Iterative development of visual control systems in a research vivarium.

The goal of this study was to test the hypothesis that reintroduction of Continuous Performance Improvement (CPI) methodology, a lean approach to management at Seattle Children's (Hospital, Research Institute, Foundation), would facilitate engagement of vivarium employees in the development and sustainment of a daily management system and a work-in-process board. Such engagement was implemented through reintroduction of aspects of the Toyota Production System. Iterations of a Work-In-Process Board were generated using Shewhart's Plan-Do-Check-Act process improvement cycle. Specific attention was given to the importance of detecting and preventing errors through assessment of the following 5 levels of quality: Level 1, customer inspects; Level 2, company inspects; Level 3, work unit inspects; Level 4, self-inspection; Level 5, mistake proofing. A functioning iteration of a Mouse Cage Work-In-Process Board was eventually established using electronic data entry, an improvement that increased the quality level from 1 to 3 while reducing wasteful steps, handoffs and queues. A visual workplace was realized via a daily management system that included a Work-In-Process Board, a problem solving board and two Heijunka boards. One Heijunka board tracked cage changing as a function of a biological kanban, which was validated via ammonia levels. A 17% reduction in cage changing frequency provided vivarium staff with additional time to support Institute researchers in their mutual goal of advancing cures for pediatric diseases. Cage washing metrics demonstrated an improvement in the flow continuum in which a traditional batch and queue push system was replaced with a supermarket-type pull system. Staff engagement during the improvement process was challenging and is discussed. The collective data indicate that the hypothesis was found to be true. The reintroduction of CPI into daily work in the vivarium is consistent with the 4P Model of the Toyota Way and selected Principles that guide implementation of the Toyota Production System.

The a3 problem solving report: a 10-step scientific method to execute performance improvements in an academic research vivarium.

The purpose of this study was to illustrate the application of A3 Problem Solving Reports of the Toyota Production System to our research vivarium through the methodology of Continuous Performance Improvement, a lean approach to healthcare management at Seattle Children's (Hospital, Research Institute, Foundation). The Report format is described within the perspective of a 10-step scientific method designed to realize measurable improvements of Issues identified by the Report's Author, Sponsor and Coach. The 10-step method (Issue, Background, Current Condition, Goal, Root Cause, Target Condition, Countermeasures, Implementation Plan, Test, and Follow-up) was shown to align with Shewhart's Plan-Do-Check-Act process improvement cycle in a manner that allowed for quantitative analysis of the Countermeasure's outcomes and of Testing results. During fiscal year 2012, 9 A3 Problem Solving Reports were completed in the vivarium under the teaching and coaching system implemented by the Research Institute. Two of the 9 reports are described herein. Report #1 addressed the issue of the vivarium's veterinarian not being able to provide input into sick animal cases during the work day, while report #7 tackled the lack of a standard in keeping track of weekend/holiday animal health inspections. In each Report, a measurable Goal that established the basis for improvement recognition was present. A Five Whys analysis identified the Root Cause for Report #1 as historical work patterns that existed before the veterinarian was hired on and that modern electronic communication tools had not been implemented. The same analysis identified the Root Cause for Report #7 as the vivarium had never standardized the process for weekend/holiday checks. Successful outcomes for both Reports were obtained and validated by robust audit plans. The collective data indicate that vivarium staff acquired a disciplined way of reporting on, as well as solving, problems in a manner consistent with high level A3 Thinking.

Identification of a bladder cancer-specific ligand using a combinatorial chemistry approach.

OBJECTIVES: To develop bladder cancer-specific ligands using a combinatorial chemistry approach. MATERIALS AND METHODS: We performed a high-throughput one-bead one-compound combinatorial chemistry approach to identify ligands that bound to bladder transitional cell carcinoma cells. The whole-cell binding assay allowed successful identification of a few peptides that bound selectively to bladder cancer cells. Single cell suspensions derived from clinical bladder cancer specimens and cell lines were used to determine the binding specificity. Studies with mouse xenografts were performed to determine the in vivo binding and targeting efficiency, specificity, and biodistribution of one of the ligands. RESULTS: One cyclic peptide named PLZ4 (amino acid sequence: cQDGRMGFc) was identified that could selectively bind to bladder cancer cell lines and all of the 5 primary bladder cancer cells from human patients, but not to normal urothelial cells, cell mixtures from normal bladder specimens, fibroblasts, and blood cells. Comparison of PLZ4 binding to cell lines of different cancer origins showed that it was bladder cancer-specific (P < 0.05). PLZ4 could bind to tumor cells treated with urine at pH 6.0, but not to noncancerous cells collected from the urine of 4 patients actively being treated with intravesical Bacillus Calmette-Guerin therapy. In vivo and ex vivo imaging studies showed that PLZ4 linked to Cy5.5 fluorescent dye administered via tail vein injection was specifically taken up in mouse xenografts developed from excised fresh human bladder cancer specimens. Several ligands contain the same DGR motif, but only PLZ4 was bladder cancer-specific. We performed alanine walk and rainbow bead coding experiments, and found that the C-terminal GF residues were also important for cell binding and modulated the binding specificity. CONCLUSIONS: PLZ4 has the potential to be used for targeted therapy and imaging detection during diagnosis and follow-up/surveillance of noninvasive and advanced bladder cancer.

The adaptor-related protein complex 2, alpha 2 subunit (AP2α2) gene is a peroxisome proliferator-activated receptor cardiac target gene.

A peroxisome proliferator-actived receptor (PPAR) response element (RE) in the promoter region of the adaptor-related protein complex 2, alpha 2 subunit (AP2α2) of mouse heart has been identified. The steroid hormone nuclear PPARs and the retinoid X receptors (RXRs) are important transcriptional factors that regulate gene expression, cell differentiation and lipid metabolism. They form homo- (RXR) and hetero- (PPAR-RXR) dimers that bind DNA at various REs. The AP2α2 gene is part of complex and process that transports lipids and proteins from the plasma membrane to the endosomal system. A PPAR activator (Wy14643) and DMSO (vehicle) was introduced into control and δ337T thyroid hormone receptor (TRß1) transgenic mice. Heart tissue was extracted and AP2α2 gene expression was compared using Affymetrix expression arrays and qRT PCR among four groups [control, control with Wy14643, δ337T TRß1 and δ337T TRß1 with Wy14643]. The gene expression of AP2α2 in the Wy14643 control and transgenic mouse groups was significantly up regulated over the vehicle mouse groups in both the array (p < 0.01) and qRT PCR (p < 0.01) studies. Duplex oligo DNAs containing the PPAR/RXR motif (AGGTCA/TCCAGT) from the AP2α2 promoter were used in EMSA to verify binding of the PPAR and RXR receptors to their REs. pGL4.0 [Luc] constructs of the AP2α2 promoter with and without the PPAR/RXR motifs were co-transfected with mouse PPARα, ß or γ1 into HepG2 cells and used in lucerifase assays to verify gene activation. In conclusion our study revealed that PPARα regulates the mouse cardiac AP2α2 gene in both the control and transgenic mouse.

Differential affinity of vitronectin versus collagen for synthetic biodegradable scaffolds for urethroplastic applications.

Cell-seeded synthetic polymer scaffolds constitute an emerging technology for urethroplastic applications. The study goal was to identify urethral proteins appropriate for cell attachment and optimize their adsorption onto two types of scaffolds: porous poly(ester urethane) with a poly(caprolactone) soft segment (PEU-PCL) and poly-(96% L/4% D)-lactic acid (P96L/4DLA). Specimens from eight men undergoing urethral reconstruction for stricture diseases were subjected to immunohistochemical analysis. Type I collagen, type IV collagen and vitronectin were detected at the interface between the epithelium and its basement membrane. Electrophoresis confirmed that polypeptide chains in the starting material were also present in fractions eluted from adsorbed scaffolds. Over a 4 week incubation assay, only vitronectin exhibited 100% retention levels for all scaffolds. The saturation point for each protein on each scaffold type was determined by titration and ELISA. The collective evidence indicates the concept that vitronectin > type IV collagen > type I collagen are preferred adsorption proteins for PEU-PCL and P96L/4DLA.

Dual sources of vitronectin in the human lower urinary tract: synthesis by urothelium vs. extravasation from the bloodstream.

Vitronectin (VN), secreted into the bloodstream by liver hepatocytes, is known to anchor epithelial cells to basement membranes through interactions with cell surface integrin receptors. We report here that VN is also synthesized by urothelial cells of urothelium in vivo and in vitro. In situ hybridization, dideoxy sequencing, immunohistochemistry, and ELISA of urothelial cell mRNA, cDNA, tissue, and protein extracts demonstrated that the VN gene is active in vivo and in vitro. The expression of VN by urothelium is hypothesized to constitute one of several pathways that anchor basal cells to an underlying substratum and explains why urothelial cells adhere to glass and propagate under serum-free conditions. Therefore, two sources of VN in the human urinary bladder are recognized: 1) localized synthesis by urothelial cells and 2) extravasation of liver VN through fenestrated capillaries. When human plasma was fractionated by denaturing heparin affinity chromatography, VN was isolated in a biologically active form that supported rapid spreading of urothelial cells in vitro under serum-free conditions. This activity was inhibited by the matricellular protein SPARC via direct binding of VN to SPARC through a Ca(+2)-dependent mechanism. A novel form of VN, isolated from the same heparin affinity chromatography column and designated as the VN(c) chromatomer, also supported cell spreading but failed to interact with SPARC. Therefore, the steady-state balance among urothelial cells, their extracellular milieu, and matricellular proteins constitutes a principal mechanism by which urothelia are anchored to an underlying substrata in the face of constant bladder cycling.

Fibroblast growth factor-10 signals development of von Brunn's nests in the exstrophic bladder.

von Brunn's nests have long been recognized as precursors of benign lesions of the urinary bladder mucosa. We report here that von Brunn's nests are especially prevalent in the exstrophic bladder, a birth defect that predisposes the patient to formation of bladder cancer. Cells of von Brunn's nest were found to coalesce into a stratified, polarized epithelium which surrounds itself with a capsule-like structure rich in types I, III, and IV collagen. Histocytochemical analysis and keratin profiling demonstrated that nested cells exhibited a phenotype similar, but not identical, to that of urothelial cells of transitional epithelium. Immunostaining and in situ hybridization analysis of exstrophic tissue demonstrated that the FGF-10 receptor is synthesized and retained by cells of von Brunn's nest. In contrast, FGF-10 is synthesized and secreted by mesenchymal fibroblasts via a paracrine pathway that targets basal epithelial cells of von Brunn's nests. Small clusters of 10pRp cells, positive for both FGF-10 and its receptor, were observed both proximal to and inside blood vessels in the lamina propria. The collective evidence points to a mechanism where von Brunn's nests develop under the control of the FGF-10 signal transduction system and suggests that 10pRp cells may be the original source of nested cells.

Extracellular matrix protein coatings for facilitation of urothelial cell attachment.

Synthetic urothelium is an important goal for the tissue-engineering field that would have great utility for treating diseases and congenital defects affecting the urinary tract. A key step in the development of synthetic tissue is optimizing the conditions for coating biomaterials with cells of interest. Initial cell attachment is an important consideration when designing tissue-engineering scaffolds. The scaffold environment must also be conducive to cell proliferation and differentiation. The most popular materials for tissue-engineering scaffold often have suboptimal properties when analyzed for cell attachment and growth. It would then be of interest to know, for urinary tract tissue-engineering applications, which extracellular matrix protein coatings can facilitate urothelial cell attachment and encourage growth. Cells grown on 96-well cycloolefin plates coated with type IV or type I collagen exhibited improved initial attachment over plates coated with fibronectin or laminin. After 20 h, deoxyribonucleic acid synthesis was found to increase in cultures grown on type IV collagen, fibronectin, and laminin. Total metabolic activity of urothelial cell cultures was also monitored, and no difference was seen between any protein-coating conditions. The development of such reliable assays will be beneficial in monitoring the fate of scaffolds seeded with human urothelial cells.

Compensatory paracrine mechanisms that define the urothelial response to injury in partial bladder outlet obstruction.

Diseases and conditions affecting the lower urinary tract are a leading cause of dysfunctional sexual health, incontinence, infection, and kidney failure. The growth, differentiation, and repair of the bladder's epithelial lining are regulated, in part, by fibroblast growth factor (FGF)-7 and -10 via a paracrine cascade originating in the mesenchyme (lamina propria) and targeting the receptor for FGF-7 and -10 within the transitional epithelium (urothelium). The FGF-7 gene is located at the 15q15-q21.1 locus on chromosome 15 and four exons generate a 3.852-kb mRNA. Five duplicated FGF-7 gene sequences that localized to chromosome 9 were predicted not to generate functional protein products, thus validating the use of FGF-7-null mice as an experimental model. Recombinant FGF-7 and -10 induced proliferation of human urothelial cells in vitro and transitional epithelium of wild-type and FGF-7-null mice in vivo. To determine the extent that induction of urothelial cell proliferation during the bladder response to injury is dependent on FGF-7, an animal model of partial bladder outlet obstruction was developed. Unbiased stereology was used to measure the percentage of proliferating urothelial cells between obstructed groups of wild-type and FGF-7-null mice. The stereological analysis indicated that a statistical significant difference did not exist between the two groups, suggesting that FGF-7 is not essential for urothelial cell proliferation in response to partial outlet obstruction. In contrast, a significant increase in FGF-10 expression was observed in the obstructed FGF-7-null group, indicating that the compensatory pathway that functions in this model results in urothelial repair.

The motif of SPARC that inhibits DNA synthesis is not a nuclear localization signal.

SPARC (secreted protein acidic and rich in cysteine), although primarily known as a secreted, matricellular protein, has also been identified in urothelial cell nuclei. Many biological activities, including inhibition of cell adhesion and repression of DNA synthesis, have been ascribed to SPARC, but the influence of its intracellular localization on each of these activities is unknown. When exposed by epitope retrieval and nuclear matrix unmasking techniques, endogenous SPARC was found to localize strongly to the nuclei and the nuclear matrix of cultured urothelial cells. Live-cell time-lapse imaging revealed that exogenous fluorescently labeled recombinant (r) SPARC was taken up from medium over a 16 h period and accumulated inside cells. Two variants of rSPARC with alterations in its putative nuclear localization signal (NLS) were generated to investigate the existence and effects of the NLS. These variants demonstrated similar biophysical characteristics as the wild-type protein. Visualization by a variety of techniques, including live-cell imaging, deconvolution microscopy, and cell fractionation, all concurred that exogenous rSPARC was not able to localize to cell nuclei, but instead accumulated as perinuclear clusters. Localization of the rSPARC NLS variants was no different than wild-type, arguing against the presence of an active NLS in rSPARC. Imaging experiments showed that only permeabilized, dead cells avidly took up rSPARC into their nuclei. The rSPARC(no NLS) variant proved ineffective at inhibiting DNA synthesis, whereas the rSPARC(strong NLS) variant was a more potent inhibitor of DNA synthesis than was wild-type rSPARC. The motif of SPARC that inhibits the synthesis of urothelial cell DNA is therefore not a nuclear localization signal, but its manipulation holds therapeutic potential to generate a "Super-SPARC" that can quiesce proliferative tissues.

Translocation of fibroblast growth factor-10 and its receptor into nuclei of human urothelial cells.

Fibroblast growth factor-10 (FGF-10), a mitogen for the epithelial cells lining the lower urinary tract, has been identified inside urothelial cells, despite its acknowledged role as an extracellular signaling ligand. Recombinant (r)FGF-10 was determined by fluorescence microscopy optical sectioning to localize strongly to nuclei inside cultured urothelial cells. To clarify the possible role of a nuclear localization signal (NLS) in this translocation, a variant of rFGF-10 was constructed which lacked this sequence. rFGF-10(no NLS) was found in cytoplasm to a far greater degree than rFGF-10, identifying this motif as a possible NLS. Furthermore, this variant displayed poor or non-existent bioactivity compared to the wild-type protein in triggering mitogenesis in quiescent urothelial cells. The presence of rFGF-10(no NLS) in the nucleus suggested that additional interactions were also responsible for the nuclear accumulation of rFGF-10. The FGF-10 receptor was observed in cell nuclei regardless of the presence or concentration of exogenous rFGF-10 ligand. Co-localization studies between rFGF-10 and the FGF-10 receptor revealed a strong intracellular relationship between the two. This co-localization was seen in nuclei for both rFGF-10 and for rFGF-10(no NLS), although the correlation was weaker for rFGF-10(no NLS). These data show that an NLS-like motif of rFGF-10 is a partial determinant of its intracellular distribution and is necessary for its mitogenic activity. These advancements in the understanding of the activity of FGF-10 present an opportunity to engineer the growth factor as a therapeutic agent for the healing of damaged urothelial tissue.

FGF-10 and its receptor exhibit bidirectional paracrine targeting to urothelial and smooth muscle cells in the lower urinary tract.

Control of the regenerative properties of urothelial tissue would greatly aid the clinician in the management of urinary tract disease and disorders. Fibroblast growth factor 10 (FGF-10) is a mitogen which is particularly promising as a protein therapy for urothelial injury. The spatial synthesis, transport, targeting, and mechanistic pathway of FGF-10 and its receptor were studied in a human urothelial cell culture model and in fixed sections of lower urinary tract tissue. Synthesis of FGF-10 was restricted to mesenchymal fibroblasts, and secreted FGF-10 exhibited paracrine transport to two proximal sites, transitional epithelium and smooth muscle cell bundles, both of which were also the exclusive sites of FGF-10 receptor synthesis. The addition of recombinant FGF-10 to quiescent urothelial cells in vitro was sufficient to stimulate DNA synthesis. This stimulation was through a pathway independent of the epidermal growth factor receptor pathway. Deconvolution, light and transmission electron microscopic studies captured FGF-10 and its receptor in association with the urothelial cell surface, in cytoplasm, and within nuclei, observations that describe the mechanism that transduces the mitogenic signal in these tissues. Localization of the FGF-10 receptor to the superficial urothelial layer is clinically significant because intravesical administration of FGF-10 may provide the clinician a means to control the turnover of transitional epithelium in bladder disorders such as interstitial cystitis.

The C-terminal Ca2+-binding domain of SPARC confers anti-spreading activity to human urothelial cells.

The anti-spreading activity of secreted protein acidic and rich in cysteine (SPARC) has been assigned to the C-terminal third domain, a region rich in alpha-helices. This "extracellular calcium-binding" (EC) domain contains two EF-hands that each coordinates one Ca2+ ion, forming a helix-loop-helix structure that not only drives the conformation of the protein but is also necessary for biological activity. Recombinant (r) EC, expressed in E. coli, was fused at the C-terminus to a His hexamer and isolated under denaturing conditions by nickel-chelate affinity chromatography. rEC-His was renatured by procedures that simultaneously (i) removed denaturing conditions, (ii) catalyzed disulfide bond isomerization, and (iii) initiated Ca2+-dependent refolding. Intrinsic tryptophan fluorescence and circular dichroism spectroscopies demonstrated that rEC-His exhibited a Ca2+-dependent conformation that was consistent with the known crystal structure. Spreading assays confirmed that rEC-His was biologically active through its ability to inhibit the spreading of freshly plated human urothelial cells propagated from transitional epithelium. rEC-His and rSPARC-His exhibited highly similar anti-spreading activities when measured as a function of concentration or time. In contrast to the wild-type and EC recombinant proteins, rSPARC(E268F)-His, a point substitution mutant at the Z position of EF-hand 2, failed to exhibit both Ca2+-dependent changes in alpha-helical secondary structure and anti-spreading activity. The collective data provide evidence that the motif of SPARC responsible for anti-spreading activity was dependent on the coordination of Ca2+ by a Glu residue at the Z position of EF-hand 2 and provide insights into how adhesive forces are balanced within the extracellular matrix of urothelial cells. .

Spreading of embryologically distinct urothelial cells is inhibited by SPARC.

The AON epitope of secreted protein acidic and rich in cysteine (SPARC) is a conserved motif expressed by human SPARC in a variety of human cell types. Through the use of a monoclonal antibody that recognizes this epitope, transitional epithelium was found to restrict expression of SPARC to the suprabasal and intermediate layer. Such intracellular expression was defined by immunoreactive signals that localized to the apical plasma membranes of suprabasal and intermediate cells. Polarization of SPARC to apical plasma membranes of suprabasal cells was retained in vitro by a subpopulation of cells that exhibited characteristics of suprabasal cells--cell-cycle quiescence, large cell volumes, and multiple nuclei. In contrast, the basal layer of transitional epithelium in vivo and cycling cells in vitro did not exhibit this apical staining pattern, but instead sequestered the SPARC polypeptide within urothelial cytoplasm and/or nuclei, as revealed by immunohistochemical analysis. Elution of soluble proteins and DNA from urothelial cells revealed the presence of SPARC within the nuclear matrix--and that SPARC colocalized with the nuclear matrix Ki-67 antigen. rSPARC activity was demonstrated and quantified with a rounding assay whereby the spreading of freshly plated cells was inhibited by recombinant SPARC in a concentration- and time-dependent manner. Inhibition of spreading was observed in urothelial cells derived from endoderm (bladder) and mesoderm (ureter) germ layers. Statistically significant differences were seen between urothelial cells from these two layers. Mesodermal cells recovered more slowly from the inhibitory effects of rSPARC, such that at hour 6 endodermal cells underwent significantly more spreading, as shown by a rounding index (RI). These experiments provide new insights about the matricellular trafficking of SPARC and suggest that intra- and extra-cellular localization patterns influence the development, homeostasis, and differentiation of transitional epithelium.

Cleavage of the matricellular protein SPARC by matrix metalloproteinase 3 produces polypeptides that influence angiogenesis.

SPARC, a matricellular protein that affects cellular adhesion and proliferation, is produced in remodeling tissue and in pathologies involving fibrosis and angiogenesis. In this study we have asked whether peptides generated from cleavage of SPARC in the extracellular milieu can regulate angiogenesis. Matrix metalloproteinase (MMP)-3, but not MMP-1 or 9, showed significant activity toward SPARC. Limited digestion of recombinant human (rhu)SPARC with purified catalytic domain of rhuMMP-3 produced three major fragments, which were sequenced after purification by HPLC. Three synthetic peptides (Z-1, Z-2, and Z-3) representing motifs from each fragment were tested in distinct assays of angiogenesis. Peptide Z-1 (3.9 kDa, containing a Cu2+-binding sequence KHGK) exhibited a biphasic effect on [3H]thymidine incorporation by cultured endothelial cells and stimulated vascular growth in the chick chorioallantoic membrane (CAM). In contrast, peptides Z-2 (6.1 kDa, containing Ca2+-binding EF hand-1) and Z-3 (2.2 kDa, containing neither Cu2+-binding motifs nor EF hands), inhibited cell proliferation in a concentration-dependent manner and exhibited no effects on vessel growth in the CAM. Reciprocal results were obtained in a migration assay in native collagen gels: peptide Z-1 was ineffective over a range of concentrations, whereas Z-2 or Z-3 stimulated cell migration. Therefore, proteolysis of SPARC by MMP-3 produced peptides that regulate endothelial cell proliferation and/or migration in vitro in a mutually exclusive manner. One of these peptides containing KHGK also demonstrated a concentration-dependent effect on angiogenesis.

Induction of urothelial cell proliferation by fibroblast growth factor-7 in RAG1-deficient mice.

Fibroblast growth factor-7 (FGF-7, keratinocyte growth factor, KGF) is a 163 amino acid glycoprotein synthesized and secreted by mesenchymal cells (e.g. fibroblasts/fibrocytes) in epithelial organs, thereby functioning as a paracrine mediator of epithelial cell proliferation. In the urinary bladder, FGF-7 is transported from the lamina propria across the urothelial basement membrane to where it ultimately binds to splice variants of the FGFR2 receptor present on the basolateral surface of transitional epithelial cells. We administered 100 micrograms/ml (i.p.) recombinant FGF-7 (rFGF-7) to RAG1-deficient mice (n = 3) for 7 days and observed a striking expansion of the urinary bladder urothelium. This expansion was characterized by a layer of stratified urothelium > 20 cells thick and by positive immunostaining for the proliferation marker Ki-67. In contrast, RAG1-deficient mice (n = 3) that received only buffer injection did not exhibit detectable urothelial expansion. rFGF-7 was detected by immunoblot analyses in the serum, but not in the urine, from RAG1-deficient mice that received the recombinant protein. Mice that have a targeted disruption in the gene encoding the V(D)J recombination activation gene RAG1 have small lymphoid organs with no mature B and T lymphocytes, due to the inability of cell progenitors to perform V(D)J recombination. The biological activity of FGF-7 in RAG-1 mice indicates that immuno-dependent mechanisms are not required for the induction of urothelial cell proliferation by this epithelial cell-specific growth factor.

Localization of SPARC in developing, mature, and chronically injured human allograft kidneys.

BACKGROUND: The matricellular protein SPARC (secreted protein acidic and rich in cysteine) is expressed during development, tissue remodeling and repair. It functions as an endogenous inhibitor of cell proliferation, regulates angiogenesis, regulates cell adhesion to extracellular matrix, binds cytokines such as platelet derived growth factor and stimulates transforming growth factor-beta (TGF-beta) production. This study describes the expression of SPARC during human renal development, in normal kidneys and during renal allograft rejection. METHODS: A total of 60 renal specimens, including normal areas from tumor nephrectomies (N = 24), fetal kidneys (N = 27) and explanted renal allografts (N = 9), were included in the study. SPARC protein was localized by immunohistochemistry using two different antibodies. On consecutive sections SPARC mRNA was detected by in situ hybridization. RESULTS: In the normal adult kidney SPARC protein was expressed by visceral and parietal epithelial cells, collecting duct epithelium (CD), urothelium, smooth muscle cells of muscular arteries and focally in interstitial cells. During renal development immature glomeruli demonstrated a polarized SPARC expression in visceral epithelial cells at their surface abutting the capillary basement membranes. In the fully differentiated glomeruli the expression pattern mirrored that of the adult kidney. Furthermore, SPARC was abundantly expressed by derivatives of the ureteric bud, and smooth muscle cells of arterial walls. During chronic allograft rejection SPARC is expressed in neointimal arterial smooth muscle cells, infiltrating inflammatory cells as well as by interstitial myofibroblasts in areas of interstitial fibrosis. SPARC mRNA synthesis detected by in situ hybridization mirrored these protein expression patterns. CONCLUSION: These studies co-localize SPARC to several sites of renal injury previously shown to be sites of PDGF B-chain expression and/or activity. We speculate that SPARC could function as an accessory molecule in chronic PDGF-mediated sclerosing interstitial and vascular injury. SPARC localization to glomerular epithelial cells corresponds to similar findings in rodents, and may reflect its role in cell adhesion and /or regulation of cell shape.

Fibroblast growth factor-10 is a mitogen for urothelial cells.

Fibroblast growth factor (FGF)-10 plays an important role in regulating growth, differentiation, and repair of the urothelium. This process occurs through a paracrine cascade originating in the mesenchyme (lamina propria) and targeting the epithelium (urothelium). In situ hybridization analysis demonstrated that (i) fibroblasts of the human lamina propria were the cell type that synthesized FGF-10 RNA and (ii) the FGF-10 gene is located at the 5p12-p13 locus of chromosome 5. Recombinant (r) preparations of human FGF-10 were found to induce proliferation of human urothelial cells in vitro and of transitional epithelium of wild-type and FGF7-null mice in vivo. Mechanistic studies with human cells indicated two modes of FGF-10 action: (i) translocation of rFGF-10 into urothelial cell nuclei and (ii) a signaling cascade that begins with the heparin-dependent phosphorylation of tyrosine residues of surface transmembrane receptors. The normal urothelial phenotype, that of quiescence, is proposed to be typified by negligible levels of FGF-10. During proliferative phases, levels of FGF-10 rise at the urothelial cell surface and/or within urothelial cell nuclei. An understanding of how FGF-10 works in conjunction with these other processes will lead to better management of many diseases of the bladder and urinary tract.