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H. Shima á H. Tazawa á P. Puri

Increased expression of ®broblast growth factorsin segmental renal dysplasia

Abstract Renal dysplasia (RD) is a disorganized devel-opment of renal parenchyma that results in a de®cit offunctional renal tissue. Dysplastic renal tissue is charac-terized by primitive tubular epithelium associated withincreased mesenchyme. Several polypeptide growth fac-tors (GF), which interact with target cells through a cell-surface membrane receptor, have been reported to beinvolved in the regulation of urothelial cell growth innormal and neoplastic states. Recent reports have dem-onstrated that basic ®broblast GF (bFGF, FGF-2) is amitogen for renal proximal-tubule epithelial cells. Kera-tinocyte GF (KGF, FGF-7), which belongs to the FGFfamily, is believed to be a paracrine mediator of epithe-lial-cell proliferation. The aim of this study was toinvestigate the immunoactivity of bFGF and KGF andtheir receptors in the dysplastic kidney in order to furtherunderstand the pathogenesis of RD. Specimens of dys-plastic upper poles of duplex kidneys were surgically re-sected from ten patients. Age-matched control materialincluded six kidney specimens taken at autopsy frompatients without evidence of urologic disease. Indirectimmunohistochemistry was performed using the Strept-ABC method with four antibodies: bFGF, KGF, FGFreceptor (¯g), and KGF receptor (bek). There was absentor weak bFGF, KGF, ¯g, and bek immunoreactivity innormal kidneys. In the dysplastic kidneys, there wasstrong immunoreactivity of bFGF and KGF and theirreceptors in the epithelium of primitive tubules. Increasedlocal expression of bFGF andKGF and their receptors inprimitive tubules suggests that bFGF and KGFmay playan important role in the development of RD.

Key words Renal dysplasia á Basic ®broblast growthfactor (bFGF, FGF-2) á Keratinocyte growth factor(KGF, FGF-7)

Introduction

Kidney development requires reciprocal interactionsbetween the ureteric bud and the metanephrogenic mes-enchyme. The ureteric bud induces mesenchymal cells toproliferate and convert to epithelia. Renal dysplasia(RD) is an abnormal, disorganized development of renalparenchyma that results in a de®cit in the amount offunctional renal tissue. Dysplastic renal tissue is charac-terized by primitive ducts associated with increasedmesenchyme, from which cartilage or muscle may de-velop [6]. This intimate association of epithelium andmesenchyme in the dysplastic kidney closely resemblesthat seen in the developing kidney, and suggests thatdysplasia represents an abnormal persistence of embry-onic renal tissue. Segmental dysplasia is much more fre-quent than total involvement of the kidney, and is usuallysituated in the upper pole of a duplex kidney. It is notclear why such embryonic-like tissue does not mature,but the frequent presence of ureteric abnormalities indi-cates that there may be partial failure of the ureteric budto communicate with the mesonephric blastema.

Several polypeptide growth factors (GF) that interactwith target cells through a cell-surface membrane re-ceptor have been reported to be involved in the regula-tion of urothelial-cell growth in normal and pathologicalconditions. It has been suggested that ®broblast GFs(FGFs) may regulate aspects of kidney organogenesis,and both acidic (aFGF, FGF-1) and basic FGFs(bFGF, FGF-2) have been isolated from kidney tissue[2, 11, 13]. Recent reports have demonstrated that FGFsmay be important kidney-derived regulators of renalproximal-tubule epithelial cells in vivo and in vitro [13].bFGF has been reported to be a mitogen for renal-tu-bule epithelial cells. Keratinocyte GF (KGF, FGF-7),which belongs to the FGF family, is believed to be aparacrine mediator of epithelial-cell proliferation [4, 10].Both bFGF and KGF interact with target cells throughspeci®c cell-surface membrane receptors: FGF (¯g) andKGF receptors (bek), respectively. The aim of this study

Pediatr Surg Int (2000) 16: 306±309 Ó Springer-Verlag 2000

H. Shima á H. Tazawa á P. Puri (&)Children's Research Centre,Our Lady's Hospital for Sick Children,Crumlin, Dublin 12, Ireland

was to investigate the immunoactivity of bFGF andKGF and their receptors in the dysplastic kidney inorder to further understand the pathogenesis of RD.

Materials and methods

Specimens of dysplastic upper poles of duplex kidneys from tenpatients (mean age 3.6 years, range 8 months to 6 years, six femaleand four male) were obtained at the time of upper-pole nephrec-tomy. All children presented with urinary tract infections.Subsequent imaging studies (ultrasonography, micturating cysto-urethography, urography, and/or nephroscintigraphy) revealed aduplex kidney with a nonfunctioning or poorly-functioning uppermoiety. Age-matched control material (age range 5 months±7 years)included six kidney specimens taken at autopsy from patientswithout evidence of urologic disease. Both dysplastic and controlkidneys had been ®xed and processed under identical conditions.

Formalin-®xed, para�n-embedded sections (4 lm) of the re-sected kidney specimens were mounted on poly-L-lysine-coatedglass sides. A demasking technique to expose the antigenic site forimmunohistochemistry of bFGF was performed with heat in citricbu�er twice for 5 min using a microwave oven. Immunohisto-chemistry was performed using the streptavidin-biotin-alkalinephosphatase complex (sABC) method and anti-human antibodiesin phosphate-bu�ered saline (PBS) with 10% normal serum: bFGFpolyclonal antibody (1:800 dilution, Santa Cruz Biotechnology,Santa Cruz, CA), KGF polyclonal antibody (1:400, Santa Cruz),¯g monoclonal antibody (1:400, Chemicon, Harrow, UK), and bekpolyclonal antibody (1:100, Santa Cruz). The sections were incu-bated in antiserum at 4 °C overnight. A biotin-labelled secondantibody at 1:300 dilution (Dako-Patts, Glostrup, Denmark) wasplaced in PBS for 1 h and the sABC steps (30 min, Dako-Patts)were performed at room temperature. Alkaline phosphatase (AP)was visualized by a new fuchsin substrate system (Dako-Patts) withan endogenous AP inhibitor, levamisole (Dako-Patts).

Results

Dysplastic kidney specimens contained varying amountsof dysplastic tissue composed of areas of loose mesenc-hyme containing primitive tubules. The dysplastic tissuein some areas was distinctly demarcated from adjoiningnormal renal tissue, but in other areas merged imper-ceptibly with normal tissue. No age-related variation inthe intensity of staining was observed.

bFGF immunohistochemistry

In the cortex of the normal kidneys, there was weakbFGF immunoreactivity in the epithelium of the prox-imal and distal tubules and Bowman's capsule (Fig. 1a).In the medulla, Henle's loop and the collecting ductsexpressed weak bFGF immunoreactivity. In the dys-plastic kidneys there was strong bFGF immunoreactiv-ity in the epithelium of the primitive tubules (Fig. 1b).

¯g immunohistochemistry

In the cortex of normal kidneys, there was absent orweak ¯g immunoreactivity in the epithelium of theproximal and distal tubules and Bowman's capsule

(Fig. 2a). In the medulla, Henle's loop and the collectingducts expressed very weak ¯g immunoreactivity. In thedysplastic kidneys, there was strong ¯g immunoreactiv-ity in the epithelium of the primitive tubules (Fig. 2b).

KGF immunohistochemistry

In the cortex of the normal kidneys, there was absentKGF immunoreactivity in the epithelium of the proxi-mal and distal tubules and Bowman's capsule (Fig. 3a).In the medulla, Henle's loop and the collecting ductsdemonstrated absent KGF immunoreactivity. In thedysplastic kidneys, there was strong KGF immunore-activity in the epithelium of the primitive tubules(Fig. 3b).

bek immunohistochemistry

In the cortex of normal kidneys, there was scant bek-immunopositive epithelium in the proximal and distaltubules and Bowman's capsule (Fig. 4a). In the medulla,Henle's loop and the collecting ducts also expressed fewbek-immunopositive cells. In the dysplastic kidneys,there was strong bek immunoreactivity in the epitheliumof the primitive tubules (Fig. 4b).

Discussion

The development of dysplastic renal tissue may be in-¯uenced by several factors including re¯ux, infection,and defective communication of the ureteric bud withthe renal blastema, but it is likely that defective ex-pression of factors involved in normal morphogenesismay play a major role. It has been well-described thatcellular growth and di�erentiation during embryogenesisand organogenesis occurs under the control of manyGFs. Several GFs play an important role in kidneygrowth and di�erentiation and recovery from injury ofrenal-tubular epithelial cells both in vivo and in vitro.They promote renal mitogenesis and motogenesis,thereby potentially accelerating the process of renal-tubular regeneration [4, 5, 7±10]. It has been reportedthat proliferation and di�erentiation of renal-tubularepithelium can be stimulated by a number of GFs,including bFGF and KGF [1, 10]. However, there islittle information on how GFs mediate both renal dif-ferentiation in the normal kidney and abnormal anddisorganized development in RD.

The most striking ®nding in this study was the strongimmunoreactivity of bFGF and KGF and their receptorsin the primitive tubules of dysplastic kidneys. In contrast,bFGF, KGF, ¯g, and bek immunoreactivity was eitherabsent or weak in the normal kidneys. To our knowledge,this is the ®rst observation that the immunoreactivity ofbFGF,KGF, and their receptors is markedly increased inthe dysplastic kidney, which shows arrested development

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at an early stage of morphogenesis. Our ®ndings suggestthat the local production of peptide GFs may be involvedin the development of RD.

Recently, Barasch et al. [3] reported that at earlystages of nephrogenesis, the ureteric bud rescues themetanephrogenic mesenchyme from apoptosis by se-creting GFs that mediate this activity. One of these GFsis bFGF, which is synthesized by the ureteric bud whenpenetrating the mesenchyme [3]. bFGF rescues threetypes of progenitors found in the mesenchyme: precur-sors of tubular epithelia, precursors of capillaries, andcells that regulate growth of the ureteric bud. Further-more, it has been suggested that the ureteric bud in vivonot only mediates mesenchymal epithelial conversion,but independently determines the number of mesenchy-mal cells available to form nephrons and, ultimately,nephron numbers [3]. We speculate that the abnormalexpression of bFGF and KGF and their receptors mayaccount for the variations in the number of nephronsfound in the human kidney, and may be responsible forcongenital RD.

References

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Fig. 1a, b Immunohistochemistry of basic ®broblast growth factor(bFGF). a Normal kidney: weak bFGF immunoreactivity inepithelium of proximal and distal tubules and Bowman's capsule(´200). b Dysplastic kidney: strong bFGF immunoreactivity inepithelium of primitive tubules (arrow) (´300)Fig. 2a, b Immunohistochemistry of ®broblast growth factor receptor(¯g). a Normal kidney: weak ¯g immunoreactivity in epithelium ofproximal and distal tubules and Bowman's capsule (´200). bDysplastic kidney: strong ¯g immunoreactivity in epithelium ofprimitive tubules (arrow) (´300)Fig. 3a, b Immunohistochemistry of keratinocyte growth factor(KGF). a Normal kidney: lack of KGF immunoreactivity inepithelium of proximal and distal tubules and Bowman's capsule(´200). b Dysplastic kidney: strong KGF immunoreactivity inepithelium of primitive tubules (arrow) (´300)Fig. 4a, b Immunohistochemistry of keratinocyte growth factorreceptor (bek). a Normal kidney: Scant few bek-immunopositiveepithelium in proximal and distal tubules and Bowman's capsule(arrowhead) (´200). b Dysplastic kidney: strong bek immunoreactivityin epithelium of primitive tubules (arrow) (´300)

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