of widespread CD44 immuno-localization in developing tubules does not support the concept that developmental CD44 expression plays a critical role in the pathogenesis of nephrocalcinosis in premature infants. Our data do not however address the possibility of renal CD44 up-regulation in sick premature infants. We demonstrate that distal tubules can be differentiated from other nephron segments by EMA immunoreactivity and Hematoxylin and eosin morphology. In premature infants, OPN immuno-localization mainly occurs in proximal tubules in a cytoplasmic and perinuclear granular pattern early in human development with less staining in distal tubules. This pattern of OPN immuno-localization may play a role in the development of nephrocalcinosis in premature infants.